Asthma Life

The Patent Description & Claims data below is from USPTO Patent Application 20130123260, Methods.

The disclosure relates to compounds of formula (I) for use in the treatment or prophylaxis of rhinovirus infection, methods of treating or preventing rhinovirus infection employing said compounds or pharmaceutical composition comprising the same. The disclosure also relates to compounds of formula (I) for use in the treatment or prophylaxis of exacerbation of respiratory disorders such as asthma, chronic obstructive pulmonary disease) (COPD), bronchitis and/or cystic fibrosis by rhinovirus infection.

BACKGROUND

Such is the impact of the relentless onslaught of viruses on living organisms, that pathogenic viral infection has been one of the principle selection processes defining the course of human evolution. At one extreme, infections lead to long term damaging changes to the affected organ resulting, for example, in liver failure in the case of hepatitis B infection, or alternatively to the onset of malignant disease, such as uterine cancer, arising from papilloma virus infection. Alternatively viral infections may stimulate exacerbations of pre-existing, underlying chronic diseases; as manifested in asthma sufferers from rhinovirus infection or even precipitate acute, life-threatening disease, as occurs from multi-organ failure seen in high risk patients infected with influenza.

Many patients diagnosed with asthma or COPD (continue to suffer from uncontrolled symptoms and from exacerbations of their medical condition which can result in hospitalisation. This occurs despite the use of the most advanced, currently available treatment regimens, comprising of combination products of an inhaled corticosteroid and a long acting β-agonist. Data accumulated over the last decade indicates that a failure to effectively manage the underlying inflammatory component of the disease in the lung is the most likely reason that treatment is already poor in such cases or becomes increasingly ineffective. Given the established efficacy of corticosteroids as anti-inflammatory agents and, in particular, of inhaled corticosteroids in the treatment of asthma, these findings have provoked intense investigation. Resulting studies have identified that some environmental assaults invoke inflammatory changes in patients which prove to be insensitive to the actions of corticosteroids. An example of such a stimulated response arises from virally-mediated upper respiratory tract infections (URTI), which have particular significance in increasing morbidity associated with asthma and COPD.

Epidemiologic investigations have revealed a strong association between the presence of viral, upper respiratory tract infections and a substantial percentage of the exacerbations suffered by patients already diagnosed with chronic respiratory diseases. Some of the most compelling data in this regard derives from longitudinal studies of children suffering from asthma (Papadopoulos N. G., Papi A., Psarras S. and Johnston S. L., Paediatr. Respir. Rev., 2004, 5(3):255-260.). A variety of additional studies support the conclusion that a viral infection can precipitate exacerbations and increase disease severity. For example, experimental clinical infections with rhinovirus have been reported to cause bronchial hyper-responsiveness to histamine in asthmatics which is unresponsive to treatment with corticosteroids (Grunberg K., Sharon R. F., et al., Am. J. Respir. Crit. Care Med., 2001, 164(10):1816-1822.). Further evidence derives from the association observed between disease exacerbations in patients with cystic fibrosis and HRV infections (Wat D., Gelder C. et al., J. Cyst. Fibros., 2008, 7:320-328.). Also consistent with this body of data is the finding that respiratory viral infections, including rhinovirus, represent an independent risk factor that correlates negatively with the 12 month survival rate in paediatric, lung transplant recipients (Liu M., Worley S. et al., Transpl. Infect. Dis., 2009, 11(4):304-312.).

Clinical research indicates that the viral load is proportionate to the observed symptoms and complications and, by implication, to the severity of inflammation. For example, lower respiratory tract symptoms and bronchial hyper-responsiveness following experimental rhinovirus infection correlated significantly with virus load (Message S. D., Laza-Stanca V. et al., PNAS, 2008, 105(36):13562-13567.). Similarly, in the absence of other viral agents, rhinovirus infections were commonly associated with lower respiratory tract infections and wheezing, when the viral load was high in immunocompetent paediatric patients (Gerna G., Piralla A. et al., J. Med. Virol., 2009, 81(8):1498-1507.).

Significantly, it has been reported recently that prior exposure to rhinovirus reduced the cytokine responses evoked by bacterial products in human alveolar macrophages (Oliver B. G. G., Lim S. et al., Thorax, 2008, 63:519-525.). Additionally, infection of nasal epithelial cells with rhinovirus has been reported to promote adhesion of bacteria, including S. aureus and H. influenzae (Wang J. H., Kwon H. J. and Yong J. J., The Laryngoscope, 2009, 119(7):1406-1411.). Such cellular effects may contribute to the increased probability of patients suffering a lower respiratory tract infection following an infection in the upper respiratory tract. Accordingly, it is therapeutically relevant to focus on the ability of novel interventions to decrease viral load in a variety of in vitro systems, as a surrogate predictor of their benefit in a clinical setting.

High risk groups, for whom a rhinovirus infection in the upper respiratory tract can lead to severe secondary complications, are not limited to patients with chronic respiratory disease. They include, for example, the immunocompromised who are prone to lower respiratory tract infection, as well as patients undergoing chemotherapy, who face acute, life-threatening fever. It has also been suggested that other chronic diseases, such as diabetes, are associated with a compromised immunodefence response. This increases both the likelihood of acquiring a respiratory tract infection and of being hospitalised as a result. (Peleg A. Y., Weerarathna T. et al., Diabetes Metab. Res. Rev., 2007, 23(1):3-13; Kornum J. B., Reimar W. et al., Diabetes Care, 2008, 31(8):1541-1545.).

While viral, upper respiratory tract infections are a cause of considerable morbidity and mortality in those patients with underlying disease or other risk factors; rhinovirus infections also represent a significant healthcare burden in the general population and are a major cause of missed days at school and lost time in the workplace. (Rollinger J. M. and Schmidtke M., Med. Res. Rev., 2010, 1:42-92.). These considerations make it clear that novel medicines, possessing improved efficacy over current therapies, are urgently required to prevent and treat rhinovirus-mediated upper respiratory tract infections. In general the strategies adopted for the discovery of improved antiviral agents have targeted various proteins produced by the virus, as the point of therapeutic intervention. However, the wide range of rhinovirus serotypes makes this a particularly challenging approach to pursue and may explain why, at the present time, a medicine for the prophylaxis and treatment of rhinovirus infections has yet to be approved by any regulatory agency.

BRIEF DESCRIPTION OF THE FIGURES

The legends for the figures are as follows: FP=fluticasone propionate; PI=pleconaril; BIRB=BIRB-796; ** indicates p<0.01, * indicates p<0.05 vs. HRV control; NT=not treated.

FIG. 1: The effects of Reference Compounds 1 and 2, pleconaril and fluticasone propionate on extracellular HRV16 load.

FIG. 2: The effects of Reference Compounds 1 and 2, pleconaril and fluticasone propionate on HRV16 mRNA detected in cellular extract.

FIG. 3: The effects of Reference Compounds 1 and 2 and of pleconaril on HRV39 viral load in air-liquid interface cultured nasal epithelial cells.

FIG. 4: The effects of delaying treatment with Reference Compounds 1 and 2 on HRV extracellular load.

FIG. 5: The effects of Reference Compound 1, Reference Compound 2 and pleconaril on HRV39-induced IL-8 release in air-liquid interface culture nasal epithelial cells.

FIG. 6: Summary of the pathways and downstream consequences of RNA virus signalling.

FIG. 7: The effects of treatment with Reference Compound 2, pleconaril, BIRB-796 and fluticasone propionate on interferon β induction (mRNA) by HRV16 in MRC-5 cells.

FIG. 8: The effects of Reference Compounds 1 and 2, BIRB-796, fluticasone propionate, BAY 61-036 and Dasatinib on HRV-16 induced activation of IRF-3.

FIG. 9: The effects of Reference Compound 1, Reference Compound 2, BIRB-796, fluticasone propionate, BAY 61-036 and Dasatinib on HRV-16 induced activation of NFκB.

FIG. 10: Plot of log [IC50 value at c-SRC×IC50 value at SYK] versus potency on HRV-16 virus load.

DETAILS OF THE INVENTION

Virus entry to the host, the first step en route to virus propagation, is associated with the activation of a number of signalling pathways in the host cell which are believed to play a prominent role in the initiation of inflammatory processes (reviewed by Ludwig S., Signal Transduction, 2007, 7:81-88.) and those of viral propagation and subsequent release. One such mechanism, which has been determined to play a role in influenza virus propagation in vitro, is activation of the phosphoinositide 3-kinase/Akt pathway. The pathway has been described as being activated by the NS1 protein of the virus (Shin Y. K., Liu Q. et al., J. Gen. Virol., 2007, 88:13-18), and its inhibition is reported to reduce the titres of progeny virus (Ehrhardt C., Marjuki H. et al, Cell Microbiol., 2006, 8:1336-1348.).

Furthermore, the MEK inhibitor U0 126 has been reported to inhibit viral propagation without detection of resistant variants of the virus (Ludwig S., Wolff T. et al., FEBS Lett., 2004, 561:37-43.). More recently, studies targeting inhibition of SYK kinase have demonstrated that the enzyme plays an important role in mediating rhinovirus cell entry and virus-induced inflammatory responses, including ICAM-1 up-regulation (Sanderson M. P., Lau C. W. et al., Inflamm. Allergy Drug Targets, 2009, 8:87-95.). SYK activity is reported to be controlled by c-SRCas an upstream kinase in HRV infection (Lau C. et al., J. Immunol., 2008, 180:870-880.). A small number of studies have reported activation of cellular SRC(SRC1 or p60-SRC) or SRC family kinase in response to infection with viruses:



Adenovirus has been reported to produce P13 kinase mediated activation of Akt through a cSRC dependent mechanism,
Rhinovirus-39 induced IL-8 production was suggested to depend upon SRC kinase activation in epithelial cells (Bentley J. K. and Newcomb D. C., J. Virol., 2007, 81:1186-1194.),
Activation of SRC kinase was suggested to be involved in the induction of mucin production by rhinovirus-14 in epithelial cells and sub-mucosal glands (Inoue D. and Yamava M., Respir. Physiol. Neurobiol., 2006, 154:484-499.).

These induced responses in host cells which relate to virus propagation are offset by the initiation of processes which are intended to prevent or limit virus propagation and are focussed on interferons and their signalling pathways. Interferons are known to (i) promote the production of anti-viral proteins by infected cells, including enzymes which degrade viral nucleic acid; (ii) enhance the expression of antigens on the surface of infected cells promoting their recognition by cytotoxic T-cells; and (iii) activate cells involved in clearing viral infection, such as natural killer T cells and macrophages. Three different classes of interferons have been identified; type I (interferon α,β,ω), type II (interferon γ) and type III (interferon A). Especially type I and III are known as anti-viral interferons. Interestingly, viruses possess mechanisms to resist host immune responses. For example, human rhinovirus (HRV) inhibits interferon production by inhibition of IRF3 transcription factor (Peng T. et al., J. Virol., 2006, 80(10):5021-31; Ling Z. et al., J. Virol., 2009, 83(8):3734-42; Spann K. M., 2005, J. Virol., 79(9):5353-5362.).

Furthermore, research has suggested that epithelial cells from asthmatics have a compromised interferon response to rhinovirus infection that renders them less able to prevent infection (Wark P. A. B., Johnston S. L. et al., J.E.M., 2005, 201:937-947.). These results have been proposed as the basis for a novel therapeutic approach in which the host\’s compromised response is boosted by administration of exogenous interferon.

As well as direct anti-viral effects of type I and III interferons, type II interferons also interact with cells from the immune system to promote their accumulation and activation at specific sites. Interferon gamma has been reported to promote the chemotaxis of both neutrophils and macrophages following viral infection (Bonville C. A., Percopo C. M. et al., B. M. C. Immunol., 2009, 10:14; Crane M. J., Hokeness-Antonelli K. L. et al., J. Immunol., 2009, 183:2810-7.) and bacterial infection (Syn K., Salmon S. L. et al., Infect. Immun., 2007, 75:1196-1202; Ruan S., Young E. et al., Pulm. Pharmacol. Ther., 2006, 19:251-257.). The 10 KDa-interferon-gamma-inducible protein, (IP)-10 (CXCL10) is known to play an important role in T-cell trafficking and homing, and recruitment of natural killer cells and macrophages (Nie C. Q., Bernard N. J., et al., Public Libraray of Science Pathog., 2009, 5, e1000369. doi:10.1371/Journal.ppat.1000369.). Intranasal administration of interferon α has been reported to protect ferrets and mice against infection with influenza virus (Kugel D., Kochs G. et al., J. Virol., 2009, 83:3843-3851; Van Hoeven N., Belser J. A. et al., J. Virol., 2009, 83:2851-2861.).

Thus clinical research suggests that there is a relationship between clinical viral load and symptoms and complications. For example, lower respiratory symptoms and bronchial hyper-responsiveness following experimental rhinovirus infection were significantly correlated with virus load (Message S. D., Laza-Stanca V. et al., PNAS, 2008, 105:13562-13567.). Similarly, rhinovirus infections were mostly associated with lower respiratory tract infections (in the absence of other viral agents) and wheezing, when viral load was high in immunocompetent pediatric patients (Gerna G., Piralla A. et al., J. Med. Virol., 2009, 81:1498-1507.). Interestingly, it has been reported recently that prior exposure to rhinovirus reduced the cytokine responses evoked in human alveolar macrophages to bacterial products (Oliver B. G. G., Lim S. et al., Thorax, 2008, 63:519-525). Additionally, infection of nasal epithelial cells with rhinovirus has been reported to promote adhesion of bacteria, including S. Aureus and H. influenzae (Wang, J. H., Kwon, H. J. et al., Laryngoscope, 2009, 119:1406-1411.). Such effects may contribute to the increased probability of lower respiratory tract infections in patients following an upper respiratory tract infection. Accordingly, it is appropriate and therapeutically relevant to focus on the ability of novel interventions to decrease viral load in a variety of systems in vitro as a surrogate predictor of clinical benefit in therapeutics.

The present disclosure provides a compound of formula (I):

wherein R1 is C1-6 alkyl optionally substituted by a hydroxyl group;

R2 is H or C1-6 alkyl optionally substituted by a hydroxyl group;

R3 is H, C1-6 alkyl or C0-3 alkylC3-6 cycloalkyl;

Ar is a naphthyl or a phenyl ring either of which may be optionally substituted by one or more (for example 1 or 2) groups independently selected from C1-6 alkyl, C1-6 alkoxy, amino, C1-4 mono or di-alkyl amino;

X is 5 or 6 membered heteroaryl group containing at least one nitrogen atom and optionally including 1 or 2 further heteroatoms selected from O, S and N;

Q is selected from:

a) a saturated or unsaturated, branched or unbranched C1-10 alkyl chain, wherein at least one carbon (for example 1, 2 or 3 carbons, suitably 1 or 2, in particular 1 carbon) is replaced by a heteroatom selected from O, N, S(O)p, wherein said chain is optionally, substituted by one or more groups (for example 1, 2 or 3 groups) independently selected from oxo, halogen, an aryl group, a heteroaryl group, a heterocyclyl group or a C3-8 cycloalkyl group,

each aryl, heteroaryl, heterocyclyl or C3-8 cycloalkyl group bearing 0 to 3 substituents selected from halogen, hydroxyl, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, C1-4 mono or di-alkyl amino, C1-4 mono or di-acyl amino, S(O)qC1-6 alkyl, C0-6 alkylC(O)C1-6 alkyl or C0-6 alkylC(O)C1-6 heteroalkyl,

with the proviso that the atom linked directly to the carbonyl in —NR3C(O)— is not an oxygen or a sulfur atom; and



b) a C0-8 alkyl-heterocycle said heterocyclyl group comprising at least one heteroatom (for example 1, 2 or 3, suitably 1 or 2, in particular 1 heteroatom) selected from O, N, and S, and is optionally substituted by one, two or three groups independently selected from halogen, hydroxyl, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, C1-4 mono and di-alkyl amino, C1-4 mono or di-acyl amino, S(O)qC1-6 alkyl, C0-6 alkylC(O)C1-6 alkyl or C0-6 alkylC(O)C1-6 heteroalkyl; and

p is 0, 1 or 2;

q is 0, 1 or 2

a pharmaceutically acceptable salt thereof, including all stereoisomers, tautomers and isotopic derivatives thereof for use in the treatment or prophylaxis of HRV infection and/or the exacerbation of respiratory disorders (such as asthma, COPD, bronchitis and/or cystic fibrosis) by rhinovirus infection.

Alkyl as used herein refers to straight chain or branched chain alkyl, such as, without limitation, methyl, ethyl, n-propyl, iso-propyl, butyl, n-butyl and tert-butyl. In one embodiment alkyl refers to straight chain alkyl.

Alkoxy as used herein refers to straight or branched chain alkoxy, for example methoxy, ethoxy, propoxy, butoxy. Alkoxy as employed herein also extends to embodiments in which the oxygen atom is located within the alkyl chain, for example —C1-3 alkylOC1-3 alkyl, such as CH2CH2OCH3 or CH2OCH3. Thus in one embodiment the alkoxy is linked through carbon to the remainder of the molecule. In one embodiment the alkoxy is linked through oxygen to the remainder of the molecule, for example —C0 alkylOC1-6 alkyl. In one embodiment the disclosure relates to straight chain alkoxy.

Heteroalkyl as employed herein is intended to refer to a branched or straight chain alkyl wherein one or more, such as 1, 2 or 3 carbons are replaced by a heteroatom, selected from N, O or S(O)q, wherein q represents 0, 1 or 2. The heteroatom may replace a primary, secondary or tertiary carbon, that is, for example, OH or NH2 for CH3, or NH or O or SO2 for —CH2— or N for a CH— or a branched carbon group, as technically appropriate.

Haloalkyl as employed herein refers to alkyl groups having 1 to 6 halogen atoms, for example 1 to 5 halogens, such as per haloalkyl, in particular perfluoroalkyl, more specifically —CF2CF3 or CF3.

C1-4 mono or di-acyl amino is intended to refer to NHC(O)C1-3 alkyl and to (—NC(O)C1-3 alkyl) C(O)C1-3 alkyl) respectively.

C1-4 mono or di-alkyl amino is intended to refer to —NHC1-4 alkyl and —N(C1-4 alkyl) (C1-4 alkyl) respectively.

Aryl as used herein refers to, for example C6-14 mono or polycyclic groups having from 1 to 3 rings wherein at least one ring is aromatic including phenyl, naphthyl, anthracenyl, 1,2,3,4-tetrahydronaphthyl and the like, such as phenyl and naphthyl.

Heteroaryl is a 6 to 10 membered aromatic monocylic ring or bicyclic ring system wherein at least one ring is an aromatic nucleus comprising one or more, for example 1, 2, 3 or 4 heteroatoms independently selected from O, N and S. Examples of heteroaryls include: pyrrole, oxazole, thiazole, isothiazole, imidazole, pyrazole, isoxazole, pyridine, pyridazine, pyrimidine, pyrazine, benzothiophene, benzofuran, or 1, 2, 3 and 1, 2, 4 triazole.

Heterocyclyl as employed herein refers to a 5 to 6 membered saturated or partially unsaturated non-aromatic ring comprising one or more, for example 1, 2, 3 or 4 heteroatoms independently selected from O, N and S optionally one or two carbons in the ring may bear an oxo substituent. The definition of C5-6 heterocycle as employed herein refers to a is a 5 to 6 membered saturated or partially unsaturated non-aromatic carbocyclic ring comprising one or more, for example 1, 2, 3 or 4 heteroatoms independently selected from O, N and S, wherein each heteroatom replaces a carbon atom and optionally one or two carbons may bear an oxo substituent. Clearly any valancies of a heteroatom not employed in forming or retaining the ring structure may be filled by hydrogen or a substituent, as appropriate. Thus substituents on heterocycles may be on carbon or on a heteroatom, such as N as appropriate. Examples of heterocycles and C5-6 heterocycles include pyrroline, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, pyrazoline, imidazoline, pyrazolidine, imidazolidine, oxoimidazolidine, dioxolane, thiazolidine, isoxazolidine, pyran, dihydropyran, piperidine, piperazine, morpholine, dioxane, thiomorpholine and oxathiane.

Halogen includes fluoro, chloro, bromo or iodo, in particular fluoro, chloro or bromo, especially fluoro or chloro.

Oxo as used herein refers to C═O and will usually be represented as C(O).

C3-8 cycloalkyl as employed herein is intended to refer to a saturated or partially unsaturated non-aromatic ring containing 3 to 8 carbon atoms.

C1-10 alkyl includes C2, C3, C4, C5, C6, C7, C8 or C9 as well as C1 and C10

C0-8 alkyl includes C1, C2, C3, C4, C5, C6, or C7 as well as C0 and C8.

In relation to a saturated or unsaturated, branched or unbranched C1-10 alkyl chain, wherein at least one carbon (for example 1, 2 or 3 carbons, suitably 1 or 2, in particular 1) is replaced by a heteroatom selected from O, N, S(O)p, wherein said chain is optionally, substituted by one or more groups independently selected from oxo, halogen, an aryl group, a heteroaryl group or a heterocyclyl group, it will be clear to persons skilled in the art that the heteroatom may replace a primary, secondary or tertiary carbon, that is —CH3, —CH2— or a CH— or a branched carbon group, as technically appropriate.

In one embodiment of the disclosure there is provided compounds of formula (I), wherein R1 is methyl, ethyl, propyl, iso-propyl, butyl or tert-butyl, in particular ethyl, iso-propyl or tert-butyl such as tert-butyl.

In one embodiment R1 is —C(CH3)2CH2OH.

In one embodiment R2 is methyl, ethyl, n-propyl, iso-propyl, n-butyl or tert-butyl, in particular methyl.

In one embodiment R2 is —CH2OH.

In one embodiment R2 is in the 2, 3, or 4 position (i.e. ortho, meta or para position), in particular the para (4) position.

In one embodiment Ar is substituted with 1 or 2 groups.

In one embodiment Ar is naphthyl.

In one embodiment Ar is not substituted with optional substituents.

In one embodiment Ar is substituted with 1 or 2 groups.

In one embodiment Ar is phenyl optionally substituted by 1 or 2 substituents independently selected from C1-3 alkyl or C1-3 alkoxy, for example tolyl, xylyl, anisoyl, di-methoxybenzene or methoxy-methylbenzene. The phenyl ring may, for example, be linked to the nitrogen of the urea through carbon 1 and to the group L through carbon 4. In such a case the optional one or two substituents selected from C1-3 alkyl or C1-3 alkoxy may be located in any of the unoccupied positions in the aromatic ring, for example in position 2 or in position 3 or in positions 2 and 3 or in positions 2 and 6 or in positions 3 and 5. Embodiments encompassing other possible regioisomers also form an aspect of the present disclosure.

In one embodiment R3 is H.

In one embodiment R3 is methyl, ethyl, n-propyl or iso-propyl.

In one embodiment p is 0 or 2.

In one embodiment X is selected from, pyrrole, oxazole, thiazole, isothiazole, imidazole, pyrazole, isoxazole, oxadiazole, pyridazine, pyrimidine, pyrazine, or 1,2,3 and 1,2,4 triazole, such as pyrazole, isoxazole, oxadiazole, pyridine, pyridazine, pyrimidine, pyrazine, or 1,2,3 and 1,2,4 triazole, in particular, pyrimidine, imidazole or pyridine, and especially pyridine or pyrimidine, more specifically pyridine.

In one embodiment 1, 2, 3 or 4 carbon atoms are replaced in the alkyl chain of Q by heteroatoms independently selected from O, N, S(O)p.

In one embodiment the heteroatom(s) replacing carbon(s) in the alkyl chain fragment of Q are selected from N and O.

In one embodiment Q is a saturated or unsaturated, branched or unbranched C1-8 alkyl chain or a C1-6 alkyl chain, wherein at least one carbon is replaced by a heteroatom selected from −O, —N, S(O)p. Alternatively, in this embodiment the alkyl chain may be a C2-8 alkyl or a C3-6 alkyl group, such as a C4 alkyl or a C5 alkyl group.

In one embodiment a nitrogen atom in the alkyl chain is directly bonded to the carbonyl of the fragment —NR3C(O) and additionally may, for example, be a terminal amino group.

In one embodiment Q represents C1-6 alkylNH2 or NH2.

In one embodiment Q represents —NHC1-6 alkyl such as —NHCH3 or —NHCH2CH3 or —NHCH(CH3)2.

In one embodiment the fragment Q is a saturated or unsaturated, branched or unbranched C1-10 alkyl chain wherein at least one carbon (for example 1, 2, 3 or 4 carbons, in particular 1 or 2 carbons) is replaced by a heteroatom selected from O, N, S(O)p, for example in such a manner as to provide a stable N-acyl group, NR3C(O)Q, wherein said chain is optionally substituted by one or more groups selected from oxo, halogen, an aryl group, a heteroaryl group or a heterocyclyl group, each aryl, heteroaryl or heterocyclyl group bearing 0 to 3 substituents independently selected from a relevant substituent listed above for compounds of formula (I), for example halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, C1-4 mono or di-alkyl amino and C1-4 mono or di-acyl amino.

In one embodiment the latter chain is optionally substituted by one or more groups selected from oxo, halogen, an aryl group, a heteroaryl group or a heterocyclyl group, each aryl, heteroaryl or heterocyclyl group bearing 0 to 3 substituents selected from halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, and C1-4 mono or di-alkyl amino.

In one embodiment Q is C1-4alkyl-V—R4, such as C1-3alkyl-V—R4 wherein:

V is a heteroatom selected from NRV, O or S(O)p;

RV represents H or C1-3 alkyl;

R4 is H or —C1-3alkyl, and p is as defined above,

with the proviso that the total alkyl chain length is not more than 10 carbon atoms, including replacement heteroatoms and that the resulting radical Q is a stable group, for example —CH2SCH3, —CH2SO2CH3, —CH2NHCH3, —CH2N(CH3)2, —C(CH3)2NHCH3, —CH(CH3)N(CH3)2, —(CH2)3CHNHCH3, —(CH2)3N(CH3)2, —CH2OH, —CH2OCH3, —CH(CH3)OCH3, or —(CH2)2OCH3.

In one embodiment Q is C1-3 alkyl-V—(C1-3 alkyl-Z—R5)k such as C1-3 alkyl-V—(C2-3 alkyl-Z—R5)k wherein:

V is a heteroatom selected from N, NH, O or S(O)p, such as N or NH (V will be selected from N in the case where k=2, or NH, O or S(O)p, in the case where k=1, in particular NH);

Z is independently selected from NH, O or S(O)p;

R5 is H or —C1-3alkyl;

k is an integer 1 or 2 (such as 1); and

p is as defined above,

with the proviso that the total alkyl chain length is not more than 10 carbon atoms, including replacement heteroatoms and that the resulting radical Q is a stable group. Suitably Q is C1-3alkyl-V—C1-3alkyl-OCH3 for example C1-3alkyl-V—C2-3alkyl-OCH3 such as C1-3alkyl-V—(CH2)2OCH3, in particular —CH2O(CH2)2OCH3 and CH2S(CH2)2OCH3, or —CH2NH(CH2)2OCH3, C1-3alkyl-V—(C1-3alkyl-OCH3)k wherein k represents 2, for example C1-3alkyl-V—(C2-3alkyl-OCH3)k such as —CH2N[((CH2)2OCH3]2.

In one embodiment Q is C1-3 alkyl-V—C1-2 alkyl-Z—C1-2 alkyl-Y—R6, or C1-3 alkyl-V—C2-3 alkyl-Z—C2-3 alkyl-Y—R6, wherein V, Z and Y are independently a heteroatom selected from NH, O or S(O)p,

R6 is H or methyl, and

p is as defined above,

with the proviso that the total alkyl chain length is not more than 10 carbon atoms, including replacement heteroatoms and that the resulting radical Q is a stable group. Suitably Q is —CH2V(CH2)2O(CH2)2OCH3, such as —CH2O(CH2)2O(CH2)2OCH3, —CH2NH(CH2)2O(CH2)2OCH3, or —CH2S(CH2)2O(CH2)2OCH3.

In one embodiment Q represents —NR7R8 and —NR3C(O)Q forms a urea, where R7 and

R8 independently represent hydrogen or a C1-9 saturated or unsaturated, branched or unbranched alkyl chain, wherein one or more carbons, such as 1, 2 or 3 are optionally replaced by a heteroatom selected from O, N or S(O)p. Said chain is optionally substituted by one or more groups independently selected from oxo, halogen, an aryl group, a heteroaryl group, a heterocyclyl or C3-8 cycloalkyl group, each aryl, heteroaryl or heterocyclyl group bearing 0 to 3 substituents independently selected from the relevant substituents listed above for compounds of formula (I), for example halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, C1-4 mono or di-alkyl amino and C1-4 mono or di-acyl amino with the proviso that the total alkyl chain length is not more than 10 carbon atoms, including replacement heteroatoms and that the resulting radical Q is a stable group.

In one embodiment Q represents —NR7R8 and —NR3C(O)Q forms a urea, where R7 and R8 independently represent hydrogen or a C1-9 saturated or unsaturated, branched or unbranched alkyl chain, wherein one or more carbons, such as 1, 2 or 3 are optionally replaced by a heteroatom selected from O, N or S(O)p. Said chain is optionally substituted by one or more groups independently selected from oxo, halogen, an aryl group, a heteroaryl group or a heterocyclyl group, each aryl, heteroaryl or heterocyclyl group bearing 0 to 3 substituents independently selected from the relevant substituents listed above for compounds of formula (I), for example halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, C1-4 mono or di-alkyl amino and C1-4 mono or di-acyl amino with the proviso that the total alkyl chain length is not more than 10 carbon atoms, including replacement heteroatoms and that the resulting radical Q is a stable group.

In this urea embodiment in one sub-embodiment R7 represents hydrogen.

Examples of ureas include those in which R7 and R8 are both hydrogen and Q is —NH2, or where Q is —NHCH3 or —N(CH3)2 to provide, for example, a fragment —NR3C(O)NH2 or —NR3C(O)NHCH3 or —NR3C(O)N(CH3)2.

Examples of ureas containing a heteroatom in the alkyl chain include those in which Q is: —NH(CH2)2OCH3 or —N[(CH2)2OCH3)]2. In one embodiment Q represents —NHC2-6alkylOC1-3alkyl, such as —NHCH2CH2OCH3.

Examples of ureas containing an oxo substituent include those in which Q is —NHCH2C(O)NH—C2-3alkyl-X1—C1-3alkyl, wherein X1 is a heteroatom selected from N, O or S(O)p and p is defined as above. Examples of the latter include those wherein Q is —NHCH2C(O)NHCH2CH2OCH3. Thus in one embodiment Q represents —NHC1-4 alkylC(O)NHC2alkylOCH3 such as —NHCH2C(O)NHCH2CH2OCH3.

In one embodiment Q represents —NHC1-4alkylC(O)R wherein RQ is selected from OH or —NR′R″ where R′ is hydrogen or C1-3 alkyl and R″ is hydrogen or C1-3 alkyl, for example —NHCH2C(O)OH, —NHCH2C(O)NH2 or —NHCH2C(O)NHCH3 such as —NHCH2C(O)OH or —NHCH2C(O)NHCH3.

In one embodiment Q represents —NHC1-4 alkylC(O)OC1-3alkyl, such as —NHCH2C(O)OCH2CH3.

In a further urea sub-embodiment Q represents —N—R9C1-3 alkyl-V—(C1-3 alkyl-Z—R10)k for example —N—R9C2-3 alkyl-V—(C2-3alkyl-Z—R10)k wherein:

V represents N, NH, O, S(O)p;

Z represents NH, O, S(O)p;

k is an integer 1 or 2;

p is an integer 0, 1 or 2

R9 represents H or C1-3 alkyl-V—(C1-3 alkyl-Z—R10)k such as C2-3 alkyl-V—(C2-3alkyl-Z—R10)k; and

R10 is H or C1-3 alkyl such as C1-3 alkyl;

with the proviso that the total alkyl chain length is not more than 10 carbon atoms, including replacement heteroatoms and that the resulting radical Q is a stable group.

In one embodiment Q is a saturated or unsaturated, branched or unbranched C1-10 alkyl chain, wherein at least one carbon is replaced by a heteroatom selected from O, N, and S(O)p, wherein said chain is substituted by an aryl group bearing 0 to 3 substituents, for example 1, 2 or 3, such as 1 or 2 substituents independently selected from the relevant substituents listed above for compounds of formula (I), for example from halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino and C1-4 mono or di-alkyl amino and C1-4 mono or di-acyl amino, such as a saturated or unsaturated, branched or unbranched C1-10 alkyl chain, wherein at least one carbon is replaced by a heteroatom selected from O, N, and S(O)p, wherein said chain is substituted by an aryl group bearing 0 to 3 substituents, for example 1, 2 or 3, such as 1 or 2 substituents independently selected from halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino and C1-4 mono or di-alkyl amino. In one embodiment the said aryl group is phenyl, for example substituted phenyl or unsubstituted phenyl.

In one embodiment Q represents —NHC0-6 alkylphenyl, such as —NHphenyl or NHbenzyl.

Examples of the fragment —NR3C(O)Q wherein Q comprises substituted benzyl include: —NR3C(O)CH2NHCH2C6H4(OCH3) such as —NHC(O)CH2NHCH2C6H4(OCH3), for example where the methoxy substituent is in the ortho, meta or para position, such as the para position.

In one embodiment Q is a saturated or unsaturated, branched or unbranched C1-10 alkyl chain, wherein at least one carbon is replaced by a heteroatom selected from O, N, and S(O)p, wherein said chain is substituted by a heteroaryl group bearing 0 to 3 substituents (for example 1, 2 or 3, such as 1 or 2 substituents) independently selected from the relevant substituents listed above for compounds of formula (I), for example halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 alkyl amino, C1-4 mono or di-alkyl amino and C1-4 mono or di-acyl amino, such as a saturated or unsaturated, branched or unbranched C1-10 alkyl chain, wherein at least one carbon is replaced by a heteroatom selected from O, N, and S(O)p, wherein said chain is substituted by a heteroaryl group bearing 0 to 3 substituents for example 1, 2 or 3, such as 1 or 2 substituents selected from halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 alkyl amino, C1-4 mono or di-alkyl amino. In one embodiment the said heteroaryl group is selected from, thiophene, oxazole, thiazole, isothiazole, imidazole, pyrazole, isoxazole, isothiazole, oxadiazole, 1,2,3 or 1,2,4 triazole, pyridine, pyridazine, pyrimidine, pyrazine and, in particular pyridine and pyrimidine, especially pyridine.

In one embodiment Q represents —NHC1-6 alkylheteroaryl, for example —NH(CH2)3imidazolyl or —NHCH2 isoxazole wherein the isoxazole is optionally substituted such as —NHCH2 isoxazole(CH3).

In one embodiment Q represents —NHC1-4 alkylC(O)NHC1-3alkylheteroaryl, for example a nitrogen containing heteroaryl group or a nitrogen and oxygen containing heteroaryl, more specifically —NHCH2C(O)NHCH2CH2pyridinyl, in particular where pyridinyl is linked through carbon, for example pyridin-4-yl or —NHCH2C(O)NHCH2CH2CH2imidazolyl, in particular where imidazolyl is linked through nitrogen.

In one embodiment Q is a saturated or unsaturated, branched or unbranched C1-10 alkyl chain, wherein at least one carbon is replaced by a heteroatom selected from O, N and S(O)p wherein said chain is substituted by a heterocyclyl group bearing 0 to 3 substituents (for example 1, 2 or 3, such as 1 or 2 substituents) independently selected from the relevant substituents listed above for compounds of formula (I), for example halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl amino, C1-4 mono or di-alkyl amino and C1-4 mono or di-acyl amino, such as a saturated or unsaturated, branched or unbranched C1-10 alkyl chain, wherein at least one carbon is replaced by a heteroatom selected from O, N and S(O)p wherein said chain is substituted by a heterocyclyl group bearing 0 to 3 substituents, for example 1, 2 or 3, such as 1 or 2 substituents selected from halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl amino, C1-4 mono or di-alkyl amino.

In one embodiment said heterocyclyl is selected, from a 5 or 6 membered saturated or partially unsaturated ring system comprising one or more (for example 1, 2 or 3 in particular 1 or 2) heteroatoms independently selected from O, N and S, for example pyrrolidine, tetrahydrofuran, tetrahydrothiophene, piperidine, piperazine, morpholine, 1,4-dioxane, pyrrolidine and oxoimidazolidine such as pyrrolidine, tetrahydrofuran, tetrahydrothiophene, piperidine, piperazine, morpholine, and 1,4-dioxane, in particular piperidine, piperazine, and morpholine.

A heterocyclic group may be linked to the alkyl chain of Q or to the carbonyl of —NR3C(O)— through carbon or nitrogen, in particular a nitrogen atom.

In one embodiment Q is —C1-3alkylheterocycle (for example —C0-1alkylheterocycle) said heterocyclyl group comprising at least one heteroatom (for example 1, 2 or 3, in particular 1 or 2, heteroatoms) selected from O, N and S, and is optionally substituted by one or two or three groups independently selected from the relevant substituents listed above for compounds of formula (I), for example halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, C1-4 mono and di-alkyl amino and C1-4 mono or di-acyl amino.

In one embodiment in which Q is —C0alkylheterocycle, the heterocycle is linked through carbon, and is, for example, a C-linked tetrahydropyran or a C-linked piperidine or a C-linked morpholine or a C-linked piperazine.

In one embodiment in which Q is —C0alkylheterocycle, the heterocyclic group containing one or more N atoms is linked through N. This embodiment provides for ureas in which one of the urea nitrogens is embedded within a heterocyclic ring. Examples of this embodiment include, but are not limited to, an N-linked morpholine or an N-linked piperidine or an N-linked piperazine, said N-linked piperizinyl group optionally bearing an additional C- or N-substituent (such as an N-methyl group or N—CH2CH2OCH3 group. In one embodiment Q is a heterocyclyl linked through nitrogen such as piperidinyl, in particular 4-hydroxypiperidinyl or piperazinyl, such as 4-methyl piperazinyl.

In one embodiment Q represents a heterocyclyl group, for example a nitrogen containing heterocyclyl group, in particular linked through N, such as morpholinyl or piperazinyl optionally substituted by methyl, especially 4-methyl, or piperizinyl.

In one embodiment Q is a —C1alkylheterocycle, for example tetrahydropyranylmethyl or a C- or N-linked piperazinylmethyl optionally bearing a substituent (for example a C1-6 alkyl substituent such as methyl or a C1-6 alkoxy substituent such as —CH2CH2OCH3). Additional examples include a C- or N-linked pyrrolidinylmethyl, or a C- or N-linked oxoimidazolinylmethyl (such as 2-oxoimidazolidinylmethyl, said heterocycle optionally bearing a substituent (such as N-methyl or N—SO2CH3).

In one embodiment Q represents —NHheterocyclyl (wherein the heterocyclyl bears 0 to 3 substituents selected from the relevant list of substituents listed above for compounds of formula (I), for example halogen, hydroxy, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, C1-4 mono or di-alkyl amino, —S(O)qC1-6 alkyl, C1-4 mono or di-acyl amino, C0-6 alkylC(O)C1-6alkyl or C0-6 alkylC(O)C1-6heteroalkyl), such as where the ring is linked through carbon, for example 2-piperidinyl or 3-piperidinyl or 4-piperidinyl, in particular 1-acetylpiperidin-4-yl, 1-methylpiperidin-4-yl, 1-(methylsulfonyl)piperidin-4-yl or 1-(2-(2-methoxyethoxy)acetyl)piperidin-4-yl

In one embodiment Q represents —NHC1-6 alkylheterocyclyl for example a nitrogen containing heterocyclyl group, in particular one linked through nitrogen, such as —NHCH2CH2morpholine, —NH(CH2)3morpholine or —NH(CH2)4morpholine.

In one embodiment Q represents —NHC1-6 alkylC(O)heterocyclyl (wherein the heterocyclyl bears 0 to 3 substituents selected from the relevant list of substituents listed above for compounds of formula (I), for example halogen, hydroxy, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, C1-4 mono or di-alkyl amino, C1-4 mono or di-acyl amino, C0-6 alkylC(O)C1-6 alkyl or C0-6 alkylC(O)C1-6heteroalkyl) for example a nitrogen containing heterocyclyl group, in particular one linked through nitrogen, such as —NHCH2C(O)-1-pyrrolindinyl, —NHCH2C(O)-1-piperidinyl, —NHCH2C(O)-4-morpholinyl or —NHCH2C(O)piperazinyl such as —NHCH2C(O)-4-methyl-1-piperazinyl.

In one embodiment Q represents —NHC1-4 alkylC(O)NHC1-3alkylheterocyclyl for example a nitrogen containing heterocyclyl group or a nitrogen and/or oxygen containing heterocyclyl, such as —NHCH2C(O)NHCH2CH2morpholinyl, in particular where morpholinyl is linked through nitrogen.

In one embodiment Q represents —N(C1-3 alkyl)C1-6alkylheterocyclyl, for example a nitrogen containing heterocyclyl group, in particular linked through nitrogen, such as —N(CH3)CH2CH2morpholine, —N(CH3)(CH2)3morpholine or —N(CH3)—(CH2)4morpholine.

In one embodiment Q is —C1-3alkyl-G-C1-3alkylheterocycle wherein G is a heteroatom selected from NH, O or S(O)p said heterocyclyl group comprising at least one heteroatom (for example 1, 2 or 3, in particular 1 or 2, heteroatoms) selected from O, N, and S, and is optionally substituted by one or two or three groups independently selected from relevant substituents listed above for compounds of formula (I), for example halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, C1-4 mono and di-alkyl amino and C1-4 mono or di-acyl amino such as one or two or three groups halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, C1-4 mono and di-alkyl amino. Suitably Q is —CH2G(CH2)2heterocycle for example —CH2G(CH2)2-tetrahydropyranyl; or —CH2G(CH2)2morpholinyl in which the heterocyclyl is linked through nitrogen or carbon; or CH2G(CH2)2piperazinyl in which the heterocyclyl is linked through nitrogen or carbon and optionally bearing a further C- or N-substituent (for example a C1-6 alkyl substituent such as methyl or a C1-6 alkoxy substituent such as —CH2CH2OCH3); or —CH2G(CH2)2pyrrolidinyl, in which the heterocyclyl is linked through nitrogen or carbon, for example linked through nitrogen; or —CH2G(CH2)2-oxoimidazolinyl (such as 2-oxoimidazolidinyl) for example linked through N and optionally bearing an additional C- or N-substituent (such as N-methyl or N—SO2CH3), and in which G is O or NH.

In one embodiment G is O.

In one embodiment G is NH.

In one embodiment Q is a saturated or unsaturated C1-10 alkyl chain wherein at least one carbon (for example 1, 2 or 3 carbons) is replaced by a heteroatom selected from O, N, S(O)p wherein said chain is substituted by a C3-8 carbocyclyl group and said alkyl chain is optionally substituted by one or more (for example 1 or 2) groups selected from oxo and halogen. In one embodiment said C3-8 carbocyclyl group bears one or more groups (for example 1, 2 or 3 groups) independently selected from halogen, hydroxyl, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, C1-4 mono or di-alkyl amino, C1-4 mono or di-acyl amino, S(O)qC1-6 alkyl, C0-6 alkylC(O)C1-6alkyl or C0-6 alkylC(O)C1-6heteroalkyl.

In one embodiment Q represents —NHC3-6 cycloalkyl, such as —NHcyclopropyl, —NHcyclopentyl or —NHcyclohexyl.

In one embodiment the aryl, heteroaryl or heterocyclyl group bears at least one —S(O)qC1-6 alkyl substituent and optionally bears one or two further relevant substituents independently selected from the list of substituents defined above for compounds of formula (I).

In one embodiment the C5-6 heterocycle bears at least one —S(O)qC1-6 alkyl substituent and optionally bears one or two further substituents independently selected from the relevant list of substituents defined above for compounds of formula (I).

In one embodiment the aryl, heteroaryl or heterocyclyl group bears at least one hydroxyl substituent and optionally bears one or two further substituents independently selected from the relevant list of substituents defined above for compounds of formula (I).

In one embodiment the C5-6heterocycle bears at least one hydroxyl substituent and optionally bears one or two further substituents independently selected from the relevant list of substituents defined above for compounds of formula (I).

In one embodiment the aryl, heteroaryl or heterocyclyl group bears at least one C1-4 mono and/or di-acyl amino substituent and optionally bears one or two further substituents independently selected from the relevant list defined above for compounds of formula (I).

In one embodiment the C5-6heterocycle bears at least one C1-4 mono and/or di-acyl amino substituent and optionally bears one or two further substituents independently selected from the relevant list defined above for compounds of formula (I).

In one embodiment the aryl, heteroaryl or heterocyclyl group bears at least one C0-6 alkylC(O)C1-6heteroalkyl substituent and optionally bears one or two further substituents independently selected from the relevant list defined above for compounds of formula (I).

In one embodiment the C5-6heterocycle bears at least one C0-6 alkylC(O)C1-6 heteroalkyl substituent and optionally bears one or two further substituents independently selected from the relevant list defined above for compounds of formula (I).

In one embodiment the aryl, heteroaryl or heterocyclyl group bears at least one C0-6 alkylC(O)C1-6alkyl substituent and optionally bears one or two further substituents independently selected from the relevant list defined above for compounds of formula (I).

In one embodiment the C5-6heterocycle bears at least one C0-6 alkylC(O)C1-6alkyl substituent and optionally bears one or two further substituents independently selected from the relevant substituents defined above for compounds of formula (I).

In one embodiment Q represents tetrahydrofuranyl, morpholinyl, piperidinyl such as piperidinyl bearing one hydroxyl substituent, piperazinyl such as piperazinyl bearing one methyl substituent or pyrrolidinyl such a pyrrolidinyl bearing one di-methyl amino substituent. The ring may be linked through the heteroatom, such as nitrogen. Alternatively, the ring may be linked through carbon. The substituent may, for example be para relative to the atom through which the ring is linked to the remainder of the molecule.

In one embodiment the alkyl chain fragment of Q does not bear any optional substituents.

In one embodiment the alkyl chain is saturated.

In one embodiment the alkyl chain is unbranched.

In one embodiment the alkyl chain fragment of Q bears 1, 2, or 3, for example 1 or 2, in particular 1 optional substituent.

It will be clear to persons skilled in the art that the heteroatom may replace a primary, secondary or tertiary carbon, that is a CH3, —CH2— or a —CH—, group, as technically appropriate.

In one embodiment p is 0 or 2.

In one embodiment p is 1.

In one embodiment compounds of the disclosure include those in which the fragment Q is:

—CH2OH;

—CH2OC1-6 alkyl, in particular —CH2OCH3;

—CH2CH2OCH3;

—CH2—O—(CH2)2OCH3;

—CH(CH3)OCH3;

—CH2NHCH3 or —CH2N(CH3)2

—CH2NHCH2CH2OCH3 or —CH2NHC(O)CH2OCH3;

—CH2SCH3, —CH2S(O)2CH3 or —CH2NHC(O)CH2S(O)2CH3; or

—CH2NHC(O)CH2.

In one embodiment compounds of the disclosure include those in which the fragment —NR3C(O)Q in formula (I) is represented by:

—NR3C(O)CH2OH, in particular —NHC(O)CH2OH;

—NR3C(O)CH2OC1-6 alkyl, in particular —NR3C(O)CH2OCH3, especially

—NHC(O)CH2OCH3;

—NR3C(O)CH2—O—(CH2)2OCH3, in particular —NHC(O)CH2—O—(CH2)2OCH3;

—NR3C(O)CH(CH3)OCH3 in particular —NHC(O)CH(CH3)OCH3;

—NR3C(O)CH(CH3)NHC1-3alkyl in particular —NHC(O)CH(CH3)NHCH3;

—NR3C(O)CH(CH3)N(C1-3alkyl)2 in particular —NHC(O)CH(CH3)N(CH3)2;

—NR3C(O)C(CH3)2NHCH3 in particular —NHC(O)C(CH3)2NHCH3;

—NR3C(O)(CH2)2OC1-6alkyl, such as —NR3C(O)(CH2)2OCH3, in particular —NHC(O)(CH2)2OCH3;

—NR3C(O)(CH2)3NHC1-3alkyl in particular —NHC(O)(CH2)3NHCH3;

—NR3C(O)(CH2)3N(C1-3alkyl)2 in particular —NHC(O)(CH2)3N(CH3)2;

—NR3C(O)CH2NHC1-3alkyl in particular —NHC(O)CH2NHCH3;

—NR3C(O)CH2NH(CH2)2OCH3 in particular —NHC(O)CH2NH(CH2)2OCH3;

—NR3C(O)CH2SCH3, in particular —NHC(O)CH2SCH3;

—NR3C(O)CH2S(CH2)2OCH3, in particular —NHC(O)CH2S(CH2)2OCH3;

—NR3C(O)CH2S(CH2)2O(CH2)2OCH3, in particular —NHC(O)CH2S(CH2)2O(CH2)2OCH3

—NR3C(O)CH2SOCH3, in particular —NHC(O)CH2SOCH3

—NR3C(O)CH2S(O)2CH3, in particular —NHC(O)CH2S(O)2CH3;

—NR3C(O)CH2N[(CH2)2OCH3]2 in particular —NHC(O)CH2N[(CH2)2OCH3]2;

—NR3C(O)NH2 in particular —NHC(O)NH2;

—NR3C(O)NHC1-9 alkyl, such as NR3C(O)NHC1-7 alkyl, in particular —NHC(O)NHCH3

—NR3C(O)N(C1-4 alkyl)C1-5 alkyl in particular —NHC(O)N(CH3)2; or

—NR3C(O)NHCH2CONH(CH2)2OCH3 in particular —NHC(O)NHCH2CONH(CH2)2OCH3.

In one embodiment compounds of the disclosure include compounds of formula (I) in which the fragment —NR3C(O)C0-8alkylheterocyclyl is represented by:

—NHC(O)-(tetrahydropyranyl), such as —NHC(O)-(tetrahydro-2H-pyran-4-yl):

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The Patent Description & Claims data below is from USPTO Patent Application 20130102607, Ureido-pyrazole derivatives for use in the treatment of rhinovirus infections.

The disclosure relates to compounds of formula (I) for use in the treatment or prophylaxis of respiratory syncitial virus (RSV) infection in particular viral exacerbation of respiratory disorders such as bronchitis, asthma, COPD and/or cystic fibrosis and to methods of treating or preventing RSV infection employing said compounds or pharmaceutical compositions comprising the same.

BACKGROUND

RSV infection is known to be transmitted through direct contact with an infection source but not through inhalation (Bont L., Paediatr. Respir. Rev., 2009, 10, Suppl 1:16-17). Consistent with this mechanism, the inoculation of RSV to the human nose or eyes has been reported to result in viral replication, during an incubation period of 4 to 5 days, which may then spread infection to the lower respiratory tract (Collins P. L., and Graham B. S., J. Virol., 2008, 82:2040-2055). In BALB/c mice instillation of RSV into the eye leads to an eye infection, followed by inflammation and subsequently to eye-to-lung viral transmission, resulting in respiratory pathology (Bitko V., Musiyenko A. and Bark S. J. Virol., 2007, 81:783-790).

Infection by RSV is a well known cause of respiratory disease in both infants and young children and has the potential to cause severe lung disease, including bronchiolitis and death. RSV infection has been cited as a central concern in the care of high-risk, pre-term infants (Bauer G., Bossie L. et al., Arch. Argent. Pediatr., 2009, 107:111-118). It remains unclear and controversial whether severe RSV infection in early infancy precipitates the development of asthma in later life or whether RSV bronchiolitis precedes asthma in children who are susceptible to becoming asthmatic (Mailaparambil B., Grychtol R. et al., Inflamm. Allergy Drug Targets, 2009, 8: 202-207). Recently the standard of care for treating RSV disease has been re-defined by the licensing and introduction of a monoclonal antibody therapeutic, palivizumab, a humanized mAb against the RSV F protein, which provides passive immunity against the virus.

Much more uncertainty exists regarding the prevalence and significance of RSV infections in the elderly. This arises, not least, because of the difficulty of differentiating infection by RSV from infection by influenza and the fact that both diseases show a similar prevalence by season. However, the recent development of new methods for detecting viruses has allowed research workers to investigate whether RSV infection is present in adults. This work has led to the conclusion that a significant proportion of upper respiratory tract infections (URTI) in adults is caused specifically by RSV (Caram L. B., Chen J. et al., J. Am. Geriatr. Soc., 2009, 57:482-485). Furthermore, in patients diagnosed with COPD, persistent RSV detection was associated with airway inflammation and accelerated decline in FEV(1) (Wilkinson T. M., Donaldson G. C. et al., Am. J. Respir. Crit. Care, 2006, 173:871-876). In addition RSV infection is also commonly regarded as being a significant cause of exacerbations in patients who suffer from asthma (Hansbro N. G., Horvat J. C. et al., Pharmacol. Ther., 2008, 117:313-353). Consistent with these clinical observations, RSV infection has been reported to produce airways hyper-responsiveness in mice and the persistence of RSV RNA correlated significantly with pulmonary function abnormalities (Estripeaut D., Torres J. P. et al., J. Infect. Dis., 2008, 198:1435-1443).

In addition to the established view that RSV is a significant cause of morbidity and mortality in young children, recently work suggests that it is also an important factor contributing to unwanted effects in patients suffering from chronic respiratory diseases. While the introduction of palivizumab has established a new standard of care, such therapy, using monoclonal antibodies, remains very expensive and effective new medicines to treat RSV-induced lung disease are still urgently required. Furthermore, given the mode of transmission of the pathogen, there is a significant opportunity to develop therapies that prevent and/or treat viral infection by targeting those mucosal surfaces which it attacks.

RSV exploits a variety of mechanisms to suppress innate cellular immunity responses and to maintain optimal growth in the infected host cells, represented by the suppression of type I interferon (IFN) and of interferon α and interferon β induction (Spann K. M., et al., J. Virol. 2005, 79:5353-5362). In this regard, RSV viral surface proteins have been implicated in the reduction of Type 1 interferon expression by signalling through the Toll-like receptor pathway (Oshansky C. M. et al., Viral Immunol., 2009, 2:147-161). In addition, RSV infection has been reported to decrease the cellular levels of key members of the interferon signalling pathway, including IKKε and TRAF3 (Sweden S. et al., J. Virol., 2009, 83:9682-9693).

Infection by RSV has been documented to up-regulate the expression of IL-1β, IL-6, IL-8, TNF-α, MIP1a, RANTES, and ICAM-1 in epithelial cells: the main targets of RSV in vivo. The elevated expression of these inflammatory molecules during RSV infection has been attributed to the activation of nuclear factor-κ B (Kong et al., BBRC 2003, 306:616-622). In this regard, RSV infection is believed to induce a time-dependent RelA phosphorylation during which reactive oxygen species are produced in parallel (Jamaluddin M. et al., J. Virol., 2009, 83:10605-10615) that are potent oxidative stressors of the intracellular glutathione redox state. In human airway epithelial cells this activates signals that increase the production of cytokines and chemokines (Mochizuki H. et al., Inflammation, 2009, 32:252-264). In A549 cells RSV has been reported to activate both ERK-1 and ERK-2 pathways within 5 min of infection, leading to the inhibition of pathways that decrease RSV infection (Kong X., et al., FEBS Lett., 2004, 559:33-38). P38MAPK activation by RSV was also confirmed in primary bronchial epithelial cells (Signh D., et al., Am. J. Physiol. Lung Cell. Mol. Physiol., 2007 293(2):L436-45.). Furthermore, RSV infection has been reported to increase the phosphorylation of PKC α/β in monocytic cells (Ennaciri J. et al., J. Leukoc. Biol., 2007, 81:625-631) and is associated with the induction of anti-apoptotic effects through a PI3 kinase-dependent mechanism (Thomas K. W. et al., J. Biol. Chem., 2002, 277:492-501).

BRIEF DESCRIPTION OF THE INVENTION

According to the invention, there is provided a compound of formula (I):

wherein R1 is C1-6 alkyl optionally substituted by a hydroxyl group;

R2 is H or C1-6 alkyl optionally substituted by a hydroxyl group;

R3 is H, C1-6 alkyl or C0-3 alkylC3-6 cycloalkyl;

Ar is a naphthyl or a phenyl ring either of which may be optionally substituted by one or more groups (for example 1 to 3, such as 1, 2 or 3 groups) independently selected from C1-6 alkyl, C1-6 alkoxy, amino, C1-4 mono or di-alkyl amino;

L is a saturated or unsaturated branched or unbranched C1-8 alkylene chain, wherein one or more carbons (for example 1 to 3, such as 1, 2 or 3 carbons) are optionally replaced by —O— and the chain is optionally substituted by one or more halogen atoms (for example 1 to 6);

X is 5 or 6 membered heteroaryl group containing at least one nitrogen atom and optionally including 1 or 2 further heteroatoms selected from O, S and N;

Q is selected from:

a) a saturated or unsaturated, branched or unbranched C1-10 alkyl chain, wherein at least one carbon (for example 1, 2 or 3 carbons, suitably 1 or 2, in particular 1 carbon) is replaced by a heteroatom selected from O, N, S(O)p, wherein said chain is optionally, substituted by one or more groups (for example 1, 2 or 3 groups) independently selected from oxo, halogen, an aryl group, a heteroaryl group, a heterocyclyl group or a C3-8 cycloalkyl group,

each aryl, heteroaryl, heterocyclyl or C3-8 cycloalkyl group bearing 0 to 3 substituents selected from halogen, hydroxyl, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, C1-4 mono or di-alkyl amino, C1-4 mono or di-acyl amino, S(O)qC1-6 alkyl, C0-6 alkylC(O)C1-6 alkyl or C0-6 alkylC(O)C1-6 heteroalkyl,


 with the proviso that the atom linked directly to the carbonyl in —NR3C(O)— is not an oxygen or a sulfur atom; and
b) a C0-8 alkyl-heterocycle said heterocyclyl group comprising at least one heteroatom (for example 1, 2 or 3, suitably 1 or 2, in particular 1 heteroatom) selected from O, N, and S, and which is optionally substituted by one, two or three groups independently selected from halogen, hydroxyl, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, C1-4 mono and di-alkyl amino, C1-4 mono or di-acyl amino, S(O)qC1-6 alkyl, C0-6 alkylC(O)C1-6 alkyl, C0-6 alkylC(O)NC0-6 alkyl C0-6 alkyl or C0-6 alkylC(O)C0-6 heteroalkyl; and

p is 0, 1 or 2;

q is 0, 1 or 2; or

a pharmaceutically acceptable salt thereof, including all stereoisomers, tautomers and isotopic derivatives thereof for use in the treatment or prophylaxis of RSV infection and/or exacerbation of a respiratory disorder for example a chronic respiratory disorder such as asthma or COPD by RSV infection.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effects of compound Example 1 on RSV Memphis 37 induced IL-8 release in primary 3D cultured nasal epithelial cells.

FIG. 2 shows the effects of compound Example 1 on RSV Memphis 37 viral load in primary 3D cultured nasal epithelial cells.

DETAILED DESCRIPTION

OF THE INVENTION

In one embodiment Q is selected from:

a) a saturated or unsaturated, branched or unbranched C1-10 alkyl chain, wherein at least one carbon (for example 1, 2 or 3 carbons, suitably 1 or 2, in particular 1) is replaced by a heteroatom selected from O, N, S(O)p, wherein said chain is optionally, substituted by one or more groups independently selected from oxo, halogen, an aryl group, a heteroaryl group or a heterocyclyl group,

each aryl, heteroaryl or heterocyclyl group bearing 0 to 3 substituents selected from halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, C1-4 mono or di-alkyl amino, C1-4 mono or di-acyl amino,


 with the proviso that the atom linked directly to the carbonyl in —NR3C(O)— is not an oxygen or a sulfur atom; and
b) a C0-8 alkylC5-6 heterocycle said heterocyclyl group comprising at least one heteroatom (for example 1, 2 or 3, suitably 1 or 2, in particular 1 heteroatom) selected from O, N and S, and is optionally substituted by one, two or three groups independently selected from halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, C1-4 mono and di-alkyl amino C1-4 mono or di-acyl amino.

For example, Q is selected from:

a) a saturated or unsaturated, branched or unbranched C1-10 alkyl chain, wherein at least one carbon (for example 1, 2 or 3 carbons,) is replaced by a heteroatom selected from O, N, S(O)p, wherein said chain is optionally, substituted by one or more groups independently selected from oxo, halogen, an aryl group, a heteroaryl group or a heterocyclyl group,

each aryl, heteroaryl or heterocyclyl group bearing 0 to 3 substituents selected from halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, and C1-4 mono or di-alkyl amino,


 with the proviso that the atom linked directly to the carbonyl in —NR3C(O)— is not an oxygen or a sulfur atom; and
b) a C0-8 alkylC5-6 heterocycle said heterocyclyl group comprising at least one heteroatom (for example 1, 2 or 3, suitably 1 or 2, in particular 1 heteroatom) selected from O, N and S, and is optionally substituted by one or two or three groups independently selected from halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, and C1-4 mono or di-alkyl amino.

In further embodiment there is provided a compound of formula (IA):

wherein R1 is C1-6 alkyl optionally substituted by a hydroxyl group;

R2 is H or C1-6 alkyl optionally substituted by a hydroxyl group;

R3 is H, C1-6 alkyl or C0-3 alkylC3-6 cycloalkyl;

Ar is a naphthyl or a phenyl ring either of which may be optionally substituted by one or more (for example 1 or 2) groups independently selected from C1-6 alkyl, C1-6 alkoxy, amino, C1-4 mono or di-alkyl amino;

X is 5 or 6 membered heteroaryl group containing at least one nitrogen atom and optionally including 1 or 2 further heteroatoms selected from O, S and N;

Q is selected from:

a) a saturated or unsaturated, branched or unbranched C1-10 alkyl chain, wherein at least one carbon (for example 1, 2 or 3 carbons, suitably 1 or 2, in particular 1 carbon) is replaced by a heteroatom selected from O, N, S(O)p, wherein said chain is optionally, substituted by one or more groups (for example 1, 2 or 3 groups) independently selected from oxo, halogen, an aryl group, a heteroaryl group, a heterocyclyl group or a C3-8 cycloalkyl group,

each aryl, heteroaryl, heterocyclyl or C3-8 cycloalkyl group bearing 0 to 3 substituents selected from halogen, hydroxyl, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, C1-4 mono or di-alkyl amino, C1-4 mono or di-acyl amino, S(O)qC1-6 alkyl, C0-6 alkylC(O)C1-6 alkyl or C0-6 alkylC(O)C1-6 heteroalkyl,


 with the proviso that the atom linked directly to the carbonyl in —NR3C(O)— is not an oxygen or a sulfur atom; and
b) a C0-8 alkyl-heterocycle said heterocyclyl group comprising at least one heteroatom (for example 1, 2 or 3, suitably 1 or 2, in particular 1 heteroatom) selected from O, N, and S, and is optionally substituted by one, two or three groups independently selected from halogen, hydroxyl, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, C1-4 mono and di-alkyl amino, C1-4 mono or di-acyl amino, S(O)qC1-6 alkyl, C0-6 alkylC(O)C1-6 alkyl or C0-6 alkylC(O)C1-6 heteroalkyl; and

p is 0, 1 or 2;

q is 0, 1 or 2

a pharmaceutically acceptable salt thereof, including all stereoisomers, tautomers and isotopic derivatives thereof for use in the treatment or prophylaxis of RSV infection and/or exacerbation of a respiratory disorder for example a chronic respiratory disorder such as asthma or COPD by RSV infection.

In one embodiment Q is:

a) a saturated or unsaturated, branched or unbranched C1-10 alkyl chain, wherein at least one carbon (for example 1, 2 or 3 carbons, suitably 1 or 2, in particular 1) is replaced by a heteroatom selected from O, N, S(O)p, wherein said chain is optionally, substituted by one or more groups (for example 1, 2 or 3) independently selected from oxo, halogen, an aryl group, a heteroaryl group, a heterocyclyl group or a C3-8 cycloalkyl,

at least one of said aryl, heteroaryl or heterocyclyl groups (such as each group) bears a substituents C1-4 mono or di-acyl amino and optionally 1 or 2 further substituents independently selected from the relevant list of substituents above for compounds of formula (I); or


b) a C0-8 alkylC5-6 heterocycle said heterocyclyl group comprising at least one heteroatom (for example 1, 2 or 3, suitably 1 or 2, in particular 1 heteroatom) selected from O, N, and S, substituted by a C1-4 mono or di-acyl amino and optionally 1 or 2 further substituents independently selected from the relevant list of substituents above for compounds of formula (I).

Alkyl as used herein refers to straight chain or branched chain alkyl, such as, without limitation, methyl, ethyl, n-propyl, iso-propyl, butyl, n-butyl and tert-butyl. In one embodiment alkyl refers to straight chain alkyl.

Alkoxy as used herein refers to straight or branched chain alkoxy, for example methoxy, ethoxy, propoxy, butoxy. Alkoxy as employed herein also extends to embodiments in which the oxygen atom is located within the alkyl chain, for example —C1-3 alkylOC1-3 alkyl, such as —CH2CH2OCH3 or —CH2OCH3. Thus in one embodiment the alkoxy is linked through carbon to the remainder of the molecule. In one embodiment the alkoxy is linked through oxygen to the remainder of the molecule, for example —C0 alkylOC1-6 alkyl. In one embodiment the disclosure relates to straight chain alkoxy.

Heteroalkyl as employed herein is intended to refer to a branched or straight chain alkyl wherein one or more, such as 1, 2 or 3 carbons are replaced by a heteroatom, selected from N, O or S(O)q, wherein q represents 0, 1 or 2. The heteroatom may replace a primary, secondary or tertiary carbon, that is, for example, SH, OH or NH2 for CH3, or NH or O or SO2 for —CH2— or N for a —CH— or a branched carbon group, as technically appropriate.

Haloalkyl as employed herein refers to alkyl groups having 1 to 6 halogen atoms, for example 1 to 5 halogens, such as per haloalkyl, in particular perfluoroalkyl, more specifically —CF2CF3 or CF3.

C1-4 mono or di-acyl amino is intended to refer to —NHC(O)C1-3 alkyl and to (—NC(O)C1-3 alkyl) C(O)C1-3 alkyl) respectively.

C1-4 mono or di-alkyl amino is intended to refer to —NHC1-4 alkyl and —N(C1-4 alkyl) (C1-4 alkyl) respectively.

Aryl as used herein refers to, for example C6-14 mono or polycyclic groups having from 1 to 3 rings wherein at least one ring is aromatic including phenyl, naphthyl, anthracenyl, 1,2,3,4-tetrahydronaphthyl and the like, such as phenyl and naphthyl.

Heteroaryl is a 6 to 10 membered aromatic monocyclic ring or bicyclic ring system wherein at least one ring is an aromatic nucleus comprising one or more, for example 1, 2, 3 or 4 heteroatoms independently selected from O, N and S. Examples of heteroaryls include: pyrrole, oxazole, thiazole, isothiazole, imidazole, pyrazole, isoxazole, pyridine, pyridazine, pyrimidine, pyrazine, benzothiophene, benzofuran, or 1, 2, 3 and 1, 2, 4 triazole.

Heterocyclyl as employed herein refers to a 5 to 6 membered saturated or partially unsaturated non-aromatic ring comprising one or more, for example 1, 2, 3 or 4 heteroatoms independently selected from O, N and S optionally one or two carbons in the ring may bear an oxo substituent. The definition of C6-6 heterocycle as employed herein refers to a is a 5 to 6 membered saturated or partially unsaturated non-aromatic carbocyclic ring comprising one or more, for example 1, 2, 3 or 4 heteroatoms independently selected from O, N and S, wherein each heteroatom replaces a carbon atom and optionally one or two carbons may bear an oxo substitutent. Clearly any valancies of a heteroatom not employed in forming or retaining the ring structure may be filled by hydrogen or a substituent, as appropriate. Thus substituents on heterocycles may be on carbon or on a heteroatom, such as N as appropriate. Examples of heterocycles and C5-6 heterocycles include pyrroline, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, pyrazoline, imidazoline, pyrazolidine, imidazolidine, oxoimidazolidine, dioxolane, thiazolidine, isoxazolidine, pyran, dihydropyran, piperidine, piperazine, morpholine, dioxane, thiomorpholine and oxathiane.

Halogen includes fluoro, chloro, bromo or iodo, in particular fluoro, chloro or bromo, especially fluoro or chloro.

Oxo as used herein refers to C═O and will usually be represented as C(O).

C3-8 cycloalkyl as employed herein is intended to refer to a saturated or partially unsaturated non-aromatic ring containing 3 to 8 carbon atoms.

C1-10 alkyl includes C2, C3, C4, C5, C6, C7, C8 or C9 as well as C0 and C10

C0-8 alkyl includes C1, C2, C3, C4, C5, C6, or C7 as well as C0 and C8.

In relation to a saturated or unsaturated, branched or unbranched C1-10 alkyl chain, wherein at least one carbon (for example 1, 2 or 3 carbons, suitably 1 or 2, in particular 1) is replaced by a heteroatom selected from O, N, S(O)p, wherein said chain is optionally, substituted by one or more groups independently selected from oxo, halogen, an aryl group, a heteroaryl group or a heterocyclyl group, it will be clear to persons skilled in the art that the heteroatom may replace a primary, secondary or tertiary carbon, that is CH3, —CH2— or a —CH— or a branched carbon group, as technically appropriate.

In one embodiment of the disclosure there is provided compounds of formula (I), wherein R1 is methyl, ethyl, propyl, iso-propyl, butyl or tert-butyl, in particular tert-butyl.

In one embodiment R1 is —C(CH3)2CH2OH.

In one embodiment R2 is methyl, ethyl, n-propyl, iso-propyl, n-butyl or tert-butyl, in particular methyl.

In one embodiment R2 is —CH2OH.

In one embodiment R2 is in the 2, 3, or 4 position (i.e. ortho, meta or para position), in particular the para (4) position.

In one embodiment Ar is naphthyl.

In one embodiment Ar is not substituted with optional substituents.

In one embodiment Ar is substituted with 1 or 2 groups.

In one embodiment Ar is phenyl optionally substituted by 1 or 2 substituents independently selected from C1-3 alkyl or C1-3 alkoxy, for example tolyl, xylyl, anisoyl, di-methoxybenzene or methoxy-methylbenzene. The phenyl ring may, for example, be linked to the nitrogen of the urea through carbon 1 and to the group L through carbon 4. In such a case the optional one or two substituents selected from C1-3 alkyl or C1-3 alkoxy may be located in any of the unoccupied positions in the aromatic ring, for example in position 2 or in position 3 or in positions 2 and 3 or in positions 2 and 6 or in positions 3 and 5. Embodiments encompassing other possible regioisomers also form an aspect of the present disclosure.

In one embodiment L is a straight chain linker, for example:

—(CH2)n— wherein n is 1, 2, 3, 4, 5, 6, 7 or 8; or

—(CH2)n—O—(CH2)m— wherein n and m are independently 0, 1, 2, 3, 4, 5, 6 or 7, with the proviso that n+m is zero or an integer from 1 to 7, for example where n is 0 and m is 1 or 2 or alternatively, for example, where n is 1 or 2 and m is 0.

In one embodiment L is —OCH2—, —OCH2CH2—, —CH2O— or —CH2CH2O—.

In one embodiment L is a branched chain linker RaO(CH2)m wherein m is zero or an integer 1, 2, 3, 4 or 5 and Ra is a C2-7 branched alkyl, with the proviso that the number of carbons in Ra+m is an integer from 2 to 7, especially where m is zero, such as —CH(CH3)O—, —C(CH3)2O—, —CH2CH(CH3)O—, —CH(CH3)CH2O—, —C(CH3)2CH2O— or —CH2C(CH3)2O, in particular —CH(CH3)O—.

In one embodiment L is a branched chain linker (CH2)nORb wherein n is zero or an integer 1, 2, 3, 4 or 5 and Rb is a C2-7 branched alkyl, with the proviso that the number of carbons in Rb+n is an integer from 2 to 7, for example n is zero, such as —OCH(CH3)—, —OC(CH3)2—, —OCH2CH(CH3)—, —OCH(CH3)CH2—, —OC(CH3)2CH2— or —OCH2C(CH3)2 in particular —OCH(CH3)— or —OC(CH3)2CH2—.

In one embodiment L is a branched chain linker RaORb wherein Ra and Rb are independently selected from a C2-7 branched alkylene with the proviso that the number of carbons in Ra+Rb is an integer from 4 to 7.

In one embodiment L is a saturated unbranched C1-C8 alkylene chain or a saturated branched or unbranched C2-8 alkylene chain.

In one embodiment at least one carbon in L is replaced by —O—.

In one embodiment L is —O—.

Alkylene as employed herein refers to branched or unbranched carbon radicals, such as methylene (—CH2—) or chains thereof. In the context of the present specification where alkyl is a linker then the latter is used interchangeably with the term alkylene.

In one embodiment the chain L includes 1, 2 or 3 halogen atom substituents, independently selected from fluoro, chloro, and bromo, for example an alkylene carbon may incorporate one or two chlorine atoms or one or two fluorine atoms and a terminal carbon atom, for example of a branch of an alkylene chain, may be bonded to one, two or three fluorine atoms or one, two or three chlorine atoms to provide a radical such as a trifluoromethyl or a trichloromethyl group.

In one embodiment the chain L does not include a halogen atom or atoms.

In one embodiment R3 is H.

In one embodiment R3 is methyl, ethyl, n-propyl or iso-propyl.

In one embodiment R3 is cyclopropyl.

In one embodiment X is selected from, pyrrole, oxazole, thiazole, isothiazole, imidazole, pyrazole, isoxazole, oxadiazole, pyridazine, pyrimidine, pyrazine, or 1,2,3 and 1,2,4 triazole, such as pyrazole, isoxazole, oxadiazole, pyridine, pyridazine, pyrimidine, pyrazine, or 1,2,3 and 1,2,4 triazole, in particular, pyrimidine, imidazole or pyridine, and especially pyridine or pyrimidine, more specifically pyridine.

In one embodiment 1, 2, 3 or 4 carbon atoms are replaced in the alkyl chain of Q by heteroatoms independently selected from O, N, S(O)p.

In one embodiment the heteroatom(s) replacing carbon(s) in the alkyl chain fragment of Q are selected from N and O.

In one embodiment Q is a saturated or unsaturated, branched or unbranched C1-8 alkyl chain or a C1-6 alkyl chain, wherein at least one carbon is replaced by a heteroatom selected from —O, —N, S(O)p. Alternatively, in this embodiment the alkyl chain may be a C2-8 alkyl or a C3-6 alkyl group, such as a C4 alkyl or a C5 alkyl group.

In one embodiment a nitrogen atom in the alkyl chain is directly bonded to the carbonyl of the fragment —NR3C(O) and additionally may, for example, be a terminal amino group.

In one embodiment Q represents C1-6 alkylNH2 or NH2.

In one embodiment Q represents —NHC1-6 alkyl such as —NHCH3 or —NHCH2CH3 or —NHCH(CH3)2.

In one embodiment the fragment Q is a saturated or unsaturated, branched or unbranched C1-10 alkyl chain wherein at least one carbon (for example 1, 2, 3 or 4 carbons, in particular 1 or 2 carbons) is replaced by a heteroatom selected from O, N, S(O)p, for example in such a manner as to provide a stable N-acyl group, NR3C(O)Q, wherein said chain is optionally substituted by one or more groups selected from oxo, halogen, an aryl group, a heteroaryl group, a heterocyclyl group, or C3-8 cycloalkyl each aryl, heteroaryl or heterocyclyl or C3-8 cycloalkyl group bearing 0 to 3 substituents independently selected from a relevant substituent listed above for compounds of formula (I).

In one embodiment the fragment Q is a saturated or unsaturated, branched or unbranched C1-10 alkyl chain wherein at least one carbon (for example 1, 2, 3 or 4 carbons, in particular 1 or 2 carbons) is replaced by a heteroatom selected from O, N, S(O)p, for example in such a manner as to provide a stable N-acyl group, NR3C(O)Q, wherein said chain is optionally substituted by one or more groups selected from oxo, halogen, an aryl group, a heteroaryl group or a heterocyclyl group, each aryl, heteroaryl or heterocyclyl group bearing 0 to 3 substituents independently selected from a relevant substituent listed above for compounds of formula (I), for example halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, C1-4 mono or di-alkyl amino and C1-4 mono or di-acyl amino.

In one embodiment the latter chain is optionally substituted by one or more groups selected from oxo, halogen, an aryl group, a heteroaryl group or a heterocyclyl group, each aryl, heteroaryl or heterocyclyl group bearing 0 to 3 substituents selected from halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, and C1-4 mono or di-alkyl amino.

In one embodiment Q is C1-4alkyl-V—R4, such as C1-3alkyl-V—R4 wherein:

V is a heteroatom selected from NRV, O or S(O)p;

RV represents H or C1-3 alkyl;

R4 is H or —C1-3 alkyl, and p is as defined above,

with the proviso that the total alkyl chain length is not more than 10 carbon atoms, including replacement heteroatoms and that the resulting radical Q is a stable group, for example —CH2SCH3, —CH2SO2CH3, —CH2NHCH3, —CH2N(CH3)2—C(CH3)2NHCH3, —CH(CH3)N(CH3)2, —(CH2)3CHNHCH3, —(CH2)3N(CH3)2, —CH2OH, —CH2OCH3, —CH(CH3)OCH3, or —(CH2)2OCH3.

In one embodiment Q is C1-3 alkyl-V—(C1-3 alkyl-Z—R5)k such as C1-3 alkyl-V—(C2-3 alkyl-Z—R5)k wherein:



V is a heteroatom selected from N, NH, O or S(O)p, such as N or NH
(V is N in the case where k=2, or will be selected from NH, O or S(O)p, in the case where k=1, in particular NH);
Z is independently selected from NH, O or S(O)p;
R5 is H or —C1-3alkyl;
k is an integer 1 or 2 (such as 1); and
p is as defined above,

with the proviso that the total alkyl chain length is not more than 10 carbon atoms, including replacement heteroatoms and that the resulting radical Q is a stable group. Suitably Q is C1-3alkyl-V—C1-3alkyl-OCH3 for example C1-3alkyl-V—C2-3alkyl-OCH3 such as C1-3alkyl-V—(CH2)2OCH3, in particular —CH2O(CH2)2OCH3 and CH2S(CH2)2OCH3, or —CH2NH(CH2)2OCH3, C1-3alkyl-V—(C1-3alkyl-OCH3)k wherein k represents 2, for example C1-3alkyl-V—(C2-3alkyl-OCH3)k such as —CH2N[(CH2)2OCH3]2.

In one embodiment Q is C1-3 alkyl-V—C1-2 alkyl-Z—C1-2 alkyl-Y—R6, or C1-3 alkyl-V—C2-3 alkyl-Z—C2-3 alkyl-Y—R6, wherein V, Z and Y are independently a heteroatom selected from NH, O or S(O)p,

R6 is H or methyl, and

p is as defined above,

with the proviso that the total alkyl chain length is not more than 10 carbon atoms, including replacement heteroatoms and that the resulting radical Q is a stable group. Suitably Q is —CH2V(CH2)2O(CH2)2OCH3, such as —CH2O(CH2)2O(CH2)2OCH3, —CH2NH(CH2)2O(CH2)2OCH3, or —CH2S(CH2)2O(CH2)2OCH3.

In one embodiment Q represents —NR7R8 and —NR3C(O)Q forms a urea, where R7 and R8 independently represent hydrogen or a C1-9 saturated or unsaturated, branched or unbranched alkyl chain, wherein one or more carbons, such as 1, 2 or 3 are optionally replaced by a heteroatom selected from O, N or S(O)p. Said chain is optionally substituted by one or more groups independently selected from oxo, halogen, an aryl group, a heteroaryl group, a heterocyclyl or C3-8 cycloalkyl group, each aryl, heteroaryl or heterocyclyl group bearing 0 to 3 substituents independently selected from the relevant substituents listed above for compounds of formula (I), for example halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, C1-4 mono or di-alkyl amino and C1-4 mono or di-acyl amino with the proviso that the total alkyl chain length is not more than 10 carbon atoms, including replacement heteroatoms and that the resulting radical Q is a stable group.

In one embodiment Q represents —NR7R8 and —NR3C(O)Q forms a urea, where R7 and R8 independently represent hydrogen or a C1-9 saturated or unsaturated, branched or unbranched alkyl chain, wherein one or more carbons, such as 1, 2 or 3 are optionally replaced by a heteroatom selected from O, N or S(O)p. Said chain is optionally substituted by one or more groups independently selected from oxo, halogen, an aryl group, a heteroaryl group or a heterocyclyl group, each aryl, heteroaryl or heterocyclyl group bearing 0 to 3 substituents independently selected from the relevant substituents listed above for compounds of formula (I), for example halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, C1-4 mono or di-alkyl amino and C1-4 mono or di-acyl amino with the proviso that the total alkyl chain length is not more than 10 carbon atoms, including replacement heteroatoms and that the resulting radical Q is a stable group.

In this urea embodiment in one sub-embodiment R7 represents hydrogen.

Examples of ureas include those in which R7 and R8 are both hydrogen and Q is —NH2, or where Q is —NHCH3 or —N(CH3)2 to provide, for example, a fragment —NR3C(O)NH2 or —NR3C(O)NHCH3 or —NR3C(O)N(CH3)2.

Examples of ureas containing a heteroatom in the alkyl chain include those in which Q is —NH(CH2)2OCH3 or —N[(CH2)2OCH3)]2. In one embodiment Q represents —NHC2-6alkylOC1-3alkyl, such as —NHCH2CH2OCH3.

Examples of ureas containing an oxo substitutent include those in which Q is NHCH2C(O)NH—C2-3alkyl-X1—C1-3 alkyl, wherein X1 is a heteroatom selected from N, O or S(O)p and p is defined as above. Examples of the latter include those wherein Q is —NHCH2C(O)NHCH2CH2OCH3. Thus in one embodiment Q represents —NHC1-4 alkylC(O)NHC2alkylOCH3 such as —NHCH2C(O)NHCH2CH2OCH3.

In one embodiment Q represents —NHC1-4alkylC(O)RQ wherein RQ is selected from OH or —NR′R″ where R′ is hydrogen or C1-3 alkyl and R″ is hydrogen or C1-3 alkyl, for example —NHCH2C(O)OH, —NHCH2C(O)NH2 or —NHCH2C(O)NHCH3 such as —NHCH2C(O)OH or —NHCH2C(O)NHCH3.

In one embodiment Q represents —NHC1-4alkylC(O)OC1-3alkyl, such as —NHCH2C(O)OCH2CH3.

In a further urea sub-embodiment Q represents —N—R9C1-3 alkyl-V—(C1-3 alkyl-Z—R10)k for example —N—R9C2-3 alkyl-V—(C2-3 alkyl-Z—R10)k wherein:



V represents N, NH, O, S(O)p;
Z represents NH, O, S(O)p;
k is an integer 1 or 2;
p is an integer 0, 1 or 2
R9 represents H or C1-3 alkyl-V—(C1-3 alkyl-Z—R10)k such as C2-3 alkyl-V—(C2-3 alkyl-Z—R10)k; and
R10 is H or C1-3 alkyl such as C1-3 alkyl;

with the proviso that the total alkyl chain length is not more than 10 carbon atoms, including replacement heteroatoms and that the resulting radical Q is a stable group.

In one embodiment Q is a saturated or unsaturated, branched or unbranched C1-10 alkyl chain, wherein at least one carbon is replaced by a heteroatom selected from O, N, and S(O)p, wherein said chain is substituted by an aryl group bearing 0 to 3 substituents, for example 1, 2 or 3, such as 1 or 2 substituents independently selected from the relevant substituents listed above for compounds of formula (I), for example from halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino and C1-4 mono or di-alkyl amino and C1-4 mono or di-acyl amino, such as a saturated or unsaturated, branched or unbranched C1-10 alkyl chain, wherein at least one carbon is replaced by a heteroatom selected from O, N, and S(O)p, wherein said chain is substituted by an aryl group bearing 0 to 3 substituents, for example 1, 2 or 3, such as 1 or 2 substituents independently selected from halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino and C1-4 mono or di-alkyl amino. In one embodiment the said aryl group is phenyl, for example substituted phenyl or unsubstituted phenyl.

In one embodiment Q represents —NHC0-6 alkylphenyl, such as —NHphenyl or NHbenzyl.

Examples of the fragment —NR3C(O)Q wherein Q comprises substituted benzyl include:

—NR3C(O)CH2NHCH2C6H4(OCH3) such as —NHC(O)CH2NHCH2C6H4(OCH3), for example where the methoxy substituent is in the ortho, meta or para position, such as the para position.

In one embodiment Q is a saturated or unsaturated, branched or unbranched C1-10 alkyl chain, wherein at least one carbon is replaced by a heteroatom selected from O, N, and S(O)p, wherein said chain is substituted by a heteroaryl group bearing 0 to 3 substituents (for example 1, 2 or 3, such as 1 or 2 substituents) independently selected from the relevant substituents listed above for compounds of formula (I), for example halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 alkyl amino, C1-4 mono or di-alkyl amino and C1-4 mono or di-acyl amino, such as a saturated or unsaturated, branched or unbranched C1-10 alkyl chain, wherein at least one carbon is replaced by a heteroatom selected from O, N, and S(O)p, wherein said chain is substituted by a heteroaryl group bearing 0 to 3 substituents for example 1, 2 or 3, such as 1 or 2 substituents selected from halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 alkyl amino, C1-4 mono or di-alkyl amino. In one embodiment the said heteroaryl group is selected from, thiophene, oxazole, thiazole, isothiazole, imidazole, pyrazole, isoxazole, isothiazole, oxadiazole, 1,2,3 or 1,2,4 triazole, pyridine, pyridazine, pyrimidine, pyrazine and, in particular pyridine and pyrimidine, especially pyridine.

In one embodiment Q represents —NHC1-6 alkylheteroaryl, for example —NH(CH2)3imidazolyl or —NHCH2 isoxazole wherein the isoxazole is optionally substituted such as —NHCH2 isoxazole(CH3).

In one embodiment Q represents —NHC1-4 alkylC(O)NHC1-3alkylheteroaryl, for example a nitrogen containing heteroaryl group or a nitrogen and oxygen containing heteroaryl, more specifically —NHCH2C(O)NHCH2CH2pyridinyl, in particular where pyridinyl is linked through carbon, for example pyridin-4-yl or —NHCH2C(O)NHCH2CH2CH2imidazolyl, in particular where imidazolyl is linked through nitrogen.

In one embodiment Q is a saturated or unsaturated, branched or unbranched C1-10 alkyl chain, wherein at least one carbon is replaced by a heteroatom selected from O, N and S(O)p wherein said chain is substituted by a heterocyclyl group bearing 0 to 3 substituents (for example 1, 2 or 3, such as 1 or 2 substituents) independently selected from the relevant substituents listed above for compounds of formula (I), for example halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl amino, C1-4 mono or di-alkyl amino and C1-4 mono or di-acyl amino, such as a saturated or unsaturated, branched or unbranched C1-10 alkyl chain, wherein at least one carbon is replaced by a heteroatom selected from O, N and S(O)p wherein said chain is substituted by a heterocyclyl group bearing 0 to 3 substituents, for example 1, 2 or 3, such as 1 or 2 substituents selected from halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl amino, C1-4 mono or di-alkyl amino.

In one embodiment said heterocyclyl is selected, from a 5 or 6 membered saturated or partially unsaturated ring system comprising one or more (for example 1, 2 or 3 in particular 1 or 2) heteroatoms independently selected from O, N and S, for example pyrrolidine, tetrahydrofuran, tetrahydrothiophene, piperidine, piperazine, morpholine, 1,4-dioxane, pyrrolidine and oxoimidazolidine such as pyrrolidine, tetrahydrofuran, tetrahydrothiophene, piperidine, piperazine, morpholine, and 1,4-dioxane, in particular piperidine, piperazine, and morpholine.

A heterocyclic group may be linked to the alkyl chain of Q or to the carbonyl of —NR3C(O)— through carbon or nitrogen, in particular a nitrogen atom.

In one embodiment Q is —C0-3alkylheterocycle (for example —C0-1alkylheterocycle) said heterocyclyl group comprising at least one heteroatom (for example 1, 2 or 3, in particular 1 or 2, heteroatoms) selected from O, N and S, and is optionally substituted by one or two or three groups independently selected from the relevant substituents listed above for compounds of formula (I), for example halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, C1-4 mono and di-alkyl amino and C1-4 mono or di-acyl amino.

In one embodiment Q is —C0alkylheterocycle, for example a tetrahydropyranyl or a pyrrolidinyl or a morpholinyl or a piperazinyl or an oxoimidazolinyl group, such as 2-oxoimidazolidinyl group.

In one embodiment in which Q is —C0alkylheterocycle, the heterocycle is linked through carbon, and is, for example, a C-linked tetrahydropyran or a C-linked piperidine or a C-linked morpholine or a C-linked piperazine.

In one embodiment in which Q is —C0alkylheterocycle, the heterocyclic group containing one or more N atoms is linked through N. This embodiment provides for ureas in which one of the urea nitrogens is embedded within a heterocyclic ring. Examples of this embodiment include, but are not limited to, an N-linked morpholine or an N-linked piperidine or an N-linked piperazine, said N-linked piperizinyl group optionally bearing an additional C- or N-substituent (such as an N-methyl group or N—CH2CH2OCH3 group. In one embodiment Q is a heterocyclyl linked through nitrogen such as piperidinyl, in particular 4-hydroxypiperidinyl or piperazinyl, such as 4-methyl piperazinyl.

In one embodiment Q represents a heterocyclyl group, for example a nitrogen containing heterocyclyl group, in particular linked through N, such as morpholinyl or piperazinyl optionally substituted by methyl, especially 4-methyl, or piperidinyl.

In one embodiment Q is a —C1alkylheterocycle, for example tetrahydropyranylmethyl or a C- or N-linked piperazinylmethyl optionally bearing a substituent (for example a C1-6 alkyl substitutent such as methyl or a C1-6 alkoxy substituent such as —CH2CH2OCH3). Additional examples include a C- or N-linked pyrrolidinylmethyl, or a C- or N-linked oxoimidazolinylmethyl (such as 2-oxoimidazolidinylmethyl, said heterocycle optionally bearing a substitutent (such as N-methyl or N—SO2CH3).

In one embodiment Q represents —NHheterocyclyl (wherein the heterocyclyl bears 0 to 3 substituents selected from the relevant list of substituents listed above for compounds of formula (I), for example halogen, hydroxy, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, C1-4 mono or di-alkyl amino, —S(O)qC1-6 alkyl, C1-4 mono or di-acyl amino, C0-6 alkylC(O)C1-6 alkyl or C0-6 alkylC(O)C1-6 heteroalkyl), such as where the ring is linked through carbon, for example

2-piperidinyl or 3-piperidinyl or 4-piperidinyl, in particular 1-acetylpiperidin-4-yl, 1-methylpiperidin-4-yl, 1-(methylsulfonyl)piperidin-4-yl or 1-(2-(2-methoxyethoxy)acetyl)piperidin-4-yl

In one embodiment Q represents —NHC1-6 alkylheterocyclyl for example a nitrogen containing heterocyclyl group, in particular one linked through nitrogen, such as —NHCH2CH2morpholine, —NH(CH2)3morpholine or —NH(CH2)4morpholine.

In one embodiment Q represents —NHC1-6 alkylC(O)heterocyclyl (wherein the heterocyclyl bears 0 to 3 substituents selected from the relevant list of substituents listed above for compounds of formula (I), for example halogen, hydroxy, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, C1-4 mono or di-alkyl amino, C1-4 mono or di-acyl amino, C0-6 alkylC(O)C1-6 alkyl or C0-6 alkylC(O)C1-6 heteroalkyl) for example a nitrogen containing heterocyclyl group, in particular one linked through nitrogen, such as —NHCH2C(O)-1-pyrrolindinyl, NHCH2C(O)-1-piperidinyl, —NHCH2C(O)-4-morpholinyl or —NHCH2C(O)piperazinyl such as —NHCH2C(O)-4-methyl-1-piperazinyl.

In one embodiment Q represents —NHC1-4 alkylC(O)NHC1-3alkylheterocyclyl for example a nitrogen containing heterocyclyl group or a nitrogen and/or oxygen containing heterocyclyl, such as —NHCH2C(O)NHCH2CH2morpholinyl, in particular where morpholinyl is linked through nitrogen.

In one embodiment Q represents —N(C1-3 alkyl)C1-6 alkylheterocyclyl, for example a nitrogen containing heterocyclyl group, in particular linked through nitrogen, such as —N(CH3)CH2CH2morpholine, —N(CH3)(CH2)3morpholine or —N(CH3)(CH2)4morpholine.

In one embodiment Q is —C1-3alkyl-G-C1-3alkylheterocycle wherein G is a heteroatom selected from NH, O or S(O)p said heterocyclyl group comprising at least one heteroatom (for example 1, 2 or 3, in particular 1 or 2, heteroatoms) selected from O, N, and S, and is optionally substituted by one or two or three groups independently selected from relevant substituents listed above for compounds of formula (I), for example halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, C1-4 mono and di-alkyl amino and C1-4 mono or di-acyl amino such as one or two or three groups halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, C1-4 mono and di-alkyl amino. Suitably Q is —CH2G(CH2)2heterocycle for example —CH2G(CH2)2tetrahydropyranyl; or —CH2G(CH2)2morpholinyl in which the heterocyclyl is linked through nitrogen or carbon; or CH2G(CH2)2piperazinyl in which the heterocyclyl is linked through nitrogen or carbon and optionally bearing a further C- or N-substituent (for example a C1-6 alkyl substitutent such as methyl or a C1-6 alkoxy substituent such as —CH2CH2OCH3); or —CH2G(CH2)2pyrrolidinyl, in which the heterocyclyl is linked through nitrogen or carbon, for example linked through nitrogen; or —CH2G(CH2)2oxoimidazolinyl (such as 2-oxoimidazolidinyl) for example linked through N and optionally bearing an additional C- or N-substitutent (such as N-methyl or N—SO2CH3), and in which G is O or NH.

In one embodiment G is O.

In one embodiment G is NH.

In one embodiment Q is a saturated or unsaturated C1-10 alkyl chain wherein at least one carbon (for example 1, 2 or 3 carbons) is replaced by a heteroatom selected from O, N, S(O)p wherein said chain is substituted by a C3-8 carbocyclyl group and said alkyl chain is optionally substituted by one or more (for example 1 or 2) groups selected from oxo and halogen. In one embodiment said C3-8 carbocyclyl group bears one or more groups (for example 1, 2 or 3 groups) independently selected from halogen, hydroxyl, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, amino, C1-4 mono or di-alkyl amino, C1-4 mono or di-acyl amino, S(O)qC1-6 alkyl, C0-6 alkylC(O)C1-6 alkyl or C0-6 alkylC(O)C1-6 heteroalkyl.

In one embodiment Q represents —NHC3-6 cycloalkyl, such as —NHcyclopropyl, —NHcyclopentyl or —NHcyclohexyl.

In one embodiment the aryl, heteroaryl or heterocyclyl group bears at least one —S(O)qC1-6 alkyl substitutent and optionally bears one or two further relevant substituents independently selected from the list of substituents defined above for compounds of formula (I).

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The Patent Description & Claims data below is from USPTO Patent Application 20130102576, Derivatives of 1-phenyl-2-pyridinyl alkyl alcohols as phosphodiesterase inhibitors.

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims priority to European Patent Application No. 11186056.5, filed on Oct. 21, 2011, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to inhibitors of the phosphodiesterase 4 (PDE4) enzyme. More particularly, the invention relates to derivatives of 1-phenyl-2-pyridinyl alkyl alcohols, methods of preparing such compounds, compositions containing them, and therapeutic use thereof.

2. Discussion of the Background

Airway obstruction characterizes a number of severe respiratory diseases including asthma and chronic obstructive pulmonary disease (COPD). Events leading to airway obstruction include oedema of airway walls, increased mucous production and inflammation.

Drugs for treating respiratory diseases such as asthma and COPD are currently administered through inhalation. One of the advantages of the inhalatory route over the systemic one is the possibility of delivering the drug directly at site of action, reducing systemic side-effects, thus resulting in a more rapid clinical response and a higher therapeutic ratio.

Inhaled corticosteroids are the current maintenance therapy of choice for asthma and together with bronchodilator beta2-agonists for acute symptom relief, they form the mainstay of current therapy for the disease. The current management of COPD is largely symptomatic by means of bronchodilating therapy with inhaled anticholinergics and inhaled beta2-adrenoceptor agonists. However, corticosteroids do not reduce the inflammatory response in COPD as they do in asthma.

Another class of therapeutic agents which has been widely investigated in view of its anti-inflammatory effects for the treatment of inflammatory respiratory diseases such as asthma and COPD is represented by the inhibitors of the enzymes phosphodiesterases (PDEs), in particular of the phosphodiesterase type 4 (hereinafter referred to as PDE4).

Various compounds acting as PDE4 inhibitors have been disclosed in the prior art. However, the usefulness of several PDE4 inhibitors of the first-generation such as rolipram and piclamilast has been limited due to their undesirable side effects. Said effects include nausea and emesis due to their action on PDE4 in the central nervous system and gastric acid secretion due to the action on PDE4 in parietal cells in the gut.

The cause of said side effects has been widely investigated. It has been found that PDE4 exists in two distinct forms representing different conformations, that were designated as high affinity rolipram binding site or HPDE4, especially present in the central nervous system and in parietal cells, and low affinity rolipram binding site or LPDE4 (Jacobitz, S et al., Mol. Pharmacol., 1996, 50, 891-899, which is incorporated herein by reference in its entirety), which is found in the immune and inflammatory cells. While both forms appear to exhibit catalytic activity, they differ with respect to their sensitivity to inhibitors. In particular compounds with higher affinity for LPDE4 appear less prone to induce side-effects such as nausea, emesis and increased gastric secretion.

The effort of targeting LPDE4 has resulted in a slight improvement in the selectivity for the second-generation PDE4 inhibitors such as roflumilast. Nonetheless, roflumilast is under dosed in order to achieve an acceptable side effect profile.

Other classes of compounds acting as PDE4 inhibitors have been disclosed in the prior art. For example, EP 1 634 606 discloses, inter alia, ketone derivatives like benzofuran or 1,3-benzodioxole derivatives.

WO 94/02465 discloses, inter alia, ketone derivatives of general formula

wherein R1 is lower alkyl and R2 may be alkyl, alkenyl, cycloalkyl, cycloalkyl, cycloalkenyl, cyclothioalkyl or cyclothioalkenyl.

WO 95/35281 in the name of Celltech Therapeutics concerns tri-substituted phenyl derivatives.

WO2009/018909 discloses derivatives of 1-phenyl-2-pyridinyl alkyl alcohols which have the following general formula

as inhibitors of phosphodiesterase 4 (PDE4) enzyme.

WO2009/077068 discloses further derivatives of 1-phenyl-2-pyridinyl alkyl alcohols which have the following general formula

as inhibitors of phosphodiesterase 4 (PDE4) enzyme.

WO2010/089107 discloses further derivatives of 1-phenyl-2-pyridinyl alkyl alcohols which have the following general formula

as inhibitors of phosphodiesterase 4 (PDE4) enzyme.

Although several PDE4 inhibitors have been disclosed so far as above reported, there is still a need for further PDE4 inhibitors. Particularly, there is still a need for further PDE4 inhibitors endowed with a high affinity for PDE4 enzyme and which show an appropriate developability profile as an inhalation treatment for example in terms of reduced side effects. Such reduction of side effects may be achieved, by way of example, through a low systemic exposure of the drug; an appropriate profile in terms of some pharmacokinetic characteristics, especially metabolic clearance, may be thus key to this goal. The present invention addresses the above mentioned need by providing the compounds of the invention.

SUMMARY

OF THE INVENTION

Accordingly, it is one object of the present invention to provide novel compounds acting as inhibitors of the phosphodiesterase 4 (PDE4) enzyme.

It is another object of the present invention to provide novel methods of preparing such a compound.

It is another object of the present invention to provide novel compositions which contain such a compound.

It is another object of the present invention to provide novel methods of preventing and/or treating certain diseases and conditions by administering such a compound.

These and other objects, which will become apparent during the following detailed description, have been achieved by the inventors\’ discovery that compounds of formula (I) set out below are useful as phosphodiesterase 4 (PDE4) inhibitors.

In particular, the present invention is directed to derivatives of 1-phenyl-2-pyridinyl alkyl alcohols of general formula (I):

wherein:

R1 and R2, which can be the same or different, are independently selected from the group consisting of:



(C1-C6) alkyl, optionally substituted by (C3-C7) cycloalkyl;
(C1-C6) haloalkyl;
(C3-C7) cycloalkyl; and
(C3-C7) heterocycloalkyl;

R3 is hydrogen, (C1-C6) alkyl or (C1-C3) alkylthio(C1-C6) alkyl;

A is a partially unsaturated or unsaturated bicyclic ring system consisting of two fused monocyclic ring systems B and C as below represented

wherein ring B contains a nitrogen atom which represents the point of attachment for ring

A to the rest of the molecule through a —(CHR3)— group and wherein ring B and C may optionally contain further heteroatoms;

p is an integer from zero to 3;

Y is an oxo group;

n is an integer from zero to 3;

K is selected from the group consisting of:



(C1-C6) alkyl, optionally substituted by one or more (C3-C7) cycloalkyl groups;
(C3-C7) heterocycloalkyl(C1-C4) alkyl;
(C3-C7) heterocycloalkyl, optionally substituted by one or more (C1-C6) alkyl groups;
(C1-C4) haloalkyl;
a group —OR4 wherein R4 is selected from the group consisting of:

H;
(C1-C10) alkyl, optionally substituted by (C3-C7) cycloalkyl or heteroaryl;


halogen atoms;
a group —CN;
a group —NO2;
NR5R6 wherein R5 and R6, which can be the same or different, are independently selected from the group consisting of:

H;
a group —OH;
NR7R8(C1-C4)alkyl wherein R7 and R8, which can be the same or different, are independently selected from the group consisting of: H; (C1-C6) alkyl, optionally substituted with (C1-C6) alkoxyl; and NR9R10(C1-C6) alkyl wherein R9 and R10, which can be the same or different, are H or (C1-C6) alkyl; or they form with the nitrogen atom to which they are linked a (C3-C7) heterocycloalkyl ring optionally substituted by (C1-C6)alkyl or (C1-C6) alkylcarbonyl;
(C1-C6) alkyl, optionally substituted by (C1-C6) alkoxyl or heteroaryl, (C3-C7) heterocycloalkylcarbonyl, heteroarylcarbonyl, all of them being optionally further substituted by one or more (C1-C6) alkyl, (C1-C6) haloalkyl or (C1-C6) alkoxyl groups, which may be the same or different and are independently selected;
a group —SO2R11, wherein R11 is (C1-C6) alkyl;
a group —C(O)R12, wherein R12 is (C1-C6) alkyl optionally substituted by (C1-C6) alkoxyl;


NR13R14(C1-C4)alkyl; wherein R13 and R14, which can be the same or different, are independently selected in the group consisting of: —SO2(C1-C6) alkyl, H, (C1-C6) alkyl, and (C3-C7)heterocycloalkyl(C1-C4) alkyl; and
—SO2NR15R16: wherein R15 and R16, which can be the same or different, are independently H or (C1-C6) alkyl;

wherein groups R4 to R16 may have the same or different meanings at each occurrence, if present in more than one group;

and pyridine N-oxides, pharmaceutically acceptable salts, and solvates thereof;

and wherein the compound of formula (I) is not: 3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetoxy)ethyl)pyridine.

The present invention also encompasses the pharmaceutically acceptable salts and/or solvates of the compounds of formula (I).

The present invention further provides the corresponding pyridine N-oxides.

Hereinafter, compounds of formula (I), pyridine N-oxides, and their pharmaceutically acceptable salts and solvates, defined in any aspect of the invention (except intermediate compounds described in the chemical processes) are referred to as “compounds of the invention”.

The present invention further provides processes for the preparation of the compounds of the invention.

The present invention also provides pharmaceutical compositions of compounds of the invention either alone or in combination, in admixture with one or more pharmaceutically acceptable carriers.

In a further aspect, the present invention provides the use of the compounds of the invention as a medicament.

In another aspect, the present invention provides the use of the compounds of the invention for the manufacture of a medicament.

In particular, the present invention provides the use of the compounds of the invention for the prevention and/or treatment of any disease characterized by phosphodiesterase 4 (PDE4) overactivity and/or wherein an inhibition of PDE4 activity is desirable.

In particular, the compounds of the invention alone or combined with other active ingredients may be administered for the prevention and/or treatment of a disease of the respiratory tract characterized by airway obstruction such as asthma and COPD.

In a further aspect, the present invention provides the use of compounds of the invention for the preparation of a medicament for the prevention and/or treatment of any disease characterized by phosphodiesterase 4 (PDE4) overactivity and/or wherein an inhibition of PDE4 activity is desirable.

Moreover, the present invention provides a method of prevention and/or treatment of any disease wherein PDE4 inhibition is desirable, said method comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound of the invention.

DETAILED DESCRIPTION

OF THE PREFERRED EMBODIMENTS

The following definitions apply:

“Halogen atoms” includes fluorine, chlorine, bromine, and iodine, preferably chlorine.

“(C1-Cx) alkyl” where x is an integer greater than 1, means straight-chained and branched alkyl groups wherein the number of constituent carbon atoms is in the range 1 to x. Examples of alkyl groups are methyl, ethyl, n-propyl, isopropyl, and t-butyl.

“(C1-Cx) alkoxyl” where x is an integer greater than 1, means straight-chained and branched alkoxy groups wherein the number of carbon atoms is in the range 1 to x. Examples of alkyl groups are methoxyl, ethoxyl, n-propoxyl, isopropoxyl, and t-butoxyl.

“(C1-Cx)haloalkyl” refers to the above defined “(C1-Cx)alkyl” groups wherein one or more hydrogen atoms are replaced by one or more halogen atoms, which can be the same or different from each other. Examples of said (C1-Cx)haloalkyl groups may include halogenated, poly-halogenated and fully halogenated alkyl groups wherein all of the hydrogen atoms are replaced by halogen atoms, e.g., trifluoromethyl or difluoro methyl groups.

“NRjRw(C1-Cx)alkyl” means the above defined “(C1-Cx)alkyl” groups wherein one hydrogen atom is replaced by one a group —NRjRw.

“(C1-Cz)alkylthio” where z is an integer greater than 1, means straight-chained and branched alkylthio groups wherein the number of constituent carbon atoms is in the range 1 to z and which are linked to other groups via the sulfur atom. Examples of alkylthio groups are methylthio, ethylthio, and so on.

“(C1-Cz)alkylthio(C1-Cx)alkyl” means the above “(C1-Cx)alkyl” group wherein one or more hydrogen atoms are replaced by one “(C1-Cz)alkylthio” group.

“(C3-Cy)cycloalkyl”, where y is an integer greater than or equal to 3, means saturated cyclic hydrocarbon groups containing from 3 to y ring carbon atoms. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

The derived expression “(C3-Cy)heterocycloalkyl” refers to saturated monocyclic (C3-Cy)cycloalkyl groups, in which at least one ring carbon atom is replaced by a heteroatom (e.g. N, NH, S, or O). Not limiting examples of (C3-Cy)heterocycloalkyl are represented by: pyrrolidinyl, thiazolidinyl, piperazinyl, piperidinyl, morpholinyl, and thiomorpholinyl.

“(C1-Cx)alkylcarbonyl” means (C1-Cx)alkylCO— groups wherein the group “(C1-Cx)alkyl” has the meaning above defined.

“(C3-Cy)heterocycloalkylcarbonyl” means “(C3-Cy)heterocycloalkylCO—” groups wherein the group “(C3-Cy)heterocycloalkyl” has the meaning above defined.

“(C3-Cy)heterocycloalkyl(C1-Cx) alkyl” means the above “(C1-Cx)alkyl” group wherein one or more hydrogen atoms are replaced by one or more “(C3-Cy)heterocycloalkyl” groups.

“Aryl” means mono or bi-cyclic ring systems which have 6 to 10 ring atoms, wherein at least one ring is aromatic.

“Heteroaryl” means mono- or bi-cyclic ring systems with 5 to 11 ring atoms, in which at least one ring is aromatic and in which at least one ring atom is a heteroatom (e.g. N, NH, S, or O).

Examples of suitable monocyclic aryl or heteroaryl systems include, for instance, phenyl, thiophene (thiophenyl), benzene (phenyl), pyrrole (pyrrolyl), pyrazole (pyrazolyl), imidazole (imidazolyl), isoxazole (isoxazolyl), oxazole (oxazolyl), isothiazole (isothiazolyl), thiazole (thiazolyl), pyridine (pyridinyl), imidazolidine (imidazolidinyl), furan (furanyl) radicals and the like.

Examples of suitable aryl or heteroaryl bicyclic systems include naphthalene (naphthyl), biphenylene (biphenylenyl), purine (purinyl), pteridine (pteridinyl), benzotriazole (benzotriazolyl), quinoline (quinolinyl), isoquinoline (isoquinolinyl), indole (indolyl), isoindole (isoindolyl), benzothiophene (benzothiophenyl), dihydrobenzo dioxin, dihydrobenzo dioxepin, benzo oxazine radicals and the like.

“Heteroarylcarbonyl” means heteroarylCO— groups wherein the group “heteroaryl” has the meaning above defined.

“Ring system” means mono- or bicyclic ring systems which may be saturated, partially unsaturated or unsaturated, such as aryl, (C3-C8)cycloalkyl, (C3-C7)-heterocycloalkyl or heteroaryl.

The present invention is directed to a class of compounds acting as inhibitors of the phosphodiesterase 4 (PDE4) enzyme. Said class of compounds inhibits the conversion of cyclic nucleotides, in particular cyclic adenosine monophosphate (cAMP), into their inactive 5′-mononucleotide forms.

In the airways, the physiological responses to elevated intracellular levels of cyclic nucleotides, in particular of cAMP, lead to the suppression of the activity of immune and pro-inflammatory cells such as mast cells, macrophages, T lymphocytes, eosinophils and neutrophils, resulting in a decrease of the release of inflammatory mediators which include cytokines such as IL-1, IL-3 and tumor necrosis factor-alpha (TNF-α). It also leads to an airway smooth muscle relaxation and a decrease in oedema.

The present invention relates to derivatives of 1-phenyl-2-pyridinyl alkyl alcohols of general formula (I), pharmaceutically acceptable salts and pyridine N-oxides thereof,

wherein R1, R2, R3, Y, K, n, p. and A are as above defined.

The term “pharmaceutically acceptable salts”, as used herein, refers to derivatives of compounds of formula (I) wherein the parent compound is suitably modified by converting any of the free acid or basic group, if present, into the corresponding addition salt with any base or acid conventionally intended as being pharmaceutically acceptable.

Suitable examples of said salts may include mineral or organic acid addition salts of basic residues such as amino groups, as well as mineral or organic basic residues such as carboxylic groups.

Suitable cations of inorganic bases for use in the preparation of said salts comprise ions of alkali or alkaline earth metals such as potassium, sodium, calcium or magnesium.

Those obtained by reacting the main compound, functioning as a base, with an inorganic or organic acid to form a salt comprise, for example, salts of hydrochloric acid, sulfuric acid, phosphoric acid, methane sulfonic acid, camphor sulfonic acid, oxalic acid, maleic acid, succinic acid and citric acid.

Compounds of general formula (I) contain at least one stereogenic center, namely represented by the carbon atom (1) with an asterisk below, and therefore exist as optical stereoisomers.

Where the compounds according to the invention have at least one stereogenic center, they may accordingly exist as enantiomers. Where the compounds according to the invention possess two or more stereogenic centers, they may additionally exist as diastereoisomers. It is to be understood that all such isomers and mixtures thereof in any proportion are encompassed within the scope of the present invention.

In a preferred embodiment, the present invention is directed to compounds of formula (I)\’, which are compounds of formula (I) as above defined where the absolute configuration of carbon (1) is that shown herebelow:

The absolute configuration for carbon (1) is assigned on the basis of Cahn-Ingold-Prelog nomenclature based on groups\’ priorities.

In one preferred embodiment, for compounds of formula (I), the absolute configuration at carbon (1) is (S).

It is to be understood that all preferred groups or embodiments described herebelow for compounds of formula (I) may be combined among each other and apply to compounds of formula (I)′ as well mutatis mutandis.

In a preferred embodiment, compounds of the invention are pyridine N-oxides.

Ring A consists of two fused monocyclic ring systems B and C as below represented

wherein ring B contains the nitrogen atom which represents the point of attachment for ring A to the rest of the molecule through a —(CHR3)— group (hereabove indicated by an asterisk) and ring B and C may optionally contain further heteroatoms (e.g. N, NH, S or O).

Ring A, consisting of two fused monocyclic ring systems B and C, may be substituted by n groups Y and/or p groups K as above defined at any suitable position of rings B and C.

Not limiting examples of ring A are represented herebelow:

In one embodiment, ring A is selected from the group consisting of:

In one preferred embodiment, ring A is selected from the group consisting of:

In one preferred embodiment, ring C is a monocyclic aryl or heteroaryl system.

In a further preferred embodiment, ring C is a phenyl group.

In a preferred embodiment, ring B contains only one nitrogen atom. In another preferred embodiment, ring B contains one further heteroatom which may be nitrogen or sulfur.

In one preferred embodiment, ring C is a monocyclic aryl or monocyclic heteroaryl ring system and ring B is a 5 or 6 membered heterocycloalkyl group.

In one preferred embodiment, zero Y groups are connected to ring C and n groups Y are connected to ring B.

In a further preferred embodiment, zero Y groups are connected to ring C, n groups Y are connected to ring B and n is an integer ranging from 0 to 3. In a still further preferred embodiment, zero Y groups are connected to ring C, n groups Y are connected to ring B and n is an integer ranging from 1 to 3.

In one preferred embodiment, ring A which is substituted by n groups Y is selected in the group consisting of:

In one preferred embodiment, R2 is (C1-C6) haloalkyl or (C1-C6) alkyl.

In another preferred embodiment, R1 is (C1-C6) alkyl which is optionally substituted by (C3-C7) cycloalkyl.

In a further preferred embodiment, R2 is (C1-C6) haloalkyl and R1 is (C1-C6) alkyl which is substituted by (C3-C7) cycloalkyl.

In one preferred embodiment, R3 is hydrogen or methyl. In another preferred embodiment, R3 is hydrogen.

In one preferred embodiment, n is zero. In another preferred embodiment, n is 1 or 2.

In one preferred embodiment, p is zero. In another preferred embodiment, p is 1 or 2.

In a preferred embodiment, K is selected from the group consisting of:



a group —OR4 wherein R4 is (C1-C10) alkyl;
NR5R6 wherein R5 and R6, which can be the same or different, are independently selected from the group consisting of:

H;
NR7R8(C1-C4)alkyl wherein R7 and R8, which can be the same or different, are independently selected from the group consisting of: H; (C1-C6) alkyl, optionally substituted with (C1-C6) alkoxyl; and NR9R10(C1-C6)alkyl wherein R9 and R10, which can be the same or different, are H or (C1-C6) alkyl; or they form with the nitrogen atom to which they are linked a (C3-C7) heterocycloalkyl ring optionally substituted by (C1-C6)alkyl or (C1-C6) alkylcarbonyl;
(C1-C6) alkyl, optionally substituted by heteroaryl;
a group —SO2R11, wherein R11 is (C1-C6) alkyl;
a group —C(O)R12, wherein R12 is (C1-C6) alkyl optionally substituted by (C1-C6) alkoxyl.

In one embodiment, a compound of formula (I) is selected from the group consisting of:

(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)acetoxy)ethyl)pyridine 1-oxide;
(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(2-(1,1-dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl)acetoxy)ethyl)pyridine 1-oxide;
(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(2-(1,3-dioxoisoindolin-2-yl)acetoxy)ethyl)pyridine 1-oxide;
(S)-4-(2-(2-(4-amino-1,3-dioxoisoindolin-2-yl)acetoxy)-2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)ethyl)-3,5-dichloropyridine 1-oxide;
(S)-4-(2-(2-(6-amino-1-oxoisoindolin-2-yl)acetoxy)-2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)ethyl)-3,5-dichloropyridine 1-oxide;
(S)-3,5-dichloro-4-(2-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)acetoxy)-2-(3,4-dimethoxyphenyl)ethyl)pyridine 1-oxide;
(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(2-(6-(methylsulfonamido)-1-oxoisoindolin-2-yl)acetoxy)ethyl)pyridine 1-oxide;
(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(2-(3,3-dimethyl-1,1-dioxidobenzo[d]isothiazol-2(3H)-yl)acetoxy)ethyl)pyridine 1-oxide;
(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(2-(5-methoxy-1H-indol-1-yl)acetoxy)ethyl)pyridine 1-oxide;
(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(2-(2,3-dioxoindolin-1-yl)acetoxy)ethyl)pyridine 1-oxide;
(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(2-(5-(hydroxyamino)-1,3-dioxoisoindolin-2-yl)acetoxy)ethyl)pyridine 1-oxide;
(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(2-(4-(hydroxyamino)-1,3-dioxoisoindolin-2-yl)acetoxy)ethyl)pyridine 1-oxide;
(S)-4-(2-(2-(5-amino-1,3-dioxoisoindolin-2-yl)acetoxy)-2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)ethyl)-3,5-dichloropyridine 1-oxide;
3,5-dichloro-4-((2S)-2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(2-(1,3-dioxoisoindolin-2-yl)propanoyloxy)ethyl)pyridine 1-oxide;
(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(2-(5-(N-(2-morpholinoethyl)methylsulfonamido)-1,3-dioxoisoindolin-2-yl)acetoxy)ethyl)-pyridine 1-oxide;
(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(2-(4-(methylsulfonamido)-1,3-dioxoisoindolin-2-yl)acetoxy)ethyl)pyridine 1-oxide;
(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(2-(2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)acetoxy)ethyl)pyridine 1-oxide;
(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(2-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)acetoxy)ethyl)pyridine 1-oxide;

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Agent: Boehringer Ingelheim International Gmbh – Ingelheim Am Rhein, DE
USPTO Applicaton #: #20130090338 – Class: 5142358 (USPTO) – 04/11/13 – Class 514 

The Patent Description & Claims data below is from USPTO Patent Application 20130090338, New ccr2 receptor antagonists, method for producing the same, and use thereof as medicaments.

  



FIELD OF INVENTION

The present invention relates to novel antagonists for CCR2 (CC chemokine receptor 2), the method for producing the same, and their use for providing medicaments for treating conditions and diseases where activation of CCR2 plays a causative role, especially pulmonary diseases like asthma and COPD, neurologic disease, especially of pain diseases, immune related diseases, especially diabetes mellitus including diabetes nephropathy, and cardiovascular diseases, especially atherosclerotic disease.

BACKGROUND OF THE INVENTION

The chemokines are a family of small, proinflammatory cytokines, with potent chemotatctic activities. Chemokines are chemotactic cytokines that are released by a wide variety of cells to attract various cells, such as monocytes, macrophages, T cells, eosinophils, basophils and neutrophils to sites of inflammation.

Chemokine receptors, such as CCR2 or CCR5 have been implicated as being important mediators of inflammatory and immunoregulatory disorders and diseases as well as autoimmune pathologies such as rheumatoid arthritis and atherosclerosis. Accordingly, agents which modulate chemokine receptors such as the CCR2 and CCR5 receptor would be useful in such disorders and diseases.

In particular it is widely accepted that numerous conditions and diseases involve inflammatory processes. Such inflammations are critically triggered and/or promoted by the activity of macrophages, which are formed by differentiation out of monocytes. It has further been found that monocytes are characterized by, e.g., a high expression of membrane-resident CCR2, whereas the CCR2 expression in macrophages is lower. CCR2 is a critical regulator of monocytes trafficking, which can be described as the movement of the monocytes towards an inflammation along a gradient of monocyte chemoattractant proteins (MCP-1, MCP-2, MCP-3, MCP-4).

Therefore, in order to reduce macrophage-induced inflammation, it would be desirable to block the monocyte CCR2 by an antagonist, so that the monocytes can be less triggered to move towards an inflammation area for conversion into macrophages.

Based on the aforesaid there is a need for providing effective antagonists for CCR2, which are pharmacologically acceptable.

DESCRIPTION OF THE INVENTION

It has now been found that such effective CCR2 inhibitors can be provided by compounds according to general formula (I),

wherein R1 is a group selected from among —H, -halogen, —CN, —O—C1-C4-alkyl, —C1-C4-alkyl, —CH═CH2, —C≡CH, —CF3, —OCF3, —OCF2H, —C5-C10-heteroaryl, and —OCFH2,

and wherein R7 is a group selected from among —H, -halogen, —CN, —O—C1-C4-alkyl, —C1-C4-alkyl, —CH═CH2, —C≡CH, —CF3, —OCF3, —OCF2H, and —OCFH2;

or wherein R7 and R1 on two neighbouring ring atoms together form a —C3-C6-alkenylene group, such that an annellated aromatic ring is formed, in which one or two or three carbon centers may optionally be replaced by 1 or 2 or 3 hetero atoms selected from N, O, and S, wherein the resulting annelated ring being optionally substituted by one or more groups selected from among —OH, —NH2, —C1-C3-alkyl, —O—C1-C6-alkyl, —CN, —CF3, —OCF3, and halogen;

wherein R4 is a group selected from among -hydrogen, —C1-C3-alkyl, —CF3, —OCF3, —OCF2H, -halogen, —CN, —O—C1-C4-alkyl, —CH═CH2, —C≡CH, and —OCFH2;

wherein A is C or N;

wherein R14 is a group selected from among -hydrogen, and —C1-C3-alkyl;

wherein R2 is selected from among —H, -halogen, —CN, —O—C1-C4-alkyl, —C1-C4-alkyl, -cyclopropyl, —CH═CH2, —C≡CH, —CF3, —OCF3, —OCF2H, and —OCFH2;

wherein R3 is selected from among —H, -methyl, -ethyl, -propyl, -i-propyl, -cyclopropyl, —OCH3, —CF3, and —CN;

wherein n is 1, 2 or 3;

wherein G and E are N,

or wherein G is N and E is C,

or wherein G is C and E is N;

wherein R5 is a group of the structure —NR8R9,

wherein R8, R9, are independently selected from among —H, —C1-C6-alkyl, and a group of the structure —C3-C6-cycloalkyl, wherein such ring is optionally substituted by one or more groups selected from among —F, and —OCH3;

or wherein R5 is a group of the structure —N(R10,R10′),

wherein R10 and R10′, together form a —C2-C6-alkylene group such that a ring is formed, wherein such ring is optionally substituted with one or more groups selected from among —OH, —OCH3, —CF3, —OCF3, —CN, -halogen, —C1-C4-alkyl, ═O, and —N(C0-C3-alkyl)-SO2—C1-C3-alkyl;

or wherein R5 is a group of the structure —N(R11,R11′),

wherein R11 and R11′ together form a —C2-C6-alkylene group such that a ring is formed, in which one ore two carbon centers may optionally be replaced by one or two hetero atoms selected from among N, O, and S,

wherein such ring is optionally substituted by one or more groups on one ring atom or on two neighbouring ring atoms selected from among —OH, —NH2, —C1-C3-alkyl, —O—C1-C6-alkyl, —CN, —CF3, —OCF3, ═O, and halogen;

or wherein R5 is a group of the structure -L1-R13,

wherein L1 is selected from among —NH— and —N(C1-C4-alkyl)-,

wherein R13 is —C3-C8-heterocyclyl,

wherein R13 is optionally substituted by one or more groups selected from among halogen, —CF3, —OCF3, —CN, —OH, —O—C1-C4-alkyl, —C1-C6-alkyl;

wherein R6 is selected from among —H, —C1-C4-alkyl, —OH, —O—C1-C4-alkyl, -halogen, —CN, —CF3, and —OCF3;

as well as in form of their acid addition salts with pharmacologically acceptable acids.

Preferred compounds of formula (I) according to the invention are compounds wherein R1 is a group selected from among —H, -halogen, —CN, —O—C1-C4-alkyl, —C1-C4-alkyl, —CH═CH2, —C≡CH, —CF3, —OCF3, —OCF2H, —C5-C10-heteroaryl, and —OCFH2,

and wherein R7 is a group selected from among —H, -halogen, —CN, —O—C1-C4-alkyl, —C1-C4-alkyl, —CH═CH2, —C≡CH, —CF3, —OCF3, —OCF2H, and —OCFH2;

or wherein R7 and R1 on two neighbouring ring atoms together form a —C3-C6-alkenylene group, such that an annellated aromatic ring is formed, in which one or two or three carbon centers may optionally be replaced by 1 or 2 or 3 hetero atoms selected from N, O, and S, wherein the resulting annelated ring being optionally substituted by one or more groups selected from among —OH, —NH2, —C1-C3-alkyl, —O—C1-C6-alkyl, —CN, —CF3, —OCF3, and halogen;

wherein R4 is a group selected from among -hydrogen, —C1-C3-alkyl, —CF3, —OCF3, —OCF2H, -halogen, —CN, —O—C1-C4-alkyl, —CH═CH2, —C≡CH, and —OCFH2;

wherein A is C or N;

wherein R14 is a group selected from among -hydrogen, and —C1-C3-alkyl;

wherein R2 is selected from among —H, -halogen, —CN, —O—C1-C4-alkyl, —C1-C4-alkyl, -cyclopropyl, —CH═CH2, —C≡CH, —CF3, —OCF3, —OCF2H, and —OCFH2;

wherein R3 is selected from among —H, -methyl, -ethyl, -propyl, -i-propyl, -cyclopropyl, —OCH3, —CF3, and —CN;

wherein n is 1, 2 or 3;

wherein G and E are N,

or wherein G is N and E is C,

or wherein G is C and E is N;

wherein R5 is a group of the structure —N(R10,R10′),

wherein R10 and R10′, together form a —C2-C6-alkylene group such that a ring is formed, wherein such ring is optionally substituted with one or more groups selected from among —OH, —OCH3, —CF3, —OCF3, —CN, -halogen, —C1-C4-alkyl, ═O, and —N(C0-C3-alkyl)-SO2—C1-C3-alkyl;

or wherein R5 is a group of the structure -L1-R13,

wherein L1 is selected from among —NH— and —N(C1-C4-alkyl)-,

wherein R13 is —C3-C8-heterocyclyl,

wherein R13 is optionally substituted by one or more groups selected from among halogen, —CF3, —OCF3, —CN, —OH, —O—C1-C4-alkyl, —C1-C6-alkyl;

wherein R6 is selected from among —H, —C1-C4-alkyl, —OH, —O—C1-C4-alkyl, -halogen, —CN, —CF3, and —OCF3.

Preferred compounds of formula (I) according to the invention are compounds with R2, R3, R5, R6, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, E, G, A, and n as herein before or below defined,

wherein R1 is a group selected from among —H, -halogen, —C1-C4-alkyl, —CF3, —OCF3, and —C6-heteroaryl comprising a N atom; and wherein R7 is a group selected from among —H, -halogen, —C1-C4-alkyl, —CF3, —OCF3, and —C6-heteroaryl comprising a N atom; and wherein R4 denotes-hydrogen.

Preferred compounds of formula (I) according to the invention are compounds with R2, R3, R5, R6, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, E, G, A, and n as herein before or below defined,

wherein R1 and R7 are a group selected from -halogen, preferably selected from —Cl; and wherein R4 denotes-hydrogen.

Preferred compounds of formula (I) according to the invention are compounds with R2, R3, R5, R6, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, E, G, A, and n as herein before or below defined,

wherein R7 and R1 on two neighbouring ring atoms together form —C3-C6-alkenylene group, such that an annellated aromatic ring is formed, in which one or two carbon centers may optionally be replaced by 1 or 2 hetero atoms selected from N; and wherein R4 is a group selected from among —H, —OCH3, and —C1-C3-alkyl, preferably selected from among -hydrogen, and —C1-C3-alkyl.

Preferred compounds of formula (I) according to the invention are compounds with R2, R3, R5, R6, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, E, G, A, and n as herein before or below defined,

wherein R7 and R1 on two neighbouring ring atoms together form —C3-C6-alkenylene group, such that an annellated aromatic ring is formed, in which one carbon centers may optionally be replaced by 1 hetero atoms selected from N; and wherein R4 is a group selected from among —H, —OCH3, and —C1-C3-alkyl, preferably selected from among -hydrogen, and —C1-C3-alkyl.

Preferred compounds of formula (I) according to the invention are compounds with R2, R3, R5, R6, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, E, G, A, and n as herein before or below defined,

wherein R7 and R1 on two neighbouring ring atoms together form —C3-C6-alkenylene group, and wherein R4 is a group selected from among —H, —OCH3, and —C1-C3-alkyl, preferably selected from among -hydrogen, and —C1-C3-alkyl.

Preferred compounds of formula (I) according to the invention are compounds with R2, R3, R5, R6, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, E, G, A, and n as herein before or below defined,

wherein R7 and R1 on two neighbouring ring atoms together form a —C4-alkenylene group, such that an annellated aromatic ring is formed, in which one or two carbon centers may optionally be replaced by 1 or 2 hetero atoms selected from N; and wherein R4 is a group selected from among -hydrogen, and —C1-C3-alkyl.

Preferred compounds of formula (I) according to the invention are compounds with R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, E, G, and n as herein before or below defined,

wherein A denotes N.

Preferred compounds of formula (I) according to the invention are compounds with R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, E, G, and n as herein before or below defined,

wherein A denotes C.

Preferred compounds of formula (I) according to the invention are compounds with R1, R3, R4, R5, R6, R7, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, E, G, A, and n as herein before or below defined,

wherein R2 is selected from among —H, —C1-C4-alkyl, -cyclopropyl, and —O—C1-C4-alkyl.

Preferred compounds of formula (I) according to the invention are compounds with R1, R3, R4, R5, R6, R7, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, E, G, A, and n as herein before or below defined,

wherein R2 is selected from among —H, —CH3, -cyclopropyl, and —OCH3.

Preferred compounds of formula (I) according to the invention are compounds with R1, R3, R4, R5, R6, R7, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, E, G, A, and n as herein before or below defined,

wherein R2 is selected from among —CH3, -cyclopropyl, and —OCH3.

Preferred compounds of formula (I) according to the invention are compounds with R1, R3, R4, R5, R6, R7, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, E, G, A, and n as herein before or below defined,

wherein R2 is selected from among -Methyl and —OCH3.

Preferred compounds of formula (I) according to the invention are compounds with R1, R3, R4, R5, R6, R7, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, E, G, A, and n as herein before or below defined,

wherein R2 denotes -Methyl.

Preferred compounds of formula (I) according to the invention are compounds with R1, R3, R4, R5, R6, R7, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, E, G, A, and n as herein before or below defined,

wherein R2 denotes —OCH3.

Preferred compounds of formula (I) according to the invention are compounds with R1, R3, R4, R5, R6, R7, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, E, G, A, and n as herein before or below defined,

wherein R2 denotes -cyclopropyl.

Preferred compounds of formula (I) according to the invention are compounds with R1, R3, R4, R5, R6, R7, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, E, G, A, and n as herein before or below defined,

wherein R2 denotes —H.

Preferred compounds of formula (I) according to the invention are compounds with R1, R2, R4, R5, R6, R7, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, E, G, A, and n as herein before or below defined,

wherein R3 is selected from among -hydrogen, —CF3, and —OCH3.

Preferred compounds of formula (I) according to the invention are compounds with R1, R2, R4, R5, R6, R7, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, E, G, A, and n as herein before or below defined,

wherein R3 is selected from among —CF3, and —OCH3.

Preferred compounds of formula (I) according to the invention are compounds with R1, R2, R4, R5, R6, R7, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, E, G, A, and n as herein before or below defined,

wherein R3 denotes -hydrogen.

Preferred compounds of formula (I) according to the invention are compounds with R1, R2, R4, R5, R6, R7, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, E, G, A, and n as herein before or below defined,

wherein R3 denotes —OCH3.

Preferred compounds of formula (I) according to the invention are compounds with R1, R2, R4, R5, R6, R7, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, E, G, A, and n as herein before or below defined,

wherein R3 denotes —CF3.

Preferred compounds of formula (I) according to the invention are compounds with R1, R2, R4, R5, R6, R7, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, E, G, A, and n as herein before or below defined,

wherein R3 denotes -cyclopropyl.

Preferred compounds of formula (I) according to the invention are compounds with R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, E, G, and A, as herein before or below defined,

wherein n is selected from among 1, and 2.

Preferred compounds of formula (I) according to the invention are compounds with R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, E, G, and A, as herein before or below defined,

wherein n is 2.

Preferred compounds of formula (I) according to the invention are compounds with R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, A, and n as herein before or below defined, wherein G and E are N or wherein G is N and E is C.

Preferred compounds of formula (I) according to the invention are compounds with R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, A, and n as herein before or below defined, wherein G and E are N.

Preferred compounds of formula (I) according to the invention are compounds with R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, A, and n as herein before or below defined, wherein G is N and E is C.

Preferred compounds of formula (I) according to the invention are compounds with R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, A, and n as herein before or below defined,

wherein G is C and E is N.

Preferred compounds of formula (I) according to the invention are compounds with R1, R2, R3, R4, R6, R7, R8, R9, R11, R11′, R14, E, G, A, and n as herein before or below defined, wherein R5 is a group of the structure —N(R10,R10′), wherein R10 and R10′, together form a —C4-alkylene group such that a ring is formed, wherein such ring is optionally substituted with one or more groups selected from among —N(C0-C3-alkyl)-SO2—C1-C3-alkyl;

or wherein R5 is a group of the structure -L1-R13, wherein L1 denotes —NH—, wherein R13 is —C6-heterocyclyl comprising an O atom, wherein R13 is optionally substituted by a group selected from among —O—C1-C4-alkyl.

Preferred compounds of formula (I) according to the invention are compounds with R1, R2, R3, R4, R6, R7, R8, R9, R11, R11′, R13, R14, L1, E, G, A, and n as herein before or below defined, wherein

R5 is a group of the structure —N(R10,R10′), wherein R10 and R10′, together form a —C4-alkylene group such that a ring is formed, wherein such ring is optionally substituted with one or more groups selected from among —N(C0-C3-alkyl)-SO2—C1-C3-alkyl.

Preferred compounds of formula (I) according to the invention are compounds with R1, R2, R3, R4, R6, R7, R8, R9, R11, R11′, R13, R14, L1, E, G, A, and n as herein before or below defined, wherein

R5 is a group of the structure —N(R10,R10′), wherein R10 and R10′, together form a —C4-alkylene group such that a ring is formed, wherein such ring is optionally substituted with one or more groups selected from among —N(CH3)—SO2—CH3.

Preferred compounds of formula (I) according to the invention are compounds with R1, R2, R3, R4, R6, R7, R8, R9, R10, R10′, R11, R11′, R14, E, G, A, and n as herein before or below defined,

wherein R5 is a group of the structure -L1-R13, wherein L1 denotes —NH—, wherein R13 is —C6-heterocyclyl comprising an O atom, wherein R13 is optionally substituted by a group selected from among —O—C1-C4-alkyl.

Preferred compounds of formula (I) according to the invention are compounds with R1, R2, R3, R4, R6, R7, R8, R9, R10, R10′, R11, R11′, R14, E, G, A, and n as herein before or below defined,

wherein R5 is a group of the structure -L1-R13, wherein L1 denotes —NH—, wherein R13 is —C6-heterocyclyl comprising an O atom, wherein R13 is optionally substituted by a group selected from -halogen.

Preferred compounds of formula (I) according to the invention are compounds with R1, R2, R3, R4, R6, R7, R8, R9, R10, R10′, R11, R11′, R14, E, G, A, and n as herein before or below defined,

wherein R5 is a group of the structure -L1-R13, wherein L1 denotes —NH—, wherein R13 is —C6-heterocyclyl comprising an O atom, wherein R13 is optionally substituted by a group selected from —F.

Preferred compounds of formula (I) according to the invention are compounds with R1, R2, R3, R4, R6, R7, R8, R9, R10, R10′, R11, R11′, R14, E, G, A, and n as herein before or below defined,

wherein R5 is a group of the structure -L1-R13, wherein L1 denotes —NH—, wherein R13 is —C6-heterocyclyl comprising an O atom, wherein R13 is optionally substituted by a group selected from among —OCH3, and -halogen, preferably selected from among —OCH3.

Preferred compounds of formula (I) according to the invention are compounds with R1, R2, R3, R6, R7, R8, R9, R10, R10′, R11, R11′, R14, E, G, A, and n as herein before or below defined,

wherein R4 denotes —H, and wherein R5 denotes a group of the structure -L1-R13, wherein L1 is a group selected from among —NH—, and —N(CH3)—, wherein R13 is a group of the structure

Preferred compounds of formula (I) according to the invention are compounds with R1, R2, R3, R4, R6, R7, R8, R9, R11, R11′, R13, R14, L1, E, G, A, and n as herein before or below defined, wherein

R5 is a group of the structure

Preferred compounds of formula (I) according to the invention are compounds with R1, R2, R3, R4, R5, R7, R8, R9, R10, R10′, R11, R11′, R13, R14, L1, E, G, A, and n as herein before or below defined,

wherein R6 denotes —H.

Preferred compounds of formula (I) according to the invention are compounds with R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R10′, R11, R11′, R13, L1, E, G, A, and n as herein before or below defined,

wherein R14 denotes —H.

All of the above embodiments under formula (I) have to be understood to optionally be present in form of their individual optical isomers, mixtures of their individual optical isomers, or racemates, as well as in form of their acid addition salts with pharmacologically acceptable acids, as well as in form of their solvates and/or hydrates.

It has now been found that such compounds as herein before or below defined could be used as a medicament.

It has been found that such compounds as herein before or below defined could be used for making a medicament for the treatment of inflammatory diseases. It has been found that such compounds as herein before or below defined could be used for making a medicament for the treatment of inflammatory diseases, wherein the inflammatory diseases are selected from inflammatory diseases of the respiratory tract. It has been found that such compounds as herein before or below defined could be used for making a medicament for the treatment of inflammatory diseases, wherein the inflammatory diseases are selected from chronic obstructive pulmonary disease, asthma, and cystic fibrosis. It has been found that such compounds as herein before or below defined could be used for making a medicament for the treatment of neurologic diseases, preferably for the treatment of pain diseases especially for the treatment of inflammatory and neuropathic pain disease, especially for the treatment of chronic pain. It has been found that such compounds as herein before or below defined could be used for making a medicament for the treatment of immune related diseases, preferably for the treatment of diabetes mellitus. It has been found that such compounds as herein before or below defined could be used for making a medicament for the treatment of cardiovascular diseases, preferably for the treatment of peripheral atherosclerotic disease. It has been found that such compounds as herein before or below defined could be used for making a medicament for the treatment of diabetic nephropathy.

Present invention encloses compounds as herein before or below defined as medicaments.

Present invention encloses compounds as herein before or below defined as medicaments for the treatment of inflammatory diseases. Present invention encloses compounds as herein before or below defined as medicaments for the treatment of inflammatory diseases, wherein the inflammatory diseases are selected from inflammatory diseases of the respiratory tract.

Present invention encloses compounds as herein before or below defined as medicaments for the treatment of inflammatory diseases, wherein the inflammatory diseases are selected from chronic obstructive pulmonary disease, asthma, and cystic fibrosis. Present invention encloses compounds as herein before or below defined as medicaments for the treatment of neurologic diseases, preferably for the treatment of pain diseases especially for the treatment of inflammatory and neuropathic pain disease, especially for the treatment of chronic pain.

Present invention encloses compounds as herein before or below defined as medicaments for the treatment of immune related diseases, preferably for the treatment of diabetes mellitus.

Present invention encloses compounds as herein before or below defined as medicaments for the treatment of cardiovascular diseases, preferably for the treatment of peripheral atherosclerotic disease. Present invention encloses compounds as herein before or below defined as medicaments for the treatment of diabetic nephropathy.

It has been found that such compounds as herein before or below defined could be used for the treatment of inflammatory diseases. It has been found that such compounds as herein before or below defined could be used for the treatment of inflammatory diseases, wherein the inflammatory diseases are selected from inflammatory diseases of the respiratory tract. It has been found that such compounds as herein before or below defined could be used for the treatment of inflammatory diseases, wherein the inflammatory diseases are selected from chronic obstructive pulmonary disease, asthma, and cystic fibrosis. It has been found that such compounds as herein before or below defined could be used for the treatment of neurologic diseases, preferably for the treatment of pain diseases especially for the treatment of inflammatory and neuropathic pain disease, especially for the treatment of chronic pain. It has been found that such compounds as herein before or below defined could be used for the treatment of immune related diseases, preferably for the treatment of diabetes mellitus. It has been found that such compounds as herein before or below defined could be used for the treatment of cardiovascular diseases, preferably for the treatment of peripheral atherosclerotic disease. It has been found that such compounds as herein before or below defined could be used for the treatment of diabetic nephropathy.

DEFINITIONS

Terms not specifically defined herein should be given the meanings that would be given to them by one of skill in the art in light of the disclosure and the context. As used in the specification, however, unless specified to the contrary, the following terms have the meaning indicated and the following conventions are adhered to.

In the groups, radicals, or moieties defined below, the number of carbon atoms is often specified preceding the group, for example, —C1-C6-alkyl means an alkyl group or radical having 1 to 6 carbon atoms. In general, for groups comprising two or more subgroups, the last named subgroup is the radical attachment point, for example, the substituent “aryl-C1-C3-alkyl-” means an aryl group which is bound to a C1-C3-alkyl-group, the latter of which is bound to the core or to the group to which the substituent is attached.

In case a compound of the present invention is depicted in form of a chemical name and as a formula in case of any discrepancy the formula shall prevail. An asterisk is may be used in sub-formulas to indicate the bond which is connected to the core molecule as defined.

For example, the term “3-carboxypropyl-group” represents the following substituent:

wherein the carboxy group is attached to the third carbon atom of the propyl group. The terms “1-methylpropyl-”, “2,2-dimethylpropyl-” or “cyclopropylmethyl-” group represent the following groups:

The asterisk may be used in sub-formulas to indicate the bond which is connected to the core molecule as defined.

Many of the following terms may be used repeatedly in the definition of a formula or group and in each case have one of the meanings given above, independently of one another.

Unless otherwise stated, all the substituents are independent of one another. If for example there might be a plurality of C1-C6-alkyl groups as substituents in one group, in the case of three substituents C1-C6-alkyl, one may represent methyl, one n-propyl and one tert-butyl.

Within the scope of this application, in the definition of possible substituents, these may also be represented in the form of a structural formula. An asterisk (*) in the structural formula of the substituent is to be understood as being the linking point to the rest of the molecule. Moreover, the atom of the substituent which follows the linking point is referred to as the atom in position number 1. Thus, for example, the groups N-piperidinyl (Piperidin-A), 4-piperidinyl (Piperidin-B), 2-tolyl (Tolyl-C), 3-tolyl (Tolyl-D), and 4-tolyl (Tolyl-E) are shown as follows:

If there is no asterisk (*) in the structural formula of the substituent, each hydrogen atom may be removed from the substituent and the valency thus freed may act as a binding site to the rest of a molecule. Thus, for example, (Tolyl-F) may represent 2-tolyl, 3-tolyl, 4-tolyl, and benzyl

The term “substituted” as used herein, means that any one or more hydrogens on the designated atom is replaced with a selection from the indicated group, provided that the designated atom\’s normal valence is not exceeded, and that the substitution results in a stable compound.

By the term “optionally substituted” is meant within the scope of the invention the above-mentioned group, optionally substituted by a lower-molecular group. Examples of lower-molecular groups regarded as chemically meaningful are groups consisting of 1-200 atoms. Preferably such groups have no negative effect on the pharmacological efficacy of the compounds. For example the groups may comprise:



Straight-chain or branched carbon chains, optionally interrupted by heteroatoms, optionally substituted by rings, heteroatoms or other common functional groups.
Aromatic or non-aromatic ring systems consisting of carbon atoms and optionally heteroatoms, which may in turn be substituted by functional groups.
A number of aromatic or non-aromatic ring systems consisting of carbon atoms and optionally heteroatoms which may be linked by one or more carbon chains, optionally interrupted by heteroatoms, optionally substituted by heteroatoms or other common functional groups.

By the term “branched or unbranched, saturated or unsaturated C1-C6-carbon chain” it is meant a chain of carbon atoms, which is constituted by 1 to 6 carbon atoms arranged in a row and which can optionally further comprise branches or one or more hetero atoms selected from N, O or S. Said carbon chain can be saturated or unsaturated by comprising double or triple bonds.

If the carbon chain is to be substituted by a group which together with one or two carbon atoms of an alkylene chain forms a carbocyclic ring with 3, 5 or 6 carbon atoms, this includes the following examples of the rings:

The term “C1-Cn-alkyl”, wherein n is an integer from 2 to n, either alone or in combination with another radical denotes an acyclic, saturated, branched or linear hydrocarbon radical with 1 to n C atoms. For example the term C1-C5-alkyl embraces the radicals H3C—, H3C—CH2—, H3C—CH2—CH2—, H3C—CH(CH3)—, H3C—CH2—CH2—CH2—, H3C—CH2—CH(CH3)—, H3C—CH(CH3)—CH2—, H3C—C(CH3)2—, H3C—CH2—CH2—CH2—CH2—, H3C—CH2—CH2—CH(CH3)—, H3C—CH2—CH(CH3)—CH2—, H3C—CH(CH3)—CH2—CH2—, H3C—CH2—C(CH3)2—, H3C—C(CH3)2—CH2—, H3C—CH(CH3)—CH(CH3)— and H3C—CH2—CH(CH2CH3)—.

By the term “C1-C6-alkyl” (including those which are part of other groups) are meant branched and unbranched alkyl groups with 1 to 6 carbon atoms and by the term “C1-C4-alkyl” are meant branched and unbranched alkyl groups with 1 to 4 carbon atoms. Alkyl groups with 1 to 4 carbon atoms are preferred. By the term “C1-C3-alkyl” are meant branched and unbranched alkyl groups with 1 to 3 carbon atoms and by the term “C2-C4-alkyl” are meant branched and unbranched alkyl groups with 2 to 4 carbon atoms. Examples for alkyl groups with 1-6 carbon atoms include: methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, iso-pentyl, neo-pentyl or hexyl. Optionally the abbreviations Me, Et, n-Pr, i-Pr, n-Bu, i-Bu, t-Bu, etc. may also be used for the above-mentioned groups. Unless stated otherwise, the definitions propyl, butyl, pentyl and hexyl include all the possible isomeric forms of the groups in question. Thus, for example, propyl includes n-propyl and iso-propyl, butyl includes iso-butyl, sec-butyl and tert-butyl etc.

The term “C1-Cn-alkylene” wherein n is an integer 2 to n, either alone or in combination with another radical, denotes an acyclic, straight or branched chain divalent alkyl radical containing from 1 to n carbon atoms. For example the term C1-C4-alkylene includes —CH2—, —CH2—CH2—, —CH(CH3)—, —CH2—CH2—CH2—, —C(CH3)2—, —CH(CH2CH3)—, —CH(CH3)—CH2—, —CH2—CH(CH3)—, —CH2—CH2—CH2—CH2—, —CH2—CH2—CH(CH3)—, —CH(CH3)—CH2—CH2—, —CH2—CH(CH3)—CH2—, —CH2—C(CH3)2—, —C(CH3)2—CH2—, —CH(CH3)—CH(CH3)—, —CH2—CH(CH2CH3)—, —CH(CH2CH3)—CH2—, —CH(CH2CH2CH3)—, —CH(CH(CH3))2— and —C(CH3)(CH2CH3)—.

By the term “C1-C8-alkylene” (including those which are part of other groups) are meant branched and unbranched alkylene groups with 1 to 8 carbon atoms. By the term “C1-C6-alkylene” are meant branched and unbranched alkylene groups with 1 to 6 carbon atoms. By the term “C2-C8-alkylene” are meant branched and unbranched alkylene groups with 2 to 8 carbon atoms. By the term “C2-C6-alkylene” are meant branched and unbranched alkylene groups with 2 to 6 carbon atoms. By the term “C4-C5-alkylene” are meant branched and unbranched alkylene groups with 4 to 5 carbon atoms. By the term “C2-C6-alkylene” are meant branched and unbranched alkylene groups with 2 to 6 carbon atoms. By the term “C1-C4-alkylene” are meant branched and unbranched alkylene groups with 1 to 4 carbon atoms. By the term “C1-C2-alkylene” are meant branched and unbranched alkylene groups with 1 to 2 carbon atoms. By the term “C1-alkylene” are meant an alkylene groups with 1 carbon atom. By the term “C5-alkylene” are meant branched and unbranched alkylene groups with 5 carbon atoms. By the term “C0-C4-alkylene” are meant branched and unbranched alkylene groups with 0 to 4 carbon atoms, thus also a single bond is encompassed. By the term “C0-C3-alkylene” are meant branched and unbranched alkylene groups with 0 to 3 carbon atoms, thus also a single bond is encompassed. By the term “C1-C3-alkylene” are meant branched and unbranched alkylene groups with 1 to 3 carbon atoms. Examples for C1-C8-alkylene include: methylene, ethylene, propylene, 1-methylethylene, butylene, 1-methylpropylene, 1,1-dimethylethylene, 1,2-dimethylethylene, pentylene, 1,1-dimethylpropylene, 2,2-dimethylpropylene, 1,2-dimethylpropylene, 1,3-dimethylpropylene, hexylene, heptylene or octylene. Unless stated otherwise, the definitions propylene, butylene, pentylene, hexylene, heptylene and octylene include all the possible isomeric forms of the groups in question with the same number of carbons. Thus, for example, propyl also includes 1-methylethylene and butylene includes 1-methylpropylene, 1,1-dimethylethylene, 1,2-dimethylethylene.

A —C1-alkylene group, which is linked to a structure on two neighbouring ring atoms such that an annellated ring is formed, results to a C3-carbocycle. A —C2-alkylene group, which is linked to a structure on two neighbouring ring atoms such that an annellated ring is formed, results to a C4-carbocycle. A —C3-alkylene group, which is linked to a structure on two neighbouring ring atoms such that an annellated ring is formed, results to a C5-carbocycle. A —C4-alkylene group, which is linked to a structure on two neighbouring ring atoms such that an annellated ring is formed, results to a C6-carbocycle. A —C5-alkylene group, which is linked to a structure on two neighbouring ring atoms such that an annellated ring is formed, results to a C7-carbocycle. A —C6-alkylene group, which is linked to a structure on two neighbouring ring atoms such that an annellated ring is formed, results to a C8-carbocycle.

In the definition of possible substituents, which are linked to such C1-C6-alkylene groups forming a C3-C8-carbocycle, it is to be understood that any of the atoms of the resulting C3-C8-carbocycles could be the linking point for such a substituent.

If the carbon chain is to be substituted by a group which together with one or two carbon atoms of the alkylene chain forms a carbocyclic ring with 3, 5 or 6 carbon atoms, this includes the following examples of the rings:

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The Patent Description & Claims data below is from USPTO Patent Application 20130078717, Binding members-513.

This application claims the benefit under 35 U.S.C. §119(e) of Application No. 61/112,381 (US) filed on 7 Nov. 2008.

This invention relates to binding members, especially antibody molecules, for the interleukin 1 receptor-1 (IL-1R1). The binding members are useful for the treatment of disorders mediated by IL-1R1 including rheumatoid arthritis, asthma and chronic obstructive pulmonary disease (COPD). The invention also relates to processes for the preparation of such binding members, methods of treatment of disorders mediated by IL-1R1 using binding members of the invention and the use of binding members of the invention in the preparation of a medicament for the treatment of disorders mediated by IL-1R1.

interleukin (IL-1) is a multifunctional cytokine, which plays a major role in inflammatory responses during immune-mediated diseases and infections. IL-1 is produced from a variety of cell types following stimulation with bacterial products, cytokines or immune complexes. IL-1 displays autocrine and paracrine activities on a variety of cell types promoting the production of inflammatory mediators such as prostaglandins, nitric oxide, cytokines, chemokines, metalloproteinases and adhesion molecules. Blocking IL-1 biological activity should be beneficial to prevent tissue damage caused by excessive production or disregulated IL-1 activity or to normalise aberrant responses to pathogens for example during an exacerbation of COPD.

The IL-1 family of cytokines currently consists of eleven individual members, IL-1alpha (IL-1α), IL-1beta (IL-1β), interleukin-18 (IL-18), Interleukin 1 antagonist (IL-1Ra), IL1F5-10 and Interleukin-33 (IL-33). Four of these, namely IL-1α, IL-1β, IL-18 & IL-1Ra (IL-1 receptor antagonist), have been characterised most fully and linked to pathological processes in a variety of diseases, and IL-1α, IL-1β, and IL-1Ra alone have clearly been shown to interact with membrane IL-1R1 (1, 2, 3). IL-1α and IL-1β are the products of separate genes. These proteins are related at the amino acid level, IL-1α and IL-1β share 22% homology, with IL-1α and IL-1Ra sharing 18% homology. IL-1β shares 26% homology with IL-1Ra. The genes for IL-1α, IL-1β & IL-1Ra members are located on a similar region in human chromosome 2q14 (4, 5).

Both IL-1α and IL-1β are synthesized as 31-kDa precursor peptides that are cleaved to generate 17-kDa mature IL-1α and IL-1β. IL-1β is produced by a variety of cell types including epithelial cells and macrophaes. It is released from cells after cleavage by the cysteine protease caspase-1 (IL-1β converting enzyme (ICE) (6)), IL-1α is cleaved by calpain proteases and can remain on the plasma membrane from where it appears to activate cells, via direct cell to cell contact (7). Pro-IL-1α contains a nuclear localization sequence in its amino terminal, which can lead to activation of a variety of cellular pathways (8).

IL-1Ra is a naturally occurring inhibitor of the IL-1 system. It is produced as four different isoforms derived from alternative mRNA splicing and alternative translation initiation. A 17 kDa secreted isoform of IL-1Ra is expressed as variably glycosylated species, of 22-25 Kda (9,10) now termed sIL-1Ra. An 18 kDa intracellular isoform is termed icIL-1Ra1 (11). The isoform icIL-1Ra2 is produced by an alternative transcriptional splice from exon located between icIL-1Ra1 and sIL-1Ra first exons (12) A third 16 kDa intracellular isoform icIL-1Ra3 has also been identified (13). Kineret® (anakinra) is a recombinant, nonglycosylated form of the soluble human interleukin-1 receptor antagonist (IL-1Ra). Kineret® differs from native human IL-1Ra in that it has the addition of a single methionine residue at its amino terminus. Kineret® consists of 153 amino acids and has a molecular weight of 17.3 kilodaltons. Kineret® is approved for the treatment of moderate to severe active rheumatoid arthritis.

IL-1α and IL-1β exert their biological effects by binding to a transmembrane receptor, IL-1R1 (RefSeg NM—00877), which belongs to the IL-1 receptor family. There are currently ten members of the IL-1 receptor family (14); IL-1Receptor I (IL-1R1 (80 kDa), IL-1RII (68 kDa) and IL-1 receptor accessory protein (IL-1RacP) being relevant to the signalling of IL-1α and β. IL-1R1 and IL1RacP complex in the cell membrane to form a high affinity receptor capable of signalling on binding of IL-1α or Il-1β. IL-1Ra binds IL-1R1 but does not interact with IL-1RAcP. IL-1α. Il-1β and IL-1Ra also bind IL-RII which does not have an intracellular signalling domain.

All three of these receptors can be expressed as membrane bound or soluble proteins. IL-1R type I (IL-1R1), IL-1RII & IL-1R accessory protein (IL-1RAcP) belong to the immunoglobulin (Ig) gene superfamily with their extracellular region containing three Ig-like domains. IL-1R1 and IL-1RacP have cytoplasmic domains (Toll-like IL-1R (TIR)) domains, which are related to the Toll-Like receptor (TLR) superfamily. IL-1R1 is termed the signalling receptor as upon ligand binding and complexing with IL-1RAcP the signal transduction is initiated via its cytoplasmic tail of 213 amino acid residues 5). Current literature suggests that IL-1RII acts only as a ‘decoy receptor’ either at the cell surface or extracellularly as a soluble form (16).

The crystal structure of the extracellular region of the IL-1R1 bound to IL-1β has been resolved to 2.5A resolution (17). The two N-terminal Ig domains appear rigid due to a disulfide linker, with the third domain showing more flexibility, The IL-1R1 appears wrapped around IL-1β, with two significant areas of contact. One of these is in a groove between domains 1 & 2, while the second area of contact is a smaller area located on the third domain. Interestingly the IL-1Ra also appears to bind to the groove region between domains 1&2 of the IL-1R1, however there does not appear to be any contact between the IL-1Ra and the third Ig domain of the IL-1R1 (18).

Once IL-1 has bound to the IL-1R1 chain the IL-1RAcP is recruited to the ligand-receptor pair and forms a high affinity receptor complex, which results in initiation of signal transduction.

A model of IL-1RAcP interaction with IL-1-IL-1R1 has been proposed based on mutagenesis and antibody studies (19, 20 & 21). It shows that the IL-1RAcP interacts with the interface between IL-1 and IL-1R1. These studies also demonstrated that the AcP could not interact with the IL-1Ra-IL-1R1 pair, which forms a more relaxed structure. Greenfeder et al, (22) have shown that the IL4R1 bound with IL-1Ra fails to recruit the IL-1RAcP and therefore fails to signal. The ILRa acts by occupying the binding site on IL-1R1 for IL-1β or IL-1α and in addition failing to form the signalling complex with IL-1RAcP.

A further member of the IL-1R family is the type II IL-1R (IL-1RII). This receptor is highly homologous to the IL-1R1 in the extracellular region and can bind IL-1α & IL-1β. Current evidence suggests that however, does not initiate signalling due to the lack of an intracytoplasmic domain. This receptor can be cleaved from the cell surface and along with the membrane form act as inhibitors of IL-1 activity by acting as decoy receptors (16). IL-1RII has a higher affinity for IL-1β and a lower affinity for the IL-1Ra, which means that IL-1RII does not block the inhibitory activity of the IL-1Ra (23). Ligand binding to the IL-1RII causes recruitment of the IL-1RAcP, however this complex remains non-signalling (2). Because the IL-1RAcP is removed in this way by the IL-1RII and prevents IL-1RAcP binding to it can also block IL-1 actions by this mechanism, and this is termed “co-receptor competition” (24). However, it has not been definitively disproved at this time that IL-1RII could recruit another signalling chain, although cells that express high levels of have been shown to become unresponsive to IL-1β (25).

The high affinity complex formed when IL-1 binds to IL-1R1 leads to the recruitment of the IL-1RAcP and initiates receptor signalling. IL-1R1 and IL-1RacP have cytoplasmic domains (Toll-like IL-1R (TIR)) domains, which are related to the Toll-Like receptor (TLR) superfamily.

During signal transduction the TIR domain of the adaptor molecule MyD88 interacts with the TIR domain of the IL-1RAcP and causes recruitment of a receptor complex containing IRAK-4 and IRAK-1. It has been proposed that the phosphorylated IRAK in turn recruits TRAF6 to the receptor complex. IRAK then brings TRAF6 to TAK1, TAB1, and TAB2, which are preassociated on the membrane before stimulation to form a membrane-associated complex II. The formation of complex II leads to the phosphorylation of TAK1 and TAB2 on the membrane by an unknown kinase, followed by the dissociation of TRAF6-TAK1-TAB1-TAB2 (complex III) from IRAK and consequent translocation of complex III to the cytosol. The formation of complex and its interaction with additional cytosolic factors leads to the activation of TAK1 Phosphorylated IRAK remains on the membrane and eventually is ubiquitinated and degraded. Activation of TAK-1 leads to the activation of IKK, degradation of IkB proteins resulting in NF-kB activation that activates transcription in the nucleus. TAK-1 has also been shown to play a role in activation of the mitogen activated protein kinase pathway (MAPK) that, via activation of p38, JNK and ERK1/2, regulates activity of transcription factors including AP1 (26). Since signalling transduction is amplified down these multiple pathways, the percentage receptor occupancy per cell by ligand only needs to be low to initiate a physiological response in the IL-1R expressing cell (perhaps as low as 10 receptors occupied per cell).

IL-1 is a major inflammatory cytokine, which has an important role in many chronic inflammatory diseases. The expression of IL-1 at the gene and protein level has been examined in a variety of diseases. Increased levels of IL-1 have been reported in type 2 diabetes (27,28, 29), HIV-1 solid tumours, leukaemias, Alzheimers disease, ischaemic disease (30) and atherosclerosis (31), asthma, COPD and OA (32). IL-1 has been shown to exert multiple biological effects by a variety of in vitro and in vivo studies. Its pleiotropic actions are related to its major role on the gene expression of a variety of inflammatory mediators, including prostanoids, nitric oxide, cytokines, chemokines, proteases & adhesion molecules and cytokine receptor expression (32). Excessive production or expression of these inflammatory mediators is associated with disease pathology and tissue remodelling and destruction. Therefore, IL-1 represents a pivotal therapeutic target for many common inflammatory disorders such as rheumatoid arthritis, osteoarthritis (OA), asthma and chronic obstructive pulmonary disease (COPD), type 2 diabetes, ischaemic disease and atherosclerosis.

The present invention provides binding members which bind to IL-1R1 and inhibit the biological activity of IL-1α and/or IL-1β, including fully human antibodies, or antigen-binding portions thereof.

Binding members directed to IL-1R1 have been disclosed in the following International Patent Applications: WO2004/022718, WO 2005/023872, WO 2007/063311, WO 2007/063308 and WO 2006/059108.

In another embodiment the invention provides an isolated binding member, for example, an antibody, specific for IL-1R1 which competes with IL-1Ra for binding to IL-1R1.

In another embodiment the invention provides an isolated binding member specific for IL-1R1 which competes with IL-1 and IL-1Ra for binding to IL-1R1 and binds Il-1R1 with a KD of 10 pM or less when measured by Kinexa™. In one embodiment IL-1 relates to IL-1α, in another embodiment Il-1 relates to IL-1β. In another embodiment Il-1 relates to both IL-1α and IL-1β.

Antibodies which block both IL-1 and IL1Ra binding are believed to be particularity efficacious. In the absence of IL-1, the IL-1R1 internalizes with a t1/2 of approximately 11 hours, however in the presence of IL-1 the receptor undergoes more rapid internalisation so that t1/2 is approximately 1.5 hours [33, 34]. In contrast, IL-1Ra binds IL-1R1 but does not induce increased internalisation of the receptor [35]. When the IL-1R1 is internalised it is not readily recycled back to the membrane surface [33] and so it is possible that antibodies binding to an epitope similar to that of IL-1 alone may be internalised readily, may be channeled into endosomal pathways as a result, and may undergo a greater rate of clearance via this receptor-mediated clearance mechanism. Antibodies to epitopes more similar to IL-1ra may be less susceptible to increase the rate of receptor internalisation and may not undergo increased clearance via a receptor mediated mechanism, and are therefore perhaps more likely to have a circulatory clearance and half-life typical of a human IgG. International patent application WO 2004/022718 disclosed a class of antibodies which blocked both IL-1 and IL-1Ra binding to IL-1R1, however, this class was much less potent than the preferred class of antibodies disclosed which bound to the third domain of Il-1R and prevented Il-1β binding. In contrast, antibodies of the present invention are able to block Il-1 and Il-1Ra binding to Il-1R1 and bind Il-1R1 with high affinity.

In another embodiment of the invention there is provided an isolated binding member specific for IL-1R1 which has a mean IC50, averaged from at least 6 different donors, of less than 1 nM for the inhibition of IL-1β induced IL-6 production in whole human blood in the presence of 30 pM IL-1β. In further embodiments the mean IC50, averaged from at least 10, 15 or 20 different donors. In further embodiments the mean IC50 is less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 300 pM, less than 200 pM, less than 100 pM or less than 50 pM.

Binding members of the invention bind to IL-1R1 and neutralize IL-1R1 with high potency. Neutralisation means inhibition of a biological activity of IL-1R1. Binding members of the invention may neutralize one or more biological activities of IL-1R1, typically binding members of the invention inhibit IL1α and IL1β binding to IL-1R1.

The binding members of the invention may also bind to and neutralize non-human IL-1R1, meaning IL-1R1 orthologs that occur naturally in species other than human.

Binding members of the invention are normally specific for IL-1R1 over other proteins, and thus bind IL-1R1 selectively. Such selectivity may be determined or demonstrated, for example, in a standard competition assay.

Suitable assays for measuring neutralisation of IL-1R1 by binding members of the invention include, for example, ligand receptor biochemical assays and surface plasmon resonance (SPR) (e.g., BIACORE™).

Binding kinetics and affinity (expressed as the equilibrium dissociation constant KD) of IL-1R1-binding members for human IL-1R1 may be determined, e.g. using surface plasmon resonance (BIACORE™), Binding members of the invention normally have an affinity for human IL-1R1 (KD) of less than about 1 nM, and in some embodiments have a KD of less than about 100 pM, in other embodiments have a KD of less than 50 pM, in other embodiments have a KD of less than 25 pM, in other embodiments have a KD of less than 10 pM, in other embodiments have a KD of less than 5 pM, in other embodiments have a KD of less than 3 pM, in other embodiments have a KD of less than 1 pM.

A number of methodologies are available for the measurement of binding affinity of an antibody to its antigens, one such methodology is KinExA™, The Kinetic Exclusion Assay (KinExA™.) is a general purpose immunoassay platform (basically a flow spectrofluorimeter) that is capable of measuring equilibrium dissociation constants, and association and dissociation rate constants for antigen/antibody interactions. Since KinExA™. is performed after equilibrium has been obtained, it is an advantageous technique to use for measuring the KD of high affinity interactions where the off-rate of the interaction may be very slow. The use of KinExA™. is particularly appropriate in this case where the affinity of antibody and antigen are higher than can be accurately predicted by surface plasmon resonance analysis, The KinExA™. methodology can be conducted as described in Drake et al (2004) Analytical Biochemistry 328, 35-43.

In one embodiment of the invention the binding members of the invention are specific for IL-1R with a KD of 300 pM or lower as measured using the KinExA™. methodology. Alternatively a of 200 pM or lower, 100 pM or lower, 50 pM or lower, 20 pM or lower or 10 pM or lower, 5 pM or lower, 3 pM or lower, 1 pM or lower.

Inhibition of biological activity may be partial or total. Binding members may inhibit an IL-1R1 biological activity, such as IL-1β induced IL-8 release in CYNOM-K1 cells or IL-1α and IL-1β induced IL-8 release in HeLa cells, by 100%, or alternatively by: at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity of a concentration of IL-1α or β that induces 50% or 80% of the maximum possible activity in absence of the binding member.

The neutralizing potency of a binding member is normally expressed as an IC50 value, in nM unless otherwise stated. In functional assays, IC50 is the concentration of a binding member that reduces a biological response by 50% of its maximum. In ligand-binding studies, IC50 is the concentration that reduces receptor binding by 50% of maximal specific binding level. IC50 may be calculated by plotting % of maximal biological response as a function of the log of the binding member concentration, and using a software program, such as Prism (GraphPad Software Inc., La Jolla, Calif., USA) to fit a sigmoidal function to the data to generate IC50 values. Potency may be determined or measured using one or more assays known to the skilled, person and/or as described, or referred to herein. The neutralizing potency of a binding member can be expressed as a Geomean.

Neutralisation of IL-1R1 activity by a binding member in an assay described herein, indicates that the binding member binds to and neutralizes IL-1R1. Other methods that may be used for determining binding of a binding member to IL-1R1 include ELISA, Western blotting, immunoprecipitation, affinity chromatography and biochemical assays.

A binding member of the invention may have a similar or stronger affinity for human IL-1R1 than for IL-1R1 of other species. Affinity of a binding member for human IL-1R1 may be, similar to or for example, within 5 or 10-fold that for cynomolgus monkey IL-1R1. Alternatively, a binding member may have a similar binding affinity for human and cynomolgus monkey IL-1R1.

A binding member of the invention comprises an IL-1R1 binding motif comprising one or more CDRs, e.g. a ‘set of CDRs’ within a framework. A set of CDRs comprises one or more CDRs selected from: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3. In one embodiment a set of CDRs comprises a HCDR3 in Table 2 optionally combined with one or more CDRs selected from: HCDR1, HCDR2, LCDR1, LCDR2 and LCDR3, for example one or more CDRs selected from: HCDR1, HCDR2, LCDR1, LCDR2 and LCDR3 in Table 2, In another embodiment of the invention a set of CDRs comprises a HCDR3 and a LCDR3 in Table 2 optionally combined with one or more CDRs selected from: HCDR1, HCDR2, LCDR1 and LCDR2, for example one or more CDRs selected from: HCDR1. HCDR2, LCDR1 and LCDR2 in Table 2. In another embodiment of the invention a set of CDRs comprises a HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 in Table 2. Whilst it is preferred, to select the one or more CDRs from the same antibody in Table 2, CDRs may be selected from one or more antibodies listed in Table 2.

In another embodiment, a binding member of the invention, for example an antibody, comprises an IL-1R1 binding motif comprising one or more CDRs selected from: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, for example as disclosed in Tables 1a and 1b, wherein said binding member specifically binds Il-1R1.

In another embodiment, a binding member of the invention, for example an antibody, comprises an IL-1R1 binding motif comprising one or more CDRs selected from: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, for example as disclosed in Tables 1a and 1b, wherein said binding member specifically binds Il-1R1 and competes with IL-1β and IL-1Ra for binding to IL-1R1 and binds Il-1R1 with a KD of 10 pM or less when measured by Kinexa™.

As described herein, a parent antibody molecule was isolated having the set of CDR sequences as shown in Table 1a (see Antibody 1). Through a process of optimisation we generated a panel of antibody clones numbered 2-3, with CDR sequences derived from the parent CDR sequences and having modifications at the positions indicated, in Table 1. Thus, for example, it can be seen from Table 1a that Antibody 2 has the parent HCDR1, HCDR2, LCDR1 and LCDR2, and has a parent HCDR3 sequence in which: Kabat residue 100E is replaced with T, Kabul residue 100F is replaced with V, Kabat residue 100G is replaced with D, Kabat residue 100H is replaced with A, Kabat residue 100I is replaced with A, Kabat residue 101 is replaced with V and Kabat residue 102 is replaced with D.

As described herein, a second parent antibody molecule was isolated having the set of CDR sequences as shown in Table 1b (see Antibody 4). Through a process of optimisation we generated a panel of antibody clones numbered 5-10 with CDR sequences derived from the parent CDR sequences and having modifications at the positions indicated in Table 1b. Thus, for example, it can be seen from Table 1b that Antibody 5 has the parent HCDR1, HCDR2, LCDR1 and LCDR2, and has a parent HCDR3 sequence in which: Kabat residue 100A is replaced with A, Kabat residue 100B is replaced with P, Kabat residue 100C is replaced with P, Kabat residue 100D is replaced with P. Kabat residue 100E is replaced with L, Kabat residue 100F is replaced with 0 and Kabat residue 100I is replaced with G. It can also be seen from Table 1b that Antibody 6 has the parent HCDR1, HCDR2, LCDR1 and LCDR2, and has a parent HCDR3 sequence in which: Kabat residue 100A is replaced with E, Kabat residue 100B is replaced with Q, Kabat residue 100C is replaced with Y, Kabat residue 100D is replaced with 0, Kabat residue 100E is replaced with V, Kabat residue 100F is replaced with V, Kabat residue 100J has been deleted, Kabat residue 101 is replaced with F and Kabat residue 102 is replaced with V.

Described herein is a binding member comprising the parent set of CDRs as shown in Table 1a (Antibody 1), in which HCDR1 is SEQ ID NO: 93 (Kabat residues 31-35), HCDR2 is SEQ ID NO: 94 (Kabat residues 50-65), HCDR3 is SEQ ID NO: 95 (Kabat residues 95-102), LCDR1 is SEQ ID NO: 98 (Kabat residues 24-34), LCDR2 is SEQ ID NO: 99 (Kabat residues 50-56) and LCDR3 is SEQ ID NO: 100 (Kabat residues 89-97). The binding member according to the invention may also be the parent binding member (Antibody 1) as shown in Table 1a, wherein one or more of the CDRs have one or more amino acid additions, substitutions, deletions, and/or insertions. In some embodiments, the binding member comprises a set of CDRs having from one to twelve additions, substitutions, deletions and/or insertions relative to the parent sequences of Antibody 1. In another embodiment from one to ten additions, substitutions, deletions and/or insertions relative to Antibody 1. In another embodiment from one to five additions, substitutions, deletions and/or insertions relative to the parent sequences of Antibody 1. In another embodiment one to three additions, substitutions, deletions and/or insertions relative to Antibody 1.

In certain embodiments the binding member of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; wherein the HCDR3 has the amino acid sequence of SEQ ID NO: 95 optionally having from 1 to 7 amino acid additions, substitutions, deletions and/or insertions; and the LCDR3 has the amino acid sequence of SEQ ID NO: 100 optionally having from 1 to 5 amino acid additions, substitutions, deletions and/or insertions. In such embodiments, the HCDR1 may have the amino acid sequence SEQ ID NO: 93; the HCDR2 may have the amino acid sequence SEQ ID NO: 94; the LCDR1 may have the amino acid sequence SEQ ID NO: 98; and the LCDR2 may have the amino acid sequence SEQ ID NO: 99. Alternatively, the HCDR1, the HCDR2, the LCDR1, and the LCDR2 may also collectively have one or more amino acid additions, substitutions, deletions, and/or insertions relative to the parent sequences (Antibody 1), such as from one to ten substitutions.

Described herein is a binding member comprising the parent set of CDRs as shown in Table 1b (Antibody 4), in which HCDR1 is SEQ ID NO: 103 (Kabat residues 31-35), HCDR2 is SEQ ID NO: 104 (Kabat residues 50-65), HCDR3 is SEQ ID NO: 105 (Kabat residues 95-102), LCDR1 is SEQ ID NO: 108 (Kabat residues 24-34), LCDR2 is SEQ ID NO: 109 (Kabat residues 50-56) and LCDR3 is SEQ ID NO: 110 (Kabat residues 89-97). The binding member according to the invention may also be the parent binding member as shown in Table 1b, wherein one or more of the CDRs have one or more amino acid additions, substitutions, deletions, and/or insertions. In some embodiments, the binding member comprises a set of CDRs having from one to fifteen additions, substitutions, deletions and/or insertions relative to the parent sequences of Antibody 4. In another embodiment one to ten additions, substitutions, deletions and/or insertions relative to Antibody 4. In another embodiment form one to five additions, substitutions, deletions and/or insertions relative to the parent sequences of Antibody 4. In another embodiment one to three additions, substitutions, deletions and/or insertions relative to Antibody 4.

In certain embodiments the binding member of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; wherein the HCDR3 has the amino acid sequence of SEQ ID NO: 105 optionally having from 1 to 9 amino acid additions, substitutions, deletions and/or insertions; and the LCDR3 has the amino acid sequence of SEQ ID NO: 110 optionally having from 1 to 6 amino acid additions, substitutions, deletions and/or insertions. In such embodiments, the HCDR1 may have the amino acid sequence SEQ ID NO: 103; the HCDR2 may have the amino acid sequence SEQ ID NO: 104; the LCDR1 may have the amino acid sequence SEQ ID NO: 1.08; and the LCDR2 may have the amino acid sequence SEQ ID NO: 109. Alternatively, the HCDR1, the HCDR2, the LCDR1, and the LCDR2 may also collectively have one or more amino acid additions, substitutions, deletions, and/or insertions relative to the parent sequences (Antibody 4), such as from one to ten substitutions.

Described herein is a binding member comprising the Antibody 6 set of CDRs as shown in Table 1b, in which HCDR1 is SEQ ID NO: 63 (Kabat residues 31-35), HCDR2 is SEQ ID NO: 64 (Kabat residues 50-65), HCDR3 is SEQ ID NO: 65 (Kabat residues 95-102), LCDR1 is SEQ ID NO: 68 (Kabat residues 24-34), LCDR2 is SEQ ID NO: 69 (Kabat residues 50-56) and LCDR3 is SEQ ID NO: 70 (Kabat residues 89-97). The binding member according to the invention may also be the Antibody 6 binding member as shown in Table 1b, wherein one or more of the CDRs have one or more amino acid additions, substitutions, deletions, and/or insertions. In some embodiments, the binding member comprises a set of CDRs having from one to seventeen additions, substitutions, deletions and/or insertions relative to the sequences of Antibody 6. In another embodiment one to ten additions, substitutions, deletions and/or insertions relative to Antibody 6. In another embodiment form one to five additions, substitutions, deletions and/or insertions relative to the sequences of Antibody 6. In another embodiment one to three additions, substitutions, deletions and/or insertions relative to Antibody 6. In another embodiment one to two additions, substitutions, deletions and/or insertions relative to Antibody 6. In another embodiment one additions, substitution, deletion or insertion relative to Antibody 6.

In certain embodiments the binding member of the invention comprises HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3; wherein the HCDR3 has the amino acid sequence of SEQ ID NO: 65 optionally having from 1 to 11 amino acid additions, substitutions, deletions and/or insertions; and the LCDR3 has the amino acid sequence of SEQ ID NO: 70 optionally having from 1 to 6 amino acid additions, substitutions, deletions and/or insertions. In such embodiments, the HCDR1 may have the amino acid sequence SEQ ID NO: 63; the HCDR2 may have the amino acid sequence SEQ ID NO: 64; the LCDR1 may have the amino acid sequence SEQ OD NO: 68; and the LCDR2 may have the amino acid sequence SEQ ID NO: 69. Alternatively, the HCDR1, the HCDR2, the LCDR1, and the LCDR2 may also collectively have one or more amino acid additions, substitutions, deletions, and/or insertions relative to the sequences of Antibody 6, such as from one to ten substitutions.

A binding member of the invention may comprise one or a combination of CDRs as described herein, For example, the binding member of the invention may comprise an HCDR1 having the amino acid sequence of SEQ ID NO: 93; an HCDR2 having the amino acid sequence of SEQ ID NO: 94; an HCDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOS: 95, 5 or 125; an LCDR1 having the amino acid sequence of SEQ ID NO: 98; an LCDR2 having the amino acid sequence SEQ ID NO: 99; and an LCDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOS: 100, 10 or 130.

A binding member of the invention may comprise one or a combination of CDRs as described herein. For example, the binding member of the invention may comprise an HCDR1 having the amino acid sequence of SEQ ID NO: 103; an HCDR2 having the amino acid sequence of SEQ ID NO: 104; an HCDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOS: 105, 15, 65, 25, 35, 75, 45, 115, 55 or 85; an LCDR1 having the amino acid sequence of SEQ ID NO: 108; an LCDR2 having the amino acid sequence SEQ ID NO: 109; and an LCDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOS: 110, 20, 70, 30, 40, 80, 50, 120, 60 or 90.

A binding member of the invention may comprise one or a combination of CDRs as described herein. For example, the binding member of the invention may comprise an HCDR1 having the amino acid sequence of SEQ ID NO: 93; an HCDR2 having the amino acid sequence of SEQ ID NO: 94; an HCDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOS:95, 5, 125; 105, 15, 65, 25, 35, 75, 45, 115, 55 or 85 an LCDR1 having the amino acid sequence of SEQ NO: 98 or 108; an LCDR2 having the amino acid sequence SEQ ID NO: 99 or 109; and an LCDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOS: 100, 10, 130, 110, 20, 70, 30, 40, 80, 50, 120, 60 or 90.

In certain embodiments, the binding member or VH domain of the invention comprises an Antibody 1 HCDR3 (SEQ ID NO:95) with one or more of the following substitutions or deletions:

Kabat residue 100E replaced by T;

Kabat residue 100F replaced V or L;

Kabat residue 100G replaced by D;

Kabat residue 100H replaced by A or P;

Kabat residue 100I replaced by A or P;

Kabat residue 101 replaced by V or G;

Kabat residue 102 replaced by D or V;

In certain embodiments, the binding member or VH domain of the invention comprises an Antibody 4 HCDR3 (SEQ ID NO:105) with one or more of the following substitutions or deletions:

Kabat residue 100A replaced by A or E;

Kabat residue 100B replaced P. Q, or A;

Kabat residue 100C replaced by P, Y, S or L;

Kabat residue 100D replaced by P, G or A;

Kabat residue 100E replaced by L or V;

Kabat residue 100F replaced by G, V or P;

Kabat residue 100G replaced by V;

Kabat residue 100H replaced by Y;

Kabat residue 100I replaced by G or D;

Kabat residue 100J replaced by A or deleted;

Kabat residue 101 replaced by F;

Kabat residue 102 replaced by V.

In some embodiments, a binding member, or a VL domain thereof may comprise the Antibody 1 LCDR3 (SEQ ID NO 100) with one or more of the following substitutions:

Kabat residue 94 replaced by H or A;

Kabat residue 95 replaced by A;

Kabat residue 95A replaced by E or R;

Kabat residue 95B replaced by Q or V;

Kabat residue 97 replaced by H or L.

In some embodiments, a binding member, or a VL domain thereof may comprise the Antibody 4 LCDR3 (SEQ ID NO 110) with one or more of the following substitutions:

Kabat residue 94 replaced by A, V, D, or R;

Kabat residue 95 replaced by G, R or A;

Kabat residue 95A replaced by G, L, A, V or D;

Kabat residue 95B replaced by H, R, A or D;

Kabat residue 96 replaced by H, P or A.

Kabat residue 97 replaced by H, V or Q.

In certain embodiments, the binding member or VU domain of the invention comprises an Antibody 6 HCDR3 (SEQ ID NO:65) with one or more of the following substitutions or additions:

Kabat residue 100A replaced by G or A;

Kabat residue 100B replaced S, P or A;

Kabat residue 100C replaced by D, P, S L;

Kabat residue 100D replaced by Y, P or A;

Kabat residue 100E replaced by T or L;

Kabat residue 100F replaced by T, G or P;

Kabat residue 100G replaced by V;

Kabat residue 100H replaced by Y;

Kabat residue 100I replaced by G or D;

Kabat residue 100J deleted in Antibody 6 is reinstated as a A or F;

Kabat residue 101 replaced by D;

Kabat residue 102 replaced by I.

In some embodiments, a binding member, or VL domain thereof may comprise the Antibody 6 LCDR3 (SEQ ID NO 70) with one or more of the following substitutions:

Kabat residue 94 replaced by S, A, D, H, L or R;

Kabat residue 95 replaced by L, G or A;

Kabat residue 95A replaced by S, El, A, V or D;

Kabat residue 95B replaced by R, A or D;

Kabat residue 96 replaced by S, P or A.

Kabat residue 97 replaced by L, H Q.

In one embodiment, the invention is a binding member comprising a set of CDRs in which: HCDR1 has amino acid sequence SEQ ID NO: 3, HCDR2 has amino acid sequence SEQ ID NO: 4, HCDR3 has amino acid sequence SEQ OD NO: 5, LCDR1 has amino acid sequence SEQ ID NO: 8, LCDR2 has amino acid sequence SEQ ID NO: 9, and LCDR3 has amino acid sequence SEQ ID NO: 10.

In one embodiment, the invention is a binding member comprising a set of CDRs in which: HCDR1 has amino acid sequence SEQ NO: 63, HCDR2 has amino acid sequence SEQ ID NO: 64, HCDR3 has amino acid sequence SEQ ID NO: 65, LCDR1 has amino acid sequence SEQ ID NO: 68, LCDR2 has amino acid sequence SEQ ID NO: 69, and LCDR3 has amino acid sequence SEQ ID NO: 70.

In one embodiment, the invention is a binding member comprising a set of CDRs in which: HCDR1 has amino acid sequence SEQ ID NO: 23, HCDR2 has amino acid sequence SEQ ID NO: 24, HCDR3 has amino acid sequence SEQ ID NO: 25, LCDR1 has amino acid sequence SEQ ID NO: 28, LCDR2 has amino acid sequence SEQ NO; 29, and LCDR3 has amino acid sequence SEQ ID NO: 20.

In one embodiment, the invention is a binding member comprising a set of CDRs in which: HCDR1 has amino acid sequence SEQ NO; 113, HCDR2 has amino acid sequence SEQ ID NO: 114, HCDR3 has amino acid sequence SEQ ID NO: 115, LCDR1 has amino acid sequence SEQ ID NO: 118, LCDR2 has amino acid sequence SEQ ID NO: 119, and LCDR3 has amino acid sequence SEQ ID NO: 120.

In one embodiment, the invention is a binding member comprising a set of CDRs in which: HCDR1 has amino acid sequence SEQ ID NO: 53, HCDR2 has amino acid sequence SEQ ID NO: 54, HCDR3 has amino acid sequence SEQ ID NO: 55, LCDR1 has amino acid sequence SEQ ID NO: 58, LCDR2 has amino acid sequence SEQ ID NO: 59, and LCDR3 has amino acid sequence SEQ ID NO: 60.

A binding member of the invention may be one which competes or cross-competes for binding to IL-1R1 with any binding member disclosed herein which both binds IL-1R1 and comprises a binding member such as VH and/or VL domain, CDR e.g. HCDR3, and/or set of CDRs disclosed herein, for example the antibodies disclosed in Table 2. Competition between binding members may be assayed easily in vitro, for example using ELISA and/or by tagging a specific reporter molecule to one binding member which can be detected in the presence of one or more other untagged binding members, to enable identification of binding members which bind the same epitope or an overlapping epitope. Such methods are readily known to one of ordinary skill in the art, and are described in more detail herein. Thus, a further aspect of the present invention provides a binding member specific for IL-1R1 that competes or cross-competes for binding to human IL-1R1 with an antibody molecule comprising a VH and/or VL domain or a CDR e.g. HCDR3 or set of CDRs of any of antibodies 1 to 10. In one embodiment, the binding member of the invention competes or cross-competes with Antibody 1 and/or Antibody 3 of Table 2.

Another embodiment of the invention provides binding members which bind to a specific region of IL-1R1, for example an epitope. Specifically the same epitope or part thereof as is bound by any one of the antibodies listed in Table 2.

Another embodiment of the invention provides an isolated binding member which binds an epitope comprised within one or more of the following sequences of Il-1R1:

(i) N123-V134;

(ii) L140-K157; and/or

(iii) K178-R180.

Another embodiment of the invention provides an isolated binding member specific for IL-1R1 according to claim 16 which binds a discontinuous epitope comprised within the following sequences of IL-1R1:

(i) N123-V134;

(ii) 1.40-K157; and

(iii) K178-R180.

In further aspects the present invention provides a binding member comprising a human antibody antigen-binding site which competes or cross-competes with an antibody antigen-binding site for binding to human wherein the antibody antigen-binding site is composed of a VH domain and a VL domain, and wherein the VH and VL domains comprise a set of CDRs of the parent (Antibody 1 or Antibody 4), or of any of antibodies 2 to 3 or 5 to 10 list in Table 2.

Any suitable method may be used to determine the sequence of residues bound by a binding member. For example, a peptide-binding scan may be used, such as a PEPSCAN-based enzyme linked immuno assay (ELISA). In a peptide-binding scan, such as the kind provided by PEPSCAN Systems, short overlapping peptides derived from the antigen are systematically screened for binding to a binding member. The peptides may be covalently coupled to a support surface to form an array of peptides. Peptides may be in a linear or constrained conformation. A constrained conformation may be produced using peptides having a terminal Cys residue at each end of the peptide sequence. The Cys residues can be covalently coupled directly or indirectly to a support surface such that the peptide is held in a looped conformation. Thus, peptides used in the method may have Cys residues added to each end of a peptide sequence corresponding to a fragment of the antigen. Double looped peptides may also be used, in which a Cys residue is additionally located at or near the middle of the peptide sequence. The Cys residues can be covalently coupled directly or indirectly to a support surface such that the peptides form a double-looped conformation, with one loop on each side of the central Cys residue. Peptides can be synthetically generated, and Cys residues can therefore be engineered at desired locations, despite not occurring naturally in the IL-1R1 sequence. Optionally, linear and constrained peptides may both be screened in a peptide-binding, assay. A peptide-binding scan may involve identifying (e.g. using ELISA) a set of peptides to which the binding member binds, wherein the peptides have amino acid sequences corresponding to fragments of IL-1R (e.g. peptides of about 5, 10 or 15 contiguous residues of IL-1R1), and aligning the peptides in order to determine a footprint of residues bound by the binding member, where the footprint comprises residues common to overlapping peptides.

Alternatively or additionally the peptide-binding scan method may involve identifying peptides to which the binding member binds with at least a given signal:noise ratio. Details of a suitable peptide-binding scan method for determining binding are known in the art. Other methods that are well known in the art and that could be used to determine the residues bound by an antibody, and/or to confirm peptide-binding scan results, include site directed mutagenesis, hydrogen deuterium exchange, mass spectrometry, NMR, and X-ray crystallography.

A binding member of the invention may be an antibody molecule or binding fragment thereof, preferably a human antibody molecule or a humanized antibody molecule or binding fragment thereof. The antibodies may be monoclonal antibodies, especially of human, murine, chimeric or humanized origin, which can be obtained according to the standard methods well known to the person skilled in the art.

Although, as noted below, CDRs can be carried by non-antibody scaffolds, the structure for carrying a CDR or a set of CDRs of the invention will generally be an antibody heavy or light chain sequence or substantial portion thereof in which the CDR or set of CDRs is located at a location corresponding to the CDR or set of CDRs of naturally occurring VH and VL antibody variable domains encoded by rearranged immunoglobulin genes. The structures and locations of immunoglobulin variable domains may be determined by reference to Kabat, et al., 1987 [36], and updates thereof findable under “Kabat” using any internet search engine.

An antibody of the invention normally comprises an antibody VH and/or VL domain. A VH domain of the invention comprises a set of HCDRs, and a VL domain comprises a set of LCDRs. An antibody molecule may comprise an antibody VH domain comprising a VH CDR1, CDR2 and CDR3 and a framework. It may alternatively or also comprise an antibody VL domain comprising a VL CDR1, CDR2 and CDR3 and a framework. AB example of an antibody VH domain of the invention is SEQ ID NO. 22, and an example of an antibody VL domain of the invention is SEQ ID NO. 27.

The invention provides binding members comprising a HCDR1 and/or HCDR2 and/or HCDR3 of any of antibodies in Table 2 and/or an LCDR1 and/or LCDR2 and/or LCDR3 of any of antibodies in Table 2. The binding member may comprise a set of VH CDRs, optionally it may also comprise a set of VL CDRs, and the VL CDRs may be from the same or a different antibody as the VH CDRs.

Typically, a VH domain is paired with a VL domain to provide an antibody antigen-binding site, although as discussed further below a VH or VL domain alone may be used to bind antigen. For example, the Antibody 1 VH domain (see Table 2) may be paired with the Antibody 1 VL domain, an that an antibody antigen-binding site is formed comprising both the antibody 1 VH and VL domains. Analogous embodiments are provided for the other VH and VL domains disclosed herein. In other embodiments, the Antibody 1 VH is paired with a VL domain other than the Antibody 1. Light-chain promiscuity is well established in the art, Again, analogous embodiments are provided by the invention for the other VH and VL domains disclosed herein. Thus, the VH of any of the antibodies in Table 2 may be paired with the VL of the same or any other antibodies in Table 2.

A further aspect of the invention is an antibody molecule comprising a VH domain that has at least 60, 70, 80, 85, 90, 95, 98 or 99% amino acid sequence identity with a VH domain of any of antibodies shown in Table 2, or comprising a set of HCDRs (e.g., HCDR1, HCDR2, and/or HCDR3) shown in Table 1a or 1b. The antibody molecule may optionally also comprise a VL domain that has at least 60, 70, 80, 85, 90, 95, 98 or 99% amino acid sequence identity with a VL domain of any of the antibodies 1 to 28, or with a set of LCDRs (e.g. LCDR1, LCDR2, and/or LCDR3) shown in Table 1a or 1b. Algorithms that can be used to calculate % identity of two amino acid sequences include e.g. BLAST [37], FASTA [38], or the Smith-Waterman algorithm [39], e.g. employing default parameters.

Binding members of the present invention may further comprise antibody constant regions or parts thereof, e.g. human antibody constant regions or parts thereof. For example, a VL domain may be attached at its C-terminal end to antibody light chain constant domains including human Cκ or Cλ chains. Similarly, a binding member based on a VH domain may be attached at its C-terminal end to all or part (e.g. a CH1 domain) of an immunoglobulin heavy chain derived from any antibody isotype, e.g. IgG, IgA, IgE and IgM and any of the isotype sub-classes, particularly IgG1, IgG2, IgG3 and IgG4, IgG1 is advantageous due to its ease of manufacture and stability, e.g., half-life. Any synthetic or other constant region variants which modulate binding member function and/or properties e.g. stabilizing variable regions, may also be useful in the present invention.

Furthermore, it may be desired according to the present invention to modify the amino acid sequences described herein, in particular those of human heavy chain constant regions to adapt the sequence to a desired allotype, e.g. an allotype found in the Caucasian population.

A binding member may comprise an antibody molecule, or binding fragment thereof, having one or more CDRs, a set of CDRs, within an antibody framework. For example, one or more CDRs or a set of CDRs of an antibody may be grafted into a framework (e.g. human framework) to provide an antibody molecule. The framework regions may be of human germline gene sequences, or be non-germlined. Thus, the framework may be germlined where one or more residues within the framework are changed to match the residues at the equivalent position in the most similar human germline framework. Thus, a binding member of the invention may be an isolated human antibody molecule having VH domain comprising a set of HCDRs in a human germline framework, e.g. human germline IgG VH framework. The binding member also has a VL domain comprising a set of LCDRs, e.g. in a human germline IgG VL framework.

VH and/or VL framework residues may be modified as discussed and exemplified herein e.g. using site-directed mutagenesis. A VH or VL domain according to the invention, or a binding member comprising such a VL domain, preferably has the NTH and/or VL domain sequence of an antibody of Table 2 and comprising a HCDR3 of the invention.

A non-germlined antibody molecule has the same CDRs, but different frameworks, compared to a germlined antibody molecule, Germlined antibodies may be produced by germlining framework regions of the VH and VL domain sequences shown herein for these antibodies.

Alterations may be made in one or more framework regions and/or one or more CDRs. The alterations normally do not result in loss of function, so a binding member comprising a thus-altered amino acid sequence should retain an ability to bind and/or neutralize IL-1R1. It may retain the same quantitative binding and/or neutralizing ability as a binding member in which the alteration is not made, e.g. as measured in an assay described herein. The binding member comprising a thus-altered amino acid sequence may have an improved ability to bind and/or neutralize IL-1R1.

Alteration may comprise replacing one or more amino acid residue(s) with a non-naturally occurring or non-standard amino acid, modifying one or more amino acid residue into a non-naturally occurring or non-standard form, or inserting one or more non-naturally occurring or non-standard amino acid into the sequence. Examples of numbers and locations of alterations in sequences of the invention are described elsewhere herein. Naturally occurring amino acids include the 20 “standard” L-amino acids identified as G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C, K, R, H, D, E by their standard single-letter codes. Non-standard amino acids include any other residue that may be incorporated into a polypeptide backbone or result from modification of an existing amino acid residue. Non-standard amino acids may be naturally occurring or non-naturally occurring. Several naturally occurring non-standard amino acids are known in the art, such as 4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, N-acetylserine, etc. [40]. Those amino acid residues that are derivatised at their N-alpha position will only be located at the N-terminus of an amino-acid sequence. Normally in the present invention an amino acid is an L-amino acid, but it may be a D-amino acid. Alteration may therefore comprise modifying an L-amino acid into, or replacing it with, a D-amino acid. Methylated, acetylated and/or phosphorylated forms of amino acids are also known, and amino acids in the present invention may be subject to such modification.

Amino acid sequences in antibody domains and binding members of the invention may comprise non-natural or non-standard amino acids described above. Non-standard amino acids (e.g. D-amino acids) may be incorporated into an amino acid sequence during synthesis, or by modification or replacement of the “original” standard amino acids after synthesis of the amino acid sequence.

Use of non-standard and/or non-naturally occurring amino acids increases structural and functional diversity, and can thus increase the potential for achieving desired IL-1R1-binding and neutralizing properties in a binding member of the invention. Additionally, D-amino acids and analogues have been shown to have better pharmacokinetic profiles compared with standard L-amino acids, owing to in vivo degradation of polypeptides having L-amino acids after administration to an animal a human.

Novel VH or VL regions carrying CDR-derived sequences of the invention may be generated using random mutagenesis of one or more selected VH and/or VL genes to generate mutations within the entire variable domain. Such a technique is described by Gram et al, [41], who used error-prone PCR. In some embodiments one or two amino acid substitutions are made within an entire variable domain or set of CDRs.

Another method that may be used is to direct mutagenesis to CDR regions of VH or VL genes. Such techniques are disclosed by Barbas et al. [42] and Schier al. [43].

All the above-described techniques are known as such in the art and the skilled person will be able to use such techniques to provide binding members of the invention using routine methodology in the art.

A further aspect of the invention provides a method for obtaining an antibody antigen-binding site for IL-1R1, the method comprising providing by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a VH domain set out herein, optionally combining the VH domain thus provided with one or more VL domains, and testing the VH domain or VH/VL combination or combinations to identify a binding member or an antibody antigen-binding site for IL-1R1 and optionally with one or more desired properties, e.g. ability to neutralize IL-1R1 activity. Said VL domain may have an amino acid sequence which is substantially as set out herein. An analogous method may be employed in which one or more sequence variants of a VL domain disclosed herein are combined with one or more VH domains.

Variable domain amino acid sequence variants of any of the VH and VL domains whose sequences are specifically disclosed herein may be employed in accordance with the present invention, as discussed. Particular variants may include one or more amino acid sequence alterations (addition, deletion, substitution and/or insertion of an amino acid residue). In certain embodiments, the variants have less than about 20, less than 15, less than 10 or less than 5 such alterations.

As noted above, a CDR amino acid sequence substantially as set out herein may be carried as a CDR in a human antibody variable domain or a substantial portion thereof. The HCDR3 sequences substantially as set out herein represent embodiments of the present invention and each of these may be carried as a HCDR3 in a human heavy chain variable domain or a substantial portion thereof, optionally in combination with a HCDR1, HCDR2, LCDR1, LCDR2 and/or LCDR3 of the invention.

Binding members of the invention also include fragments of antibodies that comprise an antibody antigen binding site. Fragments of an antibody are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Antibody fragments that comprise an antibody antigen-binding site include, but are not limited to, molecules such as Fab, Fab′, Fab′-SH, scFv, Fv, dAb, Fd and disulphide stabilized variable region (dsFv). Various other antibody molecules including one or more antibody antigen-binding sites have been engineered, including for example Fab2, Fab3, diabodies, triabodies, tetrabodies and minibodies. Antibody molecules and methods for their construction and use are described in Holliger & Hudson (44).

It has been shown that fragments of a whole antibody can perform the function of binding antigens. Examples of binding fragments are (i) the Fab fragment consisting of VL, VH, constant light chain domain (CL) and constant heavy chain domain 1 (CH1) domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment [45, 46, 47], which consists of a VH or a VL domain; (v) isolated CDR regions; (vi) F(ab′)2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site [48, 49]; (viii) bispecific single chain Fv dimers (for example as disclosed in WO 1993/011161) and (ix) “diabodies”, multivalent or multispecific fragments constructed by gene fusion (for example as disclosed in WO94/13804 and [50]. Fv, scFv or diabody molecules may be stabilized by the incorporation of disulphide bridges linking the VH and VL domains [51]. Minibodies comprising a say joined to a CM domain may also be made [52]. Other examples of binding fragments are Fab′, which differs from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain domain, including one or more cysteines from the antibody hinge region, and Fab′-SH, which is a Fab′ fragment in which the cysteine residue(s) of the constant domains bear a free thiol group.

Antibody fragments of the invention can be obtained starting from a parent antibody molecule (Antibody 1 or 4) or any of the antibody molecules 2, 3, 5 to 10, by methods such as digestion by enzymes e.g. pepsin or papain and/or by cleavage of the disulfide bridges by chemical reduction. In another manner, the antibody fragments comprised in the present invention can be obtained by techniques of genetic recombination likewise well known to the person skilled in the art or else by peptide synthesis by means of, for example, automatic peptide synthesizers, such as those supplied by for example the company Applied Biosystems Inc (Foster City, Calif., USA), or by nucleic acid synthesis and expression.

Functional antibody fragments according to the present invention include any functional fragment whose half-life is increased by a chemical modification, for example by PEGylation, or by incorporation in a liposome.

A dAb (domain antibody) is a small monomeric antigen-binding fragment of an antibody, namely the variable region of an antibody heavy or light chain [47]. VH dAbs occur naturally camelids (e.g. camel, llama) and may be produced by immunizing a camelid with a target antigen, isolating antigen-specific B cells and directly cloning dAb genes from individual B cells. dAbs are also producible in cell culture. Their small size, good solubility and temperature stability makes them particularly physiologically useful and suitable for selection and affinity maturation, Camelid VH dAbs are being developed for therapeutic use under the name “Nanobodies™”. A binding member of the present invention may be a dAb comprising a VH or VL domain substantially as set out herein, or a VH or VL domain comprising a set of CDRs substantially as set out herein.

Antibodies of the invention include bispecific antibodies. Bispecific or bifunctional antibodies form a second generation of monoclonal antibodies in which two different variable regions are combined in the same molecule [53]. Their use has been demonstrated both in the diagnostic field and in the therapy field from their capacity to recruit new effector functions or to target several molecules on the surface of tumour cells. Where bispecific antibodies are to be used, these may be conventional bispecific antibodies, which can be manufactured in a variety of ways [54], e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. These antibodies can be obtained by chemical methods [55, 56] or somatic methods [57, 58] but likewise and preferentially by genetic engineering techniques which allow the heterodimerization to be forced and thus facilitate the process of purification of the antibody sought [59]. Examples of bispecific antibodies include those of the BiTE™ technology in which the binding domains of two antibodies with different specificity can be used and directly linked via short flexible peptides. This combines two antibodies on a short single polypeptide chain. Diabodies and say can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction.

Bispecific antibodies can be constructed as entire IgG, as bispecific Fab′2, Fab′PEG, as diabodies or else as bispecific scFv. Further, two bispecific antibodies can be linked using routine methods known in the art to form tetravalent antibodies.

Bispecific diabodies, as opposed to bispecific whole antibodies, may also be particularly useful because they can be readily constructed and expressed in E. coli. Diabodies (and many other polypeptides, such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO 1994/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against IL-1R1, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected. Bispecific whole antibodies may be made by alternative engineering methods as described in Ridgeway et al, [60] or a described in WO 1996/27011, WO 1998/50431 and WO 2006/028936.

Alternatively, a binding member of the invention may comprise an antigen-binding site within a non-antibody molecule, normally provided by one or more CDRs e.g. a set of CDRs in a non-antibody protein scaffold, as discussed further below.

An antigen binding site may be provided by means of arrangement of CDRs on non-antibody protein scaffolds, such as fibronectin or cytochrome B etc. [61, 62, 63], or by randomizing or mutating amino acid residues of a loop within a protein scaffold to confer binding specificity for a desired target. Scaffolds for engineering novel binding sites in proteins have been reviewed in detail by Nyaren at. [63]. Protein scaffolds for antibody mimics are disclosed in WO200034784, which is herein incorporated by reference in its entirety, in which the inventors describe proteins (antibody mimics) that include fibronectin type III domain having at least one randomised loop. A suitable scaffold into which to graft one or more CDRs, e.g. a set of HCDRs, may be provided by any domain member of the immunoglobulin gene superfamily. The scaffold may be a human or non-human protein. An advantage of a non-antibody protein scaffold is that it may provide an antigen-binding site in a scaffold molecule that is smaller and/or easier to manufacture than at least some antibody molecules. Small size of a binding member may confer useful physiological properties, such as an ability to enter cells, penetrate deep into tissues or reach targets within other structures, or to bind within protein cavities of the target antigen. Use of antigen binding sites in non-antibody protein scaffolds is reviewed, in Wess, 2004 [64]. Typical are proteins having a stable backbone and one or more variable loops, in which the amino acid sequence of the loop or loops is specifically or randomly mutated to create an antigen-binding site that binds the target antigen. Such proteins include the IgG-binding domains of protein A from S. aureus, transferrin, tetranectin, fibronectin (e.g. 10th fibronectin type III domain), lipocalins as well as gamma-crystalline and other Affilin™ scaffolds (Scil Proteins). Examples of other approaches include synthetic “Microbodies” based on cyclotides—small proteins having intra-molecular disulphide bonds, Microproteins (Versabodies™, Amunix Inc, Mountain View, Calif., USA) and ankyrin repeat proteins (DARPins, Molecular Partners AG, Zürieh-Schlieren, Switzerland). Such proteins also include small, engineered protein domains such as, for example, immuno-domains (see for example, U.S. Patent Publication Nos. 2003/082630 and 2003/157561). Immuno-domains contain at least one complementarily determining region (CDR) of an antibody.

A binding member according to the present invention may comprise other amino acids, forming a peptide or polypeptide, such as a folded domain, or to impart to the molecule another functional characteristic in addition to ability to bind antigen. Binding members of the invention may carry a detectable label, or may be conjugated to a toxin or a targeting moiety or enzyme (e.g. via a peptidyl bond or linker). For example, a binding member may comprise a catalytic site (e.g. in an enzyme domain) as well as an antigen binding site, wherein the antigen binding site binds to the antigen and thus targets the catalytic site to the antigen. The catalytic site may inhibit biological function of the antigen, e.g. by cleavage.

The invention also comprises binding members which have been modified to change, i.e. increase, decrease or eliminate, the biological effect function of the binding members, for example antibodies with modified Fc regions. In some embodiments, the binding members or antibodies as disclosed herein can be modified to enhance their capability of fixing complement and participating in complement-dependent cytotoxicity (CDC). In other embodiments, the binding members or antibodies can be modified to enhance their capability of activating effector cells and participating in antibody-dependent cytotoxicity (ADCC). In yet other embodiments, the binding members or antibodies as disclosed herein can be modified both to enhance their capability of activating effector cells and participating in antibody-dependent cytotoxicity (ADCC) and to enhance their capability of fixing complement and participating in complement-dependent cytotoxicity (CDC).

In some embodiments, the binding members or antibodies as disclosed herein can be modified to reduce their capability of fixing complement and participating in complement-dependent cytotoxicity (CDC). In other embodiments, the binding members or antibodies can be modified to reduce their capability of activating effector cells and participating in antibody-dependent cytotoxicity (ADCC). In yet other embodiments, the binding members or antibodies as disclosed herein can be modified both to reduce their capability of activating effector cells and participating in antibody-dependent cytotoxicity (ADCC) and to reduce their capability of fixing complement and participating in complement-dependent cytotoxicity (CDC).

In certain embodiments, the half-life of a binding member or antibody as disclosed herein and of compositions of the invention is at least about 4 to 7 days. In certain embodiments, the mean half-life of a binding member or antibody as disclosed herein and of compositions of the invention is at least about 2 to 5 days, 3 to 6 days, 4 to 7 days, 5 to 8 days, 6 to 9 days, 7 to 10 days, 8 to 11 days, 8 to 12, 9 to 13, 10 to 14, 11 to 15, 12 to 16, 13 to 17, 14 to 18, 15 to 19, or 16 to 20 days. In other embodiments, the mean half-life of a binding member or antibody as disclosed herein and of compositions of the invention is at least about 17 to 21 days, 18 to 22 days, 19 to 23 days, 20 to 24 days, 21 to 25, days, 22 to 26 days, 23 to 27 days, 24 to 28 days, 25 to 29 days, or 26 to 30 days. In still further embodiments the half-life of a binding member or antibody as disclosed herein and of compositions of the invention can be up to about 50 days, in certain embodiments, the half-lives of antibodies and of compositions of the invention can be prolonged by methods known in the art. Such prolongation can in turn reduce the amount and/or frequency of dosing of the antibody compositions. Antibodies with improved in vivo half-lives and methods for preparing them are disclosed in U.S. Pat. No. 6,277,375; and International Publication Nos. WO 1998/23289 and WO 1997/3461.

In another embodiment, the invention provides an article of manufacture including a container. The container includes a composition containing a binding member or antibody as disclosed herein, and a package insert or label indicating that the composition can be used to treat disorder associated with IL-1R1.

In other embodiments, the invention provides a kit comprising a composition containing a binding member or antibody as disclosed herein, and instructions to administer the composition to a subject in need of treatment.

The present invention provides formulation of proteins comprising a variant Fc region. That is, a non-naturally occurring Fc region, for example an Fc region comprising one or more non naturally occurring amino acid residues. Also encompassed by the variant Fc regions of present invention are Fc regions which comprise amino acid deletions, additions and/or modifications.

The serum half-life of proteins comprising Fc regions may be increased by increasing the binding affinity of the Fc region for FcRn. In one embodiment, the Fc variant protein has enhanced serum half life relative to comparable molecule.

In another embodiment, the present invention provides an Fc variant, wherein the Fc region comprises at least one non naturally occurring amino acid at one or more positions selected from the group consisting of 239, 330 and 332, as numbered by the EU index as set forth in Kabat. In a specific embodiment, the present invention provides an Fc variant, wherein the Fc region comprises at least one non naturally occurring amino acid selected from the group consisting of 239D, 3301, and 332E, as numbered by the EU index as set forth in Kabat, Optionally, the Fc region may further comprise additional non-naturally occurring amino acid at one or more positions selected from the group consisting of 252, 254, and 256, as numbered by the EU index as set forth in Kabat. In a specific embodiment, the present invention provides an Fc variant, wherein the Fc region comprises at least one non naturally occurring amino acid selected from the group consisting of 239D, 330L and 332E, as numbered by the EU index as set forth in Kabat and at least one non naturally occurring amino acid at one or more positions selected from the group consisting of 252Y, 254T and 256E, as numbered by the EU index as set forth in Kabat.

In another embodiment, the present invention provides an Fc variant, wherein the Fc region comprises at least one non naturally occurring amino acid at one or more positions selected from the group consisting of 234, 235 and 331, as numbered by the EU index as set forth in Kabat. In a specific embodiment, the present invention provides an Fc variant, wherein the Fc region comprises at least one non naturally occurring amino acid selected from the group consisting of 234F, 235F, 235Y, and 331S, as numbered by the EU index as set forth in Kabat. In a further specific embodiment, an Fc variant of the invention comprises the 234F, 235F, and 331S non naturally occurring amino acid residues, as numbered by the EU index as set forth in Kabat. In another specific embodiment, an Fc variant of the invention comprises the 234F, 235Y, and 331S non naturally occurring amino acid residues, as numbered by the EU index as set forth in Kabat. In another specific embodiment, the present invention provides an Fc variant, wherein the Fc region comprises at least one non naturally occurring amino acid selected from the group consisting of 234F, 235E and 331S, as numbered by the EU index as set forth in Kabat. In another specific embodiment, the present invention provides an Fc variant, wherein the Fc region comprises the non naturally occurring amino acid consisting of 234F, 235E and 331S, as numbered by the EU index as set forth in Kabat, Optionally, the Fc region may further comprise additional non naturally occurring amino acid at one or more positions selected from the group consisting of 252, 254, and 256, as numbered by the EU index as set forth in Kabat, In a specific embodiment, the present invention provides an Fc variant, wherein the Fc region comprises at least one non naturally occurring amino acid selected from the group consisting of 234F, 235F, 235Y, and 331S, as numbered by the EU index as set forth in Kabat; and at least one non naturally occurring amino acid at one or more positions are selected from the group consisting of 252Y, 254T and 256E, as numbered by the EU index as set forth in Kabat.

In another embodiment, the present invention provides an Fc variant protein formulation, wherein the Fc region comprises at least a non naturally occurring amino acid at one or more positions selected from the group consisting of 239, 330 and 332, as numbered by the EU index as set forth in Kabat, in a specific embodiment, the present invention provides an Fc variant protein formulation, wherein the Fc region comprises at least one non naturally occurring amino acid selected from the group consisting of 239D, 3301, and 332E, as numbered by the EU index as set forth in Kabat, Optionally, the Fc region may further comprise additional non naturally occurring amino acid at one or more positions selected from the group consisting of 252, 254, and 256, as numbered by the EU index as set forth in Kabat. In a specific embodiment, the present invention provides an Fc variant protein formulation, wherein the Fc region comprises at least one non naturally occurring amino acid selected from the group consisting of 239D, 330L and 332E, as numbered by the EU index as set forth in Kabat and at least one non naturally occurring amino acid at one or more positions are selected from the group consisting of 252Y, 254T and 256E, as numbered by the EU index as set forth in Kabat.

In another embodiment, the present invention provides an Fc variant protein formulation, wherein the Fc region comprises at least one non naturally occurring amino acid at one or more positions selected from the group consisting of 234, 235 and 331, as numbered by the EU index as set forth in Kabat. In a specific embodiment, the present invention provides an Fc variant protein formulation, wherein the Fc region comprises at least one non naturally occurring amino acid selected from the group consisting of 234F, 235F, 235Y, and 331S, as numbered by the EU index as set forth in Kabat, Optionally, the Fc region may further comprise additional non naturally occurring amino acid at one or more positions selected from the group consisting of 252, 254, and 256, as numbered by the EU index as set forth in Kabat. In a specific embodiment, the present invention provides an Fc variant protein formulation, wherein the Fc region comprises at least one non naturally occurring amino acid selected from the group consisting of 234F, 235F, 235Y, and 331S, as numbered by the EU index as set forth in Kabat; and at least one non naturally occurring amino acid at one or more positions are selected from the group consisting of 252Y, 254T and 256E, as numbered by the EU index as set forth in Kabat.

Methods for generating non naturally occurring Fc regions are known in the art. For example, amino acid substitutions and/or deletions can be generated by mutagenesis methods, including, but not limited to, site-directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci, USA 82:488-492 (1985)), PCR mutagenesis (Higuehi, “PCR Protocols: A Guide to Methods and Applications”, Academic Press, San Diego, pp. 177-183 (1990)), and cassette mutagenesis (Wells et al., Gene 34:315-323 (1985)). Preferably, site-directed mutagenesis is performed by the overlap-extension PCR method (Higuchi, in “PCR Technology: Principles and Applications for DNA Amplification”, Stockton Press, New York, pp. 61-70 (1989)). The technique of overlap-extension PCR (Higuchi, ibid.) can also be used to introduce any desired mutation(s) into a target sequence (the starting DNA). For example, the first round of PCR in the overlap-extension method involves amplifying the target sequence with an outside primer (primer 1) and an internal mutagenesis primer (primer 3), and separately with a second outside primer (primer 4) and an internal primer (primer 2), yielding two PCR segments (segments A and B). The internal mutagenesis primer (primer 3) is designed to contain mismatches to the target sequence specifying the desired mutation(s). In the second round of PCR, the products of the first round of PCR (segments A and B) are amplified by PCR using the two outside primers (primers 1 and 4). The resulting full-length PCR segment (segment C) is digested with restriction enzymes and the resulting restriction fragment is cloned into an appropriate vector. As the first step of mutagenesis, the starting DNA (e.g., encoding an Fc fusion protein, an antibody or simply an Fc region), is operably cloned into a mutagenesis vector. The primers are designed to reflect the desired amino acid substitution. Other methods useful for the generation of variant Fc regions are known in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 5,885,573; 5,677,425; 6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260; 6,528,624; 6,194,551; 6,737,056; 6,821,505; 6,277,375; U.S. Patent Publication Nos. 2004/0002587 and PCT Publications WO 94/29351; WO 99/58572; WO 00/42072; WO 02/060919; WO 04/029207; WO 04/099249; WO 04/063351; WO 06/23403).

In some embodiments of the invention, the glycosylation patterns of the antibodies provided herein are modified to enhance ADCC and CDC effector function. See Shields R L et al., (2002) IBC, 277:26733; Shinkawa T et al., (2003) IBC, 278:3466 and Okazaki A et al., (2004) J. Mol, Biol., 336: 1239. In some embodiments, an Fc variant protein comprises one or more engineered glycoforms, i.e., a carbohydrate composition that is covalently attached to the molecule comprising an Fc region. Engineered glycoforms may be useful for a variety of purposes, including but not limited to enhancing or reducing effector function. Engineered glycoforms may be generated by any method known to one skilled in the art, for example by using engineered or variant expression strains, by co-expression with one or more enzymes, for example DI N-acetylglucosaminyltransferase III (GnTI11), by expressing a molecule comprising an Fc region in various organisms or cell lines from various organisms, or by modifying carbohydrate(s) after the molecule comprising Fc region has been expressed. Methods for generating engineered glycoforms are known in the art, and include but are not limited to those described in Umana et al, 1999, Nat. Biotechnol 17:176-180; Davies et al., 20017 Biotechnol Bioeng 74:288-294; Shields et al, 2002, J Biol. Chem. 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473) U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser, No. 10/113,929; PCT WO 00/61739A1; PCT WO 01/292246A PCT WO 02/311140A1; PCT WO 02/30954A1; Potillegent™ technology (Biowa, Inc. Princeton, N.J.); GlycoMAb™ glycosylation engineering technology (Glycart Biotechnology AG, Zurich, Switzerland). See, e.g., WO 00/061739; EA01229125; US 20030115614; Okazaki et al., 2004, JMB, 336: 1239-49.

It is also known in the art that the glycosylation of the Fc region can be modified to increase or decrease effector function (see for examples, Umana et al, 1999, Nat. Biotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al, 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473) U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No. 10/113,929; PCT WO 00/61739A1; PCT WO 01/292246A1; PCT WO 02/311140A1; PCT WO 02/30954A1; Potillegent™ technology (Biowa, Inc, Princeton, N.J.); GlycoMAb™ glycosylation engineering technology (Glycart Biotechnology AG, Zurich, Switzerland). Accordingly, in one embodiment the Fc regions of the antibodies of the invention comprise altered glycosylation of amino acid residues. In another embodiment, the altered glycosylation of the amino acid residues results in lowered effector function. In another embodiment, the altered glycosylation of the amino acid residues results in increased effector function. In a specific embodiment, the Fc region has reduced fucosylation. In another embodiment, the Fc region is aft/c (see for examples, U.S. Patent Application Publication No. 200570226867). In another embodiment the Fc region is sialylated, such as wherein at least one galactose moiety is connected to a respective terminal sialic acid moiety by a α 2,6 linkage (see for example: International Patent Application Publication No. WO2009079382).

The binding members are useful for treating and/or preventing disorders that are mediated by IL-1R1, especially inflammatory disorders such as rheumatoid arthritis, osteoarthritis (OA) asthma and chronic obstructive pulmonary disease (COPD). The binding members are also useful for treating and/or preventing disorders that are mediated by IL-1R1 such as HIV-1, solid tumours, leukaemias, Alzheimers disease and ischaemic disease,

Further aspects of the present invention provide for compositions containing binding members of the invention, and their use in methods of inhibiting and/or neutralizing including methods of treatment of the human or animal body by therapy.

For example, binding members according to the invention may be used in a method of treatment and/or prevention, or used in a method of diagnosis, of a biological response, disease, disorder, or condition in the human or animal body (e.g. in a human patient), or in vitro.

The method of treatment and/or prevention may comprise administering to said patient a binding member of the invention in an amount sufficient to measurably neutralize IL-1R1. Conditions treatable in accordance with the present invention include any in which IL-1R1 plays a role, such as COPD and asthma.

Binding members of the present invention may be used in methods of diagnosis or treatment in human or animal subjects, especially human. Binding member of the invention may be used in the preparation of a medicament for use in methods of diagnosis or treatment in human or animal subjects, especially human. The invention further provides the use of a binding member of the present invention for diagnosis or treatment in human or animal subjects, especially humans, Treatment comprises disorders characterized by biological effects mediated by IL-1R1, particularly inflammatory disorders such as rheumatoid arthritis, osteoarthritis (OA) asthma and COPD.

Accordingly, the invention provides a method for treating inflammatory disorders, such as rheumatoid arthritis, osteoarthritis, asthma and COPD in a mammal, comprising administering to said mammal a binding member of the invention. In another embodiment the invention provides the use of a binding member of the invention in the manufacture of a medicament for the treatment of inflammatory disorders, such as rheumatoid arthritis, osteoarthritis, asthma and COPD in a mammal. In another embodiment the invention provides the use of a binding member of the invention for the treatment of inflammatory disorders, such as rheumatoid arthritis, osteoarthritis, asthma and COPD in a mammal. In one embodiment the mammal is a human, in another embodiment the mammal is a non-human animal. In one embodiment the binding member is an antibody, VH domain, or VL domain of the invention, in an amount sufficient to neutralize IL-1R1.

Accordingly, the invention provides a method for the inhibition of neutrophil recruitment and chemotaxis into the lung in a mammal, comprising administering to said mammal a binding member of the invention. In another embodiment the invention provides the use of a binding member of the invention in the manufacture of a medicament for the inhibition of neutrophil recruitment and chemotaxis into the lung in a mammal. In another embodiment the invention provides the use of a binding member of the invention for inhibition of neutrophil recruitment and chemotaxis into the lung in a mammal. In one embodiment the mammal is a human, in another embodiment the mammal is a non-human animal. In one embodiment the binding member is an antibody, VH domain, or VL domain of the invention, in an amount sufficient to neutralize IL-1R1.

Accordingly, the invention provides a method for treating a disorder selected from HIV, solid tumours, leukaemias, Alzheimer\’s disease, type II diabetes, ischaemic disease and atherosclerosis in a mammal, comprising administering to said mammal a binding member of the invention. In another embodiment the invention provides the use of a binding member of the invention in the manufacture of a medicament for the treatment of a disorder selected from HIV, solid tumours, leukaemias, Alzheimer\’s disease, type II diabetes, ischaemic disease and atherosclerosis in a mammal. In another embodiment the invention provides the use of a binding member of the invention for the treatment of a disorder selected from HIV, solid tumours, leukaemias, Alzheimer\’s disease, type II diabetes, ischaemic disease and atherosclerosis in a mammal. In one embodiment the mammal is a human, in another embodiment the mammal is a non-human animal. In one embodiment the binding member is an antibody, VH domain, or VL domain of the invention, in an amount sufficient to neutralize IL-1R1.

When test cells are contacted with the binding member of the invention in vitro, a control cell(s) may also be used for positive controls (e.g., reactions containing no binding member) and/or negative controls (e.g., reactions containing no IL-1R1 and/or antigen),

When cells are contacted by the binding member in vivo, for example, by administering the binding member of the invention to a mammal exhibiting IL-1α- and/or IL-1β-mediated biological responses, the binding member of the invention is administered in amounts sufficient to neutralize IL-1R1.

Still further, the invention provides a method for reducing IL-1R1-mediated activity in a mammal, such as a human, comprising administering a binding member, such as an antibody, VH domain, or VL domain of the invention. In another embodiment the invention provides the use of a binding member of the invention in the manufacture of a medicament for reducing IL-1R1-mediated activity in a mammal. In another embodiment the invention provides the use of a binding member of the invention for reducing IL-1R 1-mediated activity in a mammal. In one embodiment the mammal is a human, in another embodiment the mammal is a non-human animal. In one embodiment the binding member is an antibody, VH domain, or VL domain of the invention, in an amount sufficient to neutralize IL-1R1 and reduce IL-1R1-mediated activity.

Diseases or disorders for which binding members of the invention may be used include but are not limited to:

1. Respiratory tract: obstructive diseases of the airways including: asthma, including bronchial, allergic, intrinsic, extrinsic, exercise-induced, drug-induced (including aspirin and NSAID-induced) and dust-induced asthma, both intermittent and persistent and of all severities, and other causes of airway hyper-responsiveness; chronic obstructive pulmonary disease (COPD); bronchitis, including infectious and eosinophilic bronchitis; emphysema; bronchiectasis; cystic fibrosis; sarcoidosis; farmer\’s lung and related diseases; hypersensitivity pneumonitis; lung fibrosis, including cryptogenic fibrosing alveolitis, idiopathic interstitial pneumonias, fibrosis complicating anti-neoplastic therapy and chronic infection, including tuberculosis and aspergillosis and other fungal infections; complications of lung transplantation; vasculitic and thrombotic disorders of the lung vasculature, and pulmonary hypertension; antitussive activity including treatment of chronic cough associated with inflammatory and secretory conditions of the airways, and iatrogenic cough; acute and chronic rhinitis including rhinitis medicamentosa, and vasomotor rhinitis; perennial and seasonal allergic rhinitis including rhinitis nervosa (hay fever); nasal polyposis; acute viral infection including the common cold, and infection due to respiratory syncytial virus, influenza, coronavirus (including SARS), adenovirus, and ARDS and ALI;

2. Bone and joints: arthritides associated with or including osteoarthritis/osteoarthrosis, both primary and secondary to, for example, congenital hip dysplasia; cervical and lumbar spondylitis, and low back and neck pain; rheumatoid arthritis and Still\’s disease; seronegative spondyloarthropathies including ankylosing spondylitis, psoriatic arthritis, reactive arthritis and undifferentiated spondarthropathy; septic arthritis and other infection-related arthopathies and bone disorders such as tuberculosis, including Potts\’ disease and Poncet\’s syndrome; acute and chronic crystal-induced synovitis including urate gout, calcium pyrophosphate deposition disease, and calcium apatite related tendon, bursal and synovial inflammation; Behcet\’s disease; primary and secondary Sjogren\’s syndrome; systemic sclerosis and limited scleroderma; systemic lupus erythematosus, mixed connective tissue disease, and undifferentiated connective tissue disease; inflammatory myopathies including dermatomyositits and polymyositis; polymalgia rheumatica; juvenile arthritis including idiopathic inflammatory arthritides of whatever joint distribution and associated syndromes, and rheumatic fever and its systemic complications; vasculitides including giant cell arteritis, Takayasu\’s arteritis, Churg-Strauss syndrome, polyarteritis nodosa, microscopic polyarteritis, and vasculitides associated with viral infection, hypersensitivity reactions, cryoglobulins, and paraproteins; low back pain; Familial Mediterranean fever, Muckle-Wells syndrome, and Familial Hibernian Fever, Kawasaki\’s disease, Kikuchi disease; drug-induced arthalgias, tendonititides, and myopathies;

3. Pain and connective tissue remodelling of musculoskeletal disorders due to injury, for example sports injury, or disease: arthitides (for example rheumatoid arthritis, osteoarthritis, gout or crystal arthropathy), other joint disease (such as intervertebral disc degeneration or temporomandibular joint degeneration), bone remodelling disease (such as osteoporosis, Paget\’s disease or osteonecrosis), polychondritits, scleroderma, mixed connective tissue disorder, spondyloarthropathies or periodontal disease (such as periodontitis);

4. Skin: psoriasis, parapsoriasis, atopic dermatitis, contact dermatitis or other eczematous dermatoses, and delayed-type hypersensitivity reactions; phyto- and photodermatitis; seborrhoeic dermatitis, dermatitis herpetiformis, lichen planus, lichen sclerosus et atrophica, pyoderma gangrenosum, skin sarcoid, discoid lupus erythematosus, pemphigus, pemphigoid, epidermolysis bullosa, mycosis fungoides, urticaria, angioedema, vasculitides, toxic erythemas, cutaneous eosinophilias, alopecia areata, male-pattern baldness, Sweet\’s syndrome, Weber-Christian syndrome, erythema multiforme; cellulitis, both infective and non-infective; panniculitis; cutaneous lymphomas, non-melanoma skin cancer and other dysplastic lesions; drug-induced disorders including fixed drug eruptions;

5. Eyes: blepharitis; conjunctivitis, including perennial and vernal allergic conjunctivitis; iritis; anterior and posterior uveitis; choroiditis; autoimmune; degenerative or inflammatory disorders affecting the retina; ophthalmitis including sympathetic ophthalmitis; sarcoidosis; infections including viral, fungal, and bacterial;

6. Gastrointestinal tract: glossitis, gingivitis, periodontitis; oesophagitis, including reflux; eosinophilic gastro-enteritis, mastocytosis, Crohn\’s disease, colitis e.g. ulcerative colitis, indeterminant colitis, proctitis, microscopic colitis, pruritis ani; Coeliac disease, irritable bowel syndrome, irritable bowel disorder, non-inflammatory diarrhoea and food-related allergies which may have effects remote from the gut (thr example migraine, rhinitis or eczema);

7. Abdominal: hepatitis, including autoimmune, alcoholic a d viral: fibrosis and cirrhosis of the liver; cholecystitis; pancreatitis, both acute and chronic;

8. Genitourinary: nephritis including interstitial and glomerulonephritis; nephrotic syndrome; cystitis including acute and chronic (interstitial) cystitis and thinner\’s ulcer; acute and chronic urethritis, prostatitis, epididymitis, oophoritis and salpingitis; vulvo-vaginitis; Peyronie\’s disease; erectile dysfanction (both male and female);

9. Allograft rejection: acute and chronic following, for example, transplantation of kidney, heart, liver, lung, bone marrow, skin or cornea or following blood transfusion; or acute and chronic graft versus host disease;

10. CNS: Alzheimer\’s disease and other dementing disorders including CJD and nvCJD; amyloidosis; multiple sclerosis and other demyelinating syndromes; cerebral atherosclerosis and vasculitis; temporal arteritis; myasthenia gravis; acute and chronic pain (acute, intermittent or persistent, whether of central or peripheral origin) including visceral pain, headache, migraine, trigeminal neuralgia, atypical facial pain, joint and bone pain, pain arising from cancer and tumor invasion, neuropathic pain syndromes including diabetic, post-herpetic, and HIV-associated neuropathies; tropical spastic paraparesis, neurosarcoidosis; central and peripheral nervous system complications of malignant, infectious or autoimmune processes;

11. Other auto-immune and allergic disorders (including in combination with other allergy therapies) including Hashimoto\’s thyroiditis, Graves\’ disease, Addison\’s disease, diabetes mellitus, idiopathic thrombocytopaenic purpura, eosinophilic fasciitis, hyper-IgE syndrome, antiphospholipid syndrome; pre-term labour

12. Other disorders with an inflammatory or immunological component; including acquired immune deficiency syndrome (AIDS), leprosy, Sezary syndrome, and paraneoplastic syndromes;

13. Cardiovascular: atherosclerosis, affecting the coronary and peripheral circulation; pericarditis; myocarditis, inflammatory and auto-immune cardiomyopathies including myocardial sarcoid; ischaemic reperfusion injuries; endocarditis, valvulitis, and aortitis including infective for example syphilitic); vasculitides; disorders of the proximal and peripheral veins including phlebitis and thrombosis, including deep vein thrombosis and complications of varicose veins; and

14. Oncology: treatment of common cancers including prostate, breast, lung, ovarian, pancreatic, bowel and colon, stomach, skin and brain tumours and, malignancies affecting the bone marrow (including the leukaemias) and lymphoproliferative systems, such as Hodgkin\’s and non-Hodgkin\’s lymphoma; including the prevention and treatment of metastatic disease and tumour recurrences, and paraneoplastic syndromes.

The data presented herein with respect to binding and neutralization of IL-1R1 thus indicate that binding members of the invention can be used to treat or prevent such disorders, including the reduction of severity of the disorders. Accordingly, the invention provides a method of treating or reducing the severity of at least one symptom of any of the disorders mentioned herein, comprising administering to a patient in need thereof an effective amount of one or more binding members of the present invention alone or in a combined therapeutic regimen with another appropriate medicament known in the art or described, herein such that the severity of at least one symptom of any of the above disorders is reduced,

Binding members of the invention may be used in appropriate animals and in animal models of disease, especially monkeys.

Thus, the binding members of the present invention are useful as therapeutic agents in the treatment of diseases or disorders involving IL-1R1, e.g. IL-1R1 production, expression and/or activity, especially aberrant production, expression, or activity. A method of treatment may comprise administering an effective amount of a binding member of the invention to a patient in need thereof, wherein production, expression and/or activity of IL-1R1 is thereby decreased. A method of treatment may comprise (i) identifying a patient demonstrating increased IL-1R1 or IL-\ levels or activity thereof, for instance using the diagnostic methods described above, and (ii) administering an effective amount of a binding member of the invention to the patient, wherein increased production, expression and/or activity of IL-1R1 is decreased. An alternative method of treatment may comprise (i) identifying a patient who has no apparent increase in IL-1R1-mediated activity but who is believed to benefit from administration of a binding member of the invention, and (ii) administering an effective amount of a binding member of the invention to the patient. An effective amount according to the invention is an amount that decreases the increased production, expression and/or activity of IL-1R1 so as to decrease or lessen the severity of at least one symptom of the particular disease or disorder being treated, but not necessarily cure the disease or disorder.

The invention also provides a method of antagonizing at least one effect of IL-1R1 comprising contacting with or administering an effective amount of one or more binding members of the present invention such that said at least one effect of is antagonised. Effects of IL-1R1 that may be antagonised by the methods of the invention include biological responses mediated by IL-1α and/or IL-1β, and any downstream effects that arise as a consequence of these binding reactions.

Accordingly, further aspects of the invention provide the use of an isolated binding member, such as an antibody, VH domain or VL domain of the invention for the manufacture of a medicament for treating a disorder associated with, or mediated by, IL-1R1 as discussed herein. Such use of, or methods of making, a medicament or pharmaceutical composition comprise formulating the binding member with a pharmaceutically acceptable excipient.

A pharmaceutically acceptable excipient may be a compound or a combination of compounds entering into a pharmaceutical composition not provoking secondary reactions and which allows, for example, facilitation of the administration of the active compound(s), an increase in its lifespan and/or in its efficacy in the body, an increase in its solubility in solution or else an improvement in its conservation. These pharmaceutically acceptable excipients are well known and will be adapted by the person skilled in the art as a function of the nature and of the mode of administration of the active compound(s) chosen.

Binding members of the present invention will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the binding member. Thus pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, inhaled, intra-tracheal, topical, intra-vesicular or by injection, as discussed below.

Pharmaceutical compositions for oral administration, such as for example single domain antibody molecules (e.g. “Nanobodies™”) etc are also envisaged in the present invention. Such oral formulations may be in tablet, capsule, powder, liquid or semi-solid form. A tablet may comprise a solid carrier, such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier, such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols, such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intra-venous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles, such as Sodium Chloride Injection, Ringer\’s Injection, Lactated Ringer\’s Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be employed as required including buffers such as phosphate, citrate and other organic acids; antioxidants, such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3′-pentanol; and m-cresol); low molecular weight polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagines, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions, such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants, such as TWEEN™, PUTRONICS™ or polyethylene glycol (PEG).

Binding members of the present invention may be formulated in liquid, semi-solid or solid forms depending on the physicochemical properties of the molecule and the route of delivery. Formulations may include excipients, or combinations of excipients, for example: sugars, amino acids and surfactants. Liquid formulations may include a wide range of antibody concentrations and pH. Solid formulations may be produced by lyophilisation, spray drying, or drying by supercritical fluid technology, for example. Formulations of anti-IL-1R1 will depend upon the intended route of delivery: for example, formulations for pulmonary delivery may consist of particles with physical properties that ensure penetration into the deep lung upon inhalation; topical formulations (e.g. for treatment of scarring, e.g. dermal scarring) may include viscosity modifying agents, which prolong the time that the drug is resident at the site of action. A binding member may be prepared with a carrier that will protect the binding member against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are known to those skilled in the art [65].

Anti-IL-1R1 treatment may be given orally (such as for example single domain antibody molecules (e.g. “Nanobodies™”)) by injection (for example, subcutaneously, intra-articular, intra-venously, intra-peritoneal, intra-arterial or intra-muscularly), by inhalation, intra-tracheal, by the intra-vesicular route (instillation into the urinary bladder), or topically (for example intra-ocular, intra-nasal, rectal, into wounds, on skin). The treatment may be administered by pulse infusion, particularly with declining doses of the binding member. The route of administration can be determined by the physicochemical characteristics of the treatment, by special considerations for the disease or by the requirement to optimize efficacy or to minimize side-effects. One particular route of administration is intra-venous. Another route of administering pharmaceutical compositions of the present invention is subcutaneously. It is envisaged that anti-IL-1R1 treatment will not be restricted to use in the clinic. Therefore, subcutaneous injection using a needle-free device is also advantageous.

Examples of intravenous formulations include:



25 mM histidine,
120 mM sodium chloride
pH 6.0.

A binding member for IL-1R1 or composition comprising a binding member for IL-1R1 may be used as part of a combination therapy in conjunction with an additional medicinal component. Combination treatments may be used to provide significant synergistic effects, particularly the combination of an anti-IL-1R1 binding member with one or more other drugs. A binding member for IL-1R1 may be administered concurrently or sequentially or as a combined preparation with another therapeutic agent or agents, for the treatment of one or more of the conditions listed herein.

A binding member of the invention may be formulated and/or used in combination with other available treatments for Il-1R1 mediated diseases such as obstructive diseases of the airways, asthma and allergic disorders, or other disorders involving IL-1R1 mediated effects.

A binding member according to the present invention may be provided as sole therapy or in combination or addition with one or more of the following agents:

a cytokine or agonist or antagonist of cytokine function (e.g. an agent which acts on cytokine signalling pathways, such as a modulator of the SOCS system), such as an alpha-, beta- and/or gamma-interferon; insulin-like growth factor type I (IGF-1), its receptors and associated binding proteins; interleukins (IL), e.g. one or more of IL-2 to -33, and/or an interleukin antagonist or inhibitor, such as anakinra; inhibitors of receptors of interleukin family members or inhibitors of specific subunits of such receptors, a tumour necrosis factor alpha (TNF-α) inhibitor, such as an anti-TNF monoclonal antibodies (for example infliximab, adalimumab and/or CDP-870) and/or a TNF receptor antagonist, e.g. an immunoglobulin molecule (such as etanercept) and/or a low-molecular-weight agent, such as pentoxyfylline;

a modulator of B cells, e.g. a monoclonal antibody targeting B-lymphocytes (such as CD20 (rituximab) or MRA-aIL16R) or T-lymphocytes (e.g. CTLA4-Ig, HuMax Il-15 or Abatacept);

a modulator that inhibits osteoclast activity, for example an antibody to RANKL;

a modulator of chemokine or chemokine receptor function, such as an antagonist of CCR1, CCR2, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10 and CCR11 (for the C—C family); CXCR1, CXCR2, CXCR3, CXCR4 and CXCR5 and CXCR6 (for the C—X—C family) and CX3 CR1 for the C—X3—C family;

an inhibitor of matrix metalloproteases (MMPs), i.e. one or more of the stromelysins, the collagenases and the gelatinases as well as aggrecanase, especially collagenase-1 (MMP-1), collagenase-2 (MMP-8), collagenase-3 (MMP-13), stromelysin-1 (MMP-3), stromelysin-2 (MMP-10) and/or stromelysin-3 (MMP-1) and/or MMP-9 and/or MMP-12, e.g. an agent such as doxycycline;

a leukotriene biosynthesis inhibitor, 5-lipoxygenase (5-LO) inhibitor or 5-lipoxygenase activating protein (FLAP) antagonist, such as zileuton; ABT-761; fenleuton; tepoxalin; Abbott-79175; Abbott-85761; N-(5-substituted)-thiophene-2-alkylsulfonamides; 2,6-di-tert-butylphenolhydrazones; methoxytetrahydropyrans such as Zeneca ZD-2138; the compound SB-210661; a pyridinyl-substituted 2-cyanonaphthalene compound, such as L-739,010; a 2-cyanoquinoline compound, such as L-746,530; indole and/or a quinoline compound, such as MK-591, MK-886 and/or BAY×1005;

a receptor antagonist for leukotrienes (LT) B4, LTC4, LTD4, and LTE4, selected from the group consisting of the phenothiazin-3-1s, such as L-651,392; amidino compounds, such as CGS-25019c; benzoxalamines, such as ontazolast; benzenecarboximidamides, such as BIIL 284/260; and compounds, such as zafirlukast, ablukast, montelukast, pranlukast, verlukast (MK-679), RG-12525, Ro-245913, iralukast (CGP 45715A) and BAY x 7195;

a phosphodiesterase (PDE) inhibitor, such as a methylxanthanine, e.g. theophylline and/or aminophylline; and/or a selective PDE isoenzyme inhibitor, e.g. a PDE4 inhibitor and/or inhibitor of the isoform PDE4D and/or an inhibitor of PDE5:

a histamine type 1 receptor antagonist, such as cetirizine, loratadine, desloratadine, fexofenadine, acrivastine, terfenadine, astemizole, azelastine, levocabastine, chlorpheniramine, promethazine, cyclizine, and/or mizolastine (generally applied orally, topically or parenterally);

a proton pump inhibitor (such as omeprazole) or gastroprotective histamine type 2 receptor antagonist;

an antagonist of the histamine type 4 receptor;

an alpha-1/alpha-2 adrenoceptor agonist vasoconstrictor sympathomimetic agent, such as propylhexedrine, phenylephrine, phenylpropanolamine, ephedrine, pseudoephedrine, naphazoline hydrochloride, oxymetazoline hydrochloride, tetrahydrozoline hydrochloride, xylometazoline hydrochloride, tramazoline hydrochloride and ethylnorepinephrine hydrochloride;

an anticholinergic agent, e.g. a muscarinic receptor (M1, M2, and M3) antagonist, such as atropine, hyoscine, glycopyrrrolate, ipratropium bromide, tiotropiumrn bromide, oxitropium bromide, pirenzepine and telenzepine;

a beta-adrenoceptor agonist (including beta receptor subtypes 1-4), such as isoprenaline, salbutamol, formoterol, salmeterol, terbutaline, orciprenaline, bitolterol mesylate and/or pirbuterol, e.g. a chiral enantiomer thereof;

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Agent: Boehringer Ingelheim International Gmbh – Ingelheim Am Rhein, DE
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The Patent Description & Claims data below is from USPTO Patent Application 20130079359, Novel piperidino-dihydrothienopyrimidine sulfoxides and their use for treating copd and asthma.

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20130072502 – Heterocyclic carboxylic acid derivatives having a 2,5-substituted oxazolopyrimidine ring – in which A, R1, R2, R3, X and Y are defined as indicated in the claims. The compounds of the formula I modulate the activity of the Edg-1 receptor and in particular are agonists of this receptor, and are useful for the treatment of diseases such as atherosclerosis, heart failure …

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The Patent Description & Claims data below is from USPTO Patent Application 20130052163, Human rhinovirus (hrv) antibodies.

RELATED APPLICATIONS

This application claims the benefit of provisional application U.S. Ser. No. 61/529,008, filed on Aug. 30, 2011, the contents which are herein incorporated by reference in their entirety.

INCORPORATION OF SEQUENCE LISTING

The contents of the text file named “37418-519001US_ST25.txt,” which was created on Aug. 23, 2012 and is 54 KB in size, are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to prophylaxis, therapy, diagnosis and monitoring of human rhinovirus (HRV) infection. The invention is more specifically related to human neutralizing monoclonal antibodies specific for HRV, such as broad and potent neutralizing monoclonal antibodies specific for HRV and their manufacture and use. Broad neutralization suggests that the antibodies can neutralize HRV isolates from multiple isotypes.

BACKGROUND OF THE INVENTION

Human rhinoviruses are the most common infectious agents in humans, worldwide. These viruses are also most commonly known as the primary cause of the common cold. Commensurate with their role as instigating colds, the primary route of entry for human rhinovirus is the upper respiratory tract. These viruses travel rapidly throughout the local population because they are transmitted through air, e.g. via contaminated respiratory droplets of sneezes and coughs, via contact with contaminated surfaces, and via person-to-person contact. Infection also occurs rapidly. The virus adheres to cell surface receptors within minutes of entering the respiratory tract. Symptoms appear in most individuals within days. However, the incubation time can vary from approximately 12 hours to a week.

Infection by human rhinovirus can be fatal; however, more common symptoms include, but are not limited to sore throat, runny nose, nasal congestion, sneezing, cough, muscle aches, fatigue, malaise, headache, muscle weakness, and loss of appetite. Infections frequently occur during the time of year when people spend most time indoors and, therefore, spend most time in close proximity to one another, e.g. from September to April. The consequences of the human rhinovirus infection are not only medical, but economical. Students and employees must isolate themselves from school and colleagues to prevent spread of the virus, which results in lost educational opportunity and productivity.

Despite a long-felt need in the art and ongoing attempts to cure infections caused by the human rhinovirus, including the common cold, a need still exists for an effective treatment that addresses the underlying cause of these illnesses by neutralizing the virus itself.

SUMMARY

OF THE INVENTION

The invention solves this long-felt need by providing compositions and methods for preventing and treating human rhinovirus infection.

The present invention provides a novel method for isolating potent, broadly neutralizing monoclonal antibodies against HRV. Peripheral Blood Mononuclear Cells (PBMCs) are obtained from a donor selected for HRV neutralizing activity in the plasma, and memory B cells are isolated for culture in vitro. The B cell culture supernatants are then screened by a primary neutralization assay in a high throughput format, and B cell cultures exhibiting neutralizing activity are selected for rescue of monoclonal antibodies. It is surprisingly observed that neutralizing antibodies obtained by this method do not always exhibit epitope- or viral-binding at levels that correlate with neutralization activity. The method of the invention therefore allows identification of novel antibodies with cross-isotype neutralization properties.

Specifically, the invention provides an isolated fully human monoclonal antibody, wherein the monoclonal antibody has the following characteristics a) binds to an epitope on the rhinovirus capsid protein selected from the group consisting of VP1, VP2, VP3, and VP4; b) binds to rhinovirus inside infected cells; and c) binds to rhinovirus. Alternatively, or in addition, the antibody binds to an epitope comprising a portion of two or more rhinovirus capsid proteins selected from the group consisting of VP1, VP2, VP3, and VP4. In a preferred embodiment, the epitope is non-linear. The antibody is isolated from a B-cell from a human donor.

In one aspect, the antibody binds to or cross-neutralizes rhinovirus serotypes from one or more clades selected from the group consisting of clade A (major group), clade A (minor group), clade B, and clade D. Alternatively, the antibody binds to or cross-neutralizes rhinovirus serotypes from two or more or three or more clades selected from the group consisting of clade A (major group), clade A (minor group), clade B, and clade D. In another aspect, the antibody binds to at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of HRV serotypes selected from the group consisting of HRV-12, HRV-13, HRV-16, HRV-21, HRV-23, HRV-24, HRV-28, HRV-34, HRV-36, HRV-38, HRV-40, HRV-51, HRV-54, HRV-61, HRV-63, HRV-64, HRV-67, HRV-74, HRV-75, HRV-76, HRV-88, HRV-89, HRV-29, HRV-31, HRV-49, HRV-62, HRV-14, HRV-26, HRV-37, HRV-48, HRV-52, HRV-70, HRV-83, HRV-84, HRV-86, HRV-93, HRV-08, and HRV-45. Preferably, the antibody binds to at least 90% of these HRV serotypes. Alternatively, or in addition, the antibody neutralizes at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of HRV serotypes HRV serotypes selected from the group consisting of HRV-12, HRV-13, HRV-16, HRV-21, HRV-23, HRV-24, HRV-28, HRV-34, HRV-36, HRV-38, HRV-40, HRV-51, HRV-54, HRV-61, HRV-63, HRV-64, HRV-67, HRV-74, HRV-75, HRV-76, HRV-88, HRV-89, HRV-29, HRV-31, HRV-49, HRV-62, HRV-14, HRV-26, HRV-37, HRV-48, HRV-52, HRV-70, HRV-83, HRV-84, HRV-86, HRV-93, HRV-08, and HRV-45. Preferably, the antibody neutralizes at least 40% of these HRV serotypes. Furthermore, the antibody neutralizes the HRV serotypes with a median IC50 value of equal to or less than 100 ng/mL.

Optionally, the antibody is TCN-711 (6893_E11), TCN-716 (6362_F16), TCN-717 (6358_H17), or TCN-722 (6385_L22). The isolated fully human monoclonal antibody that binds to or neutralizes HRV, comprises, (a) a VH CDR1 region comprising the amino acid sequence of DFYWT (SEQ ID NO: 5); a VH CDR2 region comprising the amino acid sequence of EIDRDGATYYNPSLKS (SEQ ID NO: 6); a VH CDR3 region comprising the amino acid sequence of RPMLRGVWGNFRSNWFDP (SEQ ID NO: 7); a VL CDR1 region comprising the amino acid sequence of SGSSSNIGYSYVS (SEQ ID NO: 14); a VL CDR2 region comprising the amino acid sequence of ENNKRPS (SEQ ID NO: 15); and a VL CDR3 region comprising the amino acid sequence of GTWDTRLFGGV (SEQ ID NO: 16); (b) a VH CDR1 region comprising the amino acid sequence of DFAMH (SEQ ID NO: 21); a VH CDR2 region comprising the amino acid sequence of SISRDGSTKYSGDSVKG (SEQ ID NO: 22); a VH CDR3 region comprising the amino acid sequence of DSPYYLDIVGYRYFHHYGMDV (SEQ ID NO: 23); a VL CDR1 region comprising the amino acid sequence of RASQILHSYNLA (SEQ ID NO: 30); a VL CDR2 region comprising the amino acid sequence of GAYNRAS (SEQ ID NO: 31); and a VL CDR3 region comprising the amino acid sequence of QQYGDSPSPGLT (SEQ ID NO: 32); (c) a VH CDR1 region comprising the amino acid sequence of QNDYHWA (SEQ ID NO: 37); a VH CDR2 region comprising the amino acid sequence of SVHYRQKSYYSPSLKS (SEQ ID NO: 38); a VH CDR3 region comprising the amino acid sequence of HNREDYYDSNAYFDE (SEQ ID NO: 39); a VL CDR1 region comprising the amino acid sequence of SGDDLENTLVC (SEQ ID NO: 46); a VL CDR2 region comprising the amino acid sequence of QDSKRPS (SEQ ID NO: 47); and a VL CDR3 region comprising the amino acid sequence of QTWHRSTAQYV (SEQ ID NO: 48); or (d) a VH CDR1 region comprising the amino acid sequence of SNDQYWA (SEQ ID NO: 53); a VH CDR2 region comprising the amino acid sequence of SVHYRRRNYYSPSLES (SEQ ID NO: 54); a VH CDR3 region comprising the amino acid sequence of HNWEDYYESNAYFDY (SEQ ID NO: 55); a VL CDR1 region comprising the amino acid sequence of SGDQLENTFVC (SEQ ID NO: 62); a VL CDR2 region comprising the amino acid sequence of QGSKRPS (SEQ ID NO: 63); and a VL CDR3 region comprising the amino acid sequence of QAWDRSTAHYV (SEQ ID NO: 64).

Alternatively, the antibody binds to the same epitope as TCN-711 (6893_E11), TCN-716 (6362_F16), TCN-717 (6358_H17), or TCN-722 (6385_L22). Alternatively stated, the invention provides an antibody that binds the same epitope as an antibody comprising, (a) a VH CDR1 region comprising the amino acid sequence of DFYWT (SEQ ID NO: 5); a VH CDR2 region comprising the amino acid sequence of EIDRDGATYYNPSLKS (SEQ ID NO: 6); a VH CDR3 region comprising the amino acid sequence of RPMLRGVWGNFRSNWFDP (SEQ ID NO: 7); a VL CDR1 region comprising the amino acid sequence of SGSSSNIGYSYVS (SEQ ID NO: 14); a VL CDR2 region comprising the amino acid sequence of ENNKRPS (SEQ ID NO: 15); and a VL CDR3 region comprising the amino acid sequence of GTWDTRLFGGV (SEQ ID NO: 16); (b) a VH CDR1 region comprising the amino acid sequence of DFAMH (SEQ ID NO: 21); a VH CDR2 region comprising the amino acid sequence of SISRDGSTKYSGDSVKG (SEQ ID NO: 22); a VH CDR3 region comprising the amino acid sequence of DSPYYLDIVGYRYFHHYGMDV (SEQ ID NO: 23); a VL CDR1 region comprising the amino acid sequence of RASQILHSYNLA (SEQ ID NO: 30); a VL CDR2 region comprising the amino acid sequence of GAYNRAS (SEQ ID NO: 31); and a VL CDR3 region comprising the amino acid sequence of QQYGDSPSPGLT (SEQ ID NO: 32); (c) a VH CDR1 region comprising the amino acid sequence of QNDYHWA (SEQ ID NO: 37); a VH CDR2 region comprising the amino acid sequence of SVHYRQKSYYSPSLKS (SEQ ID NO: 38); a VH CDR3 region comprising the amino acid sequence of HNREDYYDSNAYFDE (SEQ ID NO: 39); a VL CDR1 region comprising the amino acid sequence of SGDDLENTLVC (SEQ ID NO: 46); a VL CDR2 region comprising the amino acid sequence of QDSKRPS (SEQ ID NO: 47); and a VL CDR3 region comprising the amino acid sequence of QTWHRSTAQYV (SEQ ID NO: 48); or (d) a VH CDR1 region comprising the amino acid sequence of SNDQYWA (SEQ ID NO: 53); a VH CDR2 region comprising the amino acid sequence of SVHYRRRNYYSPSLES (SEQ ID NO: 54); a VH CDR3 region comprising the amino acid sequence of HNWEDYYESNAYFDY (SEQ ID NO: 55); a VL CDR1 region comprising the amino acid sequence of SGDQLENTFVC (SEQ ID NO: 62); a VL CDR2 region comprising the amino acid sequence of QGSKRPS (SEQ ID NO: 63); and a VL CDR3 region comprising the amino acid sequence of QAWDRSTAHYV (SEQ ID NO: 64).

The invention provides an isolated anti-HRV antibody, wherein the antibody comprises, a VH CDR1 region comprising the amino acid sequence of DFYWT (SEQ ID NO: 5); a VH CDR2 region comprising the amino acid sequence of EIDRDGATYYNPSLKS (SEQ ID NO: 6); a VH CDR3 region comprising the amino acid sequence of RPMLRGVWGNFRSNWFDP (SEQ ID NO: 7); a VL CDR1 region comprising the amino acid sequence of SGSSSNIGYSYVS (SEQ ID NO: 14); a VL CDR2 region comprising the amino acid sequence of ENNKRPS (SEQ ID NO: 15); and a VL CDR3 region comprising the amino acid sequence of GTWDTRLFGGV (SEQ ID NO: 16).

The invention provides an isolated anti-HRV antibody, wherein said antibody comprises, a VH CDR1 region comprising the amino acid sequence of DFAMH (SEQ ID NO: 21); a VH CDR2 region comprising the amino acid sequence of SISRDGSTKYSGDSVKG (SEQ ID NO: 22); a VH CDR3 region comprising the amino acid sequence of DSPYYLDIVGYRYFHHYGMDV (SEQ ID NO: 23); a VL CDR1 region comprising the amino acid sequence of RASQILHSYNLA (SEQ ID NO: 30); a VL CDR2 region comprising the amino acid sequence of GAYNRAS (SEQ ID NO: 31); and a VL CDR3 region comprising the amino acid sequence of QQYGDSPSPGLT (SEQ ID NO: 32).

The invention provides an isolated anti-HRV antibody, wherein said antibody comprises, a VH CDR1 region comprising the amino acid sequence of QNDYHWA (SEQ ID NO: 37); a VH CDR2 region comprising the amino acid sequence of SVHYRQKSYYSPSLKS (SEQ ID NO: 38); a VH CDR3 region comprising the amino acid sequence of HNREDYYDSNAYFDE (SEQ ID NO: 39); a VL CDR1 region comprising the amino acid sequence of SGDDLENTLVC (SEQ ID NO: 46); a VL CDR2 region comprising the amino acid sequence of QDSKRPS (SEQ ID NO: 47); and a VL CDR3 region comprising the amino acid sequence of QTWHRSTAQYV (SEQ ID NO: 48).

The invention provides an isolated anti-HRV antibody, wherein said antibody comprises, a VH CDR1 region comprising the amino acid sequence of SNDQYWA (SEQ ID NO: 53); a VH CDR2 region comprising the amino acid sequence of SVHYRRRNYYSPSLES (SEQ ID NO: 54); a VH CDR3 region comprising the amino acid sequence of HNWEDYYESNAYFDY (SEQ ID NO: 55); a VL CDR1 region comprising the amino acid sequence of SGDQLENTFVC (SEQ ID NO: 62); a VL CDR2 region comprising the amino acid sequence of QGSKRPS (SEQ ID NO: 63); and a VL CDR3 region comprising the amino acid sequence of QAWDRSTAHYV (SEQ ID NO: 64).

The invention provides an isolated monoclonal anti-HRV antibody comprising, a) a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 4 and a light chain sequence comprising amino acid sequence SEQ ID NO: 13, or b) a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 20 and a light chain sequence comprising amino acid sequence SEQ ID NO: 29, or c) a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 36 and a light chain sequence comprising amino acid sequence SEQ ID NO: 45, or d) a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 52 and a light chain sequence comprising amino acid sequence SEQ ID NO: 61.

The invention provides a nucleic acid molecule encoding an antibody described herein. The invention provides a vector comprising this nucleic acid molecule. The invention provides a cell comprising this vector.

The invention provides an isolated B cell clone or immortalized B-cell clone expressing an isolated monoclonal anti-HRV antibody described herein.

The invention provides an isolated epitope which binds to an isolated monoclonal anti-HRV antibody described herein. The invention further provides an immunogenic polypeptide or glycopeptide comprising this epitope.

The invention provides a composition comprising an isolated anti-HRV antibody described herein. Moreover, the invention provides a pharmaceutical composition comprising at least one isolated anti-HRV antibody described herein and a pharmaceutically acceptable carrier.

In certain embodiments, this composition or this pharmaceutical composition further comprise a second therapeutic agent. The second therapeutic agent is a second antibody, an antiviral drug, an antibiotic, a bronchodilator, a leukotriene blocker, a steroid, an antiflammatory drug, or an oxygen therapy. The second antibody may be specific for human rhinovirus, influenza, parainfluenza, coronavirus, adenovirus, respiratory syncytical virus, picornavirus, metapneumovirus, or anti-IgE antibody. If the second antibody is specific for human rhinovirus, the antibody may be an anti-HRV antibody described herein.

The second therapeutic agent is an antiviral drug. The anti-viral drug may be an entry inhibitor, a fusion inhibitor, an integrase inhibitor, a nucleoside analog, a protease inhibitor, or a reverse transcriptase inhibitor. Exemplary anti-viral drug include, but are not limited to, Abacavir, Acicolvir, Acyclovir, Adefovir, Amantadine, Amprenavir, Ampligen, Arbidol, Atazanavir, Atripla, Boceprevir, Cidofovir, Combivir, Darunavir, Delavirdine, Didanosine, Docosanol, Edoxudine, Efavirenz, Emtricitabine, Enfuvirtide, Entecavir, Famciclovir, Fomivirsen, Fosamprenavir, Foscarnet, Fosfonet, Ganciclovir, Ibacitabine, Immunovir, Idoxuridine, Imiquimod, Indinavir, Inosine, Interferon (Type I, II, or III), Interferon-alpha, Interferon-beta, Lamivudine, Lopinavir, Loviride, Maraviroc, Moroxydine, Methisazone, Nelfinavir, Nevirapine, Nexavir, Oseltamivir, Peginterferon alpha-2a, Pencicolvir, Peramivir, Pleconaril, Podophyllotoxin, Raltegravir, Ribavirin, Rimantadine, Ritonavir, Pyramidine, Saquinavir, Stavudine, Tea tree oil, Tenofovir, Tenofovir disoproxil, Tipranavir, Trifluridine, Trizivir, Tromantadine, Truvada, Valaciclovir, Valganciclovir, Vicriviroc, Vidarabine, Viramidine, Zalcitabine, Zanamivir, or Zidovudine.

The second therapeutic agent is an antibiotic. The antibiotic may be an Aminoglycoside, a Carbapenem, a Cephalosporin, a Lincosamide, a Macrolide, a Penicillin, or a Quinolone. Exemplary antibiotics include, but are not limited to, Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmycin, Tobramycin, Paromycin, Geldanamycin, Ertapenem, Dorpenem, Imipenem/Cilastatin, Meropenem, Cefadroxil, Cefazolin, Cefalotin, Cefalothin, Cefalexin, Cefaclor, Ceamandole, Cefoxitin, Cefprozil, Cefurozime, Cefixime, Cefdinir, Defditoren, Cefoperazone, Cefotaxime, Cefazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefepime, Ceftobiprole, Teicoplanin, Vancomycin, Telavancin, Clindamycin, Lincomycin, Daptomycin, Azithromyzin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Spectinomycin, Aztreonam, Furazolidone, Nitofurantoin, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Temocillin, Ticarcillin, Amoxicillin/clavulanate, Ampicillin/sulbactam, Piperacillin/tazobactam, Ticarcillin/clavulanate, Bacitracin, Colistin, Polymyxin B, Ciprofloxacin, Enoxacin, Gatifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin, Temafloxacin, Mafenide, Sulfonamidochrysoidine, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Trimethoprim, Trimethoprim-Sulfamethoxazole (Co-trumoxazole), Demeclocycline, Docycline, Minocycline, Oxytetracycline, Tetracycline, Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin, Rifampin, Rifabutin, Rifapentin, Stretomycin, Arsphenamine, Choramphenicol, Fosfomycin, Fusidic acid, Linezolid, Metonidazole, Mupirocin, Platensimycin, Quinupristin/Dalfopristin, Rifaximin, Thamphenicol, Tigecycline, Tinidazole.

The second therapeutic agent is a bronchodialator. In certain aspects, the bronchodilator is alternatively a short- or long-acting agent. Exemplary short-acting bronchodilators include, but are not limited to, a β2-agonist or an anticholinergic compound. Exemplary long-acting bronchodilators include, but are not limited to, a β2-agonist or a theophylline compound.

The second therapeutic agent is a leukotriene antagonist, inhibitor, or blocker. Leukotrienes are fatty compounds produced by the immune system that cause the inflammation found in, for example, the upper respiratory tract that results from genetic predisposition (e.g., asthma or allergy), viral infection (e.g., bronchitis or COPD), or lifestyle and environmental factors (e.g., smoking, mining, or exposure to asbestos). Regardless of the cause, leukotriene-mediated inflammation constricts airways. Accordingly, leukotriene inhibitors are often bronchodilators. Common leukotriene inhibitors (or modifiers) either inhibit the 5-lipoxygenase pathway (leukotriene synthase inhibitors) or antagonize cysteinyl-leukotriene type 1 receptors (leukotriene receptor antagonists or LTRAs). Leukotriene inhibitors, modifiers, or antagonists involved in either pathway are contemplated. Specifically, zileuton (Zyflo®) is a commercially-available drug that inhibits 5-lipoxygenase. Montelukast (Singulair®) and zafirlukast (Accolate®) are commercially-available leukotriene inhibitors that block the activity of cysteinyl leukotrienes at the CysLT1 receptor on target cells (e.g., bronchial smooth muscle).

The second therapeutic agent is a steroid. In a preferred embodiment, the steroid is a corticosteroid. Exemplary corticosteroids include, but are not limited to, hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, prednisolone, methylprednisolone, prednisone, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, halcinonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone caproate, fluocortolone pivalate, and fluprednidene acetate.

The second therapeutic agent is an anti-inflammatory agent. Exemplary anti-inflammatory agents include, but are not limited to, antihistamines and histamine receptor blockers.

The second therapeutic agent is oxygen therapy. Oxygen therapy includes, but is not limited to, supplemental oxygen gas. In a preferred embodiment, the arterial blood oxygen saturation of the subject following the oxygen treatment is greater than or equal to 85%. In a more preferred embodiment, the arterial blood oxygen saturation of the subject following the oxygen treatment is greater than or equal to 90%.

The invention further provides a method of immunizing a subject against human rhinovirus (HRV) infection, comprising administering to the subject a composition or pharmaceutical composition described herein.

The invention provides a method of preventing or treating a human rhinovirus infection, comprising administering to a subject a composition or pharmaceutical composition described herein. In certain embodiments of this method, the human rhinovirus infection causes or exacerbates the common cold, nasopharyngitis, croup, pneumonia, bronchiolitis, asthma, chronic obstructive pulmonary disease (COPD), sinusitis, bacterial superinfection, or cystic fibrosis.

The invention provides a method of preventing or treating a human rhinovirus (HRV)-related disease, comprising administering to a subject a composition or pharmaceutical composition described herein. In certain embodiments of this method, the human rhinovirus (HRV)-related disease is the common cold, nasopharyngitis, croup, pneumonia, bronchiolitis, asthma, chronic obstructive pulmonary disease (COPD), sinusitis, bacterial superinfection, or cystic fibrosis.

In certain embodiment of these methods, a subject in need of immunization, prophylaxis, or treatment for HRV-infection includes any individual who comes into frequent, routine, close, and/or direct contact with another individual who is infected with HRV. Moreover, a subject in need of the methods of the invention is an individual who is at an increased risk of infection following exposure to HRV, e.g. premature infants, those infants who do not receive their mother\’s antibodies through breast milk, infants, children, immunocompromised individuals, malnourished individuals, those individuals with inflammatory disease (and, therefore, high cell surface expression ICAM-1, the receptor for HRV), those individuals without acquired immunity to HRV (e.g., no prior exposure to HRV), and those individuals who live in areas of high density (cities), poor nutrition, and/or poor sanitation. Furthermore, a subject having asthma, bacterial infection within the upper respiratory tract or chronic obstructive pulmonary disease (COPD), is particularly susceptible to infection by HRV, because the cells of the respiratory tract in these individuals express ICAM-1 at higher levels. Subjects typically develop COPD following exposure to noxious particles or gases, which most frequently take the form of cigarette smoke. Thus, smokers have an increased risk of infection from HRV following exposure to the virus.

The invention provides a vaccine comprising either an isolated anti-HRV antibody as described herein or the epitope to which an antibody described herein binds.

The invention provides a kit comprising either an isolated anti-HRV antibody as described herein or the epitope to which an antibody described herein binds.

Other features and advantages of the invention will be apparent from and are encompassed by the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the potency of neutralization versus human rhinovirus (HRV) serotypes. The potency of neutralization is as the IC50 value on the Y-axis, which was determined in a microneutralization assay. The cross bar in each serotype indicates the median IC50 value. Data are representative of two independent experiments with similar results.

FIG. 2A-B is a pair of tables indicating the relative breadth of neutralization by TCN-717 (H17), TCN-722 (L22) and TCN-716 (F16) antibodies. The relative breadth of neutralization of each antibody is expressed as the % of virus serotypes neutralized. The 22 viruses in the major group of clade A (right panel, B) are a subset within the 38 viruses shown in the left panel (A). Neutralization by a combination of 2 antibodies is also shown.

FIG. 3 is a graph depicting the neutralization profile of TCN-717 (H17), TCN-722 (L22), and TCN-716 (F16) antibodies among 22 clade A major group serotypes and two clade D serotypes, as determined by microneutralization assay. Asterisks indicate those serotypes that were analyzed in a cytopathic effects (CPE) assay.

FIG. 4A-B is a pair of graphs depicting the direct binding of TCN-717 (H17) (A) and TCN-722 (L22) (B) to inactivated HRV virions in ELISA.

FIG. 5 is a graph depicting binding of TCN-711 (E11) to four HRV serotypes in infected HeLa cells.

DETAILED DESCRIPTION

OF THE INVENTION

Human Rhinoviruses (HRVs) are small, nonenveloped viruses that contain a single-strand RNA genome within an icosahedral capsid. Over 100 serotypes of the virus have been identified (i.e. approximately 101 serotypes). Rhinoviruses belong to the Picornaviridae family.

The primary route of entry for human rhinovirus is the upper respiratory tract, and, specifically, the nasal mucosa, mouth, and eyes. Infection also occurs locally; confined to the upper respiratory tract by the temperature and pH sensitivity of the virus. The optimal temperature for rhinovirus replication is 33-35° C., and, thus, the virus does not efficiently replicate at body temperature. There is usually no gastrointestinal involvement because the virus is unstable in such acidic conditions. Consequently, rhinovirus does not spread from the respiratory tract.

Rhinoviruses travel rapidly throughout the local population because they are transmitted through air, e.g. via contaminated respiratory droplets of sneezes and coughs, via contact with contaminated surfaces, and via person-to-person contact. Infection also occurs rapidly.

The virus adheres to cell surface receptors of cells within the respiratory epithelium within minutes of entering the respiratory tract. The major receptor for the human rhinovirus is intercellular adhesion molecule-1 (ICAM-1). The virus uses the ICAM-1 receptor for both attachment and for uncoating. Because some HRV serotypes are capable of increasing the endogenous expression of ICAM-1 within infected cells, and, therefore, increasing the individual\’s susceptibility to infection, agents that inhibit up-regulation, block translation, increase degradation, or prohibit HRV attachment to ICAM-1 are therapeutic targets. Such targets are contemplated for use in combination with the anti-HRV antibodies of the invention.

Rhinoviruses are positive strand RNA viruses with a naked nucleocapsid. Positive-sense viral RNA is similar to mRNA, and, therefore, the single-stranded RNA genome of HRV can be immediately translated by the host cell. As a further consequence, isolated and purified RNA of HRV can directly cause infection, though it may be less infectious than the whole virus particle. For this reason, isolated and purified HRV RNA is used as an immunogen to develop and screen for anti-HRV antibodies of the invention. Upon infection of a cell, the HRV replicates its own genome, initially using the machinery that is already in place to replicate and/or express genes within the host cell\’s genome. The first proteins made by HRV are enzymes, including RNA-dependent RNA polymerase, which copies the viral RNA to form a double-stranded replicative form, which forms the blueprints for the replication of new virions. The virion is composed of an outer shell, also known as the capsid or nucleocapsid, which is made of protein. The capsid protects the contents of the core, establishes to what kind of cell the virion can attach, and infects that cell. The virion also contains an interior core composed of the genome, a positive, single-stranded RNA molecule encoding the few genes required for viral reproduction which are not present in the host cell. The virus often must supply its own enzymes for initiating replication of its genome.

Symptoms appear in most individuals within days. However, the incubation time can vary from approximately 12 hours to a week depending upon the health of the subject\’s immune system, the subject\’s genetic predisposition, and the HRV serotype. Viral shedding can occur a few days before cold symptoms are recognized by the patient, typically peaks on days 2-7 of the illness, and may last as long as 3-4 weeks. What most people recognize as cold-like symptoms are actually the local inflammatory response to the virus in the respiratory tract, mediated by interferon, which produces nasal discharge, nasal congestion, sneezing, and throat irritation.

The primary HRV infection results in the production of IgA antibodies in nasal secretions and IgG antibodies in the bloodstream. Since these viruses do not enter the circulation, the mucosal IgA response may be the most important for clearing the immediate infection, and may provide immunity for 1-2 year against that particular serotype. However, an broadly neutralizing IgG antibody raised against an invariant epitope of all HRV serotypes could be used as a vaccine or treatment for HRV infection, and, this is the foundation of the present invention.

Although rhinovirus is best known as the primary cause of the common cold, this virus also causes otitis media, nasopharyngitis, croup, bronchiolitis, and pneumonia. The common cold is mild and non-life-threatening in most subjects. However, even a mild respiratory infection can become serious in an infant or young child. Antibodies to viral serotypes develop over time. Because they simply lack the time and experience required to cultivate a mature immune system, the highest incidence of HRV infection is found in infants and young children. Children may also be more contagious than adults because they tend to have higher virus concentrations in their mucosal secretions and experience a longer duration of viral shedding. Thus, infants and young children are at heightened risk for developing, and, ultimately succumbing to, severe rhinoviral infection, e.g. nasopharyngitis, croup, bronchiolitis, and pneumonia.

Individuals who are immune-compromised or malnourished are also at greater risk for developing HRV infection. Immune-compromised individuals may have an underlying medical condition such as acquired immune deficiency syndrome, may be taking medication to suppress their immune system following a transplant, may have an autoimmune condition, or may be undergoing cancer therapy such as radiation. Malnourished individuals may also be immune-compromised, and, therefore, more susceptible to infection by HRV.

Individuals who experience frequent, close, personal contact with others are also at a heightened risk of exposure to HRV, and, therefore, infection. For instance, the individual who rides public transportation versus drives alone to work would be exposed to the virus with increased frequency. Students who attend classes are more susceptible during the school year than when they are on vacation. Individuals who live in cities are more susceptible than those who live in sparsely populated suburbs. For all of these reasons, increased risk of exposure to and infection by HRV often correlates with environmental factors such as poverty and overcrowding.

Rhinovirus infection may not cause inflammatory conditions such as asthma, but it can exacerbate its effects. Similarly, HRV causes an upper respiratory tract, which causes a blockage of one or more of the eustachian tubes, and, ultimately, development of the inflammatory middle ear infection/condition, otitis media. Binding of ICAM-1 by the rhinovirus could mediate intracellular signaling cascades that trigger further inflammation in both of these conditions. Specifically, ICAM-1 signaling could produce a recruitment of inflammatory immune cells such as macrophages and granulocytes to the upper respiratory tract (where it exacerbates asthma), and furthermore, the middle ear (where it could exacerbate otitis media). Thus, treatment of a subject with a composition of the invention (which includes an anti-HRV antibody) not only neutralizes HRV, but also eliminates the HRV-mediated inflammatory response that exacerbates any underlying inflammatory conditions, such as asthma, or any secondary inflammatory condition, such as otitis media.

Rhinovirus infection can also exacerbate cystic fibrosis. Cystic fibrosis (also known as CF or mucoviscidosis) is a recessive genetic disease, which affects the entire body, causing progressive disability until death. Impaired breathing is the most serious and well-recognized symptom. Individuals with CF experience frequent lung infections.

A preceding HRV infection can also cause bacterial superinfection, and, therefore, sinusitis.

The present invention provides a novel method for isolating novel broad and potent neutralizing monoclonal antibodies against HRV. The method involves selection of a PBMC donor with high neutralization titer of antibodies in the plasma. B cells are screened for neutralization activity prior to rescue of antibodies. Novel broadly neutralizing antibodies are obtained by emphasizing neutralization as the initial screen.

Peripheral Blood Mononuclear Cells (PBMCs) were obtained from an HRV-infected donor selected for HRV neutralizing activity in the plasma. Memory B cells were isolated and B cell culture supernatants were subjected to a primary screen of neutralization assay in a high throughput format. Optionally, HRV antigen binding assays using ELISA or like methods were also used as a screen. B cell lysates corresponding to supernatants exhibiting neutralizing activity were selected for rescue of monoclonal antibodies by standard recombinant methods.

In one embodiment, the recombinant rescue of the monoclonal antibodies involves use of a B cell culture system as described in Weitcamp J-H et al., J. Immunol. 171:4680-4688 (2003). Any other method for rescue of single B cells clones known in the art also may be employed such as EBV immortalization of B cells (Traggiai E., et al., Nat. Med. 10(8):871-875 (2004)), electrofusion (Buchacher, A., et al., 1994. AIDS Res. Hum. Retroviruses 10:359-369), and B cell hybridoma (Karpas A. et al., Proc. Natl. Acad. Sci. USA 98:1799-1804 (2001).

In some embodiments, monoclonal antibodies were rescued from the B cell cultures using variable chain gene-specific RT-PCR, and transfectant with combinations of H and L chain clones were screened again for neutralization and HRV antigen binding activities. mAbs with neutralization properties were selected for further characterization.

The antibodies of the invention are able to neutralize HRV.

Monoclonal antibodies can be produced by known procedures, e.g., as described by R. Kennet et al. in “Monoclonal Antibodies and Functional Cell Lines; Progress and Applications”. Plenum Press (New York), 1984. Further materials and methods applied are based on known procedures, e.g., such as described in J. Virol. 67:6642-6647, 1993.

These antibodies can be used as prophylactic or therapeutic agents upon appropriate formulation, or as a diagnostic tool.

A “neutralizing antibody” is one that can neutralize the ability of that pathogen to initiate and/or perpetuate an infection in a host and/or in target cells in vitro. The invention provides a neutralizing monoclonal human antibody, wherein the antibody recognizes an antigen from HRV.

Preferably an antibody according to the invention is a novel monoclonal antibody referred to herein as TCN-711 (6893_E11), TCN-716 (6362_F16), TCN-717 (6358_H17), or TCN-722 (6385_L22). These antibodies were initially isolated from human samples and are produced by the B cell cultures referred to as 6893_E11, 6362_F16, 6358_H17, or 6385_L22. These antibodies have been shown to neutralize HRV in vitro. TCN-711 (6893_E11), TCN-716 (6362_F16), TCN-717 (6358_H17), and TCN-722 (6385_L22) have been shown to have broad, potent HRV neutralizing activity.

The CDRs of the antibody heavy chains are referred to as CDRH1, CDRH2 and CDRH3, respectively. Similarly, the CDRs of the antibody light chains are referred to as CDRL1, CDRL2 and CDRL3, respectively. The positions of the CDR amino acids are defined according to the IMGT numbering system as: CDR1—IMGT positions 27 to 38, CDR2—IMGT positions 56 to 65 and CDR3—IMGT positions 105 to 117. (Lefranc, M P. et al. 2003 IMGT unique numbering for immunoglobulin and T cell receptor variable regions and Ig superfamily V-like domains. Dev Comp Immunol. 27(1):55-77; Lefranc, M P. 1997. Unique database numbering system for immunogenetic analysis. Immunology Today, 18:509; Lefranc, M P. 1999. The IMGT unique numbering for Immunoglobulins, T cell receptors and Ig-like domains. The Immunologist, 7:132-136.)

The sequences of the antibodies were determined, including the sequences of the variable regions of the Gamma heavy and Kappa or Lambda light chains of the antibodies designated TCN-711 (6893_E11), TCN-716 (6362_F16), TCN-717 (6358_H17), and TCN-722 (6385_L22). In addition, the sequence of each of the polynucleotides encoding the antibody sequences was determined. Shown below are the polypeptide and polynucleotide sequences of the gamma heavy chains and kappa light chains, with the signal peptides at the N-terminus (or 5′ end) and the constant regions at the C-terminus (or 3′ end) of the variable regions, which are shown in bolded text.

TCN-711 (6893_E11) gamma heavy chain nucleotide sequence: coding sequence (leader sequence in italics, variable region in bold)

(SEQ ID NO: 1)



ATGAAACACCTGTGGTTCTTCCTCCTCCTGGCGGCAGCTCCCAGATGGGTCCTGTCCCAGGTGC






AGCTACACCAGTGGGGCACAGGAGTGTTGAAGCCTTCGGGGACCCTGTCCCTCACCTGCGGTGT





CTATGGTGGGTCCCTCACTGATTTCTACTGGACCTGGATCCGTCAGTCCCCCGCGAGGGGCCTG





GAGTGGCTTGGAGAAATCGATCGTGATGGGGCCACGTACTATAATCCGTCCCTAAAGAGTCGAA





TCACCATTTCGATAGACACGTCCAAGAAACAATTCTCCTTGAATCTGCGGTCTGTGACCGCCGC





GGACAGGGCTGTCTACTACTGTGCGAGGCGCCCTATGTTACGAGGCGTTTGGGGGAATTTTCGT





TCCAACTGGTTCGACCCCTGGGGCCAGGGAACCCAGGTCACCGTCTCGAGCGCCTCCACCAAGG





GCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGG





CTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACC





AGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGG





TGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAG





CAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCG





TGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACA





CCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCC





TGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGG





GAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGC





TGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAAC





CATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAG





GAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCG





CCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA





CTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGG





AACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCT





CCCTGTCTCCGGGTAAATGA

TCN-711 (6893_E11) gamma heavy chain variable region nucleotide sequence:

(SEQ ID NO: 2)


CAGGTGCAGCTACACCAGTGGGGCACAGGAGTGTTGAAGCCTTCGGG

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The Patent Description & Claims data below is from USPTO Patent Application 20130059878, Treatment of diseases modulated by a h4 receptor agonist.

This application claims the priority of U.S. Ser. No. 61/073,288, filed Jun. 17, 2008 and is a continuation-in-part of U.S. Ser. No. 11/784,992, filed Apr. 9, 2007 (which claims the priorities of U.S. Ser. No. 60/790,490, filed Apr. 7, 2006 and U.S. Ser. No. 60/816,754, filed Jun. 26, 2006); and U.S. Ser. No. 12/069,775, filed Feb. 12, 2008 (which claims the priorities of U.S. Ser. No. 60/889,423, filed Feb. 12, 2007, U.S. Ser. No. 60/892,325, filed Mar. 1, 2007 and U.S. Ser. No. 60/974,685, filed Sep. 24, 2007), the contents of all of which are hereby incorporated by reference, in their entirety, into this application, and from which priority is hereby claimed.

Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

The H4R is the most recently identified and characterized histamine receptor (for reviews, see de Esch J. P., et al., Trends Pharmacol. Sci. 2005, 26(9), 462-469). The receptor is found in the bone marrow and spleen and is expressed on eosinophils; basophils, mast cells (Liu, C., et al., Mol. Pharmacol. 2001, 59(3), 420-426; dendritic cells, and human synovial cells from rheumatoid arthritis patients (Ikawa, Y., et al., Biol. Pham. Bull. 2005, 28(10), 2016-2018). More recently new variants of H4 receptors were described by Richard M. van Rijn, et al. (Biochem. J. (2008) Immediate Publication, doi:10.1042/BJ20071583).

In contrast to the other histamine receptors, H4R has a distinct expression profile on immune and other cells and modulates their function (immune-modulatory role). Such cells include: mast cells, eosinophils, dendritic cells, T cells, monocytes and macrophages and antigen presenting cells in general. It is also present in endothelial and epithelial cells. The H4R appears to play a role in multiple functions of these cells, such as, activation, migration, differentiation, and cytokine and chemokine production. While the H4R has been identified and characterized, its functions and involvement in disease is still under study.

Currently, treatment of histamine related diseases generally focuses on the design of antagonists for H1R and H2R for the treatment of such diseases. For example, for allergies, antagonists of H1R such as loratadine, fexofenadine, diphenyl-hydramine, cetirizine, brompheniramine, cyproheptadine, dexchlorpheniramine, hydroxizine, ketotifen, mequitazine, oxotomide, mizolastine, ebastine, astemizole, carbinoxamide, alimemazine, buclizine, cyclizine hydrochloride, and doxylamine and others were developed. For stomach conditions exacerbated by gastric acid, antagonists of H2R such as cimetidine, ranitidine, famotidine, and nizatidine and others, were developed.

H4R antagonists have been proposed to have therapeutic potential in a number of inflammatory diseases including Inflammatory Bowel Disease (IBD), Systemic Lupus Erythematosus (SLE), atherosclerosis, allergy and asthma and others. (Zhang M, Thurmond R L, Dunford P J, The histamine H(4) receptor: a novel modulator of inflammatory and, immune disorders. Pharmacol Ther. 2007 March; 113(3):594-606).

There are very few examples of compounds, much less of marketed therapeutics, that have H1R, H2R or H3R agonist activity. An example of a compound that has some H1R agonist activity is betahistine, N-methyl-2-pyridin-2-ylethanamine (betahistine), a H3 antagonist low H1 agonist. Betahistine is not a full H1 agonist. It is a potent H3 antagonist with a low H1 agonist activity. Betahistine has a very strong affinity for histamine H3 receptors and a weak affinity for histamine H1 receptors. Betahistine seems to dilate the blood vessels within the middle ear which can relieve pressure from excess fluid and act on the smooth muscle. It is used for the treatment of Meniére syndrome (Review Article. CNS Drugs 2001: 15(11) 855-870). Examples of H3 agonists include: immepip 4-(3H-imidazol-4-ylmethyl)piperidine, imetit S-[2-(4-imidazolyl)ethyl]isothiourea. Immepip and Immetit, although not marketed drugs, are H3 receptor agonists and are currently used in studies in animals with aim to elucidate the H3 receptor function and derive, possible therapeutic utility, for brain disorders. Examples of H2 agonists include: Betazole 2-(2H-Pyrazol-3-yl)ethanamine, impromidine, N-[3-(imidazol-4-yl)-propyl]-N′-{2-[(5-methylimidazol-4-yl)methylthio]ethyl}-guanidine. Betazole and impromidine are histamine H2 agonists, used clinically as diagnostic tools to test gastric secretory function.

All examples of therapeutic molecules presented above do not utilize the H-receptor agonist activity as the major therapeutic function. In the case of Betahistine, the main therapeutic activity may not be predominantly due to the H1 agonist activity, but due to the H3 antagonist activity. The remainder agonist cases have a diagnostic utility or basic research and investigative use.

There is currently no commercially available drug to treat H4R modulated diseases that is a H4R agonist. Thus, there is an unmet and unperceived need to develop H4R agonists to treat H4R modulated diseases.

Anticholinergics

Anticholinergics are used in the treatment of COPD because they widen the airways by relaxing smooth muscle. They do this by blocking acetylcholine receptors. Acetylcholine is a chemical produced by the brain that causes muscle contraction, which in tarn constricts airways. Anticholinergics are considered first-line therapy for COPD.

Examples of anticholinergics include, but are not limited to: tiotropium bromide (Spiriva®) and ipratropium bromide (Atrovent®). Atrovent is the only inhaled anticholinergic agent available in the United States.

Combination Inhalers

Recently, a new product called Advair® was FDA approved for asthma but it may also be beneficial in the treatment of COPD. It combines two medications that have been on the market, salmeterol (a longer acting beta2-agonist) and fluticasone (a steroid). Many patients require both medications to help prevent asthma or COPD symptoms from worsening, but until now were only available as separate inhalers. Advair® cannot be used to quickly relieve asthma or COPD symptoms, it is to be taken on a scheduled basis without regard for the symptoms the patient is having at that particular moment.

Another combination inhaler is Combivent®. It contains two medications: albuterol and ipratropium. Albuterol is an inhaled beta-agonist that works in the lungs to open airways and allow for easier breathing. It does this by stimulating the beta-receptors, which are a certain type of receptor located in the lungs, which help regulate constriction and dilation of the airways. Ipratropium is an anticholinergic used in the treatment of COPD to widen the airways by relaxing and opening air passages to the lungs, making it easier to breathe.

Corticosteroids

Corticosteroids are used to treat many health conditions. This drug class is mainly used for treating asthma, but it has been used for treating COPD. Oral corticosteroids decrease inflammation in the lungs that is associated with COPD. They may take longer to work than inhaled corticosteroids, since they have to travel through the bloodstream before they get to the lungs to work. Corticosteroids are only used in COPD patients who do not respond well to other standard therapies.

Inhaled Beta-2 Agonists

Beta2-agonists work in a manner similar to adrenaline, opening airways and easing breathing. They work by binding with, and thus stimulating, “beta2-receptors” that line the cell walls of the lungs and the bronchioles. The effect of this stimulation is to relax smooth muscles and widen the airways. In COPD, beta2-agonists should be scheduled instead of taken on as needed basis. Possible side effects to the beta2-agonists include shakiness, rapid heartbeat, and upset stomach.

Until recently, all available beta2-agonists were ones that worked quickly but lasted for a relatively short time—about 4-6 hours. Longer-acting beta2-agonists have since been introduced. They cannot be used to quickly relieve symptoms, because there is a delay before they start working. Currently there are two on the market: salmeterol (Serevent®) and formoterol (Foradil®), Longer-acting beta2-agonists are prescribed as maintenance medications which are to be taken on a scheduled basis without regard for the symptoms the patient is having at that particular moment. A short-acting beta2-agonist is best to treat acute symptoms of shortness of breath.

Inhaled Corticosteroids

Corticosteroids suppress the body\’s production of substances that trigger inflammation and reduce the production of substances that maintain inflammation. This drug class is mainly used for treating asthma, but it has been used for treating COPD. Corticosteroids are only used in COPD patients who do not respond well to other standard therapies.

Mucolytics

This class of drugs is used to thin the mucus associated with cough caused by thick mucus. Mucolytics make it easier to clear the mucus, which can be irritating and cause a cough.

Oral Beta-2 Agonists

Oral beta2-agonists works in a similar fashion to inhaled beta2-agonists, but they may take longer to work than the inhaled formulation. Oral beta-agonists must be absorbed in the digestive tract and travel through the circulatory system before they begin working in the lungs, whereas the inhaled formulations go straight to the lungs.

Theophyllines

Theophyllines appear to widen airways by relaxing the smooth muscles surrounding the airways. Theophylline is also used as a long-acting bronchodilator to prevent COPD symptoms. Taken orally as tablets, capsules, or liquids, theophylline is available in immediate-release and controlled-release formulations as well as injection (aminophylline).

Tritoqualine

7-Amino-4,5,6-triethoxy-3-(5,6,7,8-tetrahydro-4-methoxy-6-methyl-1,3-dioxolo[4,5-g]isoquinolin-5-yl)phthalide or Tritoqualine (TRQ) is a drug, currently formulated in 100 mg tablets and sold in pharmacies in Europe for the treatment of allergy.

Tritoqualine is an inhibitor of the enzyme histidine decarboxylase (HDC), which catalyzes histidine decarboxylation in vivo to produce histamine, an endogenous biogenic amine, plus carbon dioxide. Inhibiting histamine production in the body is proposed to ameliorate symptoms of allergy.

Leukotriene Receptor Antagonists

Leukotriene Receptor Antagonists (LRAs), e.g., Montelukast® and Zafirlukast®) have been traditionally used for the treatment of asthma.

SUMMARY

OF THE INVENTION

The invention disclosed herein relates to the surprising discovery that H4R agonists can be used for the treatment of H4R related diseases modulated by H4R. The invention relates generally to the treatment or amelioration of H4R modulated diseases with H4R agonists.

The invention further relates to methods of using H4R agonists, alone, or in combination with one or more other active agents to achieve desirable therapeutic effects for H4R modulated diseases. Agents that can be used in combination with H4R agonists are, for example, other H4R agonists, H1R antagonists (e.g. anti-H1 drug), H2R antagonists (e.g. anti-H2 drug), H3R antagonists (e.g. anti-H3 drug), LRA and NSAIDS.

The invention also relates to methods of providing a plasma concentration of H4R and/or the other compound(s) with a peak-to-trough ratio of less than 3.5, less than 3.0, less than 2.5 or less than 2.0, over a time period spanning from about 1 hour to about 6 hours after administration to a subject.

The invention further relates to pharmaceutical formulations comprising a H4R agonist, one or more of a H1R antagonist, H2R antagonist, H3R antagonist, LRA and NSAID, and a pharmaceutically acceptable carrier.

The invention further provides pharmaceutical formulations comprising therapeutically effective amounts of an H1R antagonist, H2R antagonist, H3R antagonist, LRA and NSAID, and a pharmaceutically acceptable carrier useful to treat a H4R modulated disease.

The active compounds (e.g., H4R agonist, H1R antagonist, H2R antagonist, H3R antagonist, LRA, NSAID) in the pharmaceutical formulation may be combined in a single dosage form or for unit-dose or multi-dose administration. Pharmaceutical carriers suitable for administration of the compounds include any such carriers known to those skilled in the art to be suitable for the particular route of administration and/or suitable for a desired release rate (e.g., immediate or controlled release) of the active compounds. The multiple active ingredients can be formulated into suitable pharmaceutical preparations such as solutions, suspensions, tablets, dispersible tablets, capsules, powders, sustained release, for oral administration or in sterile solutions or suspensions for parenteral administration, as well as transdermal patch preparation, nasal formulation and dry powder inhalers et al. The compositions are formulated into pharmaceutical compositions using techniques and procedures well known in the art.

The pharmaceutical formulations of the invention provide a plasma concentration of the active compounds with a peak-to-trough ratio of less than 3.5, less than 3.0, less than 2.5 or less than 2.0 over a time period spanning from about 1 hour to about 6 hours after administration to a subject.

The invention further relates to treatment or prevention of COPD using drug combinations comprising a H4R agonist, an anti-H1 drug and an anticholinergic drug. The drug combination can further comprise any one or more of existing COPD therapies including but not limited to combination inhaler, corticosteroids, inhaled beta-2 agonists, inhaled corticosteroids, mucolytics, oral beta-2 agonists and theophyllines.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the chemical formula of Tritoqualine (7-Amino-4,5,6-triethoxy-3-(5,6,7,8-tetrahydro-4-methoxy-6-methyl-1,3-dioxolo[4,5-g]isoquinolin-5-yl)phthalide), as described in Example 3, below.

FIG. 2 illustrates the sterical structure of the Tritoqualine diastereomer D1, as described in Example 3, below.

FIG. 3 illustrates the sterical structure of the Tritoqualine diastereomer D2, as described in Example 3, below.

FIG. 4 shows a chromatogram of the separation of Tritoqualine stereoisomers via a chiral column. In the bottom part, the UV absorbance at 190 nm has been detected, while the top part depicts polarimetric detection at an averaged absorption in the range of 200-800 nm, as described in Example 3, below.

FIG. 5 shows a UV spectrum of each of the peaks of FIG. 4, as described in Example 3, below.

FIG. 6 illustrates the 3D-structures of the two stereoisomers (enantiomers) of FIGS. 4 and 5 as determined by X-Ray crystallography, as described in Example 3, below.

FIG. 7 shows the % improvement for each patient from the visit T0 to visit T1 as described in Example 12. Statistical analysis using a paired t-test on the values generated on visits T0 and T1 showed a statistically significant improvement due to Tritoqualine treatment. P-value for the paired t-test P-0.0012 calculated using GraphPad Prism®, as described in Example 3, below.

FIG. 8 shows the results of a cell proliferation assay using human TF1 cells that express H4R. When 10 μM TRQ and 10 μM CB were compared in three separate assays (n=3), TRQ was found to be superior, more potent inhibitor of cell proliferation than CB (approximately double the potency of CB). Average inhibition of cell proliferation by CB and TRQ was found to be 23% and 49%, respectively, as described in Example 13.

FIG. 9 shows a comparison between TRQ alone and, in association with Clobenpropit (TRQ+CB) on the inhibition of human TF1 cell proliferation assay; both TRQ and CB were used each, at 10 μM concentration. TRQ and Clobenpropit in 50:50-mixture produced a more potent inhibition of cell proliferation compared to Tritoqualine alone. This suggests that TRQ and CB demonstrate an additive effect on the inhibition of cell proliferation on TF1 cells, as described in Example 14.

FIG. 10 shows the effects of TRQ or CB on a cell assay using mouse medullary cells C57B1/6 expressing H4R. CB inhibits the proliferation of CFC at a dose of 10−5M (10 μM) and was compared to TRQ at the dose of 10−5M (10 μM). TRQ is a more potent inhibitor of cell proliferation than Clobenpropit, as described in Example 16.

FIG. 11 shows the effects of E2 enantiomer on a cell assay using mouse medullary cells C57B1/6 expressing H4R. Approximately 60% of haematopoietic progenitor cells are blocked by E2 enantiomer of Tritoqualine. This G0/G1 blocking of the cell cycle is reversed if a known H4R antagonist such as JNJ7777120 is used, suggesting that the inhibition of cell proliferation is due to activation of the H4R, as described in Example 15.

FIG. 12 shows the effects of E1 enantiomer on a cell assay using mouse medullary cells C57B1/6 expressing H4R. Approximately 48% of haematopoietic progenitor cells are blocked by the E1 enantiomer of Tritoqualine. This G0/G1 blocking of the cell cycle is reversed if a known H4R antagonist such as JNJ7777120 is used suggesting that the inhibition of cell proliferation is due to activation of the H4R, as described in Example 15.

FIG. 13 is a graph showing that enantiomer E2 is a more effective inhibitor of cell growth than TRQ and E1 (approximately 50% inhibition of G0/G1 phase was observed), whereas E2 inhibited G0/G1 by 57%, as described in Example 17.

FIG. 14 is a bar graph showing that tritoqualine (TRQ) is an 1-14 agonist. CB-5 is clobenpropit at 1×10−5 M (10 uM); AT-5 is a mixture of tritoqualine isomer 1 (also referred to herein as E1 or D1) and 2 (also referred to herein as E2 or D2) at 1×10−5 M (10 uM); AT-6 is a mixture of tritoqualine isomer 1 and 2 at 1×10−6 M (1 uM); E1-5 is tritoqualine isomer 1 at 1×10−5 M (10 uM); E2-5 is tritoqualine isomer 2 at 1×10−5 M (10 uM).

FIG. 15A is a scatter plot and FIG. 15B is a bar graph both showing that TRQ inhibits progenitor cell cycling. CB-5 is clobenpropit-5; AT-5 is a mixture of tritoqualine isomer 1 and 2 at 1×10−5M (10 uM); AT-6 is a mixture of tritoqualine isomer 1 and 2 at 1×10−6M (1 uM); E1-5 is tritoqualine isomer 1 at 1×10−5 M (10 uM); E1-6 is tritoqualine isomer 1 at 1×10−6 M (1 uM); E2-5 is tritoqualine isomer 2 at 1×10−5M (10 uM); E2-6 is tritoqualine isomer 2 at 1×10−6M (1 uM).

FIG. 16 is a bar graph that shows that TRQ inhibits ckit positive hematopoietic progenitor cell proliferation. The reference to CB-5, E1-5, and E2-5 is the same as FIG. 15.

FIG. 17 includes three bar graphs (FIGS. 17A, B and C) showing that TRQ promotes cells to enter and remain in the G0 and/or G1 phase of the cell cycle.

FIG. 18 is a bar graph that shows that pertussis toxin (PTX) reverses TRQ\’s effects by decreasing number of ckit positive bone marrow cells in the G0 and/or G1 phase of the cell cycle.

FIG. 19 is a bar graph that shows that TRQ maintains the hematopoietic progenitor cells in the G0 and/or G1 phase of the cell cycle so that even in the presence of Ara-C the cell CFU remains high.

FIG. 20 includes two bar graphs (FIGS. 20A and B) that shows that TRQ is an H4 agonist but does not affect organic cation transporter-3 (OCT-3) or serotonin transporter (SERT).

DETAILED DESCRIPTION

OF THE INVENTION
Definitions

As used herein “allergy” is an abnormal or altered immunologic reaction induced by an allergen in a subject who suffers from hypersensitivity to that allergen including antibody-antigen reactions that include immediate type hypersensitivity reactions such that when IgE molecules are crosslinked with an allergen(s), mast cells and basophils release mediators such as histamines. Examples of allergy symptoms include sinusitis, rhinitis, hives, headaches, post-nasal drip, coughing, sneezing, respiratory difficulties, sore throats, allergic asthma, allergic conjunctivitis, allergic rhinitis, tightness in throat and chest, and loss of voice.

For purposes of the present invention the term “controlled release” refers to a pharmaceutical dosage form which releases one or more active pharmaceutical agents over a prolonged period of time, e.g. over a period of more than 1 hour. Controlled release (CR) components can also be referred to as sustained release (BR), prolonged release (PR), or extended release (ER). When used in association with the dissolution profiles discussed herein, the term “controlled release” refers to that portion of a dosage form made according to the present invention which delivers the compositions of the invention over a period of time e.g. greater than 1 hour. The term “modified release” and “controlled release” is used interchangeably herein.

The term “immediate release” refers to a dosage form which releases the compositions of the invention substantially immediately upon contact with gastric juices and will result in substantially complete dissolution within about 1 hour. Immediate release (IR) components can also be referred to as instant release. When used in association with the dissolution profiles discussed herein, the term “immediate release” refers to that portion of a dosage form made according to the present invention which delivers the compositions of the invention over a period of time less than 1 hour.

The term “derivative” means a chemically modified compound wherein the modification is considered routine by the ordinary skilled chemist, such as an ester or an amide of an acid, protecting groups, such as a benzyl group for an alcohol or thiol, and tert-butoxycarbonyl group for an amine.

The term “effective amount” means an amount of a compound or composition according to the present invention effective in producing the desired therapeutic effect.

The term “analog” means a compound which comprises a chemically modified form of a specific compound or class thereof, and which maintains the pharmaceutical and/or pharmacological activities characteristic of said compound.

As used herein, “pharmaceutically acceptable salts” refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluensulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like.

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions; and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.

The term “about” when used in connection with percentages means±1-5%.

The term “H4R agonists” means any molecule that is an agonist to the H4 receptor. Examples of H4 agonists include, but are not limited to, Tritoqualine (TRQ) or derivative thereof (e.g. racemic versions thereof), Tritoqualine isomers (e.g., E1 and E2 enantiomers), 4-methylhistmime (4MeHA) and clobenprobit (CB).

The term “anti-H1” refers to any drag that is an antagonist to the H1 receptor. Examples of H1R antagonists include, but are not limited to, brompheniramine, cetirizine, fexofenadine, cyproheptadine, dexchlorpheniramine, hydroxyzine, ketotifen, loratadine, mequitazine, oxotomide, mizolastine, ebastine, astemizole, carbinoxamide, alimemazine, buclizine, cyclizine hydrochloride, doxylamine, mepyramine, antazoline, diphenhydramine, carbinoxamine, clemastine, dimenhydrinate, pheniramine, chlorphenamine, triprolidine, chlorcyclizine, hydroxyzine, meclizine, promethazine, and azatadine.

The term “anti-H2” refers to any drug that is an antagonist to the H2 receptor. Examples of H2R antagonists include, but are not limited to, ranitidine, cimetidine, famotidine, and nizatidine.

The term “anti-H3” refers to any drug that is an antagonist to the H3 receptor. Examples of H3R antagonists include, but are not limited to, betahistine (N-methyl-2-pyridin-2-ylethanamine), ABT-239 (4-(2-{2-[(2R)-2-Methylpyrrolidin-1-yl]ethyl}-benzofuran-5-yl)benzonitrile), Cipralisant(1R,2R)-4-(2-(5,5-dimethylhex-1-ynyl)cyclopropyl)imidazole, Ciproxifan cyclopropyl 4-(3-(1H-imidazol-4-yl)propyloxy)phenyl ketone, Clobenpropit N′-[(4-chlorophenyl)methyl]-1-[3-(3H-imidazol-4-yl)propylthio]formamidine, Thioperamide N-Cyclohexyl-4-(1H-imidazol-4-yl)-1-piperidinecarbothioamide.

The term “anti-H4” refers to any drag that is an antagonist to the H4 receptor. Examples of H4R antagonists include, but are not limited to, thioperamide, JNJ7777120 and JNJ10191584 (Zhang et al Pharmacology and therapeutics, 113 (2007) 594), N-Cyclohexyl-4-(1H-imidazol-4-yl)-1-piperidinecarbothioamide, R-α-methylhistamine. A number of other H4R antagonists are described in a review by Venable et al, (Anti Inflamm. Anti Allergy Agents Med. Chem, 2006, 5 307). None of the antagonists described in the literature are currently used as therapeutic agents.

The term H4R modulated disease includes, but is not limited to immune system diseases, and gastroinstestinal conditions ameliorated by proper histamine management. Examples include GERD; food allergies; Zollinger-Ellison Syndrome; peptic ulcer; dyspepsia; allergic eosinophilic gastroenteritis; mastocytosis with gastrointestinal symptoms; diseases provoked by CD4 Th2 lymphocytes (asthma, rhinitis conjunctivitis eczema, gastro-esophageal reflux disease and other gastric diseases exacerbated by gastric acid), CD4 Th1 lymphocytes (IBD, Crohn\’s disease, ulcerative colitis and celiac disease and autoimmune gastritis, chronic bronchitis etc.) and/or CD4 Th17 lymphocytes (Uveitis and inflammatory digestive diseases); Chronic Obstructive Pulmonary Disorder (COPD) and other pulmonary types of inflammation including asthma; disease conditions in cancer; and other diseases where histamine or the H4R is involved. H4R agonists may also be used in vaccines as adjuvant to improve immune responses to the inoculated antigens.

The gastrointestinal disease condition may be any of GERD, a food allergy, Zollinger-Ellison Syndrome, peptic ulcer, dyspepsia, allergic eosinophilic gastroenteritis, and mastocytosis with gastrointestinal symptoms.

The term “desirable therapeutic effect” means to treat a subject with the active agents of the invention in order to prevent or ameliorate a disease and/or disease condition.

The terms “therapeutic agent”, “drug” and “active agent” are used interchangeably herein and refer to the compounds disclosed herein that affect a H4R modulated disease and/or disease condition. For example, active agents of the invention include, but are not limited to, H4R agonists, H1R antagonists, H2R antagonists, H3R antagonists, LRAs, steroids, NSAIDs.

The terms “pharmaceutical formulations”, “pharmaceutical compositions” and “dosage forms” are used interchangeably herein and refer to a composition containing the active ingredient(s) of the invention in a form suitable for administration to a subject.

The term “anticholinergics” or “anticholinergic drugs” refer to compounds that block acetylcholine receptors. Acetylcholine is a chemical produced by the brain that causes muscle contraction, which in turn can constrict airways. Anticholinergics can be use for treatment of COPD.

The term “peak-to-trough ratio” refers to a comparison of the values for a peak (e.g., a high point) plasma level and a trough (e.g., a low point) plasma level of an active agent over a set amount of time. For example, a line graph with plasma levels of a drug with values ranging from 400 ng/ml (peak) to 200 ng/ml (trough) over a four hour period, gives a peak-to-trough ratio of 2 for that time. More than one peak-to-trough ratio can be illustrated in a graph.

METHODS OF THE INVENTION

The invention relates to the use of a Tritoqualine (TRQ) (including derivatives or isomers thereof (e.g. Tritoqualine isomers (E1 and E2)) as histamine H4 receptor (H4R) agonists. The invention further relates to the use of H4R agonists to treat diseases and/or disease conditions modulated by H4R agonists.

The invention relates generally to the treatment or amelioration of H4R modulated diseases or conditions with one or more compositions of the invention (e.g., H4R agonists).

In one embodiment, the invention provides methods for the treatment or amelioration of H4R modulated diseases and/or conditions in a subject, comprising administering to the subject an effective amount of a H4R agonist. In a further embodiment, the H4R agonist may not inhibit (or at least not significantly inhibit) Organic Cation Transporter-3 (OCT-3) and/or Serotonin Transporter (SERT).

The invention further provides methods for the treatment or amelioration of H4R modulated diseases or conditions in a subject, comprising administering to the subject an effective amount of a H4R agonist, in combination with one, or more other compounds to reach desirable therapeutic effects for H4R modulated diseases. Compounds that can be used in combination with H4R agonists are, for example, other H4R agonists, H1R antagonists (e.g. anti-H1 compounds or drugs), H2R antagonists (e.g. anti-H2 compounds or drugs), H3R antagonists (e.g. anti-H3 compounds or drugs), LRAs and NSAIDs. The method of the invention provides the administration of a single or a combination of H4R agonist drugs.

In one embodiment, the subject is given an effective amount of a H4R agonist(s) and an anti-H1 drug(s). In another embodiment, the subject is given an effective amount of a H4R agonist(s) and an anti-H2 drug(s). In another embodiment, the subject is given an effective amount of a H4R agonists) and an anti-H3 drug(s). In yet another embodiment, the subject is given an effective amount of a H4R agonist(s) and one or more of an anti-H1 drug, an anti-H2 drug and an anti-H3 drug. In a further embodiment, the subject may be given an effective amount of a combination of a H4R agonist(s), and any one or more of an anti-H1 drug, an anti-H2 drug and an anti-H3 drug, a LRA, a steroid and NSAID.

The H4R modulated disease and/or condition may be any of GERD, a food allergy, Zollinger-Ellison Syndrome, peptic ulcer, dyspepsia, allergic eosinophilic gastroenteritis, and mastocytosis with gastrointestinal symptoms, diseases provoked by CD4 Th2 lymphocytes (e.g., asthma, rhinitis conjunctivitis eczema, gastro-esophageal reflux disease and other gastric diseases exacerbated by gastric acid), diseases provoked CD4 Th1 lymphocytes (e.g., IBD, Crohn\’s disease, ulcerative colitis and celiac disease and autoimmune gastritis, chronic bronchitis etc.), diseases provoked CD4 Th17 lymphocytes (e.g., uveitis and inflammatory digestive diseases); Chronic Obstructive Pulmonary Disorder (COPD) and other pulmonary types of inflammation including asthma; disease conditions in cancer; and other diseases where histamine or the H4R is involved. H4R agonists may also be used in vaccines as adjuvant to improve immune responses to inoculated antigens. In a preferred embodiment, the H4R modulated disease or condition is COPD and/or asthma. To treat COPD, a preferred pharmaceutical composition comprises a tritoqualine and an anti-H4 drug such as loratadine. To treat GERD, a preferred pharmaceutical composition comprises a tritoqualine and an anti-H2 drag such as ranitidine. To treat food allergy, a preferred pharmaceutical composition comprises a tritoqualine and an antileukotriene such as Montelukast®. However other compositions of the invention may be used.

The invention also relates to methods of providing a plasma concentration of H4R agonist and/or the other active agent(s) with specific peak-to-trough ratios in a subject. In one embodiment, the peak-to-trough ratio is less than 3.5, over a time period spanning from about 1 hour to about 6, hours after administration to a subject. In another embodiment, the peak-to-trough ratio is less than 3.0, over a time period spanning from about 1 hour to about 6 hours, after administration to a subject. In yet another embodiment, the peak-to-trough ratio is less than 2.5, over a time period spanning from about 1 hour to about 6 hours, after administration, to a subject in a further embodiment, the peak-to-trough ratio is less than 2.0 over a time period spanning from about 1 hour to about 6 hours after administration to a subject.

A dose of a H4R agonist (e.g., Tritoqualine, or an isomer or derivative thereof) administered to a subject may be about 200 mg/day. In another embodiment, the dose of a H4R agonist administered to a subject may be about 1 g/day. In an additional embodiment, the dose of a H4R agonist administered to a subject may be about 2 g/day. In yet another embodiment, the dose of a H4R agonist administered to a subject may be about 3 g/day. In a further embodiment, the dose of a H4R agonist administered to a subject may be about 1-5 mg/day, about 5-10 mg/day, about 10-15 mg/day, about 15-20 mg/day, about 20-25 mg/day, about 25-30 mg/day, about 30-35 mg/day, about 35-40 mg/day, about 40-45 mg/day, about 45-50 mg/day, about 50-55 mg/day, about 55-60 mg/day, about 60-65 mg/day, about 65-70 mg/day, about 70-75 mg/day, about 75-80 mg/day, about 80-85 mg/day, about 85-90 mg/day, about 90-95 mg/day, about 95-100 mg day, about 100-105 mg/day, about 105-110 mg/day, about 110-115 mg/day, about 115-120 mg/day, about 120425 mg/clay, about 125-130 mg/day, about 130-135 mg/day, about 135-140 mg/day, about 140-145 mg/day, about 145-150 mg/day, about 150-155 mg/day, about 155-160 mg/day, about 160-165 mg/day, about 165-170 mg/day, about 170-175 mg/day, about 175-180 mg/day, about 180-185 mg/day, about 185-190 mg/day, about 190-195 mg/day, about 195-200 mg/day, about 200-205 mg/day, about 205-210 mg/day, about 210-215 mg/day, about 215-220 mg/day, about 220-225 mg/day, about 225-230 mg/day, about 230-235 mg/day, about 235-240 mg/day, about 240-245 mg/day, about 245-250 mg/day, about 250-255 mg/day, about 255-260 mg/day, about 260-265 mg/day, about 265-270 mg/day, about 270-275 mg/day, about 275-280 mg/day, about 280-285 mg/day, about 285-290 mg/day, about 290-295 mg/day, about 295-300 mg/day, about 300-305 mg/day, about 305-310 mg/day, about 310-315 mg/day, about 315-320 mg/day, about 320-325 mg/day, about 325-330 mg/day, about 330-335 mg/day, about 335-340 mg/day, about 340-345 mg/day, about 345-350 mg/day, about 350-355 mg/day, about 355-360 mg/day, about 360-365 mg/day, about 365-370 mg/day, about 370-375 mg/day, about 375-380 mg/day, about 380-385 mg/day, about 385-390 mg/day, about 390-395 mg/day, about 395-400 mg/day, about 400-405 mg/day, about 405-410 mg/day, about 410-415 mg/day, about 415-420 mg/day, about 420-425 mg/day, about 425-430 mg/day, about 430-435 mg/day, about 435-440 mg/day, about 440-445 mg/day, about 445-450 mg/day, about 1 mg/day-1 g/day, about 1 mg/day-2 g/day or about 1 mg/day-3 g/day.

Suitable examples of H4R agonists include, but are not limited to, any of Tritoqualine includes an isomer or derivative thereof, 4-methylhistmime (4MeHA) and clobenprobit (CB).

Tritoqualine maybe used successfully alone and in combinatorial therapy to treat allergy, GERD, food allergy, and COPD. The drug has two chiral centers and therefore, it exists in four isomeric forms. The commercial product includes a mixture of two isomers, one with the chiral centers at the SS configuration and the other one at the RR configuration. Examples of Tritoqualine isomers include, but are not limited to, E1 and E2 enantiomers.

Surprisingly, Tritoqualine and each of its isomers may have different activity as an H4R agonist i.e., various H4R agonists will have varying effects on. H4R. Thus, binding of various H4R agonists to the H4R can provide varying therapeutic profiles (e.g., cytokine activation profiles) in a subject.

In accordance with the practice of the invention, suitable examples of Leukotriene Receptor Antagonists (LRA) include, but are not limited to, Montelukast® (Singulair®), Pranlukast® and Zafirlukas®. The method provides the administration of a single or a combination of LRA drugs.

The dose of LRAs (e.g. Montelukast® (Singulair®)) administered to a subject may be about 10.0 mg/day. In another embodiment, the dose of a LRA(s) (e.g. Montelukast®) administered to a subject may be about 0.1 to 1.0 mg/day, about 1.0 to 2.0 mg/day, about 2.0 to 3.0 mg/day, about 3.0 to 4.0 mg/day, about 4.0 to 5.0 mg/day, about 5.0 to 6.0 mg/day, about 6.0 to 7.0 mg/day, about 7.0 to 8.0 mg/day, about 8.0 to 9.0 mg/day, about 9.0 to 10.0 mg/day, about 10.0 to 11.0 mg/day, about 11.0 to 12.0 mg/day, about 12.0 to 13.0 mg/day, about 13.0 to 14.0 mg/day, about 14.0 to 15.0 mg/day, about 15.0 to 16.0 mg/day, about 16.0 to 17.0 mg/day, about 17.0 to 18.0 mg/day, about 18.0 to 19.0 mg/day, about 19.0 to 20.0 mg/day, about 20.0 to 21.0 mg/day, about 21.0 to 22.0 mg/day, about 22.0 to 23.0 mg/day, about 23.0 to 24.0 mg/day, about 24.0 to 25.0 mg/day, about 25.0 to 26.0 mg/day, about 26.0 to 27.0 mg/day, about 27.0 to 28.0 mg/day, about 28.0 to 29.0 mg/day, about 29.0 to 30.0 mg/day, about 30.0 to 31.0 mg/day, about 31.0 to 32.0 mg/day, about 32.0 to 33.0 mg/day, about 33.0 to 34.0 mg/day, about 34.0 to 35.0 mg/day or about 1 mg/day to 35 mg/day.

In accordance with the practice of the invention, suitable examples of anti-H1 drugs includes, but are limited to, any of brompheniramine, cetirizine, fexofenadine, cyproheptadine, dexchlorpheniramine, hydroxyzine, ketotifen, loratadine, mequitazine, oxotomide, mizolastine, ebastine, astemizole, carbinoxamide, alimemazine, buclizine, cyclizine hydrochloride, doxylamine, mepyramine, antazoline, diphenhydramine, carbinoxamine, clemastine, dimenhydrinate, pheniramine, chlorphenamine, triprolidine, chlorcyclizine, hydroxyzine, meclizine, promethazine, and azatadine or analogs, equivalents, isomers, pharmaceutically acceptable salts, and solvate fauns thereof. The method provides the administration of a single or a combination of anti-H1 drugs.

The dose of an anti-H1 drug (e.g., loratadine) administered to a subject may be about 10.0 mg/day. In another embodiment, the dose of the anti-H1 drag(s) (e.g. loratadine) administered to a subject may be about 0.1 to 1.0 mg/day, about 1.0 to 2.0 mg/day, about 2.0 to 3.0 mg/day, about 3.0 to 4.0 mg/day, about 4.0 to 5.0 mg/day, about 5.0 to 6.0 mg/day, about 6.0 to 7.0 mg/day, about 7.0 to 8.0 mg/day, about 8.0 to 9.0 mg/day, about 9.0 to 10.0 mg/day, about 10.0 to 11.0 mg/day, about 11.0 to 12.0 mg/day, about 12.0 to 13.0 mg/day, about 13.0 to 14.0 mg/day, about 14.0 to 15.0 mg/day, about 15.0 to 16.0 mg/day, about 16.0 to 17.0 mg/day, about 17.0 to 18.0 mg/day, about 18.0 to 19.0 mg/day, about 19.0 to 20.0 mg/day, about 20.0 to 21.0 mg/day, about 21.0 to 22.0 mg/day, about 22.0 to 23.0 mg/day, about 23.0 to 24.0 mg/day, about 24.0 to 25.0 mg/day, about 25.0 to 26.0 mg/day, about 26.0 to 27.0 mg/day, about 27.0 to 28.0 mg/day, about 28.0 to 29.0 mg/day, about 29.0 to 30.0 mg/day, about 30.0 to 31.0 mg/clay, about 31.0 to 32.0 mg/day, about 32.0 to 33.0 mg/day, about 33.0 to 34.0 mg/day, about 34.0 to 35.0 mg/day or about 1 mg/day to about 35 mg/day.

Also, the dose of an anti-H1 drug (e.g., cetirizine) administered to a subject may be about 10 mg/day. In another embodiment, the dose of the anti-H1 drug(s) (e.g. cetirizine) administered to a subject may be about 0.1 to 0.5 mg/day, about 0.5 to 1.0 mg/day, about 1.0 to 1.5 mg/day, about 1.5 to 2.0 mg/day, about 2.0 to 2.5 mg/day, about 2.5 to 3.0 mg/day, about 3.0 to 3.5 mg/day, about 3.5 to 4.0 mg/day, about 4.0 to 4.5 mg/day, about 4.5 to 5.0 mg/day, about 5.0 to 5.5 mg/day, about 5.5 to 6.0 mg/day, about 6.0 to 6.5 mg/day, about 6.5 to 7.0 mg/day, about 7.0 to 7.5 mg/day, about 7.5 to 8.0 mg/day, about 8.5 to 9.0 mg/day, about 9.0 to 9.5 mg/day, about 9.5 to 10.0 mg/day, about 10.5 to 11.0 mg/day, about 11.0 to 11.5 mg/day, about 11.5 to 12.0 mg/day, about 12.0 to 12.5 mg/day, about 12.5 to 13.0 mg/day, about 13.0 to 13.5 mg/day, about 13.5 to 14.0 mg/day, about 14.0 to 14.5 mg/day, about 14.5 to 15.0 mg/day, about 15.0 to 15.5 mg/day, about 15.5 to 16.0 mg/day, about 16.0 to 16.5 mg/day, about 16.5 to 17.0 mg/day, about 17.0 to 17.5 mg/day, about 17.5 to 18.0 mg/day, about 18.5 to 19.0 mg/day, about 19.0 to 19.5 mg/day, about 19.5 to 20.0 mg/day, about 20.5 to 21.0 mg/day, about 21.0 to 21.5 mg/day, about 21.5 to 22.0 mg/day, about 22.0 to 22.5 mg/day, about 22.5 to 23.0 mg/day, about 23.0 to 23.5 mg/day, about 23.5 to 24.0 mg/day, about 24.0 to 24.5 mg/day, about 24.5 to 25.0 mg/day, about 25.0 to 25.5 mg/day, about 25.5 to 26.0 mg/day, about 26.0 to 26.5 mg/day, about 26.5 to 27.0 mg/day, about 27.0 to 27.5 mg/day, about 27.5 to 28.0 mg/day, about 28.5 to 29.0 mg/day, about 29.0 to 29.5 mg/day, about 29.5 to 30.0 mg/day or about 0.5 mg/day to 30.0 mg/day.

Further, the dose of an anti-H1 drug (e.g., fexofenadine) administered to a subject may be about 120.0 mg/day. In another embodiment, the dose of the anti-H1 drug(s) (e.g. fexofenadine) administered to a subject may be about 1.0 to 30.0 mg/day, about 30.0 to 50.0 mg/day, about 50.0 to about 70.0 mg/day, about 70.0 to 90.0 mg/day, about 90.0 to 110.0 mg/day, about 110.0 to 130.0 mg/day, about 130.0 to 150.0 mg/day, about 150.0 to 170.0 mg/day, about 170.0 to 190.0 mg/day, about 190.0 to 210.0 mg/day, about 210.0 to 230.0 mg/day, about 230.0 to 250.0 mg/day, about 250.0 to 270.0 mg/day, about 270.0 to 290.0 mg/day, about 290.0 to 310.0 mg/day, about 310.0 to 330.0 mg/day, about 330.0 to 350.0 mg/day, about 350.0 to 370.0 mg/day, about 370.0 to 390.0 mg/day, about 390.0 to 410.0 mg/day, about 410.0 to 430.0 mg/day, about 430.0 to 450.0 mg/day, about 450.0 to 470.0 mg/day, about 470.0 to 490.0 mg/day, about 490.0 to 510.0 mg/day or about 1 mg/day to 510 mg/day.

Suitable examples of anti-H2 drugs include, but are not limited to, any of ranitidine, cimetidine, famotidine, and nizatidine and/or analogs, equivalents, isomers, pharmaceutically acceptable salts, and solvate forms thereof. The method provides the administration of a single or combination of anti-H2 drags.

The dose of an anti-H2 drug (e.g., ranitidine) administered to a subject may be about 150 mg/day. In another embodiment, the dose of the anti-H2 drug(s) (e.g. ranitidine) administered to a subject may be about 1.0 to 20.0 mg/ml, about 20.0 to 40.0 mg/ml, about 40.0 to 60.0 mg/ml, about 60.0 to 80.0 mg/ml, about 80.0 to 100.0 mg/ml, about 100.0 to 120.0 mg/ml, about 120.0 to 140.0 mg/ml, about 140.0 to 160.0 mg/ml, about 160.0 to 180.0 mg/ml, about 180.0 to 200.0 mg/ml, about 200.0 to 220.0 mg/ml, about 220.0 to 240.0 mg/ml, about 240.0 to 260.0 mg/ml, about 260.0 to 280.0 mg/ml, about 280.0 to 300.0 mg/ml, about 300.0 to 320.0 mg/ml, about 320.0 to 340.0 mg/ml, about 340.0 to 360.0 mg/ml, about 360.0 to 380.0 mg/ml, about 380.0 to 400.0 mg/ml, about 400.0 to 420.0 mg/ml, about 420.0 to 440.0 mg/ml, about 440.0 to 460.0 mg/ml, about 460.0 to 480.0 mg/ml, about 480.0 to 500.0 mg/ml, about 500.0 to 520.0 mg/ml, about 520.0 to 540.0 mg/ml, about 540.0 to 560.0 mg/ml, about 560.0 to 580.0 mg/ml, about 580.0 to 600.0 mg/ml, about 600.0 to 620.0 mg/ml, about 620.0 to 640.0 mg/ml, about 640.0 to 660.0 mg/ml, about 660.0 to 680.0 mg/ml, about 680.0 to 700.0 mg/ml, about 700.0 to 720.0 mg/ml, about 720.0 to 740.0 mg/ml, about 740.0 to 760.0 mg/ml, about 760.0 to 780.0 mg/ml, about 780.0 to 800.0 mg/ml, about 800.0 to 820.0 mg/ml, about 820.0 to 840.0 mg/ml, about 840.0 to 860.0 mg/ml, about 860.0 to 880.0 mg/ml, about 880.0 to 900.0 mg/ml or about 1 mg/ml to about 900 mg/ml.

Suitable examples of anti-H3 drugs include, but are not limited to, any of betahistine (N-methyl-2-pyridin-2-ylethanamine), ABT-239 (4-(2-{2-[(2R)-2-Methylpyrrolidin-1-yl]ethyl}-benzofuran-5-yl)benzonitrile), Cipralisant(1R,2R)-4-(2-(5,5-dimethylhex-1-ynyl)cyclopropyl)imidazole, Ciproxifan cyclopropyl 4-(3-(1H-imidazol-4-yl)propyloxy)phenyl ketone, Clobenpropit N′-[(4-chlorophenyl)methyl]-1-[3-(3H-imidazol-4-yl)propylthio]formamidine, Thioperamide N-Cyclohexyl-4-(1H-imidazol-4-yl)-1-piperidinecarbothioamide. The method provides the administration of a single or combination of anti-H3 drugs.

The dose of an anti-H3 drug administered to a subject may be about 0.1 to 1.0 mg/day, about 1.0 to 2.0 mg/day, about 2.0 to 3.0 mg/day, about 3.0 to 4.0 mg/day, about 4.0 to 5.0 mg/day, about 5.0 to 6.0 mg/day, about 6.0 to 7.0 mg/day, about 7.0 to 8.0 mg/day, about 8.0 to 9.0 mg/day, about 9.0 to 10.0 mg/day, about 10.0 to 15.0 mg/day, about 15.0 to 20.0 mg/day, about 20.0 to 25.0 mg/day, about 25.0 to 30.0 mg/day, about 30.0 to 35.0 mg/day, about 35.0 to 40.0 mg/day, about 45.0 to 50.0 mg/day, about 50.0 to 60.0 mg/day, about 60.0 to 70.0 mg/day, about 70.0 to 80.0 mg/day, about 80.0 to 90.0 mg/day, about 90.0 to 100.0 mg/day, about 100.0 to 150.0 mg/day, about 150.0 to 200.0 mg/day, about 200.0 to 250.0 mg/day, about 250.0 to 300.0 mg/day, about 300.0 to 350.0 mg/day, about 350.0 to 400.0 mg/day, about 400.0 to 450.0 mg/day, about 450.0 to 500.0 mg/day, about 500.0 to 550.0 mg/day, about 550.0 to 600.0 mg/day, about 600.0 to 650.0 mg/day, about 650.0 to 700.0 mg/clay, about 700.0 to 750.0 mg/day, about 750.0 to 800.0 mg/day, about 800.0 to 850.0 mg/day, about 850.0 to 900.0 mg/day, about 900.0 to 950.0 mg/day, about 950.0 to 1000.0 mg/day, about 1000.0 to 1100.0 mg/day, about 1100.0 to 1200.0 mg/day, about 1200.0 to 1300.0 mg/day, about 1300.0 to 1400.0 mg/day, about 1400.0 to 1500.0 mg/day, about 1500.0 to 1600.0 mg/day, about 1600.0 to 1700.0 mg/day, about 1800.0 to 1900.0 mg/day or about 1900.0 to 2000.0 mg/day.

The invention further relates to the treatment or prevention of COPD using drug combinations comprising a H4R agonist, an anti-H1 drug and an anticholinergic drug.

In one embodiment of the invention, the invention provides methods for the treatment of COPD comprising administering to a subject an effective amount of a H4R agonist, an anti-H1 drug and an anticholinergic drug. Suitable H4R agonists and anti-H1 drugs are described above. Suitable examples of anticholinergics include, but are not limited to: tiotropium bromide (Spiriva®) and ipratropium bromide (Atrovent®).

The dose of an anticholinergic drug administered to a subject may be about 0.1 to 1.0 mg/clay, about 1.0 to 2.0 mg/day, about 2.0 to 3.0 mg/day, about 3.0 to 4.0 mg/day, about 4.0 to 5.0 mg/day, about 5.0 to 6.0 mg/day, about 6.0 to 7.0 mg/day, about 7.0 to 8.0 mg/day, about 8.0 to 9.0 mg/clay, about 9.0 to 10.0 mg/day, about 10.0 to 15.0 mg/day, about 15.0 to 20.0 mg/day, about 20.0 to 25.0 mg/day, about 25.0 to 30.0 mg/day, about 30.0 to 35.0 mg/day, about 35.0 to 40.0 mg/day, about 45.0 to 50.0 mg/day, about 50.0 to 60.0 mg/day, about 60.0 to 70.0 mg/day, about 70.0 to 80.0 mg/day, about 80.0 to 90.0 mg/day, about 90.0 to 100.0 mg/day, about 100.0 to 150.0 mg/day, about 150.0 to 200.0 mg/day, about 200.0 to 250.0 mg/day, about 250.0 to 300.0 mg/day, about 300.0 to 350.0 mg/day, about 350.0 to 400.0 mg/day, about 400.0 to 450.0 mg/day, about 450.0 to 500.0 mg/day, about 500.0 to 550.0 mg/day, about 550.0 to 600.0 mg/day, about 600.0 to 650.0 mg/day, about 650.0 to 700.0 mg/day, about 700.0 to 750.0 mg/day, about 750.0 to 800.0 mg/day, about 800.0 to 850.0 mg/day, about 850.0 to 900.0 mg/day, about 900.0 to 950.0 mg/day, about 950.0 to 1000.0 mg/day or about 1000.0. The dose of an anticholinergic drug dispensed by an inhaler to a subject may be about 0.1 mg per puff, about 0.5 mg per puff, about 1.0 mg per puff, about 2.5 mg per puff, about 5.0 mg per pug about 10.0 mg per puff, about 15.0 mg per puff, about 18.0 mg per puff, about 20.0 mg per puff, about 25.0 mg per puff, about 30.0 mg per puff, about 35.0 mg per puff, about 40.0 mg per pug about 45.0 mg per puff or about 50.0 mg per puff.

In a specific embodiment of the invention, the H4R agonist is tritoqualine, the anti-H1 drug is loratadine and the anticholinergic drug is Spiriva®.

In a further embodiment of the invention, the drug combination can further comprise any one or more of existing COPD therapies including but not limited to combination inhaler, steroids, inhaled beta-2 agonists, inhaled corticosteroids, mucolytics, oral beta-2 agonists and theophyllines.

According to the practice of the invention, suitable examples of steroids include, but are not limited to, corticosteroids, Cortisone, Hydrocortisone/cortisol, Desoxycortone, Alclometasone, Aldosterone, Amcinonide, Beclometasone, Betamethasone, Budesonide, Ciclesonide, Clobetasol, Clobetasone, Clocortolone, Cloprednol, Cortivazol, Deflazacort, Deoxycorticosterone, Desonide, Desoximetasone, Dexamethasone, Diflorasone, Diflucortolone, Difluprednate, Fluclorolone, Fludrocortisone, Fludroxycortide, Flumetasone, Flunisolide, Fluocinolone acetonide, Fluocinonide, Fluocortin, Fluocortolone, Fluorometholone, Fluperolone, Fluprednidene, Fluticasone, Formocortal, Halcinonide, Halometasone, Hydrocortisone aceponate, Hydrocortisone buteprate, Hydrocortisone butyrate, Loteprednol, Medrysone, Meprednisone, Methylprednisolone, Methylprednisolone aceponate, Mometasone furoate, Paramethasone, Prednicarbate, Prednisone, Prednisolone, Prednylidene, Rimexolone, Tixocortol, Triamcinolone and Ulobetasol. The method provides the administration of a single or combination of steroid drags.

Suitable examples of NSAIDs include, but are not limited to, acetyl salicylic acid (Aspirin), Amoxiprin, Benorilate, choline magnesium salicylate, diflunisal, Faislamine, Methyl salicylate, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors, meloxicam, tramadol, Aceclofenac, Acemetacin, Bromfenac, Etodolac, Indometacin, Nabumetone, Sulindac, Tolmetin, Ibuprofen, Carprofen, Fenbufen, Loxoprofen, Oxaprozin, Tiaprofenic acid, Suprofen, Mefenamic acid, Meclofenamicacid, Phenylbutazone, Azapropazone, Metamizole, Oxyphenbutazone, Sulfinpyrazone, Meloxicam, Piroxicam, Lornoxicam and Tenoxicam. In a preferred embodiment, NSAIDs include Aspirin, Meloxicam, Ibuprofen, Naproxen. The method provides the administration of a single or combination of NSAIDS.

In accordance with the practice of the invention, for each given daily dose of a drug, or combination of drags, listed above, the given dose may be administered once a day or multiple times a day. For example, ½ of the given dose may be administered twice a day. In another embodiment of the invention, ⅓ of the given dose may be administered 3 times a day. In a further embodiment of the invention, ¼ of the given dose may be administered 4 times a day. In yet another embodiment of the invention, ⅕ of the given dose may be administered 5 times a day. In yet another embodiment of the invention, ⅙ of the given dose may be administered 6 times a day. In yet another embodiment of the invention, 1/7 of the given, dose may be administered times a day. In yet another embodiment of the invention, ⅛ of the given dose may be is administered 8 times a day. In yet another embodiment of the invention, 1/9 of the given dose may be administered 9 times a day. In yet another embodiment of the invention, 1/10 of the given dose may be administered 10 times a day.

Additionally, for each given daily dose of a drug listed above, various fractions of the given dose may be administered to the subject at multiple times during the day, with the sum of the various fractions adding up to the given dose. For example, the amount of the fraction of the given dose administered to the subject at a given time may be any of ½ of the given dose, ⅓ of the given dose, ¼ of the given dose, ⅕ of the given dose, ⅙ of the given dose, 1/7 of the given dose, ⅛ of the given dose, 1/9 of the given dose, or 1/10 of the given dose. In another embodiment of the invention, the fraction of the given dose that is administered, and the total number of times the drug is administered can vary by day. In a further embodiment of the invention, the time of day the given dose or fraction of the given dose is administered can vary by day.

For example, a daily dose of a drug (e.g., H4R agonist) can total about 200 mg per clay. The drug can be given once a day at a dose of about 200 mg, twice a day with each dose about 100 mg, three times a day with each dose about 66.6 mg or four times a day with each dose about 50 mg.

In accordance with the practice of the invention, the drug can be administered one or more times a day, daily, weekly, monthly or yearly.

Dosage of the therapeutic agent(s) of the invention is dependant upon many factors including, but not limited to, the type of tissue affected, the type of disease being treated, the severity of the disease, a subject\’s health and response to the treatment with the agents. Accordingly, dosages of the agents can vary depending on each subject and the mode of administration.

In accordance with the practice of the invention, the subject may be a mammal. In other embodiments of the invention, the subject may be any of human, monkey, ape, dog, cat, cow, horse, sheep, rabbit, mouse, or rats.

In accordance with the practice of the invention, the administration of a given drug may be effected locally or systemically. Additionally, the route of administration of a given drug may be any of topical, enteral or parenteral. In other embodiments of the invention, the route of administration of a given drug may be any of rectal, intercisternal, bucal, intramuscular, intrasternal, intracutaneous, intrasynovial, intravenous, intraperitoneal, intraocular, periostal, intra-articular injection, infusion, oral, inhalation, subcutaneous, implantable pump, continuous infusion, gene therapy, intranasal, intrathecal, intracerebroventricular, transdermal, or by spray, patch or injection.

In accord with the practice of the invention, the route of administration of a given drug can vary during a course of treatment, or during a given day. For example, if a given drug is administered in conjunction with one or more additional drugs, each additional drug may be administered by identical or different routes compared to the other drugs.

The combination of a H4R agonist alone, or in combination with one or more of an anti-H1 drug, anti-H2 drug, anti-H3 drug, a LRA drug, a steroid and a NSAID, can be prepared in a single or multiple dosage form for administration to a subject.

The administration of a given drug to a subject can be performed daily, weekly, monthly, every other month, quarterly, or any other schedule of administration as a single dose administration, in multiple doses, or in continuous dose form. Additionally, a given drug can be administered to a subject intermittently, or at a gradual, continuous, constant, immediate or controlled rate to a subject.

In accord with the practice of the invention, if other drugs are being administered in addition to the agents of the invention, the timing of administration of each drug may be identical to or different from the timing of the other drugs. The administration of the drugs of the invention can be concurrent or at different times.

The active agents of the invention may be administered alone or in combination with other therapeutic agents. Components of the combinations may be administered either concomitantly, (e.g., as an admixture), separately but simultaneously or concurrently or sequentially. This includes presentations in which the combined active agents are administered together as a therapeutic mixture, and also procedures in which the combined active agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the active agents given first, followed by the one or more sequential active agent(s).

In accordance with the practice of the invention, the subject being administered an active agent may have any one or more of the following diseases and/or conditions: GERD; food allergies; Zollinger-Ellison Syndrome; peptic ulcer; dyspepsia; allergic eosinophilic gastroenteritis; mastocytosis with gastrointestinal symptoms; those provoked by CD4 Th2 lymphocytes (asthma, rhinitis conjunctivitis eczema, gastro-esophageal reflux disease and other gastric diseases exacerbated by gastric acid), CD4 Th1 lymphocytes (IBD, Crohn\’s disease, ulcerative colitis and celiac disease and autoimmune gastritis, chronic bronchitis etc.), CD4 Th17 lymphocytes (Uveitis and inflammatory digestive diseases); COPD and other pulmonary types of inflammation including asthma; disease conditions in cancer; and other diseases where histamine or the H4R is involved. H4R agonists may also be used in vaccines as adjuvant to improve immune responses to the inoculated antigens.

The invention provides methods for the treatment or prevention of gastrointestinal disease conditions ameliorated by histamine management in a subject, comprising administering to the subject an effective amount of a histidine decarboxylase inhibitor. In one embodiment, the subject is given an effective amount of a histamine decarboxylase inhibitor and an anti-E11 drug. In another embodiment, the subject is given an effective amount of a histidine decarboxylase inhibitor and an anti-H2 drug. In yet another embodiment, the subject is given an effective amount of a histidine decarboxylase inhibitor, an anti-H1 drug and an anti-H2 drug. In a further embodiment, the subject may be given an effective amount of a combination of a histidine decarboxylase inhibitor, and any one or both an anti-H1 drug and an anti-H2 drug together with an NSAID.

An embodiment of the invention also provides methods for the treatment or prevention of gastrointestinal disease conditions ameliorated by histamine management in a subject comprising administering to the subject an effective amount of a HDC inhibitor in combination with an effective amount a LRA. In a further embodiment, the subject is given an effective amount of a HDC inhibitor in combination with an effective amount of a LRA and an effective amount of an anti-H1 drug. In another embodiment of the invention, the subject is given an effective amount of a HDC inhibitor in combination with an effective amount of a LRA and an effective amount of an anti-H2 drug. In a further embodiment of the invention, the subject is given an effective amount of a HDC inhibitor in combination with an effective amount of a LRA, an effective amount of an anti-H1 drug and an effective amount of an anti-H2 drug.

The invention further provides methods for treatment or prevention of COPD comprising administering to the subject an effective amount of HDC inhibitor. In one embodiment, the patient is administered an effective amount of a HDC inhibitor in combination with an effective amount of an anti-H1 drug. Histidine decarboxylase inhibitors in general, as well as histamine receptor antagonists e.g. Anti-H1 drugs have never been used for the treatment of COPD. Tritoqualine (an HDC inhibitor) and Loratadine (an anti-H1 drug) were traditionally used for the treatment of allergy and primarily allergic rhinitis. The combination of Tritoqualine and Loratadine showed statistically significant effectiveness in the management of COPD when compared with the standard treatment of beta agonists, bronchodilators, steroids and oxygen. The presumed action of combining Tritoqualine and an anti-H1 drug may be by increasing the TH1 cells thus balancing the equilibrium between TH1 and TH2 cells to the detriment of TH2 cells. As a result, secretion of inflammatory cytokines such as IL4, IL5, and IL10 is reduced causing less pulmonary inflammation.

In another embodiment of the invention the patient is given HDC inhibitor in combination with any one or more of COPD therapies including but not limited to Anticholinergics, Combination Inhaler, Corticosteroids, Inhaled Beta-2 Agonists, Inhaled Corticosteroids, Mucolytics, Oral Beta-2 Agonists, Bronchodilators and Theophyllines. In a further embodiment, the patient is given HDC inhibitor and anti-H1 drug in combination with any one or more of COPD therapies including but not limited to Anticholinergics, Combination Inhaler, Corticosteroids, Inhaled Beta-2 Agonists, Inhaled Corticosteroids, Mucolytics, Oral Beta-2 Agonists, Bronchodilators and Theophyllines. In yet another embodiment of the invention, the patient is administered an effective amount of a HDC inhibitor, an anti-H1 drug and a NSAID.

Suitable examples of histidine decarboxylase inhibitors include, but are not limited to, any of Tritoqualine or an isomer thereof, alpha-fluoromethylhistidine, 3-methoxy-5,7,3′,4′-tetrahydroxyflavan, naringenin, (+)-cyanidanol-3, the dipeptide His-Phe, and 4-imidazolyl-3-amino-2-butanone, polyphenols such as catechins and related structures; these include, but are not limited to: (−)-epigallocatechin gallate, (−)-epicatechin gallate, (−)-epicatechin, (−)-epigallocatechin, and is composed of (−)-epicatechin, (−)-epigallocatechin; and other flavonoids such as O-methyl-3(+)catechin; or analogs, equivalents, isomers, pharmaceutically acceptable salts, and solvate forms of any of the above. The Tritoqualine isomer may be an SS isomer of Tritoqualine or an RR isomer of Tritoqualine. The method provides the administration of single or a combination of histidine decarboxylase inhibitors.

In accordance with the practice of the invention, the subject may have any one or more of the following: 1) a history of GERD; 2) a history of allergies (for example food allergies); 3) a history of COPD; 4) previous unsatisfactory treatment with an anti-H1 and/or anti-H2 drug; 5) previously unsatisfactory treatment with existing COPD therapies; 6) previous unsatisfactory treatment with proton pump inhibitors (PPI); 7) previous diagnosis of GERD and concurrent symptoms of allergy; and/or 8) previous unsatisfactory treatment with cromoglycate.

In one embodiment, the subject may suffer from COPD or GERD (or any of the gastrointestinal disorders or diseases disclosed herein) but does not exhibit allergy symptoms which include any one or more of allergic asthma, allergic conjunctivitis, and allergic rhinitis.

For example, the allergy may be confirmed with two positive prick tests for the same allergen or set of allergens. The allergens may be any of Dermatophagoid Pternzonysisnus, Demathophagoid Farinae, cat dander, dog dander, food allergens, fungal proteins, and pollen proteins. Additionally, the food allergen may be any of wheat, egg, soy, potato, peanut, and/or tomato proteins. The fungal protein may be any of a protein from a species in the alternaria genus, a protein from a species in the mucor genus, and a protein from a species in the aspergillus genus. The pollen protein may be any of pollen of birch tree, pollen of cypress tree, pollen of Quercus tree, pollen of lolium perenne, and pollen of ray grass.

COMPOSITIONS OF THE INVENTION

The present invention provides pharmaceutical formulations (also known as pharmaceutical compositions or dosage forms) comprising a first active agent (e.g., a H4R agonist), one or more additional active agent (e.g., a H1R antagonist, a H2R antagonist, a H3R antagonist, a LRA, a steroid, a NSAID or other active agent), and a pharmaceutically acceptable carrier or vehicle.

The present invention also provides pharmaceutical formulations for the treatment of COPD comprising a first active agent (e.g., a H4R agonist), a second active agent (e.g., a H1R antagonist), a third active agent (e.g., an anticholinergic drug) and a pharmaceutically acceptable carrier or vehicle.

Pharmaceutically acceptable carrier or vehicle refers to a non-toxic solid, semisolid (also referred to herein as softgel) or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The invention also provides methods for treating or ameliorating H4R modulated diseases using said pharmaceutical formulations.

H4R agonists include, but are not limited to, Tritoqualine or an isomer thereof, 4-methylhistmime (4MeHA) and clobenprobit (CB). The pharmaceutical formulation of the invention provides one or more H4R agonist drugs. Examples of Tritoqualine isomers include, but are not limited to, the E1 and E2 enantiomer.

H1 antagonists (anti-H1 drugs) include, but are not limited to, brompheniramine, cetirizine, levocetirizine, fexofenadine, cyproheptadine, dexchlorpheniramine, hydroxizine, ketotifen, loratadine, mequitazine, oxotomide, mizolastine, ebastine, astemizole, carbinoxamide, alimemazine, buclizine, cyclizine hydrochloride, doxylamine, mepyramine, antazoline, diphenhydramine, carbinoxamine, clemastine, dimenhydrinate, pheniramine, chlorphenamine, triprolidine, chlorcyclizine, hydroxyzine, meclizine, promethazine and azatadine and/or analogs, equivalents, isomers, salts, and solvate forms thereof. The pharmaceutical formulation of the invention provides one or more anti-H1 drugs.

H2 antagonists (anti-H2 drugs) include, but are not limited to, ranitidine, cimetidine, famotidine, and nizatidine and/or analogs, equivalents, isomers, salts, and solvate forms thereof. The pharmaceutical formulation of the invention provides one or more anti-H2 drugs.

H3 antagonists (anti-H3 drugs) include, but are not limited to, betahistine (N-methyl-2-pyridin-2-ylethanamine), ABT-239 (4-(2-{2-[(2R)-2-Methylpyrrolidin-1-yl]ethyl}-benzofuran-5-yl)benzonitrile), Cipralisant(1R,2R)-4-(2-(5,5-dimethylhex-1-ynyl)cyclopropyl)imidazole, Ciproxifan cyclopropyl 4-(3-(1H-imidazol-4-yl)propyloxy)phenyl ketone, Clobenpropit N′-[(4-chlorophenyl)methyl]-1-[3-(3H-imidazol-4-yl)propylthio]formamidine, Thioperamide N-Cyclohexyl-4-(1H-imidazol-4-yl)-1-piperidinecarbothioamide. The pharmaceutical formulation of the invention provides one or more anti-H3 drugs.

Anticholinergic drugs include, but are not limited to: tiotropium bromide (Spiriva®) and ipratropium bromide (Atrovent®).

LRAs include, but are not limited to, Montelukast® (Singulair®), Pranlukast® and Zafirlukast®. The method provides the administration of a single or a combination of LRAs.

Steroids include, but are not limited to, corticosteroids, Aldosterone, Cortisone, Hydrocortisone/cortisol, Desoxycortone, Alclometasone, Amcinonide, Beclometasone, Betamethasone, Budesonide, Ciclesonide, Clobetasol, Clobetasone, Clocortolone, Cloprednol, Cortivazol, Deflazacort, Deoxycorticosterone, Desonide, Desoximetasone, Dexamethasone, Diflorasone, Diflucortolone, Difluprednate, Fluclorolone, Fludrocortisone, Fludroxycortide, Flumetasone, Flunisolide, Fluocinolone acetonide, Fluocinonide, Fluocortin, Fluocortolone, Fluorometholone, Fluperolone, Fluprednidene, Fluticasone, Formocortal, Halcinonide, Halometasone, Hydrocortisone aceponate, Hydrocortisone buteprate, Hydrocortisone butyrate, Loteprednol, Medrysone, Meprednisone, Methylprednisolone, Methylprednisolone aceponate, Mometasone furoate, Paramethasone, Prednicarbate, Prednisone, Prednisolone, Prednylidene, Rimexolone, Tixocortol, Triamcinolone and Ulobetasol. The method provides the administration of a single or combination of steroid drugs.

NSAIDs include, but are not limited to, acetyl salicylic acid (Aspirin), Amoxiprin, Benorilate, choline magnesium salicylate, diflunisal, Faislamine, Methyl salicylate, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors, meloxicam, tramadol, Aceclofenac, Acemetacin, Bromfenac, Etodolac, Indometacin, Nabumetone, Sulindac, Tolmetin, Ibuprofen, Carprofen, Fenbufen, Loxoprofen, Oxaprozin, Tiaprofenic acid, Suprofen, Mefenamic acid, Meclofenamicacid, Phenylbutazone, Azapropazone, Metamizole, Oxyphenbutazone, Sulfinpyrazone, Meloxicam, Piroxicam, Lornoxicam and Tenoxicam. In a preferred embodiment, NSAIDs include Aspirin, Meloxicam, ibuprofen, Naproxen, Phosphodiesterase (PDE4) inhibitors Prostaglandin E4. The method provides the administration of a single or combination of NSAIDS.

In one embodiment of the invention, the pharmaceutical formulation comprises the H4R agonist and a pharmaceutically acceptable vehicle. In a particular embodiment, the H4R agonist is Tritoqualine or an isomer thereof.

In another embodiment of the invention, the pharmaceutical formulation comprises the H4R agonist, one or more other active agent and a pharmaceutically acceptable vehicle. In a particular embodiment, the H4R agonist is Tritoqualine or an isomer thereof. The active agent can include, but is not limited to, H1R antagonists, H2R antagonists, H3R antagonists, H4R agonist, LRAs, steroids and NSAIDs.

In yet another embodiment, the pharmaceutical composition comprises a tritoqualine and an anti-H2 drag.

The present invention also provides pharmaceutical formulations comprising a solid or liquid dosage form of a H4R agonist (e.g., Tritoqualine, E1 or E2) and a plurality of particles which permit the formulation of solid or liquid dosage form of the H4R agonist. The particles comprise, but are not limited to, excipients disclosed herein below, e.g., typical excipients for softgels.

In one embodiment, the solid or liquid dosage form of the H4R agonist formulation further comprises an H1R antagonist. In another embodiment, the solid or liquid dosage form of the H4R agonist formulation further comprises an H2R antagonist. In another embodiment, the solid or liquid dosage form of the H4R agonist formulation further comprises an H3 antagonist. In yet another embodiment, the solid or liquid dosage form of the H4R agonist formulation further comprises one or more of an H1R, an H2R antagonist, an H3 antagonist, a LRA drug and an NSAID.

In one aspect, the present invention provides a pharmaceutical composition for the treatment of H4R modulated diseases or conditions comprising a solid or liquid dosage form of the H4R agonist (e.g., Tritoqualine, E1 or E2), wherein the composition is an administrable formulation that allows resorption of the H4R agonist into a subject. In one embodiment, the administrable formulation can be, e.g., an inhalant or a topically administrable formulation such as an ointment or cream.

Anti-H1, anti-H2 and anti-H3 drugs are present in the various compositions of the invention in a proportion of the order of 0.1 to 2000 mg.

In the case of a pharmaceutical composition according to the invention containing an antihistamine compound (for example, anti-H1, anti-H2 or anti-H3) and a H4R agonist, these compounds are present in a proportion of the order of:



0.1 to 2000 mg of anti H2 compound (when used),
0.1 to 2000 mg of anti-H1 compound (when used),
0.1 to 2000 mg of anti-H3 compound (when used), and
0.10 to 3000 mg of a H4R agonist such as Tritoqualine or its isomers.

In the case of a composition according to the invention containing H4R agonist and one or more of an antihistamine compound (for example, anti-H1, anti-H2 or anti-H3), an LRA drug, a steroid drug and a NSAID, these compounds are present in a proportion of the order of:



0.1 to 2000 mg of anti H2 compound (when used),
0.1 to 2000 mg of anti-H1 compound (when used)
0.1 to 2000 mg of anti-H3 compound (when used),
0.1 to 2000 mg of a LRA drug (when used)
0.01 to 2000 mg of steroid compound (when used),
0.1 to 5000 mg of NSAID compound (when used), and
0.10 to 3000 mg of a H4R agonist such as Tritoqualine or its isomers.

Further, the active agents of the invention can be pegylated, phosphorylated, esterified, derivatized with amino acids and/or peptides, to improve solubility for both formulation and bioavailability. Additionally, lipid derivatization and other lipophile derivatization can be used to improve mucosal permeability, absorption and formulation of the active agents of the invention in oily vehicles.

Dosage Forms

Dosage forms can be made according to well known methods in the art. Some preferred methods are described below.

The pharmaceutical compositions of the invention may be formulated as solid dosage forms, such as capsules, pills, softgels, tablets, caplets, troches, wafer, sprinkle, chewing gum or the like, for oral administration. The pharmaceutical compositions of the invention may also be formulated as liquid dosage forms such as elixir, suspension or syrup.

The pharmaceutical compositions of the invention may also be presented in a dosage form for transdermal application, for example an ointment for children, a form for oral administration, for example a slow release product, or in gastro-resistant tablet form or gum form. They may also be in spray, bronchial form or eye lotion form, or other galenic forms with programmed mucosal and secondarily per os disintegration.

Therefore the different pharmaceutical compositions of the invention can be administered by several routes chosen in accordance with the patient\’s pathological profile and age. For children, the patch form, syrup form or tablets to be dissolved in the mouth. The other forms, eye lotion or injection may also be used. In adults all galenic forms (also known as dosage forms) can be contemplated.

The advantage of a coupled or combined galenic form also provides simplicity of treatment, patient compliance with the simplified treatment and therefore a more successful outcome.

The pharmaceutical compositions of the present invention may be mixed with pharmaceutically acceptable carriers, binders, diluents, adjuvants, excipients, or vehicles, such as preserving agents, fillers, polymers, disintegrating agents, glidants, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, lubricating agents, acidifying agents, coloring agent, dyes, preservatives and dispensing agents, or compounds of a similar nature depending on the nature of the mode of administration and dosage forms. Such ingredients, including pharmaceutically acceptable carriers and excipients that may be used to formulate oral dosage forms, are described in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (1986), incorporated herein by reference in its entirety.

Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation. Examples of pharmaceutically acceptable carriers include water, saline, Ringer\’s solution, dextrose solution, ethanol, polyols, vegetable oils, fats, ethyl oleate, liposomes, waxes polymers, including gel forming and non-gel forming polymers, and suitable mixtures thereof. The carrier may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as, ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient.

Examples of binders include, but are not limited to, microcrystalline cellulose and cellulose derivatives, gum tragacanth, glucose solution, acacia mucilage, gelatin solution, molasses, polyinylpyrrolidine, povidone, crospovidones, sucrose and starch paste.

Examples of diluents include, but are not limited to, lactose, sucrose, starch, kaolin, salt, mannitol and dicalcium phosphate.

Examples of excipients include, but are not limited to, starch, surfactants, lipophilic vehicles, hydrophobic vehicles, pregelatinized starch, Avicel, lactose, milk sugar, sodium citrate, calcium carbonate, dicalcium phosphate, and lake blend purple. Typical excipients for dosage forms such as a softgel include gelatin for the capsule and oils such as soy oil, rice bran oil, canola oil, olive oil, corn oil, and other similar oils; glycerol, polyethylene glycol liquids, vitamin E TPGS as a surfactant and absorption enhancer (Softgels: Manufacturing Considerations; Wilkinson P, Foo Sog Hom, Special Drug Delivery Systems; Drugs and the Pharmaceutical Sciences Vol 41 Praveen Tyle Editor, Marcel Dekker 1990, 409-449; Pharmaceutical Dosage Forms and Drug Delivery by Ansel, Popovich and Allen 1995, Williams and Wilkins, Chapter 5 pp 155-225). Tritoqualine and anti H1 may form either a solution in a selected oil vehicle or a suspension of fine particles (comprising any of the excipients disclosed herein, e.g., typical excipients for softgels).

Examples of disintegrating agents include, but are not limited to, complex silicates, croscarmellose sodium, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar and carboxymethylcellulose.

Examples of glidants include, but are not limited to, colloidal silicon dioxide, talc, corn starch.

Examples of wetting agents include, but are not limited to, propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene laural ether.

Examples of sweetening agents include, but are not limited to, sucrose, lactose, mannitol and artificial sweetening agents such as saccharin, and any number of spray dried flavors.

Examples of flavoring agents include, but are not limited to, natural flavors extracted from plants such as fruits and synthetic blends of compounds which produce a pleasant sensation, such as, but not limited to peppermint and methyl salicylate.

Examples of lubricants include magnesium or calcium stearate, sodium lauryl sulphate, talc, starch, lycopodium and stearic acid as well as high molecular weight polyethylene glycols.

Examples of coloring agents include, but are not limited to, any of the approved certified water soluble FD and C dyes, mixtures thereof; and water insoluble FD and C dyes suspended on alumina hydrate.

The artisan of ordinary skill in the art will recognize that many different ingredients can be used in formulations according to the present invention, in addition to the active agents, while maintaining effectiveness of the formulations in treating the H4R modulated diseases. The list provided herein is not exhaustive.

Matrix Based Dosage Forms

Dosage forms according to one embodiment of the present invention may be in the form of coated or uncoated matrices. The term matrix, as used herein, is given its well known meaning in the pharmaceutical arts as a solid material having an active agent (e.g., the components of the compositions of the invention) of the invention incorporated therein. Upon exposure to a dissolution media, channels are formed in the solid material so that the active agent can escape.

The skilled artisan will appreciate that the matrix material can be chosen from a wide variety of materials which can provide the desired dissolution profiles. Materials can include, for example, one or more gel forming polymers such as polyvinyl alcohol, cellulose ethers including, for example, hydroxypropylalkyl celluloses such as hydroxypropyl cellulose, hypromellose, prop-2-enoic acid, hydroxypropyl methyl cellulose, hydroxyalkyl celluloses such as hydroxypropyl cellulose, natural or synthetic gums such as guar gum, xanthum gum, and alginates, as well as ethyl cellulose, polyvinyl pyrrolidone, fats, waxes, polycarboxylic acids or esters such as the Carbopol R series of polymers, meth acrylic acid copolymers, and methacrylate polymers.

In addition to the above-mentioned ingredients, a controlled release matrix may also contain suitable quantities of other materials, for example, diluents, lubricants, binders, granulating aids, colorants, flavorants, and glidants that are conventional in the pharmaceutical arts. The quantities of these additional materials should be sufficient to provide the desired effect to the desired formulation. A controlled release matrix incorporating particles may also contain suitable quantities of these other materials such as diluents, lubricants, binders, granulating aids, colorants, flavorants, and glidants that are conventional in the pharmaceutical arts in amounts up to about 75% by weight of the particulate, if desired.

Methods of making matrix dosages are well known in the art and any known method of making such dosages which yields the desired immediate release and controlled release dissolution profiles can be used. One such method involves the mixture of the compositions of the invention with a solid polymeric material and one or more pharmaceutically acceptable excipients which can then be blended and compressed in controlled release tablet cores. Such tablet cores, can be used for further processing as bi-layer or multilayer tablets, press coated tablets, or film coated tablets.

In addition, the formulation of respective release components can occur by appropriate granulation methods as is well known in the art. In wet granulation, solutions of the binding agent can be added with stirring to the mixed powders. The powder mass can be wetted with the binding solution until the mass has the consistency of damp snow or brown sugar. The wet granulated material can be forced through a sieving device. Moist material from the milling step can be dried by placing it in a temperature controlled container. After drying, the granulated material can be reduced in particle size by passing it through a sieving device. Lubricant can be added, and the final blend can then be compressed into a matrix dosage form such as a matrix tablet.

In fluid-bed granulation, particles of inert material and/or active agent (e.g., the components of the compositions of the invention) can be suspended in a vertical column with a rising air stream. While the particles are suspended, a common granulating material in solution can be sprayed into the column. There will be a gradual particle buildup under a controlled set of conditions resulting in tablet granulation. Following drying and the addition of lubricant, the granulated material will be ready for compression.

In dry-granulation, the active agent (e.g., the components of the compositions of the invention), binder, diluent, and lubricant can be blended and compressed into tablets. The compressed large tablets can be comminuted through the desirable mesh screen by sieving equipment. Additional lubricant can be added to the granulated material and blended gently. The material can then be compressed into tablets.

Particle Based Dosage Forms
Immediate Release and Controlled Release Particles

Dosage forms according to another embodiment of the present invention may be in the form of coated or uncoated immediate release/controlled release dosage forms. The immediate release/controlled release dosage forms of the present invention can take the form of pharmaceutical particles. The dosage forms can include immediate release particles in combination with controlled release particles in a ratio sufficient to deliver the desired dosages of active agents (e.g., the components of the compositions of the invention). The controlled release particles can be produced by coating the immediate release particles with an enteric coat.

The particles can be produced according to any of a number of well known methods for making particles. The immediate release particles can comprise the active agent combination (the compositions of the invention) and a disintegrant. Suitable disintegrants can include, for example, starch, low-substitution hydroxypropyl cellulose, croscarmellose sodium, calcium carboxymethyl cellulose, hydroxypropyl starch, and microcrystalline cellulose.

In addition to the above-mentioned ingredients, a controlled release matrix may also contain suitable quantities of other materials, for example, diluents, lubricants, binders, granulating aids, colorants, flavorants, and glidants that are conventional in the pharmaceutical arts. The quantities of these additional materials should be sufficient to provide the desired effect to the desired formulation. A controlled release matrix incorporating particles may also contain suitable quantities of these other materials such as diluents, lubricants, binders, granulating aids, colorants, flavorants, and glidants that are conventional in the pharmaceutical arts in amounts up to about 75% by weight of the particulate, if desired.

Particles can assume any standard structure known in the pharmaceutical arts. Such structures can include, for example, matrix particles, non-pareil cores having a drug layer and active or inactive cores having multiple layers thereon. A controlled release coating can be added to any of these structures to create a controlled release particle.

The term particle as used herein means a granule having a diameter of between about 0.01 mm and about 5.0 mm, preferably between about 0.1 mm and about 2.5 mm, and more preferably between about 0.5 mm and about 2 mm. The skilled artisan will appreciate that particles according to the present invention can be any geometrical shape within this size range and so long as the mean for a statistical distribution of particles falls within the particle sizes enumerated above, they will be considered to fall within the contemplated scope of the present invention.

The release of the therapeutically active agent (e.g., the components of the compositions of the invention) from the controlled release formulation of the present invention can be further influenced, i.e., adjusted to a desired rate, by the addition of one or more release-modifying agents. The release-modifying agent may be organic or inorganic and include materials that can be dissolved, extracted, or leached from the coating in the environment of use. The pore-formers may comprise one or more hydrophilic materials such as hydroxypropyl methylcellulose. The release-modifying agent may also comprise a semi-permeable polymer. In certain preferred embodiments, the release-modifying agent is selected from hydroxypropyl methylcellulose, lactose, metal stearates, and mixtures thereof.

The controlled release particles of the present invention can slowly release the compositions of the invention when ingested. The controlled release profile of the formulations of the present invention can be altered, for example, by increasing or decreasing the thickness of a retardant coating, i.e., by varying the amount of overcoating. The resultant solid controlled release particles may thereafter be placed in a gelatin capsule in an amount sufficient to provide an effective controlled release dose when ingested and contacted by an environmental fluid, e.g., gastric fluid, intestinal fluid or dissolution media.

The dosage forms of the invention may be coated (e.g., film coated or enterically coated) as known by those of skill in the art. For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine.

Examples of enteric-coatings include, but are not limited to, phenylsalicylate, fatty acids, fats, waxes, shellac, ammoniated shellac and cellulose acetate phthalates. Film coatings include, but are not limited to, hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000 and cellulose acetate phthalate.

In one example, the dosage forms e.g., particles of the invention as described above, may be overcoated with an aqueous dispersion of a hydrophobic or hydrophilic material to modify the release profile. The aqueous dispersion of hydrophobic material preferably further includes an effective amount of plasticizer, e.g. triethyl citrate. Preformulated aqueous dispersions of ethylcellulose, such as AQUACOAT™ or SURELEASE™ products, may be used. If a SURELEASE™ product is used, it is not necessary to separately add a plasticizer.

The hydrophobic material may be selected from the group consisting of alkylcellulose, acrylic and methacrylic acid polymers and copolymers, shellac, zein, fatty oils, hydrogenated castor oil, hydrogenated vegetable oil, or mixtures thereof. In certain preferred embodiments, the hydrophobic material can be a pharmaceutically acceptable acrylic polymer including, but not limited to, acrylic acid and methacrylic acid copolymers, methyl methacrylate, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), methacrylic acid alkylamine copolymer, poly(methyl methacrylate), poly(methacrylic acid anhydride), polymethacrylate, polyacrylamide, poly(methacrylic acid anhydride), and glycidyl methacrylate copolymers. In alternate embodiments, the hydrophobic material can be selected from materials such as one or more hydroxyalkyl celluloses such as hydroxypropyl methylcellulose. The hydroxyalkyl cellulose can preferably be a hydroxy (C.sub.1 to C.sub.6) alkyl cellulose, such as hydroxypropylcellulose, hydroxypropylmethylcellulose, or preferably hydroxyethylcellulose. The amount of the hydroxyalkyl cellulose in the present oral dosage form can be determined, in part, by the precise rate of active agents (e.g., the components of the compositions of the invention) desired and may vary from about 1% to about 80%.

In embodiments of the present invention where the coating comprises an aqueous dispersion of a hydrophobic polymer, the inclusion of an effective amount of a plasticizer in the aqueous dispersion of hydrophobic polymer can further improve the physical properties of the film. For example, because ethylcellulose has a relatively high glass transition temperature and does not form flexible films under normal coating conditions, it may be necessary to plasticize the ethylcellulose before using it as a coating material. Generally, the amount of plasticizer included in a coating solution can be based on the concentration of the film-former, e.g., most often from about 1 percent to about 50 percent by weight of the film-former. Concentration of the plasticizer, however, can be preferably determined after careful experimentation with the particular coating solution and method of application.

Examples of suitable plasticizers for ethylcellulose include water-insoluble plasticizers such as dibutyl sebacate, diethyl phthalate, triethyl citrate, tributyl citrate, and triacetin, although other water-insoluble plasticizers (such as acetylated monoglycerides, phthalate esters, castor oil, etc.) may be used. Triethyl citrate may be an especially preferred plasticizer for the aqueous dispersions of ethyl cellulose of the present invention.

Examples of suitable plasticizers for the acrylic polymers of the present invention include, but are not limited to, citric acid esters such as triethyl citrate NF XVI, tributyl citrate, dibutyl phthalate, and possibly 1,2-propylene glycol. Other plasticizers which have proved to be suitable for enhancing the elasticity of the films formed from acrylic films such as EUDRAGIT™ RL/RS lacquer solutions include polyethylene glycols, propylene glycol, diethyl phthalate, castor oil, and triacetin. Triethyl citrate may be an especially preferred plasticizer for aqueous dispersions of ethyl cellulose. It has further been found that addition of a small amount of talc may reduce the tendency of the aqueous dispersion to stick during processing and acts a polishing agent.

One commercially available aqueous dispersion of ethylcellulose is the AQUACOAT™ product which is prepared by dissolving the ethylcellulose in a water-immiscible organic solvent and then emulsifying the ethylcellulose in water in the presence of a surfactant and a stabilizer. After homogenization to generate submicron droplets, the organic solvent can be evaporated under vacuum to form a pseudolatex. The plasticizer will not be incorporated into the pseudolatex during the manufacturing phase. Thus, prior to using the pseudolatex as a coating, the AQUACOAT™ product can be mixed with a suitable plasticizer.

Another aqueous dispersion of ethylcellulose is commercially available as SURELEASE™ product (Colorcon, Inc., West Point, Pa., U.S.A.). This product can be prepared by incorporating plasticizer into the dispersion during the manufacturing process. A hot melt of a polymer, plasticizer (dibutyl sebacate), and stabilizer (oleic acid) can be prepared as a homogeneous mixture which can then be diluted with an alkaline solution to obtain an aqueous dispersion which can be applied directly onto substrates.

In one embodiment, the acrylic coating can be an acrylic resin lacquer used in the form of an aqueous dispersion, such as that which is commercially available from Rohm Pharma under the trade name EUDRAGIT™. In additional embodiments, the acrylic coating can comprise a mixture of two acrylic resin lacquers commercially available from Rohm Pharma under the trade names EUDRAGIT™ RL 30 D and EUDRAGIT™RS 30 D. EUDRAGIT™ RL 30 D and EUDRAGIT™ RS 30 are copolymers of acrylic and methacrylic esters with a low content of quaternary ammonium groups, the molar ratio of ammonium groups to the remaining neutral (meth)acrylic esters being 1:20 in EUDRAGIT™ RL 30 and 1:40 in EUDRAGIT™ RS 30 D. The mean molecular weight is about 150,000 Daltons. The code designations RL (high permeability) and RS (low permeability) refer to the permeability properties of these agents. EUDRAGIT™ RL/RS mixtures are insoluble in water and in digestive fluids; however, coatings formed from them are swellable and permeable in aqueous solutions and digestive fluids.

The EUDRAGIT™ RL/RS dispersions may be mixed together in any desired ratio in order to ultimately obtain a controlled-release formulation having a desirable dissolution profile. Desirable controlled release formulations may be obtained, for instance, from a retardant coating derived from one of a variety of coating combinations, such as 100% EUDRAGIT™ RL; 50% EUDRAGIT™ RL and 50% EUDRAGIT™ RS; or 10% EUDRAGIT™ RL and EUDRAGIT™ 90% RS. Of course, one skilled in the art will recognize that other acrylic polymers may also be used, for example, others under the EUDRAGIT™ brand. In addition to modifying the dissolution profile by altering the relative amounts of different acrylic resin lacquers, the dissolution profile of the ultimate product may also be modified, for example, by increasing or decreasing the thickness of the retardant coating.

The stabilized product may be obtained by subjecting the coated substrate to oven curing at a temperature above the Tg (glass transition temperature) of the plasticized acrylic polymer for the required time period, the optimum values for temperature and time for the particular formulation being determined experimentally. In certain embodiments of the present invention, the stabilized product is obtained via an oven curing conducted at a temperature of about 45° C. for a time period from about 1 to about 48 hours. It is also contemplated that certain products coated with the controlled-release coating of the present invention may require a curing time longer than 24 to 48 hours, e.g., from about 48 to about 60 hours or more.

The coating solutions preferably contain, in addition to the film-former, plasticizer, and solvent system (i.e., water), a colorant to provide elegance and product distinction. Color may be added to the solution of the compositions of the invention instead of, or in addition to the aqueous dispersion of hydrophobic material. For example, color may be added to an AQUACOAT™ product via the use of alcohol or propylene glycol based color dispersions, milled aluminum lakes and opacifiers such as titanium dioxide by adding color with shear to the water soluble polymer solution and then using low shear to the plasticized AQUACOAT™ product.

Alternatively, any suitable method of providing color to the formulations of the present invention may be used. Suitable ingredients for providing color to the formulation when an aqueous dispersion of an acrylic polymer is used include titanium dioxide and color pigments, such as iron oxide pigments. The incorporation of pigments, may, however, increase the retardant effect of the coating.

Spheroids or beads coated with the compositions of the invention can be prepared, for example, by dissolving the compositions of the invention in water and then spraying the solution onto a substrate, for example, non pareil 18/20 beads, using a Wuster insert. Optionally, additional ingredients can also be added prior to coating the beads in order to assist the binding of the compositions of the invention to the beads, and/or to color the solution, etc. For example, a product which includes hydroxypropyl methylcellulose with or without colorant (e.g., OPADRY™ product, commercially available from Coloron, Inc.) may be added to the solution and the solution mixed (e.g., for about 1 hour) prior to application onto the beads. The resultant coated substrate, beads in this example, may then be optionally overcoated with a bather agent to separate the compositions of the invention from the hydrophobic controlled release coating. An example of a suitable barrier agent is one which comprises hydroxypropyl cellulose. However, any film-former known in the art may be used. It is preferred that the barrier agent does not affect the dissolution rate of the final product.

Immediate release particles according to the present invention may be coated with a controlled release coating in order to change the release rate to obtain the dissolution rates according to the present invention.

Press Coated, Pulsatile Dosage Form

In another embodiment of the present invention, the compositions of the invention can be administered via a press coated pulsatile drug delivery system suitable for oral administration with a controlled release component, which contains a compressed blend of an active agent (e.g., the components of the compositions of the invention) and one or more polymers, substantially enveloped by an immediate release component, which contains a compressed blend of the active agent and hydrophilic and hydrophobic polymers. The immediate-release component preferably comprises a compressed blend of active agent and one or more polymers with disintegration characteristics such that the polymers disintegrate rapidly upon exposure to the aqueous medium.

The controlled-release component preferably can comprise a combination of hydrophilic and hydrophobic polymers. In this embodiment, once administered, the hydrophilic polymer will dissolve away to weaken the structure of the controlled-release component, and the hydrophobic polymer will retard the water penetration and help to maintain the shape of the drug delivery system.

In accordance with the present invention, the term “polymer” includes single or multiple polymeric substances, which can swell, gel, degrade or erode on contact with an aqueous environment (e.g., water). Examples include alginic acid, carboxymethylcellulose calcium, carboxymethylcellulose sodium, colloidal silicon dioxide, croscarmellose sodium, crospovidone, guar gum, magnesium aluminum silicate, methylcellulose, microcrystalline cellulose, polacrilin potassium, powdered cellulose, pregelatinized starch, sodium alginate, sodium starch glycolate, starch, ethylcellulose, gelatin, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polymethacrylates, povidone, pregelatinized starch, shellac, and zein, and combinations thereof.

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