WO2013088445A1 - Pyrimidine derivatives for treatment of inflammatory diseases - Google Patents

Abstract

Small pyrimidine derivatives of the formula I useful for treatment of an inflammatory disease or a central nervous system (CNS) injury, particularly for treatment of rheumatoid arthritis, multiple sclerosis, cerebral ischemia or spinal cord injury.

Description

PYRIMIDINE DERIVATIVES FOR TREATMENT OF INFLAMMATORY

DISEASES

FIELD OF THE INVENTION

[0001] The present invention relates to small pyrimidine molecules active as POSH inhibitors and their pharmaceutical uses.

BACKGROUND OF THE INVENTION

[0002] Human POSH is a large polypeptide containing a RING domain and four SH3 domains. POSH is a ubiquitin ligase (also termed an "E3" enzyme); the RING domain mediates ubiquitination of, for example, the POSH polypeptide itself. POSH interacts with a large number of proteins, functioning as a ubiquitin ligase or a scaffold protein, and participates in a host of different biological processes, such as protein degradation, viral maturation, apoptotic activation, cell migration and axon outgrowth.

[0003] POSH intersects with and regulates a wide range of key cellular functions that may be manipulated by affecting the level of and/or activity of POSH polypeptides or POSH- AP (POSH - associated proteins) polypeptides. Many features of POSH, and particularly human POSH, are described in PCT patent publications WO03/095971A2 and WO03/078601A2, the teachings of which are incorporated by reference herein.

[0004] Small pyrimidine derivatives, which are inhibitors of the ubiquitin ligase activity of human POSH, and compositions and methods for treatment of viral infections and neurological conditions, disorders or diseases, are disclosed in WO 2008/056356, the teachings of which are incorporated by reference herein.

[0005] Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disorder that may affect many tissues and organs, but principally attacks flexible (synovial) joints. The process involves an inflammatory response of the capsule around the joints (synovium) secondary to swelling (hyperplasia) of synovial cells, excess synovial fluid, and the development of fibrous tissue (pannus) in the synovium. The pathology of the disease process often leads to the destruction of articular cartilage and ankylosis (fusion) of the joints. Rheumatoid arthritis can also produce diffuse inflammation in the lungs, membrane around the heart (pericardium), the membranes of the lung (pleura), and white of the eye (sclera), and also nodular lesions, most common in subcutaneous tissue. Although the cause of rheumatoid arthritis is unknown, autoimmunity plays a pivotal role in both its chronicity and progression, and RA is considered a systemic autoimmune disease. Various treatments are available, including non-pharmacological treatment such as physical therapy, orthoses, occupational therapy and nutritional therapy, but these do not stop the progression of joint destruction. Analgesia (painkillers) and anti-inflammatory drugs, including steroids, are used to suppress the symptoms, while disease-modifying antirheumatic drugs (DMARDs) are required to inhibit the underlying immune process.

[0006] Current therapies for treatment of chronic inflammatory diseases are unsatisfactory, probably as a result of their inability to cope with the complex mechanisms that initiate and maintain inflammatory conditions. One aspect of inflammation is the migration of immune cells to a site of injury or infection. The use of the small pyrimidine derivatives disclosed in the above-mentioned WO 2008/056356 for treatment of cell migration-related conditions, disorders or diseases is disclosed in WO 2011/064661, the teachings of which are incorporated by reference herein.

SUMMARY OF THE INVENTION

[0007] The present invention relates, in one aspect, to a POSH inhibitor compound of formula I or a pharmaceutical composition thereof for use in the treatment of an inflammatory disease or a central nervous system (CNS) injury:

wherein

X is O, S or NH;

R is H or Ci-Ce alkyl; Ri is H, Ci-C₆ alkyl, OR', SR', halogen, COOR', S0₂R', unsubstituted C₆-C₁₀ aryl or C6-C₁₀ aryl substituted by one or more radicals selected from C C6 alkyl, halogen, nitro, halogen, OR, SR, COOR and -NRR, or two adjacent R_1; together with the carbon atoms to which they are attached, form a fused saturated, partially saturated or aromatic 5- 6 membered ring, optionally containing 1 or 2 further heteroatoms selected from N, S and/or O;

R' is H, C -C alkyl or phenyl;

or an enantiomer or a pharmaceutically acceptable salt thereof.

[0008] The compounds of the formula I or the pharmaceutical composition comprising them are particularly suitable for treatment of the inflammatory diseases rheumatoid arthritis and multiple sclerosis and for treatment of the CNS injuries cerebral ischemia and spinal cord injury.

[0009] In another aspect, the present invention provides a method for treatment of an inflammatory disease or a CNS injury, wherein said inflammatory disease is rheumatoid arthritis or multiple sclerosis and said CNS injury is cerebral ischemia or spinal cord injury, which comprises administering to an individual in need a therapeutic effective amount of a compound of formula I, an enantiomer or a pharmaceutically acceptable salt thereof.

[0010] In a further aspect, the present invention provides a method for treatment of multiple sclerosis or a CNS injury selected from cerebral ischemia and spinal cord injury which comprises administering to a subject in need a therapeutically effective amount of a compound of formula I, an enantiomer or a pharmaceutically acceptable salt thereof, thus preventing or inhibiting neuronal degeneration with or without promoting axon outgrowth.

BRIEF DESCRIPTION OF DRAWINGS

[0011] Figs. 1A-B show the effects of Compound 1 (CI) or Compound 2 (C2) administered intraperitoneally (IP), once a day (QD, panel A) or twice a day (BID, panel B) in 35 Day DBA/lOlaHsd Mouse Semi-Established Type II Collagen Arthritis on the Clinical Arthritis Score - All Paws. Treatment group n=12; normal controls n=4; Empty squares: naive mice (*d28-34), black squares: vehicle control QD (A), gray squares: vehicle control BID (B), solid diamonds: MTX (0.5 mg/kg)(*d28-34), empty circles: CI (Compound 1, 10 mg/kg)(B:#d28-32), solid circles: CI (20 mg/kg)(B: #d29-34), empty triangles: C2 (Compound 2, 10 mg/kg)(*d30-31, A), solid triangles: C2 (20 mg/kg)(*d28-34, A); *p < 0.05 ANOVA to Vehicle control (QD); #p < 0.05 ANOVA to vehicle control (BID).

[0012] Fig. 2 depicts effects of Compound 1 (CI) or Compound 2 (C2) administered IP, QD or BID in 35 Day DBA/lOlaHsd Mouse Semi-Established Type II Collagen Arthritis. Clinical Arthritis on the Score with AUC Calculation - All paws. Treatment group n=12; normal controls n=4; *p < 0.05 ANOVA to Vehicle control (QD); #p < 0.05 ANOVA to Vehicle control (BID).

[0013] Figs. 3A-B show the effects of Compound 1 (CI) or Compound 2 (C2) administered IP, QD (panel A) or BID (panel B) in 35 Day DBA/lOlaHsd Mouse Semi-Established Type II Collagen Arthritis on Incidence over time. Treatment group n=12; normal controls n=4; Empty squares: naive mice, black squares: vehicle control QD (A), gray squares: vehicle control BID (B), solid diamonds: MTX (0.5 mg/kg), empty circles: CI (10 mg/kg), solid circles: CI (20 mg/kg), empty triangles: C2 (10 mg/kg, A), solid triangles: C2 (20 mg/kg, A).

[0014] Figs. 4A-B show the effects of Compound 1 (CI) administered IP, BID either alone or in combination with Methotrexate (MTX, panel A, given per os - PO) or PO, BID either alone or in combination with ketoconazole (panel B, given PO), in 35 Day DBA/lOlaHsd Mouse Semi-Established Type II Collagen Arthritis on the Clinical Arthritis Score - All Paws. Treatment group n=10; normal controls n=4; *p < 0.05 ANOVA to Grp 2; #p < 0.05 ANOVA to Grp 6; $p < 0.05 ANOVA to Grp 7; For A: empty squares: naive mice, black squares: vehicle control (MTX vehicle QD + vehicle 1 BID, Grp 2), solid diamonds: MTX (0.5 mg/kg, Grp 3)(*d27-28), empty circles: CI (20 mg/kg), solid circles: CI (20 mg/kg) & MTX(*d26-34); for B: empty squares: naive mice, black squares: vehicle control (MTX vehicle QD + vehicle 1 BID, Grp 2), gray squares: vehicle-1 (Grp 6); empty diamonds: Ketoconazole (50 mg/kg, Grp 7); solid diamonds: MTX (0.5 mg/kg)(*d27-28), empty circles: CI (20 mg/kg), solid circles: CI (20 mg/kg) & Ketoconazole (50 mg/kg)(#d25-34; $d28-34).

[0015] Fig. 5 depicts effects of Compound 1 administered IP, BID either alone or in combination with MTX given PO, or PO, BID either alone or in combination with ketoconazole given PO, in 35 Day DBA/lOlaHsd Mouse Semi-Established Type II Collagen Arthritis. Clinical Arthritis Score with AUC Calculation - All paws. CI & Ket: CI (20 mg/kg) & Ketoconazole (50 mg/kg); Treatment group n=10; normal controls n=4; groups (left to right): Naive, vehicle (MTX vehicle QD + vehicle- 1 BID, MTX (0.5 mg/kg), CI (20 mg/kg), CI (20 mg/kg) & MTX, vehicle- 1, ketoconazole (50 mg/kg), CI (20 mg/kg), CI (20 mg/kg) & ketoconazole (50 mg/kg); *p < 0.05 ANOVA to Grp 2, @p < 0.05 ANOVA to Grp3, #p < 0.05 ANOVA to Grp 6, $p < 0.05 ANOVA to Grp 7.

[0016] Figs. 6A-B show the effects of Compound 1 (CI) administered IP, BID either alone or in combination with MTX (panel A) or PO, BID either alone or in combination with ketoconazole (panel B), in 35 Day DBA/lOlaHsd Mouse Semi- Established Type II Collagen Arthritis on Incidence Over Time. Treatment group - n=10; normal control - n=4; For A: empty squares: naive mice, solid squares: vehicle control (MTX vehicle, QD; vehicle 1 BID), solid diamonds: MTX (0.5 mg/kg), empty circles: CI (20 mg/kg), solid circles: CI (20 mg/kg) & MTX; for B: empty squares: naive mice, gray squares: vehicle- 1; empty triangles: Ketoconazole (50 mg/kg); solid triangles: MTX (0.5 mg/kg), empty circles: CI (20 mg/kg), solid circles: CI (20 mg/kg) & Ketoconazole (50 mg/kg).

[0017] Figs. 7A-B show the effects of Compound 1 (CI) administered IP, BID either alone or in combination with MTX given PO (panel A) or PO, BID either alone or in combination with ketoconazole given PO (panel B), in 35 Day DBA/lOlaHsd Mouse Semi-Established Type II Collagen Arthritis on individual histopathologic parameters in six joints (four paws and two knees). Black bars: inflammation; light gray bars: pannus; white bars: cartilage damage; dark gray bars: bone resorption; scoring: 0- normal, 1-: minimal, 2- mild, 3- moderate, 4- marked, 5- severe; treatment group n=10; normal controls n=4; *p < 0.05 ANOVA to Grp 2; #p < 0.05 ANOVA to Grp 6; $p < 0.05 ANOVA to Grp 7.

[0018] Fig. 8 depicts the effects of Compound 1 (CI) and Compound 2 (C2) administration on Experimental Autoimmune Encephalitis (EAE) clinical manifestation over time, presented as group mean score (GMS). Solid circles: vehicle control; empty circles: TV-5600 (25 mg/kg); solid square: vehicle C1/C2; solid diamonds: CI (20 mg/kg); empty diamonds: C2 (20 mg/kg). DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0019] Without limiting the scope to further possible definitions, as used in the present specification, the terms hereinbelow are defined as follows:

[0020] As used herein, the terms "administering", "administration", and like terms refer to any method which, in sound medical practice, delivers an effective amount of an active agent, practically as a component or an active ingredient in a pharmaceutical composition, to a subject in need thereof in such a manner as to provide a therapeutic effect. Thus, the phrase "therapeutically effective amount" as used herein refers to an amount of the presently described active agent effective to yield a desired therapeutic response Suitable modes of delivering the active agent or a composition therefore include oral administration as well as parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal, intrathecal, intracranial, intranasal or topical routes.

[0021] As used herein, the phrases "dosage form", "pharmaceutical dosage form", "unit dosage form" and the like, refer to the combination of one or more drug substances and one or more excipients namely, a pharmaceutical composition, that is administered to a patient in need of treatment, and can be in the form of a solution, an aqueous solution, an emulsion, a suspension, tablets, capsules, patches, suppositories, a cream, a gel, a lotion, and the like.

[0022] As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline. Some examples for materials that can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyllaurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations.

[0023] A "solid carrier" as used herein refers to one or more substances which may act as lubricants, binders, tablet disintegration agents or even an encapsulating material. Solid carriers as used herein also include diluents, flavoring agents, solubilizers and suspending agents. Non-limiting examples of suitable solid carriers include magnesium carbonate, magnesium stearate, talc, cornstarch, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methyl cellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, other cellulose derivatives, a low melting wax, cocoa butter, and the like.

[0024] As used herein, the phrase "pharmaceutically acceptable salts" refers to salts of certain ingredient(s) which possess the same activity as the unmodified compound(s) and which are neither biologically nor otherwise undesirable. A salt can be formed with, for example, organic or inorganic acids, or organic or inorganic bases. Non-limiting examples of suitable acids include acetic acid, acetylsalicylic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzoic acid, benzenesulfonic acid, bisulfic acid, boric acid, butyric acid, camphoric acid, camphorsulfonic acid, carbonic acid, citric acid, cyclopentanepropionic acid, digluconic acid, dodecylsulfic acid, ethanesulfonic acid, formic acid, fumaric acid, glyceric acid, glycerophosphoric acid, glycine, glucoheptanoic acid, gluconic acid, glutamic acid, glutaric acid, glycolic acid, hemisulfic acid, heptanoic acid, hexanoic acid, hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthylanesulfonic acid, naphthylic acid, nicotinic acid, nitrous acid, oxalic acid, pelargonic acid, phosphoric acid, propionic acid, saccharin, salicylic acid, sorbic acid, succinic acid, sulfuric acid, tartaric acid, thiocyanic acid, thioglycolic acid, thiosulfuric acid, tosylic acid, undecylenic acid, and natural and synthetically derived amino acids.

[0025] Non-limiting examples of suitable bases include, for example, low molecular weight alkanolamines such as ethanolamine, diethanolamine, N-ethylethanolamine, N- methyldiethanolamine, triethanolamine, diethylaminoethanol, 2-amino-2-methyl-n- propanol, dimethylaminopropanol, 2-amino-2-methylpropanediol, and triisopropanolamine. Poorly volatile bases, for example, ethylenediamine, hexamethylenediamine, morpholine, piperidine, piperazine, cyclohexylamine, tributylamine, dodecylamine, N,N-dimethyldodecylamine, stearylamine, oleylamine, benzylamine, dibenzylamine, N-ethylbenzyl amine, dimethylstearylamine, N- methylmorpholine, N-methylpiperazine, 4-methylcyclohexylamine, and N- hydroxyethylmorpholine.

[0026] As used herein, the terms "POSH," "POSH protein(s)" or "POSH polypeptide(s)" are used interchangeably and refer to a polypeptide that includes in its amino acid sequence a RING domain and at least one SH3 domain. In some instances, the POSH protein may have 3 or 4 SH3 domains.

[0027] The terms "POSH-mediated ubiquitination" or "POSH protein-mediated ubiquitination" are used interchangeably and refer to any ubiquitination process that requires the involvement of a POSH protein.

[0028] The terms "ubiquitination inhibitor", "POSH inhibitor" or "POSH protein inhibitor" are used interchangeably and refer to a pyrimidine derivative of formula I herein that inhibits a POSH activity as defined in PCT/US02/36366 (WO 03/095972), hereby incorporated herein by reference in its entirety as if fully disclosed herein, including POSH protein-mediated ubiquitination.

[0029] As used herein, the terms "subject" or "individual" or "animal" or "patient" or "mammal," refers to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired, for example, a human.

[0030] As used herein, the terms "treatment" or "treating" of a disease or injury encompasses alleviation of at least one symptom thereof, a reduction in the severity thereof, or the delay, prevention, or inhibition of the progression thereof. Treatment need not mean that the disease or injury is totally cured. To be an effective treatment, a useful composition herein needs only to reduce the severity of the disease or injury, reduce the severity of symptoms associated therewith, delay the onset of clinical symptoms, or provide improvement to a patient or subject's quality of life.

[0031] It has been found in accordance with the present invention that when small pyrimidine derivatives defined by the formula I below, and known to act as POSH inhibitors, were administered to a mouse model for human rheumatoid arthritis, they were capable of attenuating increase in symptoms of the disease, for example as measured by the Clinical Arthritis Score, and delaying the onset of the disease.

[0032] It has been found in accordance with the present invention that treatment with small pyrimidine derivatives of formula I significantly reduced the severity of the clinical symptoms in a mouse model of human multiple sclerosis along the disease course, and that administration of such compounds significantly postponed the disease onset.

[0033] Thus, in one aspect, the present invention provides a compound of formula I or a pharmaceutical composition comprising it for use in treatment of an inflammatory disease or a central nervous system (CNS) injury, wherein said inflammatory disease is rheumatoid arthritis or multiple sclerosis and said CNS injury is cerebral ischemia or spinal cord injury:

wherein

X is O, S or NH;

R is H or Ci-Ce alkyl;

Ri is H, Ci-Ce alkyl, OR' , SR', halogen, COOR', S0₂R', unsubstituted C₆-C₁₀ aryl or C6-Cio aryl substituted by one or more radicals selected from C -C alkyl, halogen, nitro, halogen, OR, SR, COOR and -NRR, or two adjacent R_{1 ;} together with the carbon atoms to which they are attached, form a fused saturated, partially saturated or aromatic 5- 6 membered ring, optionally containing 1 or 2 further heteroatoms selected from N, S and/or O;

R' is H, CrC₆ alkyl or phenyl;

or an enantiomer or a pharmaceutically acceptable salt thereof. [0034] In certain embodiments, the alkyl of the compound of formula I is a straight or branched CrC₆ alkyl, preferably a lower alkyl of Ci-C₄ atoms such as methyl, ethyl, n- propyl, isopropyl, n-butyl, isobutyl, or tert-butyl. In one preferred embodiment the lower alkyl is methyl.

[0035] Ri may be H, Ci-C₆ alkyl, halogen, OR', SR', COOR', -S0₂R', or C₆-Ci₀ aryl which may be substituted by one or more radicals selected from CrC₆ alkyl, halogen, nitro, OR, SR, COOR and -NRR, wherein R' is H, CrC₆ alkyl, or phenyl, or two adjacent Ri together with the carbon atoms to which they are attached, form a fused saturated, partially saturated or aromatic 5-membered or 6-membered ring, optionally containing 1 or 2 further heteroatoms selected from N, S and/or O.

[0036] In certain embodiments, the group -NRR is the free amino group -NH₂, when the two Rs are both hydrogen, or a secondary amino group when one R is H and the other R is Ci-C₄ alkyl, or a tertiary amino group when both Rs are each CrC₄ alkyl.

[0037] Aryl as defined herein is a C6-Cio aromatic carbocyclic group, particularly phenyl or naphthyl that may be substituted by one or more radicals as defined herein above.

[0038] Halogen includes fluoro, chloro, bromo or iodo. In some embodiments, the halogen is chloro.

[0039] In certain preferred embodiments, the compound used in accordance with the present invention in a pyrimidine derivative of the formula I above, wherein X is S, and R and Ri each independently is H or methyl.

[0040] The presently described compounds of formula I can be readily produced by the methods disclosed in WO 2008/056356 or, alternatively, by methods well known to those of ordinary skill in the art, without undue experimentation.

[0041] In certain more preferred embodiments, particular compounds are used for treatment of an inflammatory disease or a CNS injury, identified herein as Compounds 1, 2, 3 and 4, wherein Compound 1 is:

Compound 2 is:

Compound 3 is:

[0042] In certain embodiments, the inflammatory disease treatable by any of the Compounds 1-4 is rheumatoid arthritis. [0043] It certain other embodiments, Compound 1 is for use for the treatment of an inflammatory disease or a CNS injury, most preferably for treatment of rheumatoid arthritis.

[0044] It has further been found in accordance with the present invention that the beneficial effect for treatment of RA of administering a combination of the small pyrimidine derivatives of formula I together with the anti-inflammatory methotrexate (MTX) was greater than the beneficial effect of each one of these active agents when administered separately, thus showing a synergistic effect. Hence, it can be expected that this combination may provide an equal or greater beneficial effect to that obtained with the pyrimidine derivative alone even if the pyrimidine derivative and/or methotrexate are used at sub-therapeutic dosages.

[0045] Accordingly, in certain embodiments, for the treatment of rheumatoid arthritis, the compound of formula I is combined with one or more drugs commonly used for the treatment of this disease, such as, but not limited to, corticosteroids, such as for example, dexamethasone, methylprednisolone or prednisone, disease-modifying anti-rheumatic drugs e.g. MTX and hydroxychloroquine, and/or biological treatments such as TNF-a and IL-1 blockers.

[0046] In certain embodiments, the compound of formula I or the pharmaceutical composition comprising it are used in combination with methotrexate for the treatment of rheumatoid arthritis. In particular embodiments, Compound 1 is used in a combined therapy with MTX.

[0047] According to the literature, the recommended therapeutic dose of MTX for treatment of rheumatoid arthritis is 7.5 mg/week (either single dose or three equal partial doses).

[0048] The compound of formula I, when given in combination with MTX, may be administered at a dose that is a therapeutically effective dose when given alone or a dose that is sub-therapeutic when given alone; likewise MTX, when given in combination with an compound of formula I, may be administered at a dose that is a therapeutically effective dose when given alone or at a dose that is sub-therapeutic when given alone. Since the combined effect of the two compounds is synergistic, the combination therapy when at least one of the compounds is given at a sub-therapeutic dose can be expected to produce a therapeutic effect.

[0049] Importantly, it was found that the small pyrimidine derivatives of formula I were efficacious both when injected and when given as an oral formulation with an agent, in this specific case ketoconazole, which increases their bioavailability.

[0050] In certain embodiments of the invention, treatment of rheumatoid arthritis comprises reduction of inflammation, reduction of pannus infiltration, reduction of cartilage damage, and/or reduction of bone resorption.

[0051] In some embodiments, treatment of rheumatoid arthritis with the compound of formula I or the pharmaceutical composition comprising it comprises reduction of inflammation score by at least 40%, reduction of pannus score by at least 60%, reduction of cartilage damage by at least 55%, and/or reduction of bone resorption by at least 60%. In some embodiments, combined therapy of rheumatoid arthritis, comprising the use of the compound of formula I or the pharmaceutical composition comprising it in combination with a MTX, comprises reduction of inflammation score by at least 80%, reduction in pannus by at least 90%, reduction in cartilage damage by at least 80%, and/or reduction in bone resorption by at least 90%.

[0052] In certain embodiments, the compound or a pharmaceutical composition comprising it and MTX are for either sequential or concomitant administration.

[0053] In certain embodiments, the compound of formula I or a pharmaceutical composition comprising it is for chronic administration, alone or in combination with MTX.

[0054] In certain embodiments, the compound of formula I and MTX are administered in two separate compositions, in other embodiments in a single composition. In other words, the compound and MTX may be in a binary composition comprising one unit dose comprising a compound of formula I, and one unit dose comprising methotrexate; or the two compounds may be in a unitary pharmaceutical composition (single dosage form).

[0055] In certain embodiments, the compound of formula I, when administered alone, is preferably administered in a therapeutically effective amount. In particular embodiments, the compound of formula I is administered orally to the patient. In other embodiments, the active agent is injected to the patient.

[0056] In certain embodiments, in a combined therapy with MTX, the binary composition or the single dosage form is delivered to the subject via oral administration or by injection.

[0057] In certain embodiments, the compound of formula I or the pharmaceutical composition comprising it and MTX are administered orally. Alternatively, the compound is administered by injection and MTX is administered orally.

[0058] When the compound is administered by injection, either alone or in a combined therapy with MTX it is injected to the patient once a day, twice a day or three times a day; in particular embodiments, twice a day.

[0059] Generally, the actual amount administered and the rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc., is within the responsibility of general practitioners or specialists, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington's Pharmaceutical Sciences.

[0060] Multiple sclerosis (MS) is an inflammatory autoimmune disease that attacks myelinated axons in the central nervous system leading to damage of the fatty myelin sheaths around the axons of the brain and spinal cord. The demyelination is expressed in neurological symptoms including changes in sensation such as loss of sensitivity or tingling, pricking or numbness (hypoesthesia and paresthesia), muscle weakness, clonus, muscle spasms, or difficulty in moving.

[0061] Several subtypes or patterns of progression have been described for MS. The relapsing-remitting subtype, which describes the initial course of 80% of individuals with MS, is characterized by unpredictable relapses followed by periods of months to years of relative quiet (remission) with no new signs of disease activity. Deficits suffered during attacks may either resolve or leave sequelae, the latter being more common as a function of time. [0062] Secondary progressive MS (sometimes called "galloping MS") describes around 65% of those with an initial relapsing-remitting MS, who then begin to have progressive neurologic decline between acute attacks without any definite periods of remission. Occasional relapses and minor remissions may appear. The median time between disease onset and conversion from relapsing-remitting to secondary progressive MS is 19 years. The primary progressive subtype describes the approximately 10-15% of individuals who never have remission after their initial MS symptoms. It is characterized by progression of disability from onset, with no, or only occasional and minor, remissions and improvements. The age of onset for both cases is around 40 years of age, but for the primary progressive subtype is later than for the relapsing-remitting. Progressive relapsing MS describes those individuals who, from onset, have a steady neurologic decline but also suffer clear superimposed attacks. This is the least common of all subtypes

[0063] Thus, in certain embodiments, the compound of formula I or the pharmaceutical composition comprising it is for use in treatment of multiple sclerosis.

[0064] The compound of formula I, when used for treatment of MS, may be administered in conjunction with a known drug for treatment of multiple sclerosis selected from Copaxone®, Avonex®, Betaseron®, Extavia® or Rebif®, Tysabri (Natalizumab) GyleniaTM (fingolimod) and BG-12 (dimethyl fumarate).

[0065] In some embodiments, the compound of formula I or the pharmaceutical composition comprising it, alone or in conjunction with a known drug for treatment of multiple sclerosis, is for use in treatment of relapsing-remitting multiple sclerosis. In other embodiments the compound of formula I or the pharmaceutical composition comprising it, alone or in conjunction with a known drug for treatment of multiple sclerosis, is for use in treatment of secondary progressive multiple sclerosis or primary progressive multiple sclerosis.

[0066] In certain embodiments, treatment of multiple sclerosis comprises reduction in weight loss, attenuation in increase in time of responses to stimuli, reduction in tremors, attenuation in loss of muscle tone, reduction in leg weakness and/or reduction in paralysis.

[0067] In certain embodiments, the compound of formula I said known drug for treatment of multiple sclerosis are for either sequential or concomitant administration. [0068] In certain embodiments, the compound is for chronic administration, alone or in combination with said known drug for treatment of multiple sclerosis.

[0069] In other embodiments, the compound and said known drug for treatment of multiple sclerosis are either in two separate dosage forms and the pharmaceutical composition is a binary composition, or, alternatively, in a single, unitary dosage form. The binary composition of the single unit composition may be administered orally or via injection to the subject in need thereof.

[0070] In still other embodiments, both the compound of formula I and said known drug for treatment of multiple sclerosis either in the form of a single composition or in the form of a binary composition, are administered orally. Alternatively, the compound is separately administered by injection and the known drug for treatment of multiple sclerosis is administered orally.

[0071] When the compound is administered by injection, either alone or in a combined therapy with the known drug for treatment of multiple sclerosis, it is injected to the patient once a day, twice a day or three times a day; in particular embodiments, twice a day.

[0072] In certain embodiments, the compound of formula I that is used for treatment of multiple sclerosis either alone or in combined therapy with a known drug for treatment of multiple sclerosis is Compound I.

[0073] In certain embodiments, the compound of formula I or the pharmaceutical composition comprising it is for use in the treatment of multiple sclerosis or a CNS injury selected from cerebral ischemia or spinal cord injury, by preventing or inhibiting neuronal degeneration with or without promoting axon outgrowth.

[0074] In certain embodiments, the compound of formula I, in particular compound 1, or the pharmaceutical composition comprising it, is for use in treatment that promotes axon outgrowth.

[0075] In some embodiments, the compound of formula I, in particular compound 1, or the pharmaceutical composition comprising it, is for use in treatment of an inflammatory or neurodegenerative condition associated with dysregulated inflammatory cytokine expression. [0076] In certain embodiments, the treatment of an inflammatory or neurodegenerative condition associated with dysregulated inflammatory cytokine expression is applied in order to regulate the pro-inflammatory cytokines TNF-a, IL-1 IL-6, IFN-γ or 11-17.

[0077] In a further embodiment, the compound of formula I is for use in treatment that protects against nerve degeneration. The action of the compound attenuates or inhibits both acute neurodegeneration caused by acute injury such as stroke, cerebral or hemorrhagic ischemia, or spinal cord injury, and chronic neurodegeneration that follows acute injury or disease of the nervous system.

[0078] In another aspect, the present invention provides a method for treatment of an inflammatory disease or a CNS injury, wherein said inflammatory disease is rheumatoid arthritis or multiple sclerosis and said CNS injury is cerebral ischemia or spinal cord injury, comprising administering to a subject in need a therapeutically effective amount of a compound of general formula I as defined above, in particular compound 1, or a pharmaceutical composition comprising it.

[0079] In other aspects, the present invention provides methods for promoting axon outgrowth and treatment of inflammatory or neurodegenerative conditions associated with dysregulated inflammatory cytokine expression, comprising administering to a subject in need a therapeutically effective amount of a compound of general formula I as defined above, in particular compound 1, or a pharmaceutical composition comprising it.

[0080] In certain embodiments, the method of protection against nerve degeneration or the method for promoting axon outgrowth is applied to a subject suffering from multiple sclerosis, cerebral ischemia or spinal cord injury.

[0081] In certain embodiments, the method for promoting axon outgrowth is applied, in accordance with the invention, to a subject in need that suffers from spinal cord injury.

[0082] The compound may be used, in accordance with the present invention, in the form of one of its pharmaceutically acceptable salts formed with an organic or inorganic acid, or with an organic or inorganic base. The suitable acid or base which may be used for the preparation of the pharmaceutically acceptable salt may be any of the acids or bases mentioned above, depending on the compound used. If organic bases are used, poorly volatile bases are preferably employed such as any of those mentioned above, more preferably ethanolamine.

[0083] Further pharmaceutically acceptable salts that can be used in accordance with the invention are basic salts of quaternary ammonium hydroxides such as trimethylbenzylammonium hydroxide, tetramethylammonium hydroxide, or tetraethylammonium hydroxide, as can guanidine and its derivatives, in particular its alkylation products. However, it is also possible to employ as salt-forming agents, for example, low molecular weight alkylamines such as methylamine, ethylamine, or triethylamine. Other suitable salts useful for the purpose of the invention are salts with inorganic cations, for example alkali metal salts, in particular sodium, potassium, or ammonium salts, alkaline earth metal salts such as, in particular, the magnesium or calcium salts, as well as salts with bi- or tetravalent cations, for example the zinc, aluminum, or zirconium salts.

[0084] Also contemplated are salts with organic bases, such as dicyclohexylamine salts; methyl-D-glucamine; and salts with amino acids, such as arginine, lysine, and so forth. Also, the basic nitrogen-containing groups can be quatemized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates, such as dimethyl, diethyl, dibutyl, and diamyl sulfates; long chain halides, such as decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; asthma halides, such as benzyl and phenethyl bromides; and others. Aqueous or oil-soluble or dispersible products are thereby obtained.

[0085] The pharmaceutical composition used in accordance with the invention comprises at least one active agent in its original form or in its pharmaceutically acceptable salt form, and one or more pharmaceutically acceptable carriers or excipients.

[0086] The carrier may comprise, in total, from about 0.1 % to about 99.99999% by weight of the pharmaceutical composition, and be any of the non-toxic, inert, and effective carriers defined above or known in the art.

[0087] The pharmaceutical compositions useful for the purpose of the invention are preferably in the form of oral compositions. The oral compositions contemplated herein may take the form of tablets, capsules, soft-gels, hard gels, solutions, suspensions, powders, dispersible granules, cachets, combinations thereof, or any other oral pharmaceutical dosage form as commonly known in the art. The tablets may be coated by methods well known in the art. Preparations for oral administration may be suitably formulated to give controlled release of the active agent.

[0088] Powdered formulations used in accordance with the present invention usually comprise a solid carrier. The carrier can be a finely divided solid which is in admixture with the compound. In a tablet, the active agent can be mixed with a carrier having the necessary binding properties in suitable proportions and compacted in the size and shape desired. Any of the solid carriers mentioned above may be employed in the solid pharmaceutical compositions used according to the invention.

[0089] Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p- hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

[0090] In order to improve absorption of the active agent, the oral formulation may be supplemented with solubilizers or absorption rheumatoid enhancers such as surface-active agents, e.g. surfactants, steroidal detergents, acylcarnitines, and alkanoylcholines, liposomes, mucoadhesive polymers, prodrug modification, nano- and microparticles, modifications of known bacterial intestinal toxins, n-acetylated a-amino acids and n- acetylated non-a-amino acids or ketoconazole.

[0091] For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the compounds of the formula I used in accordance with the invention can be formulated in liquid solutions, for example in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the compounds may be formulated in solid form and re-dissolved or suspended immediately prior to use. Lyophilized forms are also included. [0092] The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0093] For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetra-fluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0094] The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

[0095] Systemic administration can also be by transmucosal or trans dermal means. For transmucosal or trans dermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays or using suppositories. For topical administration, the presently described compounds are formulated into ointments, salves, gels, or creams as generally known in the art. A wash solution can be used locally to treat an injury or inflammation to accelerate healing.

[0096] In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0097] Any non-toxic, inert, excipients known in the art may be used to formulate the compositions used in accordance with the present invention. Wetting agents (such as sodium lauryl sulphate), fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate), disintegrants (e.g., potato starch or sodium starch glycolate) and lubricantssulphate (e.g., magnesium stearate, talc or silica), as well as coloring agents, flavoring agents, tabletting agents, stabilizers, antioxidants, and preservatives may also be present. Suitable pharmaceutically acceptable carriers, excipients, and diluents well known to those of skill in the art, are described in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, N.J. (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association); International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the "Inactive Ingredient Guide", U.S. Food and Drug Administration (FDA), Centre for Drug Evaluation and Research (CDER) Office of Management. Examples of pharmaceutically acceptable excipients, carriers and diluents useful in the present compositions include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO.

[0098] These additional inactive components, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks, such as Goodman and Gillman's: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergamon Press (1990), and Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990).

[0099] The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

[00100] Although the invention has been described in detail, nevertheless changes and modifications, which do not depart from the teachings of the present invention, will be evident to those skilled in the art. Such changes and modifications are deemed to come within the purview of the present invention and the appended claims.

[00101] The invention will now be illustrated by the following non-limiting examples.

EXAMPLES

Example 1. Compounds 1 and 2 are efficient in treating a type II collagen-induced arthritis model.

Introduction

[00102] Mice (DBA/llacJ, 1J or B10R111) reliably develop polyarthritis when immunized against bovine type II collagen (1) using a variety of methodologies including day 0, day 15, day 16, or day 21 immunizations with and without concurrent boosting with endotoxin or recombinant IL-1 (2). The disease that occurs is usually not symmetrical and any combination of paws/joints may be affected. Since caliper measurement of small mouse ankles is challenging, subjective clinical scoring systems are often used in conjunction with histologic scoring methods. Treatments can be prophylactic (starting before or at the time of immunization), semi-established (starting any time after immunization and up to the boost), or therapeutic (after observation of lesions) and, depending on the immunization protocol used and extent of destruction desired, can extend from 10 days to several weeks. Lesions in affected joints resemble those occurring in rat collagen arthritis (2). This model has been particularly useful in evaluating the effects of clinically used biologic agents such as Interleukin- 1 receptor antagonist (IL-lra) and the soluble TNF receptors (3-7). Enhancement of disease incidence and severity has been demonstrated in mice immunized with type II collagen and concurrently given cytokines such as IL-1 (8- 9).

[00103] The current study was conducted to determine the efficacy of Compound 1 administered twice daily (BID) or daily (QD) or Compound 2 administered QD on days 18-33 by the intraperitoneal (IP) route for inhibition of the inflammation (paw swelling), cartilage destruction, and bone resorption that occurs in 35-day semi-established type II collagen arthritis in mice. Materials and Methods

Test Article Identification and Preparation. The sponsor supplied the test articles (Compound 1, Compound 2). Bolder BioPATH, Inc. supplied the methotrexate (MTX). See protocol for calculations and specifics of dose preparation in vehicle for dosing at 5 ml/kg (10 ml/kg for MTX). The vehicle was 5% DMSO, 5% PEG400, 5% Tween 80 and 1% meglumine water solution (500 mg/g) in 30% ΗΡ-β-CD. The vehicle for MTX was 1% carboxymethylcellulose (CMC).

Test System Identification. Male DBA/lOlaHsd mice (n=112) that were 5-7 weeks old on arrival and weighed approximately 16-22 grams (mean 20 g) on study day 18 were obtained from Harlan, Inc., Indianapolis, IN. Mice were at least 6 weeks old at time of first immunization. Animals were identified by a distinct number of ink marks at the base of the tail delineating animal number. After enrollment, all cages were labeled with protocol number, group and animal numbers.

Environment and Husbandry. Upon arrival, animals were housed 4/cage in shoe-box polycarbonate cages with wire tops, wood chip bedding, and suspended food and water bottles. Animal care including room, cage, and equipment sanitation conformed to the guidelines cited in the Guide for the Care and Use of Laboratory Animals (10) and the applicable standard operating procedures of Bolder BioPATH, Inc. Animals were acclimated for 11 days prior to being placed in the study. An attending veterinarian was on site or on call during the live phase of the study. No concurrent medications were given. During the acclimation and study periods, animals were housed in a laboratory environment with temperatures ranging 67-76°F and relative humidity of 30-70%. Automatic timers provided 12 hours of light and 12 hours of dark. Animals were allowed access ad libitum to Harlan Teklad Rodent Chow and fresh municipal tap water.

Experimental Design. Animals (12/treatment group, 4/group for normal) were anaesthetized with Isoflurane, shaved at the base of the tail, and injected intradermally with 150 μΐ of Freund's Complete Adjuvant (CFA, Sigma) containing bovine type II collagen (Elastin Products, Owensville, MO) (2 mg/ml) at the base of the tail on day 0 and again on day 21. On study day 18, mice were randomized by body weight into treatment groups, and treatment (BID or QD at 12 or 24 hour intervals) was initiated. On study days 24-34, onset of arthritis occurred. Mice were terminated on day 34. Clinical scores for each of the paws (right front, left front, right rear, left rear) were given on study days 18-34.

[00104] Experimental groups were as described in Table 1.

Table 1: experimental groups

Ctrl: vehicle control; naive - non-arthritic, untreated mice; MTX: methotrexate (0.5mg/kg); cmpd: treatment (compound); NA: non applicable; dose level - mg/kg; dose - mg/ml (all 5ml/kg except for Grp 4 - 10 ml/kg); CI: compound 1; C2: compound 2

Observations, measurements, and specimens

[00105] Mice were weighed on study days 18, 20, 22, 24, 26, 28, 30, 32 and 34

(prior to necropsy). Daily clinical scores were given for each of the paws (right front, left front, right rear, left rear) on study days 18-34 using the following criteria.

0= normal

1=1 hind or fore paw joint affected or minimal diffuse erythema and swelling

2=2 hind or fore paw joints affected or mild diffuse erythema and swelling

3=3 hind or fore paw joints affected or moderate diffuse erythema and swelling 4= Marked diffuse erythema and swelling, or =4 digit joints affected)

5=Severe diffuse erythema and severe swelling entire paw, unable to flex digits). [00106] At necropsy, all animals were bled via cardiac puncture for serum and terminated via cervical dislocation. Fore paws, hind paws and knees were removed and placed in NBF. Whole blood was allowed to clot at room temperature for approximately 2 hours before being spun at 13,000 rpm for 8 minutes. Sera was frozen at -20°C and shipped to sponsor further analysis.

Morphologic Pathology Methods. After 1-2 days in fixative and 4-5 days in 5% formic acid for decalcification, tissues were trimmed, processed for paraffin embedding, sectioned at 8 μιη and stained with toluidine blue (T blue). Hind paws, fore paws, and knees were embedded and sectioned in the frontal plane. Six joints from each animal were processed for histopathology evaluation. The joints were then scored as indicated below.

[00107] When scoring paws or ankles from mice with lesions of type II collagen arthritis, severity of changes as well as number of individual joints affected must be considered. When only 1-3 joints of the paws or ankles out of a possibility of numerous metacarpal/metatarsal/digit or tarsal/tibiotarsal joints were affected, an arbitrary assignment of a maximum score of 1, 2 or 3 for parameters below was given depending on severity of changes. If more than 3 joints were involved, the criteria below were applied to the most severely affected/majority of joints.

Inflammation

• 0 = Normal

• 1 = Minimal infiltration of inflammatory cells in synovium and periarticular tissue of affected joints

• 2 = Mild infiltration of inflammatory cells. If referring to paws, generally restricted to affected joints (1-3 affected)

• 3 = Moderate infiltration with moderate edema. If referring to paws, restricted to affected joints, generally 3-4 joints + wrist or ankle

• 4 = Marked infiltration affecting most areas with marked edema, 1 or 2 unaffected joints may be present

• 5 = Severe diffuse infiltration with severe edema affecting all joints and periarticular tissues.

[00108] The inflammatory infiltrate in mice and rats with type II collagen arthritis consists of neutrophils and macrophages with smaller numbers of lymphocytes when the lesions are in the acute to subacute phase. Tissue edema and neutrophil exudates within the joint space are common in the acute to subacute phase. As the inflammation progresses to chronic, mononuclear inflammatory cells (monocytes, lymphocytes) predominate and fibroblast proliferation, often with deposition of metachromatic matrix, occurs in synovium and periarticular tissue. Exudate is less common in the joint space. Unless indicated in the comments area, the inflammation type is acute to subacute.

[00109] DBA mice have an increased incidence of dactylitis and onchyoperiostitis affecting the nail bed and distal phalynx (11). These lesions are recorded in the comments section but are not included in the CIA score.

Pannus

• 0 = Normal

• 1 = Minimal infiltration of pannus in cartilage and subchondral bone, marginal zones

• 2 = Mild infiltration with marginal zone destruction of hard tissue in affected joints

• 3 = Moderate infiltration with moderate hard tissue destruction in affected joints

• 4 = Marked infiltration with marked destruction of joint architecture, affecting most joints

• 5 = Severe infiltration associated with total or near total destruction of joint architecture, affects all joints.

Cartilage Damage

• 0 = Normal

• 1 = Minimal = generally minimal to mild loss of toluidine blue staining with no obvious chondrocyte loss or collagen disruption in affected joints

• 2 = Mild = generally mild loss of toluidine blue staining with focal areas of chondrocyte loss and/or collagen disruption in some affected joints

• 3 = Moderate = generally moderate loss of toluidine blue staining with multifocal chondrocyte loss and/or collagen disruption in affected joints, some matrix remains on any affected surface with areas of severe matrix loss

• 4 = Marked = marked loss of toluidine blue staining with multifocal marked (depth to deep zone) chondrocyte loss and/or collagen disruption in most joints, if knee-one surface with total to near total cartilage loss • 5 = Severe = severe diffuse loss of toluidine blue staining with multifocal severe (depth to tide mark) chondrocyte loss and/or collagen disruption in all joints, if knee-2 or more surfaces with total to near total cartilage loss.

Bone Resorption

• 0 = Normal

• 1 = Minimal = small areas of resorption, not readily apparent on low magnification, rare osteoclasts in affected joints, restricted to marginal zones

• 2 = Mild = more numerous areas of resorption, not readily apparent on low magnification, osteoclasts more numerous in affected joints, restricted to marginal zones

• 3 = Moderate = obvious resorption of medullary trabecular and cortical bone without full thickness defects in cortex, loss of some medullary trabeculae, lesion apparent on low magnification, osteoclasts more numerous in affected joints

• 4 = Marked = Full thickness defects in cortical bone, often with distortion of profile of remaining cortical surface, marked loss of medullary bone, numerous osteoclasts, affects most joints

• 5 = Severe = Full thickness defects in cortical bone and destruction of joint architecture of all joints.

[00110] For each animal, the inflammation, pannus, cartilage damage and bone damage scores were determined. A sum total animal score was determined as well as sums and means for each of the individual parameters. The mean scores are presented in Figs 1- 2.

[00111] Neutrophil counts were done in sections by counting the number of neutrophils in a typical heavily infiltrated 40x objective field and then estimating how many such fields were present in the worst affected section of paw on each slide (2 paw sections were present on each slide). Only neutrophils in the dermis and joint space were counted - not neutrophils in the bone marrow. Overall inflammation was also scored in the sections on a semi-quantitative scale from 0-5, as follow:

• 0 = no inflammation

• 1 = minimal inflammation

• 2 = mild inflammation

• 3 = moderate inflammation • 4 = marked inflammaton

• 5 = severe inflammation

Animal Disposal. Animal carcasses were disposed of according to Bolder BioPATH, Inc. procedures.

Statistical Analysis

[00112] Clinical data for paw scores (means for animal) were analyzed by determining the area under the dosing curve (AUC) for arthritis days 18-34. For calculation of AUC, the daily mean scores for each mouse were entered into Microsoft Excel and the area between the treatment days and the final day was computed. Means for each group were determined and % inhibition from arthritis controls was calculated by comparing values for treated and normal animals. Data were analyzed using a Student's t-test or Mann-Whitney U test (non-parametric). If applicable, data were further analyzed across all groups using a oneway analysis of variance (1-way ANOVA) or Kruskal-Wallis test (non- parametric), along with the appropriate multiple comparison post-test. ANOVAs were calculated using Prism 5.0c software (GraphPad). Unless indicated, Bolder BioPATH, Inc. performs statistical analysis on raw (untransformed) data only. Statistical tests make certain assumptions regarding the data's normality and homogeneity of variance, and further analysis may be required if testing resulted in violations of these assumptions. Significance for all tests was set at p<0.05.

% Inhibition=B/A X 100

A=Mean Normal - Mean Disease Control

B=Mean Treated - Mean Disease Control

Specimen and Raw Data Storage. Samples (slides, blocks, serum), study data, and reports were delivered to sponsor either during or after the completion of the study.

Statement of Effect of Deviations on the Quality and Integrity of the Study. There were no deviations from the study protocol.

Results

Live Phase and Necropsy Parameters

[00113] Disease parameters for mice treated BID or QD with vehicle were similar

(see Figures la-5b). [00114] Daily body weight measurements were not significantly affected for mice in any treatment group as compared to vehicle treated disease controls (not shown).

[00115] Daily clinical arthritis scores were significantly reduced (by ANOVA) toward normal for mice treated with 20 mg/kg Compound 2 (at days 28-31) or MTX (d30, 31, 33) when compared to QD vehicle controls. When compared to vehicle controls using a Mann-Whitney U test, daily clinical arthritis scores were significantly reduced for mice treated with 10 mg/kg Compound 2 (d30-31), 20 mg/kg Compound 2 (d28-34), or MTX (d28-34) as compared to QD vehicle controls, and for mice treated with 10 mg/kg Compound 1 BID (d29-32) or 20 mg/kg Compound 1 BID (d30-34) as compared to BID vehicle controls (Figs. 1A-B).

[00116] When compared to vehicle controls using a Mann-Whitney U test, clinical scores AUC were significantly reduced toward normal for mice treated with 20 mg/kg Compound 2 (43% reduction, p = 0.009), 10 mg/kg Compound 1 BID (34%, p = 0.046), or MTX (44%, p = 0.012) as compared to the respective QD or BID vehicle controls (Figs. 2A-A).

[00117] Disease control animals treated with vehicle (BID or QD) and mice treated with 10 mg/kg Compound 1 QD had 100% disease incidence by study day 28. Mice treated with 20 mg/kg Compound 1 QD or 10 mg/kg Compound 2 had 100% incidence by day 29. MTX treated mice had 100% disease incidence by day 32. Reduced disease incidence was recorded for mice treated with 10 mg/kg Compound 1 BID (92% disease incidence at study termination), 20 mg/kg Compound 1 BID (67%), or 20 mg/kg Compound 2 (92%) (see Figs. 3A-B).

Morphologic Pathology

[00118] Disease control animals had histopathology changes consistent with those seen in type II collagen-induced arthritis (CIA) in most joints with scores ranging from normal to severe. Microscopic alteration included infiltration of synovium and periarticular tissue with neutrophils and mononuclear inflammatory cells (inflammation), marginal zone pannus and bone resorption, and cartilage damage (proteoglycan loss, chondrocyte death, and collagen matrix destruction) (data not shown).

[00119] Histopathologic evaluation revealed that mice treated with MTX had significantly (by ANOVA) reduced six -joint mean inflammation (43% reduction), cartilage damage (58%), bone resorption (57%), and mean animal score (51%) as compared to QD vehicle controls. When compared to the respective (BID or QD) vehicle controls using a Mann- Whitney U test, MTX treated mice also had significantly reduced six -joint mean pannus (55% reduction, p = 0.007), and mice treated with 20 mg/kg Compound 1 BID had significantly reduced six-joint mean inflammation (49%, p = 0.019), pannus (51%, p = 0.032), cartilage damage (54%, p = 0.019), bone resorption (51%, p = 0.042), and mean animal score (51%, p = 0.019) (data not shown).

[00120] Mice treated with MTX had significantly (by ANOVA) reduced paw cartilage damage (51% reduction) as compared to QD vehicle controls. When compared to the respective (BID or QD) vehicle controls using a Mann-Whitney U test, MTX treated mice also had significantly reduced paw inflammation (38% reduction, p = 0.018), pannus (48%, p = 0.024), bone resorption (50%, p = 0.020), and mean animal score (44%, p = 0.014); and mice treated with 20 mg/kg Compound 1 BID had significantly reduced paw inflammation (49%, p = 0.016), pannus (50%, p = 0.034), cartilage damage (49%, p = 0.035), bone resorption (48%, p = 0.037), and mean animal score (49%, p = 0.022) (data not shown).

[00121] Mice treated with MTX had significantly (by ANOVA) reduced knee pannus (86% reduction), cartilage damage (70%), bone resorption (85%), and mean animal score (68%) as compared to QD vehicle controls. When compared to the respective (BID or QD) vehicle controls using a Mann-Whitney U test, MTX treated mice also had significantly reduced paw inflammation (57% reduction, p = 0.006); mice treated with 10 mg/kg Compound 1 QD had significantly reduced cartilage damage (33%, p = 0.048); mice treated with 20 mg/kg Compound 1 QD had significantly reduced cartilage damage (44%, p = 0.015); mice treated with 20 mg/kg Compound 1 BID had significantly reduced cartilage damage (65%, p = 0.028); and mice treated with 10 mg/kg Compound 2 had significantly reduced cartilage damage (44%, p = 0.029) and mean animal score (41%, p = 0.049) (data not shown).

Conclusions

[00122] Results of BID treatment with Compound 1 or QD treatment with

Compound 2 were significant by t-test for some clinical and histopathology parameters. Example 2. Compound 1 is efficient in treating a type II collagen-induced arthritis model alone or in combination with MTX.

Introduction and Objectives

[00123] Determine efficacy of potential anti-inflammatory agent administered (IP or per os (PO), QD or BID) in inhibiting the inflammation, pannus formation, cartilage destruction and bone resorption associated with semi-established type II collagen arthritis in mice.

Test System

[00124] Species/Strain or Breed: 100 (104 order 5-7 weeks of age) Male

DBA/lOlaHsd (Harlan)

[00125] Age Range at Study Initiation: At least 6 weeks at time of first immunization. -18 grams.

[00126] Acclimation: Will be acclimated for enough days after arrival at BBP that all animals are at least 6 weeks old.

[00127] Housing: 3-5/animals/cage

Materials

[00128] Agent (TX) in vehicle, Type II collagen (Elastin Products), Freund's complete adjuvant (with supplemental M. tuberculosis (MT), 4 mg/ml) (Difco).

General Study Design

[00129] Mice were anesthetized with Isoflurane, shaved at the base of the tail, and injected intradermally withl50μl of 2 mg/ml Bovine Type II collagen (Elastin Products, Owensville, MO) in Freund's complete adjuvant (Sigma) (dO and day 21). Randomization into each group by body weight was done on study day 18. Onset of arthritis occurred on days 23-34. Treatment was initiated after enrollment and continued twice daily (BID) through day 33. Mice were terminated on day 34. Clinical scores were given for each of the paws (right front, left front, right rear, left rear) on days 18-34. Experimental groups were as described in Table 2. Table 2: Study Group Designations

CI: Compound 1

Disease Induction

[00130] Mice were anesthetized with Isoflurane and given 150 μΐ of Bovine Type II collagen in Freund' s complete adjuvant injections (DO and day 21). Onset of arthritis occur on study days 18-35.

Name (Vendor): Type II Collagen (Elastin Products)

Designation: Bovine Type II Collagen

Batch #: CJ385, LOT 89033

Characteristics: Soluble, from new born calf joints

Storage Conditions: 2-8°C

Stability, Exp Date: 2 yrs, 7/21/2012

Purity: >99.6

Name (Vendor): Freund 's complete adjuvant (Difco)

Designation: Adjuvant

Characteristics:

Storage Conditions: 2-8°C • Stability, Exp Date:

• Content: 1 mg/ml

Test Article and Vehicle:

Test Article and Vehicle Formulation

[00131] * Vehicle 1 = 5% DMSO, 5% PEG400, 5% Tween 80 and 1% meglumine water solution (500 mg/g) in 30 HP-B-CD.

[00132] * Formulation preparation for PO dosing of Compound 1 (20 mg/kg, 10 mL/kg) in combination with ketoconazole (50 mg/kg, 10 mg/mL):

1) Weigh 9.62 mg of Compound 1 and 23.95 mg of ketoconazole into a clean tube.

2) Add 0.240 ml of DMSO to the tube, vortex for 1 min and warm the tube at 60 °C for 10-15 mins.

3) Add 0.240 ml of PEG 400 to the tube, vortex for 1 min and warm the tube at 60 °C for 10-15 mins.

4) Add 0.240 ml of Tween 80 to the tube, vortex for 1 min and warm the tube at 60 °C for 5-8 mins.

5) Add 0.048 ml of meglumine water solution (500 mg/g) to the tube, vortex for 1 min and warm the tube at 60 °C for 3-5 mins.

6) Add 4.032 mL of 30 HP-P-CD to the tube, vortex for 1 min.

The same formulation was used for PO and IP.

[00133] * Same vehicle for Compound 1 or keoconazole alone.

[00134] Dosing Formulation and Vehicle Handling: Formulation: Prepared daily.

Store at 25°C for the second daily dosing.

[00135] Compound Needed:

• PRT = 20*4*2*10-0.025*16*1.3= ~ 850 mg

• Ketoconazole = 100*2*10*0.025*1.3*16=1040 mg ~ lg

Live Phase Conduct results presented in mean clinical arthritic score.

[00136] Clinical Scoring Criteria for Fore and Hind Paws

[00137] 0 = normal

1 = hind or fore paw joint affected or minimal diffuse erythema and swelling

2 = hind or fore paw joints affected or mild diffuse erythema and swelling

3 = hind or fore paw joints affected or moderate diffuse erythema and swelling

4 = Marked diffuse erythema and swelling, or =4 digit joints affected)

5 = Severe diffuse erythema and severe swelling entire paw, unable to flex digits).

Humane Practice

[00138] Good science and the policies of Bolder BioPATH necessitate the limitation of pain and distress in all species. In keeping with this policy all protocols are reviewed with this in mind and the investigator is asked to provide rationale during the approval process. For Mouse Type Two Collagen-Induced Arthritis Models, animals showing signs of Morbidity according to the Bolder BioPATH IACUC Veterinary Care Program, including the loss of more than 20% body weight (within a week's time) will be removed from the study and humanly sacrificed via C02 inhalation.

[00139] For histopathologic scoring methods for mouse joints with type ii collagen arthritis and statistical analysis including clinical data and histopathologic data, see Example 1.

Results

[00140] Clinical arthritis scores were significantly reduced (p<0.05 in Student's t- test) in animals treated with Compound 1, MTX, or a combination of Compound 1 with MTX (Figs. 4-5).

[00141] Reduced disease incidence was recorded for mice treated with Compound 1,

MTX, or a combination of Compound 1 with MTX (Fig. 6A).

[00142] All six -joint mean histopathology parameters were significantly reduced toward normal for mice treated intraperitoneally with CI plus oral MTX (91% reduction of summed animal score) as compared to vehicle-treated controls. Looking at recution of specific parameters following the combination therapy, inflammation was reduced by 87%, pannus by 98%, cartilage damage was reduced by 90% and bone resorption by 98%. Six- joint scores for histopathology parameters following treatment intraperitoneally with CI alone resulted in 47% reduction in inflammation score, 64% reduction in pannus, 61% reduction in cartilage damage, and 66% reduction in bone resorption.

[00143] Six -joint mean histopathology parameters were also significantly reduced toward normal for mice treated orally with CI plus ketoconazole (61%) as compared to Vehicle-treated controls. Mice given combination therapy also had a significantly reduced six-joint summed pannus (71% reduction), cartilage damage (58%), bone resorption (71%), and summed animal score (59%) as compared to treatment with ketoconazole alone. Inflammation was reduced by 52% (Figs. 7A-B).

Conclusions

[00144] Treatment with CI (20 mg/kg) administered IP alone demonstrated significant effect in the CIA model as determined by evaluation of body weight, clinical arthritis scores, and % incidence. CI (20 mg/kg) administered in combination with MTX (0.5 mg/kg) demonstrated significant and synergistic effect in the CIA model as determined by evaluation of body weight, clinical arthritis scores, and histopathology of the joints (Figs. 4A, 5, 6A and 7A).

[00145] Treatment with CI (20 mg/kg) administered PO showed very low bioavailability and no clinical effect. In order to increase bioavailability, ketoconazole was added together with CI. Oral administration of CI (in an oral formulation containing Ketoconazole (50 mg/kg) demonstrated significant effect in the CIA model as determined by evaluation of body weight, clinical arthritis scores, and histopathology of the joints as compared to vehicle controls (Figs. 4B, 5, 6B and 7B).

[00146] Daily clinical arthritis scores were significantly reduced toward normal for mice treated with CI alone (Fig. 4A). Significant and synergistic reduction was also observed in combination with MTX (*p < 0.05 on days 26-34, Fig. 4A) as compared to vehicle controls and for mice treated orally with CI (d25-34) as compared to Vehicle 1 controls (Fig. 4B). Daily clinical scores for mice given combination therapy were also significantly reduced on days 28-34 as compared to treatment with vehicle alone. Clinical arthritis scores expressed as area under the curve (AUC) were significantly reduced toward normal for mice treated IP injection of CI plus MTX (96% reduction) as compared to vehicle controls and for mice treated orally with CI (74%) as compared to Vehicle 1 controls (Fig. 5). Clinical arthritis scores AUC for mice given combination therapy were also significantly (72%) reduced as compared to mice treated with Ketoconazole alone. Evaluation of histopathology confirmed the clinical findings; mice given combination therapy with MTX or oraly had significantly reduced histopathology parameters as compared to vehicles 1 controls (Figs. 7A-B).

Example 3. Compounds 1 and 2 are efficient in treating EAE.

Introduction

[00147] Objective: The aim of this study was to determine the efficacy of

Compound 1 and Compound 2 administered intraperitoneally in the MOG induced Experimental Autoimmune Encephalitis (EAE) in C57B1 mice.

[00148] Justifications: EAE is an accepted animal model for multiple sclerosis, and induction with MOG in C57BL/6 is a well-established model.

[00149] General Design: Disease was induced in the mice by the injection of the encephalitogenic emulsion (MOG/CFA). The compounds or the vehicle were administered from study initiation (Day 0) until termination (Day 30). Daily oral treatment with TV- 5600 (25mg/kg) was used as a positive control.

Materials

• TV-5600, batch # 650300106. CAS Reg. No. 248282-07-7. Also known as

laquinimod sodium. Available from Teva Pharmaceutical Industries Ltd.

• Purified water (Direct-Q, Millipore).

• Pertussis toxin: Sigma, Code # 2980, lot 044K1449.

• MOG 35-55: Mnf Novatide, lot # 90016-71-1.

• Complete Freund's Adjuvant (CFA): Sigma, code: F-5881, lot 104K8930.

• Saline: Mnf- DEMO S.A, Code 05029, lot # 0101610.

• Mycobacterium tuberculosis H37 RA (MT): Mnf- Difco.

• Compound 1

• Compound 2 Experimental Animals

[00150] Species, strain and supplier: Healthy, nulliparous, non-pregnant female mice of the C57BL/6 strain obtained from Harlan Animal Breeding Center, Israel were used in the study.

• The animals weighed about 15-22 g, and approximately 7-8 weeks old on arrival.

• The body weights of the animals were recorded on the day of delivery.

• Overtly healthy animals were assigned to study groups arbitrarily before treatment commences.

• The mice were individually identified by using ear tags. A color-coded card on each cage gave information including cage number, group number and identification.

• Animals housing and care conditions were maintained according to SOPs 33.22.01 and 33.22.02.

• The tested formulation were administered by one person to the respective groups. The labels on the cages of the group were coded and the clinical signs were scored by another person.

Test Procedures

[00151] EAE induction: EAE was induced by injecting the encephalitogenic mixture (emulsion) consisting of MOG (150 μg/mouse) and CFA enriched with M. tuberculosis (4 +1 = 5 mg/mL CFA). A volume of 0.2 ml of emulsion was injected subcutaneously into the flanks of the mice. Pertussis toxin was injected intraperitoneally on the day of induction and 48 hours later (total amount will be 150 + 150 =300 μg/mouse in 0.2 ml dosage volume).

[00152] Group Assignment: The mice were allocated to the treatment groups indicated in Table 3.

[00153] Preparation and administration of encephalito enic emulsion: Oil portion:

CFA (containing 1 mg/ml MT) was enriched to the concentration of 5mg/ml: 80.0 mg/MT were added to 20.0 ml CFA. Table 3

CI: compound 1; C2: compound 2 Liquid portion:

• 45.0 mg MOG was diluted in 15.0 ml Normal Saline to yield 3.0mg/ml MOG stock solution.

• 15.0 mL of 3.0mg/ml stock solution solution was diluted in 15.0 mL Normal Saline to yield 1.5 mg MOG /mL.

[00154] The emulsion was made from equal parts of oil and liquid portions (1: 1) in two syringes connected to each other with Leur lock, transferred to insulin syringe and 0.2 ml was injected to the right flank of each mouse.

[00155] Preparation and administration of Pertussis toxin: 60

Pertussis toxin (200 μg/ml) were added to 23.94 ml saline to yield 500 ng/ml. The pertussis toxin was administered intraperitoneally on the day of encephalitogen injection and 48 hours later (100.0 ng/0.2ml/mouse).

[00156] Preparation and administration of the test formulations

[00157] TV-5600: A concentration of 2.5 mg/ml TV-5600 was prepared in purified water for dose level 25.0 mg/kg. The test formulation were stored at 2 to 8 °C until use in amber colored bottles. The mice were administered with the TV-5600 at volume dose level of 200 μΐ/mouse by oral gavage to Grp2 once daily (Table 3).

[00158] The vehicle DDW (double distilled water) was administered to the negative control group (Grpl) in a similar manner. Formulation of Compound 1 and Compound 2

Dosing volume: 5 ml/kg

[00159] Steps 1-4 were performed at Proteologics and aliquots containing 210 μΐ were supplied frozen. Before each dosing an aliquot was thawed and warmed to 25°C.

1. Weigh 4 mg Compound 1 to a clean vial.

2. Add 50 μΐ of DMSO to the vial and warm at 60 °C for 5 min in a water bath to get a clear solution.

3. Add 50 μΐ of PEG400 to the vial, vortex for 1 min and warm at 60 °C for 5min in a water bath.

4. Add 50 μΐ of of Tween 80 to the vial, vortex for 1 min and warm at 60 °C for 5 min.

5. To each 210ul aliquot:

a. 14

of Melgumine water solution (500 mg/g) was added, vortexed for 1 min.

b. 1175 uL of 30% 2 1ydroxypropyl-|3-cyclodextrin (HPCD) was added to the vial step by step and vortexed

Table 4

Cmpd: Compound; Inj: Injection volume; Org vol: organic volume; Meg vol: Meglumine (500 mg/ml); NA: not applicable

Experimental Observations

[00160] Morbidity and Mortality: All animals were examined once daily to detect if any is dead or moribund.

[00161] EAE clinical signs: The mice were observed daily from the 10th day post-EAE induction and EAE clinical signs will be scored. The scores were recorded on observation cards according to the grades described in the table 5 below. [00162] All mice with score 1 and above were considered sick. When the first clinical sign appears all mice were given food soaked in water, which was spread on different places on the bedding of the cages.

Table 5: Evaluation of the EAE clinical signs

Score Signs Description^;

0 Normal behavior No neurological signs.

Limp tail Part or the whole tail is limp and

droopy.

Righting reflex Animal has difficulties rolling onto feet when laid on its back

Hind leg weakness wobbly walk - when the mouse walks

the hind legs are unsteady

Hind leg paralysis The mouse drags its hind legs but is able to move around using its fore legs

Full paralysis The mouse cannot move around, it looks thinner and emaciated.

Moribund/Death

[00163] For calculation purposes, the score of animals that died (6) will be carried forward.

Acceptance Criteria

[00164] Acceptance criteria for Negative Control Group:

• The control group should have at least 70 % incidence.

• The MMS should be more than 2.0.

Interpretation of Results

[00165] Calculation of the incidence of disease (Disease ratio)

. The number of sick animals in each group was summed.

. The incidence of disease was calculated as

No. of sick mice in treated group

INCIDENCE of DISEASE

No. of sick mice in control group

The percent inhibition according to incidence was calculated as

Number of sick mice in treated

INHIBITION (%)of INCIDENCE x lOO

v Number of sick mice in control group [00166] Calculation of the mortality/moribundity rate (mortality ratio)

The number of dead or moribund animals in each group was summed.

The mortality of disease was calculated as

No. of dead or moribound mice in treated group

MORTALITY of DISEASE =

No. of dead or moribound mice in control group

• The percent inhibition according to mortality was calculated as

Number of dead or moribound mice in treated group

INHIBITION (%) of MORTALITY x lOO

Number of dead or moribound mice in control group

[00167] Calculation of duration of disease

. The mean duration of disease expressed in days was calculated as

∑ Duration of disease of each mouse

Mean Duration - No. of mice in th e group

[00168] Calculation of mean delay in onset of disease

. The mean onset of disease expressed in days was calculated as

∑ Onset of disease of each mouse

Mean >

No. of mice in th e group

• The mean delay in onset of disease expressed in days was calculated by

subtracting the mean onset of disease in control group from test group.

[00169] Calculation of the mean maximal score and percent inhibition

The mean maximal score (MMS) of each group was calculated as

∑ Maximal Score of ea ch mouse

MMS

No. of mice in th e group

The percent inhibition according to MMS was calculated as

MMS of treated group

INHIBITION (%) of MMS x lOO

MMS of control group

[00170] Calculation of the group mean score and percent inhibition

The daily scores of each mouse in the test group was summed and the individual mean daily score (IMS) was calculated as

∑ Daily score of mouse

IMS

Observation period [days)

The mean group score (GMS) was calculated as ∑ IMS of ea ch mouse

GMS

No. of mice in th e group

The percent inhibition was calculated as

GMS of treated group

INHIBITION (%) of GMS x lOO

GMS of control group

Results

[00171] The results of Compound 1 and Compound 2 administration on EAE clinical manifestations and the disease profile of all treated groups compared to each vehicle (control group) is presented in Fig. 8 as group mean scores. The study was terminated at day 23 and not at day 30 as was planned.

[00172] TV- 5600 at 25 mg/kg was used as positive control treatment.

[00173] Incidence: 100 % incidence was observed in Vehicle DDW and Vehicle

Compound 1/ Compound 2 treated groups. Whereas in all other groups, the incidence rate was slightly lower 87-93%. In contrast, 0 % incidence was observed in TV 5600 positive control treated group.

[00174] Mortality: Groups treated with Compound 1 20 mg/kg and Compound 2 once and twice a day showed 33% and 20 % mortality, respectively.

[00175] Group Mean Score (GMS) and Mean Maximal Score (MMS): The MMS and GMS of Vehicle DDW group was 4.1 +0.8 and 2.5 +0.6 respectively. TV5600 had shown significant decrease in both MMS 2.5 + 1.5 and GMS 1.1 + 0.67.

[00176] The treatment with both Compound 1 and Compound 2 significantly reduced the GMS and MMS as compared to its relative vehicle.

[00177] Mean Onset Disease Duration: The appearance of EAE disease signs was relatively early in both Vehicle Control treated groups, day 12. Administration of TV 5600 and of Compound 1 significantly postponed the disease onset. Example 4. Effect of Compound 1 on pro-inflammatory cytokine release from THP1 cells in response to LPS.

[00178] Cells of the THP-1 human monocytic cell line release TNF-a upon activation by LPS (Lipopolysaccharide, E. Coli L-4391). This property can be used to determine the ability of compounds to inhibit TNF-a release. This assay is carried out to determine whether Compound 1 has any effect on TNF-a release.

[00179] THP-1 cells are passaged (P4), counted and plated on a 96-well flat-bottom plate at 100,000 cells/well/ ΙΟΟμΙ and allowed to settle for 15-30 minutes before addition of test compounds. Test compounds (positive control, vehicle, testing compounds) are added to plate at volume of ΙΟΟμΙ per well in 3 replicates. LPS is added to plate at volume of 50μ1 per well at O^g/ml from stock of O.lmg/ml (kept in -20°C) in RPMI-medium (LPS is added last to plate)._Test compounds/vehicle/ positive control are added 5-10 minutes prior to LPS activation.

[00180] Cells are incubated at 37°C for 3 hours in a C02 incubator. After 3 hours, the plates are spun for 5 minutes at 2000 rpm and 150μ1 supernatant is removed from each well for testing of TNF-a levels by ELISA. Supematants are stored at -20°C until TNF-a analysis. The remaining ΙΟΟμΙ is incubated with 20μ1 of Cell titer blue reagent (Promega) to determine cell viability.

[00181] Appropriate paradigms well known in the art for assessing release of for example IL1, IFN-γ, IL6, IL17 and IL4 from immune cells are employed to study the effect of Compound 1 or 2 on the release of these cytokines. ELISA is carried out according to the kits working protocols (R&D Systems, Quantikine human TNF-a). Samples will be diluted in calibrator diluent at 1:5.

Claims

1. A compound of formula I or a pharmaceutical composition comprising it for use in treatment of an inflammatory disease or a central nervous system (CNS) injury, wherein said inflammatory disease is rheumatoid arthritis or multiple sclerosis and said CNS injury is cerebral ischemia or spinal cord injury:

X is O, S or NH;

R is H or Ci-Ce alkyl;

Ri is H, Ci-C₆ alkyl, OR', SR', halogen, COOR', S0₂R', unsubstituted C6-C10 aryl or C6-C10 aryl substituted by one or more radicals selected from CrC₆ alkyl, halogen, nitro, halogen, OR, SR, COOR and -NRR, or two adjacent Ri, together with the carbon atoms to which they are attached, form a fused saturated, partially saturated or aromatic 5- 6 membered ring, optionally containing 1 or 2 further heteroatoms selected from N, S and/or O;

R' is H, CrC₆ alkyl or phenyl;

or an enantiomer or a pharmaceutically acceptable salt thereof.

2. The compound or the pharmaceutical composition according to claim 1, wherein X is S, and R and Ri each independently is H or methyl.

3. The compound or the pharmaceutical composition according to claim 2, wherein said compound is selected from: Compound 1 of the foraiula:

Compound 2 of the foraiula:

Compound 4 of the foraiula:

(4)

4. The compound or the pharmaceutical composition according to any one of claims 1 to 3, wherein said inflammatory disease is rheumatoid arthritis.

5. The compound or the pharmaceutical composition according to claim 4, wherein said compound is Compound 1.

6. The compound or the pharmaceutical composition according to claim 4, for use in combination with methotrexate.

7. The compound or the pharmaceutical composition according to claim 6, wherein said compound is Compound 1.

8. The compound or pharmaceutical composition according to claim 6, wherein said compound or pharmaceutical composition comprising it and said methotrexate are for sequential administration.

9. The compound or pharmaceutical composition according to claim 6, wherein said compound or pharmaceutical composition comprising it and said methotrexate are for concomitant administration.

10. The compound or pharmaceutical composition according to claim 6, wherein said compound or pharmaceutical composition comprising it and said methotrexate are in separate compositions.

11. The compound or pharmaceutical composition according to claim 6, wherein said compound or pharmaceutical composition comprising it and said methotrexate are in a single composition.

12. The compound or pharmaceutical composition according to claim 6, wherein said compound or pharmaceutical composition comprising it and methotrexate are administered orally.

13. The compound or pharmaceutical composition according to claim 6, wherein said compound or pharmaceutical composition comprising it is administered by injection and methotrexate is administered orally.

14. The compound or pharmaceutical composition according to claim 13, wherein said compound or pharmaceutical composition comprising it is administered by injection twice a day.

15. The compound or pharmaceutical composition according to any one of claims 8 to 14, wherein said compound is Compound 1.

16. The compound or the pharmaceutical composition according to any one of claims 1 to 3, wherein said inflammatory disease is multiple sclerosis.

17. The compound or the pharmaceutical composition according to claim 16, for administration along with a known drug for treatment of multiple sclerosis selected from Copaxone®, Avonex®, Betaseron®, Extavia® or Rebif®, Tysabri (Natalizumab) GyleniaTM (fingolimod) and BG-12 (dimethyl fumarate).

18. The compound or pharmaceutical composition according to claim 17, wherein said compound and said known drug for treatment of multiple sclerosis are for sequential administration.

19. The compound or pharmaceutical composition according to claim 17, wherein said compound and said known drug for treatment of multiple sclerosis are for concomitant administration.

20. The compound or pharmaceutical composition according to claim 17, wherein said compound and said known drug for treatment of multiple sclerosis are in separate compositions.

21. The compound or pharmaceutical composition according to claim 17, wherein said compound and said known drug for treatment of multiple sclerosis are in a single composition.

22. The compound or pharmaceutical composition according to claim 17, wherein said compound and said known drug for treatment of multiple sclerosis are administered orally.

23. The compound or pharmaceutical composition according to claim 17, wherein said compound is administered by injection and said known drug for treatment of multiple sclerosis is administered orally.

24. The compound or pharmaceutical composition according to claim 17, wherein said compound is administered by injection twice a day.

25. The compound or pharmaceutical composition according to any one of claims 17 to 24, wherein said compound is Compound 1.

26. The compound or the pharmaceutical composition according to any one of claims 1 to 3, for use in the treatment of multiple sclerosis or a CNS injury selected from cerebral ischemia or spinal cord injury by preventing or inhibiting neuronal degeneration with or without promoting axon outgrowth.