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Title:
AZACITIDINE AND ITS DERIVATIVES FOR THE TREATMENT OF MULTIPLE SCLEROSIS
Document Type and Number:
WIPO Patent Application WO/2020/176741
Kind Code:
A9
Abstract:
Provided are methods of treating multiple sclerosis, which comprises administering to a patient a therapeutically effective amount of DNA methylation blocker. Also provided are method of retarding development of multiple sclerosis by administering to a patient a therapeutically effective amount of DNA methylation blocker.

Inventors:
RAUCH TIBOR (HU)
BÓDI PÉTER (HU)
OROSZ JÁNOS (HU)
TÓTH FERENC (HU)
SOLYMOSI TAMÁS (HU)
GLAVINAS HRISTOS (HU)
Application Number:
PCT/US2020/020122
Publication Date:
October 08, 2020
Filing Date:
February 27, 2020
Export Citation:
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Assignee:
NANGENEX NANOTECHNOLOGY INCORPORATED (HU)
International Classes:
A61P25/28; A61K31/216; A61K31/341; A61K31/352; A61K31/53; C07D251/16; C07D311/02; C07H19/12
Attorney, Agent or Firm:
STEVENS, Lauren (US)
Download PDF:
Claims:
What is claimed:

1. A method of treating an autoimmune disease comprising administering to a patient in need thereof a therapeutically effective amount of at least one DNA methylation blocker.

2. The method of claim 1 , wherein the at least one DNA methylation blocker is administered by systemic administration·

3. The method of claim 1, wherein the at least one DNA methylation blocker is administered by oral administration.

4. The method of claim 1 , wherein at least one DNA methylation blocker covalently modifies bases in the DNA and amino acid residues in the histones.

5. The method of claim 4, wherein the at least one DNA methylation blocker is chosen from azacitidine and 2’, 3’, 5’-triacetyl-5-azacitidine.

6. The method of claim 5, wherein the at least one DNA methylation blocker DNA is azacitidine (4-amino- l- -D-ribofuranosyl-s-triazin-2(lH)-one).

7. The method of claim 5, wherein the at least one DNA methylation blocker DNA is tri- acetylated prodrug of azacitidine (4-amino-l- -D-ribofuranosyl-s-triazin-2(lH)-one).

8. The method of claim 7, wherein the tri-acetylated prodrug of azacitidine (4-amino- I-b-D- ribofuranosyl-s-triazin-2(lH)-one) is orally bioavailable.

9. The method of claim 1, wherein the at least one DNA methylation blocker directly blocks DNA methyltransferase activity.

10. The method of claim 9, wherein the at least one DNA methylation blocker is chosen from N-Phthalyl-L-tryptophan and (-)-epigallocatechin-3-gallate.

11. The method of any one of claims 1 to 10, wherein the autoimmune disease is multiple sclerosis.

12. The method of claim 11, wherein the multiple sclerosis is chosen from clinically isolated syndrome, relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis, progressive-relapsing multiple sclerosis, and classical variants of multiple sclerosis selected from Devic's neuromyelitis optica, acute disseminated encephalomyelitis, Marburg multiple sclerosis, Schilder's disease and Balo's concentric sclerosis.

13. The method of any one of claims 1 to 12, further comprising treating at least one comorbidity of multiple sclerosis.

14. The method of claim 13, wherein the at least one comorbidity of multiple sclerosis is chosen from vascular and cerebrovascular disease, autoimmune diseases, chronic lung disease, gastrointestinal disease, renal disease, and visual disorders.

15. The method of claim 13, wherein the at least one comorbidity of multiple sclerosis is chosen from asthma, Crohn disease, diabetes type 1, diabetes type 2, myasthenia gravis, psoriasis, rheumatoid arthritis, SLE (lupus) and thyroid disease.

16. The method of any one of claims 1 to 15, further comprising administering a SIP inhibitor and/or a monoclonal antibody.

17. The method of claim 16, wherein the SIP inhibitor is fingolimod.

18. The method of claim 16, wherein the monoclonal antibody is chosen fromanti-CD25 antibody, anti-CD52 antibody, anti-CD20 antibody and anti-VLA4 antibody.

19. The method of claim 16, wherein the monoclonal antibody is a monoclonal antibody-based therapy for the treatment of multiple sclerosis.

20. The method of any one of claims 1 to 19, wherein the treatment is prophylactic treatment before the onset of multiple sclerosis.

21. The method of any one of claims 1 to 19, wherein the treatment is therapeutic treatment after the onset of multiple sclerosis. 22. The method of any one of claims 1 to 19, wherein the treatment slows or halts progression of the multiple sclerosis disease.

23. The method of any one of claims 1 to 22, wherein the at least one DNA methylation blocker is administered for a period of time sufficient to achieve one or more of changes selected from: (a) reduced frequency of relapse in the subject, (b) reduced probability of relapse in the subject, (c) reduced annualized relapse rate in the subject, (d) reduced risk of progression in the subject, (e) reduced number of new or newly enlarging T2 lesions in the subject, (f) reduced number of new non-enhancing T1 hypointense lesions in the subject, and (g) reduced number of gadolinium (Gd+) lesions in the subject.

Description:
AZACITIDINE AND ITS DERIVATIVES FOR THE TREATMENT OF MULTIPLE

SCLEROSIS

[001] This application claims the benefit of priority of United States Provisional Patent Application Serial No. 62/811,592 filed February 28, 2019, the disclosure of which is incorporated by reference in its entirety for all purposes.

[002] Multiple sclerosis is a chronic illness involving the central nervous system. The disease usually begins between the ages of 20 and 50 and is twice as common in women as in men. It is difficult to predict the long-term outcome, but the average life expectancy is 30 years from the start of the disease.

[003] Currently there is no known cure for multiple sclerosis, available MS treatments can be divided into two categories: (i) the management of relapses and symptomatic treatments used for short-term amelioration of symptoms such as fatigue, pain and spasticity, and (ii) disease modifying treatments (DMT), which are used to reduce inflammatory disease activity and its long-term clinical consequences.

[004] The frequently used therapeutic approach is to start with a safe but moderately effective DMT, typically IRNb, glatiramer acetate, teriflunomide or dimethyl fumarate, and switch to another first-line DMT in patients with intolerable adverse effects or to a more effective DMT in those with new relapses or MRI lesions. These treatment options might include fingolimod (INN, trade name Gilenya, Novartis, SIP inhibitor), and monoclonal antibodies including Daclizumab (anti-CD25 antibody), Alemtuzumab (anti-CD52 antibody), Ocrelizumab (anti- CD20 antibody) and Natalizumab (anti-VLA4 antibody). Monoclonal antibody-based therapies reduce disease-associated inflammation by removing T cells, B cells and myeloid cells, which might trigger increased risks of severe adverse effects such as respiratory and urinary tract infections, herpesvirus reactivation and increased risk of long-term malignancy.

[005] Among patients with multiple sclerosis (MS), treatment with a disease-modifying therapy (DMT) may be associated with more rapid development of autoimmune disease than remaining untreated. Diseases that commonly present alongside multiple sclerosis, the comorbidities, include vascular and cerebrovascular disease, autoimmune diseases, chronic lung disease, gastrointestinal disease, renal disease, and visual disorders. Approximately 30% of patients with MS present with autoimmune comorbidities. The most common autoimmune diseases were thyroid disease, asthma, type 2 diabetes mellitus, psoriasis, and rheumatoid arthritis. [006] Accordingly, there is a high unmet medical need for new treatment options including different molecular targets. The present disclosure satisfies this need and provides related advantages as well. SUMMARY

[007] Provided is a method of treating an autoimmune disease comprising administering to a patient in need thereof a therapeutically effective amount of at least one DNA methylation blocker.

[008] These and other aspects of the invention disclosed herein will be set forth in greater detail as the patent disclosure proceeds.

BRIEF DESCRIPTION OF DRAWINGS

[009] Figure 1 shows a molecular modeling of the interaction between EGCG and DNMT1. Panel A shows the structure of (-)-epigallocatechin-3-gallate (EGCG). Panel B depicts the DNMT1 protein in ribbon representation and colored by secondary structures. Panel C shows the hydrogen-binding network of EGCG in DNMT1.

[010] Figure 2 Panel A shows the molecular structure of RG108 and its interaction with DNMTs active center is shown in Panel B.

[Oil] Figure 3 is a graph of EAE grade (mean) versus time (days) for both control and azacitidine treatment. The broken line indicates the end of azacitidine treatment.

[012] Figure 4. is a graph of EAE grade (mean) versus time (days) for both control and azacitidine treatment, beginning at day 15. The broken line indicates the end of azacitidine treatment.

[013] Figure 5 is a graph of MOG35-55 induced EAE (mean grade) versus days. TAC and fingolimod treatments started when first symptoms of EAE were observed (day 9), and continued on every 2nd day. ip - intraperitoneal, os - oral administration of drug.

[014] Figure 6 shows a survival curve analysis of TAC and fingolimod treatment.

[015] Figure 7 shows the effects of drug treatments on blood parameters including hematocrit (Panel A), platelet (Panel B) and RBC numbers (Panel C).

[016] Figure 8 illustrates the effect of DNA methylation blockers on white cell populations.

[017] Figure 9 is a graph of MOG35-55 induced EAE (mean grade) versus days. Combined treatment of EAE affected animals by TAC and fingolimod. [018] Figure 10 shows the effects of drug treatments on blood parameters including white blood cells numbers (Panel A), platelet (Panel B) and RBC numbers (Panel C).

DETAILED DESCRIPTION [019] As used in the present specification, the following words and phrases are generally intended to have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise.

[020] The term“DNA methylation blocker” refer to a compound that blocks methylation of DNA. The first group of inhibitors or DNA methyltrasferase (DNMT) trapping drugs are incorporated into the replicating DNA and then recognized by DNMTs conducting epigenetic modification of DNA. Incorporated DNA methylation blockers can generate covalent bonds with DNMTs resulting in trapped enzymes that ultimately lead to rapid enzyme depletion and DNA hypomethylation. The second group of DNA methylation inhibitors directly acts on DNMTs. These drugs can interact with the active center of DNMTs and inhibits catalytic activity (i.e., de novo DNA methylation).

[021] Epigallocatechin gallate (EGCG), one of the main polyphenol compounds in green tea, inhibits DNMT activity in protein extracts and in human cancer cell lines. After structural studies, it was concluded that EGCG binds to and blocks the active site of human DNA methyltransferase 1 (DNMT1) (Figure 1). Employing screening assay based on a 3D homology model for human DNMT1 catalytic domain helped to identify N-Phthalyl-L-tryptophan (RG108), a small-molecule inhibitor. The inhibitory mechanism of RG108 appears to be direct and specific for DNMTs, and has comparatively low toxicity in human cancer cell lines (Figure 2).

[022] The term“azacytidine,” also known as“5 -azacytidine” refers to a compound that is a pyrimidine nucleoside analog of cytidine having antineoplastic activity . Proper chemical names of azacytidine include 4-amino-I-[ D-ribofuranosy]-l,3,5-triazin-2(lH)-one or 4-amino-l- [3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yi]-l,3,5-triazin-2 -one. One example of a particular stereochemical configuration in which azacytidine may exist may be illustrated as shown by the formula:

[023] The empirical formula is C8H12N4O5. The molecular weight is 244. Azacitidine is a white to off-white solid. Azacitidine was found to be insoluble in acetone, ethanol, and methyl ethyl ketone; slightly soluble in ethanol/water (50/50), propylene glycol, and polyethylene glycol; sparingly soluble in water, water saturated octanol, 5% dextrose in water, N-methyl-2- pyrrolidone, normal saline and 5% Tween 80 in water; and soluble in dimethylsulfoxide (DMSO).

[024] Azacitidine is marketed as VIDAZA ® and indicated for treatment of patients with the following French- American-British (FAB) myelodysplastic syndrome subtypes: refractory anemia (RA) or refractory anemia with ringed sideroblasts (if accompanied by neutropenia or thrombocytopenia or requiring transfusions), refractory anemia with excess blasts (RAEB), refractory anemia with excess blasts in transformation (RAEB-T), and chronic myelomonocytic leukemia (CMMoL).

[025] Azacitidine is incorporated into genomic DNA and captures DNA methyl transferases during DNA replication, which results in inactivation of the enzyme that leads to DNA hypomethylation. Azacitidine is believed to exert its antineoplastic effects by causing hypomethylation of DNA and direct cytotoxicity on abnormal hematopoietic cells in the bone marrow. The concentration of azacitidine required for maximum inhibition of DNA methylation in-vitro does not cause major suppression of DNA synthesis.

Hypomethylation may restore normal function to genes that are critical for differentiation and proliferation. The cytotoxic effects of azacitidine cause the death of rapidly dividing cells, including cancer cells that are no longer responsive to normal growth control mechanisms. Non proliferating cells are relatively insensitive to azacitidine.

[026] “Tri-acetylated azacytidine” or the“tri-acetylated prodrug of azacytidine” refers to

2',3',5'-triacetyl-5-azacytidine having the following structure:

[027] Procedures for the preparation of tri-acetylated azacytidine can be prepared following procedures known in the art, e.g., U.S. Application No. US 2008/0182806, which is incorporated herein by reference for all purposes. Briefly, acetylated (acylated) derivatives of pyrimidine nucleosides are synthesized by reacting a pyrimidine nucleoside with an activated carboxylic acid. Activated carboxylic acids when treated with appropriate reagents present a carboxylate carbon more susceptible to nucleophilic attack than the original carboxylic acid, for example acid chlorides, acid anhydrides or n-hydroxysuccinimide. In the preparation of acetyl derivatives of azacitidine, because of the presence of group sensitive to esterification, e.g., hydroxyl or amino groups, these groups can be blocked with protecting groups, e.g., t- butyldimethylsilyl ethers or t-BOC groups, respectively, before preparation of the anhydride. With acids containing more than one carboxylate group (e.g., succinic, fumaric, or adipic acid) the acid anhydride of the desired dicarboxylic acid is reacted with a pyrimidine nucleoside in pyridine or pyridine plus dimethylformamide or dimethylacetamide.

[028] The term“acetylated” refers to an organic compound having at least one acetyl functional group as a result of acetylation, when such ester moiety was absent prior to the reaction of acetylation.

[029] The term“acetyl” refers to the acyl of acetic acid, having the chemical structure—

(C=0)— o— CH 3 .

[030] The term“effective amount” of a compound refers a non-toxic but sufficient amount of the compound that provides a desired effect. This amount may vary from subject to subject, depending on the species, age, and physical condition of the subject, the severity of the disease that is being treated, the particular compound used, its mode of administration, and the like. Therefore, it is difficult to generalize an exact“effective amount,” yet, a suitable effective amount may be determined by one of ordinary skill in the art.

[031] The term“pharmaceutically acceptable” refers to a compound, additive or composition that is not biologically or otherwise undesirable. For example, the additive or composition may be administered to a subject along with a compound of the invention without causing any undesirable biological effects or interacting in an undesirable manner with any of the other components of the pharmaceutical composition in which it is contained.

[032] The term“prodrug” refers to an agent that is converted into the parent drug in vivo. Prodrugs may, for instance, be bioavailable by oral administration whereas the parent drug is not. The prodrug may also have improved solubility in pharmaceutical compositions over the parent drug. A prodrug may be converted into the parent drug by various mechanisms, including enzymatic processes and metabolic hydrolysis.

[033] The term“carrier” refers to a material added to a chemical or formulation to facilitate its preparation, storage or use.

[034] The term“excipient” refers to a medicinally inactive component contained in a drug formulation, including, for example, bulking agents, stabilizing agents, preservatives, salts, or solvents.

[035] The term“patient” or“subject” refers to organisms to be treated by the methods described herein. Such organisms include, but are not limited to, humans.

[036] The term“comorbidity” refers to another medical condition present in patients with an illness. Comorbidities in patients with multiple sclerosis can delay MS diagnosis, increase disability progression, reduce quality of life, increase hospitalization, and increase the chance of death. The most common comorbidities in patients with MS are vascular and cerebrovascular disease, autoimmune diseases, chronic lung disease, gastrointestinal disease, renal disease, and visual disorders.

[037] Provided is a method of treating an autoimmune disease comprising administering to a patient in need thereof a therapeutically effective amount of at least one DNA methylation blocker.

[038] In some embodiments, the at least one DNA methylation blocker is administered by systemic administration·

[039] In some embodiments, the at least one DNA methylation blocker is administered by oral administration.

[040] In some embodiments, the at least one DNA methylation blocker covalently modifies bases in the DNA and amino acid residues in the histones. In some embodiments, the at least one DNA methylation blocker is chosen from azacitidine and 2’, 3’, 5’-triacetyl-5-azacitidine. In some embodiments, the at least one DNA methylation blocker DNA is azacitidine (4-amino- l- -D-ribofuranosyl-s-triazin-2(lH)-one). In some embodiments, the at least one DNA methylation blocker DNA is tri-acetylated prodrug of azacitidine. In some embodiments, the tri-acetylated prodrug of azacitidine is orally bioavailable. In some embodiments, the tri- acetylated prodrug of azacytidine is crystalline.

[041] In some embodiments, the at least one DNA methylation blocker directly blocks DNA methyltransferase activity. In some embodiments, the at least one DNA methylation blocker is chosen from N-Phthalyl-L- tryptophan and (-)-epigallocatechin-3-gallate.

[042] In some embodiments, the autoimmune disease is multiple sclerosis. In some embodiments, multiple sclerosis is chosen from clinically isolated syndrome, relapsing- remitting multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis, progressive-relapsing multiple sclerosis, and classical variants of multiple sclerosis selected from Devic's neuromyelitis optica, acute disseminated encephalomyelitis, Marburg multiple sclerosis, Schilder's disease and Balo's concentric sclerosis.

[043] In some embodiments, the method further comprises treating at least one comorbidity of multiple sclerosis. In some embodiments, the at least one comorbidity of multiple sclerosis is chosen from vascular and cerebrovascular disease, autoimmune diseases, chronic lung disease, gastrointestinal disease, renal disease, and visual disorders. In some embodiments, the at least one comorbidity of multiple sclerosis is chosen from asthma, Crohn disease, diabetes type 1, diabetes type 2, myasthenia gravis, psoriasis, rheumatoid arthritis, SLE (lupus) and thyroid disease.

[044] In some embodiments, the method further comprises administering a SIP inhibitor and/or a monoclonal antibody. In some embodiments, the SIP inhibitor is fingolimod. In some embodiments, the monoclonal antibody is chosen from anti-CD25 antibody (e.g.; Daclizumab), anti-CD52 antibody (e.g.; Alemtuzumab), anti-CD20 antibody (e.g.; Ocrelizumab) and anti- VLA4 antibody (e.g.; Natalizumab). In some embodiments, the monoclonal antibody is a monoclonal antibody-based therapy for the treatment of multiple sclerosis.

[045] In some embodiments, the administration of DNA methylation inhibitors such as 2’ , 3’ , 5’-triacetyl- 5 -azacitidine or azacitidine in combination with SIP inhibitor, or monoclonal antibodies, such as anti-CD25 antibody, anti-CD52 antibody, anti-CD20 antibody and anti- VLA4 antibody or monoclonal antibody-based therapies decreases the effective doses of SIP inhibitor, or monoclonal antibodies, such as anti-CD25 antibody, anti-CD52 antibody, anti- CD20 antibody and anti-VLA4 antibody or monoclonal antibody-based therapies needed to be administered for the treatment of the diseases described herein, such as multiple sclerosis.

[046] In embodiment, the administration of DNA methylation inhibitors such as 2’, 3’, 5’- triacetyl- 5 -azacitidine and/or azacitidine in combination with SIP inhibitor, or monoclonal antibodies, such as anti-CD25 antibody, anti-CD52 antibody, anti-CD20 antibody and anti- VLA4 antibody or monoclonal antibody-based therapies reduces the risks of severe adverse effects caused by the administration of SIP inhibitor, or monoclonal antibodies, such as anti- CD25 antibody, anti-CD52 antibody, anti-CD20 antibody and anti-VLA4 antibody or monoclonal antibody-based therapies used for the treatment of the diseases described herein, such as multiple sclerosis.

[047] In some embodiments, treatment is prophylactic treatment before the onset of multiple sclerosis. In some embodiments, treatment is therapeutic treatment after the onset of multiple sclerosis. In some embodiments, treatment slows or halts progression of the multiple sclerosis disease.

[048] In some embodiments, the at least one DNA methylation blocker is administered for a period of time sufficient to achieve one or more of changes selected from: (a) reduced frequency of relapse in the subject, (b) reduced probability of relapse in the subject, (c) reduced annualized relapse rate in the subject, (d) reduced risk of progression in the subject, (e) reduced number of new or newly enlarging T2 lesions in the subject, (f) reduced number of new non-enhancing T1 hypointense lesions in the subject, and (g) reduced number of gadolinium (Gd+) lesions in the subject.

[049] Also provided are pharmaceutical compositions comprising a therapeutically effective amount of at least one DNA methylation blocker and, optionally, one or more pharmaceutically acceptable carriers.

[050] In some embodiments, at least one DNA methylation blocker is administered as a raw or pure chemical, for example as a powder in capsule formulation.

[051] In some embodiments, at least one DNA methylation blocker is formulated as a pharmaceutical composition further comprising one or more pharmaceutically acceptable carriers.

[052] Pharmaceutical compositions may be prepared by any suitable method, typically by uniformly mixing the active compound(s) with liquids or finely divided solid carriers, or both, in the required proportions and then, if necessary, forming the resulting mixture into a desired shape.

[053] Conventional excipients, such as binding agents, fillers, acceptable wetting agents, tabletting lubricants and disintegrants may be used in tablets and capsules for oral administration. The compounds described herein can be formulated into pharmaceutical compositions using techniques well known to those in the art. Suitable pharmaceutically acceptable carriers, outside those mentioned herein, are known in the art; for example, see Remington, The Science and Practice of Pharmacy, 20 th Edition, 2000, Lippincott Williams & Wilkins, (Editors: Gennaro et al.)

[054] For oral administration, the pharmaceutical composition may be in the form of, for example, a tablet or capsule. The pharmaceutical composition is preferably made in the form of a dosage unit containing a particular amount of the active ingredient. Examples of such dosage units are capsules, tablets, powders, granules or suspensions, with conventional additives such as lactose, mannitol, com starch or potato starch; with binders such as crystalline cellulose, cellulose derivatives, acacia, com starch or gelatins; with disintegrators such as corn starch, potato starch or sodium carboxymethyl-cellulose; and with lubricants such as talc or magnesium stearate. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or encapsulating materials.

[055] In powders, the carrier is a finely divided solid which is in a mixture with the finely divided active component.

[056] In tablets, the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted to the desired shape and size.

[057] The powders and tablets may contain varying percentage amounts of the active compound. A representative amount in a powder or tablet may be from 0.5 to about 90 percent of the active compound. However, an artisan would know when amounts outside of this range are necessary. Suitable carriers for powders and tablets include magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethyl cellulose, a low melting wax, cocoa butter, and the like. The term “preparation” includes the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid forms suitable for oral administration.

[058] The pharmaceutical preparations are preferably in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets or capsules. Also, the unit dosage form can be a capsule or tablet itself, or it can be the appropriate number of any of these in packaged form. [059] Further embodiments include the embodiments disclosed in the following Examples, which is not to be construed as limiting in any way.

EXAMPLES

[060] Pre-clinical animal studies using a mouse model of multiple sclerosis were conducted. Efficacy of azacitidine on multiple sclerosis disease progression was investigated. Autoimmune encephalomyelitis considered to be an adequate animal model of multiple sclerosis was used for the experiments.

[061] First, prophylactic effect of azacitidine on disease development in mice with experimental autoimmune encephalomyelitis (EAE) was investigated in 30 mice (15 control [i.e., untreated] + 15 azacitidine- treated). Intraperitoneal (i.p.) administration of azacitidine was started at the time of EAE induction and repeated every second day during the course of the experiment. The sign of the disease around the expected onset time (i.e., day 13 th ) could not be observed compared to the un-treated controls, however later some very weak disease formation was noticed (Figure 3).

[062] Only 10% (2 mg/kg; 7.5 mg/m 2 ) of the clinically applied dose of azacitidine used for the treatment of chronic myelomonocytic leukemia was administered. EAE score remained around 0.5 per animals indicating that low-dose azacitidine treatment significantly protected animals from pathogenesis of EAE (Figure 3). After 40-day treatment period, EAE score was gradually increased in the lack of azacitidine treatment.

[063] Beneficial effect of azacitidine treatment on established EAE was investigated. Mice with EAE were i.p. injected with either azacitidine (15 mice) or vehicle (15 mice) on every other day following the first symptoms of disease, when the visual EAE score was at least >0.5 per animal (Figure 4).

[064] Azacitidine treatment proved to be effective; treated animals scores were significantly below compared to the un-treated controls and after the 38 th day, symptoms of EAE sharply dropped (Figure 4). Suspended azacitidine treatment (after the40 lh day) resulted in steep worsening of EAE symptoms, which supports that the observed alleviation could be traced back to targeted inhibition of DNA methylation.

[065] These experiments were conducted by using an FDA approved DNA methylation inhibitor, azacitidine (marketed as VIDAZA ® ), effectively used in cancer treatment.

[066] Azacitidine has poor oral bioavailability due to suboptimal physicochemical properties and rapid degradation of the molecule. An orally bioavailable prodrug of azacitidine was developed (2', 3', 5'-triacetyl-5-azacitidine) which exhibited -60% oral bioavailability in clinical studies (Ziemba et al, 2011). Parallel artificial membrane permeability assay showed that the permeability of 2', 3', 5'-triacetyl-5-azacitidine was 0.05 ± 0.01* 10 6 cm/s, while the PAMPA permeability of azacitidine was undetectable (<0.005 *10 6 cm/s) which further indicates that the prodrug will be more permeable, and thereby more orally bioavailable following oral administration.

[067] The 2’, 3’, 5’-triacetyl- 5 -azacitidine (TAC), prodrug of azacitidine, is acid resistant and has reasonably good physicochemical properties for oral administration. Treatment of EAE mice with orally administered TAC efficiently controls progression of the disease (Figure 5).

[068] TAC ameliorates EAE as efficiently as fingolimod, which is currently a gold standard MS drug (Figure 6.). Treatment of EAE mice with 25 mg/kg TAC has no effect on survival of experimental animals.

[069] TAC treatment has moderate effect on hematocrit, platelet and red blood cell numbers (Figure 7.).

[070] Both DNA methylation blockers (i.e., TAC and azacitidine) induce compromised white cell number in treated animals. The observed adverse effect of DNA methylation blockers is similar to the currently employed fingolimod’ s side effect (Figure 8.).

[071] The observed drug-induced leukopenia (i.e., low white cell number) might be minimized by combined treatment of animals with suboptimal dose of TAC and fingolimod. This kind of treatment strategy is based on the fact that the two drugs have different molecular target and milder side effect.

[072] The combined treatment using the two drugs (i.e., TAC and fingolimod) at suboptimal dose has synergistic effect and acts more efficiently than in case of separate drug treatments (Figure 9). Nonetheless, the combined treatment-associated leukopenia is not changed significantly (Figure 10 panel A)

[073] The combined treatment using the two suboptimal dose of drugs (i.e., TAC and fingolimod) has no significant effect of other blood parameters including platelet number and red blood cell number (Figure 10 panel B and C).

[074] Other uses of the disclosed methods will become apparent to those in the art based upon, inter alia, a review of this patent document.