FIELD OF THE INVENTION

The invention relates to pharmaceutical compositions comprising mono- and/or oligonucleotides, dosage forms, and methods for treating and preventing diseases.

BACKGROUND OF THE INVENTION

Known methods of treating human diseases of unknown etiology include the use of corticosteroids and other immunosuppressants, and cytostatics (C. Garnett, J. F. Apperley, and J. Pavlu, Treatment and management of graft-versus-host disease: improving response and survival, Adv Hematol. 2013; 4(6): 366-378); alkylating agents, and antimetabolites (DJ. Black and RB. Livingston, Antineoplastic Drugs, Review (Part 1) Drugs, 1990, 39 (4): 489-501), among others. Various synthetic oligonucleotides are under study as anti-tumor substances, immunostimulatory drugs, and potential therapies for treating viral diseases and muscular dystrophy.

Although oligonucleotides have been regarded as a new class of drugs for more than three decades, their therapeutic applications have so far failed to fully live up to the expectations. Today, there are many different subclasses of oligonucleotides, grouped mainly by different mechanisms of biological action, and include antisense, splice-switching oligonucleotides, siRNA, miRNA, aptamers and immunostimulatory oligonucleotides. Structural differences are minimal, and consequently all nucleic acid based drugs generally suffer from poor pharmacokinetics (J. Winkler, Oligonucleotide conjugates for therapeutic applications, Ther Deliv, 2013, 4(7), 791-809). Among registered drugs, Mipomersen is known (McGowan M P, TardifJ-C, Ceska R, et al. Randomized, placebo-controlled trial of mipomersen in patients with severe hypercholesterolemia receiving maximally tolerated lipid-lowering therapy. PLoS ONE. 2012; 7(11):e49006; Gelsinger C, Steinhagen-thiessen E, Kassner U. Therapeutic potential of mipomersen in the management of familial hypercholesterolaemia. Drugs. 2012; 72(11):1445-1455; Parhofer K G. Mipomersen: evidence-based review of its potential in the treatment of homozygous and severe heterozygous familial hypercholesterolemia. Core Evid. 2012; 7:29-38).

Diseases of unknown etiology, for which many explanations have been proposed, include, among others, various dissimilar diseases and conditions, in particular, schizophrenia, oncological diseases, graft versus host reaction, myocardial infarction, stroke, autism, neurodegenerative diseases (Alzheimer's disease, Parkinson disease, dementia, etc.), acute interstitial nephritis, hypertensive crises, aging, bipolar disorder, multiple sclerosis, amyotrophic lateral sclerosis, other demyelinating diseases of the central nervous system, ankylosing spondylitis, systemic lupus erythematosus, scleroderma, CADASIL Syndrome, Huntington's disease, depressive disorder, acute respiratory distress syndrome, Crohn's disease, thrombosis, cancer, cancer cachexia, gout, inflammatory bowel disease, ulcerative colitis, primary biliary cirrhosis, primary sclerosing cholangitis, and attempts to increase life expectancy, among others.

SUMMARY OF THE INVENTION

Bacterial chromosomal DNA contains a large number of methylated cytosine and adenine. For example, the DNA ofE. colicontains 19,120 6-methyladenines and 12,045 5-methylcytosines in addition to the four regular bases (EcoSal Plus. 2014 May; 6(1): doi:10.1128/ecosalplus.ESP-0003-2013. DNA Methylation M. G. Marinusl,* and A. Løbner-Olesen).

Presently, it has been surprisingly discovered that a therapeutic effect can be achieved in a number of diseases, including, without limitation, oncological diseases (e.g., cancer), neurodegenerative disease (e.g., Alzheimer's Disease), stroke, and graft versus host disease, by introducing a mixture of oligonucleotides and mononucleotides with methylated components, e.g., 5-methylcytosine, 6-methyladenine, and other nucleotides methylated at various positions.

Accordingly, the object of the invention is to provide pharmaceutical compositions, dosage forms, and methods of treatment for diseases based on mixtures of mono-, oligo- and polynucleotides which comprise methylated nucleotides.

Various non-limiting aspects and embodiments of the invention are described below.

In one aspect, a method for treating a disease and/or condition in a patient in need of such treatment comprising administering to said patient a therapeutically effective amount of a mixture of mono- and/or oligonucleotides comprising methylated nucleotides is provided.

In one embodiment, the disease and/or condition to be treated is selected from the group consisting of schizophrenia, oncological diseases, graft versus host reaction, myocardial infarction, stroke, chronic fatigue syndrome, autism, neurodegenerative diseases selected from Alzheimer's disease, Parkinson disease, and dementia, acute interstitial nephritis, hypertensive crises, aging, bipolar disorder, multiple sclerosis, amyotrophic lateral sclerosis, other demyelinating diseases of the central nervous system, ankylosing spondylitis, systemic lupus erythematosus, scleroderma, CADASIL Syndrome, Huntington's disease, depressive disorder, acute respiratory distress syndrome, Crohn's disease, thrombosis, cancer, cancer cachexia, gout, inflammatory bowel disease, ulcerative colitis, primary biliary cirrhosis, primary sclerosing cholangitis, Hashimoto's thyroiditis, Alzheimer's diseases, Parkinson' diseases, Friedreich's ataxia, Lewy body diseases, and spinal muscular atrophy.

In another embodiment, the disease and/or condition to be treated is selected from the group consisting of oncological disease, neurodegenerative disease, stroke, and graft versus host disease.

In one embodiment, the disease is a tumor. In one embodiment, the disease is cancer. In one embodiment, the disease is Graft versus host disease. In one embodiment, the disease is an acute ischemic condition. In one embodiment, the disease is a stroke. In one embodiment, the disease is Alzheimer's disease.

In another aspect, a method of increasing a life span of a subject in need thereof is provided comprising administering to said subject an effective amount of a mixture of mono- and/or oligonucleotides comprising methylated nucleotides.

In one embodiment, the mixture of mon- and/or oligonucleotides is produced the mixture of mono- and/or oligonucleotides is produced by a nuclease treatment of polymeric DNA, wherein said DNA is selected from the group consisting of eukaryotic DNA, archaeal DNA, Gram-positive bacterial DNA, Gram-negative bacterial DNA, and fungal DNA.

In one embodiment, the mixture of mono- and/or oligonucleotides is produced by a nuclease treatment of polymeric DNA, wherein said DNA is extracellular DNA. In one embodiment, the mixture of mono- and/or oligonucleotides is produced by a nuclease treatment of polymeric DNA, wherein said DNA is genomic DNA.

In one embodiment, the nuclease may be selected from the group consisting of T7 Endonuclease I, Mung Bean Nuclease, Nuclease BAL-31, Nuclease P, Deoxyribonuclease IV, Deoxyribonuclease I, Deoxyribonuclease II, frequently cutting restriction enzymes, and any combinations thereof.

In one embodiment, the nuclease treatment is conducted for 12-20 hours at room temperature in the presence of Ca+ and Mg+ ions.

In one embodiment, the mono- and/or oligonucleotides in the mixture are obtained synthetically.

In one embodiment, the mixture of mono- and oligonucleotides comprises mononucleotides, di-, tri-, tetra- and penta-nucleotides, and are essentially free of oligomers longer than 6 nucleotides.

In one embodiment, the mixture of mono- and oligonucleotides comprises 15-18% of mononucleotides.

In one embodiment, the mixture of mono- and oligonucleotides comprises methylated nucleotides. In one embodiment, the methylated nucleotides are methylated adenine and/or methylated cytosine.

In one embodiment, methylated adenine is present in an amount from 0.1% to 1% of the total nucleotide mixture. In one embodiment, methylated cytosine is present in an amount from 0.01% to 0.1% of the total nucleotide mixture.

In one embodiment, the mixture of mono- and/or oligonucleotides is administered parenterally, topically, transdermally, orally, by inhalation/pulmonary, vaginally, rectally, nasally, buccally, sublingually, subcutaneously, intradermally, intravenously, intramuscularly, intracranially, intravitreally, or intraperitoneally.

In one embodiment, the mixture of mono- and/or oligonucleotides is administered in combination with at least one compound that potentiates its activity.

In one embodiment, the mixture of mono- and/or oligonucleotides is administered as a composition which further comprises a pharmaceutically acceptable carrier or excipient.

In one embodiment, the subject is human.

In another aspect, a pharmaceutical composition comprising a mixture of mono- and/or oligonucleotides and a pharmaceutically acceptable carrier, is provided.

In one embodiment, the pharmaceutical composition comprising a mixture of mono- and/or oligonucleotides is effective for treating a disease selected from the group consisting of schizophrenia, oncological diseases, graft versus host reaction, myocardial infarction, stroke, chronic fatigue syndrome, autism, neurodegenerative diseases selected from Alzheimer's disease, Parkinson disease, and dementia, acute interstitial nephritis, hypertensive crises, aging, bipolar disorder, multiple sclerosis, amyotrophic lateral sclerosis, other demyelinating diseases of the central nervous system, ankylosing spondylitis, systemic lupus erythematosus, scleroderma, CADASIL Syndrome, Huntington's disease, depressive disorder, acute respiratory distress syndrome, Crohn's disease, thrombosis, cancer, cancer cachexia, gout, inflammatory bowel disease, ulcerative colitis, primary biliary cirrhosis, primary sclerosing cholangitis in a patient in need of such treatment.

In one embodiment, the mixture of mono- and/or oligonucleotides is produced by a nuclease treatment of polymeric DNA, wherein said DNA is selected from the group consisting of eukaryotic DNA, archaeal DNA, Gram-positive bacterial DNA, Gram-negative bacterial DNA, and fungal DNA.

In one embodiment, the mixture of mono- and/or oligonucleotides is produced by a nuclease treatment of polymeric DNA, wherein said DNA is extracellular DNA. In one embodiment, the mixture of mono- and/or oligonucleotides is produced by a nuclease treatment of polymeric DNA, wherein said DNA is genomic DNA.

In one embodiment, the nuclease is selected from the group consisting of T7 Endonuclease I, Mung Bean Nuclease, Nuclease BAL-31, Nuclease P, Deoxyribonuclease IV, Deoxyribonuclease I, Deoxyribonuclease II, frequently cutting restriction enzymes, and any combinations thereof.

In one embodiment, the nuclease treatment is conducted for 12-20 hours at room temperature in the presence of Ca+ and Mg+ ions.

In one embodiment, the mono- and/or oligonucleotides in the mixture are obtained synthetically.

In one embodiment, the mixture of mono- and oligonucleotides comprises mononucleotides, di-, tri-, tetra- and penta-nucleotides, and a negligible amount of oligomers longer than 6 nucleotides.

In one embodiment, the mixture of mono- and oligonucleotides comprises 15-18% of mononucleotides.

In one embodiment, the mixture of mono- and oligonucleotides comprises methylated nucleotides.

In one embodiment, the methylated nucleotides are methylated adenine and/or methylated cytosine.

In one embodiment, methylated adenine is present in an amount from 0.1% to 1% of the total nucleotide mixture.

In one embodiment, methylated cytosine is present in an amount from 0.01% to 0.1% of the total nucleotide mixture.

In one embodiment, the composition comprises one or more excipients selected from the group consisting of hypromellose, lactose monohydrate, carbopol, Polycarbophil, and starch-based gel.

In yet another aspect, a pharmaceutical dosage form comprising a mixture of mono- and/or oligonucleotides is provided.

In one embodiment, the pharmaceutical dosage form comprising a mixture of mono- and/or oligonucleotides is selected from a tablet, a pill, a powder, a lozenge, a sachet, a cachet, a dragee, an elixir, a suspension, an emulsion, a solution, a syrup, an aerosol (as a solid or in a liquid medium), nasal drops, oral drops, eye drops, an ointment, a cream, a lotion, a gel, a spray, a soft gelatin capsule, a hard gelatin capsule, a suppository, an enema, an encapsulated implant, a sterile injectable solution, and a sterile packaged powder.

