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WO2003030929A1 - Combination therapies using methyl donors or methyl donor enhancers and therapeutic agents for treatment of viral, proliferative and inflammatory diseases - Google Patents

Combination therapies using methyl donors or methyl donor enhancers and therapeutic agents for treatment of viral, proliferative and inflammatory diseases Download PDF

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Publication number
WO2003030929A1
WO2003030929A1 PCT/CA2002/001503 CA0201503W WO03030929A1 WO 2003030929 A1 WO2003030929 A1 WO 2003030929A1 CA 0201503 W CA0201503 W CA 0201503W WO 03030929 A1 WO03030929 A1 WO 03030929A1
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Prior art keywords
compound
interferon
pharmaceutical composition
proliferative
methyl donor
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PCT/CA2002/001503
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French (fr)
Inventor
Tony Cruz
Aleksandra Pastrak
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Transition Therapeutics Inc.
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Priority claimed from US10/167,765 external-priority patent/US6908611B2/en
Application filed by Transition Therapeutics Inc. filed Critical Transition Therapeutics Inc.
Publication of WO2003030929A1 publication Critical patent/WO2003030929A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7135—Compounds containing heavy metals
    • A61K31/714—Cobalamins, e.g. cyanocobalamin, i.e. vitamin B12
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/212—IFN-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/215—IFN-beta
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention provides pharmaceutical compositions and methods of use for the treatment of viral, inflammatory and/or proliferative diseases with methyl donor compounds in combination with anti-viral, anti-inflammatory and/or inflammatory compounds.
  • inflammatory, viral and/or proliferative diseases include multiple sclerosis, diabetes, restenosis, cancer, hepatitis C, HI AIDS and genital warts. These types of diseases share common disease processes and as a result often share or possess related common therapies.
  • Viruses are potent infectious pathogenic agents that cause important functional alterations of the invaded cells, often resulting in cellular death. It is generally acknowledged that the cell injury in viral diseases includes not only direct damages inflicted by the proliferation of viruses but also various immunologic reactions elicited by infection with viruses. In fact, the consequences of a viral disease depend upon several viral and host factors such as the quantity of infecting viral particles, the speed of viral multiplication and spread, the impact on cell functions, the host's secondary responses to the cellular injury, and both the immunologic and the non-specific defenses of the host. In general, the effects of a viral disease include asymptomatic infections, both acute and chronic clinical diseases and induction of various types of cancers.
  • hepatitis infection results in chronic or acute inflammation of the liver and can lead to hepatocellular carcinoma in some cases.
  • hepatitis viruses A, B, C, D E and G that are prevalent among the population.
  • Inflammatory Diseases are a diverse set of disorders that are characterized by the influx of certain cell types and mediators, the presence of which can lead to tissue damage and sometimes death.
  • the inflammatory cascade is a complex process that involves the triggering of immunological response, the release of chemokines, cytokines and toxic agents by the activated cells, the up-regulation of cell surface adhesion molecules and trans- endothelial cell migration.
  • inflammation occurs as a defensive response to invasion of the host by foreign material, bacteria or to mechanical trauma, toxins and neoplasia.
  • Autoimmune responses by intrinsic stimulation also can induce inflammatory responses.
  • MS multiple sclerosis
  • MS is a multi-factorial inflammatory disease of the human central nervous system resulting in the slowing of electrical conduction along the nerve. It is estimated that close to a third of a million people in the United States have MS. MS is believed to result from inflammation and breakdown in the myelin surrounding the nerve fibers of the central nervous system. The disease is characterized by an increase in the infiltration of inflammatory cells, loss of oligodendrocytes, and increased gliosis (astrocyte hypertrophy and proliferation). (For review see Amit et al., 1999; Pouly et al., 1999; Steinman et al., 1993; Miller, 1994).
  • cancer is the most well known proliferative disease. Specifically, cancer is a generic term representing a collection of diseases arising from mutations of key molecules that regulate cell proliferation, invasion, and metastasis.
  • the ability of tumor cells to metastasize involves deregulation via overproduction or mutation of genes that allow cells to invade out of the tissue of origin, survive in a contact-independent manner, escape immune recognition, lodge at a distant site, then invade to a suitable place within the new tissue and grow there.
  • the molecules that are commonly involved in tumor initiation, progression and metastasis include adhesion molecules, growth factor receptors, factors regulating the cytoskeleton, master switches regulating cell cycle, proliferation repressor genes, proteases and transcription factors.
  • the ability of most tumors to kill is directly related to their capacity to invade and ultimately to metastasize.
  • Viral, inflammatory and proliferative diseases share common disease processes There are several classes of molecules and disease processes that are common to all these diseases. These include elevated expression of adhesion molecules, cytokines and matrix metalloproteinases, increased cell proliferation and migration, increased inflammatory cell activation and infiltration, increased angiogenesis, and increased tissue destruction and dysfunctional matrix remodeling. Consequently, compounds for the treatment of these diseases are aimed at altering the immune system, cell proliferation, cell adhesion and migration, cytokine levels or activities or viral replication.
  • anti-inflammatory, anti- viral and anti-proliferative compounds are effective at treating inflammatory, viral and proliferative diseases, these compounds often cause side effects that are exacerbated when the dose is increased or if the frequency of administration is increased. Accordingly, some patients are unable to tolerate the doses or treatment regimen needed to achieve a therapeutic effect.
  • Interferon is an example of a common anti-inflammatory, anti-viral, and anti-proliferative therapeutic compound with many undesirable side effects including local injection reactions, flu-like syndrome and depression, which are often exacerbated at high doses. Accordingly, some patients are unable to tolerate the doses needed to achieve a therapeutic effect.
  • Paclitaxel another example of an anti-inflammatory and anti-proliferative therapeutic compound used to treat various cancers including breast cancer, ovarian cancer, Kaposi's sarcoma and non-small cell lung cancer, also has its share of undesirable side effects including neutropenia, leukopenia and peripheral neuropathy.
  • many antiviral, anti-inflammatory or anti-proliferative compounds may have shown promise in vitro against inflammatory, viral, or proliferative diseases, very high doses may be required in vivo; such doses often being toxic or causing severe side effects. Therefore, the limitation of these therapies may be in using sufficient levels of the compound to achieve maximal efficacy in the absence of side effects.
  • compounds that can be combined with anti-viral, anti-proliferative or anti-inflammatory compounds to increase effectiveness of treatments for inflammatory, viral or proliferative diseases are necessary.
  • compounds that can reduce the doses and/or reduce frequency of administration to reduce side effects and to maintain or improve efficacy are necessary.
  • a compound that can improve efficacy of interferon-beta in the treatment of MS would potentially reduce the amount and/or frequency of interferon-beta administration, reducing side effects induced by interferon-beta and possibly reducing the occurrence of neutralizing antibodies to interferon-beta in MS patients.
  • Neutralizing antibodies to interferon- beta occur in about one third of MS patients treated with interferon-beta and are positively correlated with a loss of therapeutic efficacy of interferon-beta (Peisenhammer et al., 1999).
  • these compounds would not only enhance the efficacy in current or potential anti- viral, anti-proliferative or anti-inflammatory therapies but would also broaden use of these therapies into many other inflammatory, proliferative and viral diseases.
  • Methyl donor compounds have been shown to be able to affect the regulation of cells in the immune system as well as the nervous system (Tamura et al, 1999, Sakane et al, 1982).
  • vitamin B12 which is commonly known as an essential nutrient or dietary supplement.
  • Vitamin B12 compounds have been known to be involved in metabolic processes; and deficiency of vitamin B12 has been known to provoke pernicious anemia, gastrointestinal and neurological disorders. It is a co-factor essential in the metabolic pathway leading to synthesis of DNA, cell division, as well as cellular metabolism. Biochemical evidence suggests that vitamin B12 compounds may up- regulate gene transcription and thereby regulate protein synthesis (Watanabe et al, 1994).
  • methyl donors such as vitamin B12 and folic acid are commonly used as nutritional supplements, they are not combined with anti-viral, anti-inflammatory or anti-proliferative agents for the treatment of viral, inflammatory or proliferative diseases.
  • methyl donors can be combined with anti-viral, anti- inflammatory or anti-proliferative agents for the treatment of viral, inflammatory or proliferative diseases.
  • Interferon a multi-faceted molecule that possesses anti-inflammatory, anti-proliferative and anti-viral activities, is an example of the combination therapy with methyl donor compounds.
  • the invention is also supported by usage of other anti-viral, anti-inflammatory and anti-proliferative compounds such as paclitaxel and P-16 with methyl donor compounds.
  • the invention comprises a pharmaceutical composition for the treatment of a disease selected from a group of viral diseases; proliferative diseases; inflammatory diseases; proliferative and inflammatory diseases; proliferative and viral diseases; viral and inflammatory diseases; and proliferative, viral and inflammatory diseases; comprising: (1) at least one methyl donor compound; and (2) at least one second compound selected from the group consisting of: antiviral compound; anti-proliferative compound; and anti-inflammatory compound.
  • Another embodiment of this invention comprises a pharmaceutical composition for the treatment of proliferative diseases, such as cancer (including breast cancer, ovarian cancer, non-small-cell lung cancer, Kaposi's sarcoma, malignant melanoma, metastatic renal cell carcinoma, chronic myelogenous leukemia, hairy cell leukemia, follicular lymphoma, astrocytoma and glioma) comprising at least one methyl donor compound and at least one anti-proliferative compound such as paclitaxel, or at least one methyl donor compound and at least one interferon compoundor at least one methyl donor compound and at least one paclitaxel compound.
  • cancer including breast cancer, ovarian cancer, non-small-cell lung cancer, Kaposi's sarcoma, malignant melanoma, metastatic renal cell carcinoma, chronic myelogenous leukemia, hairy cell leukemia, follicular lymphoma, astrocytoma and glioma
  • cancer including breast
  • Yet another embodiment of this invention comprises a pharmaceutical composition for the treatment of viral diseases, such as hepatitis B, hepatitis C, herpes, or vesticular stomatitis comprising at least one methyl donor compound and at least one anti-viral compound, or at least one methyl donor compound and at least one interferon compound.
  • the interferon compound could be one or more of interferon-beta and interferon-beta.
  • Yet another embodiment of this invention comprises a pharmaceutical composition for the treatment of inflammatory diseases comprising at least one methyl donor compound and at least one anti-inflammatory compound, or at least one methyl donor compound and at least one interferon compound, or at least one methyl donor compound and at least one paclitaxel compound.
  • Another embodiment of this invention comprises a pharmaceutical composition for the treatment of multiple sclerosis comprising at least one methyl donor compound and at least one interferon compound, such as interferon-beta, or at least one methyl donor compound and at least one paclitaxel compound.
  • Another embodiment of this invention comprises a pharmaceutical composition for the treatment of inflammatory diseases such as multiple sclerosis comprising at least one methyl donor compound and a peptide selected from the group consisting of P16, S-3, S-7, P-32, N-2 and N-3.
  • Another aspect of this invention is a pharmaceutical composition for the treatment of hepatitis B comprising methyl donor and interferon-alpha or interferon-beta.
  • Another aspect of this invention is a pharmaceutical composition for the treatment of hepatitis C comprising a methyl donor and interferon-alpha or interferon-beta.
  • Another aspect of this invention is a pharmaceutical composition for the treatment of cancers including breast cancer, ovarian cancer, non-small cell lung cancer, Kaposi's sarcoma, glioma and astrocytoma comprising a methyl donor and paclitaxel.
  • Another aspect of this invention is a pharmaceutical composition for the treatment of cancers including Kaposi's sarcoma, malignant melanoma, metastatic renal cell carcinoma, chronic myelogenous leukemia, hairy cell leukemia, follicular lymphoma, astrocytoma and glioma, comprising a methyl donor and interferon.
  • cancers including Kaposi's sarcoma, malignant melanoma, metastatic renal cell carcinoma, chronic myelogenous leukemia, hairy cell leukemia, follicular lymphoma, astrocytoma and glioma, comprising a methyl donor and interferon.
  • Another aspect of this invention is a pharmaceutical composition according to any of the aspects outlined above wherein the methyl donor compound is conjugated to the second compound.
  • Another aspect of this invention is a method of treating a viral, proliferative or inflammatory disease, including MS, hepatitis B and hepatitis C, comprising the step of administering to a patient any of the pharmaceutical compositions outlined above.
  • Another aspect of this invention is a method of treating a viral, proliferative, or inflammatory disease, including MS, hepatitis B and hepatitis C, comprising the steps of administering the methyl donor compound at a frequency selected from the group consisting of: more than once daily, daily, more than once weekly, weekly, more than once monthly and monthly, and administering the second compound at a frequency selected from the group consisting of: more than once daily, daily, more than once weekly, weekly, more than once monthly, and monthly.
  • An other aspect of this invention is a method of treating a viral, proliferative, or inflammatory disease, including MS, hepatitis B and hepatitis C, comprising the steps of administering to a patient, either together or separately, one or more methyl donor compound, and one or more anti-inflammatory, anti- viral or anti-proliferative compound.
  • Another embodiment of this invention comprises a method of administration comprising of administering at least one methyl donor compound and at least one anti-proliferative compound such as paclitaxel, or at least one methyl donor compound and at least one interferon compound or at least one methyl donor compound and at least one paclitaxel compound.
  • the compounds may be administered by any convenient route, for example by infusion (intravenous or subcutaneous) or injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.).
  • the methyl donor compound is administered by intravenous infusion.
  • the methyl donor compound is vitamin B12 which is administered by intravenous infusion.
  • the duration of infusion can range from 15 minutes to 120 minutes.
  • the concentration of the methyl donor compound can range from 10 to 2500 mg.
  • the methyl donor compound is vitamin B 12 whose concentration can range from
  • Another aspect of this invention is a method for treating a viral, proliferative, or inflammatory disease as described herein wherein paclitaxel is the paclitaxel compound.
  • Another aspect of this invention is a method for treating a viral, proliferative, or inflammatory disease as described herein wherein interferon alpha or interferon beta is the interferon compound.
  • Another aspect of this invention is a method of administration for treating a viral, proliferative, or inflammatory disease as described herein wherein the methyl donor compound is vitamin B 12.
  • the vitamin B 12 may be administered by intravenous infusion.
  • the dose of methyl donor compound is between 10 and 2500 mg, and/or the dose of anti-inflammatory, anti-viral or anti-proliferative compound is similar or less than the therapeutic dose range used for the anti-inflammatory, anti- viral or anti-proliferative compound.
  • Another aspect of this invention is the use of any of the compounds described above to treat a viral, proliferative, or inflammatory disease.
  • Anti-inflammatory compounds means a class of compounds for treating inflammatory diseases.
  • the compounds include compounds in research, in development and compounds marketed and sold. This class of compounds includes aspirin, sodium salicylate, choline salicylate, salicylsalicylic acid, diflunisal, salsalate, indomethacin, sulindac, phenylbutazone, oxyphenbutazone, tolmetin, ibuprofen, feroprofen, flurbiprofen, ketoprofen, mefanamic acid, meclofenamate, piroxicam, naproxen, hydrocortisone, prednisolone, 6-alpha- methylprednisolone, triamcinolone, dexamethasone, betaroethasone, cyclosporine, mycophenolate mofetil, cyclophosphamide, antisense ICAM-1, 6-mercaptopurine, tacrolimus, muromonab-CD3, ISA
  • Anti-proliferative compounds means a class of compounds for treating proliferative diseases.
  • the compounds include compounds in research, in development and compounds marketed and sold. This class of compounds includes but not limited to: altretamine (hexamethylmelamine, Hexalen), anastrozole (Arimidex), Exemestane (Aromasin), bicalutamide (Casodex), busulfan (Myleran), capecitabine (Xeloda), chlorambucil (Leukeran), cyclophosphamide (Cytoxan), diethylstilbestrol diphosphate (Stilphostrol), estramustine (Emcyt), etoposide (VP-16, Vepesid), flutamide (Eulexin), hydroxyurea (Droxia), Hydrea, Mylocel, letozole (Femara), leucovorin calcium (Leucovorin), levamisole (Ergamisol), lomustine
  • Anti-viral compounds means a class of compounds which includes any therapeutic compounds for treating viral diseases.
  • the compounds include compounds in research, in development and compounds marketed and sold.
  • the class of anti-viral compounds includes interferon compounds, acyclovir, adefovir, abacavir, amprenavir, cidofovir, didanosine, fomivirsen sodium, dipivoxil, adenine, arabinoside, famciclovir, ganciclovir, lopmavir, ritonavir, lamivudine, nelfinavir mesylate, stavudine, trizivir, amivudine, lobucavir, zidovudine, indinavir, nevirapine, delavirdine, saquinavir, efavirenz, ribavirin, foscarnet, n- docosanol, oseltamivir, valacyclo
  • CFA complete Freunds' adjuvant
  • DM20 means an isoprotein proteolipid protein. It is a major integral membrane protein of the central nervous system (CNS). DM20 is normally expressed in early (post-natal) stages of growth.
  • EAE or “experimental autoimmune encephalomyelitis” means a mouse model, specifically, the immunosuppressive mouse model for multiple sclerosis.
  • Enhanced means an enhanced therapeutic effect, and includes a synergistic effect.
  • “Inflammatory diseases” means a class of diverse diseases and disorders that are characterized by any one of the following: the triggering of an inflammatory response; an upregulation of any member of the inflammatory cascade; the downregulation of any member of the inflammatory cascade.
  • Inflammatory diseases include diabetes, artheriosclerosis, inflammatory aortic aneurysm, restenosis, ischemia/reperfusion injury, glomerulonephritis, sacoidosis cancer, restenosis, reperfusion injury, rheumatic fever, systemic lupus erythematosus, rheumatoid arthritis, Reiter's syndrome, psoriatic arthritis, ankylosing spondylitis, coxarthritis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, pelvic inflammatory disease, multiple sclerosis, diabetes, osteomyelitis, adhesive capsulitis, oligoarthritis, osteoarthritis, periarthritis, polyarthritis, psoriasis, Still's disease, synovitis, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, osteoporosis, inflammatory dermatosis and wound healing.
  • the singular term "inflammatory disease” includes any one
  • Interferon compounds means interferon-alpha, interferon-alpha analogues, interferon-alpha derivatives, interferon-alpha conjugates, interferon beta, interferon-beta analogues, interferon- beta derivatives, interferon-beta conjugates and mixtures thereof.
  • Interferon-alpha and interferon-beta genes may be altered by, for example, oligonucleotide directed mutagenesis to produce interferon-beta analogues thereof, such as the human recombinant cysteine depleted or cysteine replaced analogues. Further, identity or location of more than one amino acid may be changed by targeted mutagenesis.
  • the primary amino acid sequence of the protein may be augmented by glycosylation or by other supplementary molecules such as lipids, phosphate, and acetyl groups. Further, individual amino acid residues in the chain may be modified by oxidation, reduction, or other derivatization.
  • the interferon-alpha or interferon-beta protein may be cleaved to obtain the fragments which retain activity. The whole protein or its fragments can be fused with other peptides and proteins such as immunoglobulins and other cytokines.
  • Interferon-alpha and interferon-beta conjugates may represent, for example, a composition comprising interferon-beta coupled to a non-naturally occurring polymer comprising a polyalkylene glycol moiety.
  • Preferred interferon compounds include Roferon®, Intron®, Alferon®, Infergen®, Omniferon®, Alfacon-1, interferon-alpha, interferon-alpha analogues, pegylated interferon-alpha, polymerized interferon-alpha, dimerized interferon- alpha, interferon-alpha conjugated to carriers, encapsulated interferon-alpha, interferon-alpha as oral inhalant, interferon-alpha as injectable compositions, interferon-alpha as a topical composition, Roferon® analogues, Intron® analogues, Alferon® analogues, and Infergen® analogues, Omniferon® analogues, Alfacon-1 analogues, interferon beta, AvonexTM, BetaseronTM, BetaferonTM, RebifTM, interferon-beta analogues, pegylated interferon-beta, polymerized interferon-
  • interferon compound may mean any one or more compounds from the class of interferon compounds.
  • MBP myelin basic protein
  • Methyl donor compounds means any compound which enhances the levels of endogenous methyl donors (methyl donor enhancers) or provides methyl groups or methylation of proteins (methyl donors).
  • Methyl donor enhancers include agents that stimulate the expression or activity of methyltransferases, as well as any compound that enhances the levels of intracellular methyl donors or the activity of enzymes required for methyl donation.
  • Methyl donor compounds include but are not limited to arginine, homocysteine, S- adenosylmethionine, methionine, betaine, 5-methyltetra-hydrofolate, folic acid, folate, cyanocobalamin, aquacobalamin, adenosylcobalamin, methylcobalamin, hydroxocobalamin , cyanocobalamin carbanalide, and 5-o-methylbenzylcobalmin [(5-OmeB-za)CN-Cbl] and prodrugs, derivatives, analogues or conjugates of any of these compounds.
  • methyl donor compounds include polymerization, conjugation or encapsulation of any of these compounds.
  • Preferred methyl donors include vitamin B12 and its analogues, derivatives or conjugates.
  • the singular form, "methyl donor compound" means any one or more compounds from the class of methyl donor compounds.
  • ND4 mouse model means a transgenic mouse model for multiple sclerosis, produced by transformation with multiple copies of DM20; the genetic mouse model for multiple sclerosis.
  • PBS or "phosphate buffer saline” means an injectable solution that serves as a negative control because it does not have any physiological or therapeutic effects.
  • PLP means an isoprotein proteolipid protein. PLP becomes predominant in the adult.
  • Paclitaxel compounds means paclitaxel and its pro-drugs, analogues, derivatives or conjugates and mixtures thereof.
  • Paclitaxel compounds include but are not limited paclitaxel, TAXOTERE®, TAXOL®, Docetaxel, 10-desacetyl analogues of paclitaxel and 3'N- desbenzoyl-3'N-t-butoxy carbonyl analogues of paclitaxel, 7-deoxy-docetaxol, 7,8- cyclopropataxanes, N-substituted 2-azetidones, 6,7-epoxy paclitaxels, 6,7-modified paclitaxels, 10-desacetoxytaxol, 10-deacetyltaxol (from 10-deacetylbaccatin HI), phosphonooxy and carbonate derivatives of taxol, taxol 2',7-di (sodium 1,2- benzenedicarboxylate, 10-desace
  • Proliferative diseases means a class of diverse diseases and disorders characterized by a lack of control or poorly controlled cell division or proliferation.
  • Proliferative diseases include anal cancer, bile duct cancer, colon cancer, esophageal cancer, gallbladder cancer, pancreatic cancer, small intestine cancer, stomach cancer, osteosarcoma, ovarian epithelial cancer, gestational trophoblastic tumor, uterine sarcoma, vaginal cancer, vulvar cancer, ovarian germ cell tumor, ovarian cancer, soft tissue sarcoma, acute lymphoblastic leukemia, acute myeloid leukemia, small cell lung cancer, malignant mesothelioma, malignant thymoma, hypopharyngeal cancer, laryngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, parathyroid cancer, salivary gland cancer, brain tumor, glioma, cerebellar astrocytoma, cerebral astro
  • proliferative disease includes any one or more diseases selected from the class of proliferative diseases, and includes any compound or complex disease state wherein a component of the disease state includes a disease selected from the class of proliferative diseases.
  • PTX or "pertussis toxin” means the major protein toxin produced by virulent strains of Bordetella pertussis, the organism that causes whooping cough.
  • PTX is a potent ancillary adjuvant that primes macrophages used to elicit several different autoimmune diseases, including EAE.
  • RHAMM means the receptor for hyaluronic acid mediated motility.
  • RHAMM-related peptides refers to peptide sequences or related peptide sequences of RHAMM, specifically, the S-3 peptide, the S-7 peptide, the P-32 peptide, the P-16 peptide, theV-2 peptide, and the V-3 peptide, as defined herein.
  • S-3 peptide means a specific large peptide fragment of RHAMM, and includes the C- terminal or RHAMM and has the approximate size of 45Kda.
  • S-3 peptide can refer to either the mouse or human amino acid RHAMM peptide fragment, as follows:
  • S-7 peptide means a specific RHAMM peptide fragment with an approximate size of 23 Kda and represents a smaller portion of S-3 peptide.
  • S-7 peptide can refer to either the mouse or human amino acid RHAMM peptide fragment, as follows: SEQ ID No 3 Mouse S-7 (221 amino acids)
  • P-32 peptide means a specific RHAMM peptide fragment.
  • the term “P-32 peptide” can refer to either the mouse or human amino acid RHAMM peptide fragment, as follows:
  • V-2 peptide means a specific RHAMM peptide fragment which represents a large portion of the original RHAMM peptide, includes the C-terminal and has the approximate size of 58 Kda.
  • V-2 peptide can refer to either the mouse or human amino acid RHAMM peptide fragment, as follows:
  • V-3 peptide means a specific RHAMM peptide fragment.
  • V-3 peptide can refer to either the mouse or human amino acid RHAMM peptide fragment, as follows:
  • SJL/J means a mouse model. Specifically, a female SJL/ J mouse has increased susceptibility to development of autoimmune disease. A SJL/J transgenic mouse strain is susceptible to induction of EAE (more susceptible to development of EAE than most other mouse strains). Tumor development as well as autoimmunity in this mouse may result from an effective amplification of the immune response.
  • “Synergistic” means a greater anti-inflammatory, anti-proliferative and/or anti- viral effect with the use of a combination therapy of methyl donors and anti-inflammatory, anti- proliferative, and/or anti-viral compound than with the use of any of these therapeutic compounds alone.
  • This synergistic effect can work through either similar or different mechanisms or pathways of action.
  • One advantage of a combination therapy with a synergistic effect is that standard dosages can be used for a greater therapeutic effect than expected from the addition of the effect of either compound administered alone; or alternatively lower dosages or reduced frequency of administration of the therapeutic compound(s) may be used to achieve a better therapeutic effect.
  • viral diseases means a class of diverse diseases and disorders caused by or believed to be caused by viruses.
  • the class of viral diseases includes genital warts (HPV), HIV/AIDS, herpes, influenza, measles, polio, varicella-zoster, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G., meningitis, genital warts (HPV), vesticular stomatitis virus infection, and dengue fever.
  • the singular form "viral disease” includes any one or more diseases selected from the class of viral diseases, and includes any compound or complex disease state wherein a component of the disease state includes a disease selected from the class of viral diseases.
  • Vitamin B12 compounds means a class of compounds which includes vitamin B12 and its analogues, derivatives or conjugates.
  • the class of vitamin B12 compounds includes cyanocobalamin (CN-Cbl), aquacobalamin, adenosylcobalamin, methylcobalamin, hydroxycobalamin (HC), cyanocobalamin carbanalide, and 5-o-methylbenzylcobalmin [(5- OmeB-za)CN-Cbl] as well as the desdimethyl, monoethylamide and the methylamide analogues of all of the above. Also included are the various analogues and homologues of cobamamide such as coenzyme B12 and 5-deoxydenosylcobalamin.
  • vitamin B12 examples include chlorocobalamin, sulfitocobalamin, nitrocobalamin, thiocyanatocobalamin, benzimidazole derivatives such as 5,6-dichlorobenzimidazole, 5-hydroxybenzimidazole, trimethylbenzimidazole, as well as adenosylcyanocobalamin [(Ade) CN-Cbl], cobalamin lactone, cobalamin lactam and the anilide, ethylamide, monocarboxylic and dicarboxylic acid derivatives of vitamin B12 or its analogues.
  • Preferred derivatives of vitamin B12 include the mono-, di- and tricarboxylic acid derivatives or the proprionamide derivatives of vitamin B12.
  • the compositions include polymers of these analogues or vitamin B12 conjugated to other molecules or encapsulated.
  • the singular form, "vitamin B12 compound” may mean any one or more compounds from the class of vitamin B 12 compounds.
  • Figure 1 is a graph showing the effect of a methyl donor compound (vitamin B12), interferon- beta and the combination of a methyl donor compound and interferon-beta on clinical scores in the ND4 mouse model.
  • Figure 2 is a graph showing the effect of a methyl donor compound (vitamin B12), interferon- beta and the combination of a methyl donor compound and interferon-beta on clinical scores in the EAE mouse model.
  • Figure 3 is a graph showing the effect of a methyl donor compound (vitamin B12), P-16 and the combination of a methyl donor compound and P-16 on clinical scores in the ND4 mouse model.
  • Figure 4 is a graph showing the GFAP staining in brain sections from normal, ND4-untreated animals, interferon-beta treated ND4 animals, and interferon-beta/vitamin B12 treated ND4 animals.
  • Figure 5 is a graph showing the effect of a methyl donor compound (vitamin B12), TAXOL® and the combination of a methyl donor compound and TAXOL® on clinical scores in the ND4 mouse model.
  • the current invention discloses the effect of methyl donor compounds alone and in combination with anti-viral, anti-inflammatory or anti-proliferative compounds for treatment of viral, inflammatory or proliferative diseases.
  • Methyl donor compounds alone show efficacy and clearly show an enhancing or synergistic effect in combination with other anti-viral, anti- inflammatory, and anti-proliferative compounds for treating viral, inflammatory, and proliferative diseases.
  • the present invention provides pharmaceutical compositions for enhancing anti-viral, anti- proliferative and anti-inflammatory effects and pharmaceutical compositions for treatment of viral, proliferative and inflammatory diseases.
  • the pharmaceutical composition comprises a first compound that is at least one of a methyl donor compound and a second compound that is at least one of an anti- viral, anti-proliferative and/or anti-inflammatory compound.
  • compositions of the invention preferably contain a pharmaceutically acceptable carrier or excipient suitable for rendering the compound or mixture administrable orally, intranasally, or parenterally, intravenously, intradermally, intramuscularly or subcutaneously, rectally, via inhalation or via buccal administration, or transdermally.
  • the active ingredients may be admixed or compounded with any conventional, pharmaceutically acceptable carrier or excipient. It will be understood by those skilled in the art that any mode of administration, vehicle or carrier conventionally employed and which is inert with respect to the active agents may be utilized for preparing and administering the pharmaceutical compositions of the present invention. Illustrative of such methods, vehicles and carriers are those described, for example, in Remington's Pharmaceutical Sciences, 4th ed.
  • compositions of the invention may also be conjugated to transport molecules, monoclonal antibodies or transport modalities such as vesicles and micelles that preferentially target recipient cells.
  • the compounds of the present invention in the described dosages are administered orally, intranasally, by inhalation, mtraperitoneally, subcutaneously, intra-muscularly, transdermally, sublingually or intravenously.
  • the pharmaceutical composition can be prepared, for example, in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gum or the like prepared by procedures known to those skilled in the art.
  • the compounds may be administered by any convenient route, for example by infusion or injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.).
  • Anti- viral, anti-inflammatory and anti-proliferative compounds such as interferon, paclitaxel, mitoxantrone (Novantrone) can be given via intravenous infusion to patients suffering from anti-viral, anti-inflammatory and anti-proliferative diseases.
  • interferon-alpha is administered for treatment for Kaposi's sarcoma or malignant melanoma is via an intravenous infusion route.
  • Novantrone was given to MS patients in clinical trials at a dose of either 5 mg/m 2 or 12 mg/m 2 via intravenous infusion every three months.
  • Paclitaxel can also be given via intravenous infusion to cancer patients over a duration of 3 hours with a dose of about 135-175 mg/m . 9 million units (MU) of interferon-alpha were administered to patients suffering from hepatitis C by continuous subcutaneous infusion.
  • MU 9 million units
  • Preferred administration regimen for treating a patient includes an infusion for methyl donor compounds and/or anti- viral, anti-inflammatory and anti-proliferative compounds.
  • the administration of these compounds can be combined or separate.
  • the other therapeutic agent can also be administered via other administration routes including intraperitoneally, subcutaneously, intra-muscularly, transdermally, sublingually, orally, intranasally or by inhalation.
  • the methyl donor compound can be administered first, then followed by the anti-viral, anti-inflammatory and anti-proliferative compound or vice versa.
  • the methyl donors are administered via intravenous infusion.
  • the duration of infusion for each therapeutic compound can range from 15 mins to 360 mins with the optimal range being from 15 mins to 120 mins, with the option of treatment being repeated at specific or regular intervals.
  • other types of administration regimens can precede or subsequently follow the infusion regimen for methyl donor compound and/or anti- viral, anti-inflammatory and anti-proliferative compound.
  • the therapeutically effective amount of active agent to be included in the pharmaceutical composition of the invention depends, in each case, upon several factors, e.g., the type, size and condition of the patient to be treated, the intended mode of administration, the capacity of the patient to incorporate the intended dosage form, the severity of the disease progression, etc, and therefore should be decided according to the judgment of the practitioner and each patient's circumstances.
  • the dosages used for each anti- viral, anti-proliferative and anti- inflammatory compound are similar to those dosages known to those skilled in the art and used in pre-clinical and clinical studies and in commercial use.
  • the dose may also be lower than the currently used dosages as the combination of these compounds with methyl donor compounds increases the efficacy of these compounds.
  • methyl donor compounds may be combined with anti-viral, anti-proliferative and anti-inflammatory compounds with the objective to reduce the dosages and/or reduce frequency of administration of the anti-viral, anti-proliferative and anti-inflammatory compounds, in order to achieve both effective treatment and to lessen the negative side effects of the anti-viral, anti-proliferative and anti- inflammatory compounds.
  • the preferred dosage of a methyl donor compound for the present invention is the maximum a patient requires to provide an optimal enhancing effect, such maximum being tempered by the absolute upper limit of methyl donor dosage being the maximum that a patient can tolerate and not develop any serious complications.
  • Vitamin B12 an example of a methyl donor compound, has been available for many years as an injectable treatment for pernicious anemia, with doses typically in the range of lOOO ⁇ cg. Vitamin B12 also has a long history as a general oral health supplement with doses also in the " ⁇ cg" range.
  • High doses of hydroxocobalamin are used as a cyanide-poisoning antidote, called Cyanokit® (US Patent # 5,834,448). Cyanokit® is an acute one-time 5-gram dose of hydroxocobalamin administered for emergency purposes.
  • methyl donors such as vitamin B12 have been proposed for use for the therapeutic treatment for a few diseases
  • high doses of methyl donors in combination with an anti-viral, anti-inflammatory or anti-proliferative compound would achieve an enhanced or synergistic therapeutic effect.
  • high doses of methyl donors in combination with an anti-viral, anti- inflammatory or anti-proliferative compound requires less anti-viral, anti-inflammatory or anti-proliferative compound or requires a reduction in frequency of administration of antiviral, anti-inflammatory or anti-proliferative compound.
  • the dosage of the methyl donor compound for our invention is within the range of 10 mg to 2.5 g daily.
  • compositions of the invention to be administered in accordance with the invention to a patient will depend upon those factors noted above.
  • the present invention provides methods to enhance or potentiate anti- viral, anti-proliferative and anti-inflammatory compound-induced anti-viral, anti-proliferative and anti-inflammatory effects and methods of treating viral, proliferative or inflammatory diseases in patients by administering an amount of methyl donor compound in combination with anti-viral, anti- proliferative and/or anti-inflammatory compounds.
  • Methyl donor compounds can be administered simultaneously, separately or in combination with anti-viral, anti-proliferative, or anti-inflammatory compounds, under different doses, frequency of administration and route regimens, to enhance the efficacy of anti-viral, anti-proliferative and anti-inflammatory compounds in the treatment of viral, proliferative or inflammatory diseases in patients compared to when such compounds are administered alone.
  • the first method of treatment is the administration of a pharmaceutical composition including both a methyl donor compound and an anti-viral, anti-proliferative and anti-inflammatory compound.
  • An alternate method of treatment includes the step of the administration of a pharmaceutical composition including at least one of the methyl donor compound followed by the step of the administration of a second pharmaceutical composition including at least one of an anti-viral, anti-proliferative and anti-inflammatory compound.
  • the administration of at least one of the methyl donor compound can follow the administration of at least one of the anti-viral, anti-proliferative and anti-inflammatory compound.
  • the administration of the pharmaceutical compositions can occur separately or simultaneously.
  • the administration of the pharmaceutical compositions can occur sequentially.
  • Example 1 Effect of vitamin B12 (cyanocobalamin), interferon-beta and combination of vitamin B12 (cyanocobalamin) incorporating interferon-beta in ND4 mouse model
  • Vitamin B12 cyanocobalamin
  • interferon- beta an anti-inflammatory compound that is used for the treatment of inflammatory diseases such as MS
  • the ND4 model was used, as a slow progressive demyelinating transgenic mouse model where the animals demonstrate symptoms in young adults at approximately 3 months of age. In this model, the severity of the clinical signs increased until a maximum around 6 months with animals dying around 8 to 9 months of age. Clinical signs assessed included general shaking, seizures, head jerk, hind limb and tail shiver, wobbly gait and limp tail. The scale of zero (absence) to four (constant and uncontrollable movements) was used for each of the clinical signs.
  • mice (4 animals per group) were treated with cyanocobalamin alone at a dose of 15 mg/kg; weekly and in combination with interferon-beta la (RebifTM). Interferon-beta la was administered alone and in combination with cyanocobalamin at a dose of interferon-beta la of 5000 IU, tri-weekly. All treatments began when the mice reached 3 months of age at which time signs of demyelinating disease were evident. Treatment was stopped at time of sacrifice.
  • Example 2 Effect of vitamin B12, interferon-beta and combination of vitamin B12 (cyanocobalamin) with interferon-beta in EAE mouse model
  • Vitamin B12 cyanocobalamin
  • interferon- beta an example of an anti-inflammatory compound that is used for the treatment of inflammatory diseases such as multiple sclerosis
  • EAE Experimental Allergic Encephalomyelitis
  • mice were injected intravenously with 200 ng Pertussis Toxin (PT) in lOO ⁇ l total volume at a tail vein on day 1. Following 48 hours, 200 ng PT was injected a second time. Tail vein injections were done with 30.5 gauge needles. Following the PT injection was a one time MBP/CFA (Becton Dickinson) emulsion injection. Each mouse was injected subcutaneously using a 27.5 gauge needle with 200 ⁇ g/50 ⁇ l of MBP emulsified in 50ul of complete Freund's adjuvant (CFA) at the base of the tail. The total injection volume was lOO ⁇ l. The mice were observed daily after first injection and the clinical signs of disease were recorded. These included unsteady gait, shaking, tail drop, weight loss, etc. Clinical scores were evaluated every other day starting on day 7 following immunization. Animals were treated daily for 11 days.
  • mice (4 animals per group) were treated with cyanocobalamin alone at a dose of 10 mg/kg; daily and in combination with interferon-beta.
  • Interferon-beta la (RebifTM) was administered alone and in combination with cyanocobalamin at a dose of 5000 IU of interferon-beta, daily; cyanocobalamin and interferon-beta were administered as separate injections starting on the day of first immunization. Treatment was stopped at time of sacrifice. Mice were monitored daily from day 7 after immunization for clinical signs of EAE and were scored on a scale of 0 to 5. A score of 0 represented the absence of signs while a score of 5 was given to moribund animals.
  • Vitamin B12 cyanocobalamin
  • a methyl donor compound a methyl donor compound
  • interferon-beta an example of anti-inflammatory compound used for the treatment of inflammatory diseases such as multiple sclerosis
  • ND4 mice were either (a) untreated or treated with (b) interferon-beta la (RebifTM) alone (5000 IU) or (c) 1000 IU of interferon-beta la and cyanocobalamin (15mg/kg), or (d) 2500 units of interferon-beta la and cyanocobalamin (15mg/kg) or (e) 5000 IU of interferon-beta la and B12 (15mg/kg), three times a week for the duration of the experiment. Treatment was started when the animals were 3 months of age and continued for 11 weeks. The animals were evaluated twice a week for clinical signs and the scores were averaged on a weekly basis. Refer to Example 1 for more experimental details.
  • the ND4 model is a slow progressive demyelinating transgenic mouse model where the animals demonstrate symptoms in young adults at approximately 3 months of age. The severity of the clinical signs increase until a maximum around 6 months with animals dying around 8 to 9 months of age. Clinical signs assessed include general shaking, seizures, head jerk, hind limb and tail shiver, wobbly gait and limp tail. The scale of zero (absence) to four (constant and uncontrollable movements) was used for each of the clinical signs.
  • mice (4 animals per group) were treated with cyanocobalamin alone at a dose of 15 mg/kg; weekly and in combination with P-16.
  • P-16 was administered alone and in combination with cyanocobalamin at a dose of 1 mg/kg, tri-weekly. All treatments began when the mice reached 3 months of age at which time signs of demyelinating disease were evident. Treatment was stopped at time of sacrifice.
  • Example 5 Effect of folic acid, interferon-beta and combination of folic acid and interferon-beta in ND4 mouse model
  • Folic acid an example of a methyl donor compound, and interferon-beta, an anti- inflammatory compound that is used for the treatment of inflammatory diseases such as MS, were used in a demyelinating transgenic mouse model to demonstrate a synergistic effect when a combination of methyl donor compounds and anti-inflammatory compounds were used in the treatment of such diseases as compared to treatment with anti-inflammatory compounds or methyl donor compound alone.
  • the ND4 model was used, as a slow progressive demyelinating transgenic mouse model where the animals demonstrate symptoms in young adults at approximately 3 months of age. In this model, the severity of the clinical signs increased until a maximum around 6 months with animals dying around 8 to 9 months of age. Clinical signs assessed included general shaking, seizures, head jerk, hind limb and tail shiver, wobbly gait and limp tail. The scale of zero (absence) to four (constant and uncontrollable movements) was used for each of the clinical signs.
  • mice (4 animals per group) were treated with folic acid (purchased from Supelco) alone at a dose of 200 ⁇ g/kg; tri- weekly and in combination with interferon-beta la (RebifTM). Interferon-beta la was administered alone and in combination with folic acid at a dose of interferon-beta la of 5000 IU, tri- weekly. All treatments began when the mice reached 3 months of age at which time signs of demyelinating disease were evident. Treatment was stopped at time of sacrifice.
  • Example 6 Effect of vitamin B12 (methylcobalamin), interferon and combination of vitamin B12 with interferon in inhibiting cellular proliferation in vitro
  • Interferon was also known as an anti-proliferative agent that has been reported to be effective in treating proliferative diseases such as Kaposi's sarcoma, malignant melanoma, multiple myeloma, hairy cell leukemia, metastatic renal carcinoma, chronic myelogenous leukemia, HCV-related hepatocellular carcinoma, glioma, follicular lymphoma, fibrosarcoma and high- grade astrocytoma (Fine et al., 1997).
  • the following experiment was carried out to determine whether a methyl donor compound such as vitamin B12 (methylcobalamin) could enhance the anti-proliferative effects of an anti-inflammatory compound such as interferon in astrocytoma cell lines.
  • Mouse ascite astrocytoma cells were seeded in a 96-well plate and grown to 60% confluence in standard growth conditions. Prior to treatment, cells were washed and the culture medium was replaced with culture medium containing interferon-beta alone (500 IU/ml), methylcobalamin (25 ⁇ g/ml) alone, or combination of interferon-beta (500 IU/ml), and methylcobalamin (25 ⁇ g/ml), and incubated overnight.
  • interferon-beta alone 500 IU/ml
  • methylcobalamin 25 ⁇ g/ml
  • methylcobalamin 25 ⁇ g/ml
  • the CellTiter 96 ® AQ ue0Us One Solution Cell Proliferation Assay was used as a colorimetric method for determining the number of viable cells in proliferations.
  • Cell proliferation assays were performed by adding a small amount of the CellTiter 96 ® AQ ue0Us One Solution Reagent directly to culture wells, incubating for 1-4 hours and then recording absorbance at 490nm with a 96 well plate reader.
  • the quantity of formazan product as measured by the amount of 490nm absorbance is known in the art to be directly proportional to the number of living cells in the culture. Results and Conclusions:
  • Example 7 Effect of vitamin B12 (cyanocobalamin), interferon, combination of vitamin B12 with interferon on astrocyte gliosis
  • Astrocyte gliosis refers to the increased reactivity and proliferation of astrocytes in the central nervous system and is thought to play a major role in the development of lesions in the brain. Specifically, astrocyte gliosis has been observed around hematogenous metastases of the human brain and GFAP, the protein marker for reactive astrocytes, has been used as a biomedical marker for early detection for chemically induced cancer. Both the ND4 and EAE mouse models were used below to illustrate the effect of the combination therapy on astrocyte gliosis. The ND4 transgenic mice develops normally up to three months, after which demyelination progresses thereafter.
  • ND4 mice were treated as described previously in Example 1. After the various treatments, the mice were sacrificed and brain sections removed for GFAP staining and quantitation. The brain region of hippocampus, adjacent to the dentate gyrus was fixed in formalin, paraffin- embedded and sectioned at 5 ⁇ m. Immunohistochemistry with anti-GFAP antibody was used to stain for GFAP. Glial fibrillary acidic protein was a protein expressed by astrocytes (particularly reactive astrocytes) and was used as a marker for these cells.
  • EAE mice were treated as described previously in Example 2. After the various treatments, the mice were sacrificed and brain sections removed for GFAP staining and quantitation. The brain region of hippocampus, adjacent to the dentate gyrus was fixed in formalin, paraffin- embedded and sectioned at 5 ⁇ m. Lrimunohistochemistry with anti-GFAP antibody was used to stain for GFAP. Glial fibrillary acidic protein was a protein expressed by astrocytes (particularly reactive astrocytes) and was used as a marker for these cells.
  • Table 4 Total relative GFAP in brain homogenates from normal, EAE untreated animals, vitamin B12 treated interferon-beta treated; and interferon-beta and vitamin B12 treated EAE animals.
  • Interferon-beta (5000 IU/ml) 2.0 +/- 0.5
  • Interferon-beta (5000 IU/ml) 1.4 +/- 0.2
  • Example 8 Effect of vitamin B12 (cyanocobalamin), paclitaxel and combination of vitamin B12( cyanocobalamin) with paclitaxel (TAXOL®) in ND4 mouse model
  • Paclitaxel (TAXOL®) was an anti-proliferative agent that is commonly used to treat cancers such as ovarian cancer, breast cancer, non-small cell lung cancer and Kaposi's sarcoma.
  • the following experiment was carried out to determine whether vitamin B12 (cyanocobalamin), an example of a methyl donor compound, could enhance the anti-proliferative effects of anti- proliferative compounds such as paclitaxel in the ND4 mouse model.
  • the ND4 mouse model was associated with astrocyte proliferation in the brain.
  • the ND4 transgenic mice received one of the following treatments through mtraperitoneal injections: 1) TAXOL® at a dose of 20 mg/kg once a week; 2) cyanocobalamin at a dose of 10 mg/kg once a week and TAXOL® at a dose of 5 mg/kg three times per week; and 3) cyanocobalamin at a dose of 10 mg/kg once a week. All treatments started when the mice reached 3 months of age, at which time signs of demyelinating disease were evident. Results and Conclusions:
  • Example 9 Effect of folic acid, interferon and combination of folic acid with interferon in inhibiting cellular proliferation in vitro
  • Interferon was also known as an anti-proliferative agent that has been reported to be effective in treating proliferative diseases as discussed in Example 6. The following experiment was carried out to determine whether a methyl donor compound such as folic acid could enhance the anti-proliferative effects of an anti-inflammatory compound such as interferon in astrocytoma cell lines.
  • Example 10 In vitro studies demonstrating the enhancing effect of interferon compound with vitamin B12 (hydroxocobalamin) on treatment of human hepatitis B virus (HBV)
  • Hep G2.2J5 was used as a HepG2 cell line that is stably transfected with HBV genome and that continues to secrete HBV virions into the cell growth medium.
  • This cell line has been widely used to study the activities of anti-viral agents against HBV infection and was used in this study to evaluate the enhancing effect of methyl donor compounds with interferon, an example of an anti-viral compound, on treating HBV infections.
  • Interferon was used as an example of an anti- viral agent because it was used to treat both hepatitis B and C in the clinic.
  • HepG2.2J5 cells a cell line that was stably transfected with HBV genome, were cultured in RPMI-1640 with fetal bovine serum at either 2 or 4% final concentration and with no antibiotics. The cells were seeded in a 96-well plate at a density of 2xl0 4 cells/well and grown overnight. The medium was then discarded and replaced with 100 ⁇ l of culture medium containing hydroxocobalamin (a methyl donor compound) alone (at various concentrations), interferon-beta alone (at various international units), and a mixture of varying doses of hydroxocobalamin (HC) and various doses of interferon-beta vs HC. Triplicate samples were measured for each concentration. The cells were incubated at 37°C for 9 days. Drug/medium was changed daily. On day 9, the culture media containing released HBV virions were harvested and used to extract HBV DNA.
  • hydroxocobalamin a methyl donor compound
  • HC hydroxocobalamin
  • HBV DNA from drug-treated HepG2.2J5 cell culture media was extracted using QIAamp DNA mini kit - blood and body fluid spin column according to manufacturer's instructions. Briefly, 10 ⁇ l of QIAGEN proteinase K was added to 1.5 ml Eppendorf tube. 50 ⁇ l cell supernatant and 50 ⁇ l AL buffer were added to the tube. The samples were incubated at 56°C water bath for 3 h. At the end of incubation, 50 ⁇ l ethanol was added to the sample. The mixture was transferred to a QIAamp spin column and centrifuged at 14000 rpm for 1 min. The filtrate was discarded. 50 ⁇ l Buffer AW1 was then added to the column and centrifuged at 14000 rpm for 1 min. Following washing and recentrifugation, DNA was eluted in 50 ⁇ l dH 2 O.
  • HBV DNA was detected by real-time PCR using Lightcycler assay.
  • the reaction was performed in 20 ⁇ l, comprising the following: ' 6.4/4.8 ⁇ l dH 2 0, 2 ⁇ l 25 mM MgCl 2 ⁇ 1 ⁇ l 10 ⁇ M sense primer, 1 ⁇ l 10 ⁇ M antisense primer, 8/1.6 ⁇ l LightCycler Probe (fluorescein- labeled, 5 ⁇ M), 0.8/1.6 ⁇ l LightCycler Probe 2 (LC Red 640-labeled, lO ⁇ M), 2 ⁇ l LC FastStart DNA Master Hyb Probes, 1 ⁇ l uracil DNA glycosylase.
  • PCR mix was aliquoted into glass capillaries and 5 ⁇ l DNA template was then added to the PCR mix.
  • a range of HBV plasmid DNA (lxlO 8 , lxlO 7 , lxlO 6 , ...JxlO 2 , 25 copies) was used as standards for quantitative analysis. The cycling conditions are shown in Table 5.
  • the enhancing or antagonistic effect was analyzed mathematically using the median effect equation (Chou and Talalay, 1984) by computer software CalcuSyn (Biosoft, St Louis). The range of the combination index (Cl) is illustrated below in Table 6.
  • Viral activity was decreased when cells were treated with interferon-beta but not with hydroxocobalamin.
  • the combination therapy of hydroxocobalamin with interferon-beta was more effective in attenuating viral activity compared to when these compounds were used alone.
  • Example 11 In vitro studies demonstrating the enhancing effect of interferon anti- iral activity with vitamin B12 (hydroxycobalamin) on herpes simplex virus type 2 (HSV-2) and vesticular stomatitis virus (VSV)
  • MRC-5 human fibroblast cells
  • This cell line was used in this study to evaluate the enhancing effect of hydroxocobalamin, an example of a methyl donor compound with interferon-alpha and interferon beta, both examples of antiviral compounds, on treating VSV and HSV infections.
  • a CPE (cytopathic effect) inhibition assay was performed. This assay measures the ability of a drug or agent to protect cells from lysis by a virus. Briefly, confluent MRC-5 cells in a 96- well plate were incubated with various concentrations of hydroxocobalamin (HC) alone, interferon-alpha alone, interferon-beta alone, and the mixtures of varying doses of HC and various doses of interferon-alpha or interferon-beta. The test concentrations for HC were 0, 1, 10, 100, 500 and 1000 ⁇ g/ml and that of interferon-alpha and interferon-beta were 0, 5, 10, 50, 100, and 500 IU/ml.
  • HC hydroxocobalamin
  • MRC-5 cells triplicate samples were tested for each concentration in a 96- well plate format.
  • the culture medium for MRC-5 cells was recommended by ATCC (Minimum Essential Medium Eagle with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0J mM non-essential amino acids, and 1.0 mM sodium pyruvate, 10% fetal bovine serum).
  • the cells were infected with either VSV at moi (multiplicity of infection) of 0.5 pfu/cell or HSV-2 at moi of 0.2 pfu/cell at 37°C for 1 h.
  • VSV multiplicity of infection
  • HSV-2 at moi of 0.2 pfu/cell at 37°C for 1 h.
  • the infected cells were washed with PBS and covered with medium containing either no or increasing concentrations of test compounds as above for 2-3 days at 37°C until the CPE in the control wells was 100%.
  • the cells protected from CPE were measured using neutral red uptake assay. Specifically, culture medium was removed from cells in a 96-well plate by pump. The cells were then washed once with 200 ⁇ l of PBS.
  • Antiviral effect (OD ⁇ est - ODo)/(ODcontroi - ODo) whereby OD ⁇ es t is the optical density measured with a given concentration of the test compound; ODo s the optical density measured at drug concentration zero; ODcontroi is the optical density of uninfected cells.
  • the potentiating effect was analyzed mathematically using the median-effect equation (Chou and Talalay, 1984) by a computer program CalcuSyn (Biosoft, St Louis). Refer to Table 6 for correlation between combination index obtained and degree of synergism.
  • Interferon-alpha 0 0.093 0.139 0.218 0.282 0.271
  • Interferon-beta 0 0.164 0.136 0.314 0.281 0.265
  • Anti-viral activity was significantly increased when cells were treated with hydroxocobalamin or interferon-alpha or interferon-beta.
  • the combination therapy of hydroxocobalamin with interferon-alpha or interferon-beta was more effective in attenuation viral activity compared to when these compounds were used alone. See Table 11. There was an enhanced effect for the treatment of vesticular stomatitis virus infection when hydroxocobalamin and interferon, an anti-viral agent, were combined.
  • Interferon-alpha 0 0.296 0.387 0.950 0.996 0.942
  • Interferon-beta 0 0.826 0.985 0.758 0.768 0.956
  • Example 12 In vitro studies demonstrating the enhancing effect of interferon compound with folic acid on treatment of human hepatitis B virus (HBV)
  • Hep G2.2J5 was used as a HepG2 cell line that is stably transfected with HBV genome and that continues to secrete HBV virions into the cell growth medium.
  • This cell line has been widely used to study the activities of anti- viral agents against HBV infection and was used in this study to evaluate the enhancing effect of methyl donor compounds such as folic acid with interferon, an example of an anti-viral compound, on treating HBV infections.
  • Interferon was used as an example of an anti-viral agent because it was used to treat both hepatitis B and C in the clinic.
  • HepG2.2J5 cells a cell line that was stably transfected with HBV genome, were cultured in RPMI-1640 with fetal bovine serum at either 2 or 4% final concentration and with no antibiotics. The cells were seeded in a 96-well plate at a density of 2xl0 4 cells/well and grown overnight. The medium was then discarded and replaced with 100 ⁇ l of culture medium containing folic acid (a methyl donor compound) alone (at various concentrations, 0J ⁇ g/ml to 1000 ⁇ g/ml)), interferon-beta alone (at various international units, similar to that of Example 10), and a mixture of varying doses of folic acid and various doses of interferon- beta vs HC. Triplicate samples were measured for each concentration. The cells were incubated at 37°C for 9 days. Drug/medium was changed daily. On day 9, the culture media containing released HBV virions were harvested and used to extract HBV DNA.
  • folic acid a
  • HBV DNA from drug-treated HepG2.2J5 cell culture media was extracted using QIAamp
  • the enhancing or antagonistic effect was analyzed mathematically using the median effect equation (Chou and Talalay, 1984) by computer software CalcuSyn (Biosoft, St Louis).
  • the range of the combination index (Cl) is illustrated below in Table 6 in Example 10.
  • Viral activity was decreased when cells were treated with either interferon-beta or folic acid alone.
  • the combination therapy of folic acid with interferon-beta was more effective in attenuating viral activity compared to when these compounds were used alone.
  • the anti- viral effects of interferon-beta with folic acid were synergistic.
  • these examples clearly demonstrate that the combination treatment of a methyl donor compound in conjunction with a therapeutic agent such as interferon, paclitaxel or P-16 is effective in treating inflammatory, proliferative and/or viral diseases and that the methyl donor enhances the effectiveness of or has a synergistic effect with the specific therapeutic agent against the disease indicated. Furthermore, these examples demonstrate that methyl donor compounds in combination with an anti-viral, anti-inflammatory or anti-proliferative compounds, require less anti-viral, anti-inflammatory or anti-proliferative compound.
  • a therapeutic agent such as interferon, paclitaxel or P-16

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Abstract

Pharmaceutical compositions for treating viral, proliferative and inflammatory diseases are disclosed comprising an amount of pharmaceutically acceptable methyl donor compounds in combination with anti-viral, anti-proliferative and anti-inflammatory compound. Methyl donor compounds are administered separately, simultaneously or in combination with anti-viral, anti-proliferative and/or anti-inflammatory compounds to provide an enhanced therapeutic effect for treating viral, proliferative and inflammatory diseases.

Description

COMBINATION THERAPIES USING METHYL DONORS OR METHYL DONOR ENHANCERS AND
THERAPEUTIC AGENTS FOR TREATMENT OF VIRAL, PROLIFERATIVE AND INFLAMMATORY
DISEASES
Field of the Invention
The present invention provides pharmaceutical compositions and methods of use for the treatment of viral, inflammatory and/or proliferative diseases with methyl donor compounds in combination with anti-viral, anti-inflammatory and/or inflammatory compounds.
Background of the Invention
Review of viral, inflammatory and proliferative diseases
Diseases and disorders that have significant inflammatory, viral and/or proliferative components are widespread and affect millions of people worldwide. Selected examples of inflammatory, viral and/or proliferative diseases include multiple sclerosis, diabetes, restenosis, cancer, hepatitis C, HI AIDS and genital warts. These types of diseases share common disease processes and as a result often share or possess related common therapies.
Viral Diseases
Viruses are potent infectious pathogenic agents that cause important functional alterations of the invaded cells, often resulting in cellular death. It is generally acknowledged that the cell injury in viral diseases includes not only direct damages inflicted by the proliferation of viruses but also various immunologic reactions elicited by infection with viruses. In fact, the consequences of a viral disease depend upon several viral and host factors such as the quantity of infecting viral particles, the speed of viral multiplication and spread, the impact on cell functions, the host's secondary responses to the cellular injury, and both the immunologic and the non-specific defenses of the host. In general, the effects of a viral disease include asymptomatic infections, both acute and chronic clinical diseases and induction of various types of cancers. One example of a well known viral disease is the viral hepatitis infection which results in chronic or acute inflammation of the liver and can lead to hepatocellular carcinoma in some cases. To date, there are several types of hepatitis viruses that have been identified, including hepatitis viruses A, B, C, D E and G that are prevalent among the population. For instance, it has been estimated by the Liver Foundation International that there are more than 520 million individuals worldwide that suffer from either hepatitis B or hepatitis C.
Inflammatory Diseases Inflammatory diseases are a diverse set of disorders that are characterized by the influx of certain cell types and mediators, the presence of which can lead to tissue damage and sometimes death. Essentially, the inflammatory cascade is a complex process that involves the triggering of immunological response, the release of chemokines, cytokines and toxic agents by the activated cells, the up-regulation of cell surface adhesion molecules and trans- endothelial cell migration. Typically, inflammation occurs as a defensive response to invasion of the host by foreign material, bacteria or to mechanical trauma, toxins and neoplasia. Autoimmune responses by intrinsic stimulation also can induce inflammatory responses.
One example of inflammatory disease is multiple sclerosis ("MS")- MS is a multi-factorial inflammatory disease of the human central nervous system resulting in the slowing of electrical conduction along the nerve. It is estimated that close to a third of a million people in the United States have MS. MS is believed to result from inflammation and breakdown in the myelin surrounding the nerve fibers of the central nervous system. The disease is characterized by an increase in the infiltration of inflammatory cells, loss of oligodendrocytes, and increased gliosis (astrocyte hypertrophy and proliferation). (For review see Amit et al., 1999; Pouly et al., 1999; Steinman et al., 1993; Miller, 1994).
Proliferative diseases Cancer is the most well known proliferative disease. Specifically, cancer is a generic term representing a collection of diseases arising from mutations of key molecules that regulate cell proliferation, invasion, and metastasis. The ability of tumor cells to metastasize involves deregulation via overproduction or mutation of genes that allow cells to invade out of the tissue of origin, survive in a contact-independent manner, escape immune recognition, lodge at a distant site, then invade to a suitable place within the new tissue and grow there. The molecules that are commonly involved in tumor initiation, progression and metastasis include adhesion molecules, growth factor receptors, factors regulating the cytoskeleton, master switches regulating cell cycle, proliferation repressor genes, proteases and transcription factors. However, the ability of most tumors to kill is directly related to their capacity to invade and ultimately to metastasize.
Viral, inflammatory and proliferative diseases share common disease processes There are several classes of molecules and disease processes that are common to all these diseases. These include elevated expression of adhesion molecules, cytokines and matrix metalloproteinases, increased cell proliferation and migration, increased inflammatory cell activation and infiltration, increased angiogenesis, and increased tissue destruction and dysfunctional matrix remodeling. Consequently, compounds for the treatment of these diseases are aimed at altering the immune system, cell proliferation, cell adhesion and migration, cytokine levels or activities or viral replication.
Need for enhancing compounds to decrease side effects
Although many anti-inflammatory, anti- viral and anti-proliferative compounds are effective at treating inflammatory, viral and proliferative diseases, these compounds often cause side effects that are exacerbated when the dose is increased or if the frequency of administration is increased. Accordingly, some patients are unable to tolerate the doses or treatment regimen needed to achieve a therapeutic effect.
Interferon is an example of a common anti-inflammatory, anti-viral, and anti-proliferative therapeutic compound with many undesirable side effects including local injection reactions, flu-like syndrome and depression, which are often exacerbated at high doses. Accordingly, some patients are unable to tolerate the doses needed to achieve a therapeutic effect.
Paclitaxel, another example of an anti-inflammatory and anti-proliferative therapeutic compound used to treat various cancers including breast cancer, ovarian cancer, Kaposi's sarcoma and non-small cell lung cancer, also has its share of undesirable side effects including neutropenia, leukopenia and peripheral neuropathy. In addition, although many antiviral, anti-inflammatory or anti-proliferative compounds may have shown promise in vitro against inflammatory, viral, or proliferative diseases, very high doses may be required in vivo; such doses often being toxic or causing severe side effects. Therefore, the limitation of these therapies may be in using sufficient levels of the compound to achieve maximal efficacy in the absence of side effects. Thus, compounds that can be combined with anti-viral, anti-proliferative or anti-inflammatory compounds to increase effectiveness of treatments for inflammatory, viral or proliferative diseases are necessary. In addition, compounds that can reduce the doses and/or reduce frequency of administration to reduce side effects and to maintain or improve efficacy are necessary.
For example, a compound that can improve efficacy of interferon-beta in the treatment of MS would potentially reduce the amount and/or frequency of interferon-beta administration, reducing side effects induced by interferon-beta and possibly reducing the occurrence of neutralizing antibodies to interferon-beta in MS patients. Neutralizing antibodies to interferon- beta occur in about one third of MS patients treated with interferon-beta and are positively correlated with a loss of therapeutic efficacy of interferon-beta (Peisenhammer et al., 1999).
In addition, these compounds would not only enhance the efficacy in current or potential anti- viral, anti-proliferative or anti-inflammatory therapies but would also broaden use of these therapies into many other inflammatory, proliferative and viral diseases.
There have been reports of anti-inflammatory agents being tested in combination for treatment of inflammatory diseases such as multiple sclerosis. However, these combination therapies have resulted in limited efficacy or no reduction in side effects. For example, combination therapy comprising copaxone and interferon-alpha did not improve clinical scores in EAE-treated mouse (Brod et al., 2000). While novantrone, an anti-proliferative drug, is currently tested in clinical trials for combination therapy with interferon-beta, novantrone has a significant side effect profile including serious cardiac toxicity. Novantrone is restricted in its use because the risk of heart disease increases with the cumulative dose. According to the Food and Drug Administration, patients with MS should ordinarily not receive more than 8 to 12 doses of novantrone, administered over two to three years.
Accordingly, there is a need to develop therapies for viral, inflammatory or proliferative diseases that result in sufficient levels of anti-viral, anti-inflammatory and anti-proliferative compounds to achieve maximal efficacy with minimal side effects. There is a need to develop anti-viral, anti-inflammatory and anti-proliferative combination therapies for the treatment of viral, inflammatory or proliferative diseases wherein the combined elements have an enhancing therapeutic effect, while minimizing side effects. Methyl Donors
Methyl donor compounds have been shown to be able to affect the regulation of cells in the immune system as well as the nervous system (Tamura et al, 1999, Sakane et al, 1982). One well known example of a methyl donor compound is vitamin B12 which is commonly known as an essential nutrient or dietary supplement. Vitamin B12 compounds have been known to be involved in metabolic processes; and deficiency of vitamin B12 has been known to provoke pernicious anemia, gastrointestinal and neurological disorders. It is a co-factor essential in the metabolic pathway leading to synthesis of DNA, cell division, as well as cellular metabolism. Biochemical evidence suggests that vitamin B12 compounds may up- regulate gene transcription and thereby regulate protein synthesis (Watanabe et al, 1994).
Although methyl donors such as vitamin B12 and folic acid are commonly used as nutritional supplements, they are not combined with anti-viral, anti-inflammatory or anti-proliferative agents for the treatment of viral, inflammatory or proliferative diseases.
Therefore it is therefore surprising that methyl donors can be combined with anti-viral, anti- inflammatory or anti-proliferative agents for the treatment of viral, inflammatory or proliferative diseases.
It is therefore an object of the present invention to provide a combination therapy using methyl donor compounds with anti- viral, anti-inflammatory or anti-proliferative agents for the treatment of viral, inflammatory or proliferative diseases. Interferon, a multi-faceted molecule that possesses anti-inflammatory, anti-proliferative and anti-viral activities, is an example of the combination therapy with methyl donor compounds. The invention is also supported by usage of other anti-viral, anti-inflammatory and anti-proliferative compounds such as paclitaxel and P-16 with methyl donor compounds.
Summary of the Invention
The invention comprises a pharmaceutical composition for the treatment of a disease selected from a group of viral diseases; proliferative diseases; inflammatory diseases; proliferative and inflammatory diseases; proliferative and viral diseases; viral and inflammatory diseases; and proliferative, viral and inflammatory diseases; comprising: (1) at least one methyl donor compound; and (2) at least one second compound selected from the group consisting of: antiviral compound; anti-proliferative compound; and anti-inflammatory compound.
Another embodiment of this invention comprises a pharmaceutical composition for the treatment of proliferative diseases, such as cancer (including breast cancer, ovarian cancer, non-small-cell lung cancer, Kaposi's sarcoma, malignant melanoma, metastatic renal cell carcinoma, chronic myelogenous leukemia, hairy cell leukemia, follicular lymphoma, astrocytoma and glioma) comprising at least one methyl donor compound and at least one anti-proliferative compound such as paclitaxel, or at least one methyl donor compound and at least one interferon compoundor at least one methyl donor compound and at least one paclitaxel compound.
Yet another embodiment of this invention comprises a pharmaceutical composition for the treatment of viral diseases, such as hepatitis B, hepatitis C, herpes, or vesticular stomatitis comprising at least one methyl donor compound and at least one anti-viral compound, or at least one methyl donor compound and at least one interferon compound. The interferon compound could be one or more of interferon-beta and interferon-beta.
Yet another embodiment of this invention comprises a pharmaceutical composition for the treatment of inflammatory diseases comprising at least one methyl donor compound and at least one anti-inflammatory compound, or at least one methyl donor compound and at least one interferon compound, or at least one methyl donor compound and at least one paclitaxel compound.
Another embodiment of this invention comprises a pharmaceutical composition for the treatment of multiple sclerosis comprising at least one methyl donor compound and at least one interferon compound, such as interferon-beta, or at least one methyl donor compound and at least one paclitaxel compound. Another embodiment of this invention comprises a pharmaceutical composition for the treatment of inflammatory diseases such as multiple sclerosis comprising at least one methyl donor compound and a peptide selected from the group consisting of P16, S-3, S-7, P-32, N-2 and N-3.
Another aspect of this invention is a pharmaceutical composition for the treatment of hepatitis B comprising methyl donor and interferon-alpha or interferon-beta.
Another aspect of this invention is a pharmaceutical composition for the treatment of hepatitis C comprising a methyl donor and interferon-alpha or interferon-beta.
Another aspect of this invention is a pharmaceutical composition for the treatment of cancers including breast cancer, ovarian cancer, non-small cell lung cancer, Kaposi's sarcoma, glioma and astrocytoma comprising a methyl donor and paclitaxel.
Another aspect of this invention is a pharmaceutical composition for the treatment of cancers including Kaposi's sarcoma, malignant melanoma, metastatic renal cell carcinoma, chronic myelogenous leukemia, hairy cell leukemia, follicular lymphoma, astrocytoma and glioma, comprising a methyl donor and interferon.
Another aspect of this invention is a pharmaceutical composition according to any of the aspects outlined above wherein the methyl donor compound is conjugated to the second compound.
Another aspect of this invention is a method of treating a viral, proliferative or inflammatory disease, including MS, hepatitis B and hepatitis C, comprising the step of administering to a patient any of the pharmaceutical compositions outlined above.
Another aspect of this invention is a method of treating a viral, proliferative, or inflammatory disease, including MS, hepatitis B and hepatitis C, comprising the steps of administering the methyl donor compound at a frequency selected from the group consisting of: more than once daily, daily, more than once weekly, weekly, more than once monthly and monthly, and administering the second compound at a frequency selected from the group consisting of: more than once daily, daily, more than once weekly, weekly, more than once monthly, and monthly.
An other aspect of this invention is a method of treating a viral, proliferative, or inflammatory disease, including MS, hepatitis B and hepatitis C, comprising the steps of administering to a patient, either together or separately, one or more methyl donor compound, and one or more anti-inflammatory, anti- viral or anti-proliferative compound.
Another embodiment of this invention comprises a method of administration comprising of administering at least one methyl donor compound and at least one anti-proliferative compound such as paclitaxel, or at least one methyl donor compound and at least one interferon compound or at least one methyl donor compound and at least one paclitaxel compound. The compounds may be administered by any convenient route, for example by infusion (intravenous or subcutaneous) or injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.). In one embodiment, the methyl donor compound is administered by intravenous infusion. In another embodiment, the methyl donor compound is vitamin B12 which is administered by intravenous infusion. The duration of infusion can range from 15 minutes to 120 minutes.
The concentration of the methyl donor compound can range from 10 to 2500 mg. In one embodiment, the methyl donor compound is vitamin B 12 whose concentration can range from
10 to 2500 mg.
Another aspect of this invention is a method for treating a viral, proliferative, or inflammatory disease as described herein wherein paclitaxel is the paclitaxel compound.
Another aspect of this invention is a method for treating a viral, proliferative, or inflammatory disease as described herein wherein interferon alpha or interferon beta is the interferon compound.
Another aspect of this invention is a method of administration for treating a viral, proliferative, or inflammatory disease as described herein wherein the methyl donor compound is vitamin B 12. Furthermore, the vitamin B 12 may be administered by intravenous infusion. Encompassed within this invention are any of the above treatments wherein the dose of methyl donor compound is between 10 and 2500 mg, and/or the dose of anti-inflammatory, anti-viral or anti-proliferative compound is similar or less than the therapeutic dose range used for the anti-inflammatory, anti- viral or anti-proliferative compound.
Encompassed within this invention are any of the above treatments wherein the frequency of administration of the inflammatory, anti-viral or anti-proliferative compound is similar or less than the frequency of administration for the anti-inflammatory, anti-viral or anti-proliferative compound.
Another aspect of this invention is the use of any of the compounds described above to treat a viral, proliferative, or inflammatory disease.
Definitions
Prior to setting forth the invention, it may be helpful to an understanding thereof to first set forth definitions of certain terms that will be used hereinafter.
"Anti-inflammatory compounds" means a class of compounds for treating inflammatory diseases. The compounds include compounds in research, in development and compounds marketed and sold. This class of compounds includes aspirin, sodium salicylate, choline salicylate, salicylsalicylic acid, diflunisal, salsalate, indomethacin, sulindac, phenylbutazone, oxyphenbutazone, tolmetin, ibuprofen, feroprofen, flurbiprofen, ketoprofen, mefanamic acid, meclofenamate, piroxicam, naproxen, hydrocortisone, prednisolone, 6-alpha- methylprednisolone, triamcinolone, dexamethasone, betaroethasone, cyclosporine, mycophenolate mofetil, cyclophosphamide, antisense ICAM-1, 6-mercaptopurine, tacrolimus, muromonab-CD3, ISAtx247, alefacept, efalizmab, infliximab, azathioprine, methotrexate, sulfasalazine, CT-3™, COX-2 inhibitors, OMS-103HP™, CAB-2™, LDP-01™, IPL550,260™, IPL512,602™, Reliflex™, LFA-1 antagonist, IC74, interferon beta, Avonex™, Betaseron™, Betaferon™ or Rebif™, analogues of Avonex™, Betaseron™, Betaferon™ or Rebif™, interferon compounds, taxanes, paclitaxel compounds, TAXOL®, microtubule stabilizing agents, analogues of microtubule stabilizing agents, glatiramer acetate, analogues of glatiramer acetate, Novantrone™, Antergren™, Campath™, Adapalene™, nitric oxide synthase inhibitors, anti-TNF or IL-1 compounds and antagonists, antibodies to CD52, retinoic acid antagonists, diacerhein, diacerhein analogues, adhesion peptides, MAF peptides, cytokines, hyaluronic acid (HA) binding peptides, RHAMM peptides, integrins, and mixtures thereof. The singular form, "anti-inflammatory compound", may mean any one or more compounds from the class of anti-inflammatory compounds.
"Anti-proliferative compounds" means a class of compounds for treating proliferative diseases. The compounds include compounds in research, in development and compounds marketed and sold. This class of compounds includes but not limited to: altretamine (hexamethylmelamine, Hexalen), anastrozole (Arimidex), Exemestane (Aromasin), bicalutamide (Casodex), busulfan (Myleran), capecitabine (Xeloda), chlorambucil (Leukeran), cyclophosphamide (Cytoxan), diethylstilbestrol diphosphate (Stilphostrol), estramustine (Emcyt), etoposide (VP-16, Vepesid), flutamide (Eulexin), hydroxyurea (Droxia), Hydrea, Mylocel, letozole (Femara), leucovorin calcium (Leucovorin), levamisole (Ergamisol), lomustine (CCNU, CeeNU), megestrol (Megace), melphalan (Alkeran), mercaptopurine (6- MP, Purinethol), methotrexate (Methotrexate, Rheumatrex), mitotane (Lysodren), nilutamide (Nilandron), procarbazine (Matulane), tamoxifen (Nolvadex), testolactone (Teslac), thioguanine, tretinoin (Vesanoid), mechlorethamine, 5-fluorouracil, cytarabine, gemcitabine, vinblastine, vincristine, vinorelbine, paclitaxel compounds, TAXOL®, etoposide, irinotecan, topotecan; leuprolide, flutamide, doxorubicin, bleomycin, epirubicin, mitomycin, interferon compounds, carmustine, lomustine, cisplatin, mitoxantrone (Novantrone). The singular form, "anti-proliferative compound", may mean any one or more compounds from the class of anti- proliferative compounds.
"Anti-viral compounds" means a class of compounds which includes any therapeutic compounds for treating viral diseases. The compounds include compounds in research, in development and compounds marketed and sold. The class of anti-viral compounds includes interferon compounds, acyclovir, adefovir, abacavir, amprenavir, cidofovir, didanosine, fomivirsen sodium, dipivoxil, adenine, arabinoside, famciclovir, ganciclovir, lopmavir, ritonavir, lamivudine, nelfinavir mesylate, stavudine, trizivir, amivudine, lobucavir, zidovudine, indinavir, nevirapine, delavirdine, saquinavir, efavirenz, ribavirin, foscarnet, n- docosanol, oseltamivir, valacyclovir, palivizumab, doxuridine, miquimod, vidarabine, trifuridine, ritonavir, neuraminidase inhibitor, tenofovir, disoproxil fumarate and zalcitabine. The singular form, "anti- viral compound", may mean any one or more compounds from the class of anti-proliferative compounds.
"CFA" means complete Freunds' adjuvant.
"DM20" means an isoprotein proteolipid protein. It is a major integral membrane protein of the central nervous system (CNS). DM20 is normally expressed in early (post-natal) stages of growth.
"EAE" or "experimental autoimmune encephalomyelitis" means a mouse model, specifically, the immunosuppressive mouse model for multiple sclerosis.
"Enhanced", "enhancing" or "enhances", used in the context of, for example, "enhanced effectiveness", means an enhanced therapeutic effect, and includes a synergistic effect. "Inflammatory diseases" means a class of diverse diseases and disorders that are characterized by any one of the following: the triggering of an inflammatory response; an upregulation of any member of the inflammatory cascade; the downregulation of any member of the inflammatory cascade. Inflammatory diseases include diabetes, artheriosclerosis, inflammatory aortic aneurysm, restenosis, ischemia/reperfusion injury, glomerulonephritis, sacoidosis cancer, restenosis, reperfusion injury, rheumatic fever, systemic lupus erythematosus, rheumatoid arthritis, Reiter's syndrome, psoriatic arthritis, ankylosing spondylitis, coxarthritis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, pelvic inflammatory disease, multiple sclerosis, diabetes, osteomyelitis, adhesive capsulitis, oligoarthritis, osteoarthritis, periarthritis, polyarthritis, psoriasis, Still's disease, synovitis, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, osteoporosis, inflammatory dermatosis and wound healing. The singular term "inflammatory disease" includes any one or more diseases selected from the class of inflammatory diseases, and includes any compound or complex disease state wherein a component of the disease state includes a disease selected from the class of inflammatory diseases.
"Interferon compounds" means interferon-alpha, interferon-alpha analogues, interferon-alpha derivatives, interferon-alpha conjugates, interferon beta, interferon-beta analogues, interferon- beta derivatives, interferon-beta conjugates and mixtures thereof. Interferon-alpha and interferon-beta genes may be altered by, for example, oligonucleotide directed mutagenesis to produce interferon-beta analogues thereof, such as the human recombinant cysteine depleted or cysteine replaced analogues. Further, identity or location of more than one amino acid may be changed by targeted mutagenesis. The primary amino acid sequence of the protein may be augmented by glycosylation or by other supplementary molecules such as lipids, phosphate, and acetyl groups. Further, individual amino acid residues in the chain may be modified by oxidation, reduction, or other derivatization. The interferon-alpha or interferon-beta protein may be cleaved to obtain the fragments which retain activity. The whole protein or its fragments can be fused with other peptides and proteins such as immunoglobulins and other cytokines. Interferon-alpha and interferon-beta conjugates may represent, for example, a composition comprising interferon-beta coupled to a non-naturally occurring polymer comprising a polyalkylene glycol moiety. Preferred interferon compounds include Roferon®, Intron®, Alferon®, Infergen®, Omniferon®, Alfacon-1, interferon-alpha, interferon-alpha analogues, pegylated interferon-alpha, polymerized interferon-alpha, dimerized interferon- alpha, interferon-alpha conjugated to carriers, encapsulated interferon-alpha, interferon-alpha as oral inhalant, interferon-alpha as injectable compositions, interferon-alpha as a topical composition, Roferon® analogues, Intron® analogues, Alferon® analogues, and Infergen® analogues, Omniferon® analogues, Alfacon-1 analogues, interferon beta, Avonex™, Betaseron™, Betaferon™, Rebif™, interferon-beta analogues, pegylated interferon-beta, polymerized interferon-beta, dimerized interferon-beta, interferon-beta conjugated to carriers, encapsulated interferon-beta, interferon-beta as oral inhalant, interferon-beta as an injectable composition, interferon-beta as a topical composition, Avonex™analogues, Betaseron™, Betaferon™ analogues, and Rebif™ analogues. Alternatively, agents that induce interferon- alpha or interferon-beta production or mimic the action of interferon-alpha or interferon-beta may also be employed. The singular form, "interferon compound", may mean any one or more compounds from the class of interferon compounds.
"MBP" means myelin basic protein.
"Methyl donor compounds" means any compound which enhances the levels of endogenous methyl donors (methyl donor enhancers) or provides methyl groups or methylation of proteins (methyl donors). Methyl donor enhancers include agents that stimulate the expression or activity of methyltransferases, as well as any compound that enhances the levels of intracellular methyl donors or the activity of enzymes required for methyl donation. Methyl donor compounds include but are not limited to arginine, homocysteine, S- adenosylmethionine, methionine, betaine, 5-methyltetra-hydrofolate, folic acid, folate, cyanocobalamin, aquacobalamin, adenosylcobalamin, methylcobalamin, hydroxocobalamin , cyanocobalamin carbanalide, and 5-o-methylbenzylcobalmin [(5-OmeB-za)CN-Cbl] and prodrugs, derivatives, analogues or conjugates of any of these compounds. In addition, methyl donor compounds include polymerization, conjugation or encapsulation of any of these compounds. Preferred methyl donors include vitamin B12 and its analogues, derivatives or conjugates. The singular form, "methyl donor compound", means any one or more compounds from the class of methyl donor compounds.
"Mimetic peptide" means a peptide that biologically mimics active determinants on hormones, cytokines, enzyme substrates, viruses or other bio-molecules, and may stimulate, antagonize or otherwise modulate the physiological activity of natural ligands. "ND4 mouse model" means a transgenic mouse model for multiple sclerosis, produced by transformation with multiple copies of DM20; the genetic mouse model for multiple sclerosis.
"PBS" or "phosphate buffer saline" means an injectable solution that serves as a negative control because it does not have any physiological or therapeutic effects.
"PLP" means an isoprotein proteolipid protein. PLP becomes predominant in the adult.
"Paclitaxel compounds" means paclitaxel and its pro-drugs, analogues, derivatives or conjugates and mixtures thereof. Paclitaxel compounds include but are not limited paclitaxel, TAXOTERE®, TAXOL®, Docetaxel, 10-desacetyl analogues of paclitaxel and 3'N- desbenzoyl-3'N-t-butoxy carbonyl analogues of paclitaxel, 7-deoxy-docetaxol, 7,8- cyclopropataxanes, N-substituted 2-azetidones, 6,7-epoxy paclitaxels, 6,7-modified paclitaxels, 10-desacetoxytaxol, 10-deacetyltaxol (from 10-deacetylbaccatin HI), phosphonooxy and carbonate derivatives of taxol, taxol 2',7-di (sodium 1,2- benzenedicarboxylate, 10-desacetoxy-llJ2-dihydrotaxol-10J2(18)-diene derivatives, 10- desacetoxytaxol, Protaxol (2'-and/or 7-O-ester derivatives), (2'- and/or 7-O-carbonate derivatives), asymmetric synthesis of taxol side chain, fluoro taxols, 9-deoxotaxane, (13- acetyl-9-deoxobaccatine III, 9-deoxotaxol, 7-deoxy-9-deoxotaxol, 10-desacetoxy-7-deoxy-9- deoxotaxol, Derivatives containing hydrogen or acetyl group and a hydroxy and tert- butoxycarbonylamino, sulfonated 2'-acryloyltaxol and sulfonated 2'-O-acyl acid taxol derivatives, succinyltaxol, 2'-γ-aminobutyryltaxol formate, 2'-acetyl taxol, 7-acetyl taxol, 7- glycine carbamate taxol, 2'-OH-7-PEG(5000) carbamate taxol, 2'-benzoyl and 2'J-dibenzoyl taxol derivatives, other prodrugs (2'-acetyltaxol; 2',7-diacetyltaxol; 2'succinyltaxol; 2'-(beta- alanyl)-taxol); 2'gamma-aminobutyryltaxol formate; ethylene glycol derivatives of 2'- succinyltaxol; 2'-glutaryltaxol; 2'-(N,N-dimethylglycyl) taxol; 2'-(2-(N,N- dimethylamino)propionyl)taxol; 2'orthocarboxybenzoyl taxol; 2'aliphatic carboxylic acid derivatives of taxol, Prodrugs {2'(N,N-diethylaminopropionyl)taxol, 2'(N,N- dimethylglycyl)taxol, 7(N,N-dimethylglycyl)taxol, 2'J-di-(N,N-dimethylglycyl)taxol, 7(N,N- diethylaminopropionyl)taxol, 2'J-di(N,N-diethylaminopropionyl)taxol, 2'-(L-glycyl)taxol, 7- (L-glycyl)taxol, 2'J-di(L-glycyl)taxol, 2'-(L-alanyl)taxol, 7-(L-alanyl)taxol, 2'J-di(L- alanyl)taxol, 2'-(L-leucyl)taxol, 7-(L-leucyl)taxol, 2',7-di(L-leucyl)taxol, 2'-(L- isoleucyl)taxol, 7-(L-isoleucyl)taxol, 2'J-di(L-isoleucyl)taxol, 2'-(L-valyl)taxol, 7-(L- valyl)taxol, 2'7-di(L-valyl)taxol, 2'-(L-phenylalanyl)taxol, 7-(L-phenylalanyl)taxol, 2',7-di(L- phenylalanyl)taxol, 2'-(L-prolyl)taxol, 7-(L-prolyl)taxol, 2'J-di(L-prolyl)taxol, 2'-(L- lysyl)taxol, 7-(L-lysyl)taxol, 2',7-di(L-lysyl)taxol, 2'-(L-glutamyl)taxol, 7-(L-glutamyl)taxol, 2'J-di(L-glutamyl)taxol, 2'-(L-arginyl)taxol, 7-(L-arginyl)taxol, 2'J-di(L-argmyl)taxol}, Taxol analogs with modified phenylisoserine side chains, taxotere, (N-debenzoyl-N-tert- (butoxycaronyl)-lθ-deacetyltaxol, and taxanes (e.g., baccatin m, cephalomannine, 10- deacetylbaccatin III, brevifoliol, yunantaxusin and taxusin). The singular form, "paclitaxel compound", means any one or more compounds from the class of paclitaxel compounds.
"Proliferative diseases" means a class of diverse diseases and disorders characterized by a lack of control or poorly controlled cell division or proliferation. Proliferative diseases include anal cancer, bile duct cancer, colon cancer, esophageal cancer, gallbladder cancer, pancreatic cancer, small intestine cancer, stomach cancer, osteosarcoma, ovarian epithelial cancer, gestational trophoblastic tumor, uterine sarcoma, vaginal cancer, vulvar cancer, ovarian germ cell tumor, ovarian cancer, soft tissue sarcoma, acute lymphoblastic leukemia, acute myeloid leukemia, small cell lung cancer, malignant mesothelioma, malignant thymoma, hypopharyngeal cancer, laryngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, parathyroid cancer, salivary gland cancer, brain tumor, glioma, cerebellar astrocytoma, cerebral astrocytoma, ependymoma, medulloblastoma, adrenocortical carcinoma, pituitary tumor, islet cell carcinoma, bladder cancer, kidney cancer, penile cancer, Wilm's tumor, AIDS-related lymphoma, cutaneous T-cell lymphoma, hodgkin's lymphoma, Ewing's sarcoma, chronic myelogenous leukemia, hemangiomas of infancy and childhood, follicular lymphoma, mycosis funoides, hairy cell leukemia, Kaposi's sarcoma, non-hodgkin's lymphoma, multiple myeloma, basal cell carcinoma, malignant melanoma, HCV-related hepatocellular carcinoma, colorectal cancer, non-small cell lung cancer, bladder cancer, renal cell carcinoma, neuroblastoma, bladder cancer, breast cancer, cervical cancer, liver cancer, sarcomas, thyroid cancer, endometrial cancer, uterine cancer, multiple myeloma, testicular cancer, retinoblastoma, colorectal cancer, oral cancer, rectal cancer, renal cell carcinoma, metastatic renal cell carcinoma, prostate cancer, restenosis, arteriosclerosis, proliferative diabetic retinopathy. The singular form "proliferative disease" includes any one or more diseases selected from the class of proliferative diseases, and includes any compound or complex disease state wherein a component of the disease state includes a disease selected from the class of proliferative diseases. "PTX" or "pertussis toxin" means the major protein toxin produced by virulent strains of Bordetella pertussis, the organism that causes whooping cough. PTX is a potent ancillary adjuvant that primes macrophages used to elicit several different autoimmune diseases, including EAE.
"RHAMM" means the receptor for hyaluronic acid mediated motility.
"RHAMM-related peptides" refers to peptide sequences or related peptide sequences of RHAMM, specifically, the S-3 peptide, the S-7 peptide, the P-32 peptide, the P-16 peptide, theV-2 peptide, and the V-3 peptide, as defined herein.
"S-3 peptide" means a specific large peptide fragment of RHAMM, and includes the C- terminal or RHAMM and has the approximate size of 45Kda. The term "S-3 peptide" can refer to either the mouse or human amino acid RHAMM peptide fragment, as follows:
SEQ ID No. 1 Mouse S-3 (333 amino acids)
AQAE IAQEKYNDTAQSLRDVTAQLESVQEKYNDTAQSLRDVTAQLESEQEKYNDT AQSLRDVTAQLESEQEKYNDTAQSLRDVTAQLESVQEKYNDTAQSLRDVSAQLESY KSSTLKEffiDLl^ENnLTLQEKVAMAEKSVEDVQQQILTAESTNQEYARMVQDLQNRS TLKEEEIKEITSSFLEKITDLKNQLRQQDEDFRKQLEEKGKRTAEKENVMTELTMEIN KWl^LYEELYEKTKPFQQQLDAFEAEKQALLI^HGATQEQLNKIRDSYAQLLGHQN LKQKIKHVVKLKDENSQLKSEVSKLRSQLVKRKQNELRLQGELDKALGIR
SEQ ID No 2 Human S-3 (242 amino acids) QEKYDSMVQSLEDVTAQFESYKALTASEIEDLKLENSSLQEKAAKAGKNAEDVQHQI LATESSNQEYVRMLLDLQTKSALKETEIKEITVSFLQKITDLQNQLKQQEEDFRKQLE DEEGRKAEKENTTAELTEEINKWRLLYEELYNKTKPFQIQLDAFEVEKQ ALLNEHGAAQEQLNKIRDSYAKIiGHQNLKQKIKHVVKLKDENSQLKSEVSKLRCQ LAKKKQSETKLQEELNKVLGIK
"S-7 peptide" means a specific RHAMM peptide fragment with an approximate size of 23 Kda and represents a smaller portion of S-3 peptide. The term "S-7 peptide" can refer to either the mouse or human amino acid RHAMM peptide fragment, as follows: SEQ ID No 3 Mouse S-7 (221 amino acids)
KSSTLKEIEDLKLENLTLQEKVAMAEKSVEDVQQQILTAESTNQEYARMVQDLQNRS TLKEEEIKEITSSFLEKITDLKNQLRQQDEDFRKQLEEKGKRTAEKENVMTELTMEIN KWRLLYEELYEKTKPFQQQLDAFEAEKQALLNEHGATQEQLNKIRDSYAQLLGHQN LKQKIKIINNKLKDENSQLKSENSKLRSQLNKRKQNELRLQGELDKALGIR
SEQ ID No 4 Human S-7 (221 amino acids)
KAX.TASEEDLKLENSSLQEKAAKAGKNAEDVQHQILATESSNQEYVRMLLDLQTKS ALKETEIKEHNSFLQKITDLQNQLKQQEEDFRKQLEDEEGRKAEKENTTAELTEEINK WRLLYEELYNKTKPFQIQLDAΪΕNEKQALLNEHGAAQEQLNKXRDSYAKLLGHQNL KQKIKHVVKLKDENSQLKSEVSKLRCQLAKKKQSETKLQEELNKVLGIK
"P-32 peptide" means a specific RHAMM peptide fragment. The term "P-32 peptide" can refer to either the mouse or human amino acid RHAMM peptide fragment, as follows:
SEQ ID No 5: Human P-32 KQKIKHVVKLKDENSQLKSEVSKLRCQLAKKK
SEQ ID No 6 Mouse P-32 KQKIKHVVKLKDENSQLKSENSKLRSQLNKRK
"V-2 peptide" means a specific RHAMM peptide fragment which represents a large portion of the original RHAMM peptide, includes the C-terminal and has the approximate size of 58 Kda. The term "V-2 peptide" can refer to either the mouse or human amino acid RHAMM peptide fragment, as follows:
SEQ ID No 7 Mouse V-2
MQILTERLALERQEYEKLQQKELQSQSLLQQEKELSARLQQQLCSFQEEMTSEKNVF KEELKLALAELDAVQQKEEQSERLVKQLEEERKSTAEQLTRLDNLLREKEVELEKHI AAHAQAILIAQEKYNDTAQSLRDVTAQLESVQEKYNDTAQSLRDVTAQLESEQEKY NDTAQSLRDVTAQLESEQEKYNDTAQSLRDVTAQLESVQEKYNDTAQSLRDVSAQL ESYKSSTLKEffiDLKLENLTLQEKVAMAEKSVEDVQQQILTAESTNQEYARMVQDLQ NRSTLKEEEIKEITSSFLEKITDLKNQLRQQDEDFRKQLEEKGKRTAEKENVMTELTM ET-NKWiπ-LYEELYEKTKPFQQQLDAFEAEKQALLNEHGATQEQLNKIRDSYAQLLGH QNLKQKIKHVVKLKDENSQLKSEVSKLRSQLVKRKQNELRLQGELDK-ALGIRHFDPS KAFCHASKENFTPLKEGNPNCC
SEQ ID NO 8 Human V-2 MQNLKQKFILEQQEHEKLQQKELQroSLLQQEKELSSSLHQKLCSFQEEMVKEKNLF EEELKQTLDELDKLQQKEEQAEPvLVKQLEEEAKSRAEELKLLEEKLKGKEAELEKSS AAHTQATLLLQEKYDSMVQSLEDVTAQFESYKALTASEIEDLKLENSSLQEKAAKAG KNAEDVQHQJ-LATESSNQEYVRMLLDLQTKSALKETEIKEITVSFLQKITDLQNQLKQ QEEDFRKQLEDEEGRKAEKENTTAELTEEINKWRLLYEELYNKTKPFQLQLDAFEVE KQALLNEHGAAQEQLN IRDSYAKLLGHQNLKQKIKHVVKLKDENSQLKSEVSKLR CQLAKKKQSETKLQEELNKVLGIKHFDPSKAFHHESKENFALKTPLKEGNTNCYRAP MECQESWK
"V-3 peptide" means a specific RHAMM peptide fragment. The term "V-3 peptide" can refer to either the mouse or human amino acid RHAMM peptide fragment, as follows:
SEQ ID No 9 Human V-3
MQNLKQKFILEQQEREKLQQKELQIDSLLQQEKELSSSLHQKLCSFQEEMAKEKNLFE EELKQTLDELDKLQQKEEQAERLVKQLEEEAKSRAEELKLLEEKLKGKEAELEKSSA AHTQATLLLQEKYDSMVQSLEDVTAQFESYK-ALTASEIEDLKLENSSLQEKAVAKAG KNAEDVQHQILATESSNQEYVRMLLDLQTKSALKETEIKEITVSFLQKITDLQNQLKQ QEEDFRKQLEDEEGRKAEKENTTAELTEEINKWRLLYEELYNKTKPFQLQLDAFEVE KQALLNEHGAAQEQLNKIRDSYAKLLGHQI^KQKIKHVVKLKDENSQLKSEVSKLR CQLAKKKTK
SEQ ID No 10 Mouse V-3
MQILTERLALERQEYEKLQQKELQSQSLLQQEKELSARLQQQLCSFQEEMTSEKNVF KEELKLALAELDAVQQKEEQSERLVKQLEEERKSTAEQLTRLDNLLREKEVELEKHI AAHAQAILIAQEKYNDTAQSLRDVTAQLESVQEKYNDTAQSLRDVTAQLESEQEKY NDTAQSLRDVTAQLESEQEKYNDTAQSLRDVTAQLESVQEKYNDTAQSLRDVSAQL ESYKSSTLKEFFIDLKLENLTLQEKVAMAEKSVEDVQQQILTAESTNQEYARMVQDLQ NRSTLKEEEIKEITSSFLEKITDLKNQLRQQDEDFRKQLEEKGKRTAEKENVMTELTM EΓ KWRIXYEELYEKTKPFQQQLDAFEAEKQALLNEHGATQEQLNKΓRDSYAQLLGH QNLKQKIKHVVKLKDENSQLKSEVSKLRSQLVKRKQN "P-16 peptide", "P-16", and "Ha binding peptide" mean a 16 amino acid synthetic peptide which can bind hyaluronic acid with high affinity. The peptide was isolated using phage display technology. The peptide has the following amino acid sequence:
SEQ ID No 11 P-16 CSTMMSRSHKTRSHHV
"SJL/J" means a mouse model. Specifically, a female SJL/ J mouse has increased susceptibility to development of autoimmune disease. A SJL/J transgenic mouse strain is susceptible to induction of EAE (more susceptible to development of EAE than most other mouse strains). Tumor development as well as autoimmunity in this mouse may result from an effective amplification of the immune response.
"Synergistic" means a greater anti-inflammatory, anti-proliferative and/or anti- viral effect with the use of a combination therapy of methyl donors and anti-inflammatory, anti- proliferative, and/or anti-viral compound than with the use of any of these therapeutic compounds alone. This synergistic effect can work through either similar or different mechanisms or pathways of action. One advantage of a combination therapy with a synergistic effect is that standard dosages can be used for a greater therapeutic effect than expected from the addition of the effect of either compound administered alone; or alternatively lower dosages or reduced frequency of administration of the therapeutic compound(s) may be used to achieve a better therapeutic effect.
"Viral diseases" means a class of diverse diseases and disorders caused by or believed to be caused by viruses. The class of viral diseases includes genital warts (HPV), HIV/AIDS, herpes, influenza, measles, polio, varicella-zoster, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G., meningitis, genital warts (HPV), vesticular stomatitis virus infection, and dengue fever. The singular form "viral disease" includes any one or more diseases selected from the class of viral diseases, and includes any compound or complex disease state wherein a component of the disease state includes a disease selected from the class of viral diseases. "Vitamin B12 compounds" means a class of compounds which includes vitamin B12 and its analogues, derivatives or conjugates. The class of vitamin B12 compounds includes cyanocobalamin (CN-Cbl), aquacobalamin, adenosylcobalamin, methylcobalamin, hydroxycobalamin (HC), cyanocobalamin carbanalide, and 5-o-methylbenzylcobalmin [(5- OmeB-za)CN-Cbl] as well as the desdimethyl, monoethylamide and the methylamide analogues of all of the above. Also included are the various analogues and homologues of cobamamide such as coenzyme B12 and 5-deoxydenosylcobalamin. Other analogues include chlorocobalamin, sulfitocobalamin, nitrocobalamin, thiocyanatocobalamin, benzimidazole derivatives such as 5,6-dichlorobenzimidazole, 5-hydroxybenzimidazole, trimethylbenzimidazole, as well as adenosylcyanocobalamin [(Ade) CN-Cbl], cobalamin lactone, cobalamin lactam and the anilide, ethylamide, monocarboxylic and dicarboxylic acid derivatives of vitamin B12 or its analogues. Preferred derivatives of vitamin B12 include the mono-, di- and tricarboxylic acid derivatives or the proprionamide derivatives of vitamin B12. In addition, the compositions include polymers of these analogues or vitamin B12 conjugated to other molecules or encapsulated. The singular form, "vitamin B12 compound", may mean any one or more compounds from the class of vitamin B 12 compounds.
Sequence Listing
SEQ ID No. 1
AQAITIAQEKYNDTAQSLRDVTAQLESVQEKYNDTAQSLRDVTAQLESEQEKYNDT
AQSLRDVTAQLESEQEKYNDTAQSLRDVTAQLESVQEKYNDTAQSLRDVSAQLESY
KSSTLI- EffiDLKLENLTLQEKVAMAEKSVEDVQQQILTAESTNQEYARMVQDLQNRS
TLKEEEIKEITSSIT.EKITDLKNQLRQQDEDFRKQLEEKGKRTAEKENVMTELTMEIN
KWRLLYEELYEKTI^FQQQLDAFEAEKQALLNEHGATQEQLNKIRDSYAQLLGHQN
LKQKIKHVVKLKDENSQLKSEVSKLRSQLVKRKQNELRLQGELDKALGIR
SEQ ID No 2
QEKYDSMVQSLEDVTAQFESYKALTASEIEDLKLENSSLQEKAAKAGKNAEDVQHQI LATESSNQEYVRMLLDLQTKSALKETEIKEITVSFLQKITDLQNQLKQQEEDFRKQLE DEEGRKAEKENTTAELTEEINKWRLLYEELYNKTKPFQIQLDAFEVEKQ ALLNEHGAAQEQLNmRDSYAKLLGHQNLKQKIKHVVKLKDENSQLKSEVSKLRCQ LAKKKQSETKLQEELNKVLGIK
SEQ ID No 3 KSSTLKEffiDLKLENLTLQEKVAMAEKSVEDVQQQILTAESTNQEYARMVQDLQNRS TLKEEEIKEITSSIT.EKITDLKNQLRQQDEDFRKQLEEKGKRTAEKENVMTELTMEIN KWRLLYEELYEKTKPFQQQLDAFEAEKQALLNEHGATQEQLNKIRDSYAQLLGHQN LKQΩKHNNKLKDENSQLKSENSKLRSQLVKRKQNELRLQGELDKALGIR
SEQ ID No 4
KALTASEIEDLI^ENSSLQEKAAKAGKNAEDNQHQILATESSNQEYNRMLLDLQTKS ALKETEIKEITNSI QKITDLQΝQLKQQEEDFRKQLEDEEGRKAEKEΝTTAELTEEIΝK WRLLYEELYΝKTKPFQIQLDAIΕVEKQALLΝEHGAAQEQLΝKIRDSYAKLLGHQΝL KQiaKHVVKLKDEΝSQLKSEVSKLRCQLAKKKQSETKLQEELΝKVLGIK
SEQ ID No 5:
KQ1HKHVVKLKDENSQLKSEVSKLRCQLAKKK SEQ ID No 6
KQKIKHVVKLKDENSQLKSEVSKLRSQLVKRK
SEQ ID No 7 MQILTERLALERQEYEKLQQKELQSQSLLQQEKELSARLQQQLCSFQEEMTSEKNVF KEELKLALAELDAVQQKEEQSERLVKQLEEERKSTAEQLTRLDNLLREKEVELEKHI AAHAQAILIAQEKYNDTAQSLRDVTAQLESVQEKYNDTAQSLRDVTAQLESEQEKY NDTAQSLRDVTAQLESEQEKYNDTAQSLRDVTAQLESVQEKYNDTAQSLRDVSAQL ESYKSSTLKEffiDLKLENLTLQEKVAMAEKSVEDVQQQILTAESTNQEYARMVQDLQ NRSTLKEEEIKEITSSFLEKITDLKNQLRQQDEDFRKQLEEKGKRTAEKENVMTELTM EINKWRLLYEELYEKTKPFQQQLDAFEAEKQALLNEHGATQEQLNKIRDSYAQLLGH QNLKQKIKHVVKLKDENSQLKSEVSKLRSQLVKRKQNELRLQGELDKALGI-RHFDPS KAFCHASKENFTPLKEGNPNCC
SEQ ID NO 8
MQNLKQKFILEQQEHEKLQQKELQIDSLLQQEKELSSSLHQKLCSFQEEMVKEKNLF EEELKQTLDELDKLQQKEEQAERLVKQLEEEAKSRAEELKLLEEKLKGKEAELEKSS AAHTQATLLLQEKYDSMVQSLEDVTAQFESYK-ALTASEffiDLKLENSSLQEKAAKAG KNAEDVQHQlLATESSNQEYVPvMLLDLQTKSALKETEIKEITVSFLQKITDLQNQLKQ QEEDFRKQLEDEEGRKAEKENTTAELTEEINKWRLLYEELYNKTKPFQLQLDAFEVE KQALLNEHGAAQEQLNKIPΛSYAKLLGHQNLKQKIKHVVKLKDENSQLKSEVSKLR CQLAKEKQSETI.a.QEELNKVLGIKHr^PSKAFHHESKENFALKTPLKEGNTNCYRAP MECQESWK
SEQ ID No 9
MQNLKQKFILEQQEREKLQQKELQroSLLQQEKELSSSLHQKLCSFQEEMAKEKNLFE EELKQTLDELDKLQQKEEQAERLVKQLEEEAKSRAEELKLLEEKLKGKEAELEKSSA AHTQATLLLQEKYDSMVQSLEDVTAQFESYKALTASEIEDLKLENSSLQEKAVAKAG KNAEDVQHQ1LATESSNQEYVIAMLLDLQTKSALKETEIKEITVSFLQKITDLQNQLKQ QEEDFRKQLEDEEGRKAEKENTTAELTEEINKWRLLYEELYNKTKPFQLQLDAFEVE KQALLNEHGAAQEQLNKIRDSYAKLLGHQI^KQKIKXWVKLKDENSQLKSEVSKLR CQLAKKKTK
SEQ ID No 10 MQE.TERLALERQEYEKLQQKELQSQSLLQQEKELSARLQQQLCSFQEEMTSEKNVF KEELKLALAELDAVQQKEEQSERLVKQLEEERKSTAEQLTRLDNLLREKEVELEKHI AAHAQAJJ IAQEKYNDTAQSLRDVTAQLESVQEKYNDTAQSLRDVTAQLESEQEKY NDTAQSLRDVTAQLESEQEKYNDTAQSLRDVTAQLESVQEKYNDTAQSLRDVSAQL ESYKSSTLKEffiDLiπ-ENLTLQEKVAMAEKSVEDVQQQILTAESTNQEYARMVQDLQ NRSTLKEEEIKEITSSFLEKITDLKNQLRQQDEDFRKQLEEKGKRTAEKENVMTELTM EINKWRLLYEELYEKTKPFQQQLDAFEAEKQALLNEHGATQEQLNKIRDSYAQLLGH QNLKQKIKHVVKLKDENSQLKSEVSKLRSQLVKRKQN
SEQ ID No 11 P-16
CSTMMSRSHKTRSHHV
Description of Drawings
Figure 1 is a graph showing the effect of a methyl donor compound (vitamin B12), interferon- beta and the combination of a methyl donor compound and interferon-beta on clinical scores in the ND4 mouse model.
Figure 2 is a graph showing the effect of a methyl donor compound (vitamin B12), interferon- beta and the combination of a methyl donor compound and interferon-beta on clinical scores in the EAE mouse model.
Figure 3 is a graph showing the effect of a methyl donor compound (vitamin B12), P-16 and the combination of a methyl donor compound and P-16 on clinical scores in the ND4 mouse model.
Figure 4 is a graph showing the GFAP staining in brain sections from normal, ND4-untreated animals, interferon-beta treated ND4 animals, and interferon-beta/vitamin B12 treated ND4 animals.
Figure 5 is a graph showing the effect of a methyl donor compound (vitamin B12), TAXOL® and the combination of a methyl donor compound and TAXOL® on clinical scores in the ND4 mouse model.
Detailed Description of the Invention
The current invention discloses the effect of methyl donor compounds alone and in combination with anti-viral, anti-inflammatory or anti-proliferative compounds for treatment of viral, inflammatory or proliferative diseases. Methyl donor compounds alone show efficacy and clearly show an enhancing or synergistic effect in combination with other anti-viral, anti- inflammatory, and anti-proliferative compounds for treating viral, inflammatory, and proliferative diseases.
Pharmaceutical composition
The present invention provides pharmaceutical compositions for enhancing anti-viral, anti- proliferative and anti-inflammatory effects and pharmaceutical compositions for treatment of viral, proliferative and inflammatory diseases. The pharmaceutical composition comprises a first compound that is at least one of a methyl donor compound and a second compound that is at least one of an anti- viral, anti-proliferative and/or anti-inflammatory compound.
Excipients
The pharmaceutical compositions of the invention preferably contain a pharmaceutically acceptable carrier or excipient suitable for rendering the compound or mixture administrable orally, intranasally, or parenterally, intravenously, intradermally, intramuscularly or subcutaneously, rectally, via inhalation or via buccal administration, or transdermally. The active ingredients may be admixed or compounded with any conventional, pharmaceutically acceptable carrier or excipient. It will be understood by those skilled in the art that any mode of administration, vehicle or carrier conventionally employed and which is inert with respect to the active agents may be utilized for preparing and administering the pharmaceutical compositions of the present invention. Illustrative of such methods, vehicles and carriers are those described, for example, in Remington's Pharmaceutical Sciences, 4th ed. (1970). Those skilled in the art, having been exposed to the principles of the invention, will experience no difficulty in determining suitable and appropriate vehicles, excipients and carriers or in compounding the active ingredients therewith to form the pharmaceutical compositions of the invention. The compositions of the invention may also be conjugated to transport molecules, monoclonal antibodies or transport modalities such as vesicles and micelles that preferentially target recipient cells.
Administration
The compounds of the present invention in the described dosages are administered orally, intranasally, by inhalation, mtraperitoneally, subcutaneously, intra-muscularly, transdermally, sublingually or intravenously. For oral administration the pharmaceutical composition can be prepared, for example, in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gum or the like prepared by procedures known to those skilled in the art. The compounds may be administered by any convenient route, for example by infusion or injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.).
Anti- viral, anti-inflammatory and anti-proliferative compounds such as interferon, paclitaxel, mitoxantrone (Novantrone) can be given via intravenous infusion to patients suffering from anti-viral, anti-inflammatory and anti-proliferative diseases. For example, one of the administration routes that interferon-alpha is administered for treatment for Kaposi's sarcoma or malignant melanoma is via an intravenous infusion route. Novantrone was given to MS patients in clinical trials at a dose of either 5 mg/m2 or 12 mg/m2 via intravenous infusion every three months. Paclitaxel can also be given via intravenous infusion to cancer patients over a duration of 3 hours with a dose of about 135-175 mg/m . 9 million units (MU) of interferon-alpha were administered to patients suffering from hepatitis C by continuous subcutaneous infusion.
Preferred administration regimen for treating a patient includes an infusion for methyl donor compounds and/or anti- viral, anti-inflammatory and anti-proliferative compounds. In the case of both methyl donor compounds and anti-viral, anti-inflammatory and anti-proliferative compounds being administered via infusion, the administration of these compounds can be combined or separate. In the case of either one of the therapeutic agent being administered via iinfusion, the other therapeutic agent can also be administered via other administration routes including intraperitoneally, subcutaneously, intra-muscularly, transdermally, sublingually, orally, intranasally or by inhalation. In all cases, the methyl donor compound can be administered first, then followed by the anti-viral, anti-inflammatory and anti-proliferative compound or vice versa. Preferably, the methyl donors are administered via intravenous infusion. The duration of infusion for each therapeutic compound can range from 15 mins to 360 mins with the optimal range being from 15 mins to 120 mins, with the option of treatment being repeated at specific or regular intervals. In addition, other types of administration regimens can precede or subsequently follow the infusion regimen for methyl donor compound and/or anti- viral, anti-inflammatory and anti-proliferative compound.
The therapeutically effective amount of active agent to be included in the pharmaceutical composition of the invention depends, in each case, upon several factors, e.g., the type, size and condition of the patient to be treated, the intended mode of administration, the capacity of the patient to incorporate the intended dosage form, the severity of the disease progression, etc, and therefore should be decided according to the judgment of the practitioner and each patient's circumstances. The dosages used for each anti- viral, anti-proliferative and anti- inflammatory compound are similar to those dosages known to those skilled in the art and used in pre-clinical and clinical studies and in commercial use. The dose may also be lower than the currently used dosages as the combination of these compounds with methyl donor compounds increases the efficacy of these compounds. In addition, the frequency of administration of these compounds may also be reduced as the combination with methyl donor compounds increases efficacy of these compounds. Indeed, methyl donor compounds may be combined with anti-viral, anti-proliferative and anti-inflammatory compounds with the objective to reduce the dosages and/or reduce frequency of administration of the anti-viral, anti-proliferative and anti-inflammatory compounds, in order to achieve both effective treatment and to lessen the negative side effects of the anti-viral, anti-proliferative and anti- inflammatory compounds.
The preferred dosage of a methyl donor compound for the present invention is the maximum a patient requires to provide an optimal enhancing effect, such maximum being tempered by the absolute upper limit of methyl donor dosage being the maximum that a patient can tolerate and not develop any serious complications. Vitamin B12, an example of a methyl donor compound, has been available for many years as an injectable treatment for pernicious anemia, with doses typically in the range of lOOOμcg. Vitamin B12 also has a long history as a general oral health supplement with doses also in the "μcg" range. High doses of hydroxocobalamin are used as a cyanide-poisoning antidote, called Cyanokit® (US Patent # 5,834,448). Cyanokit® is an acute one-time 5-gram dose of hydroxocobalamin administered for emergency purposes.
Though methyl donors such as vitamin B12 have been proposed for use for the therapeutic treatment for a few diseases, no person has demonstrated that high doses of methyl donors in combination with an anti-viral, anti-inflammatory or anti-proliferative compound would achieve an enhanced or synergistic therapeutic effect. Furthermore, no person has demonstrated that high doses of methyl donors in combination with an anti-viral, anti- inflammatory or anti-proliferative compound, requires less anti-viral, anti-inflammatory or anti-proliferative compound or requires a reduction in frequency of administration of antiviral, anti-inflammatory or anti-proliferative compound. The dosage of the methyl donor compound for our invention is within the range of 10 mg to 2.5 g daily.
Those skilled in the art will be aware that the amounts of the various components of the compositions of the invention to be administered in accordance with the invention to a patient will depend upon those factors noted above.
Methods and uses
The present invention provides methods to enhance or potentiate anti- viral, anti-proliferative and anti-inflammatory compound-induced anti-viral, anti-proliferative and anti-inflammatory effects and methods of treating viral, proliferative or inflammatory diseases in patients by administering an amount of methyl donor compound in combination with anti-viral, anti- proliferative and/or anti-inflammatory compounds. Methyl donor compounds can be administered simultaneously, separately or in combination with anti-viral, anti-proliferative, or anti-inflammatory compounds, under different doses, frequency of administration and route regimens, to enhance the efficacy of anti-viral, anti-proliferative and anti-inflammatory compounds in the treatment of viral, proliferative or inflammatory diseases in patients compared to when such compounds are administered alone.
The first method of treatment is the administration of a pharmaceutical composition including both a methyl donor compound and an anti-viral, anti-proliferative and anti-inflammatory compound. An alternate method of treatment includes the step of the administration of a pharmaceutical composition including at least one of the methyl donor compound followed by the step of the administration of a second pharmaceutical composition including at least one of an anti-viral, anti-proliferative and anti-inflammatory compound. Optionally, the administration of at least one of the methyl donor compound can follow the administration of at least one of the anti-viral, anti-proliferative and anti-inflammatory compound. Optionally, the administration of the pharmaceutical compositions can occur separately or simultaneously. Optionally, the administration of the pharmaceutical compositions can occur sequentially.
The following are examples of acceptable regimens:
1. More than once daily, daily, more than once weekly, weekly, more than once monthly or monthly mixtures of methyl donor compounds in combination with anti- viral, anti-proliferative and anti-inflammatory compounds for the effective treatment of anti-viral, anti-proliferative and anti-inflammatory diseases;
2. More than once daily, daily, more than once weekly, weekly, more than once monthly or monthly mixtures of methyl donor compounds simultaneously with anti- viral, anti-proliferative and anti-inflammatory compounds for the effective treatment of anti-viral, anti-proliferative and anti-inflammatory diseases;
3. More than once daily, daily, more than once weekly, weekly, more than once monthly or monthly treatments with anti-viral, anti-proliferative and anti-inflammatory compounds and methyl donor compounds administered separately either more than once daily, daily, more than once weekly, weekly, more than once monthly, or monthly;
4. More than once daily, daily, more than once weekly, weekly, more than once monthly or monthly treatments with anti-viral, anti-proliferative and anti-inflammatory compounds and methyl donor compounds administered sequentially either more than once daily, daily, more than once weekly, weekly, more than once monthly, or monthly;
5. More than once daily, daily, more than once weekly, weekly, more than once monthly or monthly mixtures of anti-viral, anti-proliferative and anti- inflammatory compounds and methyl donor compounds, as well as adjunct administration of more than once daily, daily, more than once weekly, weekly, more than once monthly or monthly doses of methyl donor compounds.
Those skilled in the art will readily understand that the pathologies and disease states expressly stated herein are not intended to be limiting. Rather the compounds of the present invention may be used to treat any disease that elicits an inflammatory, proliferative or viral response.
EXAMPLES
The examples below are exemplary of the embodiments of the present invention.
(A) Examples demonstrating anti-inflammatory effects of interferon-based combination therapies with methyl donor compounds
Example 1: Effect of vitamin B12 (cyanocobalamin), interferon-beta and combination of vitamin B12 (cyanocobalamin) incorporating interferon-beta in ND4 mouse model
Rationale:
Vitamin B12 (cyanocobalamin), an example of a methyl donor compound, and interferon- beta, an anti-inflammatory compound that is used for the treatment of inflammatory diseases such as MS, were used in a demyelinating transgenic mouse model to demonstrate a synergistic effect when a combination of methyl donor compounds and anti-inflammatory compounds were used in the treatment of such diseases as compared to treatment with anti- inflammatory compounds or methyl donor compound alone.
Experimental Protocol:
The ND4 model was used, as a slow progressive demyelinating transgenic mouse model where the animals demonstrate symptoms in young adults at approximately 3 months of age. In this model, the severity of the clinical signs increased until a maximum around 6 months with animals dying around 8 to 9 months of age. Clinical signs assessed included general shaking, seizures, head jerk, hind limb and tail shiver, wobbly gait and limp tail. The scale of zero (absence) to four (constant and uncontrollable movements) was used for each of the clinical signs.
Mice (4 animals per group) were treated with cyanocobalamin alone at a dose of 15 mg/kg; weekly and in combination with interferon-beta la (Rebif™). Interferon-beta la was administered alone and in combination with cyanocobalamin at a dose of interferon-beta la of 5000 IU, tri-weekly. All treatments began when the mice reached 3 months of age at which time signs of demyelinating disease were evident. Treatment was stopped at time of sacrifice.
Results and Conclusions:
Treatment of the ND4 mice with interferon-beta or cyanocobalamin showed attenuation of clinical signs of MS, an inflammatory disease (Figure 1) but the combination therapy of interferon-beta and cyanocobalamin had a synergistic effect in reducing the clinical signs of the inflammatory disease as compared to treatment with interferon-beta or cyanocobalamin alone.
Example 2: Effect of vitamin B12, interferon-beta and combination of vitamin B12 (cyanocobalamin) with interferon-beta in EAE mouse model
Rationale: Vitamin B12 (cyanocobalamin), an example of a methyl donor compound, and interferon- beta, an example of an anti-inflammatory compound that is used for the treatment of inflammatory diseases such as multiple sclerosis, were used in an autoimmune EAE model to demonstrate an enhancing effect when a combination of methyl donor compounds and anti- inflammatory compounds were used in the treatment of such diseases as compared to treatment with either anti-inflammatory compounds or methyl donor compounds alone.
Experimental Protocol:
Experimental Allergic Encephalomyelitis (EAE) was induced in 7-8 week old female SJL mice. Each mouse was injected intravenously with 200 ng Pertussis Toxin (PT) in lOOμl total volume at a tail vein on day 1. Following 48 hours, 200 ng PT was injected a second time. Tail vein injections were done with 30.5 gauge needles. Following the PT injection was a one time MBP/CFA (Becton Dickinson) emulsion injection. Each mouse was injected subcutaneously using a 27.5 gauge needle with 200μg/50μl of MBP emulsified in 50ul of complete Freund's adjuvant (CFA) at the base of the tail. The total injection volume was lOOμl. The mice were observed daily after first injection and the clinical signs of disease were recorded. These included unsteady gait, shaking, tail drop, weight loss, etc. Clinical scores were evaluated every other day starting on day 7 following immunization. Animals were treated daily for 11 days.
Mice (4 animals per group) were treated with cyanocobalamin alone at a dose of 10 mg/kg; daily and in combination with interferon-beta. Interferon-beta la (Rebif™) was administered alone and in combination with cyanocobalamin at a dose of 5000 IU of interferon-beta, daily; cyanocobalamin and interferon-beta were administered as separate injections starting on the day of first immunization. Treatment was stopped at time of sacrifice. Mice were monitored daily from day 7 after immunization for clinical signs of EAE and were scored on a scale of 0 to 5. A score of 0 represented the absence of signs while a score of 5 was given to moribund animals.
Results and Conclusions:
Treatment of the EAE mice with interferon-beta alone showed attenuation of clinical signs of the inflammatory disease (Figure 2) but the combination therapy of interferon-beta and cyanocobalamin were more effective in reducing the clinical signs of the inflammatory disease, indicating the combination of interferon-beta and cyanocobalamin enhanced the anti- inflammatory activity of interferon-beta.
Example 3: Effect of interferon concentration on Vitamin B12 synergistic effects
Rationale: Vitamin B12 (cyanocobalamin), an example of a methyl donor compound, and varying concentrations of interferon-beta, an example of anti-inflammatory compound used for the treatment of inflammatory diseases such as multiple sclerosis, were used in the ND4 transgenic model described above to demonstrate concentration effect of anti-inflammatory compounds on cyanocobalamin synergistic effects.
Experimental Protocol:
ND4 mice were either (a) untreated or treated with (b) interferon-beta la (Rebif™) alone (5000 IU) or (c) 1000 IU of interferon-beta la and cyanocobalamin (15mg/kg), or (d) 2500 units of interferon-beta la and cyanocobalamin (15mg/kg) or (e) 5000 IU of interferon-beta la and B12 (15mg/kg), three times a week for the duration of the experiment. Treatment was started when the animals were 3 months of age and continued for 11 weeks. The animals were evaluated twice a week for clinical signs and the scores were averaged on a weekly basis. Refer to Example 1 for more experimental details.
Results and Conclusions:
The results demonstrated that the effectiveness of the combination therapy was dependent on the concentration of interferon-beta, with significant reduction of clinical scores (at interferon-beta concentration of 5000 IU). Thus, by combining cyanocobalamin and interferon-beta, a lower level of interferon was needed to achieve pharmacological action. (Table 1).
Table 1. Effect of interferon-beta concentration on synergistic effects of cyanocobalamin on the clinical signs and symptoms in the transgenic mouse at six months
Treatment Sum of Clinical Scores (+/- SD)
ND4 Untreated 22 +/- 2
Interferon-beta (5000 IU) 21 +/- 4
Interferon-beta (1000 IU) + 18 +/- 1 cyanocobalamin (15mg/kg)
Interferon-beta (2500 IU) + 16 +/- 1 cyanocobalamin (15mg/kg)
Interferon-beta (5000 IU) + 13 +/- 2 cyanocobalamin (15mg/kg)
Example : Effect of vitamin B12 (cyanocobalamin), RHAMM mimetic (P-16 peptide) and combination of vitamin B12 (cyanocobalamin) with P-16 peptide in ND4 mouse model
Rationale:
P-16 peptide, a HA mimetic peptide, had been demonstrated to possess anti-inflammatory properties in both MS and diabetes animal studies. The effects of cyanocobalamin (an example of a methyl donor compound) in combination with P-16 peptide (an example of an anti-inflammatory compound) in the treatment of inflammatory diseases were investigated. Experimental Protocol:
The ND4 model is a slow progressive demyelinating transgenic mouse model where the animals demonstrate symptoms in young adults at approximately 3 months of age. The severity of the clinical signs increase until a maximum around 6 months with animals dying around 8 to 9 months of age. Clinical signs assessed include general shaking, seizures, head jerk, hind limb and tail shiver, wobbly gait and limp tail. The scale of zero (absence) to four (constant and uncontrollable movements) was used for each of the clinical signs.
Mice (4 animals per group) were treated with cyanocobalamin alone at a dose of 15 mg/kg; weekly and in combination with P-16. P-16 was administered alone and in combination with cyanocobalamin at a dose of 1 mg/kg, tri-weekly. All treatments began when the mice reached 3 months of age at which time signs of demyelinating disease were evident. Treatment was stopped at time of sacrifice.
Results and Conclusions:
Treatment of the ND4 mice with the P-16 peptide showed attenuation of clinical signs of MS, an inflammatory disease (Figure 3) but the combination therapy of P-16 and cyanocobalamin was more effective in reducing the clinical signs of the inflammatory disease than P-16 alone.
Example 5: Effect of folic acid, interferon-beta and combination of folic acid and interferon-beta in ND4 mouse model
Rationale:
Folic acid, an example of a methyl donor compound, and interferon-beta, an anti- inflammatory compound that is used for the treatment of inflammatory diseases such as MS, were used in a demyelinating transgenic mouse model to demonstrate a synergistic effect when a combination of methyl donor compounds and anti-inflammatory compounds were used in the treatment of such diseases as compared to treatment with anti-inflammatory compounds or methyl donor compound alone.
Experimental Protocol:
The ND4 model was used, as a slow progressive demyelinating transgenic mouse model where the animals demonstrate symptoms in young adults at approximately 3 months of age. In this model, the severity of the clinical signs increased until a maximum around 6 months with animals dying around 8 to 9 months of age. Clinical signs assessed included general shaking, seizures, head jerk, hind limb and tail shiver, wobbly gait and limp tail. The scale of zero (absence) to four (constant and uncontrollable movements) was used for each of the clinical signs.
Mice (4 animals per group) were treated with folic acid (purchased from Supelco) alone at a dose of 200 μg/kg; tri- weekly and in combination with interferon-beta la (Rebif™). Interferon-beta la was administered alone and in combination with folic acid at a dose of interferon-beta la of 5000 IU, tri- weekly. All treatments began when the mice reached 3 months of age at which time signs of demyelinating disease were evident. Treatment was stopped at time of sacrifice.
Results and Conclusions: Treatment of the ND4 mice with interferon-beta or folic acid showed attenuation of clinical signs of MS, an inflammatory disease but the combination therapy of interferon-beta and folic acid had a synergistic effect in reducing the clinical signs of the inflammatory disease as compared to treatment with interferon-beta or folic acid alone.
(B) Examples demonstrating anti-proliferative effects of combination therapies incorporating methyl donor compounds
Example 6: Effect of vitamin B12 (methylcobalamin), interferon and combination of vitamin B12 with interferon in inhibiting cellular proliferation in vitro
Rationale:
Interferon was also known as an anti-proliferative agent that has been reported to be effective in treating proliferative diseases such as Kaposi's sarcoma, malignant melanoma, multiple myeloma, hairy cell leukemia, metastatic renal carcinoma, chronic myelogenous leukemia, HCV-related hepatocellular carcinoma, glioma, follicular lymphoma, fibrosarcoma and high- grade astrocytoma (Fine et al., 1997). The following experiment was carried out to determine whether a methyl donor compound such as vitamin B12 (methylcobalamin) could enhance the anti-proliferative effects of an anti-inflammatory compound such as interferon in astrocytoma cell lines.
Experimental Protocol:
Mouse ascite astrocytoma cells were seeded in a 96-well plate and grown to 60% confluence in standard growth conditions. Prior to treatment, cells were washed and the culture medium was replaced with culture medium containing interferon-beta alone (500 IU/ml), methylcobalamin (25 μg/ml) alone, or combination of interferon-beta (500 IU/ml), and methylcobalamin (25 μg/ml), and incubated overnight.
The CellTiter 96® AQue0Us One Solution Cell Proliferation Assay was used as a colorimetric method for determining the number of viable cells in proliferations. Cell proliferation assays were performed by adding a small amount of the CellTiter 96® AQue0Us One Solution Reagent directly to culture wells, incubating for 1-4 hours and then recording absorbance at 490nm with a 96 well plate reader. The quantity of formazan product as measured by the amount of 490nm absorbance is known in the art to be directly proportional to the number of living cells in the culture. Results and Conclusions:
Treatment of either mouse interferon-beta or methylcobalamin alone resulted in a decrease in proliferative activity as compared to untreated cells. However, anti-proliferative activity was significantly increased when cells were treated with a combination of methylcobalamin and interferon-beta (Table 2 below) as compared to treatment with either methylcobalamin or interferon-beta alone.
Table 2. Effect of combination therapy on cellular proliferation in a mouse astrocytoma cell line
Treatment Measure of Proliferative Activity (Absorbance at 490 nm)
No treatment 0.8955
Treatment with mouse interferon-beta 0.3855
(500 IU/ml)
Treatment with methylcobalamin 0.2985
(25μg/ml)
Treatment with mouse interferon-beta 0J435
(500 IU/ml) and methylcobalamin
(25μg/ml)
Example 7: Effect of vitamin B12 (cyanocobalamin), interferon, combination of vitamin B12 with interferon on astrocyte gliosis
Rationale:
Astrocyte gliosis refers to the increased reactivity and proliferation of astrocytes in the central nervous system and is thought to play a major role in the development of lesions in the brain. Specifically, astrocyte gliosis has been observed around hematogenous metastases of the human brain and GFAP, the protein marker for reactive astrocytes, has been used as a biomedical marker for early detection for chemically induced cancer. Both the ND4 and EAE mouse models were used below to illustrate the effect of the combination therapy on astrocyte gliosis. The ND4 transgenic mice develops normally up to three months, after which demyelination progresses thereafter. Along with primary demyelination, astrogliosis increases from about twice normal levels at three months to extensive astrogliosis by adult stage. Similarly, the EAE-induced mice had also been shown to demonstrate extensive astrogliosis. (a) ND4Model Experimental Protocol:
ND4 mice were treated as described previously in Example 1. After the various treatments, the mice were sacrificed and brain sections removed for GFAP staining and quantitation. The brain region of hippocampus, adjacent to the dentate gyrus was fixed in formalin, paraffin- embedded and sectioned at 5 μm. Immunohistochemistry with anti-GFAP antibody was used to stain for GFAP. Glial fibrillary acidic protein was a protein expressed by astrocytes (particularly reactive astrocytes) and was used as a marker for these cells.
In addition, quantification of GFAP within the brains of normal, ND4 untreated and ND4 treated mice was carried out to provide a more precise level of protein expression. Whole brain homogenates were assayed for GFAP by slot blot. Brains were homogenized in a buffer containing 50 mM Tris-HCl pH 7.6, 0.5 mM DTT, 1 mM EDTA, and 0.43 mM PMSF. The homogenate was centrifuged at 11000 rpm for 30 min at 4°C. The pellet was isolated and resuspended in a buffer containing 10 mM sodium phosphate pH 7.5, 2 mM DTT, 6 M urea and 1 mM EDTA. Samples were loaded onto the slot blot apparatus (BioDot, Biorad) under vacuum. The blots were reacted with anti-GFAP antibodies and then a secondary antibody. The blots were developed and the relative amounts of each band quantified.
Results and Conclusions:
There was a significant increase in GFAP staining of untreated ND4 animals compared to normal animals (Figure 4), indicating extensive astrocyte gliosis. This high level of GFAP staining was decreased in interferon-beta treated animals. Treatment with interferon decreased the number of activated fibrous astrocytes slightly and the levels of GFAP in brain homogenates. The treatment of interferon-beta and cyanocobalamin inhibited the levels of activated astrocytes and levels of GFAP to near normal levels (Table 3). These data clearly demonstrated that the combination treatment of interferon-beta (an example of an anti- proliferative compound) with cyanocobalamin (an example of a methyl donor compound) acted in a synergistic manner to inhibit astrocyte proliferation and activation.
Table 3. Total relative GFAP in brain homogenates from normal, ND4 untreated animals, vitamin B12 (cyanocobalamin)-treated, interferon-beta treated; and interferon- beta and vitamin B12 treated ND4 animals. Treatment Relative Amount of
GFAP (+/- SD)
Normal 1.0
Untreated ND4 mice 4.4 +/- 0.2
Cyanocobalamin (15mg/kg) 2.8 +/- 0J
Interferon-beta (5000 IU/ml) 3.1 +/- 0J
Interferon-beta (5000 IU/ml) 1.6 +/- 0.2
+cyanocobalamin (15mg/kg)
(b) EAE Model Experimental Protocol:
EAE mice were treated as described previously in Example 2. After the various treatments, the mice were sacrificed and brain sections removed for GFAP staining and quantitation. The brain region of hippocampus, adjacent to the dentate gyrus was fixed in formalin, paraffin- embedded and sectioned at 5 μm. Lrimunohistochemistry with anti-GFAP antibody was used to stain for GFAP. Glial fibrillary acidic protein was a protein expressed by astrocytes (particularly reactive astrocytes) and was used as a marker for these cells.
In addition, quantification of GFAP within the brains of normal, EAE untreated and EAE treated mice was carried out to provide a more precise level of protein expression. Whole brain homogenates were assayed for GFAP by slot blot. Brains were homogenized in a buffer containing 50 mM Tris-HCl pH 7.6, 0.5 mM DTT, 1 mM EDTA, and 0.43 mM PMSF. The homogenate was centrifuged at 11000 rpm for 30 min at 4°C. The pellet was isolated and resuspended in a buffer containing 10 mM sodium phosphate pH 7.5, 2 mM DTT, 6 M urea and 1 mM EDTA. Samples were loaded onto the slot blot apparatus (BioDot, Biorad) under vacuum. The blots were reacted with anti-GFAP antibodies and then a secondary antibody. The blots were developed and the relative amounts of each band quantified.
Results and Conclusions:
Treatment with vitamin B12 (cyanocobalamin) was not effective in decreasing the levels of GFAP while treatment of interferon-beta was able to reduce the amounts of GFAP by ~50%. However, treatment of interferon-beta and cyanocobalamin was the most effective among the treatment groups. The combination therapy inhibited the levels of activated astrocytes and levels of GFAP to near normal levels (Table 4). These data clearly demonstrated that the combination treatment of interferon-beta, an example of anti-proliferative compound, with cyanocobalamin, an example of a methyl donor compound, acted in a synergistic manner to inhibit astrocyte proliferation and activation.
Table 4. Total relative GFAP in brain homogenates from normal, EAE untreated animals, vitamin B12 treated interferon-beta treated; and interferon-beta and vitamin B12 treated EAE animals.
Treatment Relative Amount of
GFAP (+/- SD)
Normal 1.0
Untreated EAE mice 4.2 +/- 0.2
Cyanocobalamin (15mg/kg) 4.2 +/- 0.2
Interferon-beta (5000 IU/ml) 2.0 +/- 0.5
Interferon-beta (5000 IU/ml) 1.4 +/- 0.2
+cyanocobalamin (15mg/kg)
Example 8: Effect of vitamin B12 (cyanocobalamin), paclitaxel and combination of vitamin B12( cyanocobalamin) with paclitaxel (TAXOL®) in ND4 mouse model
Rationale:
Paclitaxel (TAXOL®) was an anti-proliferative agent that is commonly used to treat cancers such as ovarian cancer, breast cancer, non-small cell lung cancer and Kaposi's sarcoma. The following experiment was carried out to determine whether vitamin B12 (cyanocobalamin), an example of a methyl donor compound, could enhance the anti-proliferative effects of anti- proliferative compounds such as paclitaxel in the ND4 mouse model. As discussed earlier, the ND4 mouse model was associated with astrocyte proliferation in the brain.
Experimental Protocol:
The ND4 transgenic mice received one of the following treatments through mtraperitoneal injections: 1) TAXOL® at a dose of 20 mg/kg once a week; 2) cyanocobalamin at a dose of 10 mg/kg once a week and TAXOL® at a dose of 5 mg/kg three times per week; and 3) cyanocobalamin at a dose of 10 mg/kg once a week. All treatments started when the mice reached 3 months of age, at which time signs of demyelinating disease were evident. Results and Conclusions:
Treatment of the ND4 mice with cyanocobalamin or paclitaxel alone showed a significant attenuation of mean clinical scores. The combination therapy of cyanocobalamin with paclitaxel was more effective in attenuation of clinical signs compared to when these compounds were used alone. There was an enhanced therapeutic effect when cyanocobalamin and Taxol™ were combined. Results are shown in Figure 5.
Example 9: Effect of folic acid, interferon and combination of folic acid with interferon in inhibiting cellular proliferation in vitro
Rationale:
Interferon was also known as an anti-proliferative agent that has been reported to be effective in treating proliferative diseases as discussed in Example 6. The following experiment was carried out to determine whether a methyl donor compound such as folic acid could enhance the anti-proliferative effects of an anti-inflammatory compound such as interferon in astrocytoma cell lines.
Experimental Protocol:
Mouse ascite astrocytoma cells were seeded in a 96-well plate and grown to 60% confluence in standard growth conditions. Prior to treatment, cells were washed and the culture medium was replaced with culture medium containing interferon-beta alone (500 IU/ml), folic acid (25 μg/ml) alone, or combination of interferon-beta (500 IU/ml), and folic acid (25 μg/ml), and incubated overnight. The CellTiter 96® AQue0us One Solution Cell Proliferation Assay was carried out as explained in Example 6.
Results and Conclusions:
Treatment of either mouse interferon-beta or folic acid alone resulted in decreased proliferative activity as compared to untreated cells. However, anti-proliferative activity was significantly increased when the astrocytoma cells were treated with a combination of folic acid and interferon-beta as compared to treatment with either folic acid or interferon-beta alone. (C) Examples demonstrating anti-viral effects of combination therapies with methyl donor compounds
Example 10: In vitro studies demonstrating the enhancing effect of interferon compound with vitamin B12 (hydroxocobalamin) on treatment of human hepatitis B virus (HBV)
Rationale:
Hep G2.2J5 was used as a HepG2 cell line that is stably transfected with HBV genome and that continues to secrete HBV virions into the cell growth medium. This cell line has been widely used to study the activities of anti-viral agents against HBV infection and was used in this study to evaluate the enhancing effect of methyl donor compounds with interferon, an example of an anti-viral compound, on treating HBV infections. Interferon was used as an example of an anti- viral agent because it was used to treat both hepatitis B and C in the clinic.
Experimental Protocol:
HepG2.2J5 cells, a cell line that was stably transfected with HBV genome, were cultured in RPMI-1640 with fetal bovine serum at either 2 or 4% final concentration and with no antibiotics. The cells were seeded in a 96-well plate at a density of 2xl04 cells/well and grown overnight. The medium was then discarded and replaced with 100 μl of culture medium containing hydroxocobalamin (a methyl donor compound) alone (at various concentrations), interferon-beta alone (at various international units), and a mixture of varying doses of hydroxocobalamin (HC) and various doses of interferon-beta vs HC. Triplicate samples were measured for each concentration. The cells were incubated at 37°C for 9 days. Drug/medium was changed daily. On day 9, the culture media containing released HBV virions were harvested and used to extract HBV DNA.
HBV DNA from drug-treated HepG2.2J5 cell culture media was extracted using QIAamp DNA mini kit - blood and body fluid spin column according to manufacturer's instructions. Briefly, 10 μl of QIAGEN proteinase K was added to 1.5 ml Eppendorf tube. 50 μl cell supernatant and 50 μl AL buffer were added to the tube. The samples were incubated at 56°C water bath for 3 h. At the end of incubation, 50 μl ethanol was added to the sample. The mixture was transferred to a QIAamp spin column and centrifuged at 14000 rpm for 1 min. The filtrate was discarded. 50 μl Buffer AW1 was then added to the column and centrifuged at 14000 rpm for 1 min. Following washing and recentrifugation, DNA was eluted in 50 μl dH2O.
HBV DNA was detected by real-time PCR using Lightcycler assay. The reaction was performed in 20 μl, comprising the following:' 6.4/4.8 μl dH20, 2 μl 25 mM MgCl2ι 1 μl 10 μM sense primer, 1 μl 10 μM antisense primer, 8/1.6 μl LightCycler Probe (fluorescein- labeled, 5μM), 0.8/1.6 μl LightCycler Probe 2 (LC Red 640-labeled, lOμM), 2 μl LC FastStart DNA Master Hyb Probes, 1 μl uracil DNA glycosylase.
PCR mix was aliquoted into glass capillaries and 5 μl DNA template was then added to the PCR mix. A range of HBV plasmid DNA (lxlO8, lxlO7, lxlO6, ...JxlO2, 25 copies) was used as standards for quantitative analysis. The cycling conditions are shown in Table 5.
Table 5. The cycling conditions for Lightcycler assay
Program # cycles Target Temp Incubation Time Temp Transition Acquisition Mode
Denaturation 1 95°C 9 min 20°C/sec None
PCR 45 95°C 5 sec 20°C/sec None
54°C 10 sec 20°C/sec Single
72°C 17 sec 20°C/sec None
Melt 1 98°C 0 sec 20°C/sec None
45°C 15 sec 20°C/sec None
70°C 0 sec 0J°C/sec Continuous
Cool 1 40°C 30 sec 20°C/sec None
The antiviral effects of the test compounds were calculated using DNA copy numbers as an endpoint. Specifically, the antiviral activities were calculated based on the following formula: Antiviral effect = (CCOntroi - Ctest)/CControi whereby the Ccontr0ι is the HBV DNA copies in the control wells (without drug); the Ctest is the HBV DNA copies in the test wells with a given concentration of a test compound.
The enhancing or antagonistic effect was analyzed mathematically using the median effect equation (Chou and Talalay, 1984) by computer software CalcuSyn (Biosoft, St Louis). The range of the combination index (Cl) is illustrated below in Table 6.
Table 6. Correlation between combination index and degree of synergism
Figure imgf000045_0001
Results and Conclusions: Table 7. Antiviral effects of test compounds alone and combinations on HBV measured by HBV DNA copies using Lightcycler assay
Concentrations (μg/ml) 0 1 10 100 500
HC 0 0.001 0.001 0.001 0.112
Concentrations (IU/ml) 0 50 100 500 1000 interferon-beta 0 0.569 0.746 0.773 0.823
Concentrations 0 1/50 10/100 100/500 500/1000 (μg/ml)/(IU/ml)
HC / interferon-beta 0 0.571 0.772 0.912 0.851
Table 8. Cl values for the combinations between hydroxocobalamin (HC) and interferon-beta on HBV
HC interferon- Cl
(μg/ml) beta (IU/ml)
1 50 1.908
10 100 0.258
100 500 0.051
500 1000 0.572
Viral activity was decreased when cells were treated with interferon-beta but not with hydroxocobalamin. The combination therapy of hydroxocobalamin with interferon-beta was more effective in attenuating viral activity compared to when these compounds were used alone. There was an enhanced effect for the treatment of hepatitis B infection when hydroxocobalamin and interferon were combined. See Table 7 for more details.
Specifically, strong and moderate potentiating effects were examined for HC on interferon- beta activity against HBV at the concentrations were higher than 10 μg/ml of HC and 100 IU/ml of interferon-beta, respectively. Based on the outcome of the combination index analysis, the anti-viral effects of interferon-beta with hydroxocobalamin were synergistic. The results are summarized in Table 8 above.
Example 11: In vitro studies demonstrating the enhancing effect of interferon anti- iral activity with vitamin B12 (hydroxycobalamin) on herpes simplex virus type 2 (HSV-2) and vesticular stomatitis virus (VSV)
Rationale:
MRC-5 (human fibroblast cells) was a HSV and VSV susceptible cell line that was used widely to study the activities of anti- viral agents against VSV and HSV infections. This cell line was used in this study to evaluate the enhancing effect of hydroxocobalamin, an example of a methyl donor compound with interferon-alpha and interferon beta, both examples of antiviral compounds, on treating VSV and HSV infections.
Experimental Protocol:
A CPE (cytopathic effect) inhibition assay was performed. This assay measures the ability of a drug or agent to protect cells from lysis by a virus. Briefly, confluent MRC-5 cells in a 96- well plate were incubated with various concentrations of hydroxocobalamin (HC) alone, interferon-alpha alone, interferon-beta alone, and the mixtures of varying doses of HC and various doses of interferon-alpha or interferon-beta. The test concentrations for HC were 0, 1, 10, 100, 500 and 1000 μg/ml and that of interferon-alpha and interferon-beta were 0, 5, 10, 50, 100, and 500 IU/ml. Triplicate samples were tested for each concentration in a 96- well plate format. The culture medium for MRC-5 cells was recommended by ATCC (Minimum Essential Medium Eagle with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0J mM non-essential amino acids, and 1.0 mM sodium pyruvate, 10% fetal bovine serum).
After overnight incubation, the cells were infected with either VSV at moi (multiplicity of infection) of 0.5 pfu/cell or HSV-2 at moi of 0.2 pfu/cell at 37°C for 1 h. Following removal of virus inoculum, the infected cells were washed with PBS and covered with medium containing either no or increasing concentrations of test compounds as above for 2-3 days at 37°C until the CPE in the control wells was 100%. The cells protected from CPE were measured using neutral red uptake assay. Specifically, culture medium was removed from cells in a 96-well plate by pump. The cells were then washed once with 200 μl of PBS. 100 μl of 0.01% neutral red (in PBS) was subsequently added to each well, and incubated at 37°C for 30 min. The dye was then removed and the cells were washed twice with 200μl PBS per well. The dye was extracted by addition of 100 μl of 50% ethanol/1% glacial acetic acid in PBS to each well and incubated at room temperature for 15 min with gentles shaking at 120-150 rpm. The absorbance at 550nm was read on an ELISA reader.
The antiviral effects were calculated based on the following formula: Antiviral effect = (ODτest - ODo)/(ODcontroi - ODo) whereby ODτest is the optical density measured with a given concentration of the test compound; ODo s the optical density measured at drug concentration zero; ODcontroi is the optical density of uninfected cells.
The potentiating effect was analyzed mathematically using the median-effect equation (Chou and Talalay, 1984) by a computer program CalcuSyn (Biosoft, St Louis). Refer to Table 6 for correlation between combination index obtained and degree of synergism.
Results and Conclusions: (a) HSV-2 Infection
The combination therapy of vitamin B12 (hydroxocobalamin) with interferon-alpha or interferon-beta was more effective in attenuation viral activity compared to when these compounds were used alone (Table 9). There was an enhanced effect for the treatment of herpes simplex virus infection when hydroxocobalamin (HC), a methyl donor compound and interferon, an anti-viral agent, were combined. Specifically, a strong potentiating effect was observed for HC on interferon-alpha activity against HSV-2 when the concentrations of HC and interferon-alpha were higher than 10 μg/ml and 10 IU/ml respectively. Strong and moderate potentiating effects were observed for HC on interferon-beta activity against HSV-2 at all concentrations tested. Based on the outcome of the combination index analysis, the antiviral effects of interferon-alpha or interferon-beta with hydroxocobalamin were synergistic (Table 10).
Table 9. Antiviral effects of test compounds alone and combinations on HSV-2 determined by CPE inhibition assay
Concentrations (μg/ml) 0 1 10 100 500 lθ"θθ" HC 0 0.014 0.032 0.056 0J66 0.210
Concentrations (IU/ml) 0 5 10 50 100 500
Interferon-alpha 0 0.093 0.139 0.218 0.282 0.271 Interferon-beta 0 0.164 0.136 0.314 0.281 0.265
Concentrations 0 1/5 10/10 100/50 500/100 1000/50 (μg/ml)/(IU/ml) 0
HC / interferon-alpha 0 0.090 0.257 0.342 0.724 0.999 HC / interferon-beta 0 0J69 0.315 0.368 0.683 0.748
Table 10. Cl values for the combinations between HC and interferon-alpha or interferon-beta on HSV-2
HC interferon- Cl HC interferon- Cl
(μg/ml) alpha (IU/ml) 1 (μg/ml) beta (IU/ml)
1 5 2.292 1 5 0.746
10 10 0.07 10 5 0.026
100 50 0.117 100 50 0.065
500 100 0.009 500 100 0.012
1000 500 <0.01 1000 500 0.013
(b) VSV Infection
Anti-viral activity (as reflected by protection of CPE) was significantly increased when cells were treated with hydroxocobalamin or interferon-alpha or interferon-beta. The combination therapy of hydroxocobalamin with interferon-alpha or interferon-beta was more effective in attenuation viral activity compared to when these compounds were used alone. See Table 11. There was an enhanced effect for the treatment of vesticular stomatitis virus infection when hydroxocobalamin and interferon, an anti-viral agent, were combined. Specifically, strong potentiating effect was observed for hydroxocobalamin on interferon-beta activity against HSV at all concentrations tested and on interferon-alpha activity when the concentrations of HC and interferon-alpha were higher than 100 μg/ml and 50 IU/ml respectively. Based on the outcome of the combination index analysis, the anti- viral effects of interferon-alpha or interferon-beta with hydroxocobalamin were synergistic (Table 12).
Table 11. Antiviral effects of test compounds alone and combinations on VSV determined by CPE inhibition assay
Concentrations (μg/ml) 0 1 10 100 500 1000
HC 0 0.001 0.001 0.039 0.001 0.002 Concentrations (IU/ml) 0 5 10 50 100 500
Interferon-alpha 0 0.296 0.387 0.950 0.996 0.942 Interferon-beta 0 0.826 0.985 0.758 0.768 0.956
Concentrations 0 1/5 10/10 100/50 500/100 1000/50 (μg/ml)/(IU/ml) 0
HC / interferon-alpha 0 0.299 0.450 0.999 0.999 0.999 HC / interferon-beta 0 0.999 0.999 0.999 0.999 0.999
Table 12. Cl values for the combinations between HC and interferon-alpha or interferon-beta on VSV
HC Interferon- Cl HC Interferon- Cl
(Mg/ml) alpha (IU/ml 1 (μg/ml) beta (IU/ml)
1 5 1.6 1 5 0
10 10 1.766 10 5 0
100 50 0.013 100 50 0.002
500 100 0.027 500 100 0.004
1000 500 0.016 1000 500 0.021
Example 12: In vitro studies demonstrating the enhancing effect of interferon compound with folic acid on treatment of human hepatitis B virus (HBV)
Rationale:
Hep G2.2J5 was used as a HepG2 cell line that is stably transfected with HBV genome and that continues to secrete HBV virions into the cell growth medium. This cell line has been widely used to study the activities of anti- viral agents against HBV infection and was used in this study to evaluate the enhancing effect of methyl donor compounds such as folic acid with interferon, an example of an anti-viral compound, on treating HBV infections. Interferon was used as an example of an anti-viral agent because it was used to treat both hepatitis B and C in the clinic.
Experimental Protocol:
HepG2.2J5 cells, a cell line that was stably transfected with HBV genome, were cultured in RPMI-1640 with fetal bovine serum at either 2 or 4% final concentration and with no antibiotics. The cells were seeded in a 96-well plate at a density of 2xl04 cells/well and grown overnight. The medium was then discarded and replaced with 100 μl of culture medium containing folic acid (a methyl donor compound) alone (at various concentrations, 0J μg/ml to 1000 μg/ml)), interferon-beta alone (at various international units, similar to that of Example 10), and a mixture of varying doses of folic acid and various doses of interferon- beta vs HC. Triplicate samples were measured for each concentration. The cells were incubated at 37°C for 9 days. Drug/medium was changed daily. On day 9, the culture media containing released HBV virions were harvested and used to extract HBV DNA.
HBV DNA from drug-treated HepG2.2J5 cell culture media was extracted using QIAamp
DNA mini kit as described previously in Example 10. HBV DNA was detected by real-time
PCR using Lightcycler assay under similar conditions as described in Example 10.
The antiviral effects of the test compounds were calculated using DNA copy numbers as an endpoint. Specifically, the antiviral activities were calculated based on the following formula: Antiviral effect = (Ccontroι - Ctest)/CControi whereby the Ccontroi is the HBV DNA copies in the control wells (without drug); the est i me HBV DNA copies in the test wells with a given concentration of a test compound.
The enhancing or antagonistic effect was analyzed mathematically using the median effect equation (Chou and Talalay, 1984) by computer software CalcuSyn (Biosoft, St Louis). The range of the combination index (Cl) is illustrated below in Table 6 in Example 10.
Results and Conclusions:
Viral activity was decreased when cells were treated with either interferon-beta or folic acid alone. However, the combination therapy of folic acid with interferon-beta was more effective in attenuating viral activity compared to when these compounds were used alone. There was an enhanced effect for the treatment of hepatitis B infection when folic acid and interferon were combined. Furthermore, based on the outcome of the combination index analysis, the anti- viral effects of interferon-beta with folic acid were synergistic.
Summary of examples
In summary, these examples clearly demonstrate that the combination treatment of a methyl donor compound in conjunction with a therapeutic agent such as interferon, paclitaxel or P-16 is effective in treating inflammatory, proliferative and/or viral diseases and that the methyl donor enhances the effectiveness of or has a synergistic effect with the specific therapeutic agent against the disease indicated. Furthermore, these examples demonstrate that methyl donor compounds in combination with an anti-viral, anti-inflammatory or anti-proliferative compounds, require less anti-viral, anti-inflammatory or anti-proliferative compound.
Although the invention has been described with preferred embodiments, it is to be understood that modifications may be resorted to as will be apparent to those skilled in the art. Such modifications and variations are to be considered within the purview and scope of the present invention.
References
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Chou TC and Talalay P. Quanatitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. (1984). Advances in Enzyme Regulation 22:27-55
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Watanabe T, Kaji R, Oka N, Bara W and Kimura J. Ultra-high dose methylcobalamin promotes nerve regeneration in experimental acrylamide neuropathy. (1994) Journal of Neurological Sciences 222(2): 140-143.

Claims

WE CLAIM:
1. A pharmaceutical composition for the treatment of a disease selected from the group consisting of: viral diseases; proliferative diseases; inflammatory diseases; proliferative and inflammatory diseases; proliferative and viral diseases; viral and inflammatory diseases; and proliferative, viral and inflammatory diseases; comprising: a. At least one methyl donor compound; and b. At least one second compound selected from the group consisting of: an antiviral compound; an anti-proliferative compound; and an anti-inflammatory compound.
2. A pharmaceutical composition for the treatment of proliferative diseases comprising: a. At least one methyl donor compound; and b. At least one anti-proliferative compound.
3. A pharmaceutical composition for the treatment of viral diseases comprising: a. At least one methyl donor compound; and b. At least one anti- viral compound.
4. A pharmaceutical composition for the treatment of inflammatory diseases comprising: a. At least one methyl donor compound; and b. At least one anti-inflammatory compound, wherein the amount, the frequency of administration or both the amount and frequency of administration of the anti-inflammatory compound present in the composition is lower than the amount, frequency of administration or both the amount and frequency of administration of the anti-inflammatory compound which would be required in the absence of the methyl donor compound. 5. The pharmaceutical composition of Claim 2, wherein the anti-proliferative compound is interferon.
6. The pharmaceutical composition of Claim 3, wherein the anti-viral compound is interferon.
7. The pharmaceutical composition of Claim 4, wherein the anti-inflammatory compound is interferon.
8. The pharmaceutical composition of Claim 2, wherein the anti-proliferative compound is paclitaxel.
9. The pharmaceutical composition of Claim 4, wherein the anti-inflammatory compound is paclitaxel. 10. The pharmaceutical composition of Claim 4, wherein the anti-inflammatory compound is a peptide selected from the group consisting of P16, S-3, S-7, P-32, V-2 and V-3.
11. The pharmaceutical composition of Claim 2, wherein the methyl donor compound is vitamin B12 compound. 12. The pharmaceutical composition of Claim 3, wherein the methyl donor compound is vitamin B12 compound.
13. The pharmaceutical composition of Claim 4, wherein the methyl donor compound is vitamin B12 compound.
14. A pharmaceutical composition for the treatment of a virus selected from the group consisting of hepatitis B, hepatitis C, herpes simplex virus type 2 and vesticular stomatitis virus comprising: a. At least one methyl donor compound; and b. At least one interferon compound.
15. A pharmaceutical composition for the treatment of cancer comprising: a. At least one methyl donor compound; and b. At least one anti-proliferative compound.
16. A pharmaceutical composition for the treatment of astrocytoma and glioma comprising: a. At least one methyl donor compound; and b. At least one anti-proliferative compound.
17. A pharmaceutical composition for the treatment of hepatitis B/hepatitis C comprising: a. At least one methyl donor compound; and b. At least one interferon compound.
18. A pharmaceutical composition for the treatment of cancer comprising: a. At least one methyl donor compound; and b. At least one interferon compound.
19. The pharmaceutical composition of Claim 18, where in cancer is one selected from a group consisting of: malignant melanoma, hairy cell leukemia, metastatic renal carcinoma, chronic myeloid leukemia and follicular lymphoma, multiple myeloma, astrocytoma and glioma. 20. A pharmaceutical composition of any of Claims 17 to 19, wherein the interferon compound is interferon-alpha.
21. A pharmaceutical composition of any of Claims 17 to 19, wherein the interferon compound is interferon-beta.
22. A pharmaceutical composition for the treatment of cancer comprising: a. At least one methyl donor compound; and b. At least one paclitaxel compound.
23. The pharmaceutical composition of Claim 22, wherein the cancer is one selected from a group consisting of: astrocytoma and glioma, ovarian cancer, breast cancer, non- small cell lung cancer and Kaposi's sarcoma. 24. The pharmaceutical composition of any of Claims 22 to 23, wherein the paclitaxel compound is paclitaxel.
25. A pharmaceutical composition for the treatment of multiple sclerosis comprising: a. At least one methyl donor compound; and b. A peptide selected from the group consisting of P16, S-3, S-7, P-32, V-2 and V-3.
26. A pharmaceutical composition for the treatment of multiple sclerosis comprising: a. At least one methyl donor compound; and b. At least one interferon compound, wherein one or more of the amount, the frequency of administration or both the amount and frequency of administration of the interferon compound present in the composition is lower than the amount, the frequency of administration or both the amount and frequency of administration of the compound which would be required in the absence of the methyl donor compound.
27. A pharmaceutical composition for the treatment of multiple sclerosis comprising: a. At least one methyl donor compound; and b. Interferon-beta.
28. A pharmaceutical composition for the treatment of multiple sclerosis comprising: a. At least one methyl donor compound; and b. Paclitaxel.
29. A pharmaceutical composition for the treatment of hepatitis B/hepatitis C comprising: a. At least one methyl donor compound; and b. Interferon-alpha.
30. A pharmaceutical composition for the treatment of hepatitis C comprising: a. At least one methyl donor compound; and b. Interferon-alpha 31. The pharmaceutical composition of any of Claims 14-30, wherein the methyl donor compound is vitamin B 12 compound.
32. The pharmaceutical composition of any of Claims 14-30, wherein the methyl donor compound is folic acid.
33. A pharmaceutical composition of any of Claims 1-32 wherein one compound is conjugated to another compound.
34. The pharmaceutical composition of any of any of Claims 1-32 wherein one compound is administered simultaneously with, separately from or sequentially to the other compound.
35. The pharmaceutical composition of any of Claims 1-32, wherein both compounds are administered by infusion.
36. The pharmaceutical composition of any of Claims 1-32, wherein one compound is administered by infusion and the other compound is administered by a route other than infusion.
37. The pharmaceutical composition of Claims 1-32 wherein the compounds are administered by a route selected from a group consisting of: intravenously, intraperitoneally, subcutaneously, intra-muscularly, transdermally, sublingually, orally, intranasally or by inhalation.
38. The pharmaceutical composition of Claim 1-32, wherein at least one of the compounds is administered by intravenous infusion and the duration of infusion ranges from 15 minutes to 120 minutes.
39. A method of treating a disease selected from the group consisting of viral, proliferative, and inflammatory diseases comprising the step of administering to a patient the pharmaceutical composition of any of Claims 1 to 38.
40. A method of treating multiple sclerosis comprising the step of administering to a patient the pharmaceutical composition of any of claims 1, 4, 7, 9, 10, 13, 25, 26, 27 and 28.
41. A method of treating hepatitis B/hepatitis C comprising the step of administering to a patient the pharmaceutical composition of any of claims 1, 3, 6, 12, 17, 29 and 30.
42. A method of treating cancer comprising the step of administering to a patient the pharmaceutical composition of any of claims 1, 2, 5, 8, 11, 15, 16, 18, 19, 20, 21, 22,
23 and 24.
43. A method of treating a disease selected from the group consisting of viral, proliferative and inflammatory diseases comprising the steps of administering to a patient, either together, or separately: a. At least one methyl donor compound; and b. At least one compound selected from the group consisting of: an anti- viral compound, an anti-proliferative compound, and an anti-inflammatory compound.
44. A method of treating a disease selected from the group consisting of viral, diseases comprising the steps of administering to a patient, either together, or separately: a. At least one methyl donor compound; and b. At least one interferon compound.
45. A method of treating a disease selected from the group consisting of viral, proliferative and inflammatory diseases comprising the steps of administering to a patient, either together, or separately: a. At least one methyl donor compound; and b. At least one paclitaxel compound.
46. A method of treating a disease selected from the group consisting of viral, proliferative and inflammatory diseases, wherein either one or both of the methyl donor compounds and anti-viral, anti-inflammatory and anti-proliferative compounds are administered parenterally.
47. A method of Claim 46, wherein either one or both of the methyl donor compounds and anti- viral, anti-inflammatory and anti-proliferative compounds are administered via a route selected from a group consisting of intravenous, subcutaneous, intraperitoneal, and intramuscular. 48. The method of any of Claims 39 to 47, wherein both compounds are administered by infusion.
49. The method of any of Claims 39 to 47, wherein both compounds are administered by infusion simultaneously.
50. The method of any of Claims 39 to 47, wherein both compounds are administered by infusion separately.
51. The method of any of Claims 39 to 47, wherein both compounds are administered by infusion sequentially.
52. The method of any of Claims 39 to 47, wherein one compound is administered by infusion and the other compound is administered by a route other than infusion. 53. The method of any of Claims 39 to 47, wherein the administration of one or other or both compounds is cycled between one or more different routes of administration.
|.4gThe method of any of Claims 39 to 53, wherein the methyl donor compound is administered by intravenous infusion, where the infusion duration is from 15 minutes to 120 minutes. 55. The method of Claim 54, wherein the methyl donor compound is vitamin B12 compound.
56. The method of any of Claims 39 to 55, wherein the dose of the methyl donor compound is between 10-2500 mg.
57. A method of treating a disease selected from the group consisting of viral, proliferative and inflammatory diseases comprising the steps of administering to a patient, by infusion: a. Vitamin B 12 compound, and b. At least one interferon compound.
58. A method of treating a disease selected from the group consisting of viral, proliferative and inflammatory diseases comprising the steps of administering to a patient,by infusion: a. Vitamin B 12 compound, and b. At least one paclitaxel compound.
59. The method of Claim 57 or 58, wherein the Vitamin B12 compound is administered by intravenous infusion, where the infusion duration is from 15 minutes to 120 minutes 60. The method of Claim 57 or 58, wherein the dose of Vitamin B12 compound is from 10 mg to 2500 mg.
61. The method of Claim 57 or 58, wherein the interferon compound or paclitaxel compound is administered by intravenous infusion.
62. The method of Claim 57 or 58 56, wherein the interferon compound or paclitaxel compound is administered by subcutaneous infusion.
63. A method of treating a disease selected from the group consisting of: ovarian cancer, breast cancer, non-small cell lung cancer, Kaposi's sarcoma, astrocytoma and glioma, comprising the steps of administering to a patient either together or separately: a. At least one methyl donor compound; and b. At least one paclitaxel compound.
64. A method of treating a disease selected from the group consisting of: malignant melanoma, hairy cell leukemia, metastatic renal carcinoma, chronic myeloid leukemia and follicular lymphoma, multiple myeloma, astrocytoma and glioma, comprising the steps of administering to a patient, either together or separately: a. At least one methyl donor compound; and b. At least one interferon compound.
65. A method of treating hepatitis B hepatitis C, comprising the steps of administering to a patient, either together or separately: a. At least one methyl donor compound; and b. At least one interferon compound.
PCT/CA2002/001503 2001-10-05 2002-10-04 Combination therapies using methyl donors or methyl donor enhancers and therapeutic agents for treatment of viral, proliferative and inflammatory diseases WO2003030929A1 (en)

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US10/167,765 2002-06-11
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US10/167,752 US6894033B2 (en) 2001-06-11 2002-06-11 Combination therapies using vitamin B12 and therapeutic agents for treatment of viral, proliferative and inflammatory diseases
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