WO2006010256A1

WO2006010256A1 - Compositions and methods comprising vitamin b12 and an impdh inhibitor for treating viral, inflammatory and proliferative diseases

Info

Publication number: WO2006010256A1
Application number: PCT/CA2005/001163
Authority: WO
Inventors: Antonio Cruz
Original assignee: Transition Therapeutics Inc.
Priority date: 2004-07-26
Filing date: 2005-07-26
Publication date: 2006-02-02
Also published as: EP1793846A1; EP1793846A4

Abstract

The invention relates generally to novel compositions and methods comprising at least one Vitamin B12 compound, at least one IMPDH inhibitor, and optionally an interferon. The compositions and methods provide beneficial effects in the treatment of viral, inflammatory, immunodeficiency, IMPDH-mediated, and proliferative diseases.

Description

Compositions and methods comprising Vitamin B12 and an IMPDH inhibitor for treating viral, inflammatory and proliferative diseases

FIELD OF THE INVENTION

The present invention provides compositions and combination therapies for the treatment and/or prevention of a disease, in particular viral, inflammatory, proliferative, immunodeficiency, and/or IMPDH- 5 mediated diseases

BACKGROUND OF TH E INVENTION

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, HIV/AIDS and genital

10 warts These diseases share common disease processes and as a result often share or possess related/common therapies The disease processes include elevated expression of adhesion molecules, cytokines and matrix metalloproteinases, increased cell proliferation and migration, increased inflammatory cell activation and infiltration, increased angiogenesis, and/or increased tissue destruction and dysfunctional matrix remodeling The I MPDH enzyme may also play a regulatory role in the metabolic pathway of these diseases Consequently,

15 compounds for the treatment of these diseases are aimed at altering the immune system, cell proliferation, cell adhesion and migration, IMPDH, cytokine levels and/or activities, and/or viral replication

Although many anti-inflammatory, anti-viral and antiproliferative compounds are effective at treating inflammatory, viral and proliferative diseases, these compounds often cause side effects that are exacerbated when the dose is increased Side effects can include local injection reactions, flu-like syndrome and depression

20 Accordingly, some patients are unable to tolerate the doses needed to achieve a therapeutic effect Interferon and ribavirin are examples of common anti-viral therapeutic compounds with many undesirable side effects, which are exacerbated at high doses Thus, it would be desirable to enhance the efficacy of anti-viral, anti-proliferative and/or anti-inflammatory compounds

Vitamin Bl 2 is a cobalt-containing B complex vitamin that has various effects on biological processes

25 ;/; vivo Vitamin B12 compounds alone have been suggested to possess some or limited anti-viral (Weinberg et al, 1995, 1998, Lott et al, 2001 , Poydock, 1979, Tsao et al , 1990), antiproliferative (Nishizawa et al, 1997, Shimizu, 1987, Poydock et al , 1979, 1985) and anti-inflammatory activities (U S Pat Nos 5,508,271 , 5,964,224, 5,716,941) Vitamin Bl 2 has also been tried in combination with other therapeutic agents (EP Patent # 0835660, U S Pat No 6,096,737) for the treatment of specific inflammatory diseases or proliferative diseases

30 The citation of any reference herein is not an admission that such reference is available as prior art to the instant invention SUMMARY OF THE INVENTION

The present invention provides a multi-component composition comprising at least one Vitamin Bl 2 compound and at least one IMPDH inhibitor, and optionally at least one interferon The invention also provides a

35 method of using a composition of the invention for the prevention and/or treatment of a disease disclosed herein in particular a viral, inflammatory, immunodeficiency, and/or proliferative disease The invention further provides a combination therapy for prevention and/or treatment of a disease disclosed herein, in particular a viral, inflammatory, immunodeficiency, and/or proliferative disease comprising administering to a patient in need thercof a therapeutically effective amount of at least one Vitamin B12 compound and at least one IMPDH inhibitor and optionally at least one interferon In an aspect the disease is an IMPDH-mediated disease

The multi-component compositions of this invention, as well as the combination of compounds used in the methods of this invention may demonstrate a different pharmacologic, therapeutic and/or metabolic profile than each compound alone or in combination Because of these differences, compositions and methods of this invention and the compounds used therein may offer advantages as therapeutics for diseases including without limitation viral, inflammatory, immunodeficiency, IMPDH-mediated and/or proliferative diseases These advantages may include increased overall therapeutic benefit and reduction in deleterious side effects Other advantages may include reduced frequency of administration of IMPDH inhibitors (e g ribavirin) and/or interferon, and/or reduced dosages of IMPDH inhibitors (e g ribavirin) and/or interferon

In an aspect, a multi-component composition of the invention comprises therapeutically effective amounts of ( 1) at least one Vitamin B12 compound, (2) at least one IMPDH inhibitor, and optionally (3) at least one interferon In a particular aspect a composition of the invention comprises a Vitamin B 12 compound and an IMPDH inhibitor In another particular aspect, a composition of the invention comprises at least one Vitamin B12 compound, at least one IMPDH inhibitor, and at least one interferon

In another aspect, a multi-component composition of the invention consists essentially of (1 ) at least one Vitamin B12 compound, (2) at least one IMPDH inhibitor, and optionally (3) at least one interferon In a particular aspect a composition of the invention consists essentially of a Vitamin B 12 compound and an IMPDH inhibitor In another particular aspect, a composition of the invention consists essentially of at least one Vitamin Bl 2 compound, at least one IMPDH inhibitor, and at least one interferon

In an embodiment, the multi-component composition comprises additive amounts, in particular synergistic amounts, of at least one Vitamin B 12 compound, at least one IMPDH inhibitor, and optionally at least one interferon The ratio of Vitamin B 12 compound to IMPDH inhibitor in a composition of the invention can be from about 0 5 1 to 1 20 or 1 1 to 1 20, in particular 1 1 to 1 10 In an aspect, the invention comprises a multi-component pharmaceutical composition for the treatment of one or more disease disclosed herein, in particular a viral disease, proliferative disease, immunodeficiency disease, IMPDH-mediated disease, and inflammatory disease A pharmaceutical composition may comprise a pharmaceutically acceptable carrier, excipient or vehicle

In accordance with another aspect, a composition is provided comprising a combination of at least one Vitamin B 12 compound, at least one IMPDH inhibitor, and optionally at least one interferon effective to exert a synergistic effect in treating a disease In particular, the composition comprises a Vitamin B 12 compound and an

I MPDH inhibitor, and optionally an interferon, one or more of which are in a dose that is at least 1 to 10 fold, 2 to 10 fold, or 5 to 10 fold lower than the doses of each component required to prevent and/or treat a disease

Another aspect of this invention is a composition according to any of the aspects outlined above wherein one or more of the components are conjugated For example, a Vitamin B12 compound can be conjugated to an IMPDH inhibitor or interferon, an IMPDH inhibitor can be conjugated to an interferon, or all the components of the composition can be conjugated Another aspect of this invention is a method of treating a disease disclosed herein, in particular a viral, proliferative, immunodeficiency, IMPDH-mediated, and/or inflammatory disease, including MS, hepatitis B and hepatitis C, comprising administering to a patient any of the compositions outlined herein

Another aspect of this invention is a method of treating a disease disclosed herein, in particular a viral, proliferative, immunodeficiency, I MPDH-mediated, and/or inflammatory disease, including MS, hepatitis B and hepatitis C, comprising administering at least one Vitamin B12 compound at a frequency of more than once daily, daily, more than once weekly, weekly, or more than once monthly and monthly, and administering at least one IMPDH inhibitor at a frequency of more than once daily, daily, more than once weekly, weekly, more than once monthly, or monthly, and optionally at least one interferon at a frequency of more than once daily, daily, more than once weekly, weekly, more than once monthly, or monthly

Another aspect of this invention is a method of treating a disease disclosed herein, in particular a viral, proliferative, immunodeficiency, IMPDH-mediated, and/or inflammatory disease including MS, hepatitis B and hepatitis C, comprising administering to a patient, either together or separately, at least one Vitamin B12 compound, at least one IMPDH inhibitor, and optionally at least one interferon In an embodiment, this invention provides methods of treating an IMPDH mediated disease in a patient comprising the step of administrating to the mammal any of the compositions and combinations described herein In a particular, these methods are useful in suppressing an immune response in a mammal

In an aspect, the invention provides a method of treating a disease disclosed herein, in particular a viral, proliferative, immunodeficiency, I MPDH-mediated, and/or inflammatory disease in a patient non-responsive to treatment with an IMPDH inhibitor (e g ribavirin) alone, interferon alone, or an IMPDH inhibitor and interferon, comprising administering to the patient a therapeutically effective amount of at least one Vitamin Bl 2 compound and optionally at least one IMPDH inhibitor and/or at least one interferon In a particular embodiment, the disease is a viral disease, more particularly hepatitis B or hepatitis C

In an aspect, the invention provides a method of treating a disease disclosed herein, in particular a viral, proliferative, immunodeficiency, 1 MPDH-mediated, and/or inflammatory disease in a patient that has had a sub- optimal response to treatment with an IMPDH inhibitor (e g ribavirin) alone, interferon alone, or an IMPDH inhibitor and interferon, comprising administering to the patient an effective amount of at least one Vitamin B 12 compound and optionally at least one IMPDH inhibitor and/or at least one interferon, to provide an enhanced or optimal response In a particular embodiment, the disease is a viral disease, more particularly hepatitis B or hepatitis C

The invention contemplates a method of retreatment using a therapeutically effective amount of at least one Vitamin Bl 2 compound and at least one IMPDH inhibitor, and optionally at least one interferon, for patients suffering from a disease disclosed herein, in particular a viral, proliferative, immunodeficiency, IMPDH- mediated, and/or inflammatory disease, in particular a viral disease, more particularly hepatitis B or hepatitis C, who fail to respond to therapy with an interferon and/or an IMPDH inhibitor, or who following cessation of such therapy suffered a relapse or who relapse while on therapy

The invention includes combination treatments providing synergistic activity or delivering synergistically effective amounts of at least one Vitamin B 12 compound, at least one IMPDH inhibitor, and optionally at least one interferon Compositions suitable for use in the present invention include compositions - A- whcrcin the active ingredients are contained in a synergistically effective amount Such a composition comprises sufficient amounts of each component to achieve a desired result that is greater than the result achieved with each component on its own

Another aspect of this invention is the use of any of the compounds or compositions described herein, or the use of such compounds or compositions in the preparation of a medicament to treat a viral, proliferative, immunodeficiency and/or inflammatory disease A further aspect of this invention is the use of any of the compounds or compositions described above, or the use of such compounds or compositions in the preparation of a medicament to treat an IMPDH-mediated disease

In an embodiment, the invention relates to synergistically effective amounts of a least one Vitamin Bl 2 compound, at least one IMPDH inhibitor, and optionally at least one interferon in the preparation of a medicament for treating a disease

Encompassed within this invention are any of the above treatments and compositions wherein the dose of Vitamin B 12 compound is between about 0 1 to lO.OOOmg or 10 and 10,000 mg, in particular between about 10 and 2500 mg In some aspects using selected Vitamin B12 compounds the dose is between about 0 5 to 1500 mg or between about 10 to 1500 mg

In particular aspects of the invention the disease is a viral disease Therefore, the invention contemplates a composition or combination therapies for the treatment of viral diseases, such as hepatitis B, hepatitis C, herpes, or vesticular stomatitis comprising at least one Vitamin B 12 compound, at least one IMPDH inhibitor, and optionally at least one interferon Such compositions and combination therapies can be particularly useful in treating and/or preventing diseases associated with RNA viruses, in particular hepatitis B or hepatitis C, or retroviral diseases, such as HTLV-I and HTLV-2, HIV-I and HIV-2, nasopharyngeal carcinoma virus and Herpes viruses, such as Epstein-Barr, cytomegaloviruses and Herpes Simplex, Types 1 and 2, or Type 6 The compositions and combination therapies may also be particularly useful in treating and/or preventing respiratory syncytial virus, influenza A and B viruses, parainfluenza virus, rhinovirus, and hemorrhagic fever viruses In an embodiment, the viral disease is hepatitis C and the composition or combination therapy comprises a Vitamin B12 compound, an IMPDH inhibitor and interferon-alpha or interferon-beta

In another particular aspect the disease is a proliferative disease Therefore the invention contemplates a composition or combination therapy for the treatment of a proliferative disease, such as cancer comprising at least one Vitamin B12 compound, at least one IMPDH inhibitor, and optionally at least one interferon In an embodiment, the compositions and methods are useful for inhibiting tumors and cancer in a mammal Such methods are useful in treating and/or preventing diseases, including, tumors and malignancies, such as lymphoma, leukemia and other forms of cancer

In another particular aspect the disease is an inflammatory disease Therefore the invention contemplates a composition or combination therapy for the treatment of an inflammatory disease comprising at least one Vitamin B12 compound, at least one IMPDH inhibitor, and optionally at least one interferon Such methods may be particularly useful in treating or preventing diseases, including, osteoarthritis, acute pancreatitis, chronic pancreatitis, asthma and adult respiratory distress syndrome In an embodiment, the compositions and methods are useful for inhibiting vascular cellular hyperproiiferation in a mammal Such methods are useful in treating and/or preventing diseases, including, restenosis, stenosis, artherosclerosis and other hyperprohferative vascular disease

In an embodiment, the invention provides a composition or combination therapy for the treatment of MS comprising at least one Vitamin B12 compound, at least one IMPDH inhibitor, and optionally at least one interferon compound The pharmaceutical composition or combination therapy can additionally comprise an anti¬ proliferative agents such as paclitaxel or a RHAMM-relatcd peptide, in particular a RHAMM-related peptide selected from the group consisting of P16, S-3, S-7, P-32, V- 2 and V-3 (SEQ ID NOs 1 through 1 1)

The invention also provides a kit comprising a composition of the invention In an aspect, the invention provides a kit for preventing and/or treating a disease, containing a composition of the invention, a container, and instructions for use The composition of the kit can further comprise a pharmaceutically acceptable carrier, excipient, or vehicle

These and other aspects, features, and advantages of the present invention should be apparent to those skilled in the art from the following drawing and detailed description BRIEF DESCRIPTION OF THE DRAWING

The invention will now be described in relation to the drawings in which

Figure 1 is a graph showing antiviral synergy of combinations of mterferon-α, ribavirin, and a Vitamin B 12 compound in an in vitro model of hepatitis C DETAILED DESCRIPTION OF EMBODIMENTS Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs

Numerical ranges recited herein by endpoints include all numbers and fractions subsumed within that range (e g 1 to 5 includes 1, 1 5, 2, 2 75, 3, 3 90, 4, and 5) It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about " The term "about" means plus or minus 0 1 to 50%, 5-50%, or 10-40%, preferably 10-20%, more preferably 10% or 15%, ofthe number to which reference is being made Further, it is to be understood that "a," "an," and "the" include plural referents unless the content clearly dictates otherwise Thus, for example, reference to a composition containing "a compound" includes a mixture of two or more compounds

Compounds described herein can contain one or more asymmetric centers and may give rise to enantiomers, diasteπomers, and other stereoisomers forms which may be defined in terms of absolute stereochemistry as (R)- or (S)- Therefore, the invention includes all such possible diasteπomers and enantiomers as well as their racemic and optically pure forms Optically active (R)- and (S)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques When the compounds described herein contain centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and A geometric isomers All tautomeric forms are intended to be included within the scope ofthe invention

"Vitamin B 12 compound" refers to a member of a class of compounds which includes vitamin B 12 and its analogues, derivatives, conjugates, or pharmaceutically acceptable salts thereof The class includes cyanocobalamin (CN-CbI), aquacobalamin, adenosylcobalamin, methylcobalamin, hydroxycobalamin or hydroxocobalamin (HC), cyanocobalamin carbanalide, hydroxocobalamin acetate, 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 B 12 and 5-deoxydenosylcobalamin Other analogues include chlorocobalamin, sulfitocobalamin, nitrocobalamin, thiocyanatocobalamin, benzimidazole derivatives such as 5,6-dichlorobenzιmidazole, 5- hydroxybenzimidazole, tπmethylbenzimidazole, as well as adenosylcyanocobalamin [(Ade) CN-CbI], cobalamin lactone, cobalamin lactam and the anihde, ethylamide, monocarboxylic and dicarboxylic acid derivatives of vitamin B12 or its analogues In aspects of the invention the derivatives of vitamin Bl 2 include the mono-, di- and tricarboxylic acid derivatives or the propπonamide derivatives of vitamin B 12 In addition, the compositions include polymers of these analogues or vitamin B 12 conjugated to other molecules or encapsulated The singular form, "Vitamin B 12 compound", may mean any one or more compounds from the class of Vitamin Bl 2 compounds

In aspects of the invention the Vitamin Bl 2 compound is hydroxocobalamin or hydroxocobalamin acetate In other aspects of the invention the Vitamin B 12 compound is cyanocobalamin In still other aspects of the invention, the Vitamin B 12 compound is mcthylcobalamin

"IMPDH" refers to inosine-5'-monophosphate dehydrogenase (EC 1 1 1 205) The enzyme is involved in the de novo synthesis of guanosinc nucleotides IMPDH catalyzes the NAD-dependent oxidation of inosme-5'- monophosphate (IMP) to xanthosine-5'-monophosphate (XMP) [Jackson R C et al , Nature, 256, pp 331-333, ( 1975)] It is ubiquitous in eukaryotes, bacteria and protozoa [Y Natsumeda & S F Carr, Ann N Y Acad , 696, pp 88-93 ( 1993)] The enzyme follows an ordered Bi-Bi reaction sequence of substrate and cofactor binding and product release It first binds IMP to IMPDH, followed by binding of the cofactor NAD The reduced cofactor, NADH, is then released from the product, followed by the product, XMP [S F Carr ct al , J Biol Chem , 268, pp 27286-90 (1993), E W Holmes et al , Biochim Biophys Acta, 364, pp 209-217 (1974)]

An "IMPDH inhibitor" is an antagonist of IMPDH i e , it decreases the amount of or duration of the activity of an IMPDH The inhibition may be direct or indirect, or by a competitive or non-competitive mechanism Examples of inhibitors of IMPDH include mycophenolic acid (MPA) and derivatives thereof (see for example U S Pat Nos 5,380,879 and 5,444,072 and PCT publications WO 94/01105 and WO 94/12184), nucleoside analogs such as a l-(β-D-πbofuranosyl)- IH- 1,2,4-tπazole compound, tiazofuπn, and mizoπbine (see L Hedstrom, et al Biochemistry, 29, pp 849-854 (1990), and heterocyclic compounds described in US Patent Nos 6,919,335 and 6,916,809, or pharmaceutically acceptable salts or prodrugs thereof

As used herein the term "l -(β-D-πbofuranosyl)-l H-l ,2,4-tπazole compound" refers to compounds having the structure

wherein R is selected from the group consisting of carboxamide, carboxamidine, and thiocarboxamide, and wherein X is hydrogen or acyl, in particular C₁ -C₂o acyl

The term "acyl", alone or in combination, means a carbonyl or thiocarbonyl group bonded to a radical selected from, for example, optionally substituted, hydπdo, alkyl (e g haloalkyl), alkenyl, alkynyl, alkoxy ("acyloxy" including substituted acyloxy such as alkoxyalkyl and haloalkoxy), aryl, heterocyclyl, heteroaryl, sulfinyl (e g alkylsulfinylalkyl), sulfonyl (e g alkylsulfonylalkyl), cycloalkyl, cycloalkenyl, thioalkyl, thioaryl, amino (e g alkylamino or dialkylamino), and aralkoxy In particular Ci -C₂₀ acyl refers to a linear or branched group of about 1 -20 carbon atoms Examples of acyl groups include formyl, acetyl, propionyl, isopropionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, octanoyl, lauroyl, aroyl (benzoyl, toluoyl, α- naphthoyl, β-naphthoyl, 2-nicotinoyl, 3-nicotinoyl, 4-nicotinoyl, etc ), acryloyl, methacryloyl, oxalo, methoxalyl, ethoxalyl, cyclohexylcarbonyl, benzylcarbonyl, halo(fluoro, chloro, bromo, iodo)benzylcarbonyl, phenylbenzylcarbonyl, benzyloxycarbonyl, phenoxycarbonyl, phenylethylcarbonyl, ciπnamoyl, methylmalonyl, ethylmalonyl, and propylmalonyl In particular aspects lower acyl groups such as acetyl, propionyl, isopropionyl, butyryl, isobutyryl, and valeryl arc utilized Examples of tπazole compounds include l -(β-D-πbofuranosyl)-lH-l ,2,4-tπazole-3-carboxamidine, 1 -

(5'-O-butyryl-β-D-πbofuranosyl)- 1 H- 1 ,2,4-tπazolc-3-carboxamide, 1 -(5'-0-phosphate-β-D-πbofuranosyl)- IH- l ,2,4-tπazole-3-carboxamide, l-(2', 3', 5'-O-acetyl-^β-D-ribofuranosyl)-lH- l,2,4-tπazole-3-carboxamide, l-(β-D- πbofuranosyl)-l H-l ,2,4-tπazole-3-carboxamιdιne, and the like

In particular aspects of the invention the IMPDH inhibitor is 1 -β-D-πbofuranosyl- lH-l,2,4-tπazole-3- carboxamide (C₈Hi₂N₄Os, also known as ribavirin) or analogues or derivatives thereof Ribavirin is descπbed in the Merck Index, compound No 8199, Eleventh Edition It is commercially available from ICN Pharmaceuticals, Inc , Costa Mesa, Calif Ribavirn is commercially available in capsule and tablet forms under the trademarks Virazole®, Rebetol® (Scheπng Plough), Ribaspherc® and Copegus® (Hoffman-LaRoche) The manufacture and formulation of ribavirin are described in U S Pat No 4,21 1 ,771 In other particular aspects of the invention the IMPDH inhibitor is an analogue of ribavirin including but not limited to 2'-deoxyπbaviπn, 3'deoxyribaviπn, 2', 3' didcoxyribaviπn, 2',3'-dideoxy 2', 3' didehydroπbaviπn, 2', 3' anhydroπbaviπn, acyclovir, acyclovir sodium, valacyclovir, valacylcovir hydrochloride, Zovirax®, Valtrex®, or levoviπn

In further particular aspects of the invention, the IMPDH inhibitor is a nucleoside analog that is a prodrug of ribavirin For example, the inhibitor can be a guanosine analog that is converted into ribavirin by adenosine deaminase in the liver (e g Viramidine, Valeant Pharmaceuticals, Calif )

"Interferon" refers to a native sequence interferon polypeptide, isoform, polypeptide analogue, polypeptide derivative, chimeric polypeptide, or fragments, and variants thereof, or pharmaceutically acceptable salts thereof The term includes class 1 interferons (interferons that all bind to receptor type I such as α-, β-, δ- and ω-interferons, τ-interferons, consensus interferons and asialo-interferons), class II interferons (interferons that all bind to receptor type II such as γ-interferons) and pegylated interferons In particular, the term refers to interferon-alpha, interferon-alpha analogues, interferon-alpha derivatives, interferon-alpha conjugates, interferon beta, interferon-beta analogues, interferon-beta derivatives, interferon-beta conjugates, interferon-epsilon, interferon-epsilon analogues, interferon-epsilon derivatives, interferon-epsilon conjugates, and mixtures thereof Interferon-alpha and interferon-beta genes may be altered by, for example, by 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 deπvatization 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

Examples of interferon compounds include Roferon®, Intron®, Alferon®, Infergen®, Ommferon®, Alfacon- 1 , interferon-alpha, interferon-alpha analogues, pegylated interferon-alpha, polymerized interferon- alpha, dimeπzed interferon-alpha, interferon-alpha conjugated to carriers, interferon-alpha as oral inhalant, intcrfcron-alpha as injectable compositions, interferon-alpha as a topical composition, Roferon® analogues, Intron® analogues, Alferon® analogues, and Infergen® analogues, Ommferon® analogues, Alfacon- 1 analogues, interferon beta, Avonex®, Betaseron®, Betaferon®, Rebif®, interferon-beta analogues, pegylated interferon-beta, polymerized interferon-beta, dimeπzed interferon-beta, interferon-beta conjugated to carriers, 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 Additionally, 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" may mean any one or more compounds from the class of interferon compounds

In certain aspects of the invention, the interferon compound is alpha interferon "Alpha interferon" refers to a natural or recombinant interferon exhibiting biological properties similar to those of human leucocyte interferon A number of alpha interferon species are known which are designated by a numeral after the Greek letter, and all are contemplated for use in this invention Alpha hybrid interferons wherein fragments of two or more native alpha interferon species are joined (See for instance, EP No 51873) may also be used in the present invention Particular forms of alpha interferon for use in the compositions and methods of the present invention are alpha-1 and alpha-2 interferon, preferably alpha-2 interferon Alpha-2 interferon may be prepared by recombinant-DNA methods [see Nagata et al , Nature, VoI 284, pages 316-320 (1980)]

In particular aspects of the invention the interferon-alpha is selected from interferon alpha-2α, interferon alpha-2β, a consensus interferon, a purified interferon alpha product or a pegylated interferon-alpha, including a pegylated intcrferon-alpha-2α or a pegylated interferon alpha-2β (e g Pegasys®, Pegetron®, and Pcglntron®) More particularly, the interferon-alpha is selected from interfeion alpha-2a, interferon alpha-2b, or a purified interferon alpha product and the amount of interferon-alpha administered is from 2 to 30 million IU, 2 to 15 million IU, or 2 to 10 million IU per week on a weekly, TIW, QOD or daily basis In an embodiment, the interferon-alpha administered is interferon-alpha-2β and the amount of interferon-alpha administered is 3 million IU TIW A "native-sequence interferon polypeptide" comprises a polypeptide having the same amino acid sequence of an interferon polypeptide derived from nature Such native-sequence polypeptides can be isolated from nature or can be produced by recombinant or synthetic means The term specifically encompasses naturally occurring truncated or secreted forms of a polypeptide, polypeptide variants including naturally occurring variant forms (e g alternatively spliced forms or splice variants), and naturally occurring allelic variants

A "polypeptide analogue" refers to a polypeptide wherein one or more amino acid residues of a native or parent polypeptide have been substituted by another amino acid residue, one or more amino acid residues of a native polypeptide have been inverted, one or more amino acid residues of the native polypeptide have been deleted, and/or one or more amino acid residues have been added to the native polypeptide Such an addition, substitution, deletion, and/or inversion may be at either of the N-terminal or C-terminal end or within the native polypeptide, or a combination thereof

Mutations may be introduced into a polypeptide by standard methods, such as site-directed mutagenesis and PCR-mediated mutagenesis Conservative substitutions can be made at one or more predicted non-essential amino acid residues A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue with a similar side chain Ammo acids with similar side chains are known in the art and include amino acids with basic side chains (e g Lys, Arg, His), acidic side chains (e g Asp, GIu), uncharged polar side chains (e g GIy, Asp, GIu, Ser, Thr, Tyr and Cys), nonpolar side chains (e g Ala, VaI, Leu, Iso, Pro, Trp), beta-branched side chains (e g Thr, VaI, Iso), and aromatic side chains (e g Tyr, Phe, Tip, His) Mutations can also be introduced randomly along part or all of the native sequence, for example, by saturation mutagenesis Following mutagenesis the variant polypeptide can be recombinantly expressed

A "polypeptide derivative" refers to a polypeptide in which one or more of the amino acid residues of a native polypeptide have been chemically modified A chemical modification includes adding chemical moieties, creating new bonds, and removing chemical moieties A polypeptide may be chemically modified, for example, by alkylation, acylation, glycosylation, pegylation, ester formation, deamidation, or amide formation As used herein, the term "disease" refers to a condition in a subject, including but not limited to viral, inflammatory, immunodeficiency, proliferative and/or IMPDH-mediated diseases Certain conditions may be characterized as more than one disorder For example, certain conditions may be characterized as both proliferative disorders and inflammatory disorders

"IMPDH-mediated disease" refers to a disease state in which the IMPDH enzyme plays a regulatory role in the metabolic pathway of the disease Examples of IMPDH-mediated diseases include but are not limited to transplant rejection and autoimmune diseases, inflammatory diseases, proliferative diseases, and infectious diseases

For example, the compounds, compositions and methods of the invention may be used in the treatment of transplant rejection (e g , kidney, liver, heart, lung, pancreas (islet cells), bone marrow, cornea, small bowel and skin allografts and heart valve xenografts) and autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, juvenile diabetes, asthma, inflammatory bowel disease (Crohn's disease, ulcerative cohtus), lupus, diabetes, melhtus myasthenia gravis, psoriasis, dermatitis, eczema, seborrhoea, pulmonary inflammation, eye uveitis, hepatitis, Grave's disease, Hashimoto's thyroiditis, Behcet's or Sjorgen's syndrome (dry eyes/mouth), pernicious or immunohaemolytic anaemia, idiopathic adrenal insufficiency, polyglandular autoimmune syndrome, glomerulonephritis, scleroderma, lichen planus, viteligo (depigmentation of the skin), autoimmune thyroiditis, alveolitis, viral, bacterial, or fungal infections, inflammatory diseases, proliferative diseases, vascular diseases, such as restenosis, stenosis, rtherosclerosis and other hyperprohferative vascular diseases, and viral diseases including viral replication diseases, such as retroviral diseases and herpes In addition, IMPDH enzymes are also known to be present in bacteria and thus may regulate bacterial growth As such, the compositions and methods described herein may be useful in treatment or prevention of bacterial infection, alone or in combination with other antibiotic agents

"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, artheπosclerosis, inflammatory aortic aneurysm, acute pancreatitis, chronic pancreatitis, asthma, adult respiratory distress syndrome, restenosis, ischemia/reperfusion injury, glomerulonephritis, sacoidosis cancer, restenosis, reperfusion injury, rheumatic fever, systemic lupus erythematosus, rheumatoid arthritis, Reiter's syndrome, psoriatic arthritis, ankylosing spondylitis, coxarthπtis, 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

"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 disorders associated with an overgrowth of connective tissues, such as various fibrotic conditions, including scleroderma, arthritis, juvenile arthritis, gouty arthritis, and liver cirrhosis, and conditions such as restenosis, arteriosclerosis, vascular cellular hyperprohferation, and proliferative diabetic retinopathy Proliferative diseases also include neoplastic disorders including without limitation cancer and tumors, such as solid tumors, lymphomas and leukemia, in particular 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, 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, mycosis funoides, hairy cell leukemia, Kaposi's sarcoma, non-hodgkin's lymphoma, multiple myeloma, basal cell carcinoma, malignant melanoma, colorectal cancer, non-small cell lung carcinoma, bladder cancer, pancreatic carcinoma, 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, and prostate cancer, 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 The term also includes proliferative disorders refractory to treatment with other chemotherapeutics or that is refractory to treatment with other chemotherapeutics due to multidrug resistance

"Viral disease" means a class of diverse diseases and disorders caused by or believed to be caused by viruses The term includes any stage of a viral infection, including incubation phase, latent or dormant phase, acute phase, and development and maintenance of immunity towards a virus Consequently, the term "treatment" is meant to include aspects of generating or restoring immunity of the patient's immune system, as well as aspects of suppressing or inhibiting viral replication Viral diseases includes genital warts (HPV), HIV/AIDS, herpes, influenza, measles, polio, vaπcclla-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

Examples of viruses that can cause disease or symptoms that can be treated with a composition or treatment of the invention, include, but are not limited to the following DNA and RNA viral families Arbovirus, Adenoviπdac, Arenaviπdae, Arterivirus, Birnaviπdae, Bunyaviπdae, Caliciviπdae, Circoviπdae, Coronaviπdae, Flaviviπdae, Hepadnaviπdae (Hepatitis), Herpesviπdac (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e g , Paramyxoviπdae, Morbillivirus, Rhabdoviπdae), Orthomyxoviπdae (e g , Influenza), Papovaviπdae, Parvoviπdae, Picomaviπdae, Poxviπdae (such as Smallpox or Vaccinia), Reoviπdae (e g , Rotavirus), Retroviπdae (HTLV-I, HTLV-II, Lentivirus), and Togaviπdae (e g , Rubivirus) Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to arthritis, bronchiolitis, encephalitis, eye infections (e g , conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), meningitis, opportunistic infections (e g , AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e g , Kaposi's, warts), and viremia

In certain aspects of the invention viral diseases includes genital warts (HPV), HIV/AIDS, herpes, influenza, measles, polio, vaπcella-zoster, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G , meningitis, genital warts (HPV), vesticular stomatitis virus infection, and dengue fever

In other aspects of the invention the viral diseases are associated with respiratory syncytial virus, influenza A and B viruses, parainfluenza virus, rhinovirus, and hemorrhagic fever viruses

"Immunodeficiency diseases" refer to a group of diverse conditions caused by one or more immune system defects and characterized clinically by increased susceptibility to infections with consequent severe, acute, recurrent, or chronic disease Examples of immunodeficiency diseases include without limitation transient hypogammaglobulinemia, selective IgA deficiency, X-linked agammaglobulinemia (Bruton's agammaglobulinemia, congenital agammaglobulinemia), common variable immunodeficiency (Acquired agammaglobulinemia), hyper-IgM immunodeficiency, IgG subclass deficiency, DiGeorge Anomaly (Thymic hypoplasia, third and fourth pharyngeal pouch syndrome), chronic mucocutaneous candidiasis, combined immunodeficiency, Wiskott-Aldrich Syndrome, Ataxia-telandiectasia, X-linked lymphoproliferative syndrome, hypcr-lgE syndrome, chronic granulomatous disease, leukocyte adhesion deficiency (MAC- 1/LFA-1/CR3 Deficiency), splenic deficiency syndromes, and protein-losing immunodeficiencies

"Pharmaceutically acceptable salt(s)," refers to salts which arc suitable for use in contact with a sub_ject or patient without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio The term includes salts of acidic or basic groups which may be present in the compounds suitable for use in the present invention Pharmaceutically acceptable salts are described for example, in S M Berge, et al , J Pharmaceutical Sciences, 1977, 66 1 Examples of pharmaceutically acceptable salts include sodium, calcium, ammonium, ferric hydroxides, isopropylamine, tπethylamine, 2-ethylamine, 2- ethylamino, ethanol, histidine, procaπne, and potassium salts of carboxylic acid groups and hydrochloride salts of amino groups Other pharmaceutically acceptable salts of amino groups are hydrobromide, sulfate, hydrogen sulfate, phosphate, acetate, oxalic, hydrogen phosphate, dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate) salts

The terms "administering" or "administration" refers to the process by which a therapeutically effective amount of compounds or a composition contemplated herein are delivered to a patient for treatment purposes Compounds and compositions are administered in accordance with good medical practices taking into account the patient's clinical condition, the site and method of administration, dosage, patient age, sex, body weight, and other factors known to physicians

The term "treating" refers to reversing, alleviating, or inhibiting the progress of a disease disclosed herein, or one or more symptoms of such disease, to which such term applies Depending on the condition of the patient, the term also refers to preventing a disease, and includes preventing the onset of a disease, or preventing the symptoms associated with a disease The term also refers to reducing the seventy of a disease or symptoms associated with such disease prior to affliction with the disease Such prevention or reduction of the severity of a disease prior to affliction refers to administration of a compound or composition of the present invention to a subject that is not at the time of administration afflicted with the disease "Preventing" also refers to preventing the recurrence of a disease or of one or more symptoms associated with such disease The terms "treatment" and "therapeutically," refer to the act of treating, as "treating" is defined above A treatment may be either performed in an acute or chronic way

"Enhanced", "enhancing" or "enhances", used in the context of, for example, "enhanced efficacy", means an enhanced therapeutic effect, and includes a synergistic effect

A "beneficial effect" refers to an effect of a composition or combination therapy described herein including favorable pharmacological and/or therapeutic effects, and improved pharmacokinetic properties and biological activity In embodiments of the invention, beneficial effects include but are not limited to the following decreased or prevention of disease progression, increased survival, or treatment or reversal of a disease

A beneficial effect may be a statistically significant effect in terms of statistical analysis of an effect of two or three compounds versus the effects of each or two of the compounds as the case may be "Statistically significant" or "significantly different" effects or levels with two or three compounds compared with each compound alone or two compounds as the case may be may represent levels that are higher or lower than a standard In embodiments of the invention, the difference may be 1 5, 2, 3, 4, 5, or 6 times higher or lower compared with the effect obtained with each compound alone or two compounds as the case may be

In particular aspects, beneficial effects refer to the inhibition or reduction in virus, in particular hepatitis

C or hepatitis B, the inhibition or reduction in the spread of infection, the inhibition or reduction in the onset, development or progression of one or more symptoms associated with a viral disease In other particular aspects, beneficial effects refer to the inhibition or reduction in the proliferation of cancerous cells, the inhibition or reduction in the spread of tumor cells (metastasis), the inhibition or reduction in the onset, development or progression of one or more symptoms associated with cancer, the reduction in the size of a tumor, and/or the improvement in a patient's ECOG or Kamofsky score In still other particular aspects, such terms refer to a reduction in the swelling of one or more joints, organs or tissues, or a reduction in the pain associated with an inflammatory disorder

An "additive effect" refers to an effect that is equal to the sum of the effects of the individual compounds

"Synergistic" means a greater pharmacological or therapeutic effect, in particular a greater anti- inflammatory, antiproliferative and/or anti-viral effect, with the use of a multi-component composition or combination therapy of at least one Vitamin B 12 compound, at least one IMPDH inhibitor and optionally at least one interferon, than with the use of any of these compounds alone This synergistic effect can work through either similar or different mechanisms or pathways of action One potential advantage of a combination therapy with a synergistic effect is that standard dosages may be used for a greater therapeutic effect than expected from the addition of the effect of one, two or three compounds as the case may be 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

The terms "subject", "individual" and "patient" are used interchangeably herein and refer to an animal including a warm-blooded animal such as a mammal, which is afflicted with or suspected of having or being pre- disposed to a disease described herein Mammal includes without limitation any members of the Mammalia In general, the terms refer to a human The terms also include domestic animals bred for food or as pets, including horses, cows, sheep, poultry, fish, pigs, cats, dogs, and zoo animals, goats, apes (e g gorilla or chimpanzee), and rodents such as rats and mice The methods herein for use on subjects/individuals/patients contemplate prophylactic as well as curative use Typical subjects for treatment include persons susceptible to, suffering from or that have suffered a disease described herein

"Therapeutically effective amount" relates to the amount or dose of active compounds, composition, or combination therapy of the invention that will lead to one or more desired effect, in particular a beneficial effect A therapeutically effective amount of compounds, compositions or conjugates of the present invention can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance to elicit a desired response in the individual Dosage regime may be adjusted to provide the optimum therapeutic response (e g beneficial effects) For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation "RHAMM-rclated peptide" refers to a peptide sequence or related peptide sequence of RHAMM, in particular, the S-3 peptide, the S-7 peptide, the P-32 peptide, the P-16 peptide, the V-2 peptide, and the V- 3 peptide

"S-3 peptide" and "S-3" mean a specific large peptide fragment of RHAMM, and includes the C- terminal of RHAMM and a peptide with an approximate size of 45 Kda The term "S-3 peptide" can refer to cither the mouse or human amino acid RHAMM peptide fragment, as in SEQ ID NOs 1 and 2

"S-7 peptide" and "S-7" mean a specific RHAMM peptide fragment with an approximate size of 23 Kda and representing a smaller portion of S-3 peptide The term "S-7 peptide" can include either the mouse or human amino acid RHAMM peptide fragment, as in SEQ ID NOs 3 and 4 "P-32 peptide" and "P-32" mean a specific RHAMM peptide fragment The term "P-32 peptide" can include either the mouse or human amino acid RHAMM peptide fragment, as in SEQ ID NOs 5 and 6

"V-2 peptide" and "V-2" mean a specific RHAMM peptide fragment which represents a large portion of the original RHAMM peptide, including the C-terminal and having the approximate size of 58 Kda The term "V-2 peptide" can include either the mouse or human amino acid RHAMM peptide fragment, as in SEQ ID NOs 7 and 8

"V-3 peptide" and "V-3" mean a specific RHAMM peptide fragment The term "V-3 peptide" can include either the mouse or human amino acid RHAMM peptide fragment, as in SEQ ID NOs 9 and 10

"P- 16 peptide" and "P- 16" mean a 16 amino acid synthetic peptide which can bind hyaluronic acid with high affinity The peptide has the ammo acid sequence of SEQ ID NO 11 Compositions and Methods of Use

The present invention provides compositions for enhancing anti-viral, anti-proliferative and anti¬ inflammatory effects and compositions for prevention and/or treatment of a disease disclosed herein in particular a viral, proliferative, immunodeficiency, IMPDH-mediated and/or inflammatory disease A composition of the invention comprises at least one of a Vitamin B12 compound, at least one of an IMPDH inhibitor, and optionally an interferon In an aspect, a composition of the invention consists essentially of a Vitamin B12 compound, at least one of an IMPDH inhibitor, and optionally an interferon

In a particular aspect, for example for treating a viral disease preferably hepatitis C or hepatitis B, the Vitamin B12 compound is hydroxycobalamin, hydroxocobalamin, hydroxocobalamin acetate, or cyanocobalamin and the IMPDH compound is ribavirin In another particular aspect, for example for treating a viral disease preferably hepatitis C or hepatitis B, the Vitamin Bl 2 compound is hydroxycobalamin, hydroxocobalamin or hydroxocobalamin acetate, the IMPDH compound is ribavirin, and the interferon is interferon alpha or interferon beta

In a further particular aspect, for example for treating a viral disease preferably hepatitis C or hepatitis B, the Vitamin B 12 compound is hydroxycobalamin, hydroxocobalamin or hydroxocobalamin acetate, the IMPDH compound is ribavirin, and the interferon is interferon alpha, more particularly pegylated interferon alpha

In a still further particular aspect, a composition of the invention consists essentially of hydroxycobalamin, hydroxocolbalamin or hydroxocobalamin acetate, or cyanobalamin, ribavirin, and interferon alpha or interferon beta A composition of the invention for use in treating multiple sclerosis may additionally comprise or consist of an antiproliferative agent (e g , paclitaxel, tamoxifen, cisplatin, etc ) or a RHAMM-related peptide selected from the group consisting of P16, S-3, S-7, P-32, V-2 and V-3 (SEQ ID NOs 1 through 1 1)

The compositions of the invention preferably contain a pharmaceutically acceptable carrier, excipient, or vehicle suitable for rendering the compounds admimstrable by conventional methods including without limitation orally, intranasally, 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 a pharmaceutical composition of the invention

Compositions of the invention can be formulated as neutral or pharmaceutically acceptable salt forms

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

A composition of the invention may comprise a unit dosage of at least one Vitamin B12 compound, at least one IMPDH inhibitor, and optionally at least one interferon, in particular, to provide beneficial effects A

"unit dosage" refers to a unitary i e a single dose, which is capable of being administered to a patient, and which may be readily handled and packed, remaining as a physically and chemically stable unit dose comprising either the active agents as such or a mixture with one or more solid or liquid pharmaceutical excipients or carriers

In another aspect, an improved composition is provided comprising therapeutically effective suboptimal amounts of at least one Vitamin B12 compound, at least one IMPDH inhibitor and optionally at least one interferon in a form for chronic or acute therapy of a disease

A composition of the invention may be sterilized by, for example, by filtration through a bacteria retaining filter, addition of sterilizing agents to the composition, irradiation of the composition, or heating the composition Alternatively, the compounds or compositions of the present invention maybe provided as sterile solid preparations e g lyophihzed powder, which are readily dissolved in sterile solvent immediately prior to use

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

After compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition For administration of a composition of the invention, such labeling would include amount, frequency, and method of administration Thc present invention provides methods to enhance or potentiate the effects of an IMPDH inhibitor optionally in combination with an interferon and methods of treating a disease disclosed herein, in particular a viral, proliferative, inflammatory, immunodeficiency, and/or IMPDH-mediated disease in a patient by administering a therapeutically effective amount of at least one Vitamin B 12 compound in combination with at least one IMPDH inhibitor and optionally an interferon, or alternatively a composition of the invention Vitamin B 12 compounds can be administered simultaneously, separately or in combination with an IMPDH inhibitor and optionally interferon, under different dose and route regimens, to enhance the efficacy of an IMPDH inhibitor and optionally an interferon in the treatment of a disease disclosed herein in particular, a viral, proliferative, inflammatory, immunodeficiency, and/or IMPDH-mediated disease, more particularly hepatitis C or hepatitis B, in a patient compared to when such compounds are administered alone Greater efficacy and potency of a treatment of the invention potentially improves the therapeutic ratio of treatment, reducing untoward side effects and toxicity The methods of the invention may also enhance utility improving long-standing treatment of disease, in particular viral diseases

The invention additionally provides uses of at least one Vitamin B 12 compound, and at least one IMPDH inhibitor, and optionally at least one interferon, or a pharmaceutical composition of the invention in the preparation of medicaments for the prevention and/or treatment of a disease contemplated herein

Therapeutic efficacy and toxicity of compositions and methods of the invention may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals such as by calculating a statistical parameter such as the ED₅₀ (the dose that is therapeutically effective in 50% of the population) or LD₅₀ (the dose lethal to 50% of the population) statistics The therapeutic index is the dose ratio of therapeutic to toxic effects and it can be expressed as the ED₅₀ZLD₅₀ ratio Compositions which exhibit large therapeutic indices are preferred

A method of treatment of the invention may involve administration of a composition including at least one Vitamin Bl 2 compound and at least one IMPDH inhibitor, and optionally at least one interferon An alternate method of treatment includes the step of the administration of a composition comprising at least one Vitamin B 12 compound followed by the step of the administration of a second pharmaceutical composition comprising at least one IMPDH inhibitor, and optionally the step of administration of a third pharmaceutical composition comprising at least one interferon The administration of the Vitamin B 12 compound can follow administration of the IMPDH inhibitor, and optionally interferon The administration of the pharmaceutical compositions can occur separately or simultaneously

An aspect of this invention is the use of any of the compounds or compositions described herein, or the use of such compounds in the preparation of a medicament to treat a viral, proliferative, immunodeficiency, and/or inflammatory disease A further aspect of this invention is the use of any of the compounds or compositions described herein, or the use of such compounds or compositions in the preparation of a medicament to treat an I MPDH-mediated disease A medicament may be in a form for consumption by a subject such as a pill, tablet, caplet, soft and hard gelatin capsule, lozenge, sachet, cachet, liquid drop, elixir, suspension, emulsion, solution, syrup, aerosol (as a solid or in a liquid medium) suppository, sterile injectable solution, and/or sterile packaged powder regardless of its clinical setting Kits

The invention provides kits In an aspect, a kit comprises or consists essentially of compounds or compositions described herein The kit is a package that houses a container which contains compounds, a composition or conjugate of the invention, and also houses instructions for administering the composition to a subject In an aspect of the invention, a pharmaceutical pack or kit is provided comprising one or more containers filled with one or more of the ingredients of a composition of the invention to provide a beneficial effect Associated with such containcr(s) can be various written materials such as instructions for use, or a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use, or sale for human administration

As there may be advantages to mixing a component of a composition of the invention and a pharmaceutically acceptable carrier, excipient or vehicle near the time of use, the invention encompasses kits in which components of the compositions are packaged separately For example, the kit can contain a component (e g a vitamin B 12 compound) in a powdered or other dry form in, for example, a sterile vial or ampule and, in a separate container within the kit, a carrier, excipient, or vehicle, or a component of a carrier, excipient, or vehicle (in liquid or dry form) In an aspect, the kit can contain a component (e g a vitamin Bl 2 compound) in a dry form, typically as a powder, often in a lyophilized form in, for example, a sterile vial or ampule and, in a separate container within the kit, a carrier, excipient, or vehicle, or a component of a carrier, excipient, or vehicle Alternatively, the kit may contain a component (e g a vitamin B 12 compound) in the form of a concentrated solution that is diluted prior to administration Any of the components described herein, any of the carriers, excipients or vehicles described herein, and any combination of components and carriers, excipients or vehicles can be included in a kit

In an aspect of the invention a kit is provided preferably for treating viral diseases, more preferably hepatitis C, comprising a sterile injectable formulation of hydroxocobalamin or hydroxocobalamin acetate and a carrier (e g diluent such as water) The formulation may be characterized by one or more of the following molecular weight of Vitamin Bl 2 compound is 1406, absorption spectrum maxima exhibited at 352 + 2 nm and 525 ± 2 nm (A₃₅₂/A₅₂₅ is between 2 7 and 3 3), concentration of 150 mg/ml of hydroxocobalamin or hydroxocobalamin acetate, pH 3 5-5 0, and an osmolaπty of 375 to 525 mθsm/kg A kit may comprise 2 5, 5, 10, 15, 20, 25, or 30 ml of such a sterile injectable formulation In another aspect of the invention a kit is provided comprising a sterile injectable formulation of cyanocobalamin in a powder form and a sterile vial of saline, or comprising a sterile injectable formulation of cyanocobalamin and a carrier (e g saline) The formulation may be characterized by one or more of the following molecular weight of Vitamin B 12 compound is 1354, concentration of 10 mg/ml of cyanocobalamin, pH 4 5-7 0, and an osmolaπty of 300+100 mθsm/kg A kit may comprise 2 5, 5, 10, 15, 20, 25, or 30 ml of such a sterile injectable formulation

In the event the composition intended for administration contains a vitamin B12, an IMPOH inhibitor and optionally an interferon, the kit can also include two or more of the components (in any sufficiently stable form) The components can be combined with a carrier, excipient, or vehicle or packaged separately For example, a kit can contain a vitamin B12-containing solution, or the components thereof, and, in a separate container, an IMPDH inhibitor such as ribavirin

Optionally, a kit may also contain instructions for preparation or use (e g , written instructions printed on the outer container or on a leaflet placed therein) and one or more devices to aid the preparation of the solution and/or its administration to a patient (e g , one or a plurality of syringes, needles, filters, tape, tubing (e g , tubing to facilitate intravenous administration) alcohol swabs and/or Band-Aids®) Compositions that are more concentrated than those administered to a subject can be prepared Accordingly, such compositions can be included in the kits of the invention with, optionally, suitable materials (e g , water, saline, or other physiologically acceptable solutions) for dilution Instructions included with the kit can include, where appropriate, instructions for dilution

In other embodiments, the kits of the invention can include pre-mixed vitamin B12 compositions (with or without an additional therapeutic agent) and instructions for solubilizing any precipitate that may have formed during shipping or storage Kits containing solutions of one or more vitamin B 12 compounds and one or more carriers, excipients or vehicles may also contain any of the materials mentioned above (e g , any device to aid in preparing the composition for administration or in the administration perse) The instructions in these kits may describe suitable indications (e g , a description of patients amenable to treatment) and instructions for administering the solution to a patient

In an aspect, the invention provides a kit comprising as a first component a therapeutically effective amount of a sterile vitamin B12 compound and as a second component a therapeutically effective amount of a sterile IMPDH inhibitor for administration separately or in combination to a patient The kit may optionally comprise a sterile interferon The first and second component (and optionally interferon) may be included in a single container The components may be in sterile aqueous buffer or in the form of dry lyophihzed powder or water free concentrate Where the components are in the form of dry lyophihzed powder the kit may further comprise sterile water for reconstituting the components A kit may further comprise a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products which notice reflects approval by the agency for manufacture, use or sale of the kit for human administration, in particular to treat a disease disclosed herein The invention also provides a kit for preparing a pharmaceutical composition for administration to a patient for treatment of a viral, proliferative, inflammatory, immunodeficiency and/or IMPDH-mediated disease comprising a container comprising a therapeutically effective amount of a sterile Vitamin Bl 2 compound and a therapeutically effective amount of a sterile IMPDH inhibitor, and optionally a sterile interferon, and one or more pharmaceutically acceptable carrier or excipient capable of forming said pharmaceutical composition Administration

Compounds, compositions and conjugates of the present invention can be administered by any means that produce contact of the active agent(s) with the agent's sites of action in the body of a subject or patient to produce a desired effect , in particular a beneficial effect The compounds, compositions, and conjugates of the present invention in the described dosages may be administered by conventional methods including without limitation orally, intranasally, by inhalation, lntrapeπtoneally, subcutaneously, intramuscularly, transdermally, sublingually or intravenously The active ingredients can be administered simultaneously or sequentially and in any order at different points in time, to provide the desired effects in particular beneficial effects A compound or composition of the invention can be formulated for sustained release, for delivery locally or systemically It lies within the capability of a skilled physician or veterinarian to select a form and route of administration that optimizes the effects of the compounds, compositions and conjugates, and treatments of the present invention to provide beneficial effects

The compounds or compositions may be administered in oral dosage forms such as tablets, capsules (each of which includes sustained release or timed release formulations), troches, pills, powders, granules, elixirs, tinctures, suspensions, syrups, wafers, chewing guns, emulsions, or the like prepared by procedures known to those skilled in the art They may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular forms, all utilizing dosage forms well known to those of ordinary skill in the pharmaceutical arts The compounds, compositions, or conjugates of the invention may be administered by intranasal route via topical use of suitable intranasal vehicles, or via a transdermal route, for example using conventional transdermal skin patches A dosage protocol for administration using a transdermal delivery system may be continuous rather than intermittent throughout the dosage regimen The dosage regimen of the invention will vary depending upon known factors such as the pharmacodynamic characteristics of the agents and their mode and route of administration, the species, age, sex, health, medical condition, and weight of the patient, the nature and extent of the symptoms, the kind of concurrent treatment, the frequency of treatment, the route of administration, the renal and hepatic function of the patient, and the desired effect An amount of a compound, composition, or conjugate of the invention which will be effective in the treatment of a particular disease to provide effects, in particular beneficial effects, will depend on the nature of the disease, and can be determined by standard clinical techniques The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease, and should be decided according to the judgment of the practitioner and each patient's circumstances The dosages used for each compound may be similar to those dosages known to those skilled in the art and used in prc-clinical and clinical studies and in commercial use The concentrations may be lower than the currently used dosages as the combination of the compounds can increase efficacy of one or more of the compounds For example, Vitamin B12 compounds may be combined with an IMPDH inhibitor (e g ribavirin) with the objective to reduce the dosages and/or reduce frequency of administration of the IMPDH inhibitor in order to achieve both effective treatment and to lessen any negative effects of an IMPDH inhibitor

A particular dosage of a Vitamin B 12 compound for the present invention is the maximum a patient requires to provide an optimal enhancing effect (e g synergistic effect), such maximum being tempered by the absolute upper limit of Vitamin B12 dosage being the maximum that a patient can tolerate and not develop any serious complications A dosage of Vitamin B12 compound for use in the present invention can be within the range of about l O mg to 10 g daily to once or twice per week or month, in particular about 0 5 to 5g, 1 to 5 g, 1 to 3g, or 1 to 2g, daily to once or twice per week or month

In an aspect of the invention, for treating a viral disease, in particular hepatitis C, the Vitamin B 12 compound is hydroxocobalamin, hydroxocobalamin acetate, or cyanocobalamin, and the dosage is about 500 to 2000mg, 1000 to 2000 mg, 1000 to 1500 mg on a once or twice weekly basis, or about 5-50, 5-20, 10-30, 15-25, or 20-25 mg/kg on a once or twice weekly basis

In another aspect of the invention for treating multiple sclerosis, the Vitamin B 12 compound is cyanocobalamin, hydroxocobalamin, or hydroxocobalamin acetate, in particular cyanocobalamin, and the dosage is about 200 to l OOOmg, 200 to 500 mg, 250-350 mg, or 2-300 mg on a once or twice weekly basis, or about 2- 10, 2-5, 3-5, 3- 10, or 3- 15 mg/kg on a once or twice weekly basis

An IMPDH inhibitor such as ribavirin may be administered by injection or orally A dosage of an I MPDH inhibitor for use in the present invention can be within the range of 400 to 1600 mg per dose per day, in particular 700 to 1500 mg per dose per day or 1000 to 1500 mg per dose per day, more particularly 800 to 1200 mg per dose per day An inhibitor can be formulated with appropriate diluents and carriers to form ointments, creams, foams, and solutions having from about 0 01% to about 15% by weight, in particular from about l% to about 10% by weight of the compound For injection, the compound can be in the form of a solution or suspension, dissolved or suspended in physiologically compatible solution in particular from about 10 mg/ml to about 1500 mg/ml Injection may be intravenous, intermuscular, intracerebral, subcutaneous, or intraperitoneal For oral administration, an IMPDH inhibitor such as ribavirin may be in capsule, inhalant (lyophihzed), tablet, oral suspension, or syrup form, and in particular it can contain from about 10 to 500 mg of ribavirin, more particularly about 300 mg of ribavirin A syrup formulation may contain from about 50 mg to 200 mg of the compound per 5 ml of syrup In an aspect of the invention for treating a viral disease, in particular hepatitis C, the IMPDH inhibitor is πbavirn and the dosage of ribavirin is about 700 to 1500 mg/day, in particular about 800 to 1200 mg/day, or 1000 to 1200mg/day

When interferon is administered with an IMPDH inhibitor and Vitamin Bl 2 compound, the dosage can be from about 25 to 500 μg, 50 to 400 μg, 75 to 400 μg, or 100 to 400 μg on a weekly, TIW, QOD, or daily basis In aspects of the invention utilizing interferon-alpha-2a, more particularly pegylated interferon-alpha 2a, the amount administered can be from 100 to 400 μg, 150 to 360 μg, 175 to 360μg, 175 to 200μg, 1 -5 μg/kg, 2- 5μg/kg or 2-3 μg/kg, or 3 million IU on a weekly, TIW, QOD, or daily basis In aspects of the invention utilizing ιnterferon-alpha-2b, in particular pegylated interferon alpha-2b, the amount of interferon alpha-2b administered is from 20 - 150 μg, 25 to 125 μg, or 1 -5, 1 -4 or 1 - 3 μg/kg/week, TIW, QOD, or daily basis The dosage of interferon that can be used in particular aspects of the invention is about 2 to 30 million, 3 to 15 million, or 3 to 10 million IU once or twice weekly, TIW, QOD, or daily basis, in particular 3 million IU once or TIW In an aspect of the invention for treating a viral disease, in particular hepatitis C, the interferon is pegylated interferon alpha, in particular pegylated interferon alpha 2a, and the dosage of the interferon is about 20 to 500 μg, 50 to 400μg, 75 to 400 μg, 100 to 400 μg, 150 to 400μg, 150 to 360 μg, 180 to 360 μg, or 180μg on a weekly, TIW, QOD, or daily basis

In another aspect of the invention for treating a viral disease, in particular hepatitis C, the interferon is interferon alpha and the dosage of the interferon is about 20 to 500 μg, 25 to 200 μg, 25 to 150 μg, 25 to lOOμg, or 100 to 400 μg on a weekly, TIW, QOD, or daily basis In another aspect of the invention for treating multiple sclerosis, the interferon compound is interferon beta, and the dosage of the interferon beta is 20 to 500 μg, 30 μg to 250 μg, 30 to 200 μg, or 30 to 100 μg weekly, TlW, QOD, or daily basis

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

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 a Vitamin B 12 compound in combination with an IMPDH inhibitor, and optionally an interferon for the effective treatment of viral, proliferative, inflammatory, and IMPDH-mediated diseases,

2 More than once daily, daily, more than once weekly, weekly, more than once monthly or monthly mixtures of a Vitamin B 12 compound simultaneously with an IMPDH inhibitor, and optionally an interferon, for the effective treatment of viral, proliferative, inflammatory, and IMPDH-mediated diseases,

3 More than once daily, daily, more than once weekly, weekly, more than once monthly or monthly treatments with an IMPDH inhibitor, and Vitamin B 12 compound, and optionally an interferon, 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 mixtures of an IMPDH inhibitor and Vitamin BI 2 compound, and optionally an interferon, as well as adjunct administration of more than once daily, daily, more than once weekly, weekly, more than once monthly or monthly doses of Vitamin B 12 compounds, and 5 More than once daily, daily, more than once weekly, weekly, more than once monthly or monthly treatments with an interferon alone, a combination of interferon and an IMPDH inhibitor (e g ribavirin), and a combination of Vitamin B12 compound, an IMPDH inhibitor (e g ribavirin) and an interferon, administered separately either more than once daily, daily, more than once weekly, weekly, more than once monthly, or monthly, In an aspect of the invention, a subject with genotype 1 hepatitis C virus is treated with pegylated interferon alpha 2a at a dosage of 75 to 360 μg/week, or 100 to 200 μg/week, in particular 150 to 200 μg/week, and ribavirin at a dosage of about l OOO to 1200 mg/day followed by administration ofa Vitamin B12 compound

(e g hydroxocobalamin, hydroxocobalamin acetate, or cyanocobalamin, in particular hydroxocobalamin acetate)

In an aspect of the invention, a subject with genotype 1 hepatitis C virus is treated with pegylated interferon alpha 2b at a dosage of 1 to 5, 1 to 3 μg/kg/week, in particular 1 to 2 μg/kg/week, and ribavirin at a dosage of about 1000 to 1200 mg/day followed by administration of a Vitamin B 12 compound (e g hydroxocobalamin, hydroxocobalamin acetate, or cyanocobalamin, in particular hydroxocobalamin acetate) In another aspect of the invention, a subject with genotype 1 hepatitis C virus is treated with ribavirin and interferon alph-2a at a dosage of 150 to 400 μg/week or 180 to 400 μg/week, in particular 300 to 360 μg/weck followed by administration of a Vitamin B12 compound (e g hydroxocobalamin, hydroxocobalamin acetate, or cyanocobalamin, in particular hydroxocobalamin acetate) In a further aspect, a subject with genotype 2 or 3 hepatitis C virus is treated with pegylated interferon alpha and ribavirin at a dosage of about 700 to 1200 mg/day, in particular 700 to 900 mg/day, more particularly

800 mg/day (e g in divided doses), followed by administration of a Vitamin B 12 compound (e g hydroxocobalamin, hydroxocobalamin acetate, or cyanocobalamin, in particular hydroxocobalamin acetate)

In order that this invention be more fully understood, the following examples are set forth The examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any way Example 1

Synergistic In- Vitro Interactions between (1) Alpha Interferon and Ribavirin, (2) Alpha Interferon and Hydroxocobalamin, (3) Alpha Interferon, Ribavirin and Hydroxocobalamin against Bovine Viral Diarrhea Virus as Surrogate Models of Hepatitis C Virus (HCV) Replication Rationale:

Monotherapy of HCV-infected patients with ribavirin does not reduce viral loads or lead to sustained virologic responses (SVR), while monotherapy utilizing IFN-α leads to SVR in approximately 20% of chronically infected HCV patients The combination of IFN-α and ribavirin is much more effective clinically than either drug alone, leading to rates of SVR of approximately 40%

In order to ascertain the potential for direct synergy of antiviral effects between IFN-α & ribavirin, IFN- α & hydroxocobalamin, IFN-α, ribavirin & hydroxocobalamin, a drug-drug combination analysis was performed utilising bovine viral diarrhea virus (BVDV) HCV RNA replicons are robust systems that have shown utility in the evaluation of antiviral agents However, since replicons do not faithfully reproduce all steps of the HCV replication cycle, surrogate viruses are still widely utilized for the identification and characterization of anti-HCV agents The type member of the genus Pestivirus, bovine viral diarrhea virus (BVDV), is often utilized for this purpose Method:

The antiviral effects were assessed in a cytopathic effect assay (CPE) The assay involves the killing of cells by the cytopathic Flavivvtridae members bovine viral diarrhea virus (BVDV) and the inhibition of cell killing by active anti-viral compounds BVDV was grown in bovine kidney cells The cells were grown in an incubator On the day preceding the assay, cells were trypsinized, pelleted, counted and plated in a 96-well cell assay plate together with the appropriate growth medium The next morning the medium was removed and an aliquot of the virus was added The amount of the virus used was the maximum dilution in the appropriate assay medium that would yield complete cell killing (>80% destruction of cell monolayer) at the time of maximal CPE development Each 96-well plate contained 12 wells with complete medium alone (medium control blanks), six cell control wells (cells only), and six virus control wells (cells plus virus) Drugs were tested at multiple conccntrations, diluted in assay medium in a half-log series The highest concentration of IFN-α used was 5,000 IU/ml, while RBV was used at 82μM (20 μg/ml) and hydroxocobalamin was used at 300μg/ml

The MacSynergy II program calculated the theoretical additive interactions of the drugs based on the

Bliss Independence mathematical definition of expected effects for drug-drug interactions The Bliss Independence model is based on statistical probability and assumes that the drugs act independently to affect virus replication The calculated theoretical additive interactions were determined from the dose-response curves of the individual drugs The calculated additive effect, which represents the predicted additive interaction, was then subtracted from the observed effect to reveal effects of statistically significant greater-than-expected

(synergy) or less than-expected (antagonism) interactions The 95% confidence intervals for the experimental dose-response surfaces were used to statistically evaluate the data

A summary of the antiviral evaluation experiments is shown in Figure 1

Example 2

An open label, dose escalation study of vitamin B12 in combination with pegylated interferon and ribavirin in patients with chronic hepatitis C non-responsive to prior therapy with pegylated interferon and ribavirin.

The primary objective of the clinical trial is to evaluate the safety and tolerability of repeated intravenous (iv) infusions of vitamin B 12 in combination with pegylated interferon and ribavirin in patients with chronic hepatitis C genotype 1 Secondary objectives are to evaluate the viral response to repeated intravenous infusions of vitamin B12 in combination with pegylated interferon and ribavirin m patients with chronic hepatitis C genotype 1 , and to evaluate the pharmacokinetic profile of vitamin B 12 when used in combination with pegylated interferon plus ribavirin

In the study, a total of 28 patients with chronic hepatitis C who have failed previous treatment with pegylated interferon plus ribavirin will be treated The study treatment regimen will consist of the same type and dose of pegylated interferon and the same dose of ribavirin to which the patient failed to respond previously plus vitamin B 12 Patients' prior tolerability and viral response will serve as historical control data

During a screening period, patient eligibility for study participation will be determined All eligible patients will enter the study treatment phase and will receive pegylated interferon α 2a, or 2b and ribavirin in combination with vitamin B 12 Pegylated interferon and ribavirin will be administered according to the same dose and schedule of the patients' prior treatment Vitamin B12 will be infused intravenously twice weekly with doses escalating during the first three weeks During the first week, 500 mg vitamin B12 will be administered twice weekly and if the dose is well tolerated, 1 0 g vitamin B12 will be administered twice weekly during the second week If the second dose escalation is well tolerated, the final dose escalation to 1 5 g vitamin Bl 2 twice weekly will be administered during the third week and thereafter throughout the treatment period

Patients will receive vitamin Bl 2 by intravenous infusion twice weekly Vitamin B12 dose will be escalated during the first 3 weeks of treatment as follows 2 x 500 mg Week 0, 2 x 1 g Week 1 and 2 x 1 5 g Week 2 and thereafter The hydroxocobalamin form of vitamin B 12 is supplied in 5 mL vials at a concentration of 150 mg/mL The appropriate volume of vitamin Bl 2 will be withdrawn from the vial(s) into a syringe, and ascptically injected into a 0 9 % normal saline minibag through the injection port The total administration volume of 30 niL will be infused into the patient via an iv drip over 6 minutes and thereafter, the line will be flushed with saline

Pegylated interferon α-2a (Pegasys®) plus Ribavirin Patients will receive the same dose regimen as their previous treatment course according to standard dosing guidelines, i.e. pegylated interferon α 2a, 180 μg/week plus ribavirin 1000/1200 mg/day. Ribavirin is dosed according to body weight as follows; < 75 kg → 1000 mg/day in two doses, and >75 kg → 1200 mg/day in two doses.

Pegylated interferon α-2b (Peglntron®) plus Ribavirin. Patients will receive the same dose regimen as their previous treatment course according to standard dosing guidelines, i.e. pegylated interferon α 2b, 1.5 μg/kg/week plus ribavirin 1000/1200 mg/day. Ribavirin is dosed according to body weight as follows; < 75 kg — > 1000 mg/day in two doses, and >75 kg → 1200 mg/day in two doses

The following are the endpoints of the clinical study

• Proportion of patients achieving o a decrease in HCV-RNA of at least 2 logs at Week 12 of treatment compared to baseline o viral clearance (HCV-RNA serum negative by PCR) at Week 24 and 48 week of treatment o sustained viral response (HCV-RNA serum negative by PCR) at Week 72

• Change in mean/median HCV RNA at Week 12 of treatment compared to baseline

• PK of vitamin Bl 2

The present invention is not to be limited in scope by the specific embodiments described herein, since such embodiments are intended as but single illustrations of one aspect of the invention and any functionally equivalent embodiments are within the scope of this invention Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings Such modifications are intended to fall within the scope of the appended claims All publications, patents and patent applications referred to herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety All publications, patents and patent applications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the compounds, methodologies etc which are reported therein which might be used in connection with the invention Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention

Claims

WHAT IS CLAIM ED IS:

1 A composition for the treatment of viral, proliferative, inflammatory, immunodeficiency, and/or ιnosιne-5'-monophosphate dehydrogenase (IMPDH)-mediated diseases comprising therapeutically effective amounts of (i) at least one Vitamin B12 compound, and (ii) at least one IMPDH inhibitor, and optionally (in) at least one interferon

2 A composition according to claim 1 wherein the therapeutically effective amounts of Vitamin B 12 compound and IMPDH inhibitor arc effective to exert a synergistic effect in a subject suffering from the disease

3 A composition according to claim 1 or 2 wherein the IMPDH inhibitor and optionally interferon are in a dose that is at least 1 to 10 fold lower than the dose of the IMPDH inhibitor alone or optionally with interferon required to treat the disease

4 A composition according to claim 1, 2 or 3 wherein the ratio of Vitamin B12 compound to IMPDH inhibitor is from about 0 5 1 to 1 20 or 1 1 to 1 10

5 A composition of any preceding claim wherein the IMPDH inhibitor is a nucleoside analogue 6 A composition of any preceding claim wherein the IMPDH inhibitor is a l -(β-D-πbofuranosyl)-lH-

1 ,2,4-tπazole compound

7 A composition according to any preceding claim wherein the Vitamin B12 compound is hydroxycobalamin, cyanocobalamin, or methylcobalamin

8 A composition according to any preceding claim wherein the interferon is interferon alpha or interferon beta

9 A method of treating a viral, proliferative, inflammatory, immunodeficiency, or IMPDH-mediated disease comprising administering to a patient a composition of any preceding claim

10 A method of treating hepatitis B or hepatitis C comprising administering to a patient a composition according to any preceding claim 1 1 A method of treating a viral, proliferative, inflammatory, immunodeficiency, and/or an IMPDH- mediated disease in a patient non-responsive to treatment with an IMPDH inhibitor comprising administering to the patient a therapeutically effective amount of at least one Vitamin B12 compound and at least one IMPDH inhibitor

12 A method of treating a viral, proliferative, inflammatory, immunodeficiency, and/or an IMPDH- mediated disease in a patient non-responsive to treatment with an IMPDH inhibitor and an interferon comprising administering to the patient a therapeutically effective amount of at least one Vitamin B12 compound, at least one IMPDH inhibitor, and at least one interferon

13 A method of retreatment comprising administering to a patient a therapeutical Iy effective amount of at least one Vitamin B 12 compound and at least one IMPDH inhibitor wherein the patient suffers from a viral, proliferative, inflammatory, immunodeficiency, and/or an IMPDH-mediated disease and failed to respond to therapy with an IMPDH inhibitor, or suffered a relapse following cessation of therapy with an IMPDH inhibitor

14 A method of retreatment comprising administering to a patient a therapeutically effective amount of at least one Vitamin B12 compound, at least one IMPDH inhibitor, and at least one interferon, wherein the patient suffers from a viral, proliferative, inflammatory, immunodeficiency, and/or an IMPDH- mediated disease and failed to respond to therapy with an interferon and an IMPDH inhibitor, or suffered a relapse following cessation of therapy with an interferon and an IMPDH inhibitor

15 A method according to any one of claims 1 1 to 14 wherein the IMPDH inhibitor is a nucleoside 5 analogue

16 A method according to claim 15 wherein the nucleoside analogue is a 1 -(β-D-πbofuranosyl)- 1 H-1 ,2,4- tπazole compound

17 A method according to any preceding claim wherein the Vitamin B 12 compound is hydroxycobalamin, hydroxocobalamin, hydroxocobalamin acetate, or cyanocobalamin

I O 18 A method according to any preceding claim wherein the Vitamin B 12 compound is hydroxocobalamin or hydroxocobalamin acetate

19 A method according to any preceding claim wherein the interferon is interferon alpha or interferon beta

20 A method according to claim 12 or 14 wherein the Vitamin B12 compound is hydroxocobalamin or hydroxocobalamin acetate, the IMPDH compound is a 1 -(β-D-πbofuranosyl)- 1 H- 1,2,4-tπazole

15 compound, and the interferon is interferon alpha 2a or 2b

21 A method according to claim 19 or 20 wherein the interferon is pegylated interferon alpha 2a or 2b

22 A method according to any preceding claim wherein the Vitamin B12 compound and IMPDH inhibitor and optionally interferon are administered together or separately

23 A method according to any preceding claim wherein the dose of Vitamin Bl 2 compound is between 10- 0 1500 mg

24 A method according to any preceding claim wherein the dose of IMPDH inhibitor is less than the therapeutic dose range of the IMPDH inhibitor when used alone

25 A method according to any of claims 1 1 to 24 wherein the IMPDH-mediated disease is a viral disease

26 A method according to claim 25 wherein the viral disease is hepatitis B or hepatitis C 25 27 A method according to claim 26 wherein the disease is hepatitis C

28 A method according to claim 27 wherein the disease is associated with a genotype 1 hepatitis C virus

29 Use of a composition of any preceding claim for the preparation of a medicament for the treatment of a viral, proliferative, inflammatory, immunodeficiency and/or IMPDH-mediated disease

30 A kit for carrying out a method as claimed in any preceding claim

30 31 A kit according to claim 30 comprising as a first component a therapeutically effective amount of a sterile vitamin B 12 compound and as a second component a therapeutically effective amount of a sterile IMPDH inhibitor for administration separately or in combination to a patient

32 A kit according to claim 30 comprising a sterile injectable formulation of a hydroxocobalamin or hydroxocobalamin acetate and a pharmaceutically acceptable carrier, excipient or diluent characterized

35 by one or more of the following molecular weight of hydroxocobalamin or hydroxocobalamin acetate is 1406, absorption spectrum maxima exhibited at 352 ± 2 nm and 525 ± 2 nm (A₃₅₂AA_52S is between 2 7 and 3 3), concentration of 150 mg/ml of hydroxocobalamin or hydroxocobalamin acetate, pH 3 5-5 0, and an osmolaπty of 375 to 525 mθsm/kg