EP1244356A1 - Compositions and methods for l-nucleosides, l-nucleotides, and their analogs - Google Patents

Abstract

Nucleoside and nucleotide compounds and their analogs/prodrugs are provided. Particularly contemplated compounds include 1-β-L-ribofuranosyl-1,2,4-triazole-3-carboxamide, which may be modified and/or phosphorylated. Contemplated compounds may further be combined with other pharmacological compounds, especially including Ribavirin, antibodies, and cytokines. Preferred uses of contemplated compounds include use as an antiviral compound, anti-inflammatory compound, antineoplastic compound, and as a compound to stimulate cellular growth.

Description

COMPOSITIONS AND METHODS FOR L-NUCLEOSIDES, L-NUCLEOTIDES, AND

THEIR ANALOGS

This apphcation claims the benefit of U S provisional patent apphcation 60/173,446, filed 12^/29/1999, U S piovisional patent apphcation 60/172,097, filed 12/23/1999, U S provisional parent apphcation 60/175,111, filed 01/06/2000, U S provisional patent application 60/190,758, filed 3/20/2000, U S provisional patent apphcation 60/226,947, filed 8/22/2000, U S provisional patent application 60/226,875, filed 8/22/2000, U S provisional patent application 60/233,821, filed 9/19/2000, U S provisional patent application 60/233,823, filed 9/19/2000, U S provisional patent application 60/233,548, filed 9/19/2000, U S provisional patent apphcation 60/233,822, filed 9/19/2000, and U S provisional patent application 60/235.465, filed 9/26/2000, all of which are incorporated herein by reference

Field of The Invention

The field of the invention is pharmaceutical compositions and uses thereof

Background of The Invention There are numerous challenges to a person's health, many of which result from infection or accumulation of toxms in a vital organ, which may further result in an adverse reaction of the immune system towards the infected organ For example, an infection with the hepatitis C virus (HCV) frequently leads to a persistent inflammatory viral infection in which the organ inflammation may not be immediately attributable to the HCV vims, but lather to an infection induced imbalance m the immune response

Most known treatments of viral infections may generally be characterized as either direct antiviral treatment or indirect antiviral treatment In direct antiviral treatment, the virus is targeted with an appropriate direct antiviral drug For example, patients infected with the HIV virus typically receive a cocktail of drugs to block the virus propagation, and various classes for direct antiviral tieatment are known in the art For example, some dnect antiviral drugs block the reverse transciiptase Reveise transcπptase (RT) inhibitors are typically nucleoside analogs such as AZT, 3TC, or ddl Alternati \ ely, non-nucleoside RT inhibitois, including quercetm may be used In vitro, RT inhibitors aie typically potent antiviral drugs However, in vι\o, and especially during a period of relatively high rate of viral replication, the generation of RT inhibitor resistant virus mutants aie pi oblematic Other direct antiviral drugs block or mterfeie with the virus protem processing, and aie commonly known as protease mhibitois Piotease inhibitors aie typically highly specific towaids the viruses' proteolytic enzymes However, due to their mostly hydrophobic nature, admimstiation at desirable concentrations tends to be problematic Moreover, development of cross-resistance and severe side effects frequently compound the difficulties arising from the use protease inhibitors In order to reduce the development of multi-drug resistant virus strains, mixtures of RT inhibitors and protease inhibitors may be prescribed Although such mixtures are presently employed relatively successfully, the lelatively high occurrence of adverse side effects and the potential of generating multi-drug resistant virus strains persist

In indirect antiviral tieatment, the immune response to a viral challenge may be modulated For example, immunosuppressive drugs may be employed to reduce the inflammatory condition associated with the \ iral infection, and various immunosuppressing drugs aie known m the art Among other immunosuppressive drugs, cyclosporm A is known as a potent immunosuppressor and is frequently used to repress tissue rejection after organ transplantation However, the use of cyclosporm A tends to be problematic due to its general immunosuppressing effect, making the patient more prone to new infectious diseases Furthermore, long-term administration of cyclosporm A is frequently associated with severe side effects, including hirsutism and gmgival hyperplasia Moreover, the bioavailabihty of cyclosporm A is at least in part dependent on bile, which may pose additional problems in a hepatitis infection

To overcome at least some of the problems associated with cyclosporm A, Tacrohmus (FK506) may be employed as an immunosuppressing drug For example, Tacrohmus has found recognition in the treatment of facial atopic dermatitis Topical administration of the m muno suppressant resulted m significant improvement m 95% of all treated patients [Alaiti, S et al Tacrohmus (FK506) ointment for atopic dermatitis A phase I study m adults and childien J Am Acad Dei matol 1998, 38(1) 69-76] Furthermore, Tacrohmus appeared not to permeate through the skm baπier, thereby eliminating problems associated with systemic administration Although generally well tolerated, treatment with Tacrohmus without generally compiomismg immunity is limited to topical administration When systemically administered o\ er piolonged periods, Taciolimus frequently leads to lymphopio ferative disorders and cardiomyopathy Many known immunosuppressive drugs provide some relief for inflammatory conditions. However, the effects are not organ specific when systemically administered. Consequently, immunity towards exogenous and endogenous challenges such as bacterial and viral infections, neoplastic or malignant cells, etc. is systemically reduced. Thus, the window of a usable concentration of immunosuppressive drugs is defined by the maximum concentration that will not entirely compromise a patient's immune system, and the minimum concentration that will provide at least some desirable effect.

Although various compounds and methods for treatment of infectious and inflammatory diseases are known in the art, all or almost all of them suffer from one or more disadvantages. Therefore, there is a need to provide improved methods and compositions for treatment of those conditions.

Summary of the Invention

The present invention is directed to methods and compositions in which a nucleoside and/or nucleotide drug or its analog is administered to a subject in a concentration or dosage effective to achieve a desired pharmacological or physiological effect.

In one aspect of the inventive subject matter, contemplated compounds have a structure according to formula I, wherein R is H, a P0₃ ^2", (PO₃)₂ ^3~, or (PO )₃ ^4"group.

Formula 1

Contemplated compounds are optionally further modified with a modifying group that is covalently coupled to the carbonyl atom, and it is further contemplated that compounds according to the inventive subject matter are in a D- or L-configuration.

In another aspect of the inventive subject matter, contemplated compounds are employed to treat a viral infection, and may further be co-administered with a cytokine, preferably IFN-alpha-2b, an antibody, or Ribavirin (l-β-D-ribofuranosyl-l,2,4-triazole-3-carboxamide). In a furthei aspect of the inventive subject matter, the selectivity of contemplated compounds with respect to a pharmacological effect m a target cell is increased by modifying the compounds with a modifying group, wherem the modifying group is covalently attached to the drug via a mtiogen atom, and wherein the modifying group is enzymatically removed from the drug m the target cell Particularly contemplated modifying groups include =NH, and -N(Rι)(R₂j or =NRι, wherem Ri and R₂ are independently hydrogen, a linear alkyl, a branched alkyl, an alkenyl, an alkynyl, an ar alkyl, an aralkenyl, an aralkynyl, or an aryl, and wherem R) or R₂ may independently further comprise a nitrogen atom, an oxygen atom, a sulfur atom, or a halogen atom

In a still further aspect of the inventive subject matter, a method of treating a disease characterized by inflammation of an organ m a patient has a step in which contemplated compounds are administered to a patient at a dosage that causes systemic immunomodulation and not systemic immunosuppression of Type I and Type II responses This causes lmmuno- suppression of Type I and Type II responses m the organ of the patient due to selective accumulation of contemplated compounds m the organ

In yet a further aspect of the inventive subject matter, a method of stimulating neuronal growth has a step m which it is recognized that contemplated compounds are effective to stimulate growth of neurons withm a given concentration range In a further step, the compounds are provided to the neurons within the given concentration range

Various objects, featmes, aspects and advantages of the present mvention will become more apparent from the following detailed description of various embodiments of the invention

Brief Description of The Drawing

Figures 1A-1C are exemplary compounds according to the inventive subject matter

Figure 2 is an exemplary synthetic scheme for the synthesis of 1-β-L-πbofuranosyl- 1,2,4- tπazole-3-carboxamιde

Figure 3 is an alternah . e exemplaiy synthetic scheme for the synthesis of 1-β-L- πbofuranosyl-l,2,4-trιazole-3-carboxamιde

Figure 4 is another exemplaiy synthetic scheme for the synthesis of 1-β-L-πbofuranosyl- 1 2,4-tπazole-3-carboxamιde Figure 5 is a flow diagram depicting an exemplary method of organ-targeted immuno- suppression according to the inventive subject matter.

Figure 6 is a flow diagram depicting an exemplary method of stimulating cell growth according to the inventive subject matter.

Detailed Description

Contemplated Compounds

It is generally contemplated that all nucleotides, nucleosides, and their corresponding analogs are suitable for use in conjunction with the teachings presented herein, wherein all of the contemplated compounds may be in their respective L-configuration or D-configuration. However, particularly preferred compounds include phosphorylated and unphosphorylated Levovirin™ (l-β-L-ribofuranosyl-l,2,4-triazole-3-carboxamide, Structure 1), in which R may be hydrogen, or a phosphorous or sulfur-containing group. Where R is a phosphorous containing group, it is especially preferred that R is a monophosphate, a diphosphate, or a triphosphate as depicted in Figures 1A-1C.

Structure 1 Depending on the chemical environment (and especially depending on the pH), it should be appreciated that the phosphate groups may be in their corresponding mono-, di-, tri-, and tetra-protonated forms, and it should also be appreciated that when the phosphate groups are partially or completely deprotonated, salts may be formed with one or more mono- or multivalent cations. Especially contemplated cations are alkaline metal ions and alkaline earth metal ions such as Mg^2τ, Cs²⁺, Na⁺, etc.

In alternative aspects of the inventive subject matter, R may also be a P0₃ ^~", (PO₃)₂ ^"\ or (P0₃)₃ ^4"group in which one or more than one oxygen is replaced with a sulfur atom. While phosphate groups are generally preferred substitutents for R, other chemical groups may also be employed, and particulaily contemplated gioups mclude mono-, or polyaniomc groups, preferably with a tetragonal geometry Thus, contemplated compounds especially include modified and unmodified phosphor lated Levovirm™ Furthermore, it should be appieciated that contemplated compounds may also have a sugar moiety m the D-configuration, and an especially contemplated compound with a sugai m D-configuration is Ribavirm (1-β-D- ribofurai_osyl-l,2,4-tnazole-3-carboxamide)

It should further be appreciated that at least some of the contemplated compounds exhibit a direct antiviral effect (i e contemplated compounds immediately inhibit viral propagation) Since most organisms possess phosphatases m various compartments, it is contemplated that the compounds according to the mventn e subject matter may be gradually dephosphorylated, and one or more than one phosphate group may be removed at a time For example, a tπphosphorylated compound may be converted into a diphosphorylated, or monophosphorylated compound, or a diphosphorylated compound may be converted mto Levovirm™ in a single reaction

With respect to dephosphorylation of phosphorylated Levovirm™, it is particularly contemplated that the mode of anti . iral action shifts from a direct antiviral effect to an indirect antiviral effect The shift from a direct antiviral response to an indirect antiviral response is particularly advantageous, because even though the contemplated compounds are metabolized they retain anti-viral action over an extended period Therefoie, it should be appreciated that the mode of action of contemplated compounds, and particularly phosphorylated Levovirm™, is actually at least bimodal - comprising a direct antiviral effect portion and an indirect antiviral effect portion

With respect to the rate of dephosphorylation it is contemplated that phosphorylated Levo \ inn™ is dephosphorylated at a considerably slower rate than phosphorylated Ribavirm, an effect that is contemplated to be due to the L-configuration of the ribose in Levovirm™ With respect to the compartment or organ wheie dephosphorylation may occur, it is contemplated that dephosphorylation preferably takes place m the In er, however, other organs and compartments, including kidney, neuronal cells, and blood stieam are also contemplated

It should be especially appreciated that all known piodrug forms of contemplated compounds aie appropriate for use in conjunction with the teachings presented herein, and particulaih contemplated piodrug forms include covalent modifications that may be enzymatically (e.g., by a aminohydrolase, an oxidoreductase, or a transferase) removed from contemplated compounds. Exemplary suitable prodrug forms are described in U.S. Patent Application number 09/594,410, filed 06/16/00, incorporated herein by reference, and in "Prodrugs" by Kenneth B. Sloan (Marcel Dekker; ISBN: 0824786297), or "Design of Prodrugs" by Hans Bundgaard (AS IN: 044480675X ), also incorporated herein by reference.

Further, especially contemplated examples of suitable prodrugs include prodrugs formed by addition of a nitrogen-containing group to the carboxamide moiety of Levovirm™, which may be especially advantageous where contemplated compounds are preferentially directed to the liver. For example, the inventors have discovered (unpublished results) that the specificity of Levovirin™ with respect to its pharmacological effect in hepatocytes can be improved by modifying Levovirin™ with a nitrogen-containing modifying group that is selectively removed in hepatocytes. Structure 2 below shows Levovirin™, and Structure 3 shows Levovirin™ modified at the carboxamide group to form a carboxamidine group.

Structure 2

It is particularly contemplated that a modification of Levovirin™ with a (preferably nitrogen- containing) modifying group that can be selectively removed in a target cell (e.g., a hepatocyte) will (1) increase the selectivity of Levovirin™ with respect to the target cell, thereby (2) reducing the overall dosage to achieve a desired effective concentration, and (3) reduce potential toxicity in non-target cells. It is further contemplated that the modifying group is covalently bound to the carbonyl atom of the carboxamide group.

In further alternative aspects, the nitrogen containing modifying group need not be limited to an =NH group, but may also include various primary and secondary amines. It is generally contemplated that suitable modifying groups have the structure -N(Rι)(R₂) or =NRι, wherein Ri and R₂ are independently hydrogen, linear or branched alkyl, alkenyl, alkynyl, aralkyl, aralkenyl, or aralkynyl, aryl, all of which may further comprise heteroatoms including nitrogen, oxygen, sulfur, or a halogen. It is especially preferred, however, that alternative modifying groups are enzymatically removable from Le \ oviπn™, and particularly contemplated enzymes include ammohydrolases such as liver deammases (e g , adenosme or cytosme deammase), hvei deamidases (e g , aryl deamidase) and liver transammases (glutamate-pyruvate transaminase)

Although not limiting to the inventive concept presented herein, it is contemplated that the modifying group may inactivate Levovirm™, or prevent subsequent activation once the modified Levovirm™ is presented to a non-target cell On the other hand, the nitrogen- containing modifying group may also prevent metabolic activation of the modified Levovirm™ With respect to the step of modifying Levovirm™, it is contemplated that the modification may comprise an organo-synthetic modification, an enzymatic modification, or a de-novo synthesis to produce the modified Levovirm™

With respect to the enzymatic removal of the modification group, it is contemplated that, depending on the type of the target cell and the modifying group, the enzymatic removal may vary considerably Enzymatic removal may include enzymes from various classes, including hydrolases, transferases, lyases, and oxidoreductases, and particularly preferred subclasses are adenosme and cytosme deammases, arginases, transammases, and arylamidases It should furthei be appreciated that contemplated enzymes for the enzymatic removal of the modification group may exclusively be expressed m the target cells, however, in alternative aspects of the inventive subject matter appropriate enzymes may also be expressed in cells other than the target cells, so long as the enzyme is not ubiquitously expressed m all cells m a cell containing system It should further be appreciated that contemplated enzymes are preferably natively expressed (/ e , are non-recombinant) in the respective target cells under normal and/or pathological conditions Foi example, it is known that glutamine-pyruvate transaminase is constitutively expressed with relatively high selectivity in liver cells, and may therefore be a suitable enzyme for removal of a modification group Alternatively, it is known that cytosme deammase is expressed m relatively high quantities m colon cancer cells, but not, or only in minor quantities m normal colon cells

Synthesis of contemplated compounds

It is generally contemplated that all known methods of synthesis for D-nucleotides, D-nucleosides, and their respective analogs may be adapted for the synthesis of contemplated compounds in the L-configuration (e g , by replacing the sugar moiety m D-configuration with a sugar moiety in the corresponding L-configuration). An exemplary synthetic scheme for the synthesis of Levovirin™ (l-β-L-ribofuranosyl-l,2,4-triazole-3-carboxamide) is depicted in Figure 2.

Synthesis of 1,2,3, 5-Tetra-O-acetyl-β-L-ribofuranose (1)

To a stirred solution of L-ribose (50. Og, 333.33mmol) in anhydrous methanol (500ml) at room temperature was added freshly prepared dry methanolic HC1 (40ml, prepared by bubbling dry HC1 gas into methanol at 0°C to a weight increase of 4g) via syringe during a 15 min. period under argon atmosphere. After the addition of methanolic HCL, the reaction mixture was allowed to stir at room temperature for 3-4h. Dry pyridine (100ml) was added and evaporated to dryness under high vacuum below 40°C. This process was repeated a second time with additional dry pyridine (100ml). The residue was dissolved in dry pyridine (250ml) and cooled in an ice bath to 0°C under argon atmosphere. To this cold stirred solution was added acetic anhydride (100ml) via a dropping funnel during a 15 min. period. After the addition of acetic anhydride, the reaction was allowed to stir at room temperature under exclusion of moisture for 24h. The reaction mixture was evaporated to dryness. The residue was partitioned between ethyl acetate (400ml) and water (400ml), and extracted in EtOAc. The aqueous layer was extracted again with EtOAc (100ml). The combined EtOAc extract was washed with water (400ml), saturated NaHC0₃ (2x 300ml), water (300ml), and brine (200ml). The organic extract was dried over anhydrous Na₂S0₄, filtered, and the filtrate evaporated to dryness. The residue was co-evaporated with dry toluene (2x150ml) at high vacuum. The dried oily residue (92g, 95%) was used as such for the following reaction without further characterization.

The syrup (92 g) from the above reaction was dissolved in glacial acetic acid (300ml) and treated with acetic anhydride (75ml) at room temperature. The solution was cooled to 0-5°C in an ice bath under argon atmosphere. Concentrated H₂S0 (21ml) was added slowly during a

15min. period. After the addition of H₂SO₄, the reaction mixture was stirred at room temperature for 14h, poured on crushed ice (500g), and stirred until the ice melted. Water (500ml) was added and extracted with CHC1₃ (2x300ml). The chloroform extract was washed with water (3x400ml), saturated NaHC0₃, (2x300ml), water (200ml) and brine (200ml). The washed organic extract was dried over anhydrous MgS0₄, filtered and evaporated to dryness to give an oily residue (99g). The residue was co-evaporated with dry toluene (200ml) and dissolved in ethyl ether (200ml), which upon cooling at 10°C for a day produced colorless crystals. The crystalline solid was filtered, washed with hexanes. ether (2: 1, 50ml), and dried to give a 60.5g product.

Synthesis ofMethyl-l-(2,3,5-tri-0-acetyl-β-L-ribofιιranosyl)-l,2,4-triazole-3-carboxylate (3) and Methyl-l-(2,3,5-tri-0-acetyl-β-L-ribofιιranosyl)-l,2,4-triazole-5-carboxylate (4)

A mixture of methyl-l,2,4-triazole-3-carboxylate (25.4g, 200mmol), 1,2,3,5-tetra-O- acetyl-β-L-ribofuranose (63,66g, 200mmo_) and bis(p-nitrophenyl)phosphate (lg) were placed in an RB flask (500ml). The flask was placed in a preheated oil bath at 165-175°C under water aspirator vacuum with stirring for 25min. The acetic acid displaced was collected in an ice-cold trap that was placed between the aspirator and the RB flask. The flask was removed from the oil bath and allowed to cool. When the temperature of the flask reached roughly 60-70°C, EtOAc (300ml) and saturated NaHC0₃ (150ml) were introduced, and extracted in EtOAc. The aqueous layer was extracted again with EtOAc (200ml). The combined EtOAC extract was washed with saturated NaHC0₃ (300ml), water (300ml) and brine (200ml). The organic extract was dried over anhydrous Na₂Sθ₄, filtered and the filtrate was evaporated to dryness. The residue was dissolved in EtOH (100ml) and diluted with MeOH (60ml), which on cooling at 0°C for 12h produced colorless crystals. The solid was filtered, washed with minimum cold EtOH (20ml), and dried at high vacuum over solid NaOH to give 60g (78%>). The filtrate was evaporated to dryness and purified on a silica column using ChCl₃-> EtOAc (9:1) as the eluent. Two products were isolated from the filtrate: fast moving product 8.5g (11%) and slow moving product

5g(6.5%). The slow moving product matched with the crystallized product. The fast moving product was found to be (4) and obtained as foam. The combined yield of (3) was 65g (84%).

Synthesis of l-β-Ribofuranosyl-l,2,4-triazole-3-carboxamide (5)

Methyl-l-(2,3,5-tri-0-acetyl-β-L-ribofuranosyl)-l,2,4-triazole-3-carboxylate (62g,

161mmol) was placed in a steel bomb and treated with freshly prepared methanolic ammonia (350ml, prepared by passing dry HCL gas into dry methanol at 0°C until saturation) at 0"C. The steel bomb was closed and stirred at room temperature for 18h. The steel bomb was then cooled to 0°C, opened, and the content evaporated to dryness. The residue was treated with dry ethanol (100ml) and evaporated to dryness. The residue obtained was triturated with acetone to give a solid, which was filtered and washed with acetone. The solid was dried overnight at room temperature and dissolved in a hot EtOH (600ml) and water (10ml) mixture. The volume of the EtOH solution was reduced to 150ml by heating and stirring on a hot plate. The hot EtOH solution on cooling provided colorless crystals, which were filtered, washed with acetone, and dried under vacuum. Further concentration of the filtrate gave additional material. The total yield was 35g (89%).

In an alternative aspect of the inventive subject matter, it is contemplated that the synthesis of Levovirin™ may also employ one or more enzymatic conversions. For example, the acetylation of L-ribose may be performed with a suitable acetyl-transferase (e.g., EC 2.3.1.xx). In another example, the formation of the carboxamide group from the corresponding methylester may be facilitated by a single or dual-enzyme system involving an esterase (e. g. , EC 3.1.1.xx) and/or aminotransferase (e.g., EC 2.6.1.xx). In still another example, Levovirin™ may be enzymatically converted into the corresponding mono-, di-, or triphosphate (e.g., EC 3.1.3.xx or EC 3. L4.xx).

It is still further contemplated that various catalysts other than bis(p-itrophenyl)phosphate in quantities other than lg may be utilized. Changing the amount (i.e., the molar fraction) of the catalyst may advantageously increase the selectivity of the reaction towards a higher yield of the desired Ni isomer (L-ribose coupled to the Ni atom of the triazole ring) over the N₂ ison er. For example, appropriate amounts of bis(p-nitrophenyl)phosphate include amounts between 3-30mmol. and more. Alternatively, where appropriate, amounts lower than 3mmol (0.3mmol - 2.99mmol) may be included. In further alternative aspects of the inventive subject matter, the catalyst need not be limited to bis(p-nitrophenyl)phosphate, and alternative catalysts include p-toluenesulfonic acid, trichloro acetic acid, and p-nitrobenzoic acid.

With respect to the reaction temperature, it is particularly contemplated that lower temperatures may further increase the selectivity of the reaction towards a higher yield of the desired N] isomer over the N₂ isomer. Therefore, it is contemplated that appropriate temperatures for the coupling reaction between the triazole moiety and the ribose moiety include temperatures between about 155-165°C. more preferably between 145-165°C, and most preferably between 130-165"C.

In further alternative aspects of the inventive subject matter, it is contemplated that the selectivity of the reaction towards a higher yield of the desired Ni isomer over the N? isomer may also be favorably influenced by a chemical modification of the methyl- l ,2,4-triazole-3- carboxylate. Chemical modifications include formation of a complexmg structure that involves the N₂ atom, steπc hindrance, and direct chemical modifications of the N₂-atom For example, the free electron pair m the N? atom of the tπazole moiety and an electron donor in a modified carboxylate group may be employed to complex a metal ion, thereby reducing the availability of the N₂ atom for coupling with the ribose moiety In another example, the carboxylate group in the methyl- 1, 2.4-tπazole-3-carboxylate may be modified with a relatively bulky group that preferentially and steπcally blocks or reduces leactions occurring at the N₂ atom Alternatively, the N₂ atom may be directly modified by a protecting group, and suitable protecting groups include t-Boc, and benzyl.

Still further, it is contemplated that a higher yield of the desired Ni isomer over the N₂ isomer may also be achieved using enzymatic synthesis in which the ribose moiety (or an L-πbonucleotide) and a modified or non-modified methyl- l,2,4-tπazole-3-carboxylate serve as a substrate for a πbosyltransferase (e g , EC 2.4.2.5 or EC 2 4.2 6).

Alternatively, Levovirm™ may be synthesized via coupling of a protected L-πbose to a l,2,4-tπazole-3-nitπle, with subsequent conversion of the nitπle group to the carboxamide as shown m Figure 3. h a still further alternative synthesis, the coupling of the triazole moiety with the ribose moiety may also be achieved in a reaction m which a (e g , benzyl protected) ribose has an -NHNH₂ group coupled to the Ci atom, that is reacted with the Ni atom of the triazole carboxylate, wherein the triazole carboxylate is subsequently converted to the carboxamidme as depicted in Figure 4

With respect to the synthesis of prodrug forms of contemplated L-nucleotides, L-nucleosides and their respective analogs, it should be appreciated that a particular synthetic scheme will generally depend on the structure of the particular compound However, all manners of synthesis are considered suitable and contemplated synthetic schemes include in vitro synthesis, enzymatic synthesis, in-vivo conversions, and any chemically reasonable combination thereof Exemplaiy synthetic schemes for the formation of contemplated prodrugs are descnbed in U S Patent Application numbei 09/594,410 (supra)

Wheie contemplated L-nucleotides, L-nucleosides and their lespective analogs are phosphorylated it is contemplated that all manners of incorporating a phosphate group into a nucleotide, nucleoside or their lespective analogs are suitable Conveision of contemplated nucleosides to their corresponding phosphorylated forms can be achie \ ed synthetically (Hughes B G et al, (1983), 2', 5'-ohgoadenylated and related 2', 5-olιgonucleotιde analogues 1 Substrate specificity of the mterferon-mduced murme 2',5'-ohgoadenylate synthetase and enzymatic synthesis of ohgomers Biochemistry, 22 21 16-2126) However, various alternati . e methods are also contemplated and include enzymatic phosphoiylation (see e g , Van Rompay. A R , et al (2000). Phosphoiylation of nucleosides and nucleoside analogs by mammalian nucleoside monophosphate kinases, Pharmacol Ther 87(2-3) 189-198), and organochemical phosphoiylation m aqueous media (Schwartz, A and Ponnamperuma, C (1968), Phosphorylation of adenosme with linear polyphosphate salts in aqueous solution (Nature 218, 443)

Uses of Contemplated Compounds

It should generally be recognized that contemplated compounds may be employed in any treatment or therapy of a system that positively responds to administration of contemplated compounds However, it is particularly preferred that contemplated compounds may be employed in antiviral treatments (as a direct antiviral compound and/or as an indirect antiviral compound), m treatments to modulate the immune system, and in treatments to stimulate cellulai growth Further, particularly contemplated uses include administration of contemplated compounds m antmeoplastic treatments

An tiviral treat in en ts

It is generally contemplated that compounds accoidmg to the inventive subject mattei may be employed as a direct and/or indirect antiviral agent in a viral infection It is particularly contemplated that a method of treating a viral infection in a patient comprises a step m which composition is administered to the patient at a dosage effective to inhibit viral propagation (i e , a process involving a host cell in which one or more than one vnus causes the host cell to produce one or more copies of the virus, wherem the term "to pioduce" refers to nucleotide synthesis, protem piocessing, and protem assembly), wheiein the composition comprises at least one the contemplated compounds, and pieferably at least one of a compound according to Structures 1 and 3 Preferred dosages are m the range of bet v. een 5-2500mg/day, and moie prefei ably between 50-500mg/day Howe . ei. alternative dosages, routes, schedules and formulations are also contemplated, and suitable alternative administrations are described below While the use of contemplated compounds is not restricted to a particular virus in a particular viral infection, especially contemplated viral infections are an HIV infection, an HCV infection, an HBV infection, a RSV infection, an influenza virus infection, and a parainfluenza virus infection

Peripheral blood mononucleai cells (PBMCs) are currently used to study seveial different infections, such as Hepatitis C, HIV, Hepatitis B and varieties of the Herpes virus. (Antivir Chem Chemother 2000 July, 11(4). 291-301 , J Infect Dis 1998 Oct; 178(4): 1189-92, Virology 2000 Mar., 268(1).12-60 ) PMBCs are infected with the desired virus, and the cell lines are then studied for relevant information on how particular drugs interact with the PBMCs and how the infected PBMCs act over time or under different environmental conditions. Based on the studies conducted with infected PBMCs, models can be generated that show the effects of particular pharmaceuticals, environments, and/or conditions on the PBMC-Virus infected cells

The present inventors have discovered (unpublished results) that Ribavirm shows a positive response against PBMC-HIN infected cells Surprisingly, Levovirm™ also shows a similar immunomodulatory profile against PBMC-HIV infected cells despite the body's lack of enzymes necessary to phosphorylate the Levovirm™ present in the patient. Based on the above observations, along with other related information and tests, it is contemplated that Levovirm™, phosphorylated Levovirin™, and modified Levovirm™ according to Structure 3 can be utilized in the treatment of HIV and related viruses

Im m un omodulation

In Figure 5, a method of organ-targeted immunosuppression 500 has a first step 510 m which a drug is provided that reduces both a Type 1 response and a Type 2 response when administered above an immunosuppressive concentration, and increases the Type 1 response relative to the Type 2 response when administered below the immunosuppressive concentration, wherem the drug accumulates preferentially m a target organ In a subsequent step 520, the drug is administered to a patient m a dosage effective to accumulate the drug in the target oigan to the immunosuppiessive concentration Consequently, it is contemplated that a method of treating a disease characterized by liver inflammation in a patient may comprise one step in which a compound is provided, wherem the compound comprises Levovirm, phosphorylated Levovirm™, a modified phosphoiylated Levovirin™, or a modified Ribavirm (supra) In a further step, the compound is administered to the patient at a dosage that (a) causes systemic immunomodulation and not systemic immunosuppression of Type I and Type II responses, and (b) causes immunosuppression of Type I and Type II responses in the patient's liver

The term "immunosuppression" refeis to an event m which T and/or B cell clones of lymphocytes are depleted in size or suppressed in their reactivity, expansion or differentiation Immunosuppression may thereby arise from activation of specific or nonspecific T suppressor lymphocytes of either T or B clones, or by drugs that have generalized effects on most oi all T or B lymphocytes For example, Cyclosporm A and FK506 act relatively specifically on T cells, while alkylatmg agents such as cyclophosphamide are less specific in their action

As used herein, the term "cytokme" refers to a group of soluble protems and peptides which act as humoral regulators at nano- to picomolar concentrations and which, either under normal or pathological conditions, modulate the functional activities of individual cells and tissues Cytokines also mediate interactions between cells directly and regulate processes taking place in the extra-cellular environment

As further used herein, the term "lymphokines" refers to a subset of cytokines produced by helper T cells, and are generally considered to fall mto two subclasses, Type 1 and Type 2 Type 1 cells produce mterleukm 2 (IL-2), tumor necrosis factor (TNF ) and interferon gamma (IFNγ), and are responsible pnmarily foi cell-mediated immunity such as delayed type hypersensitivity and antiviral immunity In contiast, Type 2 cells produce mterleukms, IL4, IL-5, IL-6, IL-9, IL-10, and IL-13, and aie primanly involved in assisting humoial immune lesponses such as those seen m response to allergens (e g IgE and IgG4 antibody isotype switching)

Therefore, the terms Type 1 and Type 2 "responses" are meant to include the entire range of effects resulting from induction of Type 1 and Type 2 lymphocytes, respectively Among other things, such responses include mei eased production of the corresponding cytokines, increased proliferation of the corresponding lymphocytes, and other effects associated with increased production of cytokines, including moti ty effects A Type 1 response is generally characterized by an increase m IL-2, TNF-α, and IFN-γ, whereas a Type 2 response is typically characterized by an increase in IL4, IL-5, IL-6, and IL-10 As still further used herein, the term the drug "accumulates preferentially" m a target organ refers to a selective mechanism of a target organ resulting in an increased net uptake or retention of the drug mto the target organ relative to other tissue or organs The mechanism may thereby include active import via transporters, receptors, vesicles, etc, but may also be based on physicochemical principles, including pH dependent charge of the drug, different solubility of the drug m environments with altered ionic strength, chemical or enzymatic modification withm a target organ or target cell, and so forth

In a preferred aspect, the drug is Ribavirm, which is provided to a patient with an HCV (Hepatitis C Virus) infection, and Ribavirm is orally administered to the patient m a single dosage of 600mg/ day for a period of 180 days A single dosage of 600mg/ day is generally below a systemic immunosuppressive concentration, however is effective to preferentially accumulate m the hvei Thus, the concentration of Ribavirm m the target organ (here the liver) will significantly increase and reach an immunosuppressive concentration in the liver Ribavirm is known to increase a Type 1 response relative to a Type 2 response, and to reduce the Type 1 and Type 2 response at relatively high concentrations Examples are set forth in International Patent Application Number PCT/US98/00634 filed on January 13, 1998, incorporated herein by reference

hi alternative aspects of the inventive subject matter, the drug need not necessaπly be limited to Ribavirm, and alternative drugs include contemplated compounds (supra), particularly modified and unmodified Levovirm™ and phosphorylated Levovirm™ Further alternative drugs include contemplated compounds so long as alternative compounds reduce both a Type 1 response and a Type 2 response at an lmmunosuppressn e concentration, and increase the Type 1 response relative to the Type 2 response below the immunosuppressive concentration

With lespect to the patient, various viral infections other than HCV infection are also contemplated, including infections with arboviruses. Consequently, the target organ is not restricted to the liver, but may also include other organs such as the bram, the lung, the spleen, the thymus, the kidneys, etc In general, it is contemplated that the disease that can be treated with the method according to the subject matter presented herein, will depend on the drug's specific accumulation pattern (i e m which organ the drug prefeientially accumulates) Foi example, Ribavirm and Levov inn™ both pieferentialh accumulate in the liver, and reduce both the Type 1 and Type 2 lesponse above an immunosuppressive concentration Therefore, diseases in which suppression of an immune response in the liver is desirable are especially contemplated, and include hepatitis C, autoimmune/lupoid hepatitis, liver transplant recipients, etc

It should be especially appreciated that the method according to the inventive subject matter is not designed to provide a direct antiviral treatment, but is designed to at least partially suppress an immune response m an organ that is infected with a virus Organ targeted immune suppression is contemplated to be especially advantageous m hepatitis C, where the organ damage is not immediately attributable to the HCV virus, but rather to an infection-induced imbalance between a Type 1 response and a Type 2 response Therefore, a method of treatment with a drug that reduces specifically both the Type 1 and Type 2 response in the liver of a patient infected with HCV is contemplated to prevent hepatic damage prophylactically as well as in therapeutic approach Since Ribavirm and Levovirm™ both have excellent tolerabi ty in humans, long-term prophylaxis, and long-term treatment are particularly advantageous

With respect to the administration (route, dosage, schedule, term, etc ) of Ribavirm or alternative contemplated compounds, the same considerations as described below apply It is further contemplated that Ribavirm or alternative contemplated compounds can be employed in a general health setting, as opposed to a clinical, therapeutic setting Consequently, it is contemplated that Ribavirm or alternative contemplated compounds may also be used to improv e digestion For example, one or more of the compounds may be taken by an individual suffenng from poor digestion - whether the poor digestion is due to liver conditions such as Hepatitis B or C infections, or indeed any other conditions characterized by liver inflammation In such instances, digestion can be improved by having the person take Ribavirm or a Ribavirm like compound below an amount normally producing systemic immunosuppression, but m an amount that accumulates in the liver to a concentration that produces immunosuppression in the person's liver

Another example of a non-clinical, non-therapeutic use is for a person to take Ribavirm or alternative contemplated compounds as a means of lmpro \ mg skin color It is particularly contemplated that skin color can be improved by having the person take Ribavirm oi alternative contemplated compounds below an amount normally producing systemic immunosuppression, but m an amount that accumulates m the liver to a concentration that produces immunosuppiession m the person's liver In all of these methods it is particularly contemplated to utilize Ribavirm (l-(5-Deoxy-β- D-rιbofuranosyl)-l,2,4-tnazole-3-carboxamιde) oi Levovirin™ (l-(5-Deoxy-β-L-πbofuranosyl)- l,2,4-tπazole-3-carboxamιde), or any of their mono-, di-, or tn-phosphorylated forms The amount taken or administered is preferably sufficient to produce a systemic immunomodulation of Type I and Type II responses, and a suppression in the liver of both Type I and Type II responses Especially preferred amounts are between about 300mg/day and about 800mg/day, although in some individuals the range may be as low as about 50-100 mg/day up to as high as 2000-2400 mg/day Effects on other organs are also contemplated, including the bram or other organs m which Ribavirm is know n to significantly accumulate

Stimulation of neuronal growth

As used herein, the term "stimulating neuronal growth" refers to any process m which cell growth and/or division is either initiated from a resting cell, or accelerated in a growing and/or dividing cell, wherein "neuronal" refers to all cells that are directly or indirectly involved in the propagation of cognitive, sensory or motoi signals For example, neurons are contemplated to be directly involved m signal propagation, while myelm sheath cells or glia cells are indirectly involved by virtue of their insulating function or structural/metabolic support to a neuron Similarly, receptors are also considered neuronal cells under the scope of this definition In contrast, cells forming the inner and outer layer of the dura are not considered nemonal cells. since they are not directly or indirectly involved the propagation of cognitive, sensory or motor signals

The inventors surprisingly discovered that Ribavirm is effective to stimulate neuronal growth, and the inventors further contemplate that the various phosphorylated analogs of Ribavirm may also be effective to stimulate such growth It is still further contemplated that Levovirm™ and its phosphorylated analogs may be effective in a similar manner In a particular experiment, it has been recognized that Levovirm™ is effective to stimulate growth of unipolar neuronal cells in viti o withm a concentration lange of 0 5μM to 500μM Consequently, addition of Levov inn™ to a cultuie medium at a concentration of about 5 OμM can be employed to stimulate growth of unipolar neuronal cells It should further be appreciated that methods of stimulating neuronal growth need not be limited to unipolar neuronal cells, but may include various alternative cells, including bipolar and multipolar neuronal cells Furthermore, it is contemplated that m alternative aspects of methods of stimulating neuronal giowth, individual cell types may be targeted m a population of diverse neuronal cells For example, unipolar, bipolar and multipolar neuional cells may be targeted complex neuronal structures such as the bram, the spmal chord, or the eye Therefore, neurons may be part of a neuronal tissue that includes at least four of the following cell types an astrocyte, a dendrocyte, a myelm sheath cell, a glia cell, a unipolar neuronal cell, a bipolar neuronal cell, a multipolar neuronal cell, and a receptor cell

Consequently, contemplated methods are not necessarily limited to stimulating neuronal growth in cell culture In further alternative aspects of the inventive subject matter, it is contemplated that cells may be stimulated m a tissue culture, and it is particularly contemplated that neuronal cells may be stimulated in vivo In vivo stimulation of neuronal growth may advantageously be utilized as a prophylactic treatment, or a therapeutic treatment For example, contemplated methods according to the inventive subject matter may be utilized for prevention of demyelmating disorders or neurodegenerative diseases such as Alzheimei's disease or Parkinson's disease, or as a preventative treatment pnor to operative procedures in a patient Contemplated therapeutic treatments include reversion or attenuation of asphyxial, traumatic, toxic, infectious, degenerative, metabolic, lschemic or hypoxic insults

Consequently, as depicted m Figure 6, a method 600 of improving coordination in a patient has a first step 610 m which it is recognized that phosphorylated oi unphosphorylated Ribavirm or Levovirm™ is effective to stimulate growth of neurons in vivo withm a given concentration range h a subsequent step 620, the patient takes an amount of phosphorylated or unphosphorylated Ribavirm or Levovirm™ that is effective to stimulate growth of at least some of the person's neurons Impiovement of eye-hand coordination is especially contemplated

In a piefened method of improving coordination in a person, it is recognized that Ribavirm is effective to stimulate giowth of neuronal cells in vivo withm a concentration lange of 0 5μM to 500μM, and Ribavirm is oially administered to a patient suffering from a traumatic injury to the nervus ischiadicus m a dosage of 1200mg/day

With respect to the patient, various conditions other than traumatic injury to the nervus ischiadicus aie also contemplated, including mechanical and chemical damage to a plurality of nerv e cells, infection of neuronal cells with bactena and/or viruses, and degenerative diseases Regaidless of the nature of the patient's condition, it is contemplated that the method according to the mventn e subject matter may stimulate a wide range of neuronal cells, and it is especially contemplated that the stimulated neurons communicate between the person's bram and voluntaiy muscles, or between the persons biam and skin sensors

Anothei class of contemplated methods includes improving tactile or other sensory sensitivity m a patient Still another class of contemplated methods includes improving gross and fine motor control

While not wishing to be bound to a particular theory, it is contemplated that the admimstiation of Ribavirm, Levovirm™, oi mono-, di- and tnphosphorylated forms of

Levo \ inn™ or Ribavirm may effect a change m the Type 1 or Type 2 response m a patient which concomitantly may lead to a neuroprotective status, or a stimulation of neuronal growth Therefore, it is contemplated that compounds according to the inventive subject matter may be administered as part of a treatment of a disease m a patient in a dosage range effective to increase a Type 1 response and decrease a Type 2 response in the patient With respect to in vivo administration (route, dosage, schedule, term, etc ) of Ribavirm or alternative contemplated compounds, the same considerations as described below apply

Antineoplastic treatment

It is furthei contemplated that compounds according to the inventive subject matter may be employed as antineoplastic agent m treatment of a solid oi lymphatic tumor, and contemplated neoplasms include various carcinomas, sarcomas, and lymphomas, and particularl} include acute myeloid leukemia and chronic myeloid leukemia in blast crisis It should further be appreciated that administration of contemplated compounds in antiviral treatments will generalh follow a route, dosage, schedule and term as employed with known D-nucleotides, D-nucleosides. and then lespective analogs m antineoplastic treatments Administration of Contemplated Compounds

With respect to administration of contemplated compounds, it should be appreciated that the compounds may be admmisteied under any appropriate protocol in any appropriate pharmaceutical formulation It is generally preferred that contemplated compounds aie oially administered In alternative aspects of the mv entive subject matter, it should be appreciated that va ous alternative admmistiations aie also suitable, and it should further be recognized that a particular administration will generally depend on chemical stability, bioavailabihty, dosage, formulation, and/or desired pharmacokinetic/pharmacodynamic properties of contemplated compounds Thus, appropriate administrations will include topical delivery (e g , ointment, spray, cream, etc ), parenteral systemic delivery (e g , inhalation), and direct or indirect delivery to the blood stream (e g , l v oπ ra injection, etc )

Consequently, the formulation of contemplated compounds may vary consideiably Foi example, wheie the drug or drug composition exhibits sufficient stability to pass through the gastro-mtestinal system without undesired chemical or enzymatic modification, oral formulations may mclude syrup, tablets, gel caps, powder, etc On the other hand, wheie absorption or passage of contemplated compounds through the gastro-intestinal tract into the blood stream is problematic, suitable formulations especially include injectable solutions oi suspensions (e g , physiological salme solution buffered to a pH of about 7 2 to 7 5)

With respect to the dosage of contemplated compounds, it should be appreciated that various dosages aie suitable, and contemplated dosages typically are m the range of lmg to several lOOmg, and even moie For example, heie contemplated compounds are excieted oi metabolized at a lelatively low late, or wheie long-term tieatment is desired, dosages will typically be m the range between 5mg-200mg per day On the other hand, where bioav ailabi t of contemplated drugs is relatively low, or where metabolic conversion (e g , dephosphoiylation) is relatively fast, dosages will typically be m the range betw een 100mg-2500mg per day

W ith respect to the dosage of L-nucleosides, and especially Levovir ™, it should furthei be appreciated that Levovirm™ appeals not to be phosphor, lated in \ιvo, at least not in hepatocytes and eπ .hrocytes, and since the antivnal effect of Ribaviim appears to be dependent on phosphoiylation. one of oi dinar) skill m the art would not expect Levovirm™ to ha e a direct anti nal effect hi fact, experiments (not leported) show no direct antiviral effect The antivnal effect of Levovirm™ surprisingly appears to be at a dosage of not moie than 200 mg per da), preferably in the 10 to 200 mg range, more preferably in the 50 to 200 mg range, and even more preferably in the 50 to 100 mg range. This is supported by experimental evidence demonstrating that a given dose of Levovirin™ results in a serum level five times that of an equivalent dose of Ribavirin.

Experiments show that Ribavirin is removed from serum by becoming phosphorylated in red blood cells (see, e.g., Homma, M. et al.; High-performance liquid chromatographic determination of ribavirin in whole blood to assess disposition in erythrocytes; Antimicrob. Agents Chemother. (1999), 43(11):2716-9). Once phosphorylated, Ribavirin cannot leave the cells. Consequently, red blood cells act as a Ribavirin sink, and higher doses of Ribavirin are needed to achieve a given serum level. Levovirin™ is not phosphorylated, and therefore, tends not to accumulate in red blood cells. As a result, red blood cells do not act as a Levovirin™ sink, and lower doses of Levovirin™ are sufficient to achieve a desired serum level.

The schedule of administration may vary considerably, and contemplated schedules include a single dose over the entire course of treatment, multiple single daily doses over the entire course of treatment, multiple daily doses, and permanent dosing (e.g., permanent infusion, implanted osmotic pump, etc.) for at least part of the course of treatment. While it is generally preferred that suitable schedules sustain constant delivery of contemplated compounds, burst delivery (i.e., at least one administration at a first dose followed by at least one more administration at a dose lower than the first dose) is also appropriate. With respect to the term (i.e., duration) of treatment, it is contemplated that appropriate durations may vary between a single administration and several days, several weeks, several years, and even longer. For example, where contemplated compounds are employed in a cell culture, a single administration, or relatively short administration may be sufficient. On the other hand, where contemplated compounds are administered to treat an acute hepatic disease, appropriate treatment duration may be in the range between several days and several weeks. Similarly, where chronic hepatic diseases are treated by administration of contemplated compounds, extended administration over one or more years may be suitable.

In still further alternative aspects of the inventive subject matter, contemplated compounds may be combined with additional phanriaceutically active substances to assist in the treatment of various diseases, and particularly viral infections. Additional pharmaceutically active substances may be administered separately or together, and when administered separately,

9? administration may occm simultaneously or separately m any order Especially contemplated additional pharmaceutically active substances include antiviral agents and immune modulator substances For example, antiviral agents mclude protease inhibitors, nucleotide and/or nucleoside analogs (and especially Ribavirm), and immune modulator substances may include cytokines (e g , mterferon and γ, IL2, IL4, IL6, IL8, IL10, and IL12)

Further contemplated pharmacologically active agents include anti-fungal agents such as tolnaftate, Fungizone™, Lotnmm™, Mycelex™, Nystatin and Amphoteracin, anti-parasitics such as Mmtezol™, Niclocide™, Vermox™, and Flagyl™, bowel agents such as Immodmm™, Lomotil™, and Phazyme™, anti-tumor agents such as mterferon and γ, Adnamycm™, Cytoxan™, Imuran™, Methotrexate, Mithrac ™, Tiazofunn™, Taxol™, dermatologic agents such as Aclovate™, Cyclocort™, Denorex™, Florone™, Oxsoralen™, coal tar and salicylic acid, migraine preparations such as ergotamme compounds, steroids and immunosuppresants not listed above, including cyclosporms, Diprosone™, hydrocortisone, Floron™, Lidex™, Topicort™, and Vahsone™, and metabolic agents such as msulm, and other drugs which may not fit into the above categories

Preferred combinations of contemplated compounds with an interferon

In a particularly prefened aspect of the inventive subject matter, a synergistic combination of Levovirm ^M and at least one mterferon, preferably IFN-α-2b, is contemplated Levovirm™ is typically not, or only to a significantly lesser extent than Ribavirm, phosphorylated in erythrocytes while still exhibiting antiviral and immunomodulatory activity Consequently, it is contemplated that the pharmacological action of mterferon, and especially in the treatment of hepatic diseases, can be potentiated by co-admmistration of Levovirm at significantly lower dosages as compared to Ribavirm For example, it is contemplated that effective synergistic doses of Levovirm™ in combination with mterferon needed to treat HCV infection are projected to be in the 1-600 mg range, more preferably m the 10-400 mg range, still more preferably in the 50-300 mg range, and most preferably in the 100-300 mg range Equivalent synergistic doses of Ribavirm are considered to be 600-800 mg

In another aspect of the inventive subject matter, it is contemplated that the synergistic combination of Levovirm™ and mterferon will result m reduced toxicity relative to a combination of Ribavirm and inteiferon at equivalent effectiv e dosages, predominantly due to the lack of significant phosphorylation in eiythrocytes Viewed from yet another perspective, it is contemplated that the synergistic combination of Levovirm and mterferon specifically allows targeting of the liver due to the lack of phosphorylation of Levovirm™ in compartments other than the liver, especially erytlirocytes

With respect to co-admimstration of Levovirm and mterferon, it is contemplated that all suitable routes and protocols are appropriate, and it is especially prefened that Levovirm and mterferon are administered m a protocol similar to known administration protocols of Riba . inn and mterferon For example, Levovirm™ may be orally administered while Interferon may be subcutaneously injected In general, it is contemplated that co-admmistration of

Levov inn™ and mterferon may utilize schedules and routes independent from each other so long as both drugs are in the blood stream at measurable concentrations at the same time It is further contemplated that effective dosages of Levovirm can be projected from the effective concentiations of Ribavirm in the liver where Ribavirm was administered

While Levovirm™ is particularly contemplated, chemical modifications including prodrug forms such as modified Levovirm™ (l-beta-L-πbofuranosyl-l,2,4-trιazole-3- carboxamidme), mono- di- and tnphosphorylated Levovmn™, and stereochemical variants (e g , enantiomers, isomers, etc ) are also appropnate Examples for suitable chemical modifications and prodiug forms are descnbed in U S Patent Application Number 09/594410, (supra) It is still further contemplated, that suitable drugs may also include drugs other than Levovirm and its variants, and particularly contemplated alternative drugs include In er specific prodrugs with an amine or amide group that can be enzymatically deaminated/ deamidated m the liver

With respect to the mterferon, it is contemplated that co-administration of Levovirm need not be limited to IFN-α-2b, and co-administration may also mclude natural and synthetic fragments, isoforms, and consensus forms of mterferon-alpha Moreov er, mterferons other than interfeion-alpha are also suitable, including mterferon-beta and its natural and synthetic fragments, isoforms, and consensus forms While mterferon is particularly contemplated, cytokines other than mteifeion and chemokines are also appropriate, including IL-2, IL-12, and TNF It is especially contemplated, that pegylated forms of contemplated mteiferons (i e contemplated mterferons associated with pol ethylene glycol) aie also suitable for use in conjunction with the teachings piesented herein Combination of contemplated compounds with a second compound that binds a viral protein or a cytokine

Where the combination of contemplated compounds with other pharmacologically active agents is employed in an antiviral therapy, it is particularly contemplated that such combinations may comprise contemplated compounds with a direct and an indirect antiviral effect, and a second compound that increases the total antiviral effect (the total antiviral effect includes the direct antiviral effect and the indirect antiviral effect), wherein the second compound specifically binds a viral protein or a cytokine.

With respect to contemplated compounds in the combination, nucleoside analogs are prefened, and it is even more prefened that the nucleoside analog is Ribavirin (l-(5-Deoxy-β-D- ribofuranosyl)-l,2,4-triazole-3-carboxamide). Ribavirin is known to have a direct antiviral effect by inhibiting RNA and DNA virus replication [Huffman et al, Antimicrob. Agents Chemother (1973), 3: 235; Sidwell et al, Science (1972), 177: 705] and an indirect antiviral effect by suppressing Type 2 mediated T cell responses and promoting Type 1 mediated T cell responses as described in U.S. Patent Application Number 09/156,646, which is incorporated herein by reference. However, various compounds other than Ribavirin are also contemplated, and particularly include L-nucleoside analogs, so long as such alternative compounds have a direct and an indirect antiviral effect. For example, where especially high concentrations of an L-nucleoside analog are desirable, Levovirin™ may be employed.

It should further be appreciated that depending on the chemical nature of the first compound, the first compound may have a more pronounced direct antiviral effect or a more pronounced indirect antiviral effect. Contemplated direct antiviral effects include inhibition of viral replication, for example, an inhibition of a reverse transcriptase, whereas contemplated indirect antiviral effects include a shift in a Type 1/Type 2 balance towards a Type 1 or Type 2 response as described in U.S. Patent Application Number 09/156,646. It should also be appreciated that an indirect antiviral effect may comprise a suppression of a Type 1 and Type 2 response, which is described in greater detail in U.S. Provisional Patent Application Number 60/172,097 (supra). The shift of a Type 1/Type 2 balance towards a Type 1 or Type 2 response or suppression of the Type 1/Type 2 response may be advantageously controlled by the same first compound, wherein the dosage of the first compound determines the shift or suppression in a Type 1 or Type 2 response.

With respect to the second compound, it is prefened that the second compound comprises an antibody (e.g., a monoclonal or polyclonal antibody). It should be appreciated, however, that in alternative aspects of the inventive subject matter the antibody need not be restricted to a naturally occurring form of an antibody, but may also include a synthetic form of an antibody (e.g., mini antibodies obtained by phage panning, or other molecular evolution technology), or antibody fragments. Antibody fragments are especially desirable, where such fragments are produced by a recombinant cell, or where the molecular weight of the second compound should be relatively low (i.e., below 75kDa). Contemplated antibody fragments include an Fab, an F(ab)₂, and an scFab. Furthermore, it is contemplated that appropriate antibodies may be modified to introduce various additional features, including a reporter group, a second affinity moiety (e.g., a bispecific antibody), or a pharmacologically active molecule. For example, a reporter group may include a radioisotope, or a metal that is detectable with in vivo scanning devices (e.g., magnetic resonance imaging). Contemplated pharmacologically active molecules may include reverse transcriptase inhibitors, protease inhibitors, or cytotoxic agents. The production of recombinant and non-recombinant antibodies is well known in the art (e.g., see Current Protocols in Immunology; John Wiley & Sons (1999); Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Wanen Strober), and it is contemplated that all known methods for their production are suitable for use in conjunction with the teachings presented herein. Antibodies are typically administered by injection (e.g., i.v. injection), and the actual dose will typically lie between O.Olmg and several l Omg, however, λvhere appropriate, lower dosages are also contemplated.

It should further be appreciated that binding of the second compound to a viral protein or a cytokine is particularly advantageous where binding leads to an inactivation of a viral protein and/or a cytokine, and it is contemplated that inactivation may occur via various mechanisms. For example, inactivation of a virus may be achieved by antibody-mediated precipitation (i.e., formation of a molecular network between antibodies and viruses). Alternatively, binding of the second compound may inactivate a virus by blocking or otherwise obstructing proteins or other viral surface structures that are essential to the infectivity or propagation of the virus. Still further, binding of the second compound may occur with non-structural viral proteins, including viral polymerases and proteases. For example, contemplated binding targets (i.e., haptens) include protems such as the gp 120/41 of a HIV virus, but also protems such as the reverse transcnptase of the HIV virus Further contemplated viral protems include protems from a HIV virus, a hepatitis virus, an influenza virus, and an RSV virus With respect to cytokines it is contemplated that inactivation may be achieved by sequestration of the cytokme from the pool of cytokines For example, where the hapten for contemplated second compounds is a Type 1 cytokme, particularly contemplated cytokines include mterleukm-2, mterferon-gamma, and tumor necrosis factor-beta, whereas in cases where the hapten for the second compound is a Type 2 cytokine, particularly contemplated cytokines include ιnterleukιn-4, mterleukm-5, and ιnterleukm-10

It is generally contemplated that inactivation of a virus or a cytokine by the second compound may have a plurality of desirable effects, which may or may not exhibit an additive or synergistic effect in combination with the first compound For example, it is contemplated that m cases where the first compound has a direct antiviral effect (resulting in a significant reduction of viral titer), a second compound may even further reduce the virus titer by precipitating remaining viruses Alternatively, the second compound may reduce the number of infectious virus particles by binding to viral components that are essential for mfectivity It is further contemplated that where the second compound binds a cytokine, the second compound may shift the Type 1/Type 2 balance towards a Type 1 response by sequestenng one or more Type 2 cytokines from the pool of cytokines, and thereby helping to restore cellular immunity while the virus load is already significantly reduced In another example, it is contemplated that where the first compound has an indirect antiviral effect (also resulting m a significant reduction of virus titei), a second compound may further reduce the vims titer by precipitating remaining viruses Alternatively, one or more Type 1 and/or Type 2 cytokines may be sequestered by a second compound or mixture of second compounds thereby "fine tuning" (i e , modulating) a Type 1 and/or Type 2 response induced by the first compound

It should be appreciated that a combination of a first compound that has a direct and indirect antiviral effect with a second compound that specifically binds a virus and/or a cytokine will reduce a viral titer not only by a mechanistic (i e enzyme inhibition), but also by a systemic (i e , stimulation/modulation of immunity) action It is especially contemplated that prefened antiviral drug compositions include a first and second compound having a synergistic effect, which advantageously will help reduce the effective dosage of the first and second compound It is still further contemplated that appropriate antiviral drug compositions may also be employed in a prophylactic treatment

Combination of contemplated compounds with Ribavirin

It is particularly contemplated that co-administration of Levovmn™ with Ribavirm will reduce adverse side effects and improve tolerabi ty of Ribavirm and/or Levovirm™ With respect to the ratio of Ribavirin to Levovirm™ in the co-admmistration, it is prefened that Levov inn™ is present in at least an equimolar amount of Ribavirm However, it should be appreciated that vanous alternative ratios are also appropnate, and the particular ratio will predominantly depend on the desired effect and dosage/route of administration For example, where hemolytic anemia is of particular concern, Levovinn™ may be present in the co- admmistration in a range of about 5 lmol% to about 80mol%, or more On the other hand, where tolerabihty of Levovinn™ is limiting, Levovinn™ may be present m the co-administration m a range of about 49mol% to about 20mol%, or less

It should further be appreciated that co-administration of Ribavirm and Levovirm™ need not necessarily employ the same route of administration The term "co-admimstration" as used herein refers to any form of administration of Ribavmn and Levovinn™ such that Ribavirm and Levov inn™ are present in a measurable concentration in the system at the same time Therefore, contemplated co-admmistrations include protocols in which Ribavirin is administered in one route and Levovinn™ is administered m another route, wherem the co-admmistration may be performed simultaneously or at two different points in time For example, Ribavirm may be administered orally while Levovinn™ may be injected mtrav enously In another example, Ribav inn may be administered orally BID, and Levovirm™ may be administered orally QID

It is particularly contemplated that by variation of the molar fractions of Ribavirm and Levo \ inn™ in a co-administration protocol, particularly desirable biological effects may be tailored to the specific needs of a patient, including modulation of the Type 1/Type 2 cytokine balance, dnect antivnal effect, reduction m hematotoxic properties, etc

In another aspect of the inventive subject matter, it is contemplated that the administration or co-admimstration of Ribavirm and Levo \ inn™ will include a continuous release and/oi a reduced dosage at intervals that are moie frequent It is particularly contemplated that continuous release and/or reduced dosage at frequent intervals will reduce undesirable side effects and may increase the direct and/or indirect antiviral effect. While it is generally contemplated that compounds according to the inventive subject matter may be administered to any system, it is prefened that contemplated compounds are administered to a mammal, preferably a human, or to a cell or tissue culture.

Metabolites of Contemplated Compounds

It is generally contemplated that Levovirin™ is metabolically inert when administered to a system, however, the inventors also contemplate that Levovirin™ may have metabolites, which are shown in Structures 4-8.

Structure 6

OCOCHi

Structure 8

Structure 4 is a triazole carboxamide, Structure 5 is a triazole carboxylic acid, Structure 6 is a L-ribofuranosyl triazole carboxamide, Structure 7 is a 5'-acetyl L-ribofuranosyl triazole carboxamide, and Structure 8 is an 5'-acetyl-α-L-ribofuranosyl triazole carboxamide. While is it generally contemplated that the metabolic products of Levovirm™ aie fonried as a product of an enzymatic leaction, it should also be recognized that undei suitable mtra oi extra-cellular conditions m a cellular system, metabolites may be formed without an enzymatic reaction Thus, formation of metabolites from Levovirm™ may include redox reactions (particularly oxidation), enzymatically catalyzed reactions (e g , hydrolysis), and photochemical reactions

Contemplated reaction products are typically degradation products of Levovirm™, however, it should be recognized that metabolites may also mclude products formed by addition of chemical groups (e g , glycosylation or acetylation), and that such modified compounds may be subject to subsequent degradation m the same or different compartment While it is generally contemplated that the metabolites have a significantly reduced pharmacologically effect as compared to Levovirm™, it should be appreciated that the metabolites may have a pharmacological effect similar to Levovirm™ For example, the triazole or ribose moiety may serve as an effector (e g , allostenc inhibitor)

It is further contemplated that of a particular dose of Levovirm™ administered to a system, between 20% and 50%, preferably between 51% and 75%, more preferably between 76% and 99%, and most preferably 100% are excreted m an unmetabohzed form

Examples

Targeted hepatic immunosuppression employing Ribavirin

Three placebo-controlled studies of Ribavirm in the treatment of chronic hepatitis C weie conducted These studies included 134 patients treated with Ribavirm and 97 patients who received a placebo There were also two uncontrolled Phase II studies including a total of 23 patients treated with Ribavirin The primary response parameter was normalization oi reduction of serum ALT levels Response was also assessed m terms of elimination or reduction of serum HCV RNA levels, and improvement in liver histology as assessed by changes in Knodell scores

In all of the controlled and uncontrolled studies, using the definitions of lesponse specified m the protocols and analysis plans, Ribavirm was statistically significantly supenor to a placebo in normalizing and reducing ALT levels during treatment In the integrated analyses based on all patients m the controlled studies, using a uniform definition of response including normalization of ALT at the end of treatment or a clinically meaningful level of partial response 46%o of Ribavirm patients were responders compared to 4% of placebo patients (p<0 001) Patients generally responded after two to three months of treatment and the response was maintained as long as treatment was continued There was no evidence of loss of ALT response with inci easing duiation of treatment Following withdrawal of Ribavirm at the end of the activ e treatment phase, 11 5% of responders had a sustained response throughout the follow-up period

Regarding improvement in liver histology, m each of the controlled studies there was a non-significant trend m favor of Ribavirm in the changes in total Knodell scores and many of the component scores Analysis of the combined data by analysis of covanance using the baseline Knodell score as covanate resulted in statistically significant differences in favor of Ribavirm foi the total score and each of the component scores Thus in the controlled studies, in comparing all Ribavirin-treated patients with placebo recipients, Ribavinn had a modest but real effect in improving liver histology Withm the Ribavirm group, comparison of ALT responders and non- responders revealed that ALT responders experienced a significantly greater improvement in liver histology as compared to ALT non-responders The mean fall in total Knodell score was approximately two points for ALT responders as compared to one point for all Ribavirm-treated patients A fall in a total Knodell score of two points is generally considered by hepatologists to be clinically significant There was thus a statistically significant positive conelation between ALT response and a clinically significant degree of improvement in liver histology

In all studies, the primary endpomt was defined as a reduction m ALT level In all studies a complete ALT response was defined as normalization of the ALT level at the end of treatment A partial ALT response was defined as either a 50% or greater reduction at the end of treatment from the patient's baseline value, or a 50% or greater reduction to a level not higher than 1 5 times the upper limit of normal

In studies 92-001 and 91-DK-178, the treatment groups were compared with respect to the effect of the study medication on symptoms relev ant to hepatitis This could not be done study CT00/002 because the case report form did not permit the systematic collection of symptom data

In study 92-001, there w as a statistically significant difference m favor of Ribavirm for decreased fatigue At the end of tieatment and at the end of the follow-up period, a higher proportion of Ribavirin patients showed some improvement from baseline in fatigue compared to placebo patients (p=0.04 for end of treatment and p=0.006 for end of follow-up). In this study, there were no significant differences between the treatment groups for any other symptoms.

In study 91-DK-I78, there were isolated significant differences between the treatment groups in individual symptoms at individual visits, some favoring the placebo group and some the Ribavirin group, but there were no overall trends favoring either treatment group.

Table 1 shows the response rates in terms of improvement in liver histology. There were no statistically significant differences between the treatment groups in the changes in Knodell scores in any of the studies, although there were numerical trends in favor of Ribavirin. In study CT00/002 there was a difference in favor of Ribavirin in one of the secondary parameters (lymphoid aggregates, p=0.05).

Protocol Protocol Definition of Analysis Plan Definition of Result Response Response

92-001 Comparison of treatment groups Same No significant difference with respect to the changes from between treatment pie- to post-treatment in each groups patient's Knodell scores

91-DK-178 Long-term response: Comparison of treatment groups No significant difference Improvement in liver with respect to the changes from between treatment histopathology by "blinded pre- to post-treatment in each groups ranking of all liver biopsies for patient's Knodell scores the degree of current hepatic injury using the Wilcoxon rank sum test"

CT00/002 Improvement in degree Comparison of treatment groups No significant diffeience of inflammatory activity fiom \\ ith respect to the changes from between treatment pre- to post treatment as pre- to post- tieatment in each groups assessed by Knodell scores patient' s Knodell scores

Pre- to post treatment changes in other histological parameters Reduction m lymphoid thought to be relevant to hepatitis aggregates in Ribavirm C group

Table 1 - Comparison ot Results oi Controlled Studies - Liver Histology

Further analyses were performed in respect of the different studies. However, to make the results of these analyses more meaningful, the control studies data were combined to make the sample sizes larger. Results of Integrated Analyses

Analysis of Response to Ribavirin According to the Relevant Effectiveness Criteria -ALT Response During Ribavirin Therapy

For the purposes of the integrated effectiveness analyses, the following definitions of ALT response were used:

Complete Response: Return to within the normal range at the end of treatment.

Partial Response: 50% or greater reduction from the patient's baseline level to within 1.5 times the upper limit of normal at the end of treatment.

Responder: Meets above definitions of either complete or partial response. The definition of "responder" was determined by plotting the ALT values over time for the groups of patients fitting various definitions of response employed within each study. (The data were fitted with a cubic spline smoothing function Reinsch 1967). Three definitions of response were used:

a. Complete response = ALT in normal range at end of treatment.

b. Partial response (A) = 50% or greater reduction from the patient's baseline level to within 1.5 times the upper limit of normal at the end of treatment.

c. Partial response (B) = 50% or greater reduction from the patient's baseline level at the end of treatment.

All other patients were considered non-responders. Plots were also constructed for Ribavirin- treated non-responders within each study and for all placebo patients (responders and non- responders) within each study.

The three curves for "complete response" demonstrated that this response was achieved after approximately one third of the treatment period and was maintained thereafter. The three curves for "partial response (A)" demonstrated a similar pattern of response. The three curves for' "partial response (B)" demonstrated distinctly more variability of ALT levels during the treatment periods. The plots for Ribavirin-treated non-responders and the plots for placebo patients demonstrated, as expected, a dispersion of the data points which did not change in any recognizable pattern across the treatment and follow-up periods. It was decided that the "partial response (B)" definition was inappropriate for the purpose of the integrated effectiveness analyses. Due to the consistent pattern of response demonstrated by the "complete response" and "partial response (A)" definitions, and the fact that these definitions are clinically meaningful, it was decided that for the purpose of the integrated effectiveness analyses a "response' be defined as either "complete response" or "partial response (A)".

Table 2 displays the results for each study and for the combined database, using the above definition of ALT response. The proportions of responders in the two treatment groups were compared using either a Chi-square or Fisher's Exact test.

Study Ribavirin n/N (%) Placebo n/N (%) p Value

92-001 15 / 28 (53.6) 1/30 (3.3) O.001

91 -DK-178 11/29 (37.9) 1/29 (3 4) <0.001

CT00/002 32/70 (45.7) 2/36 (5.6) O.001

Combined database 58/ 127 (45.7) 4/95 (4.2) O.001 n = Number of patients with an ALT response (integrated definition) N = = A11 patients treated (mtent-to- treat population) minus those without valid non-missmg observations. Table 2: Percent ALT Response Rates

For identifying the ALT value conesponding to the end of treatment, the last valid, non-missing observation, going back a maximum of two visits, was canied forward for those patients missing such a true value. This same policy was employed in two out of the three controlled studies (91- DK-178 and CT00/002). Nine patients in the Ribavirin group and two placebo patients did not have such a valid non-missing observation available. The ALT response rates were consistent across the three controlled studies and ranged from 37.9 to 53.6%. When the data were combined, the ALT response rate was 45.7%. In all instances, the ALT response rates for patients treated with Ribavirin were statistically, significantly superior to the rates in patients treated with placebo. In the placebo group, ALT response rates were consistently low, and ranged from 3.3 to 5.6% and 4.2% when the data were combined.

ALT Response in the Follow-Up Period

Table 3 summarizes the rates of sustained response in studies 92-001 and 91 -DK-178 and in these two studies combined. The individual study, analysis plan definitions of sustained response are used. A sustained responder is essentially a patient with either normalization of ALT or a partial response at the end of treatment, who still meets either of these criteria throughout the follow-up period. It was not possible to provide this same analysis for study CT00/002 because too few patients had complete ALT data throughout the follow-up period. Study Ribavirin n/N (%) Placebo n/N (%)

92-001 1/15 (6.7) 0/1 (0.0)

91-DK-178 2/11 (18.2) 0/1 (0.0)

Combined database 3/'26 (11.5) 0/2 (0.0) n= Number of patients with sustained ALT response N = Number of patients with complete or partial response at the end of treatment (study analysis plan definitions)

Table 3 Percent ALT Sustained Response Rates - Protocol Definitions

In the two studies analyzed, the sustained ALT response rates were 6.7 and 18.2% for Ribavirin- treated patients compared with 0% for patients receiving placebo. Due to the low sample size and the very low number of placebo responders a statistical analysis was not performed.

Improvement in Knodell Scores

Within each of the three controlled studies, there was a consistent numerical trend in favor of Ribavirin in the changes in total scores and in many of the component scores. This same trend was thus apparent when the data from the three studies was combined. The trend applies not only to improvement of scores but also to worsening, indicating that even if no patients in either treatment group improve, then fewer patients in the Ribavirin group are worsening. This is an important observation considering that one objective of treatment is to prevent deterioration of a chronic and progressive condition. Analysis of the combined data by the CMH chi-square test does not reveal any statistically significant differences. Analysis of the combined data by analysis of variance (as used in studies 92-001 and 91 -DK-178) revealed a statistically significant difference in favor of Ribavirin for the total Knodell score but not for any of the component scores.

The liver histology data was examined further by analysis of covariance, using the baseline Knodell score as covariate. Regression analysis of the baseline Knodell scores versus the end of treatment scores for all Ribavirin-treated and placebo patients combined resulted in a slope of less than 1.0 but greater than zero. This indicated that the baseline Knodell score influenced the expectation of outcome of treatment, regardless of any difference between Ribavirin and placebo. Where the regression slope differs markedly from 1.0, analysis of covariance is a more appropriate test than analysis of variance (Fisher 1951). The result of the analysis of covariance is displayed in Table 4. The mean changes in the scores for Ribavirin- treated patients are only small, but due to small variances, the differences from placebo are statistically significant. It is of interest to note that the only Knodell sub-score that does not improve is fibrosis, and that there is less deterioration in the Ribavirin group than in the placebo group.

Mean Change in Score from Baseline to End of Treatment

Knodell Component Ribavirin N = 107 Placebo N = = 78 p Value

Periportal Activity and -0.40 -0.01 0.0004 Necrosis

Portal Inflammation -0.30 -0.10 0.0206

Lobular Necrosis -0.33 -0.13 0.0019

Fibrosis +0.07 +0.25 0.0071

Total score -1.11 -0 05 0.0091

T able 4 Compai ison of Ribavnin and Placebo in Terms of Changes in Knodell Scoies trom Baseline to End of Treatment - Analysis of Covanance Using Baseline Knodell Score as Covanate All Phase III Studies Combined

Examination of Correlation between ALT Response and Improvement in Knodell Scores

In each of the three controlled studies, Ribavirin was significantly more effective than placebo in normalizing and reducing ALT levels (An elevated serum ALT level is a biochemical indicator of hepatic inflammation). There was also a conelation between response to Ribavirin therapy in terms of normalization or reduction of ALT level within individual patients, and by an improvement in liver histology as determined by Knodell scores. We found that there was indeed a consistent trend towards a positive relationship between ALT response and improvement in Knodell scores when both parameters are treated in a categorical manner. In the combined database, this trend was statistically significant for the Total Knodell score (p = 0.008), Fibrosis (p = 0.014), and Portal Inflammation (p = 0.022) using the Cochran-Mantel-Haenszel (CMH) chi-square test (Table 5-7).

Responder Non -Responder

Protocol Response n % n % P-value'

92-001 Improved 7 46.7 4 33.3 0.099

Unchanged 7 46.7 3 25.0

Worsened 1 6.7 5 41.7

91 -DK- Improved 10 90.9 7 38.9 0.021 _* 178 Unchanged 1 9.1 5 27.8

Worsened 0 0.00 6

CT00 002 Improved 11 42 3 12 48.0 0.171

Unchanged 11 42 3 5 20.0

Worsened 4 15 4 8 32.0

Integrated Impro . ed 28 53.8 23 41.8 0.008 *

Unchanged 19 36.5 13 23.6

Worsened 5 9.6 19 34.5

N= Number of patients m study n= Number of patients in i esponse category %=(n/N)* 100 ¹ CMH statistics ^x PO.05 Table 5 Knodell Response Status bv Controlled Studies - Total bv ALT Response Responder Non -Responder

Protocol Response n % n % P-value

92-001 Improved 2 13.3 1 0.00 0.434

Unchanged 12 80.0 1 1 91.7

Worsened 1 6.7 1 8.3

91-DK-178 Improved 5 45.5 2 11.1 0.119

Unchanged 4 36.4 1 1 61.1

Worsened 2 18.2 5 27.8

CTOO/002 Improved 6 23.1 1 4.0 0.120

Unchanged 18 69.2 20 80.0

Worsened 2 7.7 4 16.0

Integrated Improved 13 25.0 3 5.5 0.014 *

Unchanged 34 65.4 42 76.4

Worsened 5 9.6 10 18.2

N= Number of patients m study n= = Number of patients in response category %= =(n/N)*100 ' CMH statistics * P<0.05

Table 6 Knodell Response by Controlled Studi es - Fibrosis by ALT response

Responder Non -Responder

Protocol Response n % n % P-value '

92-001 Improved 5 33.3 25.0 0.220

Unchanged 9 60.0 5 41.7

Worsened 1 6.7 4 33.0

91-DK-178 Improved 5 45.5 4 22.2 0.227

Unchanged 6 54.5 11 61.1

Worsened 0 0.00 3 16.7

CTOO/002 Improved 5 19.2 6 24.0 0.159

Unchanged 21 80.8 16 64.0

Worsened 0 0.00 3 12.0

Integrated Improved 15 28.8 13 23.6 0.022 *

Unchanged 36 69.2 32 58.2

Worsened 1 1.9 10 18.2

N= Number of patients in study n= ^: Number of patients m response category %= =(n/N)*100 ' CMH statistics * P<0.05

. able 7 Knodell Response Status by Controlled Studies -Portal Inflammation by ALT Response

The relationship between ALT response and improvement in liver histology was studied further in order to quantify the improvement in liver histology in patients responding to Ribavirin, and to determine if there was a subgroup of patients who derive a more substantial clinical benefit from treatment with Ribavirin. In each of the three controlled studies, Ribavirin- treated ALT responders and non-responders were compared in terms of the mean changes in total Knodell scores over the course of treatment. This analysis, displayed in Table 8 thus quantifies the directional changes displayed in Table 5.

It can be seen that in each study there was a conelation between ALT response and improvement in liver histology, in that ALT response was associated with a greater improvement in liver histology as compared to ALT non-responders. This effect was particularly striking in study 91-DK-178, where there was a mean Knodell score reduction of 4.09 in ALT responders as compared to 0.17 in ALT non-responders. A regression model was employed to examine the relationship between ALT response and Knodell score changes. This revealed that in all three studies ALT Response was a significant predictor of improvement in liver histology. Thus, ALT normalization or reduction conelates with improvement in liver histology in patients with hepatitis C treated with Ribavirin, and patients who achieve an ALT response are likely to derive a substantial clinical benefit. In patients treated with Ribavirin who do not achieve an ALT response, there does not appear to be any clinical benefit in comparison to patients who received placebo.

Study CT00/002 92-001 91-DK-178 Combined Database

Duration of treatment 24 36 48 (week)

Mean Knodell Change -1.23 -1.47 -4.09 -1.90 (Range) in ALT (-7.4) (-8.2) (-9.0) (-9.4) Responders n = 26 n = 15 n = l l n = 52

Mean Knodell Change -0.96 +0.58 -0.17 -0.36 (Range) in Non- (-9.4) (-3.6) (-6.7) (-9.7) Responders n = 25 n = 12 n = 18 n = 55

P-Value^** 0.004 0.0001 0.002 0.0001

Mean Knodell Change -1.10 -0.56 -1.65 -1.11 (Range) all Ribavirm (-9.4) (-8.6) (-9.7) (-9.7) Patients n = 51 n = 27 n = 29 n = 107

Mean Knodell Change -0.09 -+0.44 -0.52 -0.51 (Range) all Placebo (-4.4) (-3.5) (-8.4) (-8.5) Patients n = 23 n = 27 n = 27 n = 77

P-Value NS NS NS 0.01

ALT response was defined as normalization at the end of treatment or reduction of 50% or more from baseline to withm 1.5 times the upper limit of normal at the end of treatment Regression analysis

Table 8

Thus, in summary, the phase III program on Ribavirin in chronic hepatitis C consisted of three randomized, double blind, placebo-controlled, parallel group studies. 134 total patients were randomized to receive Ribavirin and 97 to receive placebo. Response to treatment was assessed using three parameters :

1. Reduction or normalization of the ALT level (elevation of ALT is a biochemical marker of hepatic inflammation). 2. Improvement in liver histology as evidenced by a reduction in the Knodell score (the Knodell scoring system quantifies the degree of liver damage by assigning scores to various relevant microscopic characteristics and summing these sub-scores to give a total score).

3. Elimination of hepatitis C virus from the blood, or a reduction in the amount of virus present.

Of the 134 patients in the phase III studies, there were 101 with complete data on ALT levels, Knodell scores and virus levels. Among these 101 patients, 24 patients met the criteria for an optimal clinical response to Ribavirin therapy. The criteria are normalization or clinically significant reduction of the ALT level, and reduction in total Knodell score of two or more points. For these 24 patients, the clinical response was obtained without an accompanying reduction in the virus level in the blood. Table 9 below summarizes the data on ALT levels and Knodell scores for the 24 responding patients as compared to the remaining 77 patients who demonstrated lesser degrees of response.

Responders N=24 Non-Responders N=77

Mean baseline ALT (range) 142.5 (52-269) 176.9 (35-629) in U/L

Mean end-of-treatment ALT (range) 36.8 (21-62) 91.7 (13-286)

U/L

Mean Knodell 4.1 (2-9) 0.15 (-7 to+9) score reduction (range)

* upper limit or normal is 40 U/L

Table 9

Thus, specific embodiments and applications of nucleosides, nucleotides, and their analogs have been disclosed. It should be apparent, however, to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the spirit of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as refening to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced.

Claims

CLAIMSWhat is claimed is:

1. A compound having the structure:

wherein R is P0₃ ^2", (P0₃)₂ ^3", or (P0₃)₃ ^4~.

2. A compound having the structure:

A compound having the structure:

wherein W is -N(Rι)(R₂) or =NRι, wherein R_t and R₂ are independently hydrogen, a linear alkyl, a branched alkyl, an alkenyl, an alkynyl, an aralkyl, an aralkenyl, an aralkynyl, or an aryl.

4. The compound of claim 3 wherein Ri or R₂ independently further comprises a nitrogen atom, an oxygen atom, a sulfur atom, or a halogen atom.

5. A method of treating a viral infection in a patient comprising administering a composition that includes a compound according to claim 1, claim 2, claim 3, or claim 4 at a dosage effective to inhibit viral propagation.

6. The method of claim 5 wherein the dosage is between 50-500mg/day.

7. The method of claim 5 wherein the dosage is between 500-2500mg/day.

8. The method of claim 5 wherein the viral infection is selected from the group consisting of an HIV infection, an HCV infection, an HBV infection, an RS V infection, an influenza virus infection, and a parainfluenza virus infection.

9. The method of claim 5 further comprising co-administering to the patient a cytokine.

10. The method of claim 9 wherein the cytokine is an interferon.

11. The method of claim 10 herein the interferon is interferon alpha-2b.

12. The method of claim 5 further comprising administering Ribavirin.

13. The compound of claim 1 wherein R is PO₃ ^".

14. A method of treating a viral infection in a patient comprising co-administering a composition comprising a compound according to claim 13 at a dosage effective to inhibit viral propagation.

15. The compound of claim 1 wherein R is (P0₃)₂ ^3~.

16. A method of treating a viral infection in a patient comprising administering a composition comprising a compound according to claim 15 at a dosage effective to inhibit viral propagation.

17. The compound of claim 1 wherein R is (P0 )₃ ^".

18. A method of treating a viral infection in a patient comprising administering a composition comprising a compound according to claim 17 at a dosage effective to inhibit viral propagation.

19. A method of increasing selectivity of a pharmacologically active molecule with respect to a pharmacological effect in a target cell, comprising:

providing a drug, wherein the drug is l-β-L-ribofuranosyl-l,2,4-triazole-3-carboxamide;

modifying the drug with a modifying group, wherein the modifying group is covalently attached to the drug via a nitrogen atom; and

wherein the modifying group is enzymatically removed from the drug in the target cell.

20. The method of claim 19 wherein the modifying group is =NH, and wherein the modifying group is covalently bound to a carbonyl atom in the drug.

21. The method of claim 19 wherein the modifying group is -N(Rι)(R₂) or =NRι, wherein Ri and R₂ are independently hydrogen, a linear alkyl, a branched alkyl, an alkenyl, an alkynyl, an aralkyl, an aralkenyl, an aralkynyl, or an aryl.

22. The method of claim 21 wherein Ri or R₂ independently further comprises a nitrogen atom, an oxygen atom, a sulfur atom, or a halogen atom.

23. A method of treating a disease characterized by inflammation of an organ in a patient, comprising:

providing a compound according to claim 1, claim 2, or claim 3; and

administering the compound to the patient at a dosage that (a) causes systemic immuno- modulation and not systemic immunosuppression of Type I and Type II responses, and (b) causes immunosuppression of Type I and Type II responses in the organ of the patient.

24. The method of claim 23 wherein the compound is a compound according to claim 1.

25. The method of claim 23 wherein the compound is a compound according to claim 2.

26. The method of claim 23 wherein the compound is a compound according to claim 3.

27. The method of claim 23 wherein the organ is a liver, and wherein the liver is infected with a virus.

28. The method of claim 27 wherein the virus is an HCV virus.

29. A method of stimulating neuronal growth, comprising:

recognizing that a compound having structure I is effective to stimulate growth of neurons within a given concentration range;

wherein Y is a 1-β-L-ribofuranosyl, a l-β-L-ribofuranosyl-5-phosphate, a 1-β-L- ribofuranosyl-5-diphosphate, a l-β-L-ribofuranosyl-5-triphosphate, a 1-β-D- ribofuranosyl, a l-β-D-ribofuranosyl-5-phosphate, a l-β-D-ribofuranosyl-5- diphosphate, or a l-β-D-ribofuranosyl-5-triphosphate; and

providing the neurons with the compound within the given concentration range.

30. The method of claim 29 wherein the compound is Ribavirin.

31. The method of claim 29 wherein the compound is a phosphorylated Ribavirin.

32. The method of claim 29 wherein the compound is Levovirin™.

33. The method of claim 29 wherein the compound is a phosphorylated Levovirin™.

34. The method of claim 29 wherein the compound is administered as part of a treatment of a disease in a patient, in a dosage range effective to increase a Type 1 response and decrease a Type 2 response in the patient.

35. The method of claim 29 further comprising targeting unipolar neuronal cells in the neurons.

36. The method of claim 29 further comprising targeting bipolar neuronal cells in the neurons.

37. The method of claim 29 wherein the neurons are part of a neuronal tissue including at least four of the following cell types: an astrocyte, a dendrocyte, a myelin sheath cell, a glia cell, a unipolar neuronal cell, a bipolar neuronal cell, a multipolar neuronal cell, and a receptor cell.

38. An antiviral drug composition, comprising:

a first compound having a direct antiviral effect and an indirect antiviral effect; and

a second compound that increases a total antiviral effect, wherein the total antiviral effect includes the direct antiviral effect and the indirect antiviral effect, and wherein the second compound specifically binds a hapten selected from the group consisting of a viral protein and a cytokine.

39. The antiviral drug composition of claim 38 wherein the first compound comprises a nucleoside analog.

40. The antiviral drug composition of claim 38 wherein the nucleoside analog is Ribavirin.

41. The antiviral drug composition of claim 38 wherein the nucleoside analog is Levovirin™.

42. The antiviral drug composition of claim 38 or claim 39 wherein the nucleoside analog is in the form of a prodrug.

43. The antiviral drug composition of claim 38 wherein the direct antiviral effect comprises an inhibition of a viral replication.

44. The antiviral drug composition of claim 38 wherein the indirect antiviral effect comprises a shift of a Type 1/Type 2 balance towards a Type 1 response.

45. The antiviral drug composition of claim 38 wherein the indirect antiviral effect comprises a suppression of a Type 1 and Type 2 response.

46. The antiviral drug composition of claim 38 wherein the second compound comprises an antibody.

47. The antiviral drug composition of claim 46 wherein the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a synthetic antibody, and an antibody fragment.

48. The antiviral drug composition of claim 38 wherein the viral protein is a protein of a virus selected from the group consisting of an HIV virus, a hepatitis virus, an influenza virus, a parainfluenza virus, and a RSV virus.

49. The antiviral drug composition of claim 38 wherein the viral protein is a reverse transcriptase.

50. The antiviral drug composition of claim 38 wherein the cytokine is a Type 1 cytokine.

51. The antiviral drug composition of claim 38 wherein the first compound and the second compound have a synergistic effect.

52. The antiviral drug composition of claim 38 wherein at least one of the first compound and the second compound selectively accumulates in an organ.

53. The antiviral drug composition of claim 52 wherein the organ is selected from the group consisting of a liver and a brain.

54. The antiviral drug composition of claim 38 wherein the first compound is Ribavirin and the second compound is an antibody that specifically binds a Type 2 cytokine.