CZ20022112A3 - Preparations and methods relating to L-nucleosides, L-nucleotides, and their analog - Google Patents

Abstract

Nucleoside and nucleotide compounds and their analogs/prodrugs are provided. Particularly contemplated compounds include 1- beta -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 relating to L-nucleosides, L-nucleotides, and analogs thereof

The present invention asserts the claims in connection with US. S. Provisional Patent Application 60/173 446, filed 12/29/1999, with U. S. Provisional Patent File 60/172 097, filed 12/23/1999, p. S. Provisional Patent Application 60/175 111, filed 01/06/2000, sU. S. Provisional Patent Specification 60/190 758, registered 3/20/2000, sU. S. Provisional Patent Application 60/226 947, registered 8/22/2000, with U. S. Provisional Patent Application 60/226 875, registered 8/22/2000, sU. S. Provisional Patent Specification 60/233 821, filed 9/19/2000, U.S. Pat. S. Provisional Patent Specification 60/223 823, filed 9/19/2000, p. S. Provisional Patent Specification 60/223 548, filed 9/19/2000, p. S. Provisional Patent Specification 60/223 822, filed 9/19/2000, and U.S. Pat. S. Provisional Patent Specification 60/235,465, filed 9/26/2000.

Technical field

The present invention relates to the field of pharmaceutical compositions and the use of pharmaceutical compositions.

• • •

BACKGROUND OF THE INVENTION

There are a large number of different effects causing reactions in relation to the health of a person, many of which result from infections or accumulations of toxic substances in a living organ, but this can result in an adverse or contradictory reaction of the immune system to that infected organ. For example, hepatitis C virus (HCV) infection often leads to a persistent viral infection where an inflamed organ cannot respond directly to the HCV virus, but rather to an infection induced by an imbalance in the immune response.

Most known methods of treating viral infections can be characterized as either a direct antiviral treatment method or an indirect antiviral treatment method. In a direct method of antiviral treatment, the virus is selected to target a suitable direct antiviral drug. For example, HIV-infected patients typically receive a mixture of medicaments to block the spread of viruses, and various groups for direct antiviral treatments are known in the art. For example, some direct antiviral drugs block reverse transcriptase. Reverse transcriptase (RT) inhibitors are typically nucleoside analogs such as AZT, 3TC, or dd1. Alternatively, non-nucleotide RT inhibitors, including quercetin, may be used. In vitro, RT inhibitors are typically potent antiviral drugs. However, in vivo, and especially during periods of relatively high viral replication rates, the generation of RT inhibitor resistant viral mutants is problematic.

Other direct antiviral drugs block or interfere with protein processing viruses and are commonly known as protease inhibitors. Protease inhibitors are typically highly specific for viral proteolytic enzymes. However, due to their mostly hydrophobic nature, their administration at the desired concentrations becomes problematic. In addition, the development of cross-resistant and often severe side effects of the compounds causes an increase in the difficulty of using protease inhibitors. In order to reduce development against many drug resistant virus strains, mixtures of RT inhibitors and protease inhibitors can be overwritten. Although such compositions have been used relatively successfully for a short period of time, there is still a relatively high incidence of adverse side effects and the theoretical possibility of generating drug-resistant strains of viruses.

In an indirect method of antiviral treatment, the immune response to eliciting a virus-induced response may be altered, modulated. For example, immunosuppressive drugs may be used to alleviate the inflammatory condition associated with a particular viral infection; Various immunosuppressive drugs are known in the art. Among others, cyclosporin A is known as an immunosuppressive drug, a potent immunosuppressive drug, and is very often used to suppress a tissue rejection reaction after an organ transplant. However, the use of cyclosporin A may lead to problems due to its overall immunosuppressive action, which makes the patient more susceptible to new infectious diseases. In addition, long-term administration of ciclosporin A is often associated with severe side effects, including excessive hair (hirsutism) and gingivitis (gingival hyperlasia). In addition, the bioavailability of cyclosporin is at least partially dependent on bile, which may present additional problems in hepatitis infection.

Tacrolimus (FK506) can be used as an immunosuppressive drug to overcome at least some of the problems associated with cyclosporin A. For example, Tacrolimus has been found to act in the treatment of atopic dermatitis on the face. Topical application of this immunosuppressive drug resulted in a significant improvement in 95% of treated patients [Alaiti, S., et al .: Tacrolimus (FK506) oitment for atopic dermatitis: A phase I study in adults and children. J. Am. Acad. Dermatol. 1993; 38 (1), pp. 69-76]. Furthermore, Tacrolimus does not appear to penetrate the skin barrier, thus eliminating any problems associated with systemic administration.

Although generally well tolerated, treatment with Tacrolimus is limited to topical administration due to the potential for overall immunodeficiency. If Tacrolimus is systematically administered over an extended period of time, lymphoproliferative diseases and cardiomyopathy can often occur.

Many known immunosuppressive drugs provide some alleviation of inflammatory conditions. However, these effects are not specific to the organ when administered systemically. Consequently, immunity to exogenous and endogenous induced responses such as bacterial and viral infections, neoplastic or malignant cells, etc. is thereby reduced. the immune system of the patient and the minimum concentration that can provide at least some desired effect.

Although various compounds and methods for treating infectious and inflammatory diseases are known in the art, all or almost all exhibit one or more disadvantages. Therefore, there is a need to provide improved methods and means for treating these conditions.

• · · ·

SUMMARY OF THE INVENTION

The present invention is directed to methods and compositions wherein the nucleoside and / or nucleotide drug or analogue thereof is administered to a subject in a concentration or dose effective to achieve the desired pharmacological or physiological effect.

According to one aspect of the invention, the present invention provides a compound of formula I:

wherein R is H, PO ₃ ² ', (PO ₃ ) 2 ³ , or (PO ₃ ) ₃ ⁴ '.

The contemplated compounds are optionally further modified by a group that is covalently attached to the carbonyl atom, said compounds of the invention being in D- or L-configuration.

In another aspect, the present invention provides the use of said compounds for the elimination of a viral infection, wherein they may further be co-administered with a scytokine, preferably IFN-alpha-2b, antibody, or Ribavirin (1pD-ribofuranosyl-1,2,4-triazole-3- carboxamide) em.

In another aspect, the present invention provides the selectivity of the compounds of interest with respect to pharmacological activity towards target cells enhanced by modifying said compounds with a modifying group, wherein said modifying group is covalently attached to said drug by a nitrogen atom, and said modifying group is enzymatically removed from said drug in a target cell. Especially then among those mentioned modifying group = NH, and N (R (R _2), or = NR _b wherein R and R ₂ are independently hydrogen, linear alkyl, branched alkyl, alkenyl, alkynyl, aralkyl, aralkenyl, aralkynyl, or aryl and wherein R 1 and R ₂ may independently further comprise a nitrogen atom, an oxygen atom, a sulfur atom, or a halogen atom.

In another aspect, the present invention provides a method of treating a disease comprising administering to a patient having an inflamed organ in the body a compound of interest in a dose that causes systemic immunomodulation rather than systematic immunosuppression of the Type I and Type II responses. The action of the Type I and Type II immunosuppression responses in a given organ of a patient is selective in view of the selectivity of the action of said compounds on that organ.

In yet another aspect, the present invention provides a method of stimulating nerve cell growth, since the compounds disclosed herein have been found to be effective in stimulating nerve cell growth within a given concentration range. In the next step, the compounds are then introduced into nerve cells within a given concentration range.

The various objects, features, features, aspects and advantages of the present invention will become more apparent from the following detailed description of various embodiments of the invention.

Brief description of the pictures

Figures 1A to 1C show examples of compounds of the present invention.

Figure 2 shows an example of a synthetic scheme for the synthesis of Ι-β-L-ribofuranosyl-1,2,4-triazole-3-carboxamide.

Figure 3 shows an alternative example of a synthetic scheme for the synthesis of 1-β-L-ribofuranosyl-1,2,4-triazole-3-carboxamide.

Figure 4 shows another example of a synthetic scheme for the synthesis of Ι-β-L-ribofuranosyl-1,2,4-triazole-3-carboxamide.

Figure 5 is a block diagram of an exemplary method of target organ immunosuppression according to the present invention.

Figure 6 is a block diagram of an example of a method of stimulating cell growth according to the present invention.

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

Detailed description

Contemplated compounds

It is generally contemplated that all nucleotides, nucleosides, and their corresponding analogs may be suitable for use in conjunction with the theory presented herein, characterized in that all compounds contemplated herein may be in either the L-configuration or the D-configuration.

However, particularly preferred compounds include phosphorylated and non-phosphorylated Levovirine ™ (1-β-L-ribofuranosyl-1,2,4-triazole-3-carboxamide, Formula I)

O-R (I) wherein R is hydrogen, or a phosphorus or sulfur-containing group. When R is a phosphorus-containing group, it is particularly preferred that R is a monophosphate, pyrophosphate, or triphosphate as shown in Figures IA to 1C.

Depending on the chemical environment (and in particular the pH), it can be appreciated that phosphate groups can be in their respective mono-, di-, and tri- and tetra-protonated forms, and it can also be positively assessed that if they are phosphate groups partially or completely deprotonated, salts with one or more mnovalent or multivalent cations may be formed. In particular, said cations are alkali metal ions and alkaline earth metal ions such as Mg ²⁺ , Cs ²⁺ , Na ⁺ , etc.

According to alternative aspects of the subject invention, R can also be a PO ₃ ² ', (PO ₃ ) 2 ³ ', or (PO ₃ ) ₃ ⁴ group wherein one or more oxygen atoms are replaced by a sulfur atom. Although phosphate groups are generally preferred substituents for R, other chemical groups may also be used, with the intended groups in particular including mono- or polyanionic groups, preferably with tetragonal geometry. These compounds include, in particular, modified and unmodified phosphorylated Levovirin ™. In addition, it can be appreciated that these compounds are: the compounds may also have a sugar moiety in the D-configuration, and in particular Ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) may be mentioned as said sugar-confluent compound.

Further, it should be appreciated that at least some of the compounds of the compounds disclosed herein exhibit a direct antiviral effect (i.e., those of the compounds that immediately prevent the spread of viruses).

Since most organisms have phosphatases in diverse portions, it is contemplated that the compounds of the present invention may be gradually dephosphorylated, and over a period of time one or more of the phosphorane groups may be removed. For example, a triphosphorylated group may be converted to a diphosphorylated or monophosphorylated compound, or the diphosphorylated compound may be converted to Levovirin ™ in a single reaction.

From the standpoint of assessing the dephosphorylation of phosphorylated Levovirine ™, a change in the mode of antiviral action from direct antiviral to indirect antiviral activity is particularly significant. A change from a direct antiviral response to an indirect antiviral response is particularly advantageous since, when the compounds are metabolized, they maintain antiviral activity for an extended period of time. It is therefore very positively appreciated that the mode of action of the compounds, and in particular phosphorylated Levovirine ™, is at least bimodally - comprising a portion of direct antiviral activity and a portion of indirect antiviral activity.

In relation to the dephosphorylation rate, it can be positively assessed that phosphorylated Levovirine ™ is dephosphorylated at a much slower rate than phosphorylated Ribavirin, and the effect is positively evaluated relative to the L-configuration of ribose in Levovirine ™. With respect to the part or organ where dephosphorylation is to take place, it is appreciated that the dephosphorylation preferably occurs in the liver, but it also applies to other organs and parts, including the kidney, nerve cells and bloodstream.

In particular, it will be appreciated that all known prodrug forms of said compounds are suitable for use in accordance with the teachings herein, and in particular said prodrug forms, including covalent modifications, which can be enzymatically (i.e., by aminohydrolase, oxidoreductase, or transferase) removed from the prodrug. of said compounds.

Furthermore, examples of particularly suitable prodrugs include prodrugs resulting from the addition of a nitrogen-containing group in the carboxyl portion of Levovirine ™, which may be particularly advantageous when said compounds are preferably for the liver. For example, Applicants have discovered (unpublished results) that the specificity of Levovirine ™ with respect to the pharmacological effect of hepatocytes can be increased by modifying Levovirine ™ with the modification ··· • t 99 9 9 9

9 98 9 9 9

9 ·

9999 99

9999 a nitrogen-containing group that is selectively removed from in hepatocytes. Structural Formula II below shows Levovirin ™, and Structural Formula III shows Levovirin ™ modified on a carboxyl group to form a carboxamidine group.

, Λλ

In particular, modification of Levovirine ™ by a modifying group (preferably containing nitrogen) that can be selectively removed from a target cell (e.g., a hepatocyte) helps (1) increase the selectivity of Levovirine ™ relative to the target cell, thereby (2) reducing the overall and (3) reducing the overall toxicity in the non-target cells. Further, the modifying group is covalently bonded to the carboxyl atom of the carboxamide group.

In other alternative aspects, the nitrogen-containing modifying group is not limited to the = NH group, but may also include various primates and secondary amines. Generally, suitable modification groups have the structure and N (R 1) (R ₂ ) or = NR ₁₅ wherein R 1 and R ₂ are independently hydrogen, linear or branched alkyl, alkenyl, alkynyl, aralkyl, aralkenyl, or aralkynyl, aryl, all of which may further comprise heteroatoms, including a nitrogen atom, an oxygen atom, a sulfur atom, or a halogen atom. However, it is particularly preferred that alternative modifying groups are enzymatically removable from Levovirin ™, and particularly when said enzymes include aminohydrolase such as liver deaminases (e.g. adenosine or cytosine deaminase), liver deamidases (e.g. deamidase), and liver transaminases (glutamate-pyruvate transaminase) ).

Without limiting the inventive concept herein, the modifying groups may inactivate Levovirin ™ or prevent subsequent activation of the once modified Levovirin ™ present in a cell that is not a target cell. On the other hand, nitrogen-containing modification groups can also prevent metabolic activation of the modified Levovirine ™. With respect to the degree of modification of Levovirine ™, it may be noted that the modification may include an organo-synthetic modification, an enzymatic modification, or a de-novo synthesis to produce a modified Levovirine ™.

• φ • φ • φ φ «

ΦΦ ΦΦ φ · φ · ··· ♦ · · φ

φφφ ΦΦ φφφφ φφφ φφφφ ··

With regard to enzymatic removal of the modifying group, it may be noted that, depending on the type of target cell and the modifying group, enzymatic removal may be very significant. Enzymatic removal may include enzymes from a variety of classes, including hydrolases, transferases, lyases, and oxidoreductases, and particularly preferred subclasses are adenosine and cytosine deaminases, arginases, transaminases, and arylamidases. Furthermore, it can be appreciated that said enzymes for enzymatic removal of the modifying group can be exclusively expressed in target cells, however, according to alternative aspects of the present invention, suitable enzymes can also be expressed in cells other than the target cells until the enzyme wherever present, it is expressed in all cells in a cell-containing system. Further, it can be appreciated that said enzymes are preferably naturally expressed (i.e., not recombinant) in optional target cells under normal and / or pathological conditions. For example, glutamate-pyruvate transaminase is known to be constitutively expressed with high selectivity in liver cells and may therefore be a suitable enzyme for removal of a modification group. Alternatively, it is known that cytosine deaminase is expressed in relatively large amounts in colon cancer cells, but not, or only in small amounts, in normal colon cells.

Synthesis of said compounds

In general, all known methods used in the synthesis of Dnucleotides, D-nucleosides and their possible analogs can be used to synthesize the compounds disclosed herein in the L-configuration (for example, by replacing the sugar moiety in the D-configuration with the corresponding sugar moiety in L -configuring). An example of a synthetic scheme for the synthesis of Levovirine ™ (1-β-L-ribofuranosyl-1,2,4-triazole-3-carboxamide) is shown in Figure 2.

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

To a stirred solution of L-ribose (50.0 g, 333.33 mmol) in anhydrous methanol (500 mL) at room temperature was added freshly prepared anhydrous methanolic HCl (40 mL, prepared by bubbling anhydrous HCl gas into methanol at 0 ° C). until the weight increased to 4 g) through a syringe within 15 minutes under an argon atmosphere. After

444 4

4 · 44 · 4 · 4 · 4

4 ··· ·· ·

• 4 • 4 ·

99

4 4

9 9 9

9 9

By adding 9999 methanolic HCl, the reaction mixture is stirred at room temperature for 3 to 4 hours. Add anhydrous pyridine (100 mL) and evaporate to dryness under very low pressure (high vacuum) below 40 ° C. This process was repeated twice with anhydrous pyridine (100 mL). The residue was dissolved in anhydrous pyridine (250 mL) and cooled with an ice bath to 0 ° C under an argon atmosphere. To this cold stirred solution was added acetic anhydride (100 mL) via a dropping funnel over 15 minutes. After the addition of acetic anhydride, the reaction was again subjected to stirring at room temperature with exclusion of moisture for 24 hours. The reaction mixture was then evaporated to dryness. The residue was partitioned between ethyl acetate (400 mL) and water (400 mL) and extracted into EtOAc. The aqueous layer was then extracted again with EtOAc (100 mL). The combined EtOAc extracts were washed with water (400 mL), saturated NaHCO ₃ (2 x 300 mL), water (300 mL), and brine (200 mL). The organic extract was dried over anhydrous Na ₂ SO ₄ , filtered and the filtrate evaporated to dryness. The residue was co-evaporated with anhydrous toluene (2 x 150 mL) under very low pressure (high vacuum). The dry oily residue (92 g, 95%) was used for the next reaction without further characterization.

The syrup (92 g) from the above reaction was dissolved in glacial acetic acid (300 mL) and treated with acetic anhydride (75 mL) at room temperature. This solution was then cooled to 0-5 ° C in an ice bath and under an argon atmosphere. Concentrated H ₂ SO ₄ (21 mL) was then slowly added over 15 minutes. After H ₂ SO ₄ was added, the reaction mixture was further stirred at room temperature for 14 hours, poured into crushed ice (500 g) and stirred until the ice melted. Water (500 mL) was then added and extracted with CHCl ₃ (2 x 300 mL). The chloroform extract was washed with water (3 x 400 mL), saturated NaHCO ₃ (2 x 300 mL), water (200 mL), and brine (200 mL). The washed organic extract was dried over anhydrous MgSO ₄ , filtered and evaporated to dryness to give an oily residue (99 g). This residue was co-evaporated with anhydrous toluene (200 mL) and dissolved in ethyl ether (200 mL) to form colorless crystals upon cooling to 10 ° C for one day. The crystalline solid was filtered, washed with hexane / ether (2: 1, 50 mL) and dried to give 60.5 g of product.

Synthesis of methyl 1- (2,3,5-tri-O-acetyl-O-L-ribofuranosyl) -1,2,4-triazole-3-carboxylate (3) and methyl-1- (2,3,5) -tri-O-acetyl-fAL-ribofuranosyl) -1,2,4-triazole-S-carboxylate (4)

A mixture of methyl 1,2,4-triazole-3-carboxylate (25.4 g, 200 mmol), 1,2,3,5-tetra-O-acetyl-β-L-ribofuranose was placed in an RB flask (500 mL). (63.66 g, 200 mmol) and bis (p-nitrophenyl) phosphate (1g). This flask was then placed in a preheated oil bath at 165-175 ° C under a water aspirator vacuum with stirring for 25 minutes. Removed

Φ · φφφφ φ φ φ φφφ

II φ φ • • • • φ φ φ φ φ oct oct φ φ • • • • • • • φ φ φ oct oct oct oct oct oct oct kyselina ová kyselina kyselina kyselina kyselina kyselina kyselina ová kyselina ová flask. The flask was then removed from the oil bath and cooled. When the temperature reached 60 ° C to 70 ° C, EtOAc (300 mL) and saturated NaHCO ₃ (150 mL) were introduced and extracted into EtOAc. The aqueous layer was then extracted again with EtOAc (200 mL). The combined EtOAc extracts were then washed with saturated Na 2 CO 4 (300 mL), water (300 mL) and brine (200 mL). The organic extract was dried over anhydrous Na ₂ SO ₄ , filtered and the filtrate evaporated to dryness. The residue was dissolved in EtOH (100 mL) and diluted with MeOH (60 mL) then cooled to 0 ° C over 12 hours; colorless crystals are obtained. The resulting solid was filtered, washed with a minimum amount of EtOH (20 mL), and then dried under high vacuum (high vacuum) over solid NaOH to give 60 g (78%). The filtrate was evaporated to dryness and purified on a silica column, eluting with CHC1 ₃ and saturated EtOAc (9: 1) as eluent. Two products are recovered from the filtrate: a fast flowing product of 8.5 g (11%) and a slow flowing product of 5 g (6.5%). The slow-moving product corresponds to the crystalline product. The fast-moving product was identified as (4) and obtained as a foam. The combined yield (3) was 65 g (84%).

Synthesis of 1-β-ribofuranosyl-1,2,4-triazole-3-carboxamide (5)

Methyl 1- (2,3,5-tri-O-acetyl-PL-ribofuranosyl) -1,2,4-triazole-3-carboxylate (62 g, 161 mmol) was placed in a steel pressure vessel, bomb, and treated with freshly prepared methanolic ammonia (350 mL, prepared by bubbling anhydrous HCl gas into anhydrous methanol at 0 ° C until saturated) at 0 ° C. The steel bomb was sealed and stirred at room temperature for 18 hours. The steel bomb was then cooled to 0 ° C, opened and the contents evaporated to dryness. The residue was then treated with anhydrous methanol (100 mL) and evaporated to dryness. The residue is then gently triturated with acetone to give a solid which is filtered and washed with acetone. The solid was then dried overnight at room temperature and dissolved in a mixture of hot EtOH (600 mL) and water (10 mL). The volume of this EtOH solution was then reduced to 150 mL and stirred on a hot plate. This hot solution upon cooling provides colorless crystals which are filtered, further washed with acetone and dried under very low pressure (vacuum). Further concentration of the filtrate yielded additional additive. The total yield is then 35 g (89%).

According to an alternative aspect of the present invention, it is disclosed that the synthesis of Levovirine ™ may also utilize one or more enzymatic conversions. For example, acetylation of L-ribose can be carried out with a suitable acetyltransferase (e.g., EC-Ti-Ti titi-Ti-Ti-Ti-Ti-Ti-Ti-Ti-Ti-Ti-Ti. · Three titi three titi

2.3.1. xx). In another example, formation of the carboxamide group from the corresponding methyl ester can be facilitated by a single or dual enzyme system comprising an esterase (e.g. EC 3.1.1.xx) and / or an aminotransferase (e.g. EC

2.6.1. XX). In another further example, Levovirin ™ may then be enzymatically converted to the corresponding mono-, di-, or triphosphate (e.g., EC 3.1.3.xx or EC3.L4.xx).

Furthermore, it is also contemplated that it is possible to use a variety of catalysts other than bis (β-mtrophenyl) phosphate in amounts other than 1 g. Changing the amount (i.e. molar proportion) of the catalyst may advantageously increase selectivity of the reaction towards higher yield of the desired N ) isomers (L-ribose attached to the Ni atom of the triazole ring) so as to exceed the N ₂ isomer. For example, suitable amounts of bis (p-nitrophenyl) phosphate include between 3 mmol and 30 mmol and more. Alternatively, amounts less than 3 mmol (0.3 mmol to 2.99 mmol) may be included as appropriate. According to other alternative aspects of the present invention, there is no need to limit the catalyst to bis (p-methenylphenyl) phosphate, and alternative catalysts include p-toluenesulfonic acid, trichloroacetic acid, and 77-nitrobenzoic acid.

In view of the reaction temperature, it is particularly contemplated that lower temperatures may further enhance the selectivity of the reaction to a higher yield of the desired Ni isomer so as to exceed the N ₂ isomer. Therefore, it is contemplated herein that suitable temperatures for the condensation reactions between the triazole moiety and the ribose moiety lie in the range of 155 ° C to 165 ° C, more preferably in the range of 145 ° C to 165 ° C, and most preferably in the range between 130 ° C to 165 ° C.

According to other alternative aspects of the present invention, the selectivity of a given reaction towards a higher yield of the desired Ni isomer to exceed the N ₂ isomer can also be favorably influenced by chemical modification of methyl 1,2,4-triazole-3-carboxylate. Chemical modifications include the formation of a complex structure in which the N ₂ atom is included, steric hindrance, and direct chemical modifications of the N ₂ atom. For example, the free electron pair at the N ₂ atom of the triazole moiety and the electron donor in the modified carboxylate group can be used for a complex metal ion compound, thereby reducing the availability of the N ₂ atom for interaction with the ribose moiety. In another case, the carboxylate group in the methyl-1,2,4-triazole-3-carboxylate can be modified by a relatively built-in group, resulting in preferential and steric blocking or reducing reactions at the N ₂ atom. Alternatively, the N ₂ atom can be directly modified with a protecting group, with suitable protecting groups including t-Boc and benzyl.

Still further, it is contemplated that a higher yield of the desired Ni isomer so as to exceed the N ₂ isomer can be achieved using enzymatic synthesis wherein the ribose moiety is * 9999

99

0 9

998 9 9

9 9

9999 99

99

9 9 9

9 9

9 0

9

0 9

9999 (or L-ribonucleotide) and modified or unmodified methyl 1,2,4-triazole-3-carboxylate serve as a substrate for ribosyl transferase (for example EC 2.4.2.5 or EC 2.4.2.6).

Alternatively, Levovirine ™ can be synthesized via the interaction of protected L-ribose to 1,2,4-triazole-3-nitrile, followed by conversion of the nitrile group to the carboxamide, as shown in Figure 3. the interaction of the triazole moiety with the ribose moiety is achieved by a reaction in which (e.g., benzyl protected) ribose has an -NHNH ₂ group attached to the C 6 atom that reacts with the Ni atom of the triazole carboxylate, where the triazole carboxylate is subsequently converted to carboxamidine as shown 4.

In view of the synthesis of prodrug forms of said L-nucleotides, L-nucleosides and their possible analogs, it would be desirable that, in particular, the synthetic scheme be generally dependent on the structure of the compound. However, all synthetic methods that may be considered appropriate and contemplated synthetic schemes include in vitro syntheses, enzymatic syntheses, in vivo syntheses, and any acceptable reasonable combinations thereof. Exemplary synthetic schemes for the formation and formation of said prodrugs are set forth in U.S. Patent Application Number 09/594 410 (supra).

Where said L-nucleotides, L-nucleosides and their possible analogs are phosphorylated, it is reported that all methods of incorporating a phosphorous group into a nucleotide, nucleoside or any analogue thereof are suitable. Conversion of contemplated nucleosides to their corresponding phosphoryl forms can be achieved synthetically (Hughes, BG A et al., (1983); 2 ', 5'-oligoadenylated and related 2', 5'-oligonucleotide analogues.) 1. Substrate specificity of the interferon- induced murine 2 ', 5'-oligoadenylate synthetase and enzymatic synthesis of oligomers. Biochemistry, 22: 2116-2126). However, a variety of methods may also be mentioned further and enzymatic phosphorylation is included (see, for example, Van Rompay, AR, et al., (2000); Phosphorylation of nucleosides and nucleoside analogues by mammalian nucleoside monophosphatekinases; Pharmacol. Ther. 87 (2 to 3) 189-198), and organochemical phosphoiylation in aqueous media (Schwartz, A. and Ponnamperuma, C. (1968); Phosphorylation of adenosine with linear polyphosphate salts in aqueous solution (Nature 218,443).

Use of said compounds

In general, the intended compounds can be used for any treatment or therapy of a system that demonstrates a positive response to the administration of the compounds disclosed herein. However, it is particularly preferred that the compounds disclosed herein are used for

• · 4 4 * * * * * *

4

4 «

4 4 4 * 4 • 44

4 «

4444 antiviral treatment (as a direct antiviral compound and / or as an indirect antiviral compound), for treatment to modulate the immune system, and for treatment related to stimulating cell growth. Furthermore, the administration of the compounds herein for antineoplastic treatment is particularly suitable.

Antiviral treatment

In general, the compounds of the present invention may be used as a direct antiviral drug and / or as an indirect antiviral drug in viral infections. In particular, the method of treating a viral infection in a patient comprises the step of administering to the patient a composition in a dosage effective to inhibit the development of a viral infection (i.e., a method which relates to a host cell in which a single virus acts or multiple viruses such that the host cell produces one or more copies of the virus, the term "producing" refers to nucleotide synthesis, protein processing, and protein folding), such composition comprising at least one of the compounds disclosed herein, and preferably at least one compound of Preferred dosages are in the range of 5 mg / day to 2500 mg / day, and more preferably in the range of 50 mg / day to 500 mg / day. However, alternative dosages, routes, methods, and formulations are also provided, but suitable alternative dosages are described below. The use of the compounds disclosed herein is not limited to individual viruses in individual viral infections, but particularly includes the viral infectious diseases HIV infection and HCV infections and HBV infections, RSV infections, influenza virus infections and para-influenza virus infections.

Peripheral blood mononuclear cells (PBMCs) are often used to study various serious infections, such as Hepatitits C, HIV, Hepatitis B and a variety of Herpes virus. (Antivir. Chem. Chemother. 2000 July; 11 (4): 291-301; Infect. Dis. 1998 Oct; 178 (4): 1189-92; Virology 2000 Mar., 268 (1): 12-60). PMBCs are infected with a given virus, and the cell line is studied for relevant information, what interactions occur between drugs and between PBMCs, and how infected PBMCs behave during the period of time under different environmental conditions. Based on studies. Carried out with infected PBMCs, models can be developed that show how the individual pharmaceutical compositions, environment and / or condition and conditions affect these PBMCs - virus-infected cells.

The inventors have discovered (unpublished results) that Ribavirin shows a positive response to PMBC-HIV infected cells. Surprisingly, Levovirm ™ also shows a similar «» · »·« <* • 9 9

9 80 ·

9

0989 99

Immunomodulatory profile to PMBC-HIV infected cells despite the loss of enzymes in the body required for phosphorylation of Levovirine ™ present in the patient. Based on the above observations, along with other information and tests relating to this, it can be stated that Levovirine, phosphorylated Levovirine and modified Levovirine according to structural formula III can be used for the treatment of HIV and similar related viruses.

Immunomodulation

Figure 5 is a method of immunosuppression of a target organ 500 having a first step 510 that provides a medicament that reduces both the Type 1 and Type 2 responses when administered at a higher concentration than the immunosuppressive concentration, and increases a Type 1 response relative to the Type 2 response when the administered concentration is lower than the immunosuppressive concentration, the drug preferably accumulating in the target organ. In the next step 520, the patient is administered the medicament at a dose that is effective to accumulate the medicament in the target organ to an immunosuppressive concentration. Subsequently, the method of treating a disease characterized herein as a patient's liver inflammation consists of two steps - in a first step, a compound is provided comprising Levovirine, phosphorylated Levovirin ™ and modified phosphorylated Levovirin ™, or modified Ribavirin (supra). In a second, second step, the patient is administered said compound at a dose that (a) causes systemic immunomodulation rather than systematic immunosuppression of the Type 1 and Type 2 responses, and (b) causes systemic immunosuppression of the Type 1 and Type 2 responses in the patient's liver. .

The term "immunosuppression" refers to a case in which the size of T and / or B cell clones of lymphocytes is reduced or their reactivity, expansion or differentiation is suppressed. Thus, immunosuppression may result from the activation of specific or non-specific T suppressor lymphocytes of either T or B clones, or most T or all T or B lymphocytes may be affected by the drug. For example, Cyclosporin A and FK506 are relatively specific to T cells, while alkylating agents such as cyclophosphamide are less specific in their action.

As used herein, the term "cytokine" refers to a group of soluble proteins and peptides that act as humoral regulators at nano- to pico-concentrations and that, either under normal or pathological conditions, modulate the functional activities of individual cells and tissues. Cytokines also attenuate cell-cell interactions directly and regulate placement patterns in the extracellular environment.

«« & * ·· «4 * · ·

Λ 0 0

0 • · 000 0 0 · ·

0 • 0

0004

As used herein, the term "Iymphokines" refers to a subset of cytokines produced by helper T cells, and can generally be considered to fall into two subclasses, namely Type 1a Type 2. Type 1 cells produce interleukin 2 (IL-2) , tumor necrosis factor (TNFα) and interferon gamma (IFNγ), and are primarily responsible for cell-mediated immunity such as delayed-type hypersensitivity and antiviral immunity. In contrast, Type 2 cells produce interleukins, IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13, and are particularly useful in assisting in cases of humoral immune responses such as those manifested in responses to allergens (e.g., IgE and IgG4 isotype shift antibodies).

Thus, the terms Type 1 and Type 2 "responses" are meant to include the entire range of effects resulting from the induction of Type 1 and optionally Type 2 lymphocytes. Among other things, these responses include increased production of the corresponding cytokines, increased proliferation of the corresponding lymphocytes, and other other effects associated with increased cytokine production, including those related to momentum. The Type 1 response is generally characterized by an increase in EL-2, TNFα and IFNγ, while the Type 2 response is generally characterized by an increase in IL-4, IL-5, IL-6 , and IL-10.

As used herein, the term drug "accumulates preferentially" in a target organ refers to a selective target organ mechanism that results in a relative increase in drug uptake or retention in that target organ compared to other other tissues or organs. This mechanism may thus involve active delivery via transporters, receptors, vesicles, etc., but may also be based on physicochemical principles, including the pH dependency of the dosed drug, the differences in solubility of the drug in different ionic strength, chemical or enzymatic modifications in target organs or target cells, and so on.

In a preferred aspect, the medicament is Ribavirin, wherein the medicament is administered to patients with HCV (hepatitis C virus) infection; Ribavirin is orally administered to a patient at a single dose of 600 mg / day for 180 days. A single dose of 600 mg / day is generally lower than the systemic immunosuppressive concentration, but is effective for preferential accumulation in the liver. Thus, the concentration of Ribavirin in the target organ (here: liver) will increase significantly and the immunosuppressive concentration in the liver will be enriched. Ribavirin is known to cause an increase in the Type 1 response compared to the Type 2 response and to reduce the Type 1 and Type 2 responses at relatively high concentrations. Examples are set forth in International Patent Application Number PCT / US98 / 00634, issued January 13, 1998, which is incorporated herein by reference.

According to alternative aspects of the present invention, it is not necessary to limit said drug to Ribavirin only; alternative drugs include the compounds disclosed herein in supra, especially modified and unmodified. Levovirin ™ and phosphorylated Levovirin ™. Other alternative drugs include the compounds disclosed herein when these alternative compounds reduce both the Type 1 response and the Type 2 response at an immunosuppressive concentration and enhance the Type 1 response compared to the Type 2 response at a lower concentration than the immunosuppressive concentration.

Various viral infections other than HCV infections, including arbovirus infections, may also be reported with respect to the patient. Consequently, the liver is not restricted to other organs, such as the brain, lungs, spleen, thymus, kidneys, etc. In general, the disease is not treated according to the subject invention, it is strongly influenced by the specific structure of the drug accumulation scheme (i.e., in which organ the drug accumulates preferentially). For example, both Ribavirin and Levovirin ™ both preferentially accumulate in the liver, and decrease both the Type 1 and Type 2 responses at a higher concentration than the immunosuppressive concentration. Particular mention may be made of those diseases in which it is desirable to suppress the immune response in the liver, such as hepatitis C, autoimmune / lupoid hepatitis, liver transplant recipients, etc.

In particular, the method of the present invention is not intended to provide direct antiviral therapy, but is intended to at least partially suppress an immune response in a target organ that is infected with a virus. The target organ of the immune response is reported to be particularly useful in hepatitis C, where the HCV virus does not directly contribute to the organ damage but rather to an imbalance between the Type 1 response and the Type 2 response that is caused by the infection. Thus, a method of treatment with a drug that specifically reduces both the Type 1 and Type 2 responses in the liver of a HCV-infected patient to prevent hepatitis damage both prolactally and therapeutically may be mentioned. Both Ribavirin and Levovirin ™ are both particularly advantageous because they exhibit excellent human tolerance, long-term prophylaxis, long-term action.

Regarding the administration (method, dosage, schedule, deadline, etc.) of Ribavirin or the alternative listed compounds, the same conditions as described herein can be applied. It can also be further noted that.) Ribavirin or alternative compounds disclosed herein may be used in a general overall health situation as well as in a clinical, therapeutic situation. Consequently, Ribavirin or the alternative compounds disclosed herein can also be used to improve digestion. For example, one or more of said compounds may be administered to individually promote poor digestion - if poor digestion is attributable to liver condition, as is the case with hepatitis B or C infections, or if other situations that are characteristic of liver inflammation occur. In such cases, digestion can be improved by administering to the patient Ribavirin or Ribavirin as the compound below in amounts that are common in systemic immunosuppression but «

the amount accumulated in the liver then reaches a concentration that corresponds to immunosuppression in the patient's liver.

As another example of a non-therapeutic or clinical use of Ribavirin or the alternative compounds mentioned in humans, the use herein is for use in improving skin coloration. In particular, Ribavirin or the alternative compounds listed below may be used to improve skin color in humans in amounts common to systemic immunosuppression, but the amount accumulated in the liver reaches a concentration that corresponds to immunosuppression in the patient's liver.

Ribavirin (1- (5-deoxy-3-D-ribofuranosyl) -1,2,4-triazole-3-carboxamide) or Levovirin ™ (1- (5-deoxy-PL- ribofuranosyl) -1,2,4-triazole-3-carboxamide, or any of the mono-, di-, or triphosphorylated forms. The amount in which it is taken or administered is preferably that sufficient to produce systematic immunomodulation of the Type 1 and Type 2 responses and suppression in the liver of both the Type 1 and Type 2 responses. Particularly preferred amounts are in the range of 300 mg / ml. day to 800 mg / day, although in some individual cases, this range may be 50 mg / day to 100 mg / day, up to as high as the range 2000 mg / day to 2400 mg / day. Effects in relation to other organs are also contemplated, including the brain or other organs known to have significant accumulation of Ribavirin.

Stimulation of neuronal growth

As used herein, the term "stimulating neuronal growth" refers to any method in which cell growth and / or cannon is either initiated from a resistant cell or accelerated growth and / or cell division, wherein the term "neuronal" refers to all cells that are directly or indirectly involved in the promotion of cognitive, sensor, or motor signals. For example, nerve cells can be said to be directly involved in signal propagation, while myelin sheath cells or supporting nerve tissue cells are indirectly included based on their isolation function or structural / metabolic support provided to nerve cells. Similarly, receptors can be considered as nerve cells within the scope of this definition. In contrast, cells that form in the lower and upper layers of the diaper are not considered nerve cells in this context and are not directly or indirectly involved in the propagation of cognitive, sensor, or motor signals.

Surprisingly, the inventors have discovered that Ribavirin is effective for stimulating neuronal growth, and the present inventors can now state here that a variety of phosphorylated analogs are described herein. · · · · · · · · · · · · · · · · · · · · · · · · ·

Ribavirin may also be effective in stimulating this growth. In addition, Levovirin ™ and its phosphorylated analogs may also be effective in a similar manner. In one experiment, Levovirin ™ was found to be effective in stimulating the growth of unipolar nerve cells in vitro at concentrations ranging from 0.5 μΜ to 500 μΜ. Subsequently, Levovirin ™ on the culture medium at a concentration of 5.0 μΜ can be used to stimulate the growth of unipolar nerve cells.

Further, methods of stimulating neuronal growth are not limited to unipolar nerve cells, but a variety of alternative cells can be included in this field, including bipolar and multipolar nerve cells. In addition, according to alternative aspects of methods of stimulating neuronal growth, individual cell types in a population of diverse diverse nerve cells may be targets. For example, the target may be unipolar, bipolar and multipolar cells in complex neuronal structures such as the brain, spinal cord, or eye. Thus, the neurons may be parts of neuronal tissue that comprise at least four of the following cell types: astrocytes, dendrocytes, myelin sheath cells, supporting nerve tissue cells, unipolar nerve cells, bipolar nerve cells, multipolar nerve cells, and receptor cells.

Consequently, said methods are not necessarily limited to stimulating neuronal growth in cell culture. According to other alternative aspects of the invention, it is further contemplated that cells may be stimulated in tissue culture, and in particular, nerve cells may be stimulated in vivo. In vivo stimulation of neuronal growth may be used as a prolactic treatment method or as therapeutic treatment. For example, the methods of the present invention can be used to prevent demyelinating diseases (diseases related to the death of the pulp nerve fibers) or neurovegetative diseases such as Alzheimer's disease or Parkinson's disease, or as a preventive treatment before surgical procedures in a patient. Methods of treatment contemplated herein also include reversing or ameliorating, attenuating asphyxia, traumatic, toxic, infectious, degenerative, metabolic, ischemic, or hypoxic insulins, or conditions of irritation.

Consequently, as shown in Figure 6, the method 600 improves coordination in a patient where, in a first step 610, it is examined whether phosphorylated or non-phosphorylated Ribavirin or Levovirin ™ is effective to stimulate neural cell growth in vivo within a given concentration range. In the next step 620, the patient receives an amount of phosphorylated or non-phosphorylated Ribavirin or Levovirine ™ that is effective to stimulate the growth of at least some of the patient's nerve cells. Particular mention may be made here of improving eye-hand coordination.

In a preferred method of improving patient coordination, it is investigated whether Ribavirin is effective in stimulating neural cell growth in the concentration range of 0.5 μ 0,5 to 500 μΜ in vivo. This is this and Ribavirin is orally administered to the patient for traumatic support in the case of ischiadicus nerve at a dose of 1200 mg / day .

However, conditions other than said traumatic condition in the nerve sciatica, including mechanical and chemical damage to a large number of nerve cells, bacterial and / or virus infections of nerve cells, and degenerative diseases are also contemplated for the patient. In view of the nature of the patient's medical condition, the method of the present invention can stimulate nerve cells to a very wide extent, in particular, that the stimulated cells pass particularly between the patient's brain and between the free muscles, or between the patient's brain and between skin sensors.

Another class of methods disclosed herein includes improving the condition of a tactile sensor or other patient sensitivity or sensitivity sensor. Yet another group of methods disclosed herein includes improved macroscopic and microscopic motor control.

Without wishing to be bound by theory or theory, administration of Ribavirin, Levovirine ™, or any of the mono-, di-, or tri-phosphorylated forms of Ribavirin or Levovirine ™ may affect changes in Type 1 response or Type 2 response in a patient, which may, under concomitant circumstances, lead to a condition where nerve cells are protected or stimulate neuronal growth. Thus, the compounds of the present invention can be administered as part of a treatment for a patient's disease, at a dosage range effective to increase the Type 1 response and reduce the Type 2 response in the patient. With respect to in vivo administration (route, dosages, schedule, deadline, etc.) of Ribavirin or alternative compounds disclosed herein are as described herein below.

Antineoplastic treatment

Further, the compounds of the present invention may be used as antineoplastic agents for the treatment of solid or lymphatic tumors, the neoplasms including various cancerous tumors, connective tissue malignancies, and lymphomas, and in particular include acute myeloid leukemia and chronic myeloid leukemia in sudden turnovers during disease. Further, administration of the compounds disclosed herein for antiviral treatment typically takes place according to the method, dosages, schedules and terms as used with known D-nucleotides, D-nucleosides, and their possible analogs in antineoplastic therapy.

• · · ·

Administration of said compounds

With respect to the administration of the compounds disclosed herein, they may be administered according to any suitable protocol in any suitable pharmaceutical formulation. It is usually preferred that the compounds are administered orally. Alternative aspects of the present invention may be appropriate for various other alternative routes of administration, however, it has been found that particular routes of administration are highly dependent on chemical stability, bioavailability, dosage, formulation, and / or the desired pharmacokinetic / pharmacodynamic properties of these herein. of said compounds. Thus, appropriate administration will depend on local delivery (e.g., in the form of an ointment, spray, cream, etc.), parenteral systemic delivery (e.g., inhalation), and whether it is direct or indirect delivery to the bloodstream (e.g., intravenous or intramuscular injection). , etc.).

Consequently, the formulations of the compounds may vary widely. For example, if the drug or drug exhibits adequate stability when passing through the gastrointestinal system without undesirable chemical or enzymatic modifications, suitable formulations may include syrup, tablets, gel capsules, powder, etc. On the other hand, when passing through of these compounds through the gastrointestinal system to absorb or penetrate the gastrointestinal system into the bloodstream, problems may arise, and suitable formulations are such as injectable solutions or suspensions (e.g., buffered saline pH 7.2 to 7.5) ).

In view of the dosages of the compounds disclosed herein, various suitable dosages may be used, the dosages typically being in the range of 1 mg to several 100 mg, and even higher. For example, if the compounds disclosed herein are excreted or metabolized at a relatively low rate, or if prolonged action is desired, then dosages are typically in the range of 5 mg per day to 200 mg per day. On the other hand, if the bioavailability of these compounds is relatively low, or if metabolic conversion (e.g., defosfolation) is relatively rapid, then dosages are typically in the range of 100 mg per day to 2500 mg per day.

Regarding the dosage of L-nucleosides, and in particular Levovirine ™, it is further noted that Levovirine ™ does not appear to be phosphorylated in vivo, at least not in hepatocytes and eiythrocytes, and since the antiviral effect of Ribavirin does not appear to be dependent on phosphorylation, a person skilled in the art would not expect Levovirm ™ to have a direct antiviral effect. Indeed, it did not show a direct antiviral effect in experiments (but not published). Surprisingly, Levovirin ™ exhibits an antiviral effect at a dose not exceeding 200 mg per day, preferably in the range 10 mg per day to 200 mg per day, more preferably in the range 50 mg per day to 200 mg per day, and even more preferably in the range 50 mg per day to 100 mg per day. Thus, it is experimentally supported that the results obtained in the Levovirin ™ dosage study correspond to five times lower serum concentrations than the equivalent dose of Ribavirin.

Experiments have shown that Ribavirin is removed from serum by appropriate phosphorylation in red blood cells (see, for example, Homma et al .: High Performance Liquid Chromatographic Determination of Ribavirin in Whole Blood to Assume Disposition in Erythrocytes; Antimicrob. Agents Chemother. (1999), 43 (11): 2716-2719). Once phosphorylated Ribavirin can no longer leave the cell. Subsequently, the red blood cells act as a Ribavirin well, and higher doses of Ribavirin are then required to reach a given serum level. Levovirin ™ is not phosphorylated and therefore does not tend to accumulate in red blood cells. As a result, red blood cells do not play the role of levovirin ™ wells, and therefore lower doses of Levovirin ™ serum are sufficient to achieve the desired serum level.

The schedule of administration may vary in different ways, with the schedules herein consisting of single single doses throughout the treatment, multiple single daily doses throughout the treatment, multiple daily doses, and permanent dosing (e.g., permanent infusion, implanted osmotic pump, etc.) for at least some of the time throughout the treatment. It is usually preferred to maintain constant delivery of the compounds according to appropriate schedules, but intermittent delivery is also appropriate (i.e., at least once the first dose is administered, followed at least once by multiple doses that are lower than the first dose). Regarding the term (i.e., duration) of treatment, it may be noted that the appropriate duration may vary between individual administrations, for several days, several weeks, several years and even longer. For example, when used herein in cell culture, single or relatively short-term administration may be appropriate. On the other hand, when administered herein for the treatment of acute hepatitis, the duration of treatment may be in the range of several days to several weeks. Similarly, when chronic hepatitis is treated by administering the compounds herein, it may be desirable to prolong the administration to one year or to several years.

According to still other alternative aspects of the present invention, the compounds disclosed herein may be combined with additional pharmaceutically active auxiliaries to assist in the treatment of a variety of diseases, and particularly in viral infections. In addition, these additional pharmaceutically active auxiliaries may also be present

9 · 9 ·

9 9 9

9 9 9

9 9 9 9 · · · ·· ·

9

The compositions may be administered separately or together, and if administered separately, then such administration may take place simultaneously or separately in any specified order. Such additional excipients include, in particular, antiviral agents and immunomodulatory components. For example, antiviral agents include protease inhibitors, nucleotide and / or nucleoside analogues (and particularly Ribavirin), and immunomodulatory components include, for example, cytokines (e.g., interferon α and γ, IL2, IL4, IL6, IL8, IL10 and IL12).

Other additional pharmaceutically active excipients mentioned herein include, for example

Til Λ η TX Λ antifungal agents such as tolnaftate, Fungizone, Lotrimin, Mycelex, Nystatin and Amphoteracin; antiparasitic agents such as Mintezol ™, Niclocide ™, Vermox ™, and Flagyl ™; gut-related agents such as Immodium, Lomotil, and Phazyme; anti-tumor τη Λ * τη δ τ / Γ τη λ agents such as interferon α and γ, Adriamycin, Cytoxan, Imuran, Methotrexate, Mithracin ™, Tiazofurin ™, Taxol ™; dermatological agents such as Aclovate ™, Cyclocort ™, Denorex ™, Florone ™, Oxsoralen ™, tar and salicylic acid; migraine preparations such as ergotamine compounds; steroids and immunosuppressants not previously listed herein, including cyclosporins, Diprosone ™, hydrocortisone; Floron ™, Lidex ™, Topicort ™, and Valisone ™; and metabolic agents such as insulin, and other medicines that do not fall into the above categories.

Preferred combinations of compounds disclosed herein with interferon

According to a particularly preferred aspect of the present invention, there may be mentioned a synergistic combination of Levovirine ™, and at least one interferon, preferably IFN-α-2b. Not only is it typical of Levovirin ™ that it acts to a significantly smaller extent than ribavirin, phosphorylated in etrythrocytes, while still showing antiviral and immunomodulatory activity. Consequently, the pharmacological action of interferon, and especially in the treatment of hepatitis, can be enhanced by concomitant administration of Levovirine ™ at significantly lower doses compared to Ribavirin. For example, effective synergistic doses of Levovirine ™ in combination with the interferon needed to treat HCV infections are proposed in the range of 1 mg to 600 mg, more preferably in the range of 10 mg to 400 mg, even more preferably in the range of 50 mg to 300 mg and most preferably between 100 mg and 300 mg. Equivalent synergistic doses of Ribavirin are reported in the range of 600 mg to 800 mg.

According to another aspect of the present invention, the synergistic combination of Levovirin ™ and interferon results in reduced toxicity when compared to the combination of Ribavirin and interferon at equivalent effective doses, in particular φφ φφφφ především především především «φφ předevšímφφφφ φφφφ due to the lack of significant phosphorylation in erythrocytes. In yet another aspect, the synergistic combination of Levovirine ™ and interferon specifically allows it to be targeted by the liver due to the lack of phosphorylation of Levovirine ™ in parts other than the liver, particularly with regard to erythrocytes.

With respect to the concomitant administration of Levovirin ™ and interferon, all suitable methods and protocols are acceptable, and it is particularly preferred that Levovirin ™ and interferon are administered according to a protocol similar to the known protocols for Ribavirin and interferon. For example, Levovirin ™ may be administered orally, while Interferon may be administered by subcutaneous injection. In general, schedules and methods that are independent of one another and independent of whether and for how long both active substances in the bloodstream are at measurable concentrations at the same time can be used for the concomitant administration of Levovirine ™ and interferon. Furthermore, it is also possible to design effective doses of Levovirine ™ according to effective doses of Ribavirin in the liver when Ribavirin was administered.

Levovirin ™ is particularly mentioned, but chemical modifications thereof, including prodrug forms such as modified Levovirin ™ (Ι-β-L-ribofuranosyl-1,2,4-triazole-3-carboxamide), mono-, di-, and triphosphorylated Levovirin ™, and stereochemical variants (e.g., enantiomers, isomers, etc.). Examples of such suitable chemical modifications and prodrug forms are described in U.S. Pat. S. Patent Application Number 09/594410, (supra). In addition, other drugs other than Levovirin ™ and its variants are also suitable drugs, and in particular alternative drugs, including specific prodrugs for the liver and an amine or amide moiety, may be mentioned. it may be enzymatically deaminated / deamidated in the liver.

With respect to interferon, it is noted that co-administration with Levovirine ™ is not limited to IFN-α-2b, but natural and synthetic fragments, isoforms, and consensus forms of inreferon-alpha may also be included in such co-administration. In addition, interferons other than interferon-alpha are also suitable, including interferon-beta and its natural and synthetic fragments, isoforms and consensus forms. In particular interferon has been reported so far, but cytokines other than interferon and chemokines, including 11-2, IL-12, and TFN may also be suitable. In particular, the pegylated forms of the interferons disclosed herein (i.e., said polyethylene glycol-associated interferons) are also suitable for use in conjunction with the teachings herein.

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

Combination of said compounds with a second viral protein or cytokine binding compound

When using combinations of the compounds disclosed herein with other pharmacologically active agents in antiviral therapy, it may be particularly noted that such combinations may comprise the compounds disclosed herein with direct and indirect antiviral activity, and a second compound which increases the overall antiviral effect consists of a direct antiviral effect and an indirect antiviral effect), wherein the second compound specifically binds a viral protein or cytokine.

As regards the compounds disclosed herein in this combination, nucleoside analogues are preferred in this respect, and even more preferably, the nucleoside analogue is Ribavirin (1- (5-deoxy-PD-furanosyl) -1,2,4-triazole) -3-carboxamide). Ribavirin is known to have a direct antiviral effect by inhibiting RNA and DNA of viral replication [Huffman et al., Antimicrob. Agents Chemother. (1973) 3: 235; Sidwell et al., Science (1972), 177: 705] and an indirect antiviral effect that suppresses the Type 2 response by T cell mediated response and supports the Type 1 response by T cell mediated activity as described in US Patent Application Number 09 / 156,646, which is incorporated herein by reference. However, various compounds other than Ribavirin, and in particular L-nucleoside analogues, as well as such alternative compounds having direct and indirect antiviral activity may also be mentioned. For example, if a particularly high concentration of L-nucleoside analogs is desired, Levovirin ™ may be used

Further, depending on the chemical nature of the first compound, the first compound may have a significantly greater direct antiviral effect or a significantly greater indirect antiviral effect. Said direct antiviral effects include inhibition of viral replication, such as reverse transcriptase inhibition, said direct antiviral effects comprising shifting the Type 1 / Type 2 equilibrium towards the Type 1 or Type 2 response as described in US Patent Application Number 09 / It should also be noted that the indirect antiviral effect may include suppression of Type 1 and Type 2 responses, as described in detail in US Patent Provisional Application Number 60/172 097 (supra). Advancing the Type 1 / Type 2 equilibrium toward the Type 1 or Type 2 response or suppressing the Type 1 / Type 2 response can be advantageously controlled or controlled by the first compound, wherein the dosage of the first compound is determined by shifting or suppressing the Type 1 or Type 2 response.

With respect to the second compound, it is preferred that the second compound comprises an antibody (e.g., a monoclonal or polyclonal antibody). However, it would be * 11 11 11 11 111 111 11

2O · * ·· 19 1 9 19

No. 9191 11 11 9 99 9999, it should be noted that, in view of alternative aspects of the present invention, it is not necessary to limit this antibody to naturally occurring forms of antibodies, but also to include synthetic forms of antibodies (e.g. mini antibodies obtained by one of the molecular developmental technology), or antibody fragments. Antibody fragments are particularly desirable where such fragments are produced by recombinant cells or where the molecular weight of the second compound could be relatively low (i.e., less than 75 kDa). Said antibody fragments include Fab, any F (ab) _2, and any scFabs. In addition, suitable antibodies may be modified by introducing a variety of additional properties, including, for example, 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 in vivo using imaging devices (e.g., magnetic resonance imaging). Said 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 (for example, see Current protocols in Immunology; John Wiley & Sons (1999); Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach , Warren Strober), and it is further possible to use all known methods for their production for use in accordance with the teachings herein. Antibodies are typically administered by injection (e.g., intravenous injections), and actual doses range from 0.01 mg to several 10 mg, but lower doses may also be considered if necessary.

Furthermore, the binding of the second compound to the viral protein or cytokine is particularly advantageous when the binding results in the inactivation of the viral protein and / or the cytokine, and it can also be said that this inactivation can take place by a variety of mechanisms. For example, virus inactivation can be achieved by antibody-mediated precipitation (i.e., the formation of a molecular network between antibodies and viruses). Alternatively, the binding of the second compound may inactivate the virus by blocking or other obstructive proteins or other viral surface structures that are essential for virus ineffectiveness or propagation. Further, the second compound may still bind to non-structural viral proteins, including viral polymerases and proteases. Said binding targets (i.e., haptens) include, for example, proteins such as HIV virus gp 120/41, but also proteins such as HIV virus reverse transcriptase. Other viral proteins also include proteins from HIV virus, hepatitis virus, influenza virus, and RSV. Regarding cytokines, it can be stated that inactivation can be achieved by sequencing the cytokine from the accumulation of cytokines. For example, if the hapten for said second compound is a Type 1 cytokine, the cytokines contemplated include interleukin-2, interferon-gamma, and tumor necrosis factor-beta, whereas the hapten for the second compound is a Type 2 cytokine, cytokines include interleukin-4, interleukin-5 and interleukin-10.

Ftft ftft ftft ftft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft

In general, inactivation of a virus or cytokine by a second compound may have a large number of desired effects, which effects may or may not be demonstrated as another additional or synergistic effect in combination with the first compound. For example, in cases where the first compound has a direct antiviral effect (resulting in a significant reduction in viral titer), the second compound may even further reduce this viral titer by precipitation of the remaining viruses. Alternatively, the second compound may then reduce the number of infectious viral particles by binding to the viral components that are essential for infectivity. Further, where the second compound binds a cytokine, the second compound shifts the Type 1 / Type 2 balance towards the Type 1 response by sequestering one or more Type 2 cytokines from the pooled cytokines, thereby helping to restoration of cellular immunity, thereby significantly reducing viral load. Alternatively, if the first compound has an indirect antiviral effect (it also results in a significant reduction in viral titer), the second compound may further reduce the viral titer by precipitation of the remaining viruses. Thus, alternatively, one or more Type 1 and / or Type 2 cytokines may be sequestered with a second compound or a mixture of second compounds, thereby fine-tuning (ie modulating) the Type 1 and / or Type 2 response induced by the first compound.

It could also be noted that the combination of a first compound having a direct and indirect antiviral effect with a second compound that specifically binds the virus and / or cytokine will reduce the viral titer not only mechanically (i.e., enzyme inhibition), but also systemically (ie, stimulation / modulation of immunity) by action. In particular, antiviral medicaments comprising a first and a second compound having a synergistic effect are preferred, which have the advantage of helping to reduce the effective dose of the first and second compound. Accordingly, suitable antiviral medicaments may also be used in prophylactic treatment.

Combination of the above compounds with Ribavirin

In particular, when co-administered with Levovirin ™ with Ribavirin, adverse side effects are reduced and the tolerability of Ribavirin and / or Levovirin ™ is improved. Regarding the ratio between Ribavirin and Levovirin ™ when co-administered, it is preferred that Levovirin ™ is present in at least an equimolar amount of Ribavirin. However, if desired, a variety of alternative ratios may be used, and in particular this ratio depends primarily on the desired effect and dosage / mode of administration. For example, 9 4 9 9 9 9 9 9 9 9 9 9 In the case of a haemolytic anemia, Levovirin ™ may be present when co-administered in a range of 51 mole% to 80 mole% or more. On the other hand, however, if the tolerability of Levovirine is limited, then Levovirine may be present when administered only in the range of 49 mole% to 20 mole% or less.

Furthermore, it is not necessary to use the same route of administration when Ribavirin and Levovirin ™ are co-administered. As used herein, the term "co-administration" refers to any form of administration of Ribavirin and Levovirin ™ where Ribavirin and Levovirin ™ are present at a measurable concentration in a given system and at a given time. Thus, co-administration includes a protocol according to which Ribavirin is administered in one way and Levovirin ™ is administered in another way, wherein the simultaneous administration can be performed simultaneously or at two different time points. For example, Ribavirin may be administered orally, while Levovirin ™ may be administered by intravenous injection. Alternatively, Ribavirin may be administered orally by BED, and Levovirin ™ may be administered orally by QID.

In particular, various variations of the Ribavirin and Levovirin ™ molar fractions in the co-administration protocol, particularly with respect to any desired biological effects, can be tailored to the specific needs of the patient, including modulation of Type 1 / Type 2 cytokine balance, direct antiviral effect, reduction of hematotoxic properties, etc.

According to another aspect of the present invention, it is further contemplated that the co-administration of Ribavirin and Levovirin ™ may include continuous release and / or dose reduction at intervals to make these intervals more frequent. In particular, continuous release and / or dose reduction at more frequent intervals may reduce unwanted side effects and may help to increase the direct and / or indirect antiviral effect. In general, the compounds of the present invention may be administered by any system, and it is preferred that the compounds are administered to mammals, preferably humans, or to cells or tissue cultures.

Metabolites of said compounds

Generally, Levovirin ™ is metabolically inert when administered into the system, however, the inventors also state that Levovirin ™ may have metabolites as shown in the following structural formulas IV-VIII:

• 0 «0 · · ·

· · · * * 4 4 4 4 9 4 9 9 4 4 4

3 «0 * 0

0 ··· 4

0 *

0 «0040

Structural formula IV represents triazolecarboxamide, structural formula V depicts triazolecarboxylic acid, structural formula VI depicts L-ribofuranosyltriazolecarboxamide, structural formula VII depicts 5'-acetyl-L-ribofuranosyltriazolecarboxamide, and structural formula VHI depicts 5'-acetyl-α-L-ribofuranamide.

Although generally speaking, the metabolic products of Levovirine ™ are formed as products of an enzymatic reaction, it has been found, however, that under certain suitable intracellular or extracellular conditions in the cellular system, metabolites may be formed without an enzymatic reaction. Formation of metabolites from Levovirin ™ may include redox reactions (particularly oxidation), enzymatically catalyzed reactions (e.g., hydrolysis), and photochemical reactions.

• · • · ··

However, the reaction products disclosed herein are typically degradation products of Levovirine ™, however, it has been found that these metabolites include products resulting from the addition of chemical groups (e.g., glycosylation or acetylation), and that the modified compounds may undergo subsequent degradation in the same or different compartments. parts. While it is generally believed that these metabolites have a significantly reduced pharmacological effect compared to Levovirine ™, it can be appreciated that these metabolites have a pharmacological effect similar to that of Levovirine ™. For example, a triazole moiety or a ribose moiety may serve as an effector (e.g., an allosteric inhibitor).

Further, a single specific dose of Levovirine ™ administered to the system is in the range of 20% to 50%, preferably 51% to 75%, more preferably 76% to 99%, and most preferably 100% excreted in an unmetabolized form.

The present invention is further illustrated by the following non-limiting examples, which are not intended to limit the scope and scope of the invention in any way.

DETAILED DESCRIPTION OF THE INVENTION

Targeted hepatitis immunosuppression using Ribavirin

Three placebo-control studies are conducted with Ribavirin in the treatment of chronic hepatitis C. These studies included 134 patients treated with Ribavirin and 97 patients receiving placebo. There are also two non-control Phase II studies that include 23 patients treated with Ribavirin. The primary response parameter is normalized or reduced by serum ALT. Response is also assessed in terms of eliminating or reducing serum levels of HCV RNA, and improving liver histology as assessed by changes in Knodell scoring.

In all control and non-control studies, using the response definitions specified in the protocols and the analysis plan, Ribavirin was statistically significantly superior to placebo at normalized and reducing ALT levels during treatment. Integrated analyzes based on all patients in control studies using a uniform response definition, including normalization of ALT at end of treatment or clinically significant level of partial response, showed that 46% of Ribavirin patients responded to treatment, whereas in placebo patients was 4% (p <0.001). Patients generally responded after two to three months of treatment and the response lasted as long as treatment continued. Thus, no loss of ALT response was found with improvement during treatment. Subsequent withdrawal of Ribavirin at the end of the active phase of treatment responded to 11.5% of patients and had a sustained response throughout the next follow-up period.

Regarding improvement in liver histology, no significant trend in favor of Ribavirin was observed in each of the control studies in terms of changes in Knodell scoring, and in terms of scoring in the individual components. Analysis of the data combination by covariance analysis using Knodell's baseline scoring as a covariant yielded a result that obtained statistically significant differences for Ribavirin for overall scoring and for scoring each of the components. Thus, in control studies, compared to all patients treated with Ribavirin and placebo, Ribavirin had a modest but real effect in improving liver histology. In the Ribavirin group, compared to ALT respondents and non-responders, it has been shown that experience with significantly greater improvement in liver histology compared to ALT respondents was confirmed in ALT respondents. The mean value in Knodell scoring was two points for ALT respondents compared to one point for all Ribavirin-treated patients. A decrease in the overall Knodell scoring of two points is generally considered from a hepatological point of view to be clinically significant. Thus, there is a statistically significant positive correlation between ALT response and clinically significant degree of improvement in liver histology.

In all studies, the primary endpoint is defined as a decrease in ALT levels. In all studies, complete overall ALT response is defined as normalization of the ALT level at the end of treatment. A partial ALT response is defined as a 50% or greater reduction at the end of patient treatment by baseline, or a 50% or greater reduction to no more than 1.5 times the upper limit of normal.

In studies 92-001 and 91-178, treatment groups were compared for the effect of study medication or hepatitis-relevant symptom. This is not done in study CT00 / 002, as this case takes the form of a report where systematic collection of symptomatic data is not used.

·························

In study 92-001, there is a statistically significant difference in favor of Ribavirin due to decreased fatigue, reduced exhaustion. At the end of treatment and at the end of the study period, patients with a higher proportion of Ribavirin showed some improvement compared to baseline in patient fatigue compared to placebo patients (p = 0.04 at the end of treatment and p = 0.006 at the end of treatment). period). In this study, there were no significant differences between the treatment groups in terms of any of the symptoms.

In study 91-DK-178, significant differences between treatment groups were isolated in individual symptoms at each visit, some in favor of the placebo group and some in favor of the Ribavirin group, but there were no overall trends in favor of one of the patient groups.

The following Table 1 shows the response rates related to the improvement of liver histology. There were no statistically significant differences between treatment groups according to the changes in Knodell scoring in any of these studies, although a numerical trend can be observed, in favor of Ribavirin. In study CT00 / 002, there is a difference in favor of Ribavirin in one of the secondary parameters (lymphoid clusters, p = 0.05).

· Ef ef f • f f e ef f f f fe fe fe fe

Table 1

Comparison of results in control studies - Histology of the liver

Protocol Response Definition Analysis Scheduled in the defined response log Result 92-001 Comparison of treatment groups Same None significant the same due to changes difference between pre- to post-treatment according to treated from Knodell's scoring groups for each patient 91-DK-178 Long-term response: improvement of liver histopathology by “blinded all” classification Comparison of the treatment groups with respect to the changes of pre- to post-treatment No significant difference between treated patients liver biopsy in terms of grade treatment according to groups current hepatitis Knodell scoring damage using Wilcox series summation test ” for each patient CT00 / 002 Improving anti-inflammatory levels Comparison of treated patients None significant activities in cases groups due to difference between pre- to post-treatment as assessment according to pre- to post-treatment changes according to treated groups Knodell scoring Knodell scoring for each patient Pre- to post-treatment, Reduction changes in other histolo- which may be relevant to hepatitis C of lymphoid clusters in Ribavirin

groups ·· · · · · ··········

For the various studies further analyzes were performed. However, to make the results of these analyzes more meaningful and more significant, the control data were combined to obtain a larger sample size and range.

Results of integrated analyzes

Analysis of Ribavirin Response by Relevant Efficacy Criteria - ALT Response During Ribavirin Therapy

For integrated efficacy analyzes, the following ALT response definitions shall apply:

Complete Response: At the end of treatment, return to normal within the range.

Partial Response: A 50% or greater reduction in patient's baseline at 1.5 times the upper limit of normal at the end of treatment.

Respondent: Meets the definition of either full or partial response. The definition of 'respondent' is intended as a time-dependent ALT value diagram for a group of patients according to the different type of response definition used in the study. (Data is presented as a graph dependence, where data is plotted as points that fit a third degree equation with a curve to obtain a smooth curve, Reinsch 1967). Three response definitions apply:

a. Full Response = ALT in the normal state within the range, at the end of treatment.

b. Partial Response (A) = 50% or greater reduction in patient's baseline at 1.5 times the upper limit of normal at the end of treatment.

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

All other patients are considered non-respondents. Graphic dependencies are also constructed for non-responders treated with Ribavirin for each study and for placebo patients (respondents and non-responders) for each study.

Three "full response" curves show that the response was achieved after one third of the treatment period and that it has continued since that time. Three curves for "partial response (A)" show a similar response display. The three "partial response (B)" curves show a markedly greater variability in ALT levels over the treatment period. Graphical dependencies for

• to · to · to · to · to · to · to · to · ·

Ribavirin-treated non-respondents and graphical dependence for placebo patients, as expected, show a variance in the data that does not exhibit such changes that would have any particular detectable character or profile during treatment and during subsequent periods of time. Accordingly, it can be concluded that the definition of 'partial response (B)' is not appropriate for the purposes of integrated efficacy analysis. Given the consistent nature of the graph dependence of the definition for 'full response' and 'partial response (A)' and the fact that these definitions are clinically meaningful, it was decided that for the purposes of an integrated efficacy analysis, 'response' would be defined either as "full response" or as "partial response (A)".

The following Table 2 shows the results for each of the studies and for the combined database using the ALT response as defined above. The differences between the respondents in the two groups of treated patients are compared using either the Chi-square test (χ-square method) or the Fischer exact test.

Table 2

Percent ALT Response Rate

Studies Ribavirin n / N% Placebo n / N% Value p 92-001 15/28 (53.6) 1/30 (3.3) <0.001 91-DK-178 11/29 (37.9) 1/29 (3.4) <0.001 CT00-002 32/70 (45.7) 2/36 (5.6) <0.001 Combined database 15/28 (53.6) 1/30 (3.3) <0.001

n = number of patients with ALT response (integrated definition).

N = all treated patients (applies to those treated according to the context herein) minus those patients whose examination was not considered valid.

· 4 4

4 4 · · · · · ·

4 4 «

4444 4 ·

44 «1 2 3 4 5«

4 4

4 * 444 4

To identify the ALT response corresponding to the end of treatment, this last valid observation returns to a maximum of two visits that successfully continue for those patients who lack such a correct value. This procedure is used in two of the three control studies (91-DK-178 and CT00 / 002). Nine patients in the Ribavirin group and two patients in the placebo group had no valid observation. ALT response rates were consistent throughout the three control studies and ranged from 37.9% to 53.6%. After combining the obtained data, the ALT response was 45.7%. In all cases, the ALT response rates in the Ribavirin-treated patients were significantly superior to those in the placebo-treated patients. In the placebo group, ALT response rates were consistently low, ranging from 3.3% to 5.6%, with a value of 4.2% when the data were combined.

ALT response in the reporting period

Table 3 summarizes steady-state response rates in studies 92-001 and 91-DK-178 and in the two combined studies. Individual studio, analytical, in accordance with the projected intended diagram of the proposed ALT definition of a sustained steady-state response is used. A responder with a sustained steady-state response is basically a patient with either a normalized ALT response or a partial response at the end of treatment that meets both of the criteria listed herein for a given reporting period. The same analysis cannot be provided for study CT00 / 002, since only a few patients can obtain complete ATL data for the entire course of the study period.

Table 3

The percentage rate of ALT lasting steady-state response - according to the logged definitions

Studies 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)

φ · • ··· • · ♦

• 944 9 • 4 • 4

. 9 4 • 4 9

4449 η = number of ALT patients with sustained steady-state response.

N = all patients with full or partial response at the end of treatment (study, analytical, in accordance with the projected intended diagram of the proposed definition).

According to the analyzes in the two studies, steady-state response rates were 6.7% and 18.2% for Ribavirin-treated patients, compared to 0% for placebo-treated patients. Due to the small sample size and the very low number of placebo respondents, no statistical analysis was used.

Improving Knodell scoring

For each of the three control studies, there is a clear numerical trend in favor of Ribavirin, with changes in overall Knodel scoring and many scoring components. The same trend is evident when data from the three studies are combined. This trend not only applies to scoring improvement but also to deterioration, which is indicated even if no improvement is observed in any of the two treatment groups, with deterioration observed in a few patients in the Ribavirin group. This is a very important observation, since one of the goals in treatment is to prevent the worsening of the chronic and progressing, developing condition. CMC analysis of the combined data using the χ-square test (Chi-square test) shows no statistically significant differences. Analysis of the combined data by analysis of variance (as used in studies 92-001 and 91-DK-178) showed a statistically significant difference in favor of Ribavirin for overall Knodell scoring, but not for scoring any of the individual components.

The liver histology data was further examined by covariance analysis using a Knodell scoring baseline as a covariant. Baseline regression analysis of Knodell scoring versus end treatment scoring for all Ribavirin and placebo treated patients showed that the slope, the slope, is less than 1.0 but greater than zero. This indicates that the baseline of Knodell scoring influences the presumption (expectation) of the outcome of the treatment, regardless of any differences between Ribavirin and placebo. Where the slope is noticeably different from 1.0, there is a more appropriate test than variance analysis (Fisher 1951). The result of the covariance analysis is shown in Table 4. Mean scoring changes for Ribavirin treated • 4

444 444 44 44 444 44

4 «4

4 4

4 • 44

The 4444 patients are only small, but due to the small variance, the differences from placebo are statistically significant. It is interesting to note that no improvement was apparent only in Knodell's sub-scoring concerning fibrosis and that there was less deterioration in the Ribavirin group compared to the placebo group.

Table 4

Comparison of Ribavirin and placebo according to changes in Knodell scoring from baseline to end of treatment - covariance analysis using Knodell scoring as covariant, taking into account all phases of the three combined studies

Mean change in scoring from baseline to end of treatment Knodell Folder Ribavirin N = 107 Placebo N = 78 Value p Periportal activity and necrosis -0.40 -0.01 0,000 4 Portal inflammation -0.30 -0.10 0.020 6 Lobular necrosis -0.33 -0.13 0,001 9 Fibrosis + 0,07 + 0.25 0,007 1 Overall scoring -1.11 -0.05 0,009 1

Investigation of correlation between ALT response and improvement of Knodell scoring

For each of the three control studies, Ribavirin is significantly more effective than placebo at normalized and decreased ALT levels (elevated serum ALT levels is a biochemical indicator of hepatitis inflammation). There is also a correlation between response to Ribavirin therapy in terms of normalization or reduction of ALT levels among patients, and with improvement in liver histology as determined by Knodell scoring. We have found that there is a truly consistent trend towards a positive relationship between ALT response and improvement in Knodell scoring and that both of these parameters are considered a decisive method, a method that is decisive for judgment.

• 4 44

4 »«

4 4 • 4 4444 4 4444 4 4 4 4 4 4 4 4 4 4 4

In the combined database, this trend is statistically significant for Knodell scoring (p = 0.008), fibrosis (p = 0.014) and portal inflammation (p = 0.022) using the Cochran-Mantel-Haenszel (CMH) χ-squared method (Chi-square test) ). (Table 5,6,7).

Table 5

Response status by Knodell rating - overall by ALT response

Respondent Nerespondent Protocol Response n % n ’% P-value ' 92-001 Improved 7 46.7 4 33.3 0,099 Unchanged 7 46.7 3 25.0 Worse 1 6.7 5 41.7 91-DK-178 Improved 10 90.9 7 38.9 0,021 * Unchanged 1 9.1 5 27.8 Worse 0 0.00 6 33.3 CT00-002 Improved 11 42.3 12 48.0 0.171 Unchanged 11 42.3 5 20.0 Worse 4 15.4 8 32.0 Integrated Improved 28 53.8 23 41.8 0.008 * Unchanged 19 Dec 36.5 13 23.6 Worse 5 9.6 19 Dec 34.5

N = number of patients in the study.

n = number of patients responding in category% = (n / N) * 100 ^] CMH statistics * P <0.05 00 0 «0« 0 0 4 4 »4 4

000 »» 0

0 · 0 • 00 0 0 0440

Table 6

Response status according to Knodell evaluation - fibrosis according to ALT response

Respondent Nerespondent Protocol Response n % n % P-value ' 92-001 Improved 2 13.3 1 0.00 0.434 Unchanged 12 80.0 11 91.7 Worse 1 6.7 1 8.3 91-DK-178 Improved 5 45.5 2 11.1 0.191 Unchanged 4 36.4 11 61.1 Worse 2 18.2 5 27.8 CT00-002 Improved 6 23.1 1 4.0 0.120 Unchanged 18 69.2 20 May 80.0 Worse 2 7.7 4 16.0 Integrated Improved 13 25.0 3 5.5 0.014 * Unchanged 34 65.4 42 76.4 Worse 5 9.6 10 18.2

N = number of patients in the study.

n = number of patients responding in the category% = (n / N) * 100. ^J CMH statistics.

* P <0.05.

· To to to to to to • • • • • • • • • • • • • • • • • • • • • • • • •

Table 7

Response status according to Knodell's assessment - portal inflammation based on ALT response

Respondent Nerespondent Protocol Response n % n % P-value ' 92-001 Improved 5 33.3 3 25.0 0.220 Unchanged 9 60.0 5 41.7 Worse 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 Worse 0 0.00 3 16.7 CT00-002 Improved 5 19.2 6 24.0 0.159 Unchanged 21 80.8 16 64.0 Worse 0 0.00 3 12.0 Integrated Improved 15 Dec 28.8 13 23.6 0,022 * Unchanged 36 69.2 32 58.2 Worse 1 1.9 10 18.2

N = number of patients in the study.

n = number of patients responding in% = (n / N) * 100 category. 'CMH statistics.

* P <0.05.

·· ·· · · · · · · · · · ·

The association between ALT response and improvement in liver histology is further studied in order to quantify the improvement in liver histology in Ribavirin-responsive patients and, therefore, to determine whether there is a subgroup of patients that can derive substantial clinical benefit from Ribavirin treatment. For each of the three observed groups, ALT responses in Ribavirin-treated respondents and non-responders are compared for the mean change in total Knodell scoring throughout the treatment period. This analysis, as shown in Table 8 below, thus quantifies direct changes, based on the data presented in Table 5.

It is evident that in each of the studies there is a correlation between ALT response and improvement in liver histology, with the ALT response being associated with a greater improvement in liver histology compared to the ALT response of non-responders. This effect is particularly surprising in study 91-DK-178, where the mean reduction in Knodell scoring is 4.7 for ALT respondents compared to 0.17 for ALT non-responders. A regression model is used to investigate the association between ALT response and changes in Kondell scoring. This showed that all three ALT response studies showed a significant predictor due to the improvement in liver histology. Thus, ALT normalization or reduction can be correlated with an improvement in liver histology in patients with hepatitis C who are treated with Ribavirin, and in patients who have achieved an ALT response and in a similar manner, a substantial clinical benefit can be derived. Patients treated with Ribavirin who did not achieve an ALT response showed no clinical benefit compared to patients receiving placebo.

·· ·· · · · · · · ·

Table 8

Studies CT00-002 92-001 91-DK-178 Combined database Duration of treatment 24 36 48 (week) Mean value of change -1.23 -1.47 -4.09 -1.90 by Knodell (range) for ALT (-7.4) (-8.2) (-9.0) (-9.4) respondents * n = 26 n = 15 n = 11 n = 52 Mean value of change -0.96 + 0.47 -0.17 -0.36 by Knodell (range) for ALT (-9.4) (-3.6) (-6.7) (-9.7) non-correspondents * n = 25 n = 12 n = 18 n = 55 P-value ** 0.004 0.000 1 0,002 0.000 1 Mean value of change -1.10 -0.56 -1.65 -1.11 by Knodell (range) for all Ribavirin. (-9.4) (-8.6) (-9.7) (-9.7) patients n = 51 n = 27 n = 29 n = 107 Mean value of change -0.09 + 0.44 -0.52 -0.51 by Knodell (range) for 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 a reduction of 50% or more relative to baseline up to 1.5 times above the normal end-of-treatment level.

** Regression analysis.

In summary, the Phase III program for Ribavirin in chronic hepatitis C consists of three randomized, two blind, placebo-controlled, parallel group studies. A total of 134 patients who received Ribavirin and 97 patients who received placebo were randomly selected. The response to said treatment is determined using the following parameters:

1. Reduction or normalization of ALT level (ALT elevation is a biochemical indicator of hepatitis).

2. Improvement of liver histology as registered by reduced Knodell scoring (Knodell scoring system quantifies the degree of liver damage by assigning points to different relevant microscopic characteristics and then summing up these subset of points to obtain a total score.

3. Elimination of hepatitis C virus from blood, or reduction of virus present.

Of the 134 patients in the phase III studies, 101 were complete with data on ALT level, Knodell scoring, and virus levels. Of these 101 patients, 24 patients met the criteria for optimal clinical response to Ribavirin therapy. These criteria are normalized or clinically significantly reduced according to the ALT level, and Knodell scoring is also reduced in terms of two or more points. A clinical response was obtained for these 24 patients without concomitant reduction in the level of viruses in the blood. Table 9 below summarizes data on ALT levels and Knodell scoring for 24 patients compared to the remaining 77 patients who have shown lower response rates.

Table 9

Respondents N = 24 Non-responders N = 77 Mean baseline ALT (range) for U / L 142.5 (52-269) 176.9 (35 - 629) Mean end of ALT treatment (range) * U / L 36.8 (21-62) 91.7 (13-286) Mean Knodell reduction scoring (range) 4.1 (2-9) 0.15 (-7 to + 9)

* The upper limit or normal state is 40 UZL.

• • •

Thus, specific embodiments and applications of nucleosides, nucleotides, and analogs thereof have been discovered and reported. However, it is to be understood that those skilled in the art are aware of the many possible variations besides those described herein, but this is without prejudice to the inventive concept herein. Accordingly, the present invention is not so limited and is further illustrated by the following appended claims. However, both the interpretation and the claim can be further interpreted in the widest possible way, as is consistent with the context herein. In particular, the terms "consisting" and "containing" may be interpreted as referring to elements, components, or steps and steps in a non-exclusive manner, indicating that they refer to elements, components or steps and steps that may be present, or used or combined with other elements, components or steps and steps, without necessarily referring or referring directly thereto.

······························

Industrial applicability

The compounds of this invention are intended to be medicaments and are therefore of importance to the pharmaceutical industry

Claims (54)

PATENT CLAIMS 1. A compound of formula I: wherein R is H, PO ₃ ² ', (PO ₃ ) 2 ³ ', or (PO ₃ ) ₃ ⁴ '. 2. Compound of structural formula EX: (IX). 3. Compound of formula (X): 9 9 • 9 9 9 • 999 ·· • 8 0 f • · ♦ 9 9 8 9 · 8 90 98 9 9 wherein W is -N (R 1) (R) ₂ or = NR _b wherein R 1 and R ₂ are independently hydrogen, linear alkyl, branched alkyl, alkenyl, alkynyl, aralkyl, aralkenyl, arylkynyl or aryl. A compound of formula X according to claim 3, wherein R 1 or R ₂ independently further comprises a nitrogen atom, an oxygen atom, a sulfur atom, or a halogen atom. A method of treating a viral infection in a patient, comprising administering a composition comprising a compound of claim 1, claim 2, claim 3, or claim 4, at a dose effective to inhibit the development of the viral infection. 6. A method of treating a viral infection in a patient according to claim 5 wherein the dose is in the range of 50 mg / day to 500 mg / day. The method of treating a viral infection in a patient according to claim 5, wherein the dose is in the range of 500 mg / day to 2500 mg / day. 8. The method of treating a viral infection in a patient according to claim 5, wherein the viral infection is selected from the group consisting of HIV infection, HCV infection, HBV infection, RSV infection, influenza virus infection and para-influenza virus infection. 9. The method of treating a viral infection in a patient of claim 5, further comprising administering to said patient a cytokine. · Φ • • • • ♦ • • • • • • • • • • · · φ φ φ φφφ <φφφ φφ φφφφ 10. A method of treating a viral infection in a patient according to claim 9, wherein the cytokine is an interferon. The method of treating a viral infection in a patient according to claim 10, wherein the interferon is alpha-2b. ’ 12. The method of treating a viral infection in a patient according to claim 5, further comprising administering Ribavirin. The compound of claim 1, wherein R is PO ₃ ² '. 14. A method of treating a viral infection in a patient comprising administering to the patient a composition comprising a compound of claim 13 in a dose effective to inhibit the development of the viral infection. The compound of claim 1, wherein R is (PO ₃ ) ₂ ³ '. 16. A method of treating a viral infection in a patient, comprising administering a composition comprising the compound of claim 15 at a dose effective to inhibit the development of the viral infection. The compound of claim 1, wherein R is (PO ₃ ) ₃ ³ '. · Tit tit tit tit tit tit tit tit tit tit tit tit tit i i i i tit tit A method of treating a viral infection in a patient, comprising administering a composition comprising the compound of claim 17 at a dose effective to inhibit the development of the viral infection. 19. A method of increasing the selectivity of a pharmacologically active molecule with respect to a pharmacological effect on target cells, comprising providing an active agent, wherein the active agent is 1-beta-L-ribofuranosyl-1,2,4-triazole-3-carboxamide ; the drug substance is modified by a modifying group, the modifying group being covalently attached to the drug substance by a nitrogen atom; wherein the modifying group is enzymatically removed from said drug in the target cell. 20. A method of increasing the selectivity of a pharmacologically active molecule with respect to a pharmacological effect on target cells, wherein the modifying group is = NH, wherein the modifying group is covalently attached to a carbonyl atom in the drug substance. 21. A method of increasing selectivity of a pharmacologically active molecule with respect to the pharmacological effect on target cells, characterized in that the modifying group is N (Ri) _(R2) or = NR !, wherein R and R ₂ are independently hydrogen, linear alkyl, branched alkyl, alkenyl, alkynyl, aralkyl, aralkenyl, aralkynyl, or aryl. 22. A method of increasing selectivity of a pharmacologically active molecule with respect to the pharmacological effect on target cells, characterized in that Ri and R ₂ independently comprise a nitrogen atom, an oxygen atom, a sulfur atom, or a halogen atom. • 4 4 4 4 4 • 444 ·· 44 ♦ · 4 4 4 4 44 4 ♦ 4 4 4 4 • 4 4 4 444 44 23. A method of treating a disease characterized by inflammation of an organ of a patient, the method comprising: provides a compound of claim 1, claim 2, or claim 3; and administering the compound to a patient at a dose that (a) causes systemic immunomodulation and not systemic immunosuppression of the Type I and Type Π responses, and (b) causes immunosuppression of the Type I and Type II responses in the patient's organ. 24. A method of treating a disease according to claim 23, wherein the compound is a compound according to claim 1. 25. A method of treating a disease according to claim 23, wherein the compound is a compound according to claim 2. 26. A method of treating a disease according to claim 23, wherein the compound is a compound according to claim 3. 27. A method of treating a disease according to claim 23, wherein the organ is a liver, wherein the liver is infected with a virus. 28. A method of treating a disease according to claim 23, wherein the virus is HCV virus. ♦ * * * * * * * * * * * * 29. A method of stimulating neuronal growth comprising: it has been found that compound of formula XI wherein Y is Ι-β-L-ribofuranosyl, lpL-ribofuranosyl-5-phosphate, lpL-ribofuranosyl-5 diphosphate, 1PL-ribofuranosyl-5-triphosphate, β-β-D-ribofuranosyl, and 1-β-D-ribofuranosyl-5-phosphate, 1β-β-β-β-D-ribofuranosyl or 1β-β-D-ribofuranosyl -triphosphate; it is effective at stimulating neuronal growth over a given concentration range; and subject to neurons with the compound within a given concentration range. 30. The method of stimulating neuronal growth of claim 29, wherein the compound is Ribavirin. 31. The method of stimulating neuronal growth of claim 29, wherein the compound is phosphorylated Ribavirin. 32. The method of stimulating neuronal growth of claim 29, wherein the compound is Levivirin ™. 33. The method of stimulating neuronal growth of claim 29, wherein the compound is phosphorylated Levivirin ™. 44 44 4 4 4 4 4 4 4 4 4 4 4 4 4 4 44 4444 ·· ·· • · 4 • 494 • 4 4 4 4 4 4 4445 44 • fcfc 44 4 4 4 4 4 4 4 4 4 4 4 4 4 34. The method of stimulating neuronal growth of claim 29, wherein the compound is administered to a patient as part of treating a disease within a dosage range effective to increase the Type I response and reduce the Type II response in the patient. 35. The method of stimulating neuronal growth of claim 29, which is directed to target unipolar nerve cells in neurons. 36. The method of stimulating neuronal growth of claim 29, which is directed to target bipolar nerve cells in neurons. 37. The method of stimulating neuronal growth according to claim 29, wherein the neurons are part of neuronal tissue comprising at least four of the following cell types: astrocytes, dendrocytes, myelin sheath cells, supporting nerve tissue cells, unipolar nerve cells, bipolar nerve cells cells, multipolar nerve cells and receptor cells. 38. An antiviral medicament comprising: a first compound having direct and indirect antiviral activity; and a second compound that enhances the overall antiviral effect, wherein the total antiviral effect comprises a direct and indirect antiviral effect, and wherein the second compound specifically binds a hapten which is selected from the group consisting of a viral protein and cytokines. 39. The antiviral medicament of claim 38, wherein the first compound comprises a nucleoside analogone. Toto · • of of of of of of of of of of of of of of of of of of of of toto toto toto toto toto toto toto toto toto · · · · To ·· · 40. The antiviral medicament of claim 38, wherein the nucleoside analog is Ribavirin. is that The antiviral medicament of claim 38, wherein the nucleoside analog is Levovirin ™. is that The antiviral medicament of claim 38 or claim 39, wherein the nucleoside analogon is in the form of a prodrug. 43. The antiviral medicament of claim 38, wherein the direct antiviral effect comprises inhibiting viral replication. 44. The antiviral medicament of claim 38, wherein the direct antiviral effect comprises a Type 1 / Type 2 equilibrium shift toward the Type 1 response. 45. The antiviral medicament of claim 38, wherein the Type 1 and Type 2 response suppression is included in the direct antiviral. 46. The antiviral medicament of claim 38, wherein the second compound comprises an antibody. »4 44 5 · · « 4 4 4 4 4 4 4 4 * 4 * 44 ·· 4 4 • 4 · · 4 4 4 47. The antiviral medicament of claim 38, 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 medicament of claim 38, wherein the viral protein is a virus protein selected from the group consisting of HIV virus, hepatitis virus, influenza virus, influenza virus, and RSV virus. 49. The antiviral medicament of claim 38, wherein the viral protein is a reverse transcriptase. 50. The antiviral medicament of claim 38, wherein the cytokine is a Type 1 cytokine. 51. The antiviral medicament of claim 38, wherein the first compound and the second compound have a synergistic effect. 52. The antiviral medicament of claim 38, wherein at least one of the first compound and the second compound is selectively accumulated in the organ. 53. The antiviral medicament of claim 38, wherein the organ is selected from the group consisting of liver and brain. • to • «• • • • • • to • to to to to to to to to to to 54. The antiviral medicament of claim 38, wherein the first compound is Ribavirin and the second compound is an antibody that specifically binds a Type 2 cytokine.