KR20030040415A - Improved specificity in treatment of diseases - Google Patents

Abstract

The present invention relates to modifying a drug by covalently binding the blocking group to the drug via a nitrogen atom in the blocking group. In modified drugs the blocking group prevents metabolic conversion and sequestration of the drug in cells other than the target cell, and the blocking group can be enzymatically removed in the target cell. In particular, the contemplated advantages of the compounds and methods of the present invention include: reduction in cytotoxicity by inhibition of metabolic conversion to potentially cytotoxic metabolites, reduction in drug dosage by reduction of intracellular sequestration other than target cells, and Increased selectivity of the drug.

Description

IMPROVED SPECIFICITY IN TREATMENT OF DISEASES

Liver disease, and in particular viral hepatitis B and hepatitis C, remains a serious threat, and various therapies have been developed. Depending on the drug used, the therapies may be classified as a combination of direct antiviral therapy, indirect antiviral therapy, or direct or indirect antiviral therapy.

Direct antiviral therapy interferes with virus replication and / or virus assembly. For example, nucleoside analogs can be used to reduce viral replication by inhibiting reverse transcriptase of the virus. However, nucleoside analogues frequently involve side effects, including anemia and / or neutropenia. In addition, prolonged exposure to nucleoside analogs resulted in drug resistance in some viral strains. To circumvent some or all of the problems associated with drug resistance, a cocktail of nucleoside analogs can be administered. Unfortunately, cocktails of nucleoside analogs typically only delay the onset of drug resistance. In addition, nucleoside analogues generally do not show selectivity between viral replication and replication in rapidly dividing cells of the host organism, thus exhibiting significant cytotoxicity to the host.

Protease inhibitors can also be used to interfere with proper assembly of viral proteins. Protease inhibitors are highly specific for viral proteases, thus avoiding the problems associated with limited selectivity between viral replication and replication in rapidly dividing host cells. However, even at relatively low doses, they tend to cause side effects including nausea, diarrhea, diabetes and nephrolithiasis. In addition, some protease inhibitors are very poorly soluble in aqueous solutions, reducing the potential amount that can be delivered to a patient. It has also been found that viral resistance to some protease inhibitors results after prolonged treatment.

Indirect antiviral therapy can be used to stimulate the immune system to recognize viral antigens, or to alter the cytokine balance of the immune system to type 1 cytokine responses, and cytokines may be used to modulate cellular immunity against cells infected with the virus. It is thought to help establish. For example, IFN-alpha can be used to treat chronic hepatitis C. However, many patients treated with IFN-alpha recurred after discontinuation of treatment, and some patients underwent a quantum progression of virus during treatment. In addition, IFN-alpha tends to cause fever, headache, lethargy, anorexia, anxiety and depression at low doses, and myelosuppression and low blood counts at high doses.

One or both of the direct and indirect antiviral treatments can be achieved by co-administration of ribavirin with interferon-alpha, which has shown a marked reduction in inflammation and serum ALT levels in many patients. Despite the relatively good therapeutic efficacy, various problems still persist with ribavirin. For example, at particularly high doses, intracellular phosphorylation of ribavirin in erythrocytes is known to contribute to hemolytic anemia. In addition, phosphorylated ribavirin tends to accumulate in red blood cells, significantly reducing the effective amount. In conclusion, relatively high amounts need to be administered to achieve a suitable amount of ribavirin in hepatocytes.

There are various compositions and methods known in the art for targeted treatment of liver disease, but all or almost all of them have one or more disadvantages. Therefore, there is still a need to provide improved compositions and methods for targeted treatment of disease.

The present invention relates to improved specificity in the treatment of diseases.

Summary of the Invention

The present invention relates to methods and compositions for increasing the selectivity of drugs. In general, drugs are covalently modified by blocking groups.

More specifically, in one aspect of the invention, the blocking group is bound to the drug via a nitrogen atom in the blocking group. The blocking group in the modified drug reduces metabolic conversion and sequestration (ie accumulation) of the drug in cells other than the target cell, and the blocking group is enzymatically removed in the target cell.

In another aspect of the invention, the metabolic shift of the drug is recognized to induce damage to the target cell, the blocking group bound to the drug via the nitrogen atom in the blocking group prevents metabolic shift, and the blocking group is the target cell It is further recognized that it is cleaved from the drug in the stomach. In conclusion, it was contemplated that the modification of the drug by the blocking group and the administration of the drug to a system comprising target cells and cells other than the target cells reduced cytotoxicity.

In a further aspect of the invention, metabolic conversion of the drug in cells other than the target cell is recognized to reduce the effective concentration of the drug, and blocking groups bound to the drug via nitrogen atoms in the blocking group prevent metabolic conversion, blocking The group is further recognized to be cleaved from the drug in the target cell. In conclusion, it was contemplated that modification of the drug using the blocking group could reduce the dose. It was also contemplated that the drug would be administered to a system comprising the target cell and cells other than the target cell.

Drugs considered include carboxamide groups, particularly drugs considered 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide and 2-beta-D-ribofura Nosyl-4-thiazolecarboxamides are included, which may also be in their respective L-isomeric forms. Although the blocking group is not limited to specific chemical properties, it is preferable that the blocking group includes a nitrogen atom. Particularly preferred blocking group is = NH. The target cells contemplated are not limited to specific cell types and may or may not be infected with the virus or they may be hyperproliferative. However, particularly preferred are hepatocytes and neurons infected with viruses or hyperproliferative and cells other than target cells include red blood cells.

Various objects, features, aspects and advantages of the invention will become more apparent from the following detailed description of the preferred embodiments of the invention, together with the accompanying drawings.

Brief description of the drawings

1A and 1B are schematic diagrams of ingestion and retention of exemplary drugs according to the present invention.

2 is an exemplary synthetic scheme for the synthesis of ribavirin.

3 is an exemplary synthetic scheme for the synthesis of modified ribavirin.

Detailed description of the invention

As used herein, the term “pharmacological effect” refers to any change in the metabolism, replication, structure or lifespan of a cell in a cell containing system, which is caused by molecules added to the system. For example, inhibition of an assimilation, catabolism or polymerase-type reaction catalyzed by an enzyme can be considered a pharmacological effect. Similarly, depolymerization of tubulin by KinI kinesin is considered a pharmacological effect within the scope of the above definition. In contrast, allosteric inhibition of enzymes by metabolites produced intracellularly of the system is not considered a pharmacological effect because no allosteric inhibitor has been added to the system.

As used herein, the term “target cell” refers to a cell in which the drug is intended to exert a pharmacological effect. For example, virus-infected hepatocytes are thought to be target cells for the drug ribavirin. In contrast, the term “cells other than target cells” includes all cells in a cell-containing system that do not target the cells.

In general, while certain drugs are intended to exert their pharmacological effects on certain cells (ie, target cells), cells other than target cells can metabolize these drugs at significant rates and are frequently undesirable and nonspecific. May cause side effects. The inventors have modified the drug into a blocking group in which this unwanted metabolic shift in cells other than the target cell (ie, intracellularly or on the surface other than the target cell is selectively removed from the target cell, resulting in a pharmacological effect of the drug. It has been found that it can be prevented by concomitantly reducing the cytotoxicity and dosage of the drug while increasing the selectivity of the drug.

In one aspect of the invention, the selectivity of the pharmacological effect of the drug is increased by a method in which the drug is identified in step 1 as having the desired pharmacological effect on the target cell. In a further step, the drug is modified to a blocking group, where the blocking group is covalently bound to the drug via the nitrogen atom in the blocking group. The blocking group further reduces the accumulation of the drug in cells other than the target cell and is enzymatically removed from the drug in the target cell. As used herein, a "selectivity" of a drug with respect to a pharmacological effect is not intended to exert a pharmacological effect on cells other than the target cell to the undesirable side effects of the drug in cells other than the target cell, but to target the cell for therapeutic purposes. It refers to the tendency of the drug to exert a pharmacological effect on.

For example, the selectivity of the pharmacological effect of 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin, formula 1) can be covalently linked to ribavirin by Increased by forming carboxamidine groups. Ribavirin is known to have antiviral properties in hepatitis cells infected with hepatitis virus. Marcellin, P. and Benhamou J .; Treatment of chronic viral hepatitis, Baillieres Clin Gastroenterol 1994 Jun; 8 (2): 233-53]. In addition, ribavirin is readily phosphorylated in erythrocytes, resulting in a significant proportion of the pharmacologically active ribavirin-phosphate. See Homma, M. et al. High-performance liquid chromatographic determination of Ribavirin in whole blood to assess disposition in erythrocytes; Antimicrob Agents Chemother 1999 Nov; 43 (11): 2716-9, which is known to reduce selective pharmacological effects. Surprisingly, we found that ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamidine, formula 2) modified with the = NH group is not phosphorylated or only algebraic in erythrocytes. Unphosphorylated, and the = NH group of 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamidine is specific and enzymatically removed from hepatocytes, thereby allowing It was found to form nosil-1,2,4-triazole-3-carboxamide.

The reduction in selective accumulation of modified ribavirin is shown in FIGS. 1A and 1B. In FIG. 1A, ribavirin (R) is provided to erythrocytes (cells other than target cells) 100. Ribavirin enters red blood cells and is phosphorylated into pharmacologically active ribavirin-phosphate (R-P), which is retained in red blood cells. Similarly, hepatocytes (target cells) 110 are provided with ribavirin (R). Ribavirin enters the hepatocytes and is phosphorylated into pharmacologically active ribavirin-phosphate (R-P), which is retained in the hepatocytes. In FIG. 1B, modified ribavirin (R *, 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamidine) is provided on erythrocytes (cells other than target cells) 101 do. The modified ribavirin enters the red blood cells, but is not phosphorylated and can therefore exit the red blood cells. Similarly, hepatocytes (target cells) 111 are provided with modified ribavirin (R *). The modified ribavirin enters the hepatocytes and is enzymatically deamined to become ribavirin, which is subsequently phosphorylated into pharmacologically active phosphorylated ribavirin (R-P), which is retained in the hepatocytes.

In another aspect of the invention, it has been contemplated that there are many drugs other than ribavirin suitable for the inventive concepts presented herein. In general, suitable drugs include drugs that are metabolized, activated and / or inactivated in cells other than the target cell, and in particular the drugs contemplated include nucleotides, nucleotides, nucleotide analogues and nucleotide analogues. For example, thiazopurin (2-beta-D-ribofuranosyl-4-thiazolecarboxamide) can be easily modified with a = NH group to produce a nucleoside having a carboxamide group that can produce the corresponding carboxamide. Cleoside analogues. In another example, another drug includes nucleoside uracil or nucleoside analog 5'-fluoro uracil (5'-FU).

In another aspect of the invention, the blocking group need not be limited to the = NH group and may include various primary and secondary amines as long as the blocking group can be covalently attached to the drug via a nitrogen atom. As used herein, the term “blocking group” refers to a chemical group that can covalently bind to a drug and, when bound to the drug, can block one or more metabolic conversions of the drug. As used herein, the term "metabolism" of a drug is caused by metabolism of the cellular or cellular system, and in particular enzymatic degradation (eg oxidative, hydrolytic cleavage) and enzymatic modification (eg glycosylation, phosphorylation) It refers to any intracellular and / or extracellular chemical change of a drug, including.

In general, suitable blocking groups have been considered to have the formula -N (R ₁ ) (R ₂ ) or = NR ₁ , where 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 nitrogen, oxygen, sulfur or halogen. However, it is particularly preferred that another blocking group can be enzymatically removed from the drug, and in particular the enzymes discussed include deaminoases (eg adenosine or cytosine deaminoases), liver deaminoases (eg nicotinamide deamination). Liver specific aminohydrolase, including aminoenzymes) and liver aminotransferases (glutamate-pyruvate aminotransferases).

The considered blocking group can be covalently attached to various positions of the drug molecule, and although it is generally desirable for the drug under consideration to be modified on a carboxamide residue, various positions other than the carboxamide group, in particular carbonyl groups (eg, Carboxylic acid and ketone-type carbonyl) have been considered. For example, the carbonyl group at the ring position of each uracil or analog 5'-FU thereof may be modified by a blocking group.

While not limited to the inventive concept presented herein, it has been contemplated that the blocking group can activate the drug or prevent subsequent activation once the modified drug is provided to cells other than the target cell. For example, when the blocking group is bound to the drug at a position necessary for the specific interaction of the drug with a target molecule (eg, a receptor or substrate binding site), the blocking group can inactivate the drug. On the other hand, the blocking group can be bound to the drug at a position that can prevent metabolic activation.

Depending on the chemical properties of the drug and / or blocking group, it was contemplated that the blocking group could substitute for the functional group or substituent, or that the blocking group would bind to the functional group or substituent. For example, if the drug is ribavirin and the blocking group is a = NH group, the oxygen atoms in the carboxamide group of ribavirin are replaced by the = NH group. On the other hand, the drug is a nucleophilic group (e.g., -O ^-) those containing, and comprises a secondary amine with a suitable leaving group is a blocking group, a secondary amine may be coupled to a nucleophilic group.

Regarding the step of modifying the drug, it was contemplated that the modification may include organic-synthetic modification, enzymatic modification or de-novo synthesis to produce the modified drug. For example, if the drug contains an activated carbonyl functional group, the amidation of the carbonyl atom can be accomplished in a single nucleophilic exchange reaction. In addition, de-novo synthesis of the modified drug may be more economically advantageous, especially if the drug has many reactive groups other than the above group bound to the blocking group. It was also particularly contemplated that suitable drugs may be enzymatically modified by introducing the blocking groups into the drug in reactions using the drug and blocking groups as enzyme substrates. If useful, it is preferred that the enzyme for such modification is derived from a target cell (eg, a homologous or heterologous source, or a recombinant source expressing a gene encoding the enzyme).

Depending on the type of cells other than the target cell and the chemical nature of the drug and / or blocking group, the prevention of accumulation of drugs in cells other than the target cell may result in a reduction in uptake via drug-specific transporters, e.g., additional or new May be achieved by one or more of a variety of mechanisms, including a reduction in metabolic conversion to a form to be retained (due to a change in electrical charge, hydrophobicity, or a change in recognition by an exporter), or (eg, within a blocking group). It is to be understood that prevention of drug accumulation can be achieved due to increased outflow from cells other than target cells (due to secretion signals). For example, if the drug is a nucleoside analog and cells other than the target cell have a nucleoside transporter that selectively introduces nucleosides that do not contain lipophilic residues; It was contemplated that addition of lipophilic moieties as blocking groups to the drug could prevent accumulation of the drug. In another example, phosphorylation (and concomitant accumulation) of various nucleosides in erythrocytes can be prevented by converting the carboxamide group to the carboxamidine group (above).

Regarding enzymatic removal of blocking groups within target cells, it has been considered that enzymatic removal can vary considerably, depending on the target cells, blocking groups and the type of drug. Enzymatic removal may include enzymes from various classes including hydrolases, transferases, lyases and oxidoreductases, and particularly preferred subclasses are adenosine and cytosine deaminoases, arginases, aminotransferases and Arylamidase. Enzymes contemplated for enzymatic removal of blocking groups may be excluded in target cells, but in another aspect of the present invention, suitable enzymes may not be expressed ubiquitously in all cells in a cell containing system. It should be further understood that it may be expressed in cells other than the target cell. It is further to be understood that the enzymes considered are natively expressed (ie, not recombinant) in each target cell under normal and / or pathological conditions. For example, it is known that glutamine-pyruvate aminotransferase is constitutively expressed with relatively high selectivity in liver cells and thus may be an enzyme suitable for the removal of blocking groups. Cytosine deaminoases are also known to be expressed in relatively high amounts in colon cancer cells, but not in normal colon cells or only in small amounts.

In another aspect of the invention, the cytotoxicity of the drug against cells other than the target cell is reduced by a method in which the metabolic conversion of the drug in cells other than the target cell induces damage to cells other than the target cell in step 1. . As used herein, the term “cytotoxic” refers to an undesired pharmacological effect on cells other than the target cell, wherein the undesired pharmacological effect includes, in particular, inhibition of replication, energy-metabolism or cell death. do. In a further step, the drug is modified to a blocking group, where the blocking group is covalently bound to the drug via a nitrogen atom in the blocking group, where the blocking group reduces metabolic conversion of the drug in cells other than the target cell, Enzymatic cleavage from the drug. In a further step, the drug is administered to a system comprising the target cell and cells other than the target cell, wherein the blocking group is covalently bound to the drug.

In a preferred aspect of reducing the cytotoxicity of the drug to cells other than the target cell, metabolic conversion comprises phosphorylation of the drug to the corresponding drug-phosphate in erythrocytes. For example, phosphorylation of the antiviral drug ribavirin in various cells to produce pharmacologically active ribavirin-5'-monophosphate is well known in the art. See Homma, M. et al. High-performance liquid chromatographic determination of Ribavirin in whole blood to assess disposition in erythrocytes; Antimicrob Agents Chemother 1999 Nov; 43 (11): 2716-9], which is involved in the inhibition of inosine monophosphate dehydrogenase (IMPDH). Unfortunately, ribavirin-5'-monophosphate has an apparent cytotoxic effect on erythrocytes [De Franceschi, et al .; Hemolytic anemia induced by ribavirin therapy in patients with chronic hepatitis C virus infection: role of membrane oxidative damage, Hepatology 2000 Apr; 31 (4): 997-1004] In conclusion, it was recognized that the prevention or reduction of the formation of ribavirin-5'-monophosphate in erythrocytes would significantly reduce the cytotoxicity of ribavirin.

However, it should be understood that various metabolic conversions other than phosphorylation of drugs in cells other than target cells have also been considered, including oxidation, reduction, hydrolytic cleavage of covalent bonds in drugs, addition or removal of pendant groups, and ring opening ( ring-opening reactions are included. For example, where cells other than target cells are hepatocytes, metabolic conversion can include various known enzymatic detoxification or lysis reactions to occur in the liver (eg, glycosylation, cytochrome P ₄₅₀ -mediated oxidation, etc. ). In another example, metabolic conversion includes phosphatase or esterase activity.

Depending on the type of metabolic conversion, the conversion may be limited to a single type of cell other than the target cell, but may occur in more than one cell type. For example, if cells other than target cells have a relatively high rate of nucleic acid synthesis, and metabolic conversion is mediated by enzymes involved in the nucleic acid synthesis, rapidly growing various types of cells may exhibit metabolic conversion. On the other hand, metabolic conversion may be locally restricted through the accessibility of the drug to a particular set of cells or organs.

It should be understood that various well known laboratory methods can be used to recognize and / or demonstrate that blocking groups covalently bound to drugs reduce metabolic conversion in cells other than target cells. For example, when cells other than the target cells are cultured in vitro, cells other than the target cells may be incubated with the corresponding radiolabeled drug, after which the metabolite of the radiolabeled drug is immunoassay, It was contemplated that this could be confirmed by various assays including thin layer chromatography or GC-MS. In addition, where cells other than the target are located in a mammal, sufficient samples for isolating and identifying metabolites of the administered drug may be provided by tissue biopsy.

It was further contemplated that the type of damage to cells other than the target cells can vary substantially, ranging from delaying cellular metabolism in cells other than the target cells to cell death. For example, when metabolic conversion produces an inhibitor of an enzyme located in that pathway, energy for cells other than the target cell may be provided at least in part via the salvage pathway. Likewise, when metabolic conversion of drugs to enzyme inhibitors proceeds at a relatively low rate, up-regulation of the expression of enzymes affected by the inhibitor can almost completely compensate for the reduction in the number of active sites. On the other hand, if metabolic conversion produces radical species, lipid peroxidation can lead to severe membrane damage and subsequent cell death.

It was further contemplated that damage resulting from metabolic turnover of the drug may occur directly or indirectly. For example, if metabolic conversion produces an enzyme inhibitor that blocks the enzyme, the damage is thought to be direct. On the other hand, if metabolic conversion produces an intermediate, the damage is considered indirect after subsequent intracellular or extracellular modification further converts the enzyme inhibitor.

Regarding the step of administering the drug to the system, it was contemplated that the suitable drug would be administered in any suitable pharmaceutical formulation under any suitable protocol. Thus, administration may be oral, parenterally (including subcutaneous injection, intravenous, intramuscular, endometrial injection or infusion techniques), non-toxic pharmaceutically acceptable carriers conventionally employed by inhalation spray, rectal, topical, and the like. It may be carried out in a dosage unit formulation containing the adjuvant and the vehicle. For example, the appropriate drug may be administered orally as a pharmacologically acceptable salt, or alternatively may be administered intravenously in physiological saline (eg, buffered to a pH of about 7.2 to 7.5). . Conventional buffers such as phosphate, bicarbonate or citrate can be used for this purpose. It was also contemplated that it would be well known to those skilled in the art to modify the route of administration and regime of administration of certain drugs to maintain the pharmacodynamics of the compounds of the present invention for maximum beneficial effect in the patient.

In certain pharmaceutical dosage forms, pro-drug forms of the drug have been considered. Those skilled in the art will recognize how to easily modify the drug under consideration in pro-drug form to facilitate delivery of the active compound to a target site in the host organism or patient. Those skilled in the art will also utilize advantageous pharmacodynamic parameters in pro-drug form, where applicable, in delivering a compound of the invention to a targeted site in a host organism or patient to maximize the intended effect of the compound of the invention.

It should be further understood that the contemplated drug may be administered alone, or in combination with other pharmacologically active agents that may be administered separately or in combination, and if administered separately, the administration may occur simultaneously or It can happen individually in any order. The pharmacologically active agents discussed include antiviral agents such as interferons (e.g., interferons α and γ), tolnaftate, Fungizone ^TM , Lotrimin ^TM , Mycelex ^TM , Nystatin and Amphotheracin Antifungal agents such as); Antiparasitic agents such as Mintezol ^™ , Niclocide ^™ , Vermox ^™ and Flagyl ^™ ; Intestinal agents such as Immodium ^™ , Lomotil ^™ and Phazyme ^™ ; Antitumor agents such as interferons α and γ, Adriamycin ^™ , Cytoxan ^™ , Imuran ^™ , methotrexate, Mithracin ^™ , Tiazofurin ^™ , Taxol ^™ ; Skin agents such as Aclovate ^™ , Cyclocort ^™ , Denorex ^™ , Florone ^™ , Oxsoralen ^™ , coal tar and salicylic acid; Migraines, such as ergotamine compounds; Steroids and immunosuppressants not described above (including cyclosporin, Diprosone ^™ , hydrocortisone; Floron ^™ , Lidex ^™ , Topicort and Valisonone; metabolic agents such as insulin), and Other drugs that may not fit into the category include cytokines such as IL2, IL4, IL6, IL8, IL10 and IL12.

Regarding the dosages of the drugs and pharmacologically active agents considered, the therapeutically effective amount will vary depending on the condition being treated, the severity, the treatment regime used, the pharmacodynamics of the agents used and the patient (animal or human) being treated. Considered. Various dosages have been further contemplated as appropriate, including not only doses of 0.5 mg / kg to 0.1 mg / kg, but also doses of 0.5 to 1.0 mg / kg or more. In general, it is preferred that the system comprising the target cell and cells other than the target cell is a mammal (most preferably human), but various alternative systems are also suitable, especially in vitro cells and tissue cultures.

Drugs, blocking groups, modifying drugs, target cells and cells other than target cells in the considered method of reducing the cytotoxicity of the drug to cells other than the target cells, are the same as the above described application. It was.

In a further aspect of the invention, the dose of drug in the system is reduced by the method in which the drug is provided, wherein metabolic conversion of the drug in a cell other than the target cell is a drug in the system comprising cells other than the target cell and the target cell. To reduce the concentration. In a further step, the drug is modified to a blocking group, where the blocking group is covalently bound to the drug via a nitrogen atom in the blocking group, where the blocking group reduces metabolic conversion of the drug in cells other than the target cell. In a subsequent step, the drug is administered to the system, where the blocking group is covalently bound to the drug, where the blocking group is enzymatically removed from the drug in the target cell.

In a preferred aspect of reducing the dose of drug in the system, the drug is ribavirin, the target cell is a virus-infected hepatocyte, and cells other than the target cell are erythrocytes. It is well known in the art (above) that ribavirin is metabolically converted to ribavirin-phosphate and that ribavirin-phosphate is retained in erythrocytes, thereby significantly lowering the concentration of ribavirin. Ribavirin is modified by covalently binding the = NH blocking group to the carboxamide carbon to replace the carbonyl oxygen in the carboxamide. Metabolic conversion of ribavirin modified with = NH blocking group was found to be significantly reduced in erythrocytes (below). It is contemplated that preferred administration of modified ribavirin is to humans in a single oral dose of 50 mg to 300 mg.

It is known that ribavirin is orally administered to humans as an antiviral drug in at least a single dose of about 600 mg to 1200 mg. Although the initial concentration of ribavirin in the system (eg, human) is between about 1 μM and several hundred μM, the ribavirin concentration is typically about 85% of the initial concentration in the system within 24 hours by sequestration into red blood cells due to phosphorylation of ribavirin in erythrocytes. To 50%. We have demonstrated that the modification of ribavirin to 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamidine significantly reduces the amount of phosphorylation of ribavirin (below). Thus, it was considered that all or almost all of the initial concentration of ribavirin is useful for the desired pharmacological effect in the target cell. In conclusion, the modification of ribavirin with the blocking group can be used to reduce the dose of ribavirin to about 5%, preferably about 10%, more preferably 25%, most preferably 50% by weight. Considered.

However, it should be understood that various dosages other than 600 mg to 1200 mg are also contemplated, including dosages of 200 mg to 600 mg, dosages of 20 mg to 200 mg and less. For example, when ribavirin is used as an immunomodulatory drug, lower doses of about 100 mg to 300 mg may be sufficient. On the other hand, when relatively high concentrations of the drug are desired, dosages of 600 mg to 1800 mg, and higher have been considered. It should also be understood that the reduction in dosage may vary significantly depending on the particular metabolic shift. For example, if metabolic conversion is relatively rapid and occurs in a large number of cells other than target cells, reductions in doses of 25% to 80% by weight, and more, have been considered. On the other hand, when metabolic conversion is relatively slow, reductions in doses of 25% to 5%, and less, have been considered.

As for the drug, blocking group, metabolic conversion, modifying the drug, administering the drug, target cells and cells other than the target cells in the considered method of reducing the dose of the drug, the same as the above described application Considered.

Example

(a) An exemplary synthesis of ribavirin is shown in FIG. 2, according to the method as outlined below.

Methyl 1- (2,3,5-tri-O-acetyl-β-D-ribofuranosyl) -1,2,4-triazole-3-carboxylate (3) and

Methyl 1- (2,3,5-tri-O-acetyl-β-D-ribofuranosyl) -1,2,4-triazole-5-carboxylate (4)

Methyl-1,2,4-triazole-3-carboxylate (25.4 g, 200 mmol) (1), 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose (63.66 g, 200 mmol) (2) and a mixture of bis (p-nitrophenyl) phosphate (1 g) were placed in an RB flask (500 mL). The flask was placed in a preheated oil bath at 165-175 ° C. under water suction vacuum with stirring for 25 minutes. The replaced acetic acid was collected in an ice cold trap located between the aspirator and the RB flask. The flask was removed from the oil bath and cooled. When the temperature of the flask reached about 60-70 ° C, EtOAc (300 mL) and saturated NaHCO ₃ (150 mL) were introduced and extracted in EtOAc. The aqueous layer was extracted again with EtOAc (200 mL). The combined EtOAc extracts were washed with saturated NaHCO ₃ (300 mL), water (200 mL) and brine (150 mL). The organic extract was dried over anhydrous Na ₂ SO ₄ , filtered and the filtrate was evaporated to dryness. The residue was dissolved in EtOH (100 mL), diluted with MeOH (60 mL) and cooled at 0 ° C. for 12 hours to give colorless crystals. The solid was filtered off, washed with minimal cold EtOH (20 mL) and dried under high vacuum on solid NaOH to give 60 g (78%). The filtrate was evaporated to dryness and purified on silica column using CHCl ₃ → EtOAc (9: 1) as eluent. Two products, about 8.5 g (11%) of fast moving product and about 5 g (6.5%) of slow moving product, were isolated from the filtrate. The slow moving product was consistent with the crystallized product. The fast moving product was found to be (4) and obtained as a foam. The combined yield of (3) was 65 g (84%); Melting point 108 to 110 ° C;

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

Methyl-1- (2,3,5-tri-O-acetyl-β-D-ribofuranosyl) -1,2,4-triazole-3-carboxylate (62 g, 161 mmol) (3) Placed in a steel bomb and treated at 0 ° C. with freshly prepared methanol ammonia (350 mL, prepared by passing anhydrous ammonia gas into the anhydrous methanol at 0 ° C. until saturated). The steel vessel was closed and stirred for 18 hours at room temperature. The steel vessel was cooled to 0 ° C., opened and the contents evaporated to dryness. The residue was treated with anhydrous EtOH (100 mL) and evaporated to dryness. The resulting residue was triturated with acetone to give a solid, which was filtered and washed with acetone. The solid was dried overnight at room temperature and dissolved in hot EtOH (600 mL) and water (10 mL) mixture. The volume of EtOH solution was reduced to 150 mL by heating and stirring on a hot plate. The hot EtOH solution was cooled to give colorless crystals which were filtered off, washed with acetone and dried under vacuum. The filtrate was further concentrated to give additional material. Total yield: 35 g (89%); Melting point 177 to 179 ° C; [α] ²⁰ _D -35.3 (c, 10, H ₂ O);

(b) An exemplary synthesis of ribavirin modified with = NH group is shown in FIG. 3, following the method as outlined below.

3-cyano-1- (2,3,5-tri-O-acetyl-β-D-ribofuranosyl) -1,2,4-triazole (7)

3-cyano-1,2,4-triazole (18.8 g, 200 mmol) (6), 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose (63.66 g, 200 mmol) and a mixture of bis (p-nitrophenyl) phosphate (1 g) were placed in an RB flask (500 mL). The flask was placed in a preheated oil bath at 165-175 ° C. under a water aspirator vacuum with stirring for 25 minutes. The replaced acetic acid was collected in an ice cold trap located between the aspirator and the RB flask. The flask was removed from the oil bath and cooled. When the temperature of the flask reached about 60-70 ° C., EtOAc (300 mL) and saturated NaHCO ₃ (150 mL) were introduced and extracted in EtOAc. The aqueous layer was extracted again with EtOAc (200 mL). The combined EtOAc extracts were washed with saturated NaHCO ₃ (300 mL), water (200 mL) and brine (150 mL). The organic extract was dried over anhydrous Na ₂ SO ₄ , filtered and the filtrate was evaporated to dryness. The residue was dissolved in ether (100 mL) and cooled at 0 ° C. for 12 h to give colorless crystals. The solid was filtered off, washed with minimal cold EtOH (20 mL) and dried over high vacuum on solid NaOH. Yield: 56.4 g (80%). Melting point 96-97 ° C.

1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamidine hydrochloride (8)

(7) (14.08 g, 40.0 mmol), NH ₄ Cl (2.14 g, 40.0 mmol) and anhydrous ammonia (150 ml) were heated in a steel vessel at 85 ° C. for 18 hours. The steel vessel was cooled, opened and the contents evaporated to dryness. The residue was crystallized from MeCN-EtOH to give 10.6 g (95%) of (8). Melting point 177 to 179 ° C.

Another exemplary route with ribavirin as starting material proceeds as follows:

2 ', 3', 5'-tri-O-acetyl-1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (9)

Suspension of 1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (28.4 g, 116.4 mmol) (ribavirin) in acetic anhydride (200 mL) and pyridine (50 mL) Was stirred at rt overnight. The resulting clear solution was concentrated in vacuo to give clear foam (43.1 g, quant.). The foam was homogeneous on TLC and used directly in the next step without purification. A small amount was purified by flash chromatography to give a sample for analysis;

3-cyano-2 ', 3', 5'-tri-O-acetyl-1-β-D-ribofuranosyl-1,2,4-triazole (10)

To a solution of (9) (43.1 g, 116.4 mmol) in chloroform (500 mL) was added triethylamine (244 mL) and the mixture was cooled to 0 ° C. in an ice-salt bath. Phosphorus oxychloride (30.7 mL, 330 mmol) was added dropwise under stirring and the solution was warmed to room temperature. After stirring the mixture at room temperature for 1 hour, TLC (hexane / acetone 3: 1) showed complete disappearance of the starting material. The brown reaction mixture was concentrated to dryness in vacuo and the residue dissolved in chloroform (500 mL). The organic solution was washed with saturated aqueous sodium bicarbonate (3 × 200 mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was chromatographed on silica gel using 20% acetone in hexane (flash chromatography) to yield 33.14 g (81% from ribavirin) as an amorphous solid (10). The solid was the same in all respects to the certified samples: melting point 101-103 ° C .; IR (calcium bromide) ν 2250 (CN), 1750 (C═O), cm ⁻¹ ;

1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamidine hydrochloride (8)

To a suspension of (10) (4.0 g, 11.4 mmol) in methanol (100 mL) was added a 1 molar solution of methanol sodium methoxide (12 mL) and the mixture was stirred at rt overnight. The solution was acidified with methanol washed Dowex H + resin to pH 4, the resin was filtered and the filtrate was concentrated to dryness in vacuo. The residue was dissolved in a minimum amount of methanol (15 mL) and transferred to a pressure bottle. A methanol solution saturated at 0 ° C. with ammonium chloride (0.61 g, 11.4 mmol) and anhydrous ammonia gas (75 mL) was added, the bottle was sealed and the solution was stirred overnight at room temperature. The solution was concentrated to dryness in vacuo and the resulting residue was crystallized from acetonitrile / ethanol to give (8) (2.95 g, 93%) as a crystalline solid. The sample was identical in all respects to the authentication sample.

In another alternative route, 1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamidine hydrochloride (8) is a microorganism as an enzyme source (under microbial non-proliferative conditions). It can be produced by the enzymatic reaction using the culture of, the original cell or cell extract of the microorganism. 3-cyano-1- (2,3,5-tri-O-acetyl-β-D-ribofuranosyl) -1,2,4-triazole (7) is 3-cyano-1,2, It can be produced by contacting a 4-triazole or salt thereof with a ribose donor in the presence of a microbial based enzyme source. Then compound (7) will be converted to (8) by treating (7) with aqueous ammonia solution. Alternatively, 1,2,4-triazole-3-carboamidine hydrochloride can be reacted with a ribose donor in the presence of an enzyme to produce (8) directly.

(c) deaminoation of liver modified ribavirin to ribavirin

In mice, a dose of 300mg / kg per day for 8 days ³ H- ribavirin and ³ H - (= NH) after repeated oral administration of the modified Ribavirin, medium and minimum concentration C _min radioactivity in the liver is a modified Ribavirin and In comparison, it was significantly lower for ribavirin. This indicates that ribavirin accounts for about 90% of liver radioactivity in mice treated with ribavirin and ribofuranosyl triazole carboxylic acid (RTCA) accounts for about 10%. In contrast, modified ribavirin accounts for about 30% of liver radioactivity and ribavirin accounts for about 70% in mice treated with modified ribavirin (see Table 1).

³ H-ribavirin ³ H-(= NH) modified ribavirin Whole Liver Radioactivity RTCA Ribavirin Modified Ribavirin 18.4 μg equivalent / g about 1.8 μg equivalent / g about 16.6 μg equivalent / g Not detected 23.8 μg Equivalent / g Not Detected About 16.6 μg Equivalent / g About 7.2 μg Equivalent / g

Table 1: Liver Radioactivity in Mice

(d) Differential radioactivity distribution of ribavirin and (= NH) modified ribavirin in red blood cells (RBC)

Ribavirin was found to be phosphorylated in RBC, and it was further suggested that phosphorylated ribavirin is a causative agent in hemolytic anemia observed in humans at high doses or long-term treatment of ribavirin. Notably, modified (= NH) -modified ribavirin is not directly transported into the RBC as demonstrated by in vitro studies (data not shown), and as shown in Table 2 below, consequently modified ribavirin It was contemplated that it would accumulate in RBC only after deamination in liver to ribavirin and subsequent phosphorylation with the corresponding phosphate.

In mice, 8 days at a dose of 300mg / kg on day ³ H- ribavirin and ³ H for - (= NH) after repeated oral administration of the modified Ribavirin, medium and minimum activity concentration C _min in the RBC is modified than ribavirin Remarkably low for ribavirin. As judged from the differential data set forth in Table 1 and Table 2, the therapeutic index for modified ribavirin (ie, the ratio between liver ribavirin concentration and RBC ribavirin concentration) is about three times the concentration of ribavirin.

After inserting the cannula into the vessels of the portal vein of cynomolgus monkeys and administering a single oral dose of 30 mg / kg of ³ H ribavirin or (= NH) -modified ³ H ribavirin, the peak radioactivity concentration in RBC was reached after 24 hours. Since then. Peak radioactivity concentrations for ³ H ribavirin and (= NH) -modified ³ H ribavirin have a half-life T _1/2 of about 1998 hours and 577 hours, respectively. After multiple doses at 30 mg / kg, steady-state radioactivity concentrations were significantly higher for ribavirin compared to (= NH) -modified ³ H ribavirin (Table 2).

³ H-ribavirin ³ H-(= NH) modified ribavirin Medium RBC radioactivity (mouse) Medium RBC radioactivity (monkey-single dose) Medium RBC radioactivity (monkey-multiple doses) 1.36 μg equivalent / g about 41 μg equivalent / g about 5089 μg equivalent / g 0.38 μg equivalent / g about 17 μg equivalent / g about 606 μg equivalent / g

Table 2: Differential Radioactivity Distribution of Ribavirin and (= NH) Modified Ribavirin in RBC

The data presented in Table 2 are also consistent with the results of the toxicity studies. Rhesus monkeys receiving ribavirin 60 mg / kg followed by 30 mg / kg per day for 10 days developed hemolytic anemia and showed a significant decrease in RBC. In contrast, monkeys receiving the same amount of modified ribavirin did not show any significant changes in RBC.

Based on the difference between portal portal vein and systemic plasma in monkeys with cannula in the portal vein following oral administration of ribavirin or modified ribavirin, hepatic radioactivity concentration after oral administration of modified ribavirin is about 50% higher than oral administration of ribavirin It was estimated to be high. Thus, only about 66% of the ribavirin dose will be needed to achieve the same liver concentration of ribavirin. Based on the lower RBC radioactivity (about 12%) and higher liver concentration (about 50%) for modified ribavirin compared to ribavirin, the treatment rate for modified ribavirin is estimated to be about 12 times that of ribavirin. Thus, modified ribavirin can be administered at a dose of about 65% of ribavirin to achieve approximately the same efficacy as ribavirin without substantially hemolytic anemia; It was contemplated that ribavirin modified to achieve higher efficacy than ribavirin without substantially hemolytic anemia can be administered at the same dose as ribavirin. Also, ribavirin modified to achieve the same therapeutic effect as ribavirin is only about 5% to 50%, preferably 20% to 50%, more preferably 10% to 15%, most preferably 5 of the ribavirin dose. It is contemplated that it may be administered at a dosage of% to 6%.

(e) In vitro deaminoation of (= NH) modified ribavirin with ribavirin

Adenosine deaminoase (ADA), isolated from calf intestine, was purchased from Boehringer Mannheim. The assay was performed in Dulbecco PBS buffer (Na ₂ HPO ₄ , 8 mM; KH ₂ PO ₄ , 1.5 mM; KCl, 2.7 mM; NaCl, 138 mM; pH 7.2) at room temperature (23 ° C.). UV spectra of (= NH) modified ribavirin and ribavirin (0.2 mM) were obtained and followed by hydrolytic deamination of (= NH) modified ribavirin to ribavirin using an absorbance difference at 240 nm. In the absence of enzymes, no spontaneous hydrolysis of modified ribavirin for 1.5 hours (= NH) was observed in buffer (pH 7.2) (data not shown), suggesting that the compound is very stable. Due to the limitations of the UV assay, spontaneous hydrolysis of viramidine will be less than 2.5 × 10 ⁻⁵ min ⁻¹ . Further experiments revealed that the addition of zinc ions into the buffer did not improve the spontaneous hydrolysis rate (data not shown).

In the presence of 0.2 μM ADA, deamination of (= NH) modified ribavirin was accelerated. The number of enzyme turnovers was estimated at about 2.5 min ⁻¹ under current assay conditions. Quadruple mass spectral analysis of the enzyme reaction product suggested that more than 75% (= NH) modified ribavirin was converted to ribavirin after overnight incubation of 0.2 mM (= NH) modified ribavirin with 0.5 μM ADA.

As such, certain embodiments and applications for improved specificity in the treatment of disease have been described. However, it will be apparent to those skilled in the art that many modifications other than those described above are possible without departing from the spirit of the invention. Accordingly, the content of the invention is not limited except as to the spirit of the appended claims. In addition, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted to refer to a component, component or step in a non-exclusive manner, which means that the referenced component, component or step is not explicitly referenced. It may be present or used in combination with a component, component or step.

Claims (19)

Identifying a drug with the desired pharmacological effect on the target cell; A method of increasing the selectivity of a drug with respect to pharmacological effects, including modifying the drug using a blocking group, wherein the blocking group is covalently bound to the drug through the nitrogen atoms in the blocking group, wherein the blocking group is Reducing the accumulation of drugs in cells other than the target cells and enzymatically removing them from the drug in the target cells. The method of claim 1, wherein the drug is selected from the group consisting of nucleotides, nucleotides, nucleotide analogues, and nucleotide analogues. The drug of claim 2, wherein the drug is 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide or 2-beta-D-ribofuranosyl-4-thiazolecarboxamide How to feature. The method of claim 1 wherein the blocking group comprises either or both of the primary amine and the secondary amine. The method of claim 1, wherein the blocking group is = NH. The method of claim 1 wherein the target cell is a hepatocyte. The method of claim 1, wherein the target cell is infected with a virus. The method of claim 1, wherein the target cell is a hyperproliferative cell. The method of claim 1, wherein the accumulation of the drug in cells other than the target cell comprises phosphorylation of the drug in cells other than the target cell. The method of claim 1, wherein the cells other than the target cells are red blood cells. The method of claim 1 wherein the enzymatic removal of the blocking group from the drug is catalyzed by an aminohydrolase. Recognizing that metabolic conversion of drugs in cells other than target cells causes damage to cells other than target cells; The blocking group is used to modify the drug (where the blocking group is covalently bound to the drug via nitrogen atoms in the blocking group, reduces metabolic conversion of the drug in cells other than the target cell, and enzymes from the drug in the target cell). Is cut into the enemy); A method of reducing cytotoxicity of a drug to cells other than the target cell, including administering the drug to a system comprising the target cell and cells other than the target cell, wherein the blocking group is covalently bound to the drug. . 13. The method of claim 12, wherein the drug is 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide and metabolic conversion comprises phosphorylation of the drug. The method of claim 12, wherein the cell other than the target cell is red blood cells. 13. The method of claim 12, wherein the blocking group is = NH. 13. The method of claim 12, wherein the damage comprises inhibition of inosine-5'-monophosphate dehydrogenase. Providing a drug, wherein metabolic conversion of the drug in a cell other than the target cell reduces the concentration of the drug in a system comprising cells other than the target cell and the target cell; The blocking group is used to modify the drug, wherein the blocking group is covalently bound to the drug via nitrogen atoms in the blocking group and reduces metabolic conversion of the drug in cells other than target cells; Drugs in a system comprising target cells and cells other than the target cells, including administering the drug to the system, wherein the blocking group is covalently bound to the drug and is enzymatically removed from the drug in the target cell How to reduce the dosage of. 18. The method of claim 17, wherein the system comprises a mammal. 18. The method of claim 17, wherein the drug is 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide and the blocking group is = NH.