WO2001054692A1 - Use of castanospermine and substituted-castanospermine compounds for treating hepatitis virus infections - Google Patents

Abstract

Compositions and methods are provided for treating hepatitis, flavivirus or pestivirus infections, especially hepatitis B or C virus infections, in mammals, especially humans.

Description

USE OF CASTANOSPERMINE AND SUBSTITUTED-CASTANOSPERMINE COMPOUNDS FOR TREATING HEPATITIS VIRUS INFECTIONS

CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority to provisional U.S. Appln. No. 60/178,777, filed

January 28, 2000.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to compositions and methods for treating hepatitis, flavivirus or pestitvirus infections, especially hepatitis B or C virus infections, in mammals, especially humans.

Description of Related Art

More than 40 million people worldwide are chronically infected with the hepatitis C virus (HCV), and this represents one of the most serious threats to public health in developed nations (Hoofnagle et al., New Engl. J. Med., 336:347-356, 1997). Hepatitis C infection is the cause of more than 10,000 deaths annually in the United States (Hepatitis C Treatment, Washington Post, November 11, 1997, at A2), a number that is expected to triple in the next twenty years in the absence of effective intervention. Chronic HCV also increases the risk of liver cancer. Persistent infection develops in as many as 85% of HCV patients and in at least 20% of these patients the chronic infection leads to cirrhosis within twenty years of onset of infection. With an estimated 3.9 million North Americans chronically infected, complications from hepatitis C infection are now the leading reasons for liver transplantation in the United States.

Therapeutic interventions that are effective for treatment of HCV infection are limited in number and effectiveness. Standard treatment for HCV infection includes administration of interferon-alpha. However, interferon-alpha is of limited use in about 20% of the HCV- infected population (Hoofnagle et al., New Engl. J. Med., 336:397-356, 1997) and treatment with this compound results in long-term improvement in only 5% of patients. Furthermore, the complications and limitations of interferon-alpha seriously limit the applicability of the treatment. An experimental treatment comprising administration of interferon-alpha and ribavirin (l-β-D-ribofuranosyl-lH-l,2,4-triazole-3-carboxamide) resulted in long-term improvement in only half of the patients suffering a relapse of HCV infection (Hepatitis C Treatment, Washington Post, November 11,1997, at A2). Clearly, the disappointing results with interferon must prompt a search for more effective and less toxic therapeutics. Thus, a critical need remains for a therapeutic intervention that effectively treats HCV infection or supplements those otherwise available.

In addition to those people chronically infected with HCV, there are more than 350 million people chronically infected with hepatitis B virus (HBV). More than 150 million of these people are likely to die from liver disease in the absence of intervention. As many as

20 million HBV carriers reside in developed nations, as do most HCV carriers. A large number of individuals who are infected with HCV are also infected with

HBV. The therapy for combined HBV/HCV infection is particularly challenging because the HBV and HCV viruses differ from one another in therapeutically significant ways.

HBV is a hepadnavirus, while HCV is a pestivirus. HBV is a DNA-containing virus, the genome of which is replicated in the nucleus of the infected cell using a combination of a DNA-dependent RNA polymerase and an RNA-dependent DNA polymerase (i.e., a reverse transcriptase). HCV is an RNA containing virus, the genome of which is replicated in the cytoplasm of the infected cell using one or more types of RNA-dependent RNA polyme- rases. Despite the frequent concurrence of HBV infection and HCV infection, a number of compounds known to be effective for treating HBV infection are not effective against HCV. For example, lamivudine (the nucleoside analog 3TC) is useful for treating HBV infection, but is not useful for treating HCV infection. The difference in the susceptibility of HBV and HCV to antiviral agents no doubt relates to their genetically based replicative differences. There remains a particularly critical need for a therapeutic intervention that effectively treats both HBV and HCV infection. Hepatitis B virus (HBV or HepB) is a causative agent of acute and chronic liver disease including liver fibrosis, cirrhosis, inflammatory liver disease, and hepatic cancer that can lead to death in some patients (Joklik, Virology, 3^rd Ed., Appleton & Lange, Norwalk, Connecticut, 1988). Although effective vaccines are available, there are still more than 300 million people worldwide, i.e., 5% of the world's population, chronically infected with the virus (Locarnini et al., Antiviral Chemistry & Chemotherapy, 7:53-64, 1996). Such vaccines have no therapeutic value for those already infected with the virus. In Europe and North America, between 0.1% to 1% of the population is infected. Estimates are that 15% to 20% of individuals who acquire the infection develop cirrhosis or another chronic disability from HBV infection. Once liver cirrhosis is established, morbidity and mortality are substantial, with about a 5 -year patient survival period (Blume et al., Advanced Drug Delivery Reviews, 17:321-331, 1995). It is therefore necessary and of high priority to find improved and effective anti-HBV anti-hepatitis therapies (Locarnini et al., Antiviral Chemistry & Chemotherapy, 7:53-64, 1996).

Other hepatitis viruses significant as agents of human disease include hepatitis A, hepatitis Delta, hepatitis E, hepatitis F, and hepatitis G (Coates et al., Exp. Opin. Ther. Patents, 5:747-756, 1995). In addition, there are animal hepatitis viruses that are species specific. These include, for example, those infecting ducks, woodchucks, and mice. Other similarly related animal viruses can cause significant losses to the livestock industry (Sullivan et al., Virus Res., 38:231-239, 1995). These animal viruses include pestiviruses and flaviviruses such as bovine viral diarrhea virus (BVDV), classical swine fever virus, border disease virus, and hog cholera virus.

SUMMARY OF THE INVENTION The present invention discloses the use of castanospermine and substituted castano- spermine compounds disclosed herein.

Compositions of such compounds or pharmaceutically acceptable salts thereof are provided. Pharmaceutically acceptable salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydro- bromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmoate, pectinate, persulfate, 3-phenyl- propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, mesylate, and undecanoate .

Methods of treating a virus infection in a mammal or cell, inhibiting production (e.g., at least one of replication of viral genomes, transcription of viral genes, translation of viral proteins, and assembly of virus) of virus in an infected mammal or cell, or improving one or more symptoms of a virus-infected mammal are also provided comprising administering to the mammal or cell in need of treatment an anti-virus effective amount of at least one of a castanospermine compound (Formula I), substituted castanospermine compound (Formula II), and pharmaceutically acceptable salts thereof.

The virus can be a flavivirus, pestivirus, or hepatitis virus. Flaviviruses include, but are not limited to, a yellow fever virus, a dengue virus (e.g., dengue viruses 1-4), a Japanese encephalitis virus, a Murray Valley encephalitis virus, a Rocio virus, a West Nile fever virus, a St. Louis encephalitis virus, a tick-borne encephalitis virus, a Louping ill virus, a Powassan virus, an Omsk hemorrhagic fever virus, and a Kyasanur forest disease virus. Pestiviruses include, but are not limited to, hepatitis C virus (HCV), rubella virus, a bovine viral diarrhea virus (BVDV), a classical swine fever virus, a border disease virus, and a hog cholera virus.

Formula I

Formula II

or a pharmaceutically acceptable salt thereof wherein R is alkenyl, alkynyl, substituted aryl- alkyl, aryloxyalkyl, substituted aryloxyalkyl, haloalkyl, hydroxyalkyl, haloalkyloxyalkyl, carbonyl, cycloalkyloxyalkyl, cycloalkylalkyloxyalkyl, alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, arylalkylcarbonyl, substituted arylalkylcarbonyl, arylalkyloxycarbonyl, aryloxyalkylcarbonyl, substituted aryloxyalkylcarbonyl, haloalkylcarbonyl, hydroxyalkyl- carbonyl, haloalkyloxyalkylcarbonyl, cycloalkyloxyalkylcarbonyl, alkoxyalkylcarbonyl, or alkyloxycarbonyl.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the inhibiting effect of castanospermine (open circles) and N-nonyl- deoxynojirimycin (filled circles) on production of bovine viral diarrhea virus (BVDV) in Madin Darby bovine kidney (MDBK) cells. DESCRIPTION OF SPECIFIC EMBODIMENTS

Castanospermine (see Structure 1) (also known as CST) and certain derivatives are known inhibitors of the N-linked oligosaccharide processing enzymes alpha glucosidase I and II (Saunier et al., J. Biol.Chem., 257:14155-14161, 1982; Elbein, Ann. Rev. Biochem., 56:997-534, 1987).

Structure 1 As glucose analogs, they also have potential to inhibit glucose transport, glucosyl- transferases, and/or glycolipid synthesis (Newbrun et al., Arch. Oral Biol., 28:516-536, 1983; Wang et al., Tetrahedron Lett., 34:403-406, 1993). Their inhibitory activity against α- glucosidases has led to the development of these compounds as antiviral agents. See, for example, Antiviral Agents Bulletin, Volume 7, Number 3, March 1994, and Glycobiology, 5:243-247, 1995.

Yield Assay:

Madin Darby bovine kidney (MDBK) cells (2 x 10⁵ cells/well) were infected with bovine viral diarrhea virus (BVDV) at a multiplicity of infection (MOI) of 1 PFU/cell. At one hour post-infection (HPI) the virus innoculum was removed and the cells were washed twice with 1 ml of MDBK medium. After the second wash, 1 ml of MDBK medium supplemented with 0, 10, 25, 50, 100 or 200 μM N-nonyl-DNJ or 0, 5, 10, 50, 100 or 500 μM castanospermine (Sigma) was added to the cultures. The cultures were incubated at 37°C for 24 hrs. At 24 hours after infection, the cultures were harvested and stored at -70°C. The virus yield was measured by standard plaque assay on fresh MDBK monolayers.

Results

BVDV yield decreased with increasing concentrations of both N-nonyl-DNJ and castanospermine (Figure 1). The level of inhibition was approximately the same for both compounds. The IC50 values for castanospermine and N-nonyl-DNJ were approximately 4 μM and 8 μM, respectively.

Castanospermine decreased BVDV yield in infected MDBK cells in another two separate trials. The MOI was 0.01 PFU/cell. As seen below, inhibition was concentration dependent.

In Trial II, there was a 42-fold reduction in production of BVDV and a 1.63 log decrease at 100 μM. The IC50 was 7 μ and the IC90 was 42 μM. In Trial III, there was a 34- fold reduction in production of BVDV and a 1.53 log decrease at 100 μM. The IC50 was 4 μ and the IC90 was 65 μM. Combining the results from these two plaque assays, the log drop at 100 μM castanospermine was 1.58 and there was a 38-fold reduction in plaque production. The IC50 was 5.5 μM and the IC90 was 53.5 μM. Toxicity data using an MTS assay resulted in a CC10 of 100 μM, a CC50 of greater than 1 mM, and a CC90 of much greater than 1 mM for castanospermine. A PI assay resulted in 1.83% of cells dead with 1 mM castanospermine.

Conclusions

The results show that compounds of the invention are effective in inhibiting bovine viral diarrhea virus (BVDV) production in MDBK cells. BVDV, like HCV, is a small enveloped positive-stranded RNA virus that has generally been used by researchers as a surrogate virus for HCV due to the fact that HCV cannot be reliably propagated in tissue culture, nor in animals other than humans and chimpanzees. Like HCV, BVDV is a pestivirus that buds from the endoplasmic reticulum (Harasawa et al., Microb. Immunol., 39:979-985, 1995). BVDC is considered by virologists to be the closest biochemical surrogate of HCV for use in cell culture (Suzich et al., J. Virol., 67:6152-6158, 1993; Donis, Vet. Clinics N. Amer., 11 :393-423, 1995), and is recognized by leading experts, including informal statements from experts within the U.S. Food and Drug Administration, as an acceptable surrogate for HCV.

Compounds, pharmaceutically acceptable salts thereof, and compositions containing them may be used as active ingredients to make medicaments or other pharmaceutical formu- lations by known processes. They may be combined with a pharmaceutically acceptable carrier or vehicle, as well as any combination of optional additives (e.g., one or more binders, colorings, desiccants, diluents, excipients, stabilizers, or preservatives). See, Ullmann 's Encyclopedia of Industrial Chemistry, 6^th Ed. (electronic edition, 1998); Remington's Pharmaceutical Sciences, 22" (Gennaro, Mack Publishing, 1990); Pharmaceutical Dosage Forms, 2^nd Ed. (various editors, Marcel Dekker, 1989-1998); and Pharmaceutical Dosage Forms and Drug Delivery Systems (Ansel et al., Williams & Wilkins, 1994). The physical form of the formulation may be solid (e.g., granulate, powder, tablet, lozenge) or liquid (e.g., cream, emulsion, gel, lotion, ointment, paste, solution, suspension, syrup). Such formulations may contain other components to facilitate administration and/or enhance uptake (e.g., buffered saline, dimethyl sulfoxide, liposomes, micronized particles, nanoparticles). Good manufacturing practices are known in the pharmaceutical industry and regulated by government agencies (e.g., Food and Drug Administration). A sterile formulation may be prepared by dissolving a component of the formulation in a sufficient amount of an appropriate solvent, followed by filter sterilization to remove any contaminating microbes. Generally, dispersions are prepared by incorporating the various sterilized components of the formulation into a sterile vehicle which contains the basic dispersion medium. For production of solid forms that are required to be sterile, vacuum- or freeze- drying can be used.

Suitable choices in formulation, administration, and dosing can be made with the goals of achieving a favorable response in individuals with respect to the treated metabolic disorder (i.e., efficacy) and avoiding undue toxicity or other harm thereto (i.e., safety). The formulation is administered to an individual in an amount effective to treat the viral infection or achieve a desire effect (e.g., slow or reduce growth of a tumor or other neoplasm). The phrase "effective amount" refers to that amount of formulation necessary to achieve treatment or other desired effect. The term "treatment" refers to both therapeutic and prophylactic treatments. For example, one or more of slowing or reducing production of virus, reducing or alleviating symptoms in an individual, or preventing symptoms from worsening or progressing. For an individual, whether or not any of the foregoing objectives is achieved may be determined by any objective or subjective measure. Efficacy of the treatment may also be measured as an improvement in morbidity or mortality (e.g., lengthening of survival curve for a selected population). Prophylactic methods are also considered treatment. Effective treatment of cells may be determined by inhibition of virus production, lysis of cells, or other cell or biologic assays. The amount which is administered to an individual is preferably an amount that does not induce toxic effects which outweigh the advantages which result from its administration. Dosage levels of active ingredients in a pharmaceutical composition can also be varied so as to achieve a transient or sustained concentration of the compound or pharmaceutically acceptable salt thereof in an individual and to result in a desired biological response. But it is also possible to start doses at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. It should be understood that the specific dose level for any particular individual may depend on a variety of factors, including age, body weight, diet, gender, general health, and medical history. The dose level selected for use may also depend on the activity, bioavailability, hydrophobicity, molecular weight, pharmacokinetics, solubility, and stability of the compound or derivative thereof. Other factors include the type of viral infection being treated, route and scheduling of administration, severity of the disease being treated, and other drug or medical regimens being used.

A bolus administered over a short time once a day is a convenient dosing schedule. Alternatively, the effective daily dose may be divided into multiple doses for purposes of administration, for example, two to twelve doses per day. It is envisioned that a daily dosage may be between about one microgram to about one gram, or any range therebetween (e.g., about 1-50 mg, about 100-500 mg, or about 10-250 mg), of the compound or derivative thereof per kilogram body weight. Such quantities may be formulated as a unit dose (i.e., a dose sufficient for a single use once to several times per day).

A pharmaceutical composition may be administered by a mucosal, pulmonary, topi- cal, or other localized or systemic route (e.g., enteral or parenteral). The term "parenteral" includes subcutaneous, intraarterial, intraarticular, intradermal, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, and other injection or infusion techniques. Pharmaceutical compositions that are useful in the present invention may be administered as an aerosol, ophthalmic, oral, suppository, topical, or other formulation. All references (e.g., articles, books, patents, and patent applications) cited above are indicative of the level of skill in the art and are incorporated by reference.

All modifications and substitutions that come within the meaning of the claims and the range of their legal equivalents are to be embraced within their scope. Moreover, the term "comprising" allows the inclusion of other elements in the claim, the phrase "comprising essentially of allows the inclusion of other elements in the claim that do not materially affect operation of the invention, and no particular relationship between or among elements of a claim is meant unless such limitation is explicitly recited (e.g., arrangement of components in a product claim, order of steps in a method claim).

From the foregoing, it would be apparent to a skilled person that the invention can be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments should be considered only as illustrative, not restrictive, because the scope of the legal protection provided for the invention will be indicated by the appended claims rather than by this specification.

Claims

WHAT IS CLAIMED:

1. A method for treating a flavivirus or pestivirus infection in a mammal, comprising administering to the mammal an anti -virus effective amount of at least one compound of Formula II or a pharmaceutically acceptable salt thereof,

Formula II wherein R is selected from the group consisting of H, alkenyl, alkynyl, substituted arylalkyl, aryloxyalkyl, substituted aryloxyalkyl, haloalkyl, hydroxyalkyl, haloalkyloxyalkyl, carbonyl, cycloalkyloxyalkyl, cycloalkylalkyloxyalkyl, alkylcarbonyl, alkenylcarbonyl, alkynyl- carbonyl, arylalkylcarbonyl, substituted arylalkylcarbonyl, arylalkyloxycarbonyl, aryloxyalkylcarbonyl, substituted aryloxyalkylcarbonyl, haloalkylcarbonyl, hydroxyalkylcarbonyl, haloalkyloxyalkylcarbonyl, cycloalkyloxyalkylcarbonyl, alkoxyalkylcarbonyl, and alkyloxy- carbonyl.

2. The method of claim 1 , wherein the mammal is infected with a flavivirus.

3. The method of claim 1, wherein the mammal is infected with a virus selected from the group consisting of a yellow fever virus, a dengue virus, a Japanese encephalitis virus, a Murray Valley encephalitis virus, a Rocio virus, a West Nile fever virus, a St. Louis encepha- litis virus, a tick-borne encephalitis virus, a Louping ill virus, a Powassan virus, an Omsk hemorrhagic fever virus, and a Kyasanur forest disease virus.

4. The method of claim 1, wherein the mammal is infected with a pestivirus.

5. The method of claim 1, wherein the mammal is infected with hepatitis B virus.

6. The method of claim 1, wherein the mammal is infected with hepatitis C virus or a bovine diarrhea virus.

7. The method of any one of claims 1-6, wherein the mammal is administered an anti- virus effective amount of at least castanospermine.

8. The method of any one of claims 1-6, wherein the mammal is administered an anti- virus effective amount of at least a substituted castanospermine.

9. The method of claim 1 , wherein the pharmaceutically acceptable salt is selected from the group consisting of acetate, adipate, alginate, citrate, aspartate, benzoate, benzene- sulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxy- ethanesulfonate, lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, mesylate, and undecanoate.

10. A pharmaceutical composition is comprised of an anti-virus effective amount of at least one compound of Formula II or a pharmaceutically acceptable salt thereof, wherein R is selected from the group consisting of alkenyl, alkynyl, substituted arylalkyl, aryloxyalkyl, substituted aryloxyalkyl, haloalkyl, hydroxyalkyl, haloalkyloxyalkyl, carbonyl, cycloalkyl- oxyalkyl, cycloalkylalkyloxyalkyl, alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, arylalkylcarbonyl, substituted arylalkylcarbonyl, arylalkyloxycarbonyl, aryloxyalkylcarbonyl, substituted aryloxyalkylcarbonyl, haloalkylcarbonyl, hydroxyalkylcarbonyl, haloalkyloxy- alkylcarbonyl, cycloalkyloxyalkylcarbonyl, alkoxyalkylcarbonyl, and alkyloxycarbonyl.

11. The composition of claim 10, wherein the composition is comprised of an anti-virus effective amount of at least castanospermine.

12. The composition of claim 10, wherein the composition is comprised of an anti-virus effective amount of at least a substituted castanospermine.

13. The composition of claim 10, wherein the pharmaceutically acceptable salt is selected from the group consisting of acetate, adipate, alginate, citrate, aspartate, benzoate, benzene- sulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxy- ethanesulfonate, lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, mesylate, and undecanoate.