- Email: [email protected]
3rd International Conference on Therapies for Viral Hepatitis
3rd International Conference on Therapies for Viral Hepatitis
3rd International Conference on Therapies for Viral Hepatitis Maui HI, USA, 12–16 December 1999
gives rise to mutations; mutations give rise to genetic diversity; selective pressure applied by drug therapy selects those polymorphs whose replication fitness is increased in this particular environment (survival of the fittest); the drug-resistant variants become the predominant virus as long as the selective pressure is there; once the selective pressure is removed, the wild-type virus again becomes the fittest species and emerges as the predominant virus.To reduce the risk of selecting drug-resistant variants in patients, one must: 1.
Robert W. King Virology Group, DuPont Pharmaceuticals Co.,Wilmington, DE, USA
2. Abstract This meeting was attended by several hundred delegates and covered, in lecture and poster presentations, recent developments in basic, applied and clinical research as they concern antiviral drug development for hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. Below are summaries of presentations that are relevant to the development of antiviral drug resistance. 2000 Harcourt Publishers Ltd
3.
Raise the virological barrier: use potent drugs and/or drug combinations to inhibit virus replication as much as possible. Using monotherapy or a therapy regimen that is barely effective is just asking for trouble. Raise the genetic barrier: use drugs that require the virus to acquire multiple mutations in order to develop resistance or use combination therapy with compounds that have different resistance patterns (multiple targets or different sites on the same target). Raise the pharmacological barrier: use drugs or combinations of drugs that give the patient best chance for compliance (i.e. fewer pills, fewer times a day).Treat patients early, the healthier the patient, the less chance that they will develop resistance.
ANTIVIRAL DRUG RESISTANCE oug Richman (University of California at San Diego, USA), a well-known AIDS clinician, reviewed what has been learned from studying drug resistance in human immunodeficiency virus (HIV) and compared that to what is being done in the viral hepatitis fields. His conclusion was that those developing antiviral drugs for HBV and HCV infection need to better apply the lessons that were learned from HIV. Using HCV as his example, Dr Richman stated that the virus-encoded RNA-dependent RNA polymerase (RDRP), having a mutation rate of 1 × 10–5 nucleotides/replication cycle, causes at least one mutation/genome/replication cycle. Most of these mutations give rise to variants that are genetically unfit resulting in an inability or greatly reduced ability to replicate. However, those mutations that are not lethal to the virus result in a genetically mixed population known as the quasispecies. Those polymorphs that are selected for by antiviral drug treatment are then classified as drug resistant variants. Drug resistant viruses can be classified into three categories. The first are variants with a single mutation that causes a major reduction in the virus’ susceptibility to the drug.The second is the variant that has more than one mutation, all of which are required to a change in a virus’ susceptibility to the drug. Historically, the mutations in categories one and two usually also result in a reduced replication fitness of the virus.The third class of variants is characterized by having multiple mutations, like the second class; however, one or more of these mutations are compensatory in that they relieve the fitness disadvantage of the mutation responsible for drug resistance. What affects the likelihood that a drug resistant variant will be selected? According to Richman, this depends on two factors: (1) the level of selective pressure applied; and (2) the magnitude of viral replication. Selection of drug resistant variants follows simple Darwinian evolution.Viral replication
D
ANTIVIRAL DRUG RESISTANCE IN THE CLINICAL SETTING Interferon resistance and HCV The only two approved treatments for chronic hepatitis are interferon-α (IFNα) alone or in combination with the nucleoside analog, ribavirin.A sustained antiviral response is seen in approximately 25% of patients after 12 months of treatment with IFN alone and is increased to approximately 40% when ribavirin is added to the treatment regimen.Avidan Neumann (BarIlan University, Israel) discussed that the success or failure of a patient to respond to treatment has been attributed to virus genotype, and race and sex of the patient. It is thought that people chronically infected with HCV genotype 1a have a higher incidence of failure to IFN than those infected with genotype 1b. Lieven Stuyver (Pharmasset Inc., USA) speculated that the amino-acid sequence of the HCV NS5A protein, for which no function has yet been described, might be important for determining IFN responsiveness. No single amino-acid change has been shown to be able to affect IFN responsiveness and it may be that several amino-acid change must take place in order to affect IFN responsiveness. Recent clinical reports have shown that African-American and Asians have a reduced response rate to treatment with IFN relative to Caucasians. It is thought that the lower percentage of responders to IFN in the HCV-infected Asian population may be due more to geographical variations in the virus than genetic differences in the patient population. However, this is not the case for the chronically infected African-American population. When the percentage of responders is compared in HCV-infected African-Americans and Caucasians from the same geographical area, thereby eliminating regional differences in virus genotypes, there is a statistically significant discrepancy with African-Americans 2000 Harcourt Publishers Ltd Drug Resistance Updates (2000) 3, 55–57 DOI: 10.1054/drup.2000.0118, available online at http://www.idealibrary.com on
55
King Table 1 Sensitivity of different HBV variants to nucleoside analog inhibitors of the HBV reverse transcriptase Reduced sensitivity to nucleoside analogue Compound Lamivudine Emtricitabine Clevudine DAPD
Wild-type
L528M
M552V/I
L528M/M552V/I
No No No No
Yes Yes No No
Yes Yes No No
Yes Yes Yes No
responding at a lower rate than Caucasians. Studies performed at Loyola University at Chicago have shown that this discrepancy disappears if patients are treated with a combination of IFN and ribavirin. Moreover,African-Americans and Caucasians with chronic HCV that did not respond to initial therapy with IFN alone had a similar and positive response to retreatment wi th IFN plus ribavirin. However, AfricanAmerican chronic carriers of HCV also experienced a higher incidence of severe side-effects to the combination therapy than their Caucasian counterparts. The response to IFN therapy in chronic carriers of HCV has been shown to be better in females than in males. The mechanisms responsible for this difference are unclear. A recent study with pre- and post-menopausal females and agematched males has led to the hypothesis that the elevated level of estrogen in women may be responsible for their increased responsiveness to IFN treatment. In this study, the response rate for men and women at the end of treatment was 57% and 69%, respectively. However, when the female population was separated on the basis of ovarian function, 74% of the pre-menopausal women responded to IFN treatment, whereas only 47% of the post-menopausal women did, suggesting that the estrogen levels in chronic carriers of HCV may influence responsiveness. Nucleoside analogue resistance and HBV The discovery of novel inhibitors of HBV replication, as well as the study of drug-resistance, has been hampered by the lack of a good in vitro system to model HBV replication. Other than IFN, which is approved for the treatment of chronic carriers of HBV, the only other compounds in the clinic are nucleoside analogs that were originally developed as antiviral agents against other unrelated viruses. Lamivudine. Lamivudine ([–]-β-L-2′, 3′-dideoxy-3′-thiacytidine; 3TC) is a cytosine analog that has potent antiviral activity against the reverse transcriptase (RT) of HIV and the hepadnaviruses. In chronic carriers of HBV, lamivudine produces a rapid and profound decrease in the level of serum HBV DNA and a normalization of alanine amino transferase (ALT) levels. However, between 15 and 40% of patients develop resistance to the drug after long-term therapy (1–3 years of treatment, respectively). The mutations responsible for lamivudine resistance have been mapped to the highly conserved YMDD nucleotidebinding motif in the RT gene of HBV.These mutations cause a 56
Drug Resistance Updates (2000) 3, 55–57
2000 Harcourt Publishers Ltd
valine or isoleucine substitution for the methionine normally found in the YMDD motif (M552V/I). In addition, this substitution can be found in conjunction with L528M or F514C/L528M substitutions. Nathaniel Brown (Glaxo Wellcome, USA) explained that the substitution at M552 alters the shape of the nucleotide-binding pocket, which reduces the binding affinity for lamivudine, thereby decreasing drug potency. Interestingly, Dr Brown pointed out that even though long-term lamivudine therapy may select for genotypic and phenotypic resistance, we do not yet know the clinical relevance of these variants. On average it takes 25 years to see a clinical manifestation of wild type HBV and we have only had an opportunity to study the lamivudine-resistant variants for less than 5 years. Emtricitabine. Emtricitabine (an oxanthiolane analog of lamivudine; -FTC) exhibits similar potency as lamivudine against wild-type HBV in cell culture; however, it is more potent against the M5521 and L528M/M5521 variants. In 2.2.15 cells, a hepatoblastoma cell line that constitutively produces HBV, emtricitabine acted synergistically with the nucleoside analogs (–) -β-D-2,6-diaminopurine dioxolane (DAPD), 2′-fluoro-5-methyl-β-L-arabinofuranosyl uracil (clevudine; LFMAU), and penciclovir to inhibit HBV replication. In the clinic, chronic carriers of HBV treated with 300 mg/day of emtricitabine showed a rapid improvement in serum viral load and ALT levels.The clinical trials with emtricitabine have not been conducted long enough to make any conclusions concerning the development of resistance. Clevudine. Clevudine (2′-fluoro-5-methyl-β-L-arabinofuranosyul uracil; L-FMAU), a uracil analogue, has been shown to be a potent inhibitor of HBV and Epstein-Barr virus; however, it has no activity against other herpesvirus or HIV. In cell culture, clevudine acted synergistically with lamivudine, emtricitabine, DAPD and penciclovir to inhibit HBV replication. In addition, clevudine is active against the L528M and M552V/I single mutation variants but demonstrated reduced potency against the L528M/M552V/I double mutation variant (Table 1).This compound is in preclinical development. Dioxolane nucleoside. DAPD, which is a prodrug of (–)-β-Ddioxolane-guanine (DXG), is a guanosine analogue with activity against HBV and HIV. It does not show a reduction in potency against the L528M, M552V/I or L528M/M552V/I variants. In 2.2.15 cells DAPD acted synergistically with lamivudine, emtricitabine and clevudine, but antagonistically with penciclovir to inhibit HBV replication.This compound is in preclinical development. CONCLUSION As we have learned from the development of antiviral drug resistance in HIV, the sooner we understand the development of resistance to a particular compound or class of compounds, the better off the patient will be. One of the main factors contributing to the development of resistant HBV and HCV variants in the clinic is that, as of yet, we have very few drugs for treating people chronically infected with either virus. Therefore, we do not have the opportunity to treating patients with a combination of compounds that will enhance the beneficial aspects of treatment and reduce the chances of developing resistant strains.
3rd International Conference on Therapies for Viral Hepatitis For HCV, a lot of effort is directed to finding compounds that inhibit the viral proteases, helicase, RNA-dependent RNA polymerase, as well as inhibiting the cap-independent translation process that the virus uses to produce its polyprotein. As this work comes to fruition, it is hoped that there will be several potent and specific compounds that clinicians can choose from to treat their patients. Likewise, for HBV, there is a need for a more diverse selection of inhibitors of virus replication.Along with the lack of a complete cell-based system to model HBV replication, the limited number of targets available for drug discovery for HBV has hampered progress in this area (the HBV genome only encodes one enzymatic protein, the polymerase). Screening non-nucleoside chemical libraries in the 2.2.15 or HepAD38 cell lines may provide some interesting classes of HBV inhibitors. In fact, Stephen Locarnini (Victorian Infectious Disease Reference Laboratories, Australia) discussed a compound, AT-61, that was discovered at Avid Therapeutics Inc., by screening their chemical library in the
HepAD38 cell line.This compound, presently under development by Triangle Pharmaceuticals, is a potent and specific inhibitor of HBV replication and is thought to work by inhibiting the packaging of viral RNA into immature Dane particle. In addition,AT-61 has been shown to act synergistically with lamivudine to inhibit HBV replication in HepAD38 cells and its potency is not affected by the amino-acid substitutions that are responsible for lamivudine resistance. Compounds like AT-61 may be ideal to use in combination with a nucleoside analog to treat chronically infected patients such that the therapeutic value of the compounds is enhanced and the development of resistance is prevented.
Correspondence to: Robert W. King, DuPont Pharmaceuticals Co., PO Box 80336,Wilmington, DE 19880–0336, USA,Tel: +1 302 695 9354; Fax: +1 302 695 3934; E-mail: [email protected]
2000 Harcourt Publishers Ltd Drug Resistance Updates (2000) 3, 55–57
57
3rd International Conference on Therapies for Viral Hepatitis Maui HI, USA, 12–16 December 1999
gives rise to mutations; mutations give rise to genetic diversity; selective pressure applied by drug therapy selects those polymorphs whose replication fitness is increased in this particular environment (survival of the fittest); the drug-resistant variants become the predominant virus as long as the selective pressure is there; once the selective pressure is removed, the wild-type virus again becomes the fittest species and emerges as the predominant virus.To reduce the risk of selecting drug-resistant variants in patients, one must: 1.
Robert W. King Virology Group, DuPont Pharmaceuticals Co.,Wilmington, DE, USA
2. Abstract This meeting was attended by several hundred delegates and covered, in lecture and poster presentations, recent developments in basic, applied and clinical research as they concern antiviral drug development for hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. Below are summaries of presentations that are relevant to the development of antiviral drug resistance. 2000 Harcourt Publishers Ltd
3.
Raise the virological barrier: use potent drugs and/or drug combinations to inhibit virus replication as much as possible. Using monotherapy or a therapy regimen that is barely effective is just asking for trouble. Raise the genetic barrier: use drugs that require the virus to acquire multiple mutations in order to develop resistance or use combination therapy with compounds that have different resistance patterns (multiple targets or different sites on the same target). Raise the pharmacological barrier: use drugs or combinations of drugs that give the patient best chance for compliance (i.e. fewer pills, fewer times a day).Treat patients early, the healthier the patient, the less chance that they will develop resistance.
ANTIVIRAL DRUG RESISTANCE oug Richman (University of California at San Diego, USA), a well-known AIDS clinician, reviewed what has been learned from studying drug resistance in human immunodeficiency virus (HIV) and compared that to what is being done in the viral hepatitis fields. His conclusion was that those developing antiviral drugs for HBV and HCV infection need to better apply the lessons that were learned from HIV. Using HCV as his example, Dr Richman stated that the virus-encoded RNA-dependent RNA polymerase (RDRP), having a mutation rate of 1 × 10–5 nucleotides/replication cycle, causes at least one mutation/genome/replication cycle. Most of these mutations give rise to variants that are genetically unfit resulting in an inability or greatly reduced ability to replicate. However, those mutations that are not lethal to the virus result in a genetically mixed population known as the quasispecies. Those polymorphs that are selected for by antiviral drug treatment are then classified as drug resistant variants. Drug resistant viruses can be classified into three categories. The first are variants with a single mutation that causes a major reduction in the virus’ susceptibility to the drug.The second is the variant that has more than one mutation, all of which are required to a change in a virus’ susceptibility to the drug. Historically, the mutations in categories one and two usually also result in a reduced replication fitness of the virus.The third class of variants is characterized by having multiple mutations, like the second class; however, one or more of these mutations are compensatory in that they relieve the fitness disadvantage of the mutation responsible for drug resistance. What affects the likelihood that a drug resistant variant will be selected? According to Richman, this depends on two factors: (1) the level of selective pressure applied; and (2) the magnitude of viral replication. Selection of drug resistant variants follows simple Darwinian evolution.Viral replication
D
ANTIVIRAL DRUG RESISTANCE IN THE CLINICAL SETTING Interferon resistance and HCV The only two approved treatments for chronic hepatitis are interferon-α (IFNα) alone or in combination with the nucleoside analog, ribavirin.A sustained antiviral response is seen in approximately 25% of patients after 12 months of treatment with IFN alone and is increased to approximately 40% when ribavirin is added to the treatment regimen.Avidan Neumann (BarIlan University, Israel) discussed that the success or failure of a patient to respond to treatment has been attributed to virus genotype, and race and sex of the patient. It is thought that people chronically infected with HCV genotype 1a have a higher incidence of failure to IFN than those infected with genotype 1b. Lieven Stuyver (Pharmasset Inc., USA) speculated that the amino-acid sequence of the HCV NS5A protein, for which no function has yet been described, might be important for determining IFN responsiveness. No single amino-acid change has been shown to be able to affect IFN responsiveness and it may be that several amino-acid change must take place in order to affect IFN responsiveness. Recent clinical reports have shown that African-American and Asians have a reduced response rate to treatment with IFN relative to Caucasians. It is thought that the lower percentage of responders to IFN in the HCV-infected Asian population may be due more to geographical variations in the virus than genetic differences in the patient population. However, this is not the case for the chronically infected African-American population. When the percentage of responders is compared in HCV-infected African-Americans and Caucasians from the same geographical area, thereby eliminating regional differences in virus genotypes, there is a statistically significant discrepancy with African-Americans 2000 Harcourt Publishers Ltd Drug Resistance Updates (2000) 3, 55–57 DOI: 10.1054/drup.2000.0118, available online at http://www.idealibrary.com on
55
King Table 1 Sensitivity of different HBV variants to nucleoside analog inhibitors of the HBV reverse transcriptase Reduced sensitivity to nucleoside analogue Compound Lamivudine Emtricitabine Clevudine DAPD
Wild-type
L528M
M552V/I
L528M/M552V/I
No No No No
Yes Yes No No
Yes Yes No No
Yes Yes Yes No
responding at a lower rate than Caucasians. Studies performed at Loyola University at Chicago have shown that this discrepancy disappears if patients are treated with a combination of IFN and ribavirin. Moreover,African-Americans and Caucasians with chronic HCV that did not respond to initial therapy with IFN alone had a similar and positive response to retreatment wi th IFN plus ribavirin. However, AfricanAmerican chronic carriers of HCV also experienced a higher incidence of severe side-effects to the combination therapy than their Caucasian counterparts. The response to IFN therapy in chronic carriers of HCV has been shown to be better in females than in males. The mechanisms responsible for this difference are unclear. A recent study with pre- and post-menopausal females and agematched males has led to the hypothesis that the elevated level of estrogen in women may be responsible for their increased responsiveness to IFN treatment. In this study, the response rate for men and women at the end of treatment was 57% and 69%, respectively. However, when the female population was separated on the basis of ovarian function, 74% of the pre-menopausal women responded to IFN treatment, whereas only 47% of the post-menopausal women did, suggesting that the estrogen levels in chronic carriers of HCV may influence responsiveness. Nucleoside analogue resistance and HBV The discovery of novel inhibitors of HBV replication, as well as the study of drug-resistance, has been hampered by the lack of a good in vitro system to model HBV replication. Other than IFN, which is approved for the treatment of chronic carriers of HBV, the only other compounds in the clinic are nucleoside analogs that were originally developed as antiviral agents against other unrelated viruses. Lamivudine. Lamivudine ([–]-β-L-2′, 3′-dideoxy-3′-thiacytidine; 3TC) is a cytosine analog that has potent antiviral activity against the reverse transcriptase (RT) of HIV and the hepadnaviruses. In chronic carriers of HBV, lamivudine produces a rapid and profound decrease in the level of serum HBV DNA and a normalization of alanine amino transferase (ALT) levels. However, between 15 and 40% of patients develop resistance to the drug after long-term therapy (1–3 years of treatment, respectively). The mutations responsible for lamivudine resistance have been mapped to the highly conserved YMDD nucleotidebinding motif in the RT gene of HBV.These mutations cause a 56
Drug Resistance Updates (2000) 3, 55–57
2000 Harcourt Publishers Ltd
valine or isoleucine substitution for the methionine normally found in the YMDD motif (M552V/I). In addition, this substitution can be found in conjunction with L528M or F514C/L528M substitutions. Nathaniel Brown (Glaxo Wellcome, USA) explained that the substitution at M552 alters the shape of the nucleotide-binding pocket, which reduces the binding affinity for lamivudine, thereby decreasing drug potency. Interestingly, Dr Brown pointed out that even though long-term lamivudine therapy may select for genotypic and phenotypic resistance, we do not yet know the clinical relevance of these variants. On average it takes 25 years to see a clinical manifestation of wild type HBV and we have only had an opportunity to study the lamivudine-resistant variants for less than 5 years. Emtricitabine. Emtricitabine (an oxanthiolane analog of lamivudine; -FTC) exhibits similar potency as lamivudine against wild-type HBV in cell culture; however, it is more potent against the M5521 and L528M/M5521 variants. In 2.2.15 cells, a hepatoblastoma cell line that constitutively produces HBV, emtricitabine acted synergistically with the nucleoside analogs (–) -β-D-2,6-diaminopurine dioxolane (DAPD), 2′-fluoro-5-methyl-β-L-arabinofuranosyl uracil (clevudine; LFMAU), and penciclovir to inhibit HBV replication. In the clinic, chronic carriers of HBV treated with 300 mg/day of emtricitabine showed a rapid improvement in serum viral load and ALT levels.The clinical trials with emtricitabine have not been conducted long enough to make any conclusions concerning the development of resistance. Clevudine. Clevudine (2′-fluoro-5-methyl-β-L-arabinofuranosyul uracil; L-FMAU), a uracil analogue, has been shown to be a potent inhibitor of HBV and Epstein-Barr virus; however, it has no activity against other herpesvirus or HIV. In cell culture, clevudine acted synergistically with lamivudine, emtricitabine, DAPD and penciclovir to inhibit HBV replication. In addition, clevudine is active against the L528M and M552V/I single mutation variants but demonstrated reduced potency against the L528M/M552V/I double mutation variant (Table 1).This compound is in preclinical development. Dioxolane nucleoside. DAPD, which is a prodrug of (–)-β-Ddioxolane-guanine (DXG), is a guanosine analogue with activity against HBV and HIV. It does not show a reduction in potency against the L528M, M552V/I or L528M/M552V/I variants. In 2.2.15 cells DAPD acted synergistically with lamivudine, emtricitabine and clevudine, but antagonistically with penciclovir to inhibit HBV replication.This compound is in preclinical development. CONCLUSION As we have learned from the development of antiviral drug resistance in HIV, the sooner we understand the development of resistance to a particular compound or class of compounds, the better off the patient will be. One of the main factors contributing to the development of resistant HBV and HCV variants in the clinic is that, as of yet, we have very few drugs for treating people chronically infected with either virus. Therefore, we do not have the opportunity to treating patients with a combination of compounds that will enhance the beneficial aspects of treatment and reduce the chances of developing resistant strains.
3rd International Conference on Therapies for Viral Hepatitis For HCV, a lot of effort is directed to finding compounds that inhibit the viral proteases, helicase, RNA-dependent RNA polymerase, as well as inhibiting the cap-independent translation process that the virus uses to produce its polyprotein. As this work comes to fruition, it is hoped that there will be several potent and specific compounds that clinicians can choose from to treat their patients. Likewise, for HBV, there is a need for a more diverse selection of inhibitors of virus replication.Along with the lack of a complete cell-based system to model HBV replication, the limited number of targets available for drug discovery for HBV has hampered progress in this area (the HBV genome only encodes one enzymatic protein, the polymerase). Screening non-nucleoside chemical libraries in the 2.2.15 or HepAD38 cell lines may provide some interesting classes of HBV inhibitors. In fact, Stephen Locarnini (Victorian Infectious Disease Reference Laboratories, Australia) discussed a compound, AT-61, that was discovered at Avid Therapeutics Inc., by screening their chemical library in the
HepAD38 cell line.This compound, presently under development by Triangle Pharmaceuticals, is a potent and specific inhibitor of HBV replication and is thought to work by inhibiting the packaging of viral RNA into immature Dane particle. In addition,AT-61 has been shown to act synergistically with lamivudine to inhibit HBV replication in HepAD38 cells and its potency is not affected by the amino-acid substitutions that are responsible for lamivudine resistance. Compounds like AT-61 may be ideal to use in combination with a nucleoside analog to treat chronically infected patients such that the therapeutic value of the compounds is enhanced and the development of resistance is prevented.
Correspondence to: Robert W. King, DuPont Pharmaceuticals Co., PO Box 80336,Wilmington, DE 19880–0336, USA,Tel: +1 302 695 9354; Fax: +1 302 695 3934; E-mail: [email protected]
2000 Harcourt Publishers Ltd Drug Resistance Updates (2000) 3, 55–57
57