New HCV Drugs Can Lead to Rapid Drug Resistance

[Guest guest]

New HCV Drugs Can Lead to Rapid Drug Resistance

3-Drug Synergistic Interactions of Small Molecular Inhibitors of

Hepatitis C Virus Replication

The Journal of Infectious Diseases Jan 1, 2008;197:42-45

Christian Grünberger, L. Wyles, A. Kaihara, and

T. Schooley

Department of Medicine, Division of Infectious Diseases, University

of California, San Diego, La Jolla

“….small molecular inhibitors of HCV have demonstrated

substantial potency but also a propensity for rapid (i.e., within a

few days) viral escape when agents are used singly…. it is quite

conceivable that >3 small molecular inhibitors will ultimately be

required for successful HCV therapy….â€

Note from Jules: it is important to bear in mind that a poor response

to peginterferon+ribavirin when used in combination with 1 oral HCV

drug may be a set u for the patient to develop rapid resistance to

the oral drug and viral failure. Ongoing phase III studies in

treatment-experienced patients with the new HCV protease inhibitor

VX950 will examine this question and provide further information.

Small molecular inhibitors of hepatitis C virus (HCV) replication

provide remarkable potency, but the rapid selection of resistance

mutations will require that these agents be used in combination for

clinical treatment. Using a model HCV replicon system, we have

extended prior in vitro studies of double combinations of candidate

small molecular inhibitors to studies evaluating the simultaneous use

of 3 agents. This was done in an effort to anticipate conditions that

might ultimately be required clinically. We formally demonstrate

synergistic antiviral activity with 3-drug combinations in this

model, further supporting the concept of clinical investigations of

combination therapy for HCV infection.

Hepatitis C virus (HCV) infection is a major cause of chronic

hepatitis, liver cirrhosis, and hepatocellular carcinoma, with >170

million individuals infected worldwide. A substantial increase in

hospitalizations and medical costs related to chronic HCV is

predicted over the next 1-2 decades. Therapy with pegylated

interferon plus ribavirin can clear the virus in 50% of persons

infected with genotype 1 HCV infection, the most commonly encountered

genotype in North America [1]. Toxicities and contraindications to

interferon-based therapy prevent most patients who would otherwise be

candidates for treatment from initiating and/or completing a

treatment course. Thus, success rates for potential treatment

candidates (as opposed to those completing a course of therapy) are

<50% [2]. These limitations in interferon-based regimens have spurred

an active search for small molecular inhibitors of HCV that could

increase response rates over the short term when used with current

therapy. Ultimately, it is possible that combinations of small

molecular inhibitors could lead to efficacious interferon-free

regimens. Clinical trials with several candidate molecules have

demonstrated substantial short-term reductions in the levels of HCV

RNA in plasma [3, 4]. As expected, viral isolates with reduced

susceptibility have emerged rapidly [5]. The avoidance or delay of

drug resistance in HIV therapeutics has been achieved by the

simultaneous use of several potent agents that collectively require

the virus to develop multiple resistance mutations [6]. Clinical

trials that established the contemporary treatment paradigm were

anticipated by in vitro studies that modeled 2- or 3-drug combination

therapy in cell culture [7, 8]. These studies were critical in the

planning of clinical trials of combination therapy and provided early

indications that some combinations of agents were likely to be

antagonistic when used together [9]. In similar studies of the HCV

replicon system, we have recently reported in vitro additivity or

synergy between pairs of agents directed at HCV. In particular,

combinations that targeted distinct viral proteins showed greater

synergy [10]. In the present report, we extend our 2-drug-combination

studies, to examine whether additivity and/or synergy among anti-HCV

agents could be demonstrated in cell culture when used in 3-drug

combinations. Because the available protease inhibitors (PIs) all

target the active site of the NS3-4A serine protease, whereas the

available polymerase inhibitors target different sites on the NS5B

polymerase, we analyzed the interactions of combinations of 1 PI and

2 polymerase inhibitors.

Materials and methods. To evaluate the synergistic effects of

triplet combinations of small molecular inhibitors of HCV, we used

the BM4-5 replicon system [11], previously used in studies in our

laboratory [10]. The firefly luciferase gene was inserted into the

BM4-5 replicon, in a manner described elsewhere [12], to create a

luciferase/neomycin phosphotransferase fusion protein (designated

“Feoâ€) and the replicon (BM4-5-Feo). Experiments were performed

exactly as described by Wyles et al. [10]. In brief, cells were

seeded into 96-well plates and incubated with compounds for 48 h. The

luciferase assay (Bright-Glo; Promega) was performed according to the

manufacturer's instructions. Relative light units for each condition

were determined by use of a microplate luminometer (Veritas

Microplate Luminometer; Biosystems) and were reported as the

mean ± SE for 3 wells. The tested compounds included 2

peptidomimetic HCV PIs-BILN 2061 and a Vertex PI (kindly provided by

Vicki Sato of Vertex Pharmaceuticals); 1 GlaxoKline trans-lactam

PI active-site mimic (kindly provided by Romines of

GlaxoKline); 1 nucleoside analog HCV-RNA-dependent RNA

polymerase inhibitor, 2'-C-methyladenosine (kindly provided by

Lee of Gilead Sciences); 1 GlaxoKline benzo-thiadiazine

RNA polymerase nonnucleoside inhibitor (NNI) (kindly provided by

Romines of GlaxoKline); and 1 Wyeth benzofuran RNA

polymerase NNI (kindly provided by Daria Hazuda of Merck) [13]. The

Wyeth and GlaxoKline NNIs occupy unique sites in the polymerase.

The IC50 of each compound was determined independently and was used

to determine the range of concentrations used for the synergy

experiments. Each compound was tested both singly and in combination,

at 2 2-fold serial dilutions above and below the IC50. The ratio of

the 3 tested compounds remained fixed across the dosing range.

Determinations of compound interactions were based on the median-

effect principle and on the multiple-drug-effect equation, as

described by Chou and Talalay [14]. Combination indices were

determined, by use of Calcusyn (Biosoft), at the IC50, IC70, and IC90

levels. A total of 9 combinations were evaluated, with 3-5 replicates

per condition. A combination index of <0.9 was considered

synergistic, a combination index of >0.9 or <1.1 was considered

additive, and a combination index of >1.1 was considered

antagonistic. The 50% , 70%, and 90% combination indices are at the

IC50, IC70, and IC90 concentrations of each drug, respectively.

Cytotoxicity was assessed for each 3-compound combination, at the

highest concentrations tested, by use of an MTS assay (CellTiter 96;

Promega).

Results. Each inhibitor showed antiviral activity in our genotype 1

replicon system (figure 1). The observed IC50 value for each of the

individual compounds is listed in figure 1. Each combination that was

tested demonstrated synergy at the IC50, IC70, and IC90

concentrations (figure 2). At the highest concentrations used in the

studies, none of the compounds or combinations exhibited cytotoxicity

(figure 3).

Conclusions.

HCV shares several key biological similarities with HIV-1, thereby

allowing for the rapid evolution of viral-resistance mutations under

selective pressure [15]; however, unlike HIV-1, HCV replication does

not involve a DNA intermediate, a feature that has redirected

therapeutic strategies toward viral elimination rather than life-long

suppressive therapy. The profound reductions in morbidity and

mortality that accompanied the advent of highly active antiretroviral

therapy in the mid-1990s depended on the development of combination

antiretroviral regimens that were capable of durable viral

suppression [16]. Early clinical trials with small molecular

inhibitors of HCV have demonstrated substantial potency but also a

propensity for rapid (i.e., within a few days) viral escape when

agents are used singly [5]. On the basis of these results of

antiretroviral therapy, we investigated triple combinations of small

molecular inhibitors of HCV replication, focusing on combinations of

drugs targeting different sites of action.

We have demonstrated that these 3-drug combinations exhibit synergy

in vitro, as was the case in analogous studies that preceded the

clinical investigation of highly active antiretroviral therapy [8].

Of the compounds used in these studies, several are in the same

chemical class and possess a mechanism of action that is similar to

that found in the compounds being developed for clinical use. The

Vertex PI used in these studies is a close structural analog of VX-

950, which has progressed through phase 2 human trials. The NS5B

polymerase inhibitors used in these studies, both nucleoside and

nonnucleosides, are members of chemical classes of compounds that are

being actively developed as HCV therapeutics. They share common

targets and resistance mutations with therapeutic candidates and, as

such, are likely to be reasonable surrogates for use in in vitro

studies.

Although there are many similarities between HCV and HIV, there are

also substantial differences in biology, and approaches that were

successful in the case of HIV therapy will undoubtedly require

modification for treatment of HCV infection. HCV replicates to a

higher level, and its protease pocket represents a target that is

much less attractive than the HIV protease; it is quite conceivable

that >3 small molecular inhibitors will ultimately be required for

successful HCV therapy. Nonetheless, as in the case of HIV

therapeutics, systematic exploitation of relevant in vitro systems

are likely to provide extremely useful information that can be used

for generating hypotheses for clinical trials. These studies provide

further support for the concept that combinations of multiple small

molecular inhibitors of HCV replication should be evaluated in

rigorous clinical trials, with the view that this approach might

ultimately lead to the development of interferon-free regimens that

can eliminate HCV from persons infected with it.

[Guest guest]

Oh aint this great?!! NOT wrote: New HCV

Drugs Can Lead to Rapid Drug Resistance3-Drug Synergistic Interactions of Small Molecular Inhibitors of Hepatitis C Virus ReplicationThe Journal of Infectious Diseases Jan 1, 2008;197:42-45Christian Grünberger, L. Wyles, A. Kaihara, and T. SchooleyDepartment of Medicine, Division of Infectious Diseases, University of California, San Diego, La Jolla“….small molecular inhibitors of HCV have demonstrated substantial potency but also a propensity for rapid (i.e., within a few days) viral escape when agents are used singly…. it is quite conceivable that >3 small molecular inhibitors will ultimately be required for successful HCV therapy….â€Note from Jules: it is important to bear in mind that a poor response to peginterferon+ribavirin when used in combination with 1 oral HCV drug may be a set u for the patient to develop rapid resistance to the oral drug

and viral failure. Ongoing phase III studies in treatment-experienced patients with the new HCV protease inhibitor VX950 will examine this question and provide further information.Small molecular inhibitors of hepatitis C virus (HCV) replication provide remarkable potency, but the rapid selection of resistance mutations will require that these agents be used in combination for clinical treatment. Using a model HCV replicon system, we have extended prior in vitro studies of double combinations of candidate small molecular inhibitors to studies evaluating the simultaneous use of 3 agents. This was done in an effort to anticipate conditions that might ultimately be required clinically. We formally demonstrate synergistic antiviral activity with 3-drug combinations in this model, further supporting the concept of clinical investigations of combination therapy for HCV infection.Hepatitis C virus (HCV)

infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma, with >170 million individuals infected worldwide. A substantial increase in hospitalizations and medical costs related to chronic HCV is predicted over the next 1-2 decades. Therapy with pegylated interferon plus ribavirin can clear the virus in 50% of persons infected with genotype 1 HCV infection, the most commonly encountered genotype in North America [1]. Toxicities and contraindications to interferon-based therapy prevent most patients who would otherwise be candidates for treatment from initiating and/or completing a treatment course. Thus, success rates for potential treatment candidates (as opposed to those completing a course of therapy) are <50% [2]. These limitations in interferon-based regimens have spurred an active search for small molecular inhibitors of HCV that could increase response rates over the

short term when used with current therapy. Ultimately, it is possible that combinations of small molecular inhibitors could lead to efficacious interferon-free regimens. Clinical trials with several candidate molecules have demonstrated substantial short-term reductions in the levels of HCV RNA in plasma [3, 4]. As expected, viral isolates with reduced susceptibility have emerged rapidly [5]. The avoidance or delay of drug resistance in HIV therapeutics has been achieved by the simultaneous use of several potent agents that collectively require the virus to develop multiple resistance mutations [6]. Clinical trials that established the contemporary treatment paradigm were anticipated by in vitro studies that modeled 2- or 3-drug combination therapy in cell culture [7, 8]. These studies were critical in the planning of clinical trials of combination therapy and provided early indications that some combinations of

agents were likely to be antagonistic when used together [9]. In similar studies of the HCV replicon system, we have recently reported in vitro additivity or synergy between pairs of agents directed at HCV. In particular, combinations that targeted distinct viral proteins showed greater synergy [10]. In the present report, we extend our 2-drug-combination studies, to examine whether additivity and/or synergy among anti-HCV agents could be demonstrated in cell culture when used in 3-drug combinations. Because the available protease inhibitors (PIs) all target the active site of the NS3-4A serine protease, whereas the available polymerase inhibitors target different sites on the NS5B polymerase, we analyzed the interactions of combinations of 1 PI and 2 polymerase inhibitors.Materials and methods. To evaluate the synergistic effects of triplet combinations of small molecular inhibitors of HCV, we used the BM4-5

replicon system [11], previously used in studies in our laboratory [10]. The firefly luciferase gene was inserted into the BM4-5 replicon, in a manner described elsewhere [12], to create a luciferase/neomycin phosphotransferase fusion protein (designated “Feoâ€) and the replicon (BM4-5-Feo). Experiments were performed exactly as described by Wyles et al. [10]. In brief, cells were seeded into 96-well plates and incubated with compounds for 48 h. The luciferase assay (Bright-Glo; Promega) was performed according to the manufacturer's instructions. Relative light units for each condition were determined by use of a microplate luminometer (Veritas Microplate Luminometer; Biosystems) and were reported as the mean ± SE for 3 wells. The tested compounds included 2 peptidomimetic HCV PIs-BILN 2061 and a Vertex PI (kindly provided by Vicki Sato of Vertex Pharmaceuticals); 1 GlaxoKline trans-lactam PI

active-site mimic (kindly provided by Romines of GlaxoKline); 1 nucleoside analog HCV-RNA-dependent RNA polymerase inhibitor, 2'-C-methyladenosine (kindly provided by Lee of Gilead Sciences); 1 GlaxoKline benzo-thiadiazine RNA polymerase nonnucleoside inhibitor (NNI) (kindly provided by Romines of GlaxoKline); and 1 Wyeth benzofuran RNA polymerase NNI (kindly provided by Daria Hazuda of Merck) [13]. The Wyeth and GlaxoKline NNIs occupy unique sites in the polymerase. The IC50 of each compound was determined independently and was used to determine the range of concentrations used for the synergy experiments. Each compound was tested both singly and in combination, at 2 2-fold serial dilutions above and below the IC50. The ratio of the 3 tested compounds remained fixed across the dosing range. Determinations of compound interactions were based on the median-effect

principle and on the multiple-drug-effect equation, as described by Chou and Talalay [14]. Combination indices were determined, by use of Calcusyn (Biosoft), at the IC50, IC70, and IC90 levels. A total of 9 combinations were evaluated, with 3-5 replicates per condition. A combination index of <0.9 was considered synergistic, a combination index of >0.9 or <1.1 was considered additive, and a combination index of >1.1 was considered antagonistic. The 50% , 70%, and 90% combination indices are at the IC50, IC70, and IC90 concentrations of each drug, respectively. Cytotoxicity was assessed for each 3-compound combination, at the highest concentrations tested, by use of an MTS assay (CellTiter 96; Promega).Results. Each inhibitor showed antiviral activity in our genotype 1 replicon system (figure 1). The observed IC50 value for each of the individual compounds is listed in figure 1. Each combination

that was tested demonstrated synergy at the IC50, IC70, and IC90 concentrations (figure 2). At the highest concentrations used in the studies, none of the compounds or combinations exhibited cytotoxicity (figure 3).Conclusions. HCV shares several key biological similarities with HIV-1, thereby allowing for the rapid evolution of viral-resistance mutations under selective pressure [15]; however, unlike HIV-1, HCV replication does not involve a DNA intermediate, a feature that has redirected therapeutic strategies toward viral elimination rather than life-long suppressive therapy. The profound reductions in morbidity and mortality that accompanied the advent of highly active antiretroviral therapy in the mid-1990s depended on the development of combination antiretroviral regimens that were capable of durable viral suppression [16]. Early clinical trials with small molecular inhibitors of HCV have

demonstrated substantial potency but also a propensity for rapid (i.e., within a few days) viral escape when agents are used singly [5]. On the basis of these results of antiretroviral therapy, we investigated triple combinations of small molecular inhibitors of HCV replication, focusing on combinations of drugs targeting different sites of action.We have demonstrated that these 3-drug combinations exhibit synergy in vitro, as was the case in analogous studies that preceded the clinical investigation of highly active antiretroviral therapy [8]. Of the compounds used in these studies, several are in the same chemical class and possess a mechanism of action that is similar to that found in the compounds being developed for clinical use. The Vertex PI used in these studies is a close structural analog of VX-950, which has progressed through phase 2 human trials. The NS5B polymerase inhibitors used in these studies,

both nucleoside and nonnucleosides, are members of chemical classes of compounds that are being actively developed as HCV therapeutics. They share common targets and resistance mutations with therapeutic candidates and, as such, are likely to be reasonable surrogates for use in in vitro studies.Although there are many similarities between HCV and HIV, there are also substantial differences in biology, and approaches that were successful in the case of HIV therapy will undoubtedly require modification for treatment of HCV infection. HCV replicates to a higher level, and its protease pocket represents a target that is much less attractive than the HIV protease; it is quite conceivable that >3 small molecular inhibitors will ultimately be required for successful HCV therapy. Nonetheless, as in the case of HIV therapeutics, systematic exploitation of relevant in vitro systems are likely to provide extremely

useful information that can be used for generating hypotheses for clinical trials. These studies provide further support for the concept that combinations of multiple small molecular inhibitors of HCV replication should be evaluated in rigorous clinical trials, with the view that this approach might ultimately lead to the development of interferon-free regimens that can eliminate HCV from persons infected with it.Jackie