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In Vitro Assessment of Re-treatment Options for Patients with Hepatitis C Virus Genotype 1b Infection Resistant to Daclatasvir Plus Asunaprevir.

Friborg J, Zhou N, Han Z, Yang X, Falk P, Mendez P, McPhee F - Infect Dis Ther (2014)

Bottom Line: Drug concentrations representing multiple 50% effective concentrations (EC50) derived in vitro and trough plasma concentrations observed in a clinical setting were utilized.Our in vitro results highlight a number of potential all-oral treatment options for patients who do not achieve a sustained virologic response following therapy with daclatasvir plus asunaprevir.These results require further evaluation in clinical studies.

View Article: PubMed Central - PubMed

Affiliation: Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, CT, 06492, USA.

ABSTRACT

Introduction: Daclatasvir is a non-structural protein 5A (NS5A) inhibitor with activity against hepatitis C virus (HCV) genotypes 1-6 in vitro, and asunaprevir is a non-structural protein 3 (NS3) protease inhibitor with activity against genotypes 1, 4, 5, and 6. This study evaluates potential options for the re-treatment of HCV genotype 1b-infected patients who have failed combination therapy with daclatasvir plus asunaprevir.

Methods: The antiviral activity of drug combination regimens in HCV subgenomic replicon cell lines representing genotype 1b (Con1 strain) wild-type or a variant with specific NS5A and NS3 amino acid substitutions conferring resistance to daclatasvir and asunaprevir were compared using replicon elimination assays. Drug concentrations representing multiple 50% effective concentrations (EC50) derived in vitro and trough plasma concentrations observed in a clinical setting were utilized.

Results: At multiple EC50 values of each drug (3×, 10×, and 30× EC50), combinations of daclatasvir plus sofosbuvir, sofosbuvir plus ledipasvir, sofosbuvir plus simeprevir, and sofosbuvir plus either a next-generation NS3 or NS5A inhibitor demonstrated comparable activity in wild-type and daclatasvir/asunaprevir-resistant cell lines. At clinically relevant drug trough concentrations, combination regimens of daclatasvir plus asunaprevir plus beclabuvir (±ribavirin), and daclatasvir plus asunaprevir plus beclabuvir plus sofosbuvir efficiently cleared daclatasvir + asunaprevir-resistant replicons from cells within 5 days of treatment.

Conclusion: Our in vitro results highlight a number of potential all-oral treatment options for patients who do not achieve a sustained virologic response following therapy with daclatasvir plus asunaprevir. These results require further evaluation in clinical studies.

No MeSH data available.


Related in: MedlinePlus

HCV replicon elimination assays with single agents and combination regimens using concentrations representing Ctrough values observed in a clinical setting in wild-type GT-1b (a and c) and DCV + ASV-resistant (NS3-D168V, NS5A-L31M-Y93H) replicon cell lines (b and d). Alfa peginterferon alfa, ASV asunaprevir, BCV beclabuvir, Ctrough trough plasma concentrations, DCV daclatasvir, DMSO, dimethyl sulfoxide, GT genotype, HCV hepatitis C virus, LDV ledipasvir, NS3 non-structural protein 3, NS5A non-structural protein 5A, RBV ribavirin, SMV simeprevir, SOF sofosbuvir
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Related In: Results  -  Collection


getmorefigures.php?uid=PMC4363215&req=5

Fig2: HCV replicon elimination assays with single agents and combination regimens using concentrations representing Ctrough values observed in a clinical setting in wild-type GT-1b (a and c) and DCV + ASV-resistant (NS3-D168V, NS5A-L31M-Y93H) replicon cell lines (b and d). Alfa peginterferon alfa, ASV asunaprevir, BCV beclabuvir, Ctrough trough plasma concentrations, DCV daclatasvir, DMSO, dimethyl sulfoxide, GT genotype, HCV hepatitis C virus, LDV ledipasvir, NS3 non-structural protein 3, NS5A non-structural protein 5A, RBV ribavirin, SMV simeprevir, SOF sofosbuvir

Mentions: Phenotypic analyses indicated that DCV + ASV-resistant replicon cell lines conferred high levels of resistance to DCV, LDV, ASV and SMV, relative to the wild-type reference replicon (Table 1). In contrast, EC50 values for BCV, peginterferon alfa, RBV and SOF were similar in both cell lines. The next-generation NS5A (BMS-1) and NS3 (BMS-2) protease inhibitors demonstrated improved potency (39-fold and 6-fold reduction in anti-HCV activity, respectively, relative to wild type) in the DCV + ASV-resistant replicon when compared to the activities of DCV and ASV (24,500-fold and 201-fold reduction in anti-HCV activity, respectively). HCV replicon elimination results for days 3, 7 and 14 are shown in Fig. 1 (complete results for Days 1, 3, 7, 11 and 14 are provided in Supplementary Fig. 1). With 14 days of treatment, two-DAA regimens of DCV + ASV, DCV + SOF, SOF + LDV and SOF + SMV demonstrated comparable activity in eliminating wild-type replicons at 10 × EC50 values (Fig. 1a). The three-DAA regimen of DCV + ASV + BCV and the four-DAA regimen of DCV + ASV + BCV + SOF demonstrated increased efficacy with complete clearance of wild-type replicons observed with the four-DAA regimen by day 11 at 3 × EC50 values. Similar results were observed with SOF in combination with either the next-generation NS3 protease inhibitor or a next-generation NS5A inhibitor (Supplementary Fig. 1). As expected, DCV + ASV did not eliminate replicons harboring NS5A-L31M-Y93H and NS3-D168V, which confer reduced susceptibilities to both compounds (Fig. 1b). The elimination of replicons by DCV + SOF, SOF + LDV, SOF + SMV, and SOF + next-generation NS3 protease or NS5A inhibitor was comparable in wild-type and DCV + ASV-resistant cell lines. The combination of DCV + ASV + BCV showed reduced activity against DCV + ASV-resistant replicons compared with wild type. To further evaluate the use of these combination regimens, replicon elimination assays were performed at drug concentrations based on clinically relevant Ctrough concentrations (Table 1). Monotherapy at Ctrough concentrations demonstrated the high potency of the NS5A inhibitors, DCV and LDV, compared with the other agents tested (Fig. 2a). With EC50 values in the picomolar range that are well below the high plasma Ctrough concentrations obtained in clinical settings, treatment with these DAAs was sufficient in eliminating wild-type replicons within 7 days. Conversely, none of the NS5A inhibitors and NS3 protease inhibitors tested at Ctrough concentrations were able to eliminate DCV + ASV-resistant replicons (Fig. 2b). Moreover, SOF as a single agent exhibited low clearance activity in this assay. Although the Ctrough of SOF (1,100 nM) is higher than the EC50 (147 nM), it is below the estimated SOF EC90 (1,230 nM). Furthermore, the metabolism and efficiency of phosphorylation of SOF appear to be lower in hepatoma cell lines compared with primary hepatocytes. An analysis of the mechanism of activation of SOF and its analogs has demonstrated that some enzymes in these metabolic pathways, such as CES1, are expressed at significantly lower levels in Huh7 cells compared with primary hepatocytes [11]; thus, the anti-HCV activity of SOF in replicon-based assays may not correlate with its activity in vivo. Similar instances of low activity with nucleosides in hepatoma-derived Huh7 cells harboring replicons have been reported [12, 13]. However, SOF has a high barrier to resistance and has demonstrated efficacy in combination regimens. For wild-type replicons, all DAA combinations at Ctrough concentrations that included an NS5A inhibitor eliminated replicons with high efficiency (Fig. 2c). In comparison, elimination of wild-type replicons was less efficient with SOF + SMV and peginterferon alfa-based combinations (Fig. 2c and Supplementary Fig. 2). Complete elimination of DCV + ASV-resistant replicons occurred by day 7 with the three-DAA regimen (DCV + ASV + BCV) ± RBV or with the four-DAA regimen (DCV + ASV + BCV + SOF) (Fig. 2d). Replicon elimination profiles were comparable in wild-type and DCV + ASV-resistant cell lines following treatment with peginterferon alfa/RBV-based regimens combined with SOF or BCV, or the DAA-only combination of SOF with a next-generation NS3 protease inhibitor (Supplementary Fig. 2).Table 1


In Vitro Assessment of Re-treatment Options for Patients with Hepatitis C Virus Genotype 1b Infection Resistant to Daclatasvir Plus Asunaprevir.

Friborg J, Zhou N, Han Z, Yang X, Falk P, Mendez P, McPhee F - Infect Dis Ther (2014)

HCV replicon elimination assays with single agents and combination regimens using concentrations representing Ctrough values observed in a clinical setting in wild-type GT-1b (a and c) and DCV + ASV-resistant (NS3-D168V, NS5A-L31M-Y93H) replicon cell lines (b and d). Alfa peginterferon alfa, ASV asunaprevir, BCV beclabuvir, Ctrough trough plasma concentrations, DCV daclatasvir, DMSO, dimethyl sulfoxide, GT genotype, HCV hepatitis C virus, LDV ledipasvir, NS3 non-structural protein 3, NS5A non-structural protein 5A, RBV ribavirin, SMV simeprevir, SOF sofosbuvir
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4363215&req=5

Fig2: HCV replicon elimination assays with single agents and combination regimens using concentrations representing Ctrough values observed in a clinical setting in wild-type GT-1b (a and c) and DCV + ASV-resistant (NS3-D168V, NS5A-L31M-Y93H) replicon cell lines (b and d). Alfa peginterferon alfa, ASV asunaprevir, BCV beclabuvir, Ctrough trough plasma concentrations, DCV daclatasvir, DMSO, dimethyl sulfoxide, GT genotype, HCV hepatitis C virus, LDV ledipasvir, NS3 non-structural protein 3, NS5A non-structural protein 5A, RBV ribavirin, SMV simeprevir, SOF sofosbuvir
Mentions: Phenotypic analyses indicated that DCV + ASV-resistant replicon cell lines conferred high levels of resistance to DCV, LDV, ASV and SMV, relative to the wild-type reference replicon (Table 1). In contrast, EC50 values for BCV, peginterferon alfa, RBV and SOF were similar in both cell lines. The next-generation NS5A (BMS-1) and NS3 (BMS-2) protease inhibitors demonstrated improved potency (39-fold and 6-fold reduction in anti-HCV activity, respectively, relative to wild type) in the DCV + ASV-resistant replicon when compared to the activities of DCV and ASV (24,500-fold and 201-fold reduction in anti-HCV activity, respectively). HCV replicon elimination results for days 3, 7 and 14 are shown in Fig. 1 (complete results for Days 1, 3, 7, 11 and 14 are provided in Supplementary Fig. 1). With 14 days of treatment, two-DAA regimens of DCV + ASV, DCV + SOF, SOF + LDV and SOF + SMV demonstrated comparable activity in eliminating wild-type replicons at 10 × EC50 values (Fig. 1a). The three-DAA regimen of DCV + ASV + BCV and the four-DAA regimen of DCV + ASV + BCV + SOF demonstrated increased efficacy with complete clearance of wild-type replicons observed with the four-DAA regimen by day 11 at 3 × EC50 values. Similar results were observed with SOF in combination with either the next-generation NS3 protease inhibitor or a next-generation NS5A inhibitor (Supplementary Fig. 1). As expected, DCV + ASV did not eliminate replicons harboring NS5A-L31M-Y93H and NS3-D168V, which confer reduced susceptibilities to both compounds (Fig. 1b). The elimination of replicons by DCV + SOF, SOF + LDV, SOF + SMV, and SOF + next-generation NS3 protease or NS5A inhibitor was comparable in wild-type and DCV + ASV-resistant cell lines. The combination of DCV + ASV + BCV showed reduced activity against DCV + ASV-resistant replicons compared with wild type. To further evaluate the use of these combination regimens, replicon elimination assays were performed at drug concentrations based on clinically relevant Ctrough concentrations (Table 1). Monotherapy at Ctrough concentrations demonstrated the high potency of the NS5A inhibitors, DCV and LDV, compared with the other agents tested (Fig. 2a). With EC50 values in the picomolar range that are well below the high plasma Ctrough concentrations obtained in clinical settings, treatment with these DAAs was sufficient in eliminating wild-type replicons within 7 days. Conversely, none of the NS5A inhibitors and NS3 protease inhibitors tested at Ctrough concentrations were able to eliminate DCV + ASV-resistant replicons (Fig. 2b). Moreover, SOF as a single agent exhibited low clearance activity in this assay. Although the Ctrough of SOF (1,100 nM) is higher than the EC50 (147 nM), it is below the estimated SOF EC90 (1,230 nM). Furthermore, the metabolism and efficiency of phosphorylation of SOF appear to be lower in hepatoma cell lines compared with primary hepatocytes. An analysis of the mechanism of activation of SOF and its analogs has demonstrated that some enzymes in these metabolic pathways, such as CES1, are expressed at significantly lower levels in Huh7 cells compared with primary hepatocytes [11]; thus, the anti-HCV activity of SOF in replicon-based assays may not correlate with its activity in vivo. Similar instances of low activity with nucleosides in hepatoma-derived Huh7 cells harboring replicons have been reported [12, 13]. However, SOF has a high barrier to resistance and has demonstrated efficacy in combination regimens. For wild-type replicons, all DAA combinations at Ctrough concentrations that included an NS5A inhibitor eliminated replicons with high efficiency (Fig. 2c). In comparison, elimination of wild-type replicons was less efficient with SOF + SMV and peginterferon alfa-based combinations (Fig. 2c and Supplementary Fig. 2). Complete elimination of DCV + ASV-resistant replicons occurred by day 7 with the three-DAA regimen (DCV + ASV + BCV) ± RBV or with the four-DAA regimen (DCV + ASV + BCV + SOF) (Fig. 2d). Replicon elimination profiles were comparable in wild-type and DCV + ASV-resistant cell lines following treatment with peginterferon alfa/RBV-based regimens combined with SOF or BCV, or the DAA-only combination of SOF with a next-generation NS3 protease inhibitor (Supplementary Fig. 2).Table 1

Bottom Line: Drug concentrations representing multiple 50% effective concentrations (EC50) derived in vitro and trough plasma concentrations observed in a clinical setting were utilized.Our in vitro results highlight a number of potential all-oral treatment options for patients who do not achieve a sustained virologic response following therapy with daclatasvir plus asunaprevir.These results require further evaluation in clinical studies.

View Article: PubMed Central - PubMed

Affiliation: Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, CT, 06492, USA.

ABSTRACT

Introduction: Daclatasvir is a non-structural protein 5A (NS5A) inhibitor with activity against hepatitis C virus (HCV) genotypes 1-6 in vitro, and asunaprevir is a non-structural protein 3 (NS3) protease inhibitor with activity against genotypes 1, 4, 5, and 6. This study evaluates potential options for the re-treatment of HCV genotype 1b-infected patients who have failed combination therapy with daclatasvir plus asunaprevir.

Methods: The antiviral activity of drug combination regimens in HCV subgenomic replicon cell lines representing genotype 1b (Con1 strain) wild-type or a variant with specific NS5A and NS3 amino acid substitutions conferring resistance to daclatasvir and asunaprevir were compared using replicon elimination assays. Drug concentrations representing multiple 50% effective concentrations (EC50) derived in vitro and trough plasma concentrations observed in a clinical setting were utilized.

Results: At multiple EC50 values of each drug (3×, 10×, and 30× EC50), combinations of daclatasvir plus sofosbuvir, sofosbuvir plus ledipasvir, sofosbuvir plus simeprevir, and sofosbuvir plus either a next-generation NS3 or NS5A inhibitor demonstrated comparable activity in wild-type and daclatasvir/asunaprevir-resistant cell lines. At clinically relevant drug trough concentrations, combination regimens of daclatasvir plus asunaprevir plus beclabuvir (±ribavirin), and daclatasvir plus asunaprevir plus beclabuvir plus sofosbuvir efficiently cleared daclatasvir + asunaprevir-resistant replicons from cells within 5 days of treatment.

Conclusion: Our in vitro results highlight a number of potential all-oral treatment options for patients who do not achieve a sustained virologic response following therapy with daclatasvir plus asunaprevir. These results require further evaluation in clinical studies.

No MeSH data available.


Related in: MedlinePlus