Hamza Kandoussi

Assessment of pharmacokinetic interactions of the HCV NS5A replication complex inhibitor daclatasvir with antiretroviral agents: ritonavir-boosted atazanavir, efavirenz and tenofovir.

BACKGROUND Approximately one-third of all HIV-infected individuals are coinfected with HCV, many of whom will receive concomitant treatment for both infections. With the advent of direct-acting antivirals (DAAs) for HCV, potential drug interactions between antiretrovirals and DAAs require evaluation prior to co-therapy. METHODS Three open-label studies were conducted in healthy subjects to assess potential interactions between the investigational first-in-class HCV NS5A replication complex inhibitor daclatasvir and representative antiretrovirals atazanavir/ritonavir, efavirenz and tenofovir disoproxil fumarate. RESULTS Target exposure was that of 60 mg daclatasvir alone. Dose-normalized (60 mg) geometric mean ratios of daclatasvir AUCτ for 20 mg ± atazanavir/ritonavir (2.10 [90% CI 1.95, 2.26]) and 120 mg ± efavirenz (0.68 [0.60, 0.78]) showed less than the three-fold elevation and two-fold reduction, respectively, in systemic exposure predicted by prior interaction studies with potent inhibitors/inducers of CYP3A4. Daclatasvir dose adjustment to 30 mg once daily with atazanavir/ritonavir and 90 mg once daily with efavirenz is predicted to normalize AUCτ relative to the target exposure (geometric mean ratios 1.05 [0.98, 1.13] and 1.03 [0.90, 1.16], respectively). Atazanavir exposure (Cmax, AUCτ and C24 trough) and efavirenz Ctrough under coadministration were similar to historical data without daclatasvir. No clinically relevant interactions between daclatasvir and tenofovir disoproxil fumarate were observed for either drug, and no dosing adjustments were indicated. Daclatasvir was well tolerated in all three studies. CONCLUSIONS The pharmacokinetic data support coadministration of daclatasvir with atazanavir/ritonavir, efavirenz and/or tenofovir. A Phase III study in HIV-HCV coinfection has commenced using the described dose modifications.

Single-dose pharmacokinetics and safety of daclatasvir in subjects with renal function impairment.

BACKGROUND Daclatasvir (DCV) is a pangenotypic inhibitor of the HCV NS5A replication complex approved in Japan and Europe for combination treatment of HCV. AI444-063 was an open-label, two-stage, adaptive study assessing DCV pharmacokinetics and safety in HCV-uninfected subjects with renal impairment. METHODS Stage 1 included 12 subjects with end-stage renal disease (ESRD) on dialysis and 12 healthy controls (creatinine clearance [CLcr]≥90 ml/min, Cockcroft-Gault) matched by sex, age and weight. All participants received a single DCV 60-mg dose on day 1. DCV plasma levels were measured through day 4. Prespecified criteria for study expansion (ESRD versus control area under the curve extrapolated to infinite time [AUC∞] geometric mean ratio [GMR] upper 90% CI>1.5) were met; therefore, 12 subjects with moderate or severe renal impairment (estimated glomerular filtration rate, 30-59 and 15-29 ml/min/1.73m(2), respectively) were included in stage 2. RESULTS All 36 participants (30 male, mean age 54 years) completed the study. DCV AUC∞ was higher in ESRD subjects versus controls (GMR 1.264, 90% CI 0.989, 1.616). Compared with normal CLcr (90 ml/min), GMR and 90% CI for unbound DCV AUC∞ at CLcr 60, 30 and 15 ml/min were 1.18 (1.07, 1.30), 1.39 (1.14, 1.70) and 1.51 (1.18, 1.94), respectively. DCV was generally well tolerated in subjects with normal renal function, ESRD, or moderate or severe renal impairment. CONCLUSIONS Observed DCV exposure increases were within the normal range of variability and were not associated with an elevated risk of adverse events. DCV can be administered in subjects with renal impairment, including ESRD, without dose modification. ClinicalTrials.gov NCT01830205.

Multiplexed LC-MS/MS method for the simultaneous quantitation of three novel hepatitis C antivirals, daclatasvir, asunaprevir, and beclabuvir in human plasma

Dual or triple combination regimens of novel hepatitis C direct-acting antivirals (DAA, daclatasvir, asunaprevir, or beclabuvir) provide high sustained virological response rates and reduced frequency of resistance compared to clinical monotherapy. To support pharmacokinetic (PK) assessments in clinical studies, a multiplexed liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantitation of daclatasvir, asunaprevir, beclabuvir (BMS-791325) and its active metabolite (BMS-794712) in human plasma was developed and validated. Human plasma samples were extracted with methyl-t-butyl ether followed by an LC-MS/MS analysis, which was conducted in a multiple reaction monitoring (MRM) mode. The lower limits of quantitation (LLOQ) were 1 ng/mL for daclatasvir, asunaprevir, and BMS-794712, and 2 ng/mL for beclabuvir. Intra-run precision (≤4.5% CV), inter-run precision (≤2.9% CV), and accuracy (±5.3% deviation) based on different concentration levels (low, geometric mean, mid and high) of the quality control samples (QCs) provided evidence of the methods accuracy and precision. Selectivity and matrix effect on LC-MS/MS detection, stability in plasma, and potential interference of coadministered drugs (ribavirin and interferon) were all evaluated and the results were acceptable. Method reproducibility was demonstrated by the reanalysis of a portion of study samples. The cross-validation results for QCs demonstrated the equivalency between this method and two single-analyte methods which were previously validated for quantitation of daclatasvir in human plasma. This approach of using a multiplexed LC-MS/MS method for the simultaneous quantitation of three DAAs is time- and cost-effective, and can maintain good data quality in sample analysis.

The effect of hepatic impairment on the pharmacokinetics of asunaprevir, an HCV NS3 protease inhibitor.

BACKGROUND It is necessary to evaluate the impact of hepatic impairment on the pharmacokinetic profile of direct-acting antiviral agents for the treatment of HCV infection. METHODS In this open-label, parallel group, multiple-dose study subjects (aged 18-70 years with a body mass index <35 kg/m(2)) with mild (n=6), moderate (n=6) and severe hepatic impairment (n=4) received asunaprevir 200 mg twice daily; healthy subjects (n=12) were matched (age, weight, gender) 1:1 to the first 4 subjects in each hepatic impairment group to act as controls. Pharmacokinetic sampling and analyses were performed on days 1 and 7 of dosing. Pharmacokinetic parameters were derived by non-compartmental methods. Geometric mean ratios (GMRs) and 90% CIs were used to assess the impact of hepatic impairment on the pharmacokinetics of asunaprevir, relative to healthy matched controls. RESULTS Compared with healthy subjects, mild hepatic impairment did not result in meaningful alterations in asunaprevir exposure (day 7 maximal plasma concentration [Cmax] GMR: 0.58 [90% CI 0.35, 0.98]; area under the plasma concentration-time curve in one dosing interval [AUCtau] GMR: 0.79 [90% CI 0.55, 1.15]); clinically significant increases in asunaprevir exposure were observed in subjects with moderate (Cmax GMR: 5.03 [90% CI 2.99, 8.47]; AUCtau GMR: 9.83 [90% CI 6.76, 14.28]) and severe hepatic impairment (Cmax GMR: 22.92 [90% CI 12.57, 41.81]; AUCtau GMR: 32.08 [90% CI 20.84, 49.40]). Correlation between increased asunaprevir exposure and all individual components of the Child-Pugh classification system was observed in subjects with moderate and severe hepatic impairment. CONCLUSIONS Mild hepatic impairment does not meaningfully affect the pharmacokinetic profile of asunaprevir. The dosing of asunaprevir in patients with moderate-to-severe hepatic impairment is not recommended. Clinicaltrials.gov identifier NCT01019070.

Practical and Efficient Strategy for Evaluating Oral Absolute Bioavailability with an Intravenous Microdose of a Stable Isotopically-Labeled Drug Using a Selected Reaction Monitoring Mass Spectrometry Assay

A strategy of using selected reaction monitoring (SRM) mass spectrometry for evaluating oral absolute bioavailability with concurrent intravenous (i.v.) microdosing a stable isotopically labeled (SIL) drug was developed and validated. First, the isotopic contribution to SRM (ICSRM) of the proposed SIL drug and SIL internal standard (IS) was theoretically calculated to guide their chemical synthesis. Second, the lack of an isotope effect on drug exposure was evaluated in a monkey study by i.v. dosing a mixture of the SIL and the unlabeled drugs. Third, after the SIL drug (100 μg) was concurrently i.v. dosed to humans, at T(max) of an oral therapeutic dose of the unlabeled drug, both drugs in plasma specimens were simultaneously quantified by a sensitive and accurate SRM assay. This strategy significantly improves bioanalytical data quality and saves time, costs, and resources by avoiding a traditional absolute bioavailability study or the newer approach of microdoses of a radio-microtracer measured by accelerator mass spectrometry.

A convenient strategy for quantitative determination of drug concentrations in tissue homogenates using a liquid chromatography/tandem mass spectrometry assay for plasma samples.

Quantitative determination of drug concentrations in tissue homogenates via liquid chromatography-tandem mass spectrometry (LC-MS/MS) is commonly conducted using the standards and analytical quality controls (QCs) prepared in the same matrix (tissue homogenates), to keep the matrix and its effects consistent on the analytes during sample extraction and analysis. In this manuscript, we proposed to analyze tissue homogenate samples using an LC-MS/MS assay with the standards and analytical QCs prepared in plasma after tissue homogenate samples were appropriately diluted with plasma. BMS-650032 was used as a model compound, and its validated dog plasma assay was used for dog liver sample analyses. The tissue matrix effect was evaluated by diluting liver homogenate QCs with drug-free plasma at different dilution factors to determine the minimum required dilution factor (MRDF) at which tissue matrix has insignificant impact to the plasma assay. The percentage deviation of the measured concentration from the nominal concentration was used as an indicator of the tissue matrix effect. The results suggested that the tissue matrix effect was decreased as the plasma dilution factor increased. Based on the results of the tissue matrix effect evaluation, liver homogenate samples were analyzed after appropriate dilutions with plasma at the MRDF or greater dilution factors. The results confirmed that this approach generates accurate data, and the process is very convenient and economic. This approach has been used on the analyses of different tissues (liver and brain) and biofluid (bile) to support several drug development programs.

A sensitive and accurate liquid chromatography–tandem mass spectrometry method for quantitative determination of the novel hepatitis C NS5A inhibitor BMS-790052 (daclastasvir) in human plasma and urine

A liquid-liquid extraction (LLE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been developed and validated for the quantification of BMS-790052 (daclastasvir) in human plasma and urine. The samples were extracted with methyl-t-butyl ether (MTBE) before analyzing by an API 4000 mass spectrometer which was operated in a multiple reaction monitoring (MRM) mode for detection of positively charged ions of BMS-790052 and its internal standard, ¹³C₁₀-BMS-790052. The standard curves ranged from 0.050 to 50.0 ng/mL for BMS-790052 in human plasma, and 1.00-1000 ng/mL in human urine. The intra-assay precision (%CV), based on four levels of analytical QCs (low, geometric mean, mid and high), was within 8.6%; inter-assay precision (%CV) was within 6.7% for both plasma and urine methods, and the mean assay accuracy (%Dev) was within ±3.0% for both plasma and urine methods. The ruggedness of this accurate, precise, and selective LLE-LC-MS/MS method has been demonstrated in the successful analysis of several thousand clinical study samples.

Effect of the coadministration of daclatasvir on the pharmacokinetics of a combined oral contraceptive containing ethinyl estradiol and norgestimate.

BACKGROUND Daclatasvir is a highly selective NS5A replication complex inhibitor currently in development for the treatment of chronic hepatitis C infection. Daclatasvir is active at picomolar concentrations and demonstrates in vitro activity against a broad range of HCV genotypes. The primary objective of this study was to assess the effect of daclatasvir on the pharmacokinetics of a combined oral contraceptive containing ethinyl estradiol and norgestimate (Ortho Tri-Cyclen(®)). METHODS In this open-label single-sequence study, 20 healthy female subjects received ethinyl estradiol and norgestimate for three cycles, with coadministration of daclatasvir in cycle 3. Pharmacokinetics of ethinyl estradiol and the active metabolites of norgestimate (norelgestromin and norgestrel) were assessed in cycles 2 and 3. RESULTS Adjusted ratios of geometric means and 90% CIs were estimated for the maximum observed plasma concentration (ethinyl estradiol 1.11 [1.02, 1.20], norelgestromin 1.06 [0.99, 1.14] and norgestrel 1.07 [0.99, 1.16]) and area under the plasma concentration-time curve in one dosing interval (ethinyl estradiol 1.01 [0.95, 1.07], norelgestromin 1.12 [1.06, 1.17] and norgestrel 1.12 [1.02, 1.23]). CONCLUSIONS Coadministration of daclatasvir resulted in no clinically relevant effects on exposure to ethinyl estradiol, norelgestromin or norgestrel.

Cynomolgus Monkey as a Clinically Relevant Model to Study Transport Involving Renal Organic Cation Transporters: In Vitro and In Vivo Evaluation

Organic cation transporter (OCT) 2, multidrug and toxin extrusion protein (MATE) 1, and MATE2K mediate the renal secretion of various cationic drugs and can serve as the loci of drug-drug interactions (DDI). To support the evaluation of cynomolgus monkey as a surrogate model for studying human organic cation transporters, monkey genes were cloned and shown to have a high degree of amino acid sequence identity versus their human counterparts (93.7, 94.7, and 95.4% for OCT2, MATE1, and MATE2K, respectively). Subsequently, the three transporters were individually stably expressed in human embryonic kidney (HEK) 293 cells and their properties (substrate selectivity, time course, pH dependence, and kinetics) were found to be comparable to the corresponding human form. For example, six known human cation transporter inhibitors, including pyrimethamine (PYR), showed generally similar IC50 values against the monkey transporters (within sixfold). Consistent with the in vitro inhibition of metformin (MFM) transport by PYR (IC50 for cynomolgus OCT2, MATE1, and MATE2K; 1.2 ± 0.38, 0.17 ± 0.04, and 0.25 ± 0.04 µM, respectively), intravenous pretreatment of monkeys with PYR (0.5 mg/kg) decreased the clearance (54 ± 9%) and increased in the area under the plasma concentration-time curve of MFM (AUC ratio versus control = 2.23; 90% confidence interval of 1.57 to 3.17). These findings suggest that the cynomolgus monkey may have some utility in support of in vitro-in vivo extrapolations (IVIVEs) involving the inhibition of renal OCT2 and MATEs. In turn, cynomolgus monkey-enabled IVIVEs may inform human DDI risk assessment.

A Review of Daclatasvir Drug-Drug Interactions

AbstractThe treatment of hepatitis C virus (HCV) infection has been revolutionized in recent years by the development of direct-acting antiviral regimens that do not contain peginterferon (pegIFN) and/or ribavirin (RBV). While direct-acting antiviral-based regimens have been shown to be greatly superior to pegIFN/RBV-based regimens in terms of efficacy and safety, they have a greater susceptibility to drug–drug interactions (DDIs). Daclatasvir (DCV)—the benchmark pangenotypic nonstructural protein 5A inhibitor—has been shown to be efficacious and generally well tolerated in partnership with other HCV direct-acting antivirals, including sofosbuvir, asunaprevir (ASV), and ASV plus beclabuvir. DCV may be the object of a DDI via the induction or inhibition of cytochrome P450 (CYP) 3A4 and/or P-glycoprotein (P-gp) by the concomitant medication, or the precipitant of a DDI via DCV-based induction/inhibition of CYP 3A4 or inhibition of P-gp, organic anion transporting polypeptide 1B1/B3, and/or breast cancer resistance protein. This article presents an overview of the drug interaction studies conducted during the clinical development of DCV, the findings of these studies that led to the guidance on concomitant medication use and dosage along with any required DCV dose modifications, and the use of the known metabolic pathway of DCV to guide concomitant dosing where direct drug–drug studies have not been conducted. The robust characterization of the DCV clinical pharmacology program has demonstrated that DCV has few or no clinically relevant DDIs with medications with which it is likely to be co-administered, and the majority of DDIs that do occur can be predicted and easily managed. Funding: Bristol-Myers Squibb.