Detection of Weak Organic Anion-Transporting Polypeptide 1B Inhibition by Probenecid with Plasma-Based Coproporphyrin in Humans.

Probenecid (PROB) is a clinical probe inhibitor of renal organic anion transporter (OAT) 1 and OAT3 that inhibits in vitro activity of hepatic drug transporters OATP1B1 and OATP1B3. It was hypothesized that PROB could potentially affect the disposition of OATP1B drug substrates. The plasma levels of the OATP1B endogenous biomarker candidates, including coproporphyrin I (CPI), CPIII, hexadecanedioate (HDA), and tetradecanedioate (TDA), were examined in 14 healthy subjects treated with PROB. After oral administration with 1000 mg PROB alone and in combination with furosemide (FSM), AUC (0-24 h) values were 1.39 ± 0.21-fold and 1.57 ± 0.41-fold higher than predose levels for CPI and 1.34 ± 0.16-fold and 1.45 ± 0.57-fold higher for CPIII. Despite increased systemic exposures, no decreases in CPI and CPIII renal clearance were observed (0.97 ± 0.38-fold and 1.16 ± 0.51-fold for CPI, and 1.34 ± 0.53-fold and 1.50 ± 0.69-fold for CPIII, respectively). These results suggest that the increase of CP systemic exposure is caused by OATP1B inhibition. Consistent with this hypothesis, PROB inhibited OATP1B1- and OATP1B3-mediated transport of CPI in a concentration-dependent manner, with IC50 values of 167 ± 42.0 and 76.0 ± 17.2 µM, respectively, in transporter-overexpressing human embryonic kidney cell assay. The inhibition potential was further confirmed by CPI and CPIII hepatocyte uptake experiments. In contrast, administration of PROB alone did not change AUC (0-24 h) of HDA and TDA relative to prestudy levels, although the administration of PROB in combination with FSM increased HDA and TDA levels compared with FSM alone (1.02 ± 0.18-fold and 0.90 ± 0.20-fold vs. 1.71 ± 0.43-fold and 1.62 ± 0.40-fold). Taken together, these findings indicate that PROB displays weak OATP1B inhibitory effects in vivo and that coproporphyrin is a sensitive endogenous probe of OATP1B inhibition. This study provides an explanation for the heretofore unknown mechanism responsible for PROB's interaction with other xenobiotics. SIGNIFICANCE STATEMENT: This study suggested that PROB is a weak clinical inhibitor of OATP1B based on the totality of evidence from the clinical interaction between PROB and CP and the in vitro inhibitory effect of PROB on OATP1B-mediated CP uptake. It demonstrates a new methodology of utilizing endogenous biomarkers to evaluate complex drug-drug interaction, providing explanation for the heretofore unknown mechanism responsible for PROB's inhibition. It provides evidence to strengthen the claim that CP is a sensitive circulating endogenous biomarker of OATP1B inhibition.

Further Studies to Support the Use of Coproporphyrin I and III as Novel Clinical Biomarkers for Evaluating the Potential for Organic Anion Transporting Polypeptide 1B1 and OATP1B3 Inhibition.

In a recent study, limited to South Asian Indian subjects (n = 12), coproporphyrin (CP) I and CPIII demonstrated properties appropriate for an organic anion-transporting polypeptide (OATP) 1B endogenous probe. The current studies were conducted in healthy volunteers of mixed ethnicities, including black, white, and Hispanic subjects, to better understand the utility of these biomarkers in broader populations. After oral administration with 600 mg rifampin, AUC(0-24h) values were 2.8-, 3.7-, and 3.6-fold higher than predose levels for CPI and 2.6-, 3.1-, and 2.4-fold higher for CPIII, for the three populations, respectively. These changes in response to rifampin were consistent with previous results. The sensitivity toward OATP1B inhibition was also investigated by evaluating changes of plasma CP levels in the presence of diltiazem and itraconazole [administered as part of an unrelated drug-drug interaction (DDI) investigation], two compounds that were predicted to have minimal inhibitory effect on OATP1B. Administration of diltiazem and itraconazole did not increase plasma CPI and CPIII concentrations relative to prestudy levels, in agreement with predictions from in vitro parameters. Additionally, the basal CP concentrations in subjects with SLCO1B1 c.521TT genotype were comparable to those with SLCO1B1 c.521TC genotype, similar to studies with probe substrates. However, subjects with SLCO1B1 c.388AG and c.388GG genotypes (i.e., increased OATP1B1 transport activity for certain substrates) had lower concentrations of CPI than those with SLCO1B1 c.388AA. Collectively, these findings provide further evidence supporting the translational value of CPI and CPIII as suitable endogenous clinical probes to gauge OATP1B activity and potential for OATP1B-mediated DDIs.

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.

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.

LC-MS/MS bioanalysis of plasma 1, 14-tetradecanedioic acid and 1, 16-hexadecanedioic acid as candidate biomarkers for organic anion-transporting polypeptide mediated drug-drug interactions.

AIM: A robust LC-MS/MS assay was developed to quantify endogenous 1, 14-tetradecanedioic acid (TDA) and 1, 16-hexadecanedioic acid (HDA) in human plasma as potential biomarkers for evaluating drug-drug interactions mediated by the hepatic drug transporters, organic anion-transporting polypeptides. RESULTS: This assay was validated using fit-for-purpose approach over standard curve range of 2.5-1000 nM for TDA and HDA using analyte-free charcoal-stripped human plasma as the surrogate matrix. Chromatographic separation condition was successfully optimized to separate TDA from an interference peak while maintaining both analytes in neutral forms to minimize carryover issue. CONCLUSION: The described assay is currently applied to a clinical study for evaluating TDA/HDA as potential substitute biomarkers for drug-drug interaction studies.

UHPLC-MS/MS bioanalysis of human plasma coproporphyrins as potential biomarkers for organic anion-transporting polypeptide-mediated drug interactions.

AIM: Coproporphyrins (CP-I and CP-III) have been identified as possible biomarkers to predict human hepatic organic anion-transporting polypeptides-mediated-drug-interactions for a new drug entering clinical development. RESULTS: The method is applicable to quantify plasma CP-I and CP-III within 0.078-15.0 nM. The results identify and address a number of challenges encountered with porphyrin assays such as photodegradation and interferences. To overcome interferences from ubiquitous porphyrins, a surrogate matrix was used to prepare calibration standards. Quality controls were prepared in plasma and surrogate matrix to ensure parallelism between surrogate matrix and plasma. CONCLUSION: A robust UHPLC-MS/MS assay was developed and validated for CP-I and CP-III in plasma, and is currently applied to clinical studies to confirm suitability of Coproporphyrins as a potential substitute for drug-drug interaction study.

A simple, fast, sensitive and robust LC-MS/MS bioanalytical assay for evaluating 7α-hydroxy-4-cholesten-3-one biomarker in a clinical program.

AIM: Serum 7α-hydroxy-cholesten-3-one (C4) has been reported as a biomarker to assess CYP7A1 enzyme activity and bile acid synthesis. To support a clinical program, a sensitive and reliable assay without derivatization was required for the analysis of C4 in human serum. Methodology & results: A systematic approach was used to optimize mass spectrometry, LC and sample extraction conditions, therefore, significantly improved assay sensitivity, and achieved the required quantification limit without derivatization. A surrogate matrix approach was used to overcome the interference from endogenous C4. A stable isotope-labeled C4 was used as internal standard. The samples were extracted using a simple protein precipitation method with 2% formic acid in acetonitrile. CONCLUSION: A simple, fast, sensitive and robust UHPLC-MS/MS method for the quantification of 0.50 ng/ml C4 in 100 µl human serum was developed and fit for purpose validated. The method was successfully applied to the bioanalysis of C4 in a clinical study.

A Review of Daclatasvir Drug-Drug Interactions.

The 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.

UHPLC-MS/MS bioanalysis of urinary DHEA, cortisone and their hydroxylated metabolites as potential biomarkers for CYP3A-mediated drug-drug interactions.

AIM: A UHPLC-MS/MS assay was developed to quantify urinary dehydroepiandrosterone (DHEA), 7ß-hydroxy-DHEA, cortisone and 6ß-hydroxycortisone as potential biomarkers to predict CYP3A activity. RESULTS: A sensitive assay at LLOQ of 0.500 ng/ml with good accuracy and precision was developed for the four analytes in human urine. This UHPLC-MS/MS assay was optimized by eliminating nonspecific loss of the analytes in urine, ensuring complete hydrolysis of the conjugates to unconjugated forms and use of the product ions of [M+H-H2O]+ for multiple reaction monitoring detection of DHEA and 7ß-hydroxy-DHEA. CONCLUSION: This assay was successfully applied to a pilot clinical study. It is also suitable for future drug-drug interaction studies to continue evaluating the potential of these steroids as biomarkers for CYP3A inhibition and induction.

A highly sensitive and selective LC-MS/MS method to quantify asunaprevir, an HCV NS3 protease inhibitor, in human plasma in support of pharmacokinetic studies.

Asunaprevir (BMS-650032) is a selective hepatitis C virus (HCV) NS3 protease inhibitor with potent activity against HCV genotypes 1, 4, 5 and 6. It has been developed in conjunction with direct-acting antiviral agents, in interferon- and ribavirin-free regimen, to improve existing therapies for HCV infection. To support the pharmacokinetic analyses in asunaprevir clinical studies, we have developed and validated a highly sensitive and robust LC-MS/MS method to quantify asunaprevir in human EDTA plasma with an LLOQ of 0.05ng/mL, which was a 20-fold sensitivity improvement over a previously reported assay for asunaprevir. A deuterated labeled [D9]-asunaprevir was used as the internal standard (IS). The analyte and the IS were extracted using a semi-automated liquid-liquid extraction (LLE) at pH 7 with methyl-t-butyl ether (MTBE) in a 96-well plate containing 10µL of 10% CHAPS as the surfactant to prevent non-specific binding issue. Chromatographic separation was achieved on a Genesis C8 column (2.1×50mm, 4µm) with a gradient elution using 0.1% formic acid in water as mobile phase A and a mixture of methanol: acetone: formic acid (95:5:0.1; v/v/v) as the mobile phase B. Positive electrospray ionization was performed using multiple reaction monitoring (MRM) with transitions of m/z 748→648 for asunaprevir and m/z 757→649 for [D9]-asunaprevir,and a collision energy of 30 electron Volts (eV). The assay was validated over a standard curve range from 0.05 to 50ng/mL for asunaprevir in human plasma. The intra- and inter assay precisions were within 7.1% CV, and the % deviation was within 5.5% of their nominal concentrations. This assay has been successfully applied to multiple clinical studies with excellent assay ruggedness and reproducibility.

An open-label investigation into drug-drug interactions between multiple doses of daclatasvir and single-dose cyclosporine or tacrolimus in healthy subjects.

BACKGROUND AND OBJECTIVE: Chronic hepatitis C virus (HCV) infection is a major cause of liver transplantation. Drug-drug interactions (DDIs) with cyclosporine and tacrolimus hindered the use of first-generation protease inhibitors in transplant recipients. The current study investigated DDIs between daclatasvir-a pan-genotypic HCV NS5A inhibitor with clinical efficacy in multiple regimens (including all-oral)-and cyclosporine or tacrolimus in healthy subjects. METHODS: Healthy fasted subjects (aged 18-49 years; body mass index 18-32 kg/m(2)) received single oral doses of cyclosporine 400 mg on days 1 and 9, and daclatasvir 60 mg once daily on days 4-11 (group 1, n = 14), or a single oral dose of tacrolimus 5 mg on days 1 and 13, and daclatasvir 60 mg once daily on days 8-19 (group 2, n = 14). Blood samples for pharmacokinetic analysis [by liquid chromatography with tandem mass spectrometry (LC-MS/MS)] were collected on days 1 and 9 for cyclosporine (72 h), on days 1 and 13 for tacrolimus (168 h) and on days 8 and 9 (group 1) or on days 12 and 13 (group 2) for daclatasvir (24 h). Plasma concentrations were determined by validated LC-MS/MS methods. RESULTS: Daclatasvir did not affect the pharmacokinetic parameters of cyclosporine or tacrolimus, and tacrolimus did not affect the pharmacokinetic parameters of daclatasvir. Co-administration of cyclosporine resulted in a 40 % increase in the area under the concentration-time curve of daclatasvir but did not affect its maximum observed concentration. CONCLUSION: On the basis of these observations in healthy subjects, no clinically relevant DDIs between daclatasvir and cyclosporine or tacrolimus are anticipated in liver transplant recipients infected with HCV; dose adjustments during co-administration are unlikely to be required.

Sensitive and accurate liquid chromatography-tandem mass spectrometry methods for quantitative determination of a novel hepatitis C NS5B inhibitor BMS-791325 and its active metabolite in human plasma and urine.

BMS-791325 is a novel hepatitis C NS5B inhibitor which is currently in clinical development. To support pharmacokinetic (PK) assessments, sensitive, accurate, precise, and reproducible liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been developed and validated for the quantitation of BMS-791325 and its active N-demethyl metabolite (BMS-794712) in human plasma and urine. Plasma and urine 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 for the simultaneous detection of the two analytes in human plasma (0.1-50 ng/mL) and in human urine (5-2500 ng/mL). Intra-run precision (3.0% R.S.D.), inter-run precision (5.3% R.S.D.), and accuracy (±4.7% deviation) from plasma and urine quality control samples provide evidence of the methods accuracy and precision. Selectivity, stability in matrices, extraction recovery, matrix effect on LC-MS detection, and interference of coadministered drugs (famotidine and ritonavir) were all acceptable. Reproducibility of the plasma method was demonstrated by reanalysis of a portion of study samples. The results of cross-validations demonstrated the equivalency of two methods validated in two labs. The plasma method was applied to the analysis of several thousand clinical study samples for PK evaluations of the drug in normal healthy subjects and in patients. The urine method was used in the first in human study to evaluate renal clearance and urinary recovery.

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.

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.

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.

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.

Evaluation of drug-drug interaction between daclatasvir and methadone or buprenorphine/naloxone.

INTRODUCTION: Daclatasvir (DCV) is a potent hepatitis C virus (HCV) NS5A replication complex inhibitor with pangenotypic (1-6) activity in vitro. Methadone (MET) and buprenorphine (BUP) are opioid medications used to treat opioid addiction; patients on HCV therapy may require MET or BUP treatment. The effect of DCV on the pharmacokinetics (PK) of MET or BUP/naloxone (NLX) was assessed in subjects on stable MET or BUP. MATERIALS AND METHODS: An open-label, two-part study assessed the effect of steady-state oral administration of DCV on the PK of MET (Part 1, P1) or BUP/NAL (Part 2, P2). Safety/tolerability and pharmacodynamics (PD, opioid withdrawal scales/overdose assessment) were also assessed. Subjects (P1, N=14; P2, N=11) received daily single-dose oral MET (40-120mg) or BUP/NLX (8/2-24/6mg) based on their prescribed stable dose throughout, in addition to DCV (60mg QD) on Days 2-9. Serial PK sampling occurred predose and postdose till 24 hours on Day 1 (MET/BUP) and Day 10 (MET/BUP/DCV). Noncompartmental PK were derived. Geometric mean ratios (GMR) and 90% confidence intervals (90% CI) for MET/BUP/norBUP Cmax and AUCTAU were derived from linear mixed effects models. RESULTS: Subjects were aged 19-39 years, mostly white (P1, 93%; P2, 100%) and male (P1, 71%; P2, 91%). All subjects completed the study. No clinically meaningful effect was demonstrated as the GMR and 90% CIs fell within the prespecified interval (P1, 0.7-1.4; P2, 0.5-2.0: see Table 1). DCV coadministration was well-tolerated: overall, six (43%) subjects had adverse events (AEs) (all mild and resolved without treatment). DCV had no clinically significant effect on the PD of MET or BUP/NLX. CONCLUSIONS: Steady-state administration of DCV 60mg QD had no clinically meaningful effect on the PK of MET or BUP/NLX and was generally well-tolerated, suggesting that no dose adjustments will be required.

The pharmacokinetics of daclatasvir and asunaprevir administered in combination in studies in healthy subjects and patients infected with hepatitis C virus.

BACKGROUND AND OBJECTIVES: The combination of direct-acting antiviral agents in patients with chronic hepatitis C virus (HCV) infection has demonstrated clinical benefit; however, evaluation of potential drug-drug interactions is required prior to therapy. METHODS: An open-label study assessed the pharmacokinetics and tolerability of the HCV NS5A replication complex inhibitor daclatasvir and the HCV NS3 protease inhibitor asunaprevir when co-administered in healthy subjects. Daclatasvir 60 mg once daily and asunaprevir 600 mg twice daily were dosed for 7 days alone followed by combination dosing for 14 days at 30 mg once daily and 200 mg twice daily, respectively. Further assessments were provided comparing exposures from the current study with those from studies in HCV-infected patients receiving either the same or higher doses of daclatasvir or asunaprevir administered alone or together. RESULTS: Dose-normalized daclatasvir and asunaprevir morning exposures were comparable with control in healthy subjects, with geometric mean area under the concentration-time curve ratios of 1.202 (90 % CI 1.113-1.298) and 0.868 (90 % CI 0.726-1.038), respectively. In HCV patients daclatasvir and asunaprevir exposures were largely comparable, when administered together or alone. CONCLUSIONS: Additional data support the conclusion that there is no clinically meaningful interaction between daclatasvir and asunaprevir in either healthy subjects or HCV-infected patients, including those also receiving peginterferon-α/ribavirin, and that the combination of daclatasvir 60 mg once daily and asunaprevir 200 mg twice daily is generally well-tolerated.

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.