Bioequivalence of a dolutegravir, abacavir, and lamivudine fixed-dose combination tablet and the effect of food.

BACKGROUND: The integrase inhibitor dolutegravir and nucleoside analogues abacavir and lamivudine are once-daily treatment options for HIV. This study (NCT01622790) evaluated, first, the bioequivalence (BE) of a fixed-dose combination (FDC) tablet containing dolutegravir 50 mg, abacavir 600 mg, and lamivudine 300 mg (dolutegravir/abacavir/lamivudine FDC) vs coadministered dolutegravir 50 mg and abacavir/lamivudine combination tablets (Epzicom) and, second, the effect of food on the dolutegravir/abacavir/lamivudine FDC tablet. METHODS: Study part A (66 healthy subjects) was a single-dose, open-label, randomized, 2-period crossover study to evaluate the BE of the dolutegravir/abacavir/lamivudine FDC tablet and dolutegravir + abacavir/lamivudine tablets in the fasted state. In study part B, 12 subjects from part A received the dolutegravir/abacavir/lamivudine FDC tablet with a high-fat meal. BE and food effect were assessed by analysis of variance to determine the ratio of geometric least squares means and associated 90% confidence intervals for key pharmacokinetic parameters for each of dolutegravir, abacavir, and lamivudine. RESULTS: Sixty-two subjects completed part A. The dolutegravir/abacavir/lamivudine tablet was bioequivalent to the dolutegravir + abacavir/lamivudine tablets; 90% confidence intervals for the geometric least squares mean ratios fell within the 0.8-1.25 BE criteria. The effect of food on the dolutegravir/abacavir/lamivudine FDC tablet was similar to previous food effects observed with the separate formulations. The safety profile was comparable between treatments, with no observed serious or grade 3/4 adverse events. CONCLUSIONS: The BE of the dolutegravir/abacavir/lamivudine FDC tablet was demonstrated; it may be administered without regard to meals.

Validation of high-performance liquid chromatography methods for determination of zidovudine, stavudine, lamivudine and indinavir in human plasma.

OBJECTIVE: Simple methods for the determination of zidovudine (AZT), stavudine (d4T), lamivudine (3TC) and indinavir (INV) in human plasma by reversed-phase liquid chromatography (HPLC) with UV detection were described and validated. METHOD: Solid-liquid extraction procedures were applied to the samples prior to analysis. Chromatography was performed on a C-18 analytical columns and the retention time ranged from 6.8 to 8.0 min for stavudine, 7.5 to 9.0 min for lamivudine, 11.2 to 11.9 min for zidovudine and indinavir. Four methods were validated for specificity, inter-and intra-assay precision and accuracy, absolute recovery and stability. RESULTS: Analytical curve ranged from 10-1600 ng/ml for stavudine, 50-3200 ng/ml for lamivudine, 0.05-5.0 microg/ml for zidovudine and 0.1-10.0 microg/ml for indinavir. Analytes stability during sampling processing and storage were established. Extraction recoveries are higher than 89% for all formulations. CONCLUSIONS: These methods proved to be simple, accurate and precise, and are currently in use in our laboratory for the quantitative analysis of antiretrovirals products in plasma, and for further pharmacokinetics and bioequivalence studies.

Validation of high-performance liquid chromatography methods for determination of zidovudine, stavudine, lamivudine and indinavir in human plasma

Objetivo: Fueron descritos y validados m¨¦todos anal¨ªticos simplespara la determinaci¨®n de zidovudina (AZT), estavudina (d4T),lamivudina (3TC), e indinavir (INV) en plasma humano por cromatograf¨ªal¨ªquida de alta resoluci¨®n (HPLC) con detecci¨®n UV.M¨¦todo: Se aplic¨® la extracci¨®n en fase s¨®lida para la preparaci¨®nde las muestras previo al an¨¢lisis. La corrida cromatogr¨¢ficase realiz¨® en una columna anal¨ªtica C-18 y el tiempo de retenci¨®nse movi¨® en un rango de 6,8-11,9 min para d4T, 7,5-9,0para 3TC y 11,2-11,9 para AZT e INV. Se validaron 4 m¨¦todosen cuanto a especificidad, precisi¨®n y exactitud entre d¨ªas y en eld¨ªa, as¨ª como recobrado y estabilidad.Resultados: Los rangos de concentraciones de las curvasanal¨ªticas eran de 10-1600 ng/ml para d4T, 50-3200 ng/ml para3TC, 0,05-5,0 ¦Ìg/ml para AZT y 0,1-10,0 ¦Ìg/ml para INV. Sedemostr¨® la estabilidad del analito durante el procesamiento de lasmuestras y el almacenamiento. Para las 4 formulaciones los resultadosdel por ciento de recobrado fue superior al 89%.Conclusiones: Estos m¨¦todos demostraron ser simples, exactos,precisos y son los utilizados actualmente en nuestro laboratoriopara el an¨¢lisis cuantitativo de productos antirretrovirales enplasma, as¨ª como para posteriores estudios de farmacocin¨¦tica ybioequivalencia(AU)

Objective: Simple methods for the determination of zidovudine(AZT), stavudine (d4T), lamivudine (3TC) and indinavir (INV)in human plasma by reversed-phase liquid chromatography(HPLC) with UV detection were described and validated.Method: Solid-liquid extraction procedures were applied tothe samples prior to analysis. Chromatography was performed ona C-18 analytical columns and the retention time ranged from 6.8to 8.0 min for stavudine, 7.5 to 9.0 min for lamivudine, 11.2 to11.9 min for zidovudine and indinavir. Four methods were validatedfor specificity, inter-and intra-assay precision and accuracy,absolute recovery and stability.Results: Analytical curve ranged from 10-1600 ng/ml forstavudine, 50-3200 ng/ml for lamivudine, 0.05-5.0 ¦Ìg/ml forzidovudine and 0.1-10.0 ¦Ìg/ml for indinavir. Analytes stabilityduring sampling processing and storage were established. Extractionrecoveries are higher than 89% for all formulations.Conclusions: These methods proved to be simples, accurateand precise, and are currently in use in our laboratory for thequantitative analysis of antiretrovirals products in plasma, and forfurther pharmacokinetics and bioequivalence studies(AU)

Effect of ribavirin on intracellular and plasma pharmacokinetics of nucleoside reverse transcriptase inhibitors in patients with human immunodeficiency virus-hepatitis C virus coinfection: results of a randomized clinical study.

The intracellular triphosphorylation and plasma pharmacokinetics of lamivudine (3TC), stavudine (d4T), and zidovudine (ZDV) were assessed in a pharmacokinetic substudy, in 56 human immunodeficiency virus-hepatitis C virus (HIV-HCV) coinfected patients receiving peginterferon alfa-2a (40KD) 180 microg/week plus either placebo or ribavirin (RBV) 800 mg/day in the AIDS PEGASYS Ribavirin International Coinfection Trial. There were no significant differences between patients treated with RBV and placebo in plasma pharmacokinetics parameters for the nucleoside reverse transcriptase inhibitors (NRTIs) at steady state (weeks 8 to 12): ratios of least squares mean of area under the plasma concentration-time curve (AUC(0-12 h)) were 1.17 (95% confidence interval, 0.91 to 1.51) for 3TC, 1.44 (95% confidence interval, 0.58 to 3.60) for d4T and 0.85 (95% confidence interval, 0.50 to 1.45) for ZDV, and ratios of least squares mean plasma C(max) were 1.33 (95% confidence interval, 0.99 to 1.78), 1.06 (95% confidence interval, 0.68 to 1.65), and 0.84 (95% confidence interval, 0.46 to 1.53), respectively. Concentrations of NRTI triphosphate (TP) metabolites in relation to those of the triphosphates of endogenous deoxythymidine-triphosphate (dTTP) and deoxcytidine-triphosphate (dCTP) were similar in the RBV and placebo groups. Differences (RBV to placebo) in least squares mean ratios of AUC(0-12 h) at steady state were 0.274 (95% confidence interval, -0.37 to 0.91) for 3TC-TP:dCTP, 0.009 (95% confidence interval, -0.06 to 0.08) for d4T-TP:dTTP, and -0.081 (95% confidence interval, -0.40 to 0.24) for ZDV-TP:dTTP. RBV did not adversely affect HIV-1 replication. In summary, RBV 800 mg/day administered in combination with peginterferon alfa-2a (40KD) does not significantly affect the intracellular phosphorylation or plasma pharmacokinetics of 3TC, d4T, and ZDV in HIV-HCV-coinfected patients.

Determination of lamivudine in human plasma by HPLC and its use in bioequivalence studies.

A simple, accurate, precise and sensitive high-performance liquid chromatographic (HPLC) method with ultraviolet detection was developed to quantificate lamivudine (3-TC) in human plasma samples from bioequivalence studies. 3-TC and stavudine (internal standard, I.S.) were extracted from 0.5 ml of human plasma by acetonitrile protein precipitation. The method was validated over a concentration range of 0.05-3.00 microg/ml and used in a bioequivalence trial between two lamivudine formulations, to assess its usefulness in this kind of study. FURP-lamivudine (Fundação para o Remédio Popular, Brazil, as test formulation) and Epivir (GlaxoSmithKline, Brazil, as reference formulation) were evaluated following a single 150 mg oral dose to 24 healthy volunteers of both genders. The dose was administered after an overnight fast according to a two-way crossover design. Bioequivalence between the products was determined by calculating 90% confidence intervals (90% CI) for the ratio of Cmax, AUC0-t and AUC0-inf values for the test and reference products, using logarithmic transformed data. The 90% confidence intervals for the ratio of Cmax (0.86-1.06), AUC0-t (0.96-1.04) and AUC0-inf (0.97-1.05) values for the test and reference products are within the 0.80-1.25 interval proposed by FDA and EMEA. It was concluded that the two 3-TC formulations are bioequivalent in their rate and extent of absorption, and thus, may be used interchangeably.

A rapid and sensitive method for simultaneous determination of lamivudine and zidovudine in human serum by on-line solid-phase extraction coupled to liquid chromatography/tandem mass spectrometry detection.

A method based on solid-phase extraction (SPE) coupled to high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection was developed for the simultaneous determination of lamivudine (3TC) and zidovudine (AZT) in human serum, using didanosine (ddI) as internal standard. The acquisition was performed in multiple reaction monitoring (MRM) mode, monitoring the transitions m/z 230.0 --> 111.8 for 3TC, m/z 268.1 --> 126.8 for AZT, and m/z 237.2 --> 136.8 for ddI. The limits of detection and quantitation were 3 and 10 ng/mL for 3TC, and 5 and 15 ng/mL for AZT. The method was linear in the studied ranges (10-1500 ng/mL for 3TC and 15-3000 ng/mL for AZT), with r(2) > 0.99 for each drug, and the run time was 4 min. The intra-assay precisions (%) were in the ranges 1.9-8.7 (3TC) and 2.2-8.9 (AZT), the inter-assay precisions were in the ranges 2.6-9.0 (3TC) and 4.2-8.1 (AZT), and the intra- and inter-assay accuracies were >97% for both drugs. The absolute recoveries were 95-99% for 3TC (45, 600 and 1200 ng/mL) and 104-112% for AZT (45, 1000 and 2400 ng/mL). The analytical method was applied to a bioequivalence study in which 24 healthy adult volunteers received single oral doses of the reference formulation and two test combined AZT/3TC tablets, in an open, three-period, balanced, randomized, crossover protocol. Based on the 90% confidence interval of the individual ratios (test formulation/reference formulation) for C(max) (peak serum concentration) and AUC(0-inf) (extrapolated area under the serum concentration vs. time curve from time zero to infinity), it was concluded that the two test formulations are bioequivalent to the reference formulation with respect to the rate and extent of absorption of both 3TC and AZT.

Determination of didanosine in human serum by on-line solid-phase extraction coupled to high-performance liquid chromatography with electrospray ionization tandem mass spectrometric detection: application to a bioequivalence study.

A method based on solid-phase extraction coupled to liquid chromatography with positive ion electrospray ionization and tandem mass spectrometric detection was developed for the determination of didanosine in human serum, using lamivudine as internal standard. The acquisition was performed in the multiple reaction monitoring mode, monitoring the transitions m/z 237 --> 136.7 for didanosine and m/z 230 --> 111.7 for lamivudine. The method was linear over the range studied (10-1500 ng ml(-1)), with r(2) > 0.98, and the run time was 5 min. The intra- and inter-assay precisions were < or =10% and the intra- and inter-assay accuracies were >95%. The absolute recoveries were 99.8% (10 ng ml(-1)), 98.4% (30 ng ml(-1)), 91.5% (700 ng ml(-1)) and 94.7% (1200 ng ml(-1)). The limits of detection and quantitation were 5 and 10 ng ml(-1), respectively. The method was applied to a bioequivalence study, in which 24 healthy adult volunteers (12 men) received single oral doses (200 mg) of reference and test didanosine formulations (buffered powder for oral solutions), in an open, two-way, randomized, crossover protocol. The 90% confidence interval of the individual ratios (test formulation/reference formulation) for C(max) (peak serum concentration) and AUC(0-inf) (area under the serum concentration versus time curve from time zero to infinity) were within the range 80-125%, which supports the conclusion that the two formulations are bioequivalent regarding the rate and extent of didanosine absorption.

Simultaneous quantitation of intracellular zidovudine and lamivudine triphosphates in human immunodeficiency virus-infected individuals.

Highly active antiretroviral therapy (HAART) is the standard treatment for infection with human immunodeficiency virus (HIV). The most common HAART regimen consists of the combination of at least one protease inhibitor (PI) with two nucleoside reverse transcriptase inhibitors (NRTIs). Contrary to PIs, NRTIs require intracellular activation from the parent compound of their triphosphate moiety to suppress HIV replication. Simultaneous intracellular determination of two NRTI triphosphates is difficult to accomplish due to their relatively small concentrations in peripheral blood mononuclear cells (PBMCs), requiring large amounts of blood from HIV-positive patients. Recently, we described a method to determine intracellular zidovudine triphosphate (ZDV-TP) concentrations in HIV-infected patients by using solid-phase extraction and tandem mass spectrometry. The limit of quantitation (LOQ) for ZDV-TP was 0.10 pmol, and the method was successfully used for the determination of ZDV-TP in HIV-positive patients. In this study, we enhanced the aforementioned method by the simultaneous quantitation of ZDV-TP and lamivudine triphosphate (3TC-TP) in PBMCs from HIV-infected patients. The LOQ for 3TC-TP was 4.0 pmol, with an interassay coefficient of variation and an accuracy of 7 and 12%, respectively. This method was successfully applied to the simultaneous in vivo determination of the ZDV-TP and 3TC-TP pharmacokinetic profiles from HIV-infected patients receiving HAART.

Intracellular studies of the nucleoside reverse transcriptase inhibitor active metabolites: a review.

Nucleoside reverse transcriptase inhibitors (NRTIs) plasma concentrations do not correlate with clinical efficacy or toxicity. These agents need to be phosphorylated to become active against HIV-infection. Thus, the characterization of the NRTIs intracellular metabolite pharmacological parameters will provide a better understanding that could lead to the development of more rational dose regimens in the HIV-infected population. Furthermore, intracellular measurements of NRTIs may provide a better marker with respect to clinical efficacy and toxicity than plasma concentrations. Thus, in this article we review the latest information regarding the intracellular pharmacological parameters of zidovudine (ZDV) and lamivudine (3TC) active metabolites in HIV-infected patients including the results from our recent clinical studies. We will start the discussion with ZDV and 3TC clinical efficacy, followed by systemic pharmacokinetics studies. We will then discuss the in vitro and in vivo intracellular studies with particular emphasis in the method development to measure these metabolites and we will conclude with the most current data from our clinical trials.

Intracellular studies of the nucleoside reverse transcriptase inhibitor active metabolites: a review

Nucleoside reverse transcriptase inhibitors (NRTIs) plasma concentrations do not correlate with clinical efficacy or toxicity. These agents need to be phosphorylated to become active against HIV-infection. Thus, the characterization of the NRTIs intracellular metabolite pharmacological parameters will provide a better understanding that could lead to the development of more rational dose regimens in the HIV-infected population. Furthermore, intracellular measurements of NRTIs may provide a better marker with respect to clinical efficacy and toxicity than plasma concentrations. Thus, in this article we review the latest information regarding the intracellular pharmacological parameters of zidovudine (ZDV) and lamivudine (3TC) active metabolites in HIV infected patients including the results from our recent clinical studies. We will start the discussion with ZDV and 3TC clinical efficacy, followed by systemic pharmacokinetics studies. We will then discuss the in vitro and in vivo intracellular studies with particular emphasis in the method development to measure these metabolites and we will conclude with the most current data from our clinical trials.