A fast-screening dispersive liquid-liquid microextraction-gas chromatography-mass spectrometry method applied to the determination of efavirenz in human plasma samples.

We demonstrate the suitability of a fast, green, easy-to-perform, and modified sample extraction procedure, i.e., dispersive liquid-liquid microextraction (DLLME) for the determination of efavirenz (EFV) in human plasma. Data acquisition was done by gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring (SIM) mode. The simplicity of the method lies in, among others, the avoidance of the use of large organic solvent volumes as mobile phases and non-volatile buffers that tend to block the plumbing in high-performance liquid chromatography (HPLC). Chromatographic and mass spectral parameters were optimized using bovine whole blood for matrix matching due to insufficient human plasma. Method validation was accomplished using the United States Food and Drug Administration (USFDA) 2018 guidelines. The calibration curve was linear with a dynamic range of 0.10-2.0 µg/mL and an R2 value of 0.9998. The within-run accuracy and precision were both less than 20% at the lower limit of quantification (LLOQ) spike level. The LLOQ was 0.027 µg/mL which compared well with some values but was also orders of magnitude better than others reported in the literature. The percent recovery was 91.5% at the LLOQ spike level. The DLLME technique was applied in human plasma samples from patients who were on treatment with EFV. The human plasma samples gave concentrations of EFV ranging between 0.14-1.00 µg/mL with three samples out of seven showing concentrations that fell within or close to the recommended therapeutic range.

Meta-analysis of the effect of CYP2B6, CYP2A6, UGT2B7 and CAR polymorphisms on efavirenz plasma concentrations.

BACKGROUND: Efavirenz primary metabolism is catalysed by CYP2B6 with minor involvement of CYP2A6. Subsequently, phase I metabolites are conjugated by UGT2B7, and constitutive androstane receptor (CAR) has been shown to transcriptionally regulate many relevant enzymes and transporters. Several polymorphisms occurring in the genes coding for these proteins have been shown to impact efavirenz pharmacokinetics in some but not all studies. OBJECTIVES: A meta-analysis was performed to assess the overall effect of CYP2B6 rs3745274, CYP2A6 (rs28399454, rs8192726 and rs28399433), UGT2B7 (rs28365062 and rs7439366) and NR1I3 (rs2307424 and rs3003596) polymorphisms on mid-dose efavirenz plasma concentrations. METHODS: Following a literature review, pharmacokinetic parameters were compiled and a meta-analysis for these variants was performed using Review Manager and OpenMetaAnalyst. A total of 28 studies were included. RESULTS: Unsurprisingly, the analysis confirmed that individuals homozygous for the T allele for CYP2B6 rs3745274 had significantly higher efavirenz concentrations than those homozygous for the G allele [weighted standard mean difference (WSMD) = 2.98; 95% CI 2.19-3.76; P < 0.00001]. A subgroup analysis confirmed ethnic differences in frequency but with a similar effect size in each ethnic group (P = 0.96). Associations with CYP2A6 and UGT2B7 variants were not statistically significant, but T homozygosity for CAR rs2307424 was associated with significantly lower efavirenz concentrations than in C homozygotes (WSMD = -0.32; 95% CI -0.59 to -0.06; P = 0.02). CONCLUSIONS: This meta-analysis provides the overall effect size for the impact of CYP2B6 rs3745274 and NR1I3 rs2307424 on efavirenz pharmacokinetics. The analysis also indicates that some previous associations were not significant when interrogated across studies.

Simultaneous quantification of intracellular lamivudine and abacavir triphosphate metabolites by LC-MS/MS.

Nucleoside reverse transcriptase inhibitors (NRTIs) require intracellular phosphorylation to active triphosphate (TP) nucleotide metabolites before they can inhibit the HIV reverse transcriptase. However, monitoring these pharmacologically active TP metabolites is challenging due to their instability and their low concentrations at the pg/ml levels in blood and tissues. The combination of lamivudine (3TC) and abacavir (ABC) is one of the first lines for HIV therapy. Therefore, a sensitive, selective, accurate, and precise LC-MS/MS method was developed and validated for the simultaneous quantification of 3TC- and ABC-TP metabolites in mouse blood and tissues. Calibration curves were linear over the range of 10-100,000 pg/ml for 3TC-TP and 4-40,000 pg/ml for carbovir-TP (CBV-TP; phosphorylated metabolite of ABC). This corresponds to 2.1-21,322 fmol/106 cells for 3TC-TP and 0.8-8000 fmol/106 cells for CBV-TP. Accuracy and precision were less than 15% for all quality control sample (QCs), and absolute extraction recovery of were >65% for 3TC-TP and >90% for CBV-TP. The method was optimized to ensure stability of TP samples and standards during sample collection, preparation, analysis, and storage conditions. This method has enhanced sensitivity and requires smaller amounts of blood and tissue samples compared to previous LC-MS/MS methods for 3TC- and CBV-TP quantification. The developed method was successfully applied to characterize the pharmacokinetic profile of TP metabolites in mouse peripheral blood mononuclear cells (PBMCs), spleen, lymph nodes, and liver cells. In addition, another direct, simple, and high-throughput method for the quantification of TP standards was developed and used for the analysis of stability samples.

Efavirenz Inhibits the Human Ether-A-Go-Go Related Current (hERG) and Induces QT Interval Prolongation in CYP2B6*6*6 Allele Carriers.

BACKGROUND: Efavirenz (EFV) has been associated with torsade de pointes despite marginal QT interval lengthening. Since EFV is metabolized by the cytochrome P450 (CYP) 2B6 enzyme, we hypothesized that EFV would lengthen the rate-corrected QT (QTcF) interval in carriers of the CYP2B6*6 decreased functional allele. OBJECTIVE: The primary objective of this study was to evaluate EFV-associated QT interval changes with regard to CYP2B6 genotype and to explore mechanisms of QT interval lengthening. METHODS: EFV was administered to healthy volunteers (n = 57) as a single 600 mg dose followed by multiple doses to steady-state. Subjects were genotyped for known CYP2B6 alleles and ECGs and EFV plasma concentrations were obtained serially. Whole-cell, voltage-clamp experiments were performed on cells stably expressing hERG and exposed to EFV in the presence and absence of CYP2B6 expression. RESULTS: EFV demonstrated a gene-dose effect and exceeded the FDA criteria for QTcF interval prolongation in CYP2B6*6/*6 carriers. The largest mean time-matched differences ∆∆QTcF were observed at 6 hours (14 milliseconds; 95% CI [1; 27]), 12 hours (18 milliseconds; 95% CI [-4; 40]), and 18 hours (6 milliseconds; 95% CI [-1; 14]) in the CYP2B6*6/*6 genotype. EFV concentrations exceeding 0.4 µg/mL significantly inhibited outward hERG tail currents (P < 0.05). CONCLUSIONS: This study demonstrates that homozygous carriers of CYP2B6*6 allele may be at increased risk for EFV-induced QTcF interval prolongation via inhibition of hERG.

Quantification of darunavir and etravirine in human peripheral blood mononuclear cells using high performance liquid chromatography tandem mass spectrometry (LC-MS/MS), clinical application in a cohort of 110 HIV-1 infected patients and evidence of a potential drug-drug interaction.

OBJECTIVES: To describe the validation of a sensitive high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method allowing the simultaneous quantification of darunavir (DRV) and etravirine (ETR) in peripheral blood mononuclear cells (PBMCs) and its application in a cohort of HIV-1 infected patients. METHODS: Blood samples were obtained from 110 patients. PMBCs were isolated using density gradient centrifugation. Drug extraction from PBMCs was performed with a 60:40 methanol-water (MeOH-H2O) solution containing deuterated IS (DRV-d9 and ETR-d8). The chromatographic separation was performed on a RP18 XBridge™ column. RESULTS: The geometric mean (GM) of cell associated concentration ([DRV]CC) and plasmatic concentration ([DRV]plasma) were 360.5ng/mL (CI95%:294.5-441.2) and 1733ng/mL (CI95%:1486-2021), respectively. A geometric mean intracellular (IC)/plasma ratio (GMR) of 0.21 (CI95%:0.18-0.24) was calculated. Adjusted for dose/body surface area and post-intake time, a statistically significant correlation was observed between [DRV]Plasma and the eGFR (p=0.002) and between [DRV]Plasma and the concomitant use of ETR (p=0.038). For the 10 patients receiving ETR in addition to DRV, the GM of [ETR]Plasma (available for 8 out of 10 patients) and [ETR]CC were 492.3ng/mL and 2951ng/mL respectively. The GMR of ETR was 7.6 (CI95%: 3.61-13.83). CONCLUSIONS: A handy and sensitive high performance LC-MS/MS method allowing the simultaneous quantification of DRV and ETR in PBMCs has been described and successfully applied in the largest cohort of DRV-treated patients reported to date. ETR accumulates more efficiently in PBMCs compared to DRV. We have also highlighted a possible impact of ETR on DRV plasma concentrations requiring further investigations.

In Vivo Profiling and Distribution of Known and Novel Phase I and Phase II Metabolites of Efavirenz in Plasma, Urine, and Cerebrospinal Fluid.

Efavirenz (EFV) is principally metabolized by CYP2B6 to 8-hydroxy-efavirenz (8OH-EFV) and to a lesser extent by CYP2A6 to 7-hydroxy-efavirenz (7OH-EFV). So far, most metabolite profile analyses have been restricted to 8OH-EFV, 7OH-EFV, and EFV-N-glucuronide, even though these metabolites represent a minor percentage of EFV metabolites present in vivo. We have performed a quantitative phase I and II metabolite profile analysis by tandem mass spectrometry of plasma, cerebrospinal fluid (CSF), and urine samples in 71 human immunodeficiency virus patients taking efavirenz, prior to and after enzymatic (glucuronidase and sulfatase) hydrolysis. We have shown that phase II metabolites constitute the major part of the known circulating efavirenz species in humans. The 8OH-EFV-glucuronide (gln) and 8OH-EFV-sulfate (identified for the first time) in humans were found to be 64- and 7-fold higher than the parent 8OH-EFV, respectively. In individuals (n = 67) genotyped for CYP2B6, 2A6, and CYP3A metabolic pathways, 8OH-EFV/EFV ratios in plasma were an index of CYP2B6 phenotypic activity (P < 0.0001), which was also reflected by phase II metabolites 8OH-EFV-glucuronide/EFV and 8OH-EFV-sulfate/EFV ratios. Neither EFV nor 8OH-EFV, nor any other considered metabolites in plasma were associated with an increased risk of central nervous system (CNS) toxicity. In CSF, 8OH-EFV levels were not influenced by CYP2B6 genotypes and did not predict CNS toxicity. The phase II metabolites 8OH-EFV-gln, 8OH-EFV-sulfate, and 7OH-EFV-gln were present in CSF at 2- to 9-fold higher concentrations than 8OH-EFV. The potential contribution of known and previously unreported EFV metabolites in CSF to the neuropsychological effects of efavirenz needs to be further examined in larger cohort studies.

Secondary metabolism pathway polymorphisms and plasma efavirenz concentrations in HIV-infected adults with CYP2B6 slow metabolizer genotypes.

OBJECTIVES: Efavirenz is widely prescribed for HIV-1 infection, and CYP2B6 polymorphisms 516G→T and 983T→C define efavirenz slow metabolizer genotypes. To identify genetic predictors of higher plasma efavirenz concentrations beyond these two common functional alleles, we characterized associations with mid-dosing interval efavirenz concentrations in 84 HIV-infected adults, all carrying two copies of these major loss-of-function CYP2B6 alleles. METHODS: Study participants had been randomized to efavirenz-containing regimens in prospective clinical trials and had available plasma efavirenz assay data. Analyses focused on secondary metabolism pathway polymorphisms CYP2A6 -48T→G (rs28399433), UGT2B7 735A→G (rs28365062) and UGT2B7 802T→C (rs7439366). Exploratory analyses also considered 196 polymorphisms and 8 copy number variants in 41 drug metabolism/transport genes. Mid-dosing interval efavirenz concentrations at steady-state were obtained ≥8 h but <19 h post-dose. Linear regression was used to test for associations between polymorphisms and log-transformed efavirenz concentrations. RESULTS: Increased efavirenz concentrations were associated with CYP2A6 -48T→G in all subjects (P = 3.8 × 10(-4)) and in Black subjects (P = 0.027) and White subjects (P = 0.0011) analysed separately; and with UGT2B7 735 G/G homozygosity in all subjects (P = 0.006) and in Black subjects (P = 0.046) and White subjects (P = 0.062) analysed separately. In a multivariable model, CYP2A6 -48T→G and UGT2B7 735 G/G homozygosity remained significant (P < 0.05 for each). No additional polymorphisms or copy number variants were significantly associated with efavirenz concentrations. CONCLUSIONS: Among individuals with a CYP2B6 slow metabolizer genotype, CYP2A6 and possibly UGT2B7 polymorphisms contribute to even higher efavirenz concentrations.

Association of higher plasma vitamin D binding protein and lower free calcitriol levels with tenofovir disoproxil fumarate use and plasma and intracellular tenofovir pharmacokinetics: cause of a functional vitamin D deficiency?

Tenofovir disoproxil fumarate (TDF) causes bone, endocrine, and renal changes by an unknown mechanism(s). Data are limited on tenofovir pharmacokinetics and these effects. Using baseline data from a multicenter study of HIV-infected youth on stable treatment with regimens containing TDF (n = 118) or lacking TDF (n = 85), we measured cross-sectional associations of TDF use with markers of renal function, vitamin D-calcium-parathyroid hormone balance, phosphate metabolism (tubular reabsorption of phosphate and fibroblast growth factor 23 [FGF23]), and bone turnover. Pharmacokinetic-pharmacodynamic associations with plasma tenofovir and intracellular tenofovir diphosphate concentrations were explored among those receiving TDF. The mean age was 20.9 (standard deviation [SD], 2.0) years; 63% were male; and 52% were African American. Compared to the no-TDF group, the TDF group showed lower mean estimated glomerular filtration rates and tubular reabsorption of phosphate, as well as higher parathyroid hormone and 1,25-dihydroxy vitamin D [1,25-OH(2)D] levels. The highest quintile of plasma tenofovir concentrations was associated with higher vitamin D binding protein, lower free 1,25-OH(2)D, higher 25-OH vitamin D, and higher serum calcium. The highest quintile of intracellular tenofovir diphosphate concentration was associated with lower FGF23. Higher plasma tenofovir concentrations were associated with higher vitamin D binding protein and lower free 1,25-OH(2)D, suggesting a functional vitamin D deficiency explaining TDF-associated increased parathyroid hormone. The finding of lower FGF23 accompanying higher intracellular tenofovir diphosphate suggests that different mechanisms mediate TDF-associated changes in phosphate handling. Separate pharmacokinetic properties may be associated with distinct TDF toxicities: tenofovir with parathyroid hormone and altered calcium balance and tenofovir diphosphate with hypophosphatemia and FGF23 regulation. (The clinical trial registration number for this study is NCT00490412 and is available online at http://clinicaltrials.gov/ct2/show/NCT00490412.).

Pharmacokinetics and safety of a new paediatric fixed-dose combination of zidovudine/lamivudine/nevirapine in HIV-infected children.

BACKGROUND: Alternatives to the available stavudine-containing paediatric fixed-dose combination (FDC) tablets are rapidly needed due to concerns regarding the cumulative toxicity of long-term stavudine exposure. We report the bioavailability and short-term safety of a novel paediatric FDC tablet of zidovudine (ZDV)/lamivudine (3TC)/nevirapine (NVP; 30/15/28 mg) in HIV-infected children. METHODS: In this Phase I/II open-label pharmacokinetic study, 42 children weighing 6-30 kg treated with NVP-based HAART for ≥4 weeks were randomized to receive the FDC tablets (GPO-VIR Z30) or the liquid formulations. Dosing was weight-based. Intensive 12-h blood sampling was performed after 2 weeks; subjects then crossed-over to the alternate formulation at equal doses and sampling repeated 2 weeks later. Pharmacokinetic parameters were determined by non-compartmental analysis. Buccal-swab samples were collected for cytochrome P450 (CYP)2B6 polymorphism analysis. RESULTS: With the FDC tablet, the geometric mean (90% CI) area under the curve (AUC) for ZDV, 3TC and NVP was 1.58 (1.49-1.68), 7.78 (7.38-8.19) and 68.88 (62.13-76.36) µg•h/ml, respectively. Rules for NVP therapeutic inadequacy were defined a priori, and despite lower NVP exposure with the tablet (P<0.001), the levels remained therapeutically adequate. ZDV AUC was similar between formulations. 3TC exposure was significantly higher with the tablet but comparable to historical data in adults and children taking branded tablets. While receiving the tablet, NVP AUC in children with CYP2B 516 GG (45%), GT (45%) and TT (10%) genotypes were 67.0, 74.5 and 106.4 µg•h/ml, respectively (P=0.04). CONCLUSIONS: Disparities in drug exposure between formulations were observed; however, the FDC tablet delivered therapeutically adequate exposures of each drug and could well play an important role in simplifying antiretroviral treatment for children.

Influence of host genetic factors on efavirenz plasma and intracellular pharmacokinetics in HIV-1-infected patients.

BACKGROUND: Efavirenz (EFV) is characterized by interindividual pharmacokinetic variability causing inconsistent clinical responses. Previous studies have identified some possible genetic determinants of the variability in plasma concentrations. However, their impact on EFV intracellular pharmacokinetics remains mostly unexplored. AIMS: To confirm previous observations concerning the influence of genetic polymorphisms on EFV plasma concentrations and to assess their effect on the intracellular pharmacokinetics of EFV. MATERIALS & METHODS: EFV concentrations in plasma ([EFV](Cmin)) and in peripheral blood mononuclear cells ([EFV](CC)) were determined in 50 HIV-infected patients. Subjects were genotyped for 13 polymorphisms in 5 different genes (CYP2A6, CYP2B6, CYP3A5, UGT2B7 and ABCB1). Relationships between genetic status and [EFV](Cmin), [EFV](CC) or EFV accumulation in peripheral blood mononuclear cells (EFV accumulation ratio or accumulation ration [AR]) were then evaluated. RESULTS: CYP2B6 allelic status was associated with differences in [EFV](Cmin) but also in [EFV](CC). Patients carrying at least one mutated allele showed significantly higher [EFV](Cmin) and [EFV](CC) than homozygous wild-type (mutated homozygous [m/m] >heterozygous [wt/m]>homozygous wild-type [wt/wt], p<0.001). ABCB1 rs3842T>C was significantly associated with higher EFV AR (p = 0.032). Finally, the ABCB1 3435C>T SNP was associated with a lower increase in CD4-cell count after EFV therapy initiation. CONCLUSION: Our study corroborates previous findings indicating that knowledge of CYP2B6 genetic status should be taken into account for an EFV treatment. Our results also constitute the first demonstration of the significant influence of CYP2B6 genetic polymorphisms on [EFV](CC) and suggest that ABCB1 SNPs may also influence the clinical impact of EFV treatment.

CYP2B6, CYP2A6 and UGT2B7 genetic polymorphisms are predictors of efavirenz mid-dose concentration in HIV-infected patients.

OBJECTIVE: UDP-glucuronosyltransferase (UGT) 2B7 was recently identified as the main enzyme mediating efavirenz N-glucuronidation. In this study, we determined whether selected UGT2B7 polymorphisms could be used to enhance the prediction of efavirenz plasma concentrations from CYP2B6 and CYP2A6 genotypes. METHODS: Mid-dose efavirenz plasma concentrations were determined in 94 HIV-infected Ghanaian patients at 2-8 weeks of antiretroviral therapy. CYP2B6 and CYP2A6 genotypes had been previously reported. UGT2B7 exon 2 single-nucleotide polymorphisms (SNPs) c.735A>G (UGT2B7*1c; rs28365062) and c.802C>T (H268Y; UGT2B7*2; rs7439366) were determined by direct sequencing with UGT2B7*1a defined as the reference allele. Relationships between efavirenz plasma concentrations, demographic variables and genotypes were evaluated by univariate and multivariate statistical approaches. RESULTS: The mean (+/-SD) mid-dose efavirenz plasma concentration was 3218 (+/-3905) ng/ml with coefficient of variation of 121%. Independent predictors of efavirenz concentration included CYP2B6 c.516TT genotype (4030 ng/ml increase; 95% confidence interval 2882-5505 ng/ml, P < 0.001), UGT2B7*1a carrier status (475 ng/ml increase; 95% confidence interval 138-899 ng/ml, P = 0.004) and CYP2A6*9 and/or *17 carrier status (372 ng/ml increase; 95% confidence interval 74-742 ng/ml, P = 0.013). Overall, CYP2B6 c.516TT genotype, UGT2B7*1a carrier status and CYP2A6*9 or *17 carrier status accounted for 45.2, 10.1 and 8.6% of the total variance, respectively. CONCLUSION: Our findings demonstrate independent effects of CYP2A6 and UGT2B7 genetic variation on efavirenz disposition beyond that of the CYP2B6 polymorphisms. The development and testing of a pharmacogenetic algorithm for estimating the appropriate dose of efavirenz should incorporate genotypic data from both the oxidative and glucuronidation pathways.

Investigation of the role of breast cancer resistance protein (Bcrp/Abcg2) on pharmacokinetics and central nervous system penetration of abacavir and zidovudine in the mouse.

Many anti-human immunodeficiency virus 1 nucleoside reverse-transcriptase inhibitors have low central nervous system (CNS) distribution due in part to active efflux transport at the blood-brain barrier. We have previously shown that zidovudine (AZT) and abacavir (ABC) are in vitro substrates for the efflux transport protein breast cancer resistance protein (Bcrp) 1. We evaluated the influence of Bcrp1 on plasma pharmacokinetics and brain penetration of zidovudine and abacavir in wild-type and Bcrp1-deficient (Bcrp1-/-) FVB mice. There was no difference in either area under the concentration-time profiles for plasma (AUC(plasma)) or brain (AUC(brain)) for zidovudine between the wild-type and Bcrp1-/- mice. The AUC(plasma) of abacavir was 20% lower in the Bcrp1-/- mice, whereas the AUC(brain) was 20% greater. This difference resulted in a 1.5-fold increase in abacavir brain exposure in the Bcrp1-/- mice. The effect of selective and nonselective transport inhibitors on the ABC brain/plasma ratio at a single time point was evaluated. 3-(6-Isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6, 7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)-propionicacid tert-butyl ester (Ko143), N[4[2-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)ethyl]phenyl]-5-methoxy-9-oxo-10H-acridine-4-carboxamide (GF120918), probenecid, and Pluronic P85 increased abacavir plasma concentrations in the wild-type mice. Abacavir plasma concentrations in Bcrp1-/- mice were increased by (R)-4-((1aR,6R,10bS)-1,2-difluoro-1,1a,6,10b-tetrahydrodibenzo (a,e)cyclopropa(c)cycloheptan-6-yl)-alpha-((5-quinoloyloxy)methyl)-1-piperazineethanol trihydrochloride (LY335979), GF120918, and probenecid, but not by Ko143. Brain/plasma concentration ratios in both the wild-type and Bcrp1-/- mice were increased by the P-glycoprotein inhibitors LY335979 and GF120918, but not by BCRP-selective inhibitors. These data indicate that deletion of Bcrp1 has little influence on the pharmacokinetics or brain penetration of AZT. However, for abacavir, deletion of Bcrp1 reduces plasma exposure and enhances brain penetration. These findings suggest that Bcrp1 does not play a significant role in limiting the CNS distribution of zidovudine and abacavir; however, brain penetration of abacavir is dependent on P-glycoprotein-mediated efflux.

Development of a population simulation model for HIV monotherapy virological outcomes using lamivudine.

Current highly active antiretroviral therapy (HAART) requires the use of combinations of three drugs to minimize the early emergence of drug-resistant HIV strains. Therefore, long-term monotherapy data with new agents are unavailable. However, the development of computer models for Monte-Carlo-type simulations of antiviral monotherapy, which incorporate HIV infection dynamic distributions from previously studied populations, together with pharmacokinetics and pharmacodynamic parameters of the new agent, could serve as an important tool. The nucleoside lamivudine (3TC) was used as a representative drug to standardize an improved pharmacodynamic and infection dynamic monotherapy model. 3TC plasma concentration versus time profiles was used to drive the cellular accumulation of 3TC-triphosphate (TP) in primary human lymphocytes in the model, over a 16 week period. The fraction of HIV reverse transcription inhibited was calculated using the median inhibitory concentration and intracellular 3TC-TP levels. Virus loads and activated CD4+ T-cell counts were generated for 2,200 theoretical individuals and compared with the outcomes of an actual 3TC monotherapy trial at the same dose. Pharmacokinetic variance alone did not account for the interindividual HIV-load variability. However, selection of appropriate distributions of the various pharmacokinetic and infection dynamics parameters produced a similar range of virus load reductions to actual observations. Therefore, once parameter and variance distributions are standardized, this modelling approach could be helpful in planning clinical trials and predicting the antiviral contribution of each agent in a HAART modality.

2'-Deoxy-4'-C-ethynyl-2-fluoroadenosine: a nucleoside reverse transcriptase inhibitor with highly potent activity against all HIV-1 strains, favorable toxic profiles and stability in plasma.

A working hypothesis to solve the critical problems of existing HAART was proposed. The study based on the hypothesis proved the validity of the hypothesis and resulted in the development of 2'-deoxy-4'-C-ethynyl-2-fluoro-adenosine (4'Ed2FA), a nucleoside reverse transcriptase inhibitor (NRTI) with highly potent activity against all HIV-1 strains, very favourable toxic profiles, and stability in plasma.

Efavirenz in plasma from HIV-infected patients does not directly block reverse transcriptase activity in cell-free assays but inhibits HIV replication in cellular assays.

The aim of this study was to characterize the potent nonimmunoglobulin (Ig) inhibitory activity defected in plasma from some HIV-infected, efavirenz (EFV)-treated patients. Concentration of EFV in plasma was measured by HPLC and correlation with reverse transcriptase (RT) inhibition or decrease in virus replication in cellular assays was searched. After plasma protein elimination by ethanol extraction, an inhibitory activity is measurable on RT in vitro that correlates with EFV concentration determined by HPLC. However, total plasma-containing EFV does not inhibit RT activity in cell-free assay, but it does efficiently inhibit virus replication in cell culture assays. Thus, despite being bound to plasma proteins (retention of EFV after extensive dialysis), EFV in plasma conserves its antiviral activity on infected cells. This observation precludes the use of crude sera and plasmas from EFV-treated patients for the study of antibody-mediated neutralizing activity.

Intracellular and plasma efavirenz concentrations in HIV-infected patients switching from successful protease inhibitor-based highly active antiretroviral therapy (HAART) to efavirenz-based HAART (SUSTIPHAR Study).

OBJECTIVES: To assess intracellular and plasma efavirenz concentrations in HIV-infected patients who switched to efavirenz-based highly active antiretroviral therapy (HAART) from successful protease inhibitor-based HAART. PATIENTS AND METHODS: A total of 49 patients were included in this observational cohort study. At inclusion, all patients had plasma HIV-RNA levels<50 copies/mL and switched to efavirenz combined with two nucleoside reverse transcriptase inhibitors. Intracellular and plasma concentrations were measured 12 h post-dose, 1 month (M1) and 6 months (M6) after starting efavirenz. Virological success (VS) was defined as plasma HIV-RNA level<50 copies/mL within the first 12 months and remaining undetectable at the end of the follow-up. RESULTS: VS was documented in 48 patients for at least 12 months (range 12-78 months). High inter-patient variabilities of intracellular and plasma efavirenz concentrations were observed (coefficients of variation>40%). At M1 and M6, respectively, median [Q1; Q3] intracellular efavirenz concentrations were 5300 [2830; 11 530] and 6790 [3870; 8790] ng/mL, median plasma efavirenz concentrations were 2050 [1600; 3100] and 2100 [1410; 2500] ng/mL. No correlation was found between intracellular and plasma concentrations. Plasma efavirenz levels exceeded the proposed threshold of 1000 ng/mL in 96% of patients from M1. CONCLUSIONS: For moderately pre-treated HIV-infected patients with few mutations who switched to efavirenz from previous successful HAART, the proposed plasma efficacy-threshold was reached without any dosage adaptation. VS was maintained beyond 12 months, despite high inter-patient and intra-patient variabilities of intracellular and plasma efavirenz concentrations.

Marked intraindividual variability in antiretroviral concentrations may limit the utility of therapeutic drug monitoring.

BACKGROUND: Effective therapeutic drug monitoring for antiretrovirals requires a better understanding of intraindividual variability in pharmacokinetics. METHODS: We determined concentrations of human immunodeficiency virus (HIV) protease and nonnucleoside reverse-transcriptase inhibitors for 10 patients with undetectable plasma HIV RNA levels who had been receiving stable regimens for > or = 11 months. Plasma samples were collected at the same time of day 3 times per week for up to 4 months. Patients were instructed to take their antiretrovirals at the same time every day. Plasma protease and nonnucleoside reverse-transcriptase inhibitor concentrations were determined using high-performance liquid chromatographic methods. Pharmacokinetic variability was expressed as intraindividual percentage coefficient of variation (ICV), which was calculated as the patient's standard deviation divided by the mean drug concentration for that patient. RESULTS: ICV was determined for 6 drugs for 10 patients, for a total of 17 different patient-drug combinations, using 600 total samples. ICV was unexpectedly high for most patients who were receiving protease inhibitors (ICVs for individual patients taking lopinavir/ritonavir were 24%, 33%, 51%, and 92%; for patients taking nelfinavir/M8 metabolite, they were 30%/44% and 39%/54%; for patients taking ritonavir, they were 34% and 43%; for patients taking saquinavir, they were 52% and 55%). ICVs for patients receiving nonnucleoside reverse-transcriptase inhibitors were lower (for patients receiving efavirenz, they were 7%, 13%, 29%, and 51%; for a patient receiving nevirapine, it was 25%). The median ICV for all patients receiving protease inhibitors (n = 12) was 43.5%, and for all patients receiving nonnucleoside reverse-transcriptase inhibitors (n = 5), the median ICV was 25%. CONCLUSIONS: Intraindividual variability in concentrations of antiretrovirals was surprisingly high in virologically suppressed patients. Possible contributors include food effects, concomitant use of prescription and herbal medications, assay variability, or medication timing, which was assessed by self-report. High intraindividual pharmacokinetic variability may limit the utility of single measurements in therapeutic drug monitoring for some antiretroviral agents.

Simultaneous determination of the HIV nucleoside analogue reverse transcriptase inhibitors lamivudine, didanosine, stavudine, zidovudine and abacavir in human plasma by reversed phase high performance liquid chromatography.

A reversed phase high performance liquid chromatography method was developed for the simultaneous quantitative determination of the nucleoside reverse transcriptase inhibitors (NRTIs) lamivudine, didanosine, stavudine, zidovudine and abacavir in plasma. The method involved solid-phase extraction with Oasis MAX cartridges from plasma, followed by high performance liquid chromatography with a SymmetryShield RP 18 column and ultraviolet detection set at a wavelength of 260 nm. The assay was validated over the concentration range of 0.015-5 mg/l for all five NRTIs. The average accuracies for the assay were 92-102%, inter- and intra-day coefficients of variation (CV) were <2.5% and extraction recoveries were higher than 97%. This method proved to be simple, accurate and precise, and is currently in use in our laboratory for the quantitative analysis of NRTIs in plasma.

Design, synthesis, anti-HIV activities, and metabolic stabilities of alkenyldiarylmethane (ADAM) non-nucleoside reverse transcriptase inhibitors.

The alkenyldiarylmethane (ADAM) HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) are effective anti-HIV agents in cell culture. However, the potential clinical utility of the ADAMs is expected to be limited by the presence of methyl ester moieties that are likely to be metabolized by nonspecific esterases in blood plasma to biologically inactive carboxylic acid derivatives. The present investigation was therefore undertaken to investigate the anti-HIV activities of the ADAMs versus HIV-1(IIIB) and HIV-2(ROD) in MT-4 cells and the stabilities of the biologically active ADAMs in rat plasma. The ADAMs displayed a wide range of metabolic stabilities in rat plasma, with half-lives ranging from 0.9 to 76.6 min. A wide assortment of structural modifications was tolerated, with 18 of the 32 compounds tested displaying EC(50) values between 0.3 and 3.7 microM versus HIV-1(IIIB) in MT-4 cells, 3 compounds in the EC(50) = 13.2-35.4 microM range, and the remaining compounds inactive. Consistent with the mechanism of action of the ADAMs as NNRTIs, they were inactive or displayed comparatively low activity versus HIV-2(ROD). The replacement of the two aromatic methyl ester substituents in one of the most active ADAMs (EC(50) = 0.6 microM) with two methyl thioester groups resulted in an increase in plasma half-life from 5.8 to 55.3 min, while maintaining the antiviral potency at the EC(50) = 1.8 microM level. At the same time, the bis(thioester) modification was less cytotoxic to uninfected MT-4 cells, with a CC(50) of >224 microM versus 160 microM for the parent compound.

Liquid chromatographic-mass spectrometric determination of the metabolism and disposition of the anti-retroviral nucleoside analogs zidovudine and lamivudine in C57BL/6N and B6C3F1 mice.

Transmission of HIV from mother to infant can be effectively prevented by zidovudine (3'-azido-3'-deoxythymidine; AZT) alone or in combination with other anti-retroviral drugs; however, significant evidence for genotoxicity, including transplacental carcinogenicity in mice, has been reported for AZT. A method, based upon solid phase extraction (SPE) in the 96-well format, gradient liquid chromatography (LC), and electrospray mass spectrometry (MS), was developed and validated to measure serum concentrations in maternal C57BL/6N and fetal B6C3F1 mice of the nucleoside analogs AZT, lamivudine ((-)2',3'-dideoxy-3'-thiacytidine; 3TC), and several metabolites selected based on importance in detoxification and bioactivation reactions. After intravenous (i.v.) and oral dosing with either 400 mg/kg AZT or 200 mg/kg 3TC, pharmacokinetics were determined for AZT, AZT-5'-glucuronide, 3'-amino-3'-deoxythymidine (AMT), AZT-5'-phosphate, 3TC, and 3TC-5'-phosphate in serum of adult female mice. Pharmacokinetics were also determined in spleen for AZT-5'-phosphate and 3TC-5'-phosphate following i.v. dosing. In addition, a preliminary assessment was made of placental transfer of AZT and 3TC and the presence of metabolites in the fetal compartment. The method described provides a means to evaluate thoroughly metabolism and disposition of anti-retroviral nucleoside analogs in maternal and fetal mice for comprehensive studies of genotoxicity.