External validation of the bilirubin-atazanavir nomogram for assessment of atazanavir plasma exposure in HIV-1-infected patients.

Atazanavir increases plasma bilirubin levels in a concentration-dependent manner. Due to less costly and readily available assays, bilirubin has been proposed as a marker of atazanavir exposure. In this work, a previously developed nomogram for detection of suboptimal atazanavir exposure is validated against external patient populations. The bilirubin nomogram was validated against 311 matching bilirubin and atazanavir samples from 166 HIV-1-infected Norwegian, French, and Italian patients on a ritonavir-boosted regimen. In addition, the nomogram was evaluated in 56 Italian patients on an unboosted regimen. The predictive properties of the nomogram were validated against observed atazanavir plasma concentrations. The use of the nomogram to detect non-adherence was also investigated by simulation. The bilirubin nomogram predicted suboptimal exposure in the patient populations on a ritonavir-boosted regimen with a negative predictive value of 97% (95% CI 95-100). The bilirubin nomogram and monitoring of atazanavir concentrations had similar predictive properties for detecting non-adherence based on simulations. Although both methods performed adequately during a period of non-adherence, they had lower predictive power to detect past non-adherence episodes. Using the bilirubin nomogram for detection of suboptimal atazanavir exposure in patients on a ritonavir-boosted regimen is a rapid and cost-effective alternative to routine measurements of the actual atazanavir exposure in plasma. Its application may be useful in clinical settings if atazanavir concentrations are not available.

Assessment of the pharmacokinetic interaction between eltrombopag and lopinavir-ritonavir in healthy adult subjects.

Eltrombopag is an orally bioavailable thrombopoietin receptor agonist that is approved for the treatment of chronic idiopathic thrombocytopenic purpura. It is being developed for other medical disorders that are associated with thrombocytopenia. Patients with human immunodeficiency virus (HIV) may suffer from thrombocytopenia as a result of their HIV disease or coinfection with hepatitis C virus (HCV). HIV medications, particularly ritonavir (RTV)-boosted HIV protease inhibitors, are involved in many drug interactions. This study evaluated the potential drug-drug interaction between eltrombopag and lopinavir (LPV)/RTV. Forty healthy adult subjects enrolled in this open-label, three-period, single-sequence crossover study received a single 100-mg dose of eltrombopag (period 1), LPV/RTV at 400/100 mg twice daily (BID) for 14 days (period 2), and LPV/RTV at 400/100 mg BID (2 doses) with a single 100-mg dose of eltrombopag administered with the morning LPV/RTV dose (period 3). There was a 3-day washout between periods 1 and 2 and no washout between periods 2 and 3. Serial pharmacokinetic samples were collected during 72 h in periods 1 and 3 and during 12 h in period 2. The coadministration of 400/100 mg LPV/RTV BID with a single dose of 100 mg eltrombopag decreased the plasma eltrombopag area under the plasma concentration-time curve from time zero extrapolated to infinity (AUC(0-∞)) by 17%, on average, with no change in plasma LPV/RTV exposure. Adverse events (AEs) reported in period 2 were consistent with known LPV/RTV AEs, such as diarrhea, abdominal pain, nausea, vomiting, rash, and fatigue. No subjects withdrew due to AEs, and no serious AEs were reported. These study results suggest that platelet counts should be monitored and the eltrombopag dose adjusted accordingly if LPV/RTV therapy is initiated or discontinued.

Influence of body weight on achieving indinavir concentrations within its therapeutic window in HIV-infected Thai patients receiving indinavir boosted with ritonavir.

Indinavir boosted with ritonavir (IDV/r) dosing with 400/100 mg, twice daily, is preferred in Thai adults, but this dose can lead to concentrations close to the boundaries of its therapeutic window. The objectives of this analysis were to validate a population pharmacokinetic model to describe IDV/r concentrations in HIV-infected Thai patients and to investigate the impact of patient characteristics on achieving adequate IDV concentrations. IDV/r concentration data from 513 plasma samples were available. Population means and variances of pharmacokinetic parameters were estimated using a nonlinear mixed effects regression model (NONMEM Version VI). Monte Carlo simulations were performed to estimate the probability of achieving IDV concentrations within its therapeutic window. IDV/r pharmacokinetics were best described by a one-compartment model coupled with a single transit compartment absorption model. Body weight influenced indinavir apparent oral clearance and volume of distribution and allometric scaling significantly reduced the interindividual variability. Final population estimates (interindividual variability in percentage) of indinavir apparent oral clearance and volume of distribution were 21.3 L/h/70 kg (30%) and 90.7 L/70 kg (22%), respectively. Based on model simulations, the probability of achieving an IDV trough concentration greater than 0.1 mg/L was greater than 99% for 600/100 mg and greater than 98% for 400/100 mg, twice daily, in patients weighing 40 to 80 kg. However, the probability of achieving IDV concentrations associated with an increased risk of drug toxicity (greater than 10.0 mg/L) increased from 1% to 10% with 600/100 mg compared with less than 1% with 400/100 mg when body weight decreased from 80 to 40 kg. The validated model developed predicts that 400/100 mg of IDV/r, twice daily, provides indinavir concentrations within the recommended therapeutic window for the majority of patients. The risk of toxic drug concentrations increases rapidly with IDV/r dose of 600/100 mg for patients less than 50 kg and therapeutic drug monitoring of IDV concentrations would help to reduce the risk of IDV-induced nephrotoxicity.

Atazanavir metabolism according to CYP3A5 status: an in vitro-in vivo assessment.

The current study was a follow-up to an in vivo study in which atazanavir oral clearance was shown to be dependent on genetically determined CYP3A5 expression status, but only in non-African Americans. The aim of this study was to identify atazanavir metabolites generated by CYP3A5 and to evaluate this metabolite pattern in the African-American versus non-African-American CYP3A5 expressors from the previous study. First, the in vitro metabolism of atazanavir was evaluated using human liver microsomes (HLM) and CYP3A4 and CYP3A5 isoforms. Second, the metabolite pattern generated by CYP3A5 was evaluated in human plasma samples from the previous study. Atazanavir metabolites were analyzed using liquid chromatography-tandem mass spectrometry methods. Metabolite areas under the time-concentration curves (AUCs) were normalized to atazanavir AUC to generate an AUC ratio. Sixteen metabolites were observed in human liver microsomal incubations representing five "phase I" biotransformation pathways. Mono-oxidation products (M1 and M2) were formed by CYP3A5 at a faster rate than CYP3A4 by 32- and 2.6-fold, respectively. This finding was replicated in HLM from a genetically determined CYP3A5 expressor versus nonexpressor. In the in vivo samples, the M1 and M2 AUC ratios were approximately 2-fold higher in CYP3A5 expressors versus nonexpressors (P < 0.05), and the difference was similar in African Americans and non-African Americans. Thus, CYP3A5 produced a unique metabolite "signature" for atazanavir in vitro and in vivo, independent of race. Therefore, other pharmacological factors are likely to explain the apparent lack of effect of genetically determined CYP3A5 expressor status on atazanavir oral clearance in African Americans from the previous study.

Quality assessment for therapeutic drug monitoring in AIDS Clinical Trials Group (ACTG 5146): a multicenter clinical trial.

In a randomized trial, AIDS Clinical Trials Group (ACTG) protocol 5146 (A5146) investigated the use of therapeutic drug monitoring (TDM) to adjust doses of HIV-1 protease inhibitors (PIs) in patients with prior virologic failure on PI-based therapy who were starting a new PI-based regimen. The overall percentage of "PI trough repeats" such as rescheduled visits or redrawn PI trough specimens increased from 2% to 5% to 10% as the process progressed from the clinical sites, the pharmacology specialty laboratory, and the study team, respectively. Cumulatively, this represents a 17% rate of failure to obtain adequate PI trough sample. While targeting a turnaround of 7 days or less from sample receipt to a drug concentration report, 12% of the received specimens required a longer period to report concentrations. The implementation of dosing changes in the TDM arm were achieved within 7 days or less for 56% of the dose change events and within 14 days or less for 77% of dose change events. This quality assurance analysis provides a valuable summary of the specific points in the TDM process that could be improved during a multicenter clinical trial including: 1) shortening the timeline of sample shipment from clinical site to the laboratory; 2) performing the collection of PI trough specimen within the targeted sampling window by careful monitoring of the last dose times and collection times by the clinicians; 3) increasing patient adherence counseling to reduce the number of samples that are redrawn due to suspecting inconsistent adherence; and 4) decreasing the time to successful TDM-based dose adjustment. The application of some of these findings may also be relevant to single-center studies or clinical TDM programs within a hospital.

Estimation of population pharmacokinetic parameters of saquinavir in HIV patients with the MONOLIX software.

In nonlinear mixed-effects models, estimation methods based on a linearization of the likelihood are widely used although they have several methodological drawbacks. Kuhn and Lavielle (Comput. Statist. Data Anal. 49:1020-1038 (2005)) developed an estimation method which combines the SAEM (Stochastic Approximation EM) algorithm, with a MCMC (Markov Chain Monte Carlo) procedure for maximum likelihood estimation in nonlinear mixed-effects models without linearization. This method is implemented in the Matlab software MONOLIX which is available at http://www.math.u-psud.fr/~lavielle/monolix/logiciels. In this paper we apply MONOLIX to the analysis of the pharmacokinetics of saquinavir, a protease inhibitor, from concentrations measured after single dose administration in 100 HIV patients, some with advance disease. We also illustrate how to use MONOLIX to build the covariate model using the Bayesian Information Criterion. Saquinavir oral clearance (CL/F) was estimated to be 1.26 L/h and to increase with body mass index, the inter-patient variability for CL/F being 120%. Several methodological developments are ongoing to extend SAEM which is a very promising estimation method for population pharmacockinetic/pharmacodynamic analyses.

Full validation of an analytical method for the HIV-protease inhibitor atazanavir in combination with 8 other antiretroviral agents and its applicability to therapeutic drug monitoring.

Because HIV medications are used in combination, it is important to develop multiplex assays to streamline the therapeutic drug monitoring process and provide rapid turnaround. This article reports full validation of an analytical method that combines atazanavir with 6 HIV-protease inhibitors (indinavir, amprenavir, saquinavir, nelfinavir, ritonavir, and lopinavir) and 2 nonnucleoside reverse transcriptase inhibitors (nevirapine and efavirenz). Using 200 microL of plasma and a simple liquid-liquid extraction method, this analytical method achieved a clean baseline and high extraction efficiencies (90.0% to 99.5%). A Zorbax C-18 (150 x 4.6 mm, 3.5 microm) analytical column was used along with a 27-minute linear gradient elution of the mobile phase to provide sharp peaks at 210 nm. This method was validated over a range of 25 to 10,000 ng/mL and is accurate (90.4% to 110.5%) and precise (precision within a day and between days ranged from 2.3% to 8.3%). Because this method is simple and inexpensive, it may have applicability in countries with low resources.

Determination of lopinavir and nevirapine by high-performance liquid chromatography after solid-phase extraction: application for the assessment of their transplacental passage at delivery.

An adaptation of the HPLC method previously described for the simultaneous assay of amprenavir, ritonavir, indinavir, saquinavir, nelfinavir and efavirenz after solid-phase extraction is proposed here for the separate analysis of the newer PI lopinavir (LPV) and the NNRTI nevirapine (NVP). After viral inactivation by heat (60 degrees C for 60 min), plasma (600 microl), with clozapine added as internal standard, is diluted 1+1 with phosphate buffer pH 7 and subjected to a solid-phase extraction on a C(18) cartridge. Matrix components are eliminated with 2 x 500 microl of a solution of 0.1% H(3)PO(4) neutralised with NaOH to pH 7. LPV and NVP are eluted with 3 x 500 microl MeOH. The resulting eluate is evaporated under nitrogen at room temperature and is reconstituted in 100 microl MeOH 50%. A 40-microl volume is injected onto a Nucleosil 100, 5 microm C(18) AB column. LPV and NVP are analysed separately using a gradient elution program with solvents constituted of MeCN and phosphate buffer adjusted to pH 5.07 and containing 0.02% sodium heptanesulfonate. LPV and NVP are detected by UV at 201 and 282 nm, respectively. The calibration curves are linear up to 10 microg/ml. The mean absolute recovery of LPV and NVP is 91% and 88%, respectively. The method is precise with mean inter-day C.V.s within 2.1-6.6% and 0.9-1.7% for LPV and NVP, and accurate (range of inter-day deviations -1.1 to +2.4%, and -1.9 to +0.8%, for LPV and NVP, respectively). The method has been validated and is currently applied to the monitoring of LPV and NVP in HIV patients, and has been notably applied in a study aimed at assessing the extent of transplacental passage of nevirapine and PIs, notably lopinavir, at the time of delivery in pregnant HIV-infected women.

[Simultaneous quantitative determination of amprenavir and indinavir in human plasma by high-performance liquid chromatography]. / Détermination simultanée de la concentration plasmatique de l'amprénavir et de l'indinavir par chromatographie liquide haute performance.

A reversed-phase high-performance liquid chromatographic assay for the determination of the HIV protease inhibitors amprenavir (Agenerase) and indinavir (Crixivan) in human plasma is described, using a mobile phase consisting of 0.50 M phosphate buffer (adjusted to pH 5,5) - Milli-Q water - acetonitrile (120: 1,080: 800, v/v/v). A solid-phase extraction using C18 extraction columns (Discovery columns 100 mg, 1 ml Supelco) and a liquid-liquid extraction with 0.5 ml hydrogenocarbonate/carbonate buffer (adjusted to pH 10.6) and 6 ml methyl ter-butyl ether have been compared. The liquid-liquid extraction has been chosen to be easier and cheaper. The method has been validated over the range of 60 to 3,000 ng/ml for amprenavir and 20 to 3,000 ng/ml for indinavir using a 0.5 ml sample volume. The specificity, linearity, accuracy and precision have been studied. The limit of detection was respectively for amprenavir and indinavir 15 and 4 ng, and the limit of quantification was 60 and 20 ng/ml. Stability tests under various conditions were performed. This assay can readily be used in a hospital laboratory for the routine monitoring of plasma concentrations of amprenavir in HIV-infected patients. The trough plasma concentrations average has been determined in patients treated by amprenavir and indinavir for seven months.

Therapeutic drug monitoring of antiretrovirals in human immunodeficiency virus infection.

The era of antiviral therapy directed against HIV-1 has now entered its second decade. In the twelve years since the FDA approved the first antiretroviral drug zidovudine there have been a number of seminal developments that have revolutionized the approach to therapy. These advances converged to change the treatment paradigm from one of therapeutic nihilism to that of cautious optimism. First, several trials demonstrated that combination therapy of nucleoside reverse transcriptase inhibitors (NRTIs) is superior to monotherapy in extending survival and delaying disease progression. Second, the concept of virologic latency in asymptomatic HIV-infected patients was revised. Mathematic modelling demonstrated that there is an ongoing high level of virus production driving a rapid turnover of CD4 cells at all stages of infection. Hence it was concluded that the aim of antiretroviral therapy (ART) should be to "hit early and hit hard." Third, significant advances in molecular virology facilitated the development of quantitative methods to measure the circulating HIV plasma RNA. HIV viral load has been shown to be a sensitive predictor of disease progression and a valuable marker of response to therapy. However, none of these developments would have translated into improved patient care without the advent of two new classes of drugs-the protease inhibitors (PIs) and the nonnucleoside reverse transcriptase inhibitors (NNRTIs).