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The objective of the present work was to conduct an "in vivo" analysis of the atazanavir-bilirubin interaction. We developed a new mathematical approach to PK/PDPK models for competitive interaction based on the Michaelis-Menten equation, which was applied to patients with polymorphisms in the gene for UDP-glucuronosyltransferase 1A1 (UGT1A1). Atazanavir is known to induce concentration-dependent increases in bilirubin plasma levels. Thus, we employed our mathematical model to analyse rises in steady state atazanavir and bilirubin concentrations, ultimately plotting a nomogram for detection of suboptimal atazanavir exposure. Application of our model revealed that an absolute value or a steady state increase in bilirubin falling below 3.8Φ µmol/L (where Φ is a correction factor, =1 for UGT1A1 wild type and ≠1 for UGT1A1 variants) could be used to predict suboptimal atazanavir exposure and treatment failure. Thus, we have successfully established a new mathematical approach for pharmacodynamic-pharmacokinetic modelling of the interaction between atazanavir and bilirubin, as it relates to genetic variants of UGT1A1. Taken together, our findings indicate that bilirubin plasma levels represent a valuable marker of atazanavir exposure., (© 2013, The American College of Clinical Pharmacology.)

The blood-testis barrier and blood-brain barrier are responsible for protecting the male genital tract and central nervous system from xenobiotic exposure. In HIV-infected patients, low concentrations of antiretroviral drugs in cerebrospinal fluid and seminal fluid have been reported. One mechanism that may contribute to reduced concentrations is the expression of ATP-binding cassette drug efflux transporters, such as P-glycoprotein (P-gp). The objective of this study was to investigate in vivo the tissue distribution of the HIV protease inhibitor atazanavir in wild-type (WT) mice, P-gp/breast cancer resistance protein (Bcrp)-knockout (Mdr1a-/-, Mdr1b-/-, and Abcg2-/- triple-knockout [TKO]) mice, and Cyp3a-/- (Cyp) mice. WT mice and Cyp mice were pretreated with a P-gp/Bcrp inhibitor, elacridar (5 mg/kg of body weight), and the HIV protease inhibitor and boosting agent ritonavir (2 mg/kg intravenously [i.v.]), respectively. Atazanavir (10 mg/kg) was administered i.v. Atazanavir concentrations in plasma (Cplasma), brain (Cbrain), and testes (Ctestes) were quantified at various times by liquid chromatography-tandem mass spectrometry. In TKO mice, we demonstrated a significant increase in atazanavir Cbrain/Cplasma (5.4-fold) and Ctestes/Cplasma (4.6-fold) ratios compared to those in WT mice (P<0.05). Elacridar-treated WT mice showed a significant increase in atazanavir Cbrain/Cplasma (12.3-fold) and Ctestes/Cplasma (13.5-fold) ratios compared to those in vehicle-treated WT mice. In Cyp mice pretreated with ritonavir, significant (P<0.05) increases in atazanavir Cbrain/Cplasma (1.8-fold) and Ctestes/Cplasma (9.5-fold) ratios compared to those in vehicle-treated WT mice were observed. These data suggest that drug efflux transporters, i.e., P-gp, are involved in limiting the ability of atazanavir to permeate the rodent brain and genital tract. Since these transporters are known to be expressed in humans, they could contribute to the low cerebrospinal and seminal fluid antiretroviral concentrations reported in the clinic.

Physiological effects of aging make the older population more susceptible to adverse drug events and drug-drug interactions. We evaluated the impact of aging and gender on the pharmacokinetics (PK) of atazanavir/ritonavir (ATV/r) 300/100 mg once daily (qd) in 22 well-suppressed HIV-infected patients. This was a 24-h intensive PK study. Subjects were HIV-1-infected adults aged ≥18 years with HIV RNA <50 copies/ml and treated with ATV/r 300/100 mg once daily plus two nucleoside reverse transcriptase inhibitors (NRTIs) for at least 2 weeks. Atazanavir and ritonavir plasma concentrations were measured by validated high-performance liquid chromatography (HPLC). Plasma PK parameters were calculated using noncompartmental methods. Since 50% of the patients were older than 42 years, age 42 was selected as the cut-off point for the older (>42 years) group. Gender, weight, duration of ATV/r therapy, and proportion treated with tenofovir disoproxil fumarate (TDF)-containing regimens did not differ between both groups. Patients from the aging group had a reduced creatinine clearance (91 versus 76 ml/min). The older group had a higher atazanavir exposure with median AUC(0-24) 71.2 vs. 53.1 mg·h/liter, C(max) 8.5 vs. 5.5 mg/liter, and C(trough) 1.17 vs. 0.78 mg/liter, and slower apparent clearance (3.5 vs. 4.8 liter/h). Ten patients (91%) from the older group and 36% from the younger group had ATV C(trough) levels higher than the proposed upper limit for toxicity of 0.85 mg/liter. Females had a lower body weight (BW) (46 versus 63 kg) than the males, but atazanavir concentrations in females were greater. However, in multivariate analysis, older age was the only significant predictor for higher atazanavir concentrations. Parameter estimate for age and atazanavir AUC after adjusting for gender and BW was 2.17 (95% CI 1.01-3.33). That is, for every year increase in age, AUC increases by approximately 2 mg·h/liter. Age seems to be an important factor influencing atazanavir pharmacokinetics. Patients from the aging group appeared to have higher atazanavir exposure compared to the younger group. Further PK explorations of ATV in the extremely aged population are warranted.

Atazanavir (Reyataz(®)) is an important member of the HIV protease inhibitor class. Because of the complexity of its chemical structure, metabolite identification and structural elucidation face serious challenges. So far, only seven non-conjugated metabolites in human plasma have been reported, and their structural elucidation is not complete, especially for the major metabolites produced by oxidations. To probe the exact sites of metabolism and to elucidate the relationship among in vivo metabolites of atazanavir, we designed and performed two sets of experiments. The first set of experiments was to determine atazanavir metabolites in human plasma by LC-MS, from which more than a dozen metabolites were discovered, including seven new ones that have not been reported. The second set involved deuterium labeling on potential metabolic sites to generate D-atazanavir analogs. D-atazanavir analogs were dosed to human in parallel with atazanavir. Metabolites of D-atazanavir were identified by the same LC-MS method, and the results were compared with those of atazanavir. A metabolite structure can be readily elucidated by comparing the results of the analogs and the pathway by which the metabolite is formed can be proposed with confidence. Experimental results demonstrated that oxidation is the most common metabolic pathway of atazanavir, resulting in the formation of six metabolites of monooxidation (M1, M2, M7, M8, M13, and M14) and four of dioxidation (M15, M16, M17, and M18). The second metabolic pathway is hydrolysis, and the third is N-dealkylation. Metabolites produced by hydrolysis include M3, M4, and M19. Metabolites formed by N-dealkylation are M5, M6a, and M6b., (Copyright © 2013 John Wiley & Sons, Ltd.)

Long-acting injectable nanoformulated antiretroviral therapy (nanoART) was developed with the explicit goal of improving medicine compliance and for drug targeting of viral tissue reservoirs. Prior nanoART studies completed in humanized virus-infected mice demonstrated sustained antiretroviral responses. However, the pharmacokinetics (PK) and tissue distribution of nanoART were not characterized. To this end, the PK and tissue distribution of nanoformulated atazanavir (ATV) and ritonavir (RTV) injected subcutaneously or intramuscularly in mice and monkeys were evaluated. Fourteen days after injection, ATV and RTV levels were up to 13-, 41-, and 4,500-fold higher than those resulting from native-drug administration in plasma, tissues, and at the site of injection, respectively. At nanoART doses of 10, 50, 100, and 250 mg/kg of body weight, relationships of more- and less-than-proportional increases in plasma and tissue levels with dose increases were demonstrated with ATV and RTV. Multiple-dose regimens showed serum and tissue concentrations up to 270-fold higher than native-drug concentrations throughout 8 weeks of study. Importantly, nanoART was localized in nonlysosomal compartments in tissue macrophages, creating intracellular depot sites. Reflective data were obtained in representative rhesus macaque studies. We conclude that nanoART demonstrates blood and tissue antiretroviral drug levels that are enhanced compared to those of native drugs. The sustained and enhanced PK profile of nanoART is, at least in part, the result of the sustained release of ATV and RTV from tissue macrophases and at the site of injection.

Stepwise multiple regression analyses were applied to 44 atazanavir pharmacokinetic profiles from 44 HIV-1 infected patients concomitantly treated with raltegravir with the goal of identifying limited sampling strategies for the prediction of drug AUC(0-12) . Atazanavir trough-based equations failed to reliably predict daily drug exposure in patients with low drug bioavailability. Conversely, different algorithms based on few samples and associated with good correlation, acceptable bias and imprecision with the measured atazanavir AUC(0-12) were identified. These models could be used to predict atazanavir exposure for clinic or research purposes., (© 2011 The Authors Fundamental and Clinical Pharmacology © 2011 Société Française de Pharmacologie et de Thérapeutique.)

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.

Limited data on the pharmacokinetics and pharmacodynamics (PK/PD) of unboosted atazanavir (uATV) in treatment-experienced patients are available. The aim of this work was to study the PK/PD of unboosted atazanavir in a cohort of HIV-infected patients. Data were available for 58 HIV-infected patients (69 uATV-based regimens). Atazanavir concentrations were analyzed by using a population approach, and the relationship between atazanavir PK and clinical outcome was examined using logistic regression. The final PK model was a linear one-compartment model with a mixture absorption model to account for two subgroups of absorbers. The mean (interindividual variability) of population PK parameters were as follows: clearance, 13.4 liters/h (40.7%), volume of distribution, 71.1 liters (29.7%), and fraction of regular absorbers, 0.49. Seven subjects experienced virological failure after switch to uATV. All of them were identified as low absorbers in the PK modeling. The absorption rate constant (0.38 ± 0.20 versus 0.75 ± 0.28 h(-1); P = 0.002) and ATV exposure (area under the concentration-time curve from 0 to 24 h [AUC(0-24)], 10.3 ± 2.1 versus 22.4 ± 11.2 mg · h · liter(-1); P = 0.001) were significantly lower in patients with virological failure than in patients without failure. In the logistic regression analysis, both the absorption rate constant and ATV trough concentration significantly influenced the probability of virological failure. A significant relationship between ATV pharmacokinetics and virological response was observed in a cohort of HIV patients who were administered unboosted atazanavir. This study also suggests that twice-daily administration of uATV may optimize drug therapy.

Most antiretrovirals are metabolized in the liver, and overexposure could be more common in human immunodeficiency virus (HIV)-infected patients with hepatic impairment. Careful monitoring of potential drug-related liver injury in clinical practice is necessary. The aim of our study was to analyze the trough concentrations (C (trough)) of atazanavir (ATV) in the plasma of HIV/hepatitis C virus (HCV)-co-infected patients and to compare the values with those of a HIV-infected control population. C (trough) values (22-26 h after last intake) of atazanavir, following the administration of atazanavir/ritonavir 300/100 mg once daily as part of antiretroviral therapy, were assessed by HPLC. We also collected data on dosing of atazanavir, and on demographic (age, gender, and ethnicity), physiological (weight and body mass index), and clinical parameters (CD4+ cell count, HIV-RNA viremia, co-medication, and hepatitis C co-infection). A total of 28 Caucasian HIV-infected adults were studied, of whom 13 were HIV/HCV co-infected. No baseline characteristics differed between the two cohorts, except statistically significant differences regarding ALT, AST, and total bilirubin. The median (range) plasma ATV C (trough) levels were 0.62 (0.05-3.22) μg/ml in HIV patients and 0.32 (0.04-3.37) μg/ml in HIV/HCV patients. Thus, there was no significant difference in plasma trough levels of atazanavir in the two cohorts. In our patients with mild impairment of hepatic function caused by HCV infection, atazanavir C (trough) was comparable in HIV-infected and HIV/HCV-co-infected patients.

Atazanavir is a selective and potent inhibitor of the HIV-1 protease. The drug is administered in combination with low-dose ritonavir, to boost atazanavir pharmacokinetics (i.e. ritonavir-boosted atazanavir), and other antiretroviral agents. The efficacy of once-daily ritonavir-boosted atazanavir plus background therapy (BT) in establishing virologic suppression in treatment-naive pediatric patients (aged 6 to <18 years) infected with HIV-1 was demonstrated in an ongoing, open-label, multicenter, phase I/II trial (PACTG 1020A). HIV-1 RNA levels of <50 or <400 copies/mL were achieved by the majority of patients (>70%) after 24 weeks' therapy, with these benefits maintained at week 48. Some treatment-experienced pediatric patients (aged 6 to <18 years) also achieved HIV-1 RNA levels of <50 or <400 copies/mL in the PACTG 1020A trial after 24 (≤45% of patients) and 48 (≤32%) weeks of treatment with ritonavir-boosted atazanavir plus BT, although the benefits of the regimen in this patient population appeared to be limited by as few as one or two protease inhibitor resistance mutations. Treatment-experienced pediatric patients (aged 10-19 years) infected with HIV-1 had mixed success in establishing/maintaining virologic suppression when they were switched from their current antiretroviral treatment regimen to once-daily ritonavir-boosted atazanavir plus BT in a small, single-center, observational study. However, some patients may have received atazanavir at a suboptimal dosage or had suboptimal susceptibility to BT agents. In the PACTG 1020A trial, use of atazanavir (with or without ritonavir) in pediatric patients aged 6 to <18 years was associated with a similar safety profile to that reported in adults.

Objective: To study the impact on the neonate of maternal antiretroviral therapy with atazanavir (ATV)., Study Design: An observational study of 22 HIV-infected women receiving, for clinical indications, antiretroviral therapy with ATV 300 mg and ritonavir 100mg during pregnancy and their 23 HIV infants (including a twin pair)., Results: Mothers had received ATV for a median duration of 19 months [range 3-49] by delivery. At delivery, plasma HIV-RNA was <40 copies/mL in all patients. Liver enzymes were normal in 19/22 patients, but one woman had grade 3-4 liver toxicity. Maternal serum bilirubin concentrations were above the upper limit of normal in most patients, with grade 3 toxicity in 5 patients. All but one woman had trough ATV concentrations during pregnancy above the minimum effective concentration. The median cord blood ATV concentration was 130 ng/mL [range<30-758]; the cord/maternal ratio was 21%. All neonates were born at term [median 38.2 weeks]. Three neonates had mildly elevated AST transaminase levels. Bilirubin concentrations at birth were significantly higher than maternal concentrations, with a median of 44 μm/L [range 24-129]; values on days 2-3 were 63 [8-212]. Five neonates had jaundice requiring phototherapy, without liver damage, and recovered without sequelae., Conclusion: Neonates whose mothers were treated with ATV should be monitored for hyperbilirubinemia, which may be due to placental transfer of unconjugated bilirubin from the mother and/or a direct effect of transplacental ATV on bilirubin metabolism in the fetus., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Background: Adequate plasma trough concentrations (Ctrough) of protease inhibitors are required to maintain antiviral activity throughout the dosing interval. Therapeutic drug monitoring is used in clinical practice to optimize dosage and avoid toxic or subtherapeutic drug exposure. The pharmacokinetic variability of Atazanavir (ATV) can be relatively large, as a result of several factors. One of the affecting factors may be hepatic impairment due to hepatitis C virus (HCV) coinfection., Methods: We collected trough plasma samples from human immunodeficiency virus (HIV)-1-infected outpatients, with and without HCV coinfection and/or cirrhosis, receiving stable highly active antiretroviral therapy containing ATV. In the total population, we mainly compared the 2 regimens: 300ATV + 100RTV OD [ritonavir (RTV), once daily (OD)] versus 400ATV OD. We used a threshold value of 0.15 μg/mL, based on the proposed therapeutic range (0.15-0.85 μg/mL). Plasma concentrations of ATV were determined by a validated assay using high-performance liquid chromatography with ultraviolet detection. A total of 214 HIV-infected outpatients were included. For each regimen, we compared 3 groups of subjects: HIV+/HCV-, HIV+/HCV+, and HIV+/HCV+ with cirrhosis., Results: In the whole study population, we observed a large variability and found suboptimal Ctrough levels (<0.15 μg/mL) in 23 subjects (2 belonging to the 300/100 OD group and 21 to the 400 OD group). For the standard dosage regimen of 300ATV + 100RTV OD, we did not find a statistical difference between HIV-infected patients without HCV coinfection versus HIV-infected patients with HCV coinfection: median 0.85 (interquartile range 0.53-1.34) and 0.95 (0.70-1.36) μg/mL, respectively. In HIV+/HCV+-infected patients with cirrhosis, we found a median Ctrough of 0.70 (0.43-1.0) μg/mL, with no statistical difference when compared with HIV+/HCV- infected patients. For the 400ATV OD (n=90) dosage regimen, the total median ATV Ctrough was 0.40 (0.23-1.0) μg/mL. In this group, we found a statistically significant difference between HIV+/HCV- and HIV+/HCV+-infected patients: median Ctrough was 0.23 (0.11-0.42) and 0.52 (0.20-1.0) μg/mL, respectively. In HIV+/HCV+ subjects with cirrhosis, the Ctrough median value was 0.42 (0.13-0.75) μg/mL, and there was a significant difference when compared with HIV patients without coinfection., Conclusions: Therapeutic drug monitoring of ATV in patients receiving unboosted regimen may be useful to identify those HIV-infected subjects, with or without HCV coinfection, who may benefit from adding low RTV doses, or the subset of patients in whom removal of RTV could be attempted without the risk of suboptimal plasma ATV exposure.

Atazanavir, a potent protease inhibitor of human immunodeficiency virus (HIV), exhibits pH-dependent solubility. Previous studies have indicated that coadministration with omeprazole 40 mg once daily significantly decreased atazanavir exposure by approximately 75%. Concomitant use of omeprazole and atazanavir is currently not recommended. This study investigated a clinically effective, low dose of omeprazole (20 mg daily) on atazanavir pharmacokinetics in 56 healthy volunteers given atazanavir/ritonavir 300/100 and 400/100 mg once daily. All atazanavir/ritonavir plus omeprazole combinations resulted in atazanavir area under the concentration-time curve (AUC) and trough concentrations (C(min)) comparable to or exceeding those observed with atazanavir 400 mg without omeprazole. Compared with atazanavir/ritonavir 300/100 mg without omeprazole, atazanavir/ritonavir 300/100 mg plus omeprazole reduced atazanavir AUC and C(min) by 42% and 46%, respectively. Increasing the atazanavir/ritonavir dose to 400/100 mg attenuated the effect of omeprazole, resulting in approximately 30% lower atazanavir C(min), with all individual C(min) values exceeded by greater than 10-fold the population mean protein binding-adjusted EC(90) against wild-type HIV. The effect of omeprazole on atazanavir/ritonavir 400/100 mg was similar whether given 1 hour prior to atazanavir/ritonavir or separated by 12 hours. No unexpected adverse events were noted. This study found that omeprazole 20 mg once daily has significantly less profound effects on atazanavir pharmacokinetics than previously observed with omeprazole 40 mg.

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.

Raltegravir (RAL) is primarily metabolized by uridine diphosphate-glucorunosyl transferase 1A1 (UGT1A1). Atazanavir (ATV), a strong inhibitor of UGT1A1, has been shown to increase plasma concentrations of RAL by approximately 50% in healthy volunteers. However, the extent of this interaction has not been studied in HIV-infected patients. A pharmacokinetic study was performed in 22 HIV-infected adults treated with 400 mg RAL plus 300 mg ATV 300 twice a day. Both drugs showed high pharmacokinetic variability (RAL AUC 0-12 7649 ± 4862 ng*h/mL; ATV AUC 0-12 = 19237 ± 13136 ng*h/mL). Notably, RAL trough concentrations were significantly higher compared with those measured in HIV subjects (n = 24) on RAL plus nucleoside reverse transcriptase inhibitors (506 ± 411 versus 177 ± 262 ng/mL, P < 0.01). A significant correlation was found between RAL and ATV area under the curve (AUC) (r = 0.611, P = 0.005). Notably, patients with ATV AUC 0-12 above the mean or with concentrations exceeding the half maximal inhibitory concentration for UGT1A1 had twofold higher RAL AUCs compared with patients with lower ATV exposure. Coadministration of ATV significantly increased plasma concentrations of RAL, especially in HIV-1-infected patients exposed to high concentrations of the protease inhibitor. This pharmacokinetic drug interaction could be handled by routine measurements of ATV trough concentrations and by the assessment of plasma RAL concentrations 2 to 3 hours after the morning drug intake.

Current procedures for obtaining and measuring plasma concentrations of HIV protease inhibitors (PIs) are technically challenging. Dried blood spot (DBS) assays offer a way to overcome many of the obstacles. We sought to develop a DBS assay for quantitation of the PI atazanavir (ATV) and to compare this method with a previously validated plasma assay. We prospectively enrolled 48 patients with well-controlled HIV disease who had been on ATV for at least 7 days. ATV was quantified from plasma by use of high-performance liquid chromatography (HPLC). A reversed-phase ultrahigh-performance liquid chromatography (UPLC) assay was utilized for DBS samples. The concentrations of ATV quantified in a DBS matrix showed very strong agreement with those measured in plasma (r(2) = 0.988). The mean difference in ATV concentration between the two methods was -10.8% (95% confidence interval [95% CI], -7.65% to -13.95%), indicating that the DBS method has a slight negative bias. A majority (97.8%) of the differences in concentration between the two assays fell within ±2 standard deviations. ATV concentrations were lower in subjects who had detectable HIV RNA in plasma (mean, 543 ng/ml) than in those with HIV RNA of <50 copies/ml (mean, 1,582 ng/ml) (P = 0.03, Wilcoxon rank-sum test). In conclusion, our study demonstrated that ATV quantitation in a DBS matrix is feasible and accurate. DBS use offers a convenient alternative for measuring plasma concentrations of ATV and may have utility in monitoring of drug concentrations in clinical practice and in future studies.

An efficient HPLC method for the determination of atazanavir in human plasma has been developed and validated. A relatively simple mobile phase consisting of acetonitrile-ammonium formate buffer (pH 3; 10 mM) (45:55, v/v) was pumped at a low flow rate of 0.3 ml/min through a reverse phase Phenomenex Luna C18 (2) (5 microm, 150 mm x 2.0 mm i.d.) column maintained at 30 degrees C. Diazepam was used as an internal standard and the eluent was monitored at 210 nm. The major advantage of this method over previously reported procedures is that the narrow-bore HPLC column used resulted in relatively short retention times for the internal standard (6.8 min) and atazanavir (8.3 min) with excellent peak resolution and associated reduction in solvent usage. Sample preparation involved liquid-liquid extraction using 400 microl plasma treated with sodium carbonate (2M) and extracted with a mixed organic solvent consisting of ethyl acetate-n-hexane (50:50, v/v). The organic layer was removed and evaporated to dryness under nitrogen. Samples were reconstituted in mobile phase (100 microl) and 20 microl was injected onto the column. The procedures were validated according to international standards with good reproducibility and linear response with correlation coefficients (r) consistently > or =0.999. The intra- and inter-day accuracies were 97.1+/-5.04 and 98.0+/-11.3 respectively at the LLOQ and between 101+/-4.48% and 104+/-2.09% for the QC samples. The intra- and inter-day precision were < or =11.6% RSD at the LLOQ and < or =6.78% RSD across the entire QC concentration range. Mean recovery based on high, medium and low quality control standards ranged between 94.4+/-1.07% and 100+/-2.22%. Plasma samples were evaluated under short-term (ambient temperature for 6h) and long-term (-10+/-2 degrees C for 2 months) storage conditions and were found to be stable. The method described is efficient and has the necessary accuracy and precision for the rapid quantitative determination of atazanavir in human plasma and is thus highly suitable for use in pharmacokinetic/bioavailability/bioequivalence studies in healthy human subjects., (Copyright 2010 Elsevier B.V. All rights reserved.)

Objective: Protease inhibitors may not penetrate into the central nervous system in therapeutic concentrations, which may allow ongoing HIV replication and injury. The objective of this study was to determine atazanavir penetration into cerebrospinal fluid (CSF)., Design: Single random plasma or paired plasma and CSF samples were drawn from participants enrolled in a multicenter, observational cohort study and taking atazanavir with or without ritonavir between October 2003 and October 2005., Methods: Plasma samples were assayed by high performance liquid chromatography and immunoassay; lower limit of detection was 45 ng/ml. CSF samples were assayed by immunoassay (ARK ATV-test); lower limit of detection was 5 ng/ml., Results: One hundred and seventeen participants (43 +/- 7.7 years, 79% men, 81 +/- 15 kg) had plasma or plasma and CSF paired samples drawn a median (interquartile range) of 10 (5-17) h postdose. Median (interquartile range) plasma atazanavir concentrations with or without ritonavir were 1278 (525-2265) and 523 (283-1344) ng/ml. The median (interquartile range) CSF concentrations with or without ritonavir were 10.3 (<5-21.1) and 7.9 (6.6-22) ng/ml. Nineteen of 79 (24%) CSF samples were less than 5 ng/ml. CSF concentrations were less than 1% of plasma concentrations and near the atazanavir wild-type IC50 of 1-11 ng/ml., Conclusion: Atazanavir CSF concentrations are highly variable and 100-fold lower than plasma concentrations, even with ritonavir boosting. CSF concentrations of atazanavir do not consistently exceed the wild-type IC50 of atazanavir and may not protect against HIV replication in the CSF.

Background: Most HPLC-UV methods for therapeutic drug monitoring of anti-HIV drugs have long run times, which reduce their applicability for high-throughput analysis. We developed an ultra-performance liquid chromatography (UPLC)-diode array detection method for the simultaneous quantification of the HIV-protease inhibitors (PIs) amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, and tipranavir (TPV), and the nonnucleoside reverse transcriptase inhibitors (NNRTIs) efavirenz and nevirapine., Methods: Solid-phase extraction of 1 mL plasma was performed with Waters HLB cartridges. After 3 wash steps, we eluted the drugs with methanol, evaporated the alcohol, and reconstituted the residue with 50 microL methanol. We injected a 4-microL volume into the UPLC system (Waters ACQUITY UPLC BEH C8 column maintained at 60 degrees C) and used a linear gradient of 50 mmol/L ammonium acetate and 50 mmol/L formic acid in water versus acetonitrile to achieve chromatographic separation of the drugs and internal standard (A-86093). Three wavelengths (215, 240, and 260 nm) were monitored., Results: All drugs were eluted within 15 min. Calibration curves with concentrations of 0.025-10 mg/L (1.875-75 mg/L for TPV) showed coefficients of determination (r(2)) between 0.993 and 0.999. The lower limits of quantification were well below the trough concentrations reported in the literature. Inter- and intraassay CVs and the deviations between the nominal and measured concentrations were <15%. The method was validated by successful participation in an international interlaboratory QC program., Conclusions: This method allows fast and simultaneous quantification of all commercially available PIs and NNRTIs for therapeutic drug monitoring.

Atazanavir (ATV) is an HIV protease inhibitor (IP) with a high in vitro activity against HIV-1, that demonstrates a high additive activity in the presence of other antiretrovirals and a synergic activity with other PI. Oral absorption is greater than 68%, maximum concentration (C(max)) being reached approximately 2 to 3 h after its administration. Its absorption is dependent on gastric pH, its administration being recommended after meals. The pharmacokinetics (PK) of ATV are non-linear; that is to say, its plasma concentrations (C(p)) do not increase in proportion to the dose. ATV is approximately 86% bound to plasma proteins. Its entry into the cerebrospinal fluid, semen or genital secretions varies but is generally less than 10-20%. Its passage across the placenta, measured as the mean of the ratios between the C(p) in umbilical cord and maternal blood, is 0.13. ATV is metabolised by oxidation by cytochrome P450 enzymes, subsequently being eliminated by the bile duct in the free or glucuronide form (80%) and by the urine. ATV is a weak competitive inhibitor of CYP3A4 and a strong inhibitor of uridine diphosphate-glucuronosyltransferase 1A1, which is the cause of the frequent high plasma bilirubin after its administration and of its pharmacological interactions.

This study investigated pregnane X receptor polymorphisms in relation to unboosted atazanavir plasma concentrations in 2 cohorts of patients. The polymorphism 63396T-->C predicted concentrations below the minimum effective concentration (150 ng/mL) with odds ratios of 18 (P = .008) and 5.13 (P = .02). Prospective studies determining potential clinical usefulness are now warranted.

A high-performance liquid chromatographic method for the determination of atazanavir (ATV) in human plasma is developed and validated. The method involves a rapid and simple solid-phase extraction (SPE) of ATV using Bond-elut C18 3 mL cartridge. The separation of ATV from internal standard and endogenous components is achieved using an isocratic elution on an octyl column and an UV detector set at 260 nm. The method is linear from 20 to 10,000 ng/mL (mean r2 = 0.9991, n = 10). The observed intra- and inter-day assay precision ranged from 2.2% to 14.7%[at the lower limit of quantitation (LOQ)], whereas accuracy varies between 1.0% and 14% (at LOQ). Mean drug recovery is 80.5% for ATV and 78.4% for IS. The method is found to be precise and accurate, practical enough for therapeutic drug monitoring in routine clinical practice and is applied for the assessment of 24-h ATV plasma concentration-time profiles in HIV-infected pregnant women.

Objective: To evaluate the predictive value of the genotypic inhibitory quotient (GIQ) on the atazanavir response in treatment-experienced HIV-1-infected patients., Patients and Methods: Thirty-six patients receiving an atazanavir-containing regimen were enrolled in the study. Atazanavir plasma concentrations were measured at month (M) 1, and genotype was performed at baseline. Virologic response was defined as a viral load <400 copies/mL or a decrease > or =1 log10., Results: The median numbers (range) of previous regimens, baseline protease inhibitors, and atazanavir resistance mutations were 8 (0 to 20), 3 (0 to 15), and 1 (0 to 10), respectively. The atazanavir-GIQ was associated with virologic response at M6, with a median value (range) of 365 (50 to 1172) in responder patients compared with 126 (23 to 1126) in nonresponders (P = 0.05). The cutoff value estimated for the atazanavir-GIQ was 183 (receiver operating characteristic curve test: 60% specificity, 74% sensitivity). Virologic response was achieved in 74% of patients with an atazanavir-GIQ >183 compared with only 26% of patients with an atazanavir-GIQ <183 (P = 0.02). Neither the number of mutations nor the atazanavir trough concentration was predictive of the virologic response., Conclusion: In pretreated patients, the atazanavir-GIQ might be useful to predict early virologic response and allow the determination of the target atazanavir trough concentration required to achieve virologic response and overcome drug resistance emergence in a given patient.

The human immunodeficiency virus protease inhibitor combination of atazanavir (ATV)-lopinavir-ritonavir was reported to exhibit a mutual pharmacoenhancement of plasma lopinavir and ATV concentrations which may be beneficial for salvage patients. We identified 17 patients in our pharmacokinetic database taking this combination and found conflicting results. Plasma concentrations of both ATV and lopinavir were modestly, although not significantly, decreased when the drugs were coadministered. Therefore, patients should be selected carefully for this regimen and frequent clinical and therapeutic drug monitoring is strongly advised.

Objectives: Studies on the pharmacokinetic interaction between atazanavir and lopinavir with ritonavir (lopinavir/ritonavir) report contradictory results. We aimed to establish the in vivo interaction between these two protease inhibitors as well as the variables influencing drug exposure., Methods: Pharmacokinetic parameters were investigated in HIV-infected patients treated with atazanavir 300 mg with ritonavir 100 mg q24h (group A) or lopinavir/ritonavir 400/100 mg q12h (group B) or atazanavir 300 mg q24h with lopinavir/ritonavir 400/100 mg q12h (group C). Patients receiving other concomitant protease inhibitors or non-nucleoside reverse transcriptase inhibitors were excluded., Results: In group A (n=10), mean +/- standard deviation atazanavir C(min) was 390 +/- 460 ng/mL, C(max) 3051 +/- 1996 ng/mL and AUC(24) 29 913 +/- 17 686 ng/mL/h. In group B (n=9), lopinavir C(min) was 7562 +/- 4292 ng/mL, C(max) 12 944 +/- 4838 ng/mL and AUC(0-12) 122 313 +/- 38 225 ng/mL/h. In group C (n=7), atazanavir C(min) was 876 +/- 460 ng/mL (P=0.039 vs. group A), C(max) 3421 +/- 3399 ng/mL and AUC(0-24) 65 055 +/- 49 843 ng/mL/h (two-sided P>0.05 for each comparison with group A), lopinavir C(min) was 7471 +/- 3745 ng/mL, C(max) 10 143 +/- 5217 ng/mL and AUC(0-12) 104 501 +/- 43 565 ng/mL/h (P>0.05 for each comparison with group B). When analysing all the groups, including controls from routine clinical practice, higher body mass index was associated with lower atazanavir C(min) and with lower lopinavir C(max). Atazanavir C(min) showed a correlation with total bilirubin levels., Conclusions: Combination with lopinavir/ritonavir provides higher atazanavir C(min) than combination with ritonavir alone, possibly because of an effect of the additional ritonavir dose. Low BMI may be associated with higher drug exposure.

Background: Adequate antiretroviral exposure during pregnancy is critical to prevent the vertical transmission of HIV and for maternal health. Pregnancy can alter drug kinetics. We assessed the pharmacokinetics of atazanavir/ritonavir (300/100 mg a day) during pregnancy., Methods: An intensive steady-state 24-h pharmacokinetic profile of atazanavir was performed in the third trimester of pregnancy and postpartum. Maternal and umbilical cord blood samples were obtained at delivery. We measured atazanavir by reverse-phase high-performance liquid chromatography., Results: Seventeen women completed the study. Antepartum, the atazanavir geometric mean area under the plasma concentration-time curve from 0 to 24 h (AUC0-24) was 28 510 ng.h/l, the maximum observed plasma concentration (Cmax) was 2 591 ng/ml and the 24-h postdose concentration (Ctrough) was 486 ng/ml. The same postpartum parameters were 30 465 ng.h/l, 2 878 ng/ml and 514 ng/ml, respectively. The antepartum to postpartum ratio for AUC0-24 was 0.94 and for Ctrough was 0.96, indicating equivalence, whereas Cmax values were slightly although not significantly lower. The ratio of cord blood/maternal atazanavir concentration in 14 paired samples was 0.13., Conclusion: Atazanavir exposure during the third trimester of pregnancy is similar to that observed in the non-pregnant period. Over the whole dosing interval, therapeutic drug concentrations well above the wild-type HIV 90% inhibitory concentration are maintained. Atazanavir crosses the placenta, potentially providing further protection for the newborn. As pregnancy does not appear to alter atazanavir exposure, no dose adjustment is required in pregnant women. Results suggest that atazanavir is a reasonable component of HAART during pregnancy.

Objective: Our objective was to evaluate the steady-state pharmacokinetics of ritonavir-boosted atazanavir when coadministered with tenofovir in HIV-1-infected adult patients., Design: Forty adult HIV-1-infected patients received either atazanavir/ritonavir 300/100 mg once daily and nucleoside reverse transcriptase inhibitors with (n = 20) or without (n = 20) tenofovir-disoproxil fumarate (tenofovir-DF) 300 mg once daily. Twenty-four-hour pharmacokinetics were assessed after at least 2 weeks of therapy according to a standardised therapeutic drug monitoring protocol., Methods: Atazanavir/ritonavir plasma concentrations were measured by liquid chromatography tandem mass spectrometry, and the geometric means of minimum and maximum concentrations (C(min), C(max)), the area under the time-concentration curve (AUC), half-life (t(1/2)) and total clearance (CL(tot)) were subject to a matched pairs-analysis. Patients' pairs were matched for gender, ethnicity, weight and Center for Disease Control and Prevention (CDC) status., Results: The respective geometric means (90% CI) for atazanavir C(min), C(max) and AUC with tenfovir vs. without tenofovir were 405 (314-523) vs. 417 (304-572) ng/ml, 3,022 (2,493-3,664) vs. 2,817 (2,341-3,390) ng/ml and 34,822 (29,315-41,363) vs. 32,101 (26,206-39,321) ng x h/ml showing no significant differences between the groups. Atazanavir plasma concentrations measured at week 5 of therapy or later were lower than in the first 4 weeks (T-test for C(max), p = .080; AUC, p = .050 and CL(tot), p = .051)., Conclusions: The coadministration of tenofovir-DF did not impair the plasma concentrations of ritonavir-boosted atazanavir in a pharmacokinetic analysis of patient pairs matched for gender, ethnicity, weight and CDC status.

Atazanavir (ATV) is a low oral bioavailability (BA) compound and, clinically, is generally coadministrated with ritonavir (RTV), which boosts the oral BA of ATV by inhibiting cytochrome P450 (CYP) 3A, and P-glycoprotein (Pgp) via the same metabolic pathway. However, depending on pharmacokinetic interaction, RTV-boosted ATV has great potential for other comedication. In this study we demonstrated the pharmaceutical approach to BA improvement of ATV without RTV in rats, based on the solid dispersion system using sodium lauryl sulfate (SLS) as a carrier and Gelucire 50/13 as an absorption enhancer. ATV solid dispersions in SLS were prepared by a conventional solvent method and, at ratios of ATV to SLS of 1 : 2 and 1 : 3, were demonstrated to form an amorphous state in powder X-ray diffraction (PXRD) analysis and exhibited 2.26- and 2.36-fold improvement in a dissolution test in comparison to bulk ATV, respectively. After oral administration to rats, ATV solid dispersion in SLS at a ratio of 1 : 2 showed a 3.5-fold increase in BA compared with bulk ATV. Moreover, the addition of Gelucire 50/13 to ATV solid dispersion, at a total ratio of Gelucire 50/13, ATV and SLS 1 : 1 : 2 gave 7.0- and 4.7-fold increase in Cmax and BA compared with bulk ATV, respectively, when the relative BA to RTV-boosted ATV reached 93%. The results in this study proved that a pharmaceutical approach could improve the bioavailability of ATV without pharmacokinetic interaction with RTV.

An efficient, isocratic high performance liquid chromatography (HPLC) method for determining human immunodeficiency virus (HIV) non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) in plasma is advantageous for laboratories participating in clinical trials and therapeutic drug monitoring (TDM) programs, or conducting small animal research. The combination of isocratic reversed phase chromatography using an S-3, 3.0 mm x 150 mm column along with low plasma volume (200 microl), rapid liquid-liquid extraction, and detection at a single wavelength (212 nm) over a short run time makes this method valuable. Within and between assay variability ranges from 0.8 to 3.5% and 1.2-6.2%, respectively. Accuracy ranges from 91.0 to 112.8% for four quality controls (50, 100, 1000, and 10,000 ng/ml) for all drugs measured (efavirenz, nevirapine, amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir).

An accurate, sensitive and simple reverse-phase (RP) high-performance liquid chromatography (HPLC) assay has been developed and validated for the simultaneous quantitative determination of tipranavir with nine other antiretroviral drugs in plasma. A liquid-liquid extraction of the drugs in tert-butylmethylether (TBME) from 200 microL of plasma is followed by a reversed phase gradient HPLC assay with UV detection at 210 nm. The standard curve for the drug was linear in the range of 80-80,000 ng/mL for tipranavir; 10-10,000 ng/mL for nevirapine, indinavir, efavirenz, and saquinavir; and 25-10,000 ng/mL for amprenavir, atazanavir, ritonavir, lopinavir, and nelfinavir. The regression coefficient (r(2)) was greater than 0.998 for all analytes. This method has been fully validated and shown to be specific, accurate and precise. Due to an excellent extraction procedure giving good recovery and a clean baseline, this method is simple, rapid, accurate and provides excellent resolution and peak shape for all analytes. Thus this method is very suitable for therapeutic drug monitoring.

Atazanavir (ATV) is a widely used human immunodeficiency virus (HIV)-1 protease inhibitor (PI) that, like other approved PIs, has been considered as a candidate for therapeutic drug monitoring (TDM). To provide ATV assay results that can be applied to patient management through TDM, the assay would need to perform in a manner consistent with Clinical Laboratory Improvement Amendments (CLIA) standards. To quantitate ATV concentrations in human plasma, the authors added ATV to a previously published reversed-phase high-performance liquid chromatography (HPLC) method from their laboratory. Detection was effected with use of a photodiode-array detector (PDA) collecting spectra at 248 nm. This method allows for detection of ATV to a lower limit of quantitation of 0.05 microg/mL, with an intra-assay coefficient of variation (CV%) of 8.9% or less over 5 days of testing and an interassay CV% ranging from 1.4 to 6.4%. The assay has met passing requirements for interlaboratory proficiency testing for 2 years nationally and internationally, with accuracy within +/-15% over all test samples. During 2 years, more than 100 batches of analyses have been performed and have proved the method is rugged, specific, and accurate. This assay method is currently used in the authors' clinical research program in TDM.

ASPIRE I and II were prospective, 3-way sequential crossover studies in healthy volunteers to compare the safety and pharmacokinetics of saquinavir/ritonavir (SQV/RTV) with saquinavir/atazanavir (SQV/ATV) administered either once daily (QD, ASPIRE I) or twice daily (BID, ASPIRE II). Treatments were separated by 10 days, and pharmacokinetic analyses were performed on days 11, 32, and 53. SQV pharmacokinetics were significantly higher when dosed with RTV compared to ATV (P < .05 for all comparisons). ATV pharmacokinetics were similar within treatment arms. ATV Cmin increased approximately 60%, and Cmax decreased approximately 35% with BID dosing compared with QD dosing. Women had higher exposure for all 3 protease inhibitors (PIs) compared with men after adjusting for weight. Adverse effects were primarily gastrointestinal-related with SQV/RTV and hyperbilirubinemia with SQV/ATV. Although SQV plasma concentrations were higher when coadministered with RTV, a combination of SQV/ATV administered BID may be a viable alternative in HIV-infected, PI-naive subjects intolerant to RTV.

We have developed an enzyme immunoassay to measure atazanavir (ATV) levels in plasma and cells. Anti-ATV polyclonal antibodies were raised in rabbits by using a synthetic ATV derivative coupled to bovine serum albumin as the immunogen, and the enzyme tracer was prepared by chemically coupling the ATV derivative with acetylcholinesterase. These reagents were used to develop a sensitive competitive enzyme immunoassay performed in microtitration plates, and the lowest limit of quantification was 150 pg/ml, which is about 10 times more sensitive than previously published techniques. The plasma assay was performed, after a simple methanol extraction, with a minimum of 30 microl of plasma. This assay showed good precision and efficiency, since the rates of recovery from human plasma and cell extracts spiked with ATV ranged form 93 to 113%, with coefficients of variation of less than 10%. ATV concentrations were measured in peripheral blood mononuclear cells incubated with various ATV concentrations and in CEM cells in the absence or presence of antiretroviral drugs and drug transporter inhibitors. The results indicated a dose-dependent uptake (intracellular concentration/extracellular concentration ratio range, 0.04 to 19). A significant increase in the accumulation of ATV was noticed in the presence of P-glycoprotein and MRP1 inhibitors (dipyridamole, inter alia). Interestingly, efavirenz significantly increased the baseline accumulation of ATV, whereas nevirapine induced a marked reduction. This new enzyme immunoassay for measuring plasma and intracellular ATV levels was fully validated and provides an inexpensive and useful tool for routine therapeutic drug monitoring. Moreover, in vitro results suggested the implication of drug transporters and interactions with other antiviral drugs that should be further explored in human immunodeficiency virus-infected patients.

Background: Hyperbilirubinemia is frequently seen in patients treated with atazanavir (ATV). Polymorphisms at the uridin-glucoronosyl-transferase 1A1 (UGT1A1) and multidrug resistance 1 (MDR1) genes may influence, respectively, bilirubin and ATV plasma concentrations., Patients and Methods: HIV-infected individuals receiving ATV 300 mg daily plus ritonavir 100 mg daily at one clinic were examined. ATV plasma concentrations were measured at steady state. MDR1-3435C>T and UGT1A1 polymorphisms were examined in DNA extracted from blood mononuclear cells., Results: A total of 118 patients (all Caucasian) were analysed. The median ATV plasma concentration was 465 ng/ml [interquartile range (IQR), 233-958]. MDR1-3435 genotypes were as follows: CC (32%), CT (47%) and TT (21%). CC patients showed higher ATV minimum concentration than those with CT/TT genotypes: 939 ng/ml (IQR, 492-1266) versus 376 ng/ml (IQR, 221-722) (P = 0.001). In multivariate analyses, having at least one T allele at MDR1-3435 was independently associated with lower ATV plasma concentrations (beta: -427 [95% confidence interval (CI), -633 to -223]; P < 0.001). The proportion of patients with grade 3-4 hyperbilirubinemia varied with distinct UGT1A1 genotypes: 80% for 7/7, 29% for 6/7 and 18% for 6/6 (P = 0.012). In the multivariate analysis, having at least one 7 allele at UGT1A1 was independently associated with severe hyperbilirubinemia (odds ratio, 2.96; 95% CI, 1.29-6.78; P = 0.01)., Conclusions: Polymorphisms at MDR1-3435 significantly influence ATV plasma concentrations, as does being Caucasian patients with CT/TT genotypes, having lower ATV levels, even using ritonavir boosting. On the other hand, although ATV plasma concentrations directly correlate with bilirubin levels, the risk of severe hyperbilirubinemia is further increased in the presence of the UGT1A1-TA7 allele.

A simple, accurate and fast method was developed for determination of the commonly used HIV protease inhibitors (PIs) amprenavir, indinavir, atazanavir, ritonavir, lopinavir, nelfinavir, M8-nelfinavir metabolite and saquinavir in human plasma. Liquid-liquid extraction was used with hexane/ethylacetate from buffered plasma samples with a borate buffer pH 9.0. Isocratic chromatographic separation of all components was performed on an Allsphere hexyl HPLC column with combined UV and fluorescence detection. Calibration curves were constructed in the range of 0.025-10 mg/l. Accuracy and precision of the standards were all below 15% and the lowest limit of quantitation was 0.025 mg/l. Stability of quality control samples at different temperature conditions was found to be below 20% of nominal values. The advantages of this method are: (1) inclusion and determination of the newly approved atazanavir, (2) simultaneous isocratic HPLC separation of all compounds and (3) increased specificity and sensitivity for amprenavir by using fluorescence detection. This method can be used for therapeutic drug monitoring of all PIs currently commercialised and is now part of current clinical practice.

Background: Significant interactions between abacavir and other antiretrovirals have not been reported. This study investigated the steady-state plasma pharmacokinetics of abacavir when co-administered with atazanavir/ritonavir or lopinavir/ritonavir in HIV-infected individuals., Methods: HIV-infected subjects on abacavir (600 mg once daily) plus two nucleoside reverse transcriptase inhibitors (NRTIs) (excluding tenofovir) underwent a 24 h pharmacokinetic assessment for plasma abacavir concentrations. Atazanavir/ritonavir (300/100 mg once daily; arm (1) or lopinavir/ritonavir (400/100 mg twice daily; arm (2) were then added and the 24 h pharmacokinetic assessment repeated. Arm 3 included subjects stable on atazanavir/ritonavir or lopinavir/ritonavir and two NRTIs (excluding tenofovir or abacavir). These patients underwent a pharmacokinetic assessment for atazanavir/ritonavir or lopinavir/ritonavir concentrations on day 1, abacavir (600 mg once daily) was then added to the regimen and the pharmacokinetic assessment repeated. Within-subject changes in drug exposure were evaluated by geometric mean (GM) ratios and 95% confidence intervals (CI)., Results: Twenty-four patients completed the study. GM (95% CI) abacavir area under the curve (AUC) was 18,621 (15,900-21,807) and 15,136 (13,339-17,174) ng.h/ml without and with atazanavir/ritonavir and 15,136 (12,298-18,628) and 10,471 (9,270-11,828) ng.h/ml without and with lopinavir/ritonavir. GM (95% CI) atazanavir AUC without and with abacavir was 26,915 (13,252-54,666) and 28,840 (19,213-43,291) ng.h/ml; lopinavir AUC without and with abacavir was 60,253 (48,084-75,509) and 63,096 (48,128-82,718) ng.h/ml., Conclusions: No changes in atazanavir or lopinavir exposures were observed following the addition of abacavir; however, decreases in abacavir plasma exposure of 17% and 32% were observed following the addition of atazanavir/ritonavir or lopinavir/ritonavir, respectively.

Highly active antiretroviral therapy (HAART) of human immunodeficiency virus-infected patients is associated with adverse effects, such as lipodystrophy and hyperlipidemia. The lipodystrophic syndrome is characterized by a peripheral lipoatrophy and/or fat accumulation in the abdomen and neck. In order to get insights into the physiopathological mechanisms underlying this syndrome, we treated mice with protease inhibitors (PIs) over a long period of time. Although atazanavir-treated mice presented the same circulating triglyceride concentration as control mice, lopinavir-ritonavir-treated mice rapidly became hypertriglyceridemic, with triglyceride levels of 200 mg/dl, whereas control and atazanavir-treated animals had triglyceride levels of 80 mg/dl. These results obtained with mice reproduce the metabolic disorder observed in humans. White adipose tissue (WAT) was analyzed after 8 weeks of treatment. Compared to the control or atazanavir treatment, lopinavir-ritonavir treatment induced a significant 25% weight reduction in the peripheral inguinal WAT depot. By contrast, the profound epididymal WAT depot was not affected. This effect was associated with a 5.5-fold increase in SREBP-1c gene expression only in the inguinal depot. Our results demonstrate that the long-term treatment of mice with PIs constitutes an interesting experimental model with which some aspects of the lipoatrophy induced by HAART in humans may be studied.

Objectives: To examine cellular and plasma concentrations of atazanavir when given in combination with saquinavir/ritonavir in HIV+ patients., Methods: Twelve HIV+ patients were receiving saquinavir/atazanavir/ritonavir 1600/200/100 mg once daily and venous blood samples were taken to determine cellular and plasma concentrations of each protease inhibitor at 2, 6, 12 and 24 h. Peripheral blood mononuclear cells were separated by density gradient centrifugation. The ratio of the cellular AUC0-24/plasma AUC0-24 was calculated to determine cellular drug accumulation. Lymphocyte P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance-associated protein (MRP1) expression was determined by flow cytometry. Nine of the patients had previously received a regimen of saquinavir/ritonavir 1600/100 mg; therefore the effect of atazanavir on the cellular and plasma pharmacokinetics of saquinavir and ritonavir was examined., Results: In vivo cellular and plasma determinations of saquinavir, atazanavir and ritonavir gave accumulation ratios of 4.9, 1.2 and 1.7, respectively. There was no relationship between saquinavir, atazanavir or ritonavir accumulation and P-gp, MRP1 or BCRP expression. When comparing pharmacokinetic values in the nine patients receiving saquinavir/ritonavir with and without atazanavir, the median cellular saquinavir AUC0-24 was significantly increased (34.9-117.2 mg.h/L) on addition of atazanavir (P=0.004). The C24 of saquinavir in plasma and cells was significantly higher with atazanavir (plasma C24 0.05 versus 0.14 mg/L with atazanavir; cellular C24 0.61 versus 2.03 mg/L with atazanavir, P=0.02)., Conclusions: The mechanism of differential intracellular protease inhibitor accumulation is unclear. Co-administration of atazanavir caused an increase in both the plasma and cellular exposure (AUC0-24) and C24 of saquinavir but not ritonavir.

A simple high-performance liquid chromatography method for the determination of the human immunodeficiency virus protease inhibitor atazanavir in human plasma samples was developed and validated. The method involved a rapid and simple solid-phase extraction of atazanavir using Oasis HLB 1cc cartridges, an isocratic reversed-phase liquid chromatography on an XTerra RP18 (150mmx4.6mm, 3.5microm) column, and ultraviolet detection at 203nm. The mobile phase consisted of phosphate buffer (pH 6, 52.5mM) and acetonitrile (43:57, v/v). Up to 48 samples could be measured in one day since the run-time of one sample was 30min. The assay was linear from 0.04 to 10microg/ml with a lower limit of quantification of 0.04microg/ml. The mean absolute recovery of ATV was 98.1%. The method was precise, with both intra-day and inter-day coefficients of variation < or =3.0%, and accurate (deviations ranged from -3.0% to 4.5% and from -3.6% to 4.7% for intra-day and inter-day analysis, respectively). There was no interference from 35 tested potentially co-administrated drugs. This method provides a simple, sensitive, precise and reproducible assay for the therapeutic drug monitoring of atazanavir in clinical routine of laboratories with standard equipment.

Background: The HIV protease inhibitor (PI) atazanavir does not impair insulin sensitivity acutely but ritonavir and lopinavir induce insulin resistance at therapeutic concentrations., Objective: To test the hypothesis that atazanavir combined with a lower dose of ritonavir would have significantly less effect on glucose metabolism than lopinavir/ritonavir in vitro and clinically., Methods: Glucose uptake was measured following insulin stimulation in differentiated human adipocytes in the presence of ritonavir (2 micromol/l) combined with either atazanavir or lopinavir (3-30 micromol/l). These data were examined clinically using the hyperinsulinemic euglycemic clamp and oral glucose tolerance testing (OGTT) in 26 healthy HIV-negative men treated with atazanavir/ritonavir (300/100 mg once daily) and lopinavir/ritonavir (400/100 mg twice daily) for 10 days in a randomized cross-over study., Results: Atazanavir inhibited glucose uptake in vitro significantly less than lopinavir and ritonavir at all concentrations. Ritonavir (2 micromol/l) combined with either atazanavir or lopinavir (3-30 micromol/l) did not further inhibit glucose uptake. During euglycemic clamp, there was no significant change from baseline insulin sensitivity with atazanavir/ritonavir (P = 0.132), while insulin sensitivity significantly decreased with lopinavir/ritonavir from the baseline (-25%; P < 0.001) and from that seen with atazanavir/ritonavir (-18%; P = 0.023). During OGTT, the HOMA insulin resistance index significantly increased from baseline at 120 min with atazanavir/ritonavir and at 150 min with lopinavir/ritonavir. The area under the curve of glucose increased significantly with lopinavir/ritonavir but not with atazanavir/ritonavir., Conclusions: Both glucose uptake in vitro and clinical insulin sensitivity in healthy volunteers demonstrate differential effects on glucose metabolism by the combination PI atazanavir/ritonavir and lopinavir/ritonavir.

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.

Objective: The influence of nevirapine, efavirenz and tenofovir co-administration on ritonavir-boosted atazanavir pharmacokinetics was investigated in HIV (human immunodeficiency virus)-infected patients., Methods: A population pharmacokinetic analysis was performed in the context of therapeutic drug monitoring (87 patients, 121 samples)., Results: A significant increase of atazanavir clearance (Cl/F) was found when either tenofovir (group B), efavirenz (group C), or nevirapine (group D) were co administered with atazanavir/ritonavir in comparison with patients treated with atazanavir/ritonavir and nucleoside reverse transcriptase inhibitors (group A): 6.24+/-0.36 l h(-1) (group A) versus 7.42+/-0.25 l h(-1) (group B) versus 9.60+/-0.27 l h(-1) (group C) versus 17.53+/-0.57 l h(-1) (group D) (P<0.001). However, the decrease of the mean trough plasma concentration of atazanavir was significant only in group D: 1.02+/-0.86 mg/l (group A) versus 0.21+/-013 mg/l (group D) (P<0.001)., Conclusion: The increase in atazanavir clearance when it is used in combination with nevirapine, efavirenz and/or tenofovir suggests that therapeutic drug monitoring of atazanavir should be performed in such circumstances.

Objective: To assess the pharmacokinetics and tolerability of lopinavir (LPV), ritonavir (RTV) and atazanavir (ATV) as a double-boosted protease inhibitor regimen in HIV-infected adults., Methods: Sixteen patients who started LPV/RTV (400/100 mg b.i.d.) and ATV (300 mg q.d.) were enrolled in the study group (arm A). LPV pharmacokinetics were compared to those of two historical groups: arm B, 15 patients who received LPV/RTV (400/100 mg b.i.d.); and arm C, 25 patients who received LPV/RTV/saquinavir (SQV) (400/100/1000 mg b.i.d.). ATV pharmacokinetics were compared to those of 15 consecutive patients who received ATV and RTV (300/100 mg q.d.) (arm D). Drug concentrations were measured by HPLC., Results: LPV concentrations were significantly higher in arm A than in arms B and C. Median (interquartile range) LPV area under the curve (AUC)0-12 values were 115.7 (99.8-136.5), 85.2 (68.3-109.2) and 85.1 (60.6-110.1) microg/h/ml, respectively. C(max) values were 12.2 (10.7-14.5), 9.5 (6.8-13.9) and 10.0 (6.9-13.6) microg/ml, respectively. C(min) values were 9.1 (7.1-10.4), 5.6 (4.7-8.2) and 5.5 (4.2-7.5) microg/ml, respectively. No difference was observed for ATV AUC0-24 or C(max) between arms A and D. ATV C(min) values were 1.07 (0.61-1.79) in arm A and 0.58 (0.32-0.83) in arm D (P = 0.001). Treatment was not discontinued in any patient because of adverse effects. At 24 weeks, viral load was < 50 copies/ml in 13 of 16 patients., Conclusions: The combination of ATV and LPV/RTV provided high plasma concentrations of both PI, which seemed to be appropriate for patients with multiple prior therapeutic failures, yielding good tolerability and substantial antiviral efficacy.

Study Objective: To determine whether the pharmacokinetics of atazanavir, a protease inhibitor used to treat human immunodeficiency virus (HIV) infection, are altered by its coadministration with lansoprazole, a proton pump inhibitor., Design: Single-dose, open-label, complete-crossover study., Setting: Clinical research center., Subjects: Ten healthy adult volunteers., Measurements and Main Results: In phase A, subjects received a single oral dose of atazanavir 400 mg alone. In phase B, the same subjects received oral lansoprazole 60 mg, and after 24 hours they were given a second dose of oral lansoprazole 60 mg with atazanavir 400 mg. Eleven blood samples were collected from each subject over a 24-hour period for determination of atazanavir plasma concentrations by a validated high-performance liquid chromatography assay. Pharmacokinetic analysis was performed by standard noncompartmental methods. Nine subjects completed the study, and no significant adverse events were reported. Absorption of atazanavir was significantly reduced when it was coadministered with lansoprazole, as evidenced by a 94% decline in mean area under the concentration-time curve during the 24 hours after administration (AUC(0-24)) (p<0.01). The mean +/- SD AUC(0-24) for phase A was 16.3 +/- 9.0 microM x hour versus 0.95 +/- 1.8 microM x hour for phase B (p<0.01). The mean +/- SD maximum concentration of atazanavir was 3.2 +/- 1.7 microM for phase A and 0.13 +/- 0.19 microM for phase B (p<0.01)., Conclusion: Acid suppression markedly reduced the bioavailability of atazanavir in this group of healthy volunteers. Based on these results, atazanavir should not be coadministered with lansoprazole or other proton pump inhibitors.

Atazanavir is the most recently introduced protease inhibitor for the suppression of the anti-human immunodeficiency virus. A sensitive and selective reversed-phase liquid chromatographic assay for this drug in human plasma has been developed and validated. Atazanavir was isolated from a 500 microL plasma sample using liquid-liquid extraction with dichloromethane. After evaporation and reconstitution of the extract the sample was analysed using liquid chromatography and ultraviolet detection at 280 nm. In the evaluated concentration range (44-4395 ng/mL atazanavir), intra-day precisions were < or =7% and inter-day precisions were < or =14%. Accuracies between 96 and 106% were found. The lower limit of quantification was 44 ng/mL with an intra-day precision of 7%, an inter-day precision of 14% and an accuracy of 87%. There was no interference from 32 tested potentially co-administrated drugs and metabolites. The usefulness of the assay was demonstrated for samples obtained from an HIV-infected patient treated with atazanavir., (Copyright 2005 John Wiley & Sons, Ltd.)

Background: Atazanavir (ATV) is recommended to be dosed at 400 mg once daily or 300 mg daily coadministered with 100 mg ritonavir (RTV)., Method: 31 male patients receiving ATV either alone or boosted with RTV for more than 2 weeks had ATV concentration measured by high performance liquid chromatography (HPLC). ATV concentrations were adjusted to obtain a 24-hour trough level using a standard pharmacokinetic formula., Results: 25 samples were taken from patients who received 300 mg ATV, 6 with 200 mg, 3 with 400 mg, and 2 with 150 mg, all boosted with 100 mg RTV. In the unboosted group, patients received 400 mg (7) or 600 mg (2). The median adjusted 24-hour trough ATV concentration was 630 ng/mL (interquartile range [IQR] 355-1034) in the boosted and 113 ng/mL (IQR 50-225) in the unboosted group (p = .001). Median serum bilirubin concentration was 34 IU/L (IQR 27.5-49) and 41 IU/L (IQR 31-45) in the boosted and unboosted groups, respectively. In the boosted group, high ATV concentrations were significantly correlated with increased serum bilirubin concentrations (p = .003)., Conclusion: ATV concentrations showed considerable interpatient variability. Bilirubin concentrations are an indicator of high ATV concentrations and may prove to be useful in selecting patients for therapeutic drug monitoring (TDM).