HIV Protease Inhibitor Atazanavir Research Articles

OII-B-4Effect of HIV protease inhibitor administration on ABC transporter expression in human peripheral blood mononuclear cells (PBMCS)

BACKGROUND/AIMS Efflux transporters in the ATP binding cassette (ABC) superfamily have been implicated in multidrug resistance (MDR). Characterization of ABC transporters at the site of drug action is essential to overcoming MDR. In the current study, we examined the expression of seven ABC transporters in humanPBMCs in response to administration of the HIV protease inhibitors atazanavir (ATZ) and saquinavir (SQV). METHODS Fourteen healthy volunteers received 1500 mg SQV and 200 mg ATZ orally twice daily for 11 days. PBMCs were isolated from each subject at baseline and on the 11th day of treatment. Quantitative RT-PCR was used to measure PBMC expression of ABCB1, ABCC1, ABCC2, ABCC3, ABCC4, ABCC6, and ABCG2. RESULTS Preliminary results show an overall increase in ABCB1 mRNA expression following SQV-ATZ administration. Interestingly, 86% of subjects demonstrated an increase in ABCB1 mRNA expression, while the remaining subjects exhibited a decrease. The effect of SQV-ATZ treatment on mRNA expression of ABCC1, ABCC2, ABCC3, ABCC4, ABCC6, and ABCG2 was variable, with induction of any gene observed in no more than 63% of individuals. (Table) Effect of SQV-ATZ administration on ABCB1 mRNA expression ABCB1 expression change N Mean change (%) Range (%) P-value Upregulated 12 55 1 to 131 <0.01 Downregulated 2 −33 −51 and −7 <0.05 Total 14 36 −51 to 131 <0.05 CONCLUSIONS Coadministration of the HIV protease inhibitors SQV and ATZ induced ABCB1 mRNA expression in the majority of individuals tested. The effect of this induction on P-glycoprotein function is being assessed. The variability in response to treatment may be due to genetic differences in these ABC genes or their regulatory partners. Clinical Pharmacology & Therapeutics (2005) 79, P6–P6; doi: 10.1016/j.clpt.2005.12.017

Practical Guidelines to Interpret Plasma Concentrations of Antiretroviral Drugs

Several relationships have been reported between antiretroviral drug concentrations and the efficacy of treatment, and toxicity. Therefore, therapeutic drug monitoring (TDM) may be a valuable tool in improving the treatment of HIV-1-infected patients in daily practice. In this regard, several measures of exposure have been studied, e.g. trough and maximum concentrations, concentration ratios and the inhibitory quotient. However, it has not been unambiguously established which pharmacokinetic parameter should be monitored to maintain optimal viral suppression. Each pharmacokinetic parameter has its pros and cons. Many factors can affect the pharmacokinetics of antiretroviral agents, resulting in variability in plasma concentrations between and within patients. Therefore, plasma concentrations should be considered on several occasions. In addition, the interpretation of the drug concentration of a patient should be performed on an individual basis, taking into account the clinical condition of the patient. Important factors herewith are viral load, immunology, occurrence of adverse events, resistance pattern and comedication. In spite of the described constraints, the aim of this review is to provide a practical guide for TDM of antiretroviral agents. This article outlines pharmacokinetic target values for the HIV protease inhibitors amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir, and the non-nucleoside reverse transcriptase inhibitors efavirenz and nevirapine. Detailed advice is provided on how to interpret the results of TDM of these drugs.

The effects of HIV protease inhibitors atazanavir and lopinavir/ritonavir on insulin sensitivity in HIV-seronegative healthy adults

Therapy with some HIV protease inhibitors (PI) contributes to insulin resistance and type 2 diabetes mellitus, by inhibition of insulin-sensitive glucose transporters. Atazanavir (ATV) is a new PI with substantially less in vitro effect on glucose transport than observed with other PI, including lopinavir (LPV) or ritonavir (RTV). Randomized, double-blind, crossover study of the effect of 5 days of administering ATV, lopinavir/ritonavir (LPV/r) or placebo on insulin-stimulated glucose disposal in 30 healthy HIV-negative subjects. Each subject was studied on two of three possible treatments with a wash-out period between treatments. The mean insulin-stimulated glucose disposal (mg/min per kg body weight) per unit insulin (microU/ml) (M/I) was 9.88, 9.80 and 7.52 for placebo, ATV and LPV/r, respectively (SEM, 0.84 for all). There was no significant difference between ATV and placebo. The M/I for LPV/r was 23% lower than that for ATV (P = 0.010) and 24% lower than that for placebo (P = 0.008). The mean glycogen storage rates were 3.85, 4.00 and 2.54 mg/min per kg for placebo, ATV and LPV/r, respectively (SEM, 0.39 for all). There was no significant difference between ATV and placebo. The glycogen storage rate for LPV/r was 36% lower than ATV (P = 0.003) and 34% lower than placebo (P = 0.006). ATV given to healthy subjects for 5 days did not affect insulin sensitivity, while LPV/r induced insulin resistance. This observation is consistent with differential in vitro effects of these PI on glucose transport. Further data are needed to assess clinical implications for body composition.

Determination of the new HIV-protease inhibitor atazanavir by liquid chromatography after solid-phase extraction

Determination of the new HIV-protease inhibitor atazanavir by liquid chromatography after solid-phase extraction

Determination of the new HIV-protease inhibitor atazanavir by liquid chromatography after solid-phase extraction

An HPLC method previously described for the simultaneous assay of amprenavir, ritonavir, indinavir, saquinavir, nelfinavir and efavirenz is proposed here for the simultaneous analysis of the new HIV protease inhibitor atazanavir (ATV) in human plasma, by off-line solid-phase extraction (SPE) followed by HPLC coupled with UV-diode array detection. After viral inactivation by heat (60 °C for 60 min), plasma (600 μl) with clozapine (internal standard) is diluted 1 + 1 with phosphate buffer pH 7 and subjected to a SPE on a C18 cartridge. Matrix components are eliminated with 2 × 500 μl of a solution of 0.1% H 3PO 4 neutralised with NaOH to pH 7. ATV is eluted with 3 × 500 μl MeOH. The resulting eluate is evaporated under nitrogen at room temperature and is reconstituted in 100 μl MeOH/H 2O 50/50. A 40 μl volume is injected onto a Nucleosil 100–5 μm C18 AB column. ATV is analysed by UV detection at 201 nm using a gradient elution program with solvents constituted of MeCN and phosphate buffer adjusted to pH 5.14. The mobile phase also contains 0.02% sodium heptanesulfonate, enabling an excellent separation of ATV from the other HIV protease inhibitors (PIs) amprenavir, indinavir, saquinavir, ritonavir, lopinavir, nelfinavir and the non-nucleoside reverse transcriptase inhibitors (NNRTIs) efavirenz and nevirapine. The calibration curves are linear up to 10 μg/ml, with a lower limit of quantification of 0.2 μg/ml. The mean absolute recovery of ATV is 96.4 ± 3.2%. The method is precise with mean inter-day CVs within 1.1–6.1%, and accurate (range of inter-day deviations +0.3 to +2.3%). The method has been validated and is currently applied to the monitoring of ATV in HIV patients.

ANTI-HIV CHEMOTHERAPY: CURRENT STATE OF THE ART

In recent years, significant progress has been made towards the chemotherapy (and ?prophylaxis) of HIV infections. This progress is situated at three different levels. (i) New anti-HIV drugs have been approved for clinical use and have entered the market: the virus entry inhibitor enfuvirtide (Fuzeon™), the nucleoside reverse transcriptase inhibitor (NRTI) emtricitabine (Emtriva™), the nucleotide reverse transcriptase inhibitor (NtRTI) tenofovir disoproxil fumarate (Viread™) and the HIV protease inhibitor (PI) atazanavir (Reyataz™). These compounds have joined the current anti-HIV drug armamentarium comprising the NRTIs zidovudine, didanosine, zalcitabine, stavudine, lamivudine and abacavir, the non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine, delavirdine and efavirenz, and the PIs saquinavir, ritonavir, indinavir, nelfinavir, amprenavir and lopinavir. This has brought the total of licensed anti-HIV drugs to nineteen. (ii) Other compounds have proceeded through preclinical and/or clinical development: CXCR4 antagonists (i.e. AMD070 and KRH-1636), CCR5 antagonists (i.e. SCH-C and TAK-220), NRTIs (i.e. amdoxovir and Reverset™), NNRTIs (i.e. capravirine and dapivirine), integrase inhibitors (such as S-1360) and PIs (such as tipranavir). (iii) Yet other compounds, acting by novel mechanisms, have recently been identified as anti-HIV agents that seem worthy of further (pre)clinical development: cell receptor CD4 downmodulators (i.e. cyclotriazadisulfonamides), viral envelope gp120-binding agents such as plant lectins and glycopeptide antibiotics, HIV integrase inhibitors such as the pyranodipyrimidine V-165, and two new classes of compounds (i.e. N-aminoimidazoles and pyridine oxide derivatives) which seem to interfere with a post-integration, transcription transactivation event. Taken together, it is obvious that in recent years both more diverse and more efficient approaches have become available for the treatment of HIV infections.

Simultaneous quantification of the new HIV protease inhibitors atazanavir and tipranavir in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

We have developed and validated an assay, using liquid chromatography coupled with electrospray tandem mass spectrometry (LC–MS/MS), for the quantification of the novel protease inhibitors (PIs) atazanavir and tipranavir. The sample pre-treatment consisted of protein precipitation with a mixture of methanol and acetronitrile using 100 μl plasma for atazanavir and 50 μl for tipranavir. Chromatographic separation was achieved on an Inertsil ODS3 column ( 50 mm×2.0 mm i.d., particle size 5 μm), with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.5 ml/min. The analytical run time was 5.5 min. The triple quadrupole mass spectrometer operated in the positive ion-mode and multiple reaction monitoring (MRM) was used for drug quantification. The assay was linear over a concentration range of 0.05–10 μg/ml for atazanavir and 0.1–75 μg/ml for tipranavir. Saquinavir-d5 was used as internal standard. The intra- and inter-day coefficients of variation were less than 3.8% for atazanavir and less than 10.4% for tipranavir. Accuracies were within ±7.3 and ±7.2% for atazanavir and tipranavir, respectively. Both drugs were stable under various relevant storage conditions. The validated concentration ranges proved to be adequate to measure concentrations of human immunodeficiency virus type-1 (HIV-1)-infected individuals. The developed method could easily be combined with a previously developed LC–MS/MS assay for the quantification of protease inhibitors.

Practical Guidelines to Interpret Plasma Concentrations of Antiretroviral Drugs

Several relationships have been reported between antiretroviral drug concentrations and the efficacy of treatment, and toxicity. Therefore, therapeutic drug monitoring (TDM) may be a valuable tool in improving the treatment of HIV-1-infected patients in daily practice. In this regard, several measures of exposure have been studied, e.g. trough and maximum concentrations, concentration ratios and the inhibitory quotient. However, it has not been unambiguously established which pharmacokinetic parameter should be monitored to maintain optimal viral suppression. Each pharmacokinetic parameter has its pros and cons. Many factors can affect the pharmacokinetics of antiretroviral agents, resulting in variability in plasma concentrations between and within patients. Therefore, plasma concentrations should be considered on several occasions. In addition, the interpretation of the drug concentration of a patient should be performed on an individual basis, taking into account the clinical condition of the patient. Important factors herewith are viral load, immunology, occurrence of adverse events, resistance pattern and comedication. In spite of the described constraints, the aim of this review is to provide a practical guide for TDM of antiretroviral agents. This article outlines pharmacokinetic target values for the HIV protease inhibitors amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir, and the non-nucleoside reverse transcriptase inhibitors efavirenz and nevirapine. Detailed advice is provided on how to interpret the results of TDM of these drugs.

Diastereoselective microbial reduction of ( S)-[3-chloro-2-oxo-1-(phenylmethyl)propyl]carbamic acid, 1,1-dimethylethyl ester

The chiral intermediate (1 S,2 R)-[3-chloro-2-hydroxy-1-(phenylmethyl)propyl]carbamic acid, 1,1-dimethylethyl ester 2a was prepared for the total synthesis of the HIV protease inhibitor Atazanavir. The diastereoselective reduction of (1 S)-[3-chloro-2-oxo-1-(phenylmethyl)propyl] carbamic acid, 1,1-dimethyl-ethyl ester 1 was carried out using microbial cultures among, which Rhodococcus, Brevibacterium, and Hansenula strains reduced 1 to 2a. Three strains of Rhodococcus gave >90% yield. A diastereomeric purity of >98% and enantiomeric excess of >99.3% were obtained for alcohol 2a.

Liquid chromatography-tandem mass spectrometric quantitative determination of the HIV protease inhibitor atazanavir (BMS-232632) in human peripheral blood mononuclear cells (PBMC): practical approaches to PBMC preparation and PBMC assay design for high-throughput analysis

A selective, accurate, and reproducible LC/MS/MS assay was developed and validated for the determination of the HIV protease inhibitor atazanavir (BMS-232632) in human peripheral blood mononuclear cells (PBMC) samples. In addition to the details of the validated LC/MS/MS method, a practical procedure is described in great detail for the preparation of large supplies of control (blank) PBMC from units of blood (each unit of blood is about 500 ml) for making the calibration standards and quality control (QC) samples. The PBMC assay design, intended for high-throughput sample analysis, is also described in some detail in regards to the composition and concentration expressions of the calibration standards and QC samples, the lysing procedure of the PBMC samples, and the final analysis/quantitation procedure. The method involved automated solid-phase extraction (SPE) of atazanavir and a stable isotope analog internal standard (I.S.) using 3M Empore ® C2-SD 96-well plates. A portion of the reconstituted sample residue was injected onto a YMC Basic analytical column which was connected to a triple quad mass spectrometer for analyte determination by positive-ion electrospray in the selected reaction monitoring (SRM) mode. The standard curve, which ranged from 5 to 2500 fmol per one million cells (fmol/10 6 cells), was fitted to a quadratic regression model weighted by 1/concentration. The lower limit of quantitation (LLOQ) was 5 fmol/10 6 cells. The inter- and intra-run coefficients of variation (CV) for the assay were <9% and the accuracy was 94–104%. Atazanavir was stable in PBMC for at least 24 h at room temperature and for at least 129 days at −15 °C.

Quantitative determination of the HIV protease inhibitor atazanavir (BMS-232632) in human plasma by liquid chromatography–tandem mass spectrometry following automated solid-phase extraction

A selective, accurate, and reproducible LC–MS–MS assay was developed for the determination of the HIV protease inhibitor atazanavir (BMS-232632) in human plasma samples. The method involved automated solid-phase extraction of atazanavir and a stable isotope analog internal standard (I.S.) using Oasis HLB 10 mg 96-well SPE plates. A portion of the reconstituted sample residue was injected onto a C 18 HDO analytical column which was configured with a triple quad mass spectrometer for analyte determination by positive ion electrospray. The assay was linear from 1.00 to 1000 ng/ml with a lower limit of quantitation of 1.00 ng/ml. The inter- and intra-day coefficients of variation (C.V.) for the assay were <4%, and the accuracy was 99–102%. Atazanavir was stable in human plasma for at least 109 h at room temperature and for at least 1 year at −20 °C.