Protease Inhibitor Atazanavir Research Articles

Neutralizing antibody and anti-retroviral drug sensitivities of HIV-1 isolates resistant to small molecule CCR5 inhibitors

The small molecule CCR5 inhibitors are a new class of drugs for treating infection by human immunodeficiency virus type 1 (HIV-1). They act by binding to the CCR5 co-receptor and preventing its use during HIV-1–cell fusion. Escape mutants can be raised against CCR5 inhibitors in vitro and will arise when these drugs are used clinically. Here, we have assessed the responses of CCR5 inhibitor-resistant viruses to other anti-retroviral drugs that act by different mechanisms, and their sensitivities to neutralizing antibodies (NAbs). The rationale for the latter study is that the resistance pathway for CCR5 inhibitors involves changes in the HIV-1 envelope glycoproteins (Env), which are also targets for NAbs. The escape mutants CC101.19 and D1/85.16 were selected for resistance to AD101 and vicriviroc (VVC), respectively, from the primary R5 HIV-1 isolate CC1/85. Each escape mutant was cross-resistant to other small molecule CCR5 inhibitors (aplaviroc, maraviroc, VVC, AD101 and CMPD 167), but sensitive to protein ligands of CCR5: the modified chemokine PSC-RANTES and the humanized MAb PRO-140. The resistant viruses also retained wild-type sensitivity to the nucleoside reverse transcriptase inhibitor (RTI) zidovudine, the non-nucleoside RTI nevirapine, the protease inhibitor atazanavir and other attachment and fusion inhibitors that act independently of CCR5 (BMS-806, PRO-542 and enfuvirtide). Of note is that the escape mutants were more sensitive than the parental CC1/85 isolate to a subset of neutralizing monoclonal antibodies and to some sera from HIV-1-infected people, implying that sequence changes in Env that confer resistance to CCR5 inhibitors can increase the accessibility of some NAb epitopes. The need to preserve NAb resistance may therefore be a constraint upon how escape from CCR5 inhibitors occurs in vivo.

The Compound 6-Chloro-1,4-Dihydro-4-Oxo-1-(β-D-Ribofuranosyl) Quinoline-3-Carboxylic Acid Inhibits HIV-1 Replication by Targeting the Enzyme Reverse Transcriptase

We describe in this paper that the chloroxoquinolinic ribonucleoside 6-chloro-1,4-dihydro-4-oxo-1-(beta-D-ribofuranosyl)-quinoline-3-carboxylic acid (compound A) inhibits the HIV-1 replication in human primary cells. We initially observed that compound A inhibited HIV-1 infection in peripheral blood mononuclear cells (PBMCs) in a dose-dependent manner, resulting in an EC(50) of 1.5 +/- 0.5 microM and in a selective index of 1134. Likewise, compound A blocked HIV-1(BA-L) replication in macrophages in a dose-dependent manner, with an EC(50) equal to 4.98 +/- 0.9 microM. The replication of HIV-1 isolates from subtypes C and F was also inhibited by compound A with the same efficiency. Compound A inhibited an early event of the HIV-1 replicative cycle, since it prevented viral DNA synthesis in PBMCs exposed to HIV-1. Kinetic assays demonstrated that compound A inhibits the HIV-1 enzyme reverse transcriptase (RT) in dose-dependent manner, with a K(I) equal to 0.5 +/- 0.04 microM. Using a panel of HIV-1 isolates harboring NNRTI resistance mutations, we found a low degree of cross-resistance between compound A and clinical available NNRTIs. In addition, compound A exhibited additive effects with the RT inhibitors AZT and nevirapine, and synergized with the protease inhibitor atazanavir. Our results encourage continuous studies about the kinetic impact of compound A towards different catalytic forms of RT enzyme, and suggest that our nucleoside represents a promising molecule for future antiretroviral drug design.

Quantification of protease inhibitors and non-nucleoside reverse transcriptase inhibitors in dried blood spots by liquid chromatography–triple quadrupole mass spectrometry

A bioanalytical method for the determination of most commonly prescribed protease inhibitors (atazanavir, darunavir, lopinavir and ritonavir) and non-nucleoside reverse transcriptase inhibitors (efavirenz and nevirapine) was developed and validated according to FDA guidelines. In brief, dried blood spots were punched out of a collection paper with a 0.25 in. diameter punch. The analytes were extracted from the punched-out disc using a mixture of acetonitrile, methanol and 0.2M zinc sulphate in water (1:1:2, v/v/v) containing the internal standards dibenzepine, 13C6-efavirenz and D5-saquinavir. 20 μL of the extract was injected onto the reversed-phase C18 column (150 mm × 2.0 mm) for separation from endogenous compounds and the analytes were quantified using a triple quadrupole mass spectrometer. The analytical run time was only 10 min. Validated concentration ranges covered the ranges encountered in routine clinical practice. The assay was linear over the concentration ranges tested (0.1–20 mg/L for atazanavir, lopinavir, nevirapine and efavirenz and 0.05–10 mg/L for darunavir and ritonavir). Accuracies and inter- and intra-run precisions at all levels ranged from 96.2 to 113.9% and 3.1 to 13.3%, respectively. Analytes in dried blood spots were stable for at least 7 days at 30 °C. The method enabled patient-friendly sample collection, easy and cheap sample shipment and non-hospital based sampling for therapeutic drug monitoring and pharmacokinetic studies.

Inhibition of HIV‐2 Protease by HIV‐1 Protease Inhibitors in Clinical Use

Over the past 10 years, protease inhibitors have been a key component in antiretroviral therapies for HIV/AIDS. While the vast majority of HIV/AIDS cases in the world are due to HIV-1, HIV-2 infection must also be addressed. HIV-2 is endemic to Western Africa, and has also appeared in European countries such as Portugal, Spain, and Estonia. Current protease inhibitors have not been optimized for treatment of HIV-2 infection; therefore, it is important to assess the effectiveness of currently FDA-approved protease inhibitors against the HIV-2 protease, which shares only 50% sequence identity with the HIV-1 protease. Kinetic inhibition assays were performed to measure the inhibition constants (K(i)) of the HIV-1 protease inhibitors indinavir, nelfinavir, saquinavir, ritonavir, amprenavir, lopinavir, atazanavir, tipranavir, and darunavir against the HIV-2 protease. Lopinavir, saquinavir, tipranavir, and darunavir exhibit the highest potency with K(i) values of 0.7, 0.6, 0.45, and 0.17 nm, respectively. These K(i) values are 84, 2, 24, and 17 times weaker than the corresponding values against the HIV-1 protease. In general, inhibitors show K(i) ratios ranging between 2 and 80 for the HIV-2 and HIV-1 proteases. The relative drop in potency is proportional to the affinity of the inhibitor against the HIV-1 protease and is related to specific structural characteristics of the inhibitors. In particular, the potency drop is high when the maximum cap size of the inhibitors consists of very few atoms. Caps are groups located at the periphery of the molecule that are added to core structures to increase the specificity of the inhibitor to its target. The caps positioned on the HIV-1 protease inhibitors affect selectivity through interactions with distinct regions of the binding pocket. The flexibility and adaptability imparted by the higher number of rotatable bonds in large caps enables an inhibitor to accommodate changes in binding pocket geometry between HIV-1 and HIV-2 protease.

An Efficient and Practical Synthesis of the HIV Protease Inhibitor Atazanavir via a Highly Diastereoselective Reduction Approach

An efficient and practical synthesis of the HIV-1 protease inhibitor Atazanavir was developed by employing the diastereoselective reduction of ketomethylene aza-dipeptide isostere 10 as the key and final step. The high diastereoselectivity of the amino ketone reduction by lithium tri-tert-butoxyaluminum hydride in diethyl ether to afford the desired syn-1,2-amino alcohol structure was achieved by Felkin−Anh control as a result of the bulky and chiral N-(methoxycarbonyl)-l-tert-leucinyl moiety as the nitrogen protecting group. The coupling of the two key intermediates, N-(methoxycarbonyl)-l-tert-leucine acylated benzyl hydrazine 7 and chloromethyl ketone 9, via an SN2 reaction furnished the amino ketone 10 in high yield under our optimized conditions. Our new methodology features the late introduction of the S-hydroxyl group and the early acylation of benzyl hydrazine and chloromethyl ketone with N-(methoxycarbonyl)-l-tert-leucine, respectively, which confers high efficiency and easy purification.

L76V – clinically relevant resensitization of the protease inhibitors (PIs) saquinavir (SQV) and atazanavir (ATV)

L76V – clinically relevant resensitization of the protease inhibitors (PIs) saquinavir (SQV) and atazanavir (ATV)

Protease inhibitor atazanavir induces leukocyte-endothelial cell interactions in the microvasculature

Protease inhibitor atazanavir induces leukocyte-endothelial cell interactions in the microvasculature

HIV-1 Protease Inhibitors Nelfinavir and Atazanavir Induce Malignant Glioma Death by Triggering Endoplasmic Reticulum Stress

HIV type 1 (HIV-1) protease inhibitors (PI) have been shown to have anticancer activity in non-HIV-associated human cancer cells. The underlying mechanism of this effect is unclear. Here, we show that the PIs nelfinavir and atazanavir cause cell death in various malignant glioma cell lines in vitro. The underlying mechanism of this antitumor effect involves the potent stimulation of the endoplasmic reticulum (ER) stress response (ESR), as indicated by increased expression of two ESR markers, GRP78 and CHOP, and activation of ESR-associated caspase-4. Induction of ESR seems to play a central role in PI-induced cell death because small interfering RNA-mediated knockdown of the protective ER chaperone GRP78 sensitizes cells; whereas knockdown of proapoptotic caspase-4 protects cells from PI-induced cell death. Furthermore, the treatment of cells with PIs leads to aggresome formation and accumulation of polyubiquitinated proteins, implying proteasome inhibition. Thus, our results support a model whereby PIs cause tumor cell death via triggering of the ESR, inhibition of proteasome activity, and subsequent accumulation of misfolded proteins. Inhibition of glioma growth via ESR takes place in the in vivo setting as well, as nelfinavir inhibits the growth of xenografted human malignant glioma, with concomitant induction of the proapoptotic ER stress marker CHOP. Because ER stress has also been reported as the mechanism for insulin resistance and diabetes, our ER stress model of PI function may also explain why these drugs may induce insulin resistance as one of their most common side effects.

The dolabellane diterpene Dolabelladienetriol is a typical noncompetitive inhibitor of HIV-1 reverse transcriptase enzyme

We recently described that a dollabelane diterpene isolated from the marine algae Dictyota pfaffii (Dolabelladienetriol) inhibits the human immunodeficiency virus type 1 (HIV-1) enzyme reverse transcriptase (RT), and HIV-1 replication in primary cells. Based on these findings, we investigated additional antiretroviral properties of Dolabelladienetriol. Here, we describe that Dolabelladienetriol blocked the synthesis and integration of HIV-1 provirus and completely abrogated viral replication in primary cells. Also, studies of kinetic mode of action revealed that the Dolabelladienetriol is a nonnucleoside RT inhibitor (NNRTI), acting as a noncompetitive inhibitor, with a K i value equal to 7.2 μM. To assess whether Dolabelladienetriol could potentiate the anti-HIV-1 effects of other HIV-1 inhibitors, HIV-1-infected cells were treated with Dolabelladienetriol at its EC 50 dose plus sub-optimal concentrations of classical antiretrovirals. Dolabelladienetriol provided an additive effect with the nucleoside RT inhibitor AZT, and a synergistic effect with the protease inhibitor atazanavir sulphate. There was no increment of the anti-HIV-1 effect resulting from the combination between Dolabelladienetriol and the NNRTI nevirapine. Using a large panel of HIV-1 isolates harboring NNRTI resistance mutations, we found no cross-resistance between Dolabelladienetriol and clinical available NNRTIs. Thus, Dolabelladienetriol is an NNRTI, with potent activity against HIV-1 isolates carrying common NNRTI-associated resistance mutations. Dolabelladienetriol may be considered as a potential new agent for anti-HIV-1 therapy.

Interaction between buprenorphine and atazanavir or atazanavir/ritonavir

Opioid addiction and HIV disease frequently co-occur. Adverse drug interactions have been reported between methadone and some HIV medications, but less is known about interactions between buprenorphine, an opioid partial agonist used to treat opioid dependence, and HIV therapeutics. This study examined drug interactions between buprenorphine and the protease inhibitors atazanavir and atazanavir/ritonavir. Opioid-dependent, buprenorphine/naloxone-maintained, HIV-negative volunteers ( n = 10 per protease inhibitor) participated in two 24-h sessions to determine pharmacokinetics of (1) buprenorphine and (2) buprenorphine and atazanavir (400 mg daily) or atazanavir/ritonavir (300/100 mg daily) following administration for 5 days. Objective opiate withdrawal scale scores and mini-mental state examination were determined prior to and following antiretroviral administration to examine pharmacodynamic effects. Pharmacokinetics of atazanavir and atazanavir/ritonavir were compared in subjects and matched, healthy controls ( n = 10 per protease inhibitor) to determine effects of buprenorphine. With atazanavir and atazanavir/ritonavir, respectively concentrations of buprenorphine ( p < 0.001, p < 0.001), norbuprenorphine ( p = 0.026, p = 0.006), buprenorphine glucuronide ( p = 0.002, p < 0.001), and norbuprenorphine glucuronide (NS, p = 0.037) increased. Buprenorphine treatment did not significantly alter atazanavir or ritonavir concentrations. Three buprenorphine/naloxone-maintained participants reported increased sedation with atazanavir/ritonavir. Atazanavir or atazanavir/ritonavir may increase buprenorphine and buprenorphine metabolite concentrations and might require a decreased buprenorphine dose.

Electrospray tandem mass spectroscopic characterisation of 18 antiretroviral drugs and simultaneous quantification of 12 antiretrovirals in plasma

The determination of antiretroviral drug concentrations in patients treated with highly active antiretroviral therapy (HAART) is an essential part of optimum patient management because of the multitude of pharmacokinetic drug interactions between these drugs and the risk of treatment failure or viral resistance if therapeutic concentrations are not reached. Currently, 21 different antiretrovirals are used in various combinations rendering therapeutic drug monitoring a laborious task. We therefore aimed to simultaneously determine as many antiretrovirals as possible using triple quadrupole mass spectroscopy with electrospray ionisation. For this purpose, spectra and fragmentation patterns of the protease inhibitors amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir, the non-nucleoside reverse transcriptase inhibitors delavirdine, efavirenz, and nevirapine, the nucleoside reverse transcription inhibitors abacavir, didanosine, emtricitabine, lamivudine, stavudine, zalcitabine, and zidovudine, and the nucleotide reverse transcriptase inhibitor tenofovir were evaluated. A bioanalytical method to determine all protease and non-nucleoside reverse transcriptase inhibitors, and zalcitabine and zidovudine concentrations in biological matrices was developed. Samples were prepared by protein precipitation with methanol after addition of three different internal standards. Antiretrovirals were separated by high-performance liquid chromatography on a Nucleosil C18-100 Nautilus column using a gradient of 20 mM ammonium acetate including 0.1% aqueous acetic acid and acetonitrile and detected by electrospray ionisation/tandem mass spectrometry in the negative (efavirenz, stavudine, zidovudine) or positive ionisation mode (all other compounds). The bioanalytical method was successfully validated according to FDA guidelines and applied to plasma and cerebrospinal fluid samples of patients treated for acquired immunodeficiency syndrome (AIDS).

Therapeutic drug monitoring of the HIV protease inhibitor atazanavir in clinical practice

Therapeutic drug monitoring (TDM) is being applied for a number of antiretroviral agents. Little is known about the use of TDM for atazanavir. This is a retrospective cohort analysis on the use of TDM of atazanavir at three clinical sites in The Netherlands. Patients were divided into three groups: (i) all patients with evaluable data of plasma atazanavir concentrations and its relationship with hyperbilirubinaemia; (ii) patients who started atazanavir without documented evidence of protease inhibitor (PI) mutations; (iii) patients who started atazanavir with documented evidence of PI mutations. The genotypic inhibitory quotient (GIQ) was calculated by dividing the mean atazanavir plasma trough concentration by the number of PI mutations. A total of 108 patients were included; 70 (65.8%) were using atazanavir/ritonavir (300/100 mg once daily). No significant relationship was observed between atazanavir plasma trough concentration and antiviral response in patients starting atazanavir without PI mutations (group 2; n = 82). In contrast, a significant relationship was observed between atazanavir GIQ and treatment response in patients starting atazanavir while having PI mutations (group 3; n = 26). The cut-off value for GIQ most predictive of virological failure was 0.23 mg/L/mutation: patients (n = 8) with a GIQ equal to or below this value had 50% virological failure whereas patients (n = 18) with a GIQ above 0.23 mg/L/mutation had only 11% virological failure (chi(2): P = 0.030). Atazanavir plasma trough concentrations were significantly related with the occurrence of increased total bilirubin concentrations. TDM of atazanavir might be beneficial for patients with documented PI resistance or patients with hyperbilirubinaemia.

Effect of Concomitantly Administered Rifampin on the Pharmacokinetics and Safety of Atazanavir Administered Twice Daily

The potent induction of hepatic cytochrome P450 3A isoforms by rifampin complicates therapy for coinfection with human immunodeficiency virus (HIV) and Mycobacterium tuberculosis. We performed an open-label, single-arm study to assess the safety and pharmacokinetic interactions of the HIV protease inhibitor atazanavir coadministered with rifampin. Ten healthy HIV-negative subjects completed pharmacokinetic sampling at steady state while receiving 300 mg atazanavir every 12 h without rifampin (period 1), 300 mg atazanavir every 12 h with 600 mg rifampin every 24 h (period 2), and 400 mg atazanavir every 12 h with 600 mg rifampin every 24 h (period 3). During period 1, the mean concentration of drug in serum at 12 h (C(12 h)) was 811 ng/ml (range, 363 to 2,484 ng/ml) for atazanavir, similar to historic seronegative data for once-daily treatment with 300 mg atazanavir boosted with 100 mg ritonavir. During periods 2 and 3, the mean C(12 h) values for atazanavir were 44 ng/ml (range, <25 to 187 ng/ml) and 113 ng/ml (range, 39 to 260 ng/ml), respectively, well below historic seronegative data for once-daily treatment with 400 mg atazanavir without ritonavir. Although safe and generally well tolerated, 300 mg or 400 mg atazanavir administered every 12 h did not maintain adequate plasma exposure when coadministered with rifampin.

Penetration of Atazanavir in Seminal Plasma of Men Infected with Human Immunodeficiency Virus Type 1

In this exploratory study, the human immunodeficiency virus (HIV) protease inhibitor atazanavir was detected in seminal plasma in 15 out of 15 HIV-infected men taking an atazanavir-containing regimen. However, this penetration was limited and variable, and the median seminal/blood plasma ratio was only 0.1.

Simultaneous determination of 8 HIV protease inhibitors in human plasma by isocratic high-performance liquid chromatography with combined use of UV and fluorescence detection: Amprenavir, indinavir, atazanavir, ritonavir, lopinavir, saquinavir, nelfinavir and M8-nelfinavir metabolite

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.

Effects of atazanavir/ritonavir and lopinavir/ritonavir on glucose uptake and insulin sensitivity: demonstrable differences in vitro and clinically

The HIV protease inhibitor (PI) atazanavir does not impair insulin sensitivity acutely but ritonavir and lopinavir induce insulin resistance at therapeutic concentrations. 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. 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. 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. 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.

An update on HIV-1 antiretroviral resistance

Emergence of antiretroviral drug resistance limits the benefit of antiretroviral therapy for many patients. Despite considerable progress, much remains to be learned about drug resistance. Studies published over the last year have contributed significantly to the understanding of HIV-1 drug resistance. The prevalence of antiretroviral drug resistance is rising among persons newly infected with HIV-1 in the US and Europe. In this setting, resistance testing prior to starting antiretroviral therapy has been shown to be cost-effective. Immunologic stability despite emergence of drug resistance on a failing antiretroviral regimen may be due to lower levels of immune activation by drug-resistant virus. Recent studies show that continued use of lamivudine contributes to partial viral suppression despite the presence of lamivudine resistance. New, more sensitive assays for detecting drug resistance mutations highlight the problem of resistance to nonnucleoside reverse transcriptase inhibitors following use of single-dose nevirapine for prevention of mother-to-child transmission. Data from laboratory and clinical studies provide a more complete understanding of resistance to the protease inhibitor atazanavir and to the fusion inhibitor enfuvirtide. New data on HIV-1 drug resistance continue to improve our understanding of the epidemiology, pathogenesis and management of antiretroviral drug resistance.

Simple determination of the HIV protease inhibitor atazanavir in human plasma by high-performance liquid chromatography with UV detection

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 1 cc cartridges, an isocratic reversed-phase liquid chromatography on an XTerra RP18 (150 mm × 4.6 mm, 3.5 μm) column, and ultraviolet detection at 203 nm. The mobile phase consisted of phosphate buffer (pH 6, 52.5 mM) and acetonitrile (43:57, v/v). Up to 48 samples could be measured in one day since the run-time of one sample was 30 min. The assay was linear from 0.04 to 10 μg/ml with a lower limit of quantification of 0.04 μg/ml. The mean absolute recovery of ATV was 98.1%. The method was precise, with both intra-day and inter-day coefficients of variation ≤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.

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

Gilbert's disease and atazanavir: From phenotype to UDP-glucuronosyltransferase haplotype

Gilbert's disease leads to intermittent non-hemolytic hyperbilirubinemia by a reduction of hepatic bilirubin glucuronidation associated with the presence of the UDP-glucuronosyltransferase (UGT) 1A1*28 polymorphism. It is considered benign because it does not result in hepatocellular damage. However, pharmacogenetic analyses have linked UGT1A1*28 to drug toxicity and cancer predisposition. The protease inhibitor atazanavir (ATV) is an inhibitor of hepatic UGT activity leading to hyperbilirubinemia in individual patients. Whether this is linked specifically to UGT1A1*28 or to more complex variants influencing glucuronidation is unclear. One hundred and six ATV-treated patients were characterized and genotyped for UGT1A1*28, the UGT1A3 (-66C) and UGT1A7 (-57G) promoter variants, and UGT1A7(129K/131K). ATV treatment increased median bilirubin levels from 10 to 41 micromol/L (P = .001) with hyperbilirubinemia exceeding 43 micromol/L in 37%. Hyperbilirubinemia over 43 micromol/L was significantly associated not only with UGT1A1*28 but also with UGT1A3-66C, UGT1A7-57G, and UGT1A7(129K/131K), although these variants do not naturally occur in linkage dysequilibrium in blood donors. Homozygous combinations of UGT1A1*28 with the other variants increased from 7.4% (normal bilirubin to 42 micromol/L) to 41% to 46.1% (43 to >85 micromol/L), and 100% (>85 micromol/L). All six patients with hyperbilirubinemia greater than 85 micromol/L were homozygous for all four variants identifying a haplotype inherited on a single allele. In conclusion, the genetic variant associated with Gilbert's disease is identified as part of a haplotype of four UGT1A variants spanning three genes at the UGT1A gene locus. This haplotype predisposes to hyperbilirubinemia in ATV treatment and may have an additional role as a pharmacogenomic risk factor for drug therapy.