JAC Advance Access originally published online on May 17, 2007
Journal of Antimicrobial Chemotherapy 2007 60(1):61-67; doi:10.1093/jac/dkm135
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Interindividual variability in the effect of atazanavir and saquinavir on the expression of lymphocyte P-glycoprotein
1 Department of Biopharmaceutical Sciences, University of California San Francisco, 1550 4th Street, San Francisco, CA 94158, USA 2 Pacific Horizon Medical Group, 2351 Clay Street, San Francisco, CA 94115, USA 3 Institute for Human Genetics, University of California San Francisco, 1550 4th Street, San Francisco, CA 94158, USA
* Correspondence address. Department of Biopharmaceutical Sciences, 1550 4th Street, Rm RH584E, Box 2911, San Francisco, CA 94143-2911, USA. Tel: +1-415-476-1159; Fax: +1-415-514-4361; E-mail: deanna.kroetz{at}ucsf.edu
Received 12 December 2006; returned 22 February 2007; revised 27 March 2007; accepted 11 April 2007
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Abstract |
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Objectives: ABCB1 encodes the efflux transporter P-glycoprotein (P-gp), which regulates the intracellular concentration of many xenobiotics, including several HIV protease inhibitors (PIs). Exposure to some xenobiotics, such as the antibiotic rifampicin, increases P-gp expression. In the present study, we investigated the effect of the HIV PIs saquinavir and atazanavir on the expression and function of ABCB1 and P-gp in primary and cultured lymphocytes, as well as the molecular interactions between these drugs and P-gp. ABCB1 and P-gp expression and function were examined in lymphocyte samples from healthy subjects before and after atazanavir-boosted saquinavir treatment. Expression and function were also studied in CEM cells following exposure to atazanavir and saquinavir. The inhibitory effects of these drugs were investigated in ABCB1-transfected HEK293T cells.
Methods: P-gp expression and function were measured by flow cytometry. ABCB1 mRNA expression was evaluated using quantitative RT–PCR.
Results: There were no overall changes in ABCB1 or P-gp expression or function after saquinavir–atazanavir treatment in primary lymphocyte samples. However, there was considerable interindividual variability in baseline lymphocyte ABCB1 expression, as well as in the degree of change in ABCB1 expression after saquinavir–atazanavir administration. In cell culture, 5 µM saquinavir increased ABCB1 levels, although it did not affect P-gp expression. Atazanavir inhibited P-gp function at concentrations above therapeutic levels.
Conclusions: Differences in lymphocyte ABCB1 expression, which may be caused by genetic polymorphisms in ABCB1 or its regulatory partners, are a likely cause of interindividual variation in the disposition and efficacy of clinically relevant P-gp substrates, including HIV PIs.
Keywords: antivirals , protease inhibitors , ABCB1
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Introduction |
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Viral resistance continues to impede the development of effective HIV antiretroviral therapies (ART).1 Interindividual variability in ART response presents a real challenge; evaluating the efficacy of multiple antiretroviral drugs for each patient comes at a considerable economic cost,2 as well as a significant cost to the patient's physical and emotional health.3 In HIV, viral resistance is caused by inherent or acquired viral mutations, which may be influenced by host factors.4 Xenobiotic transporters, which prevent compounds from crossing physiological barriers, are thought to be one of the causal host factors in HIV resistance.5 Many of these xenobiotic transporters, which utilize the energy from ATP hydrolysis to move substrate molecules across cell membranes,6 belong to the ATP-binding cassette (ABC) superfamily.7
In this superfamily, the most extensively studied xenobiotic transporter is P-glycoprotein (P-gp), which is encoded by ABCB1. P-gp was discovered in cells resistant to the anticancer vinca alkaloids and anthracyclines,8 but P-gp also transports a wide variety of structurally unrelated xenobiotics.9 The effect of P-gp on pharmacokinetics (plasma levels) and pharmacodynamics (efficacy) is routinely investigated during drug development.10 P-gp can affect pharmacokinetics by limiting bioavailability or increasing clearance11 and can regulate the drug concentration at the site of action, thereby influencing pharmacodynamics.12
Several HIV protease inhibitors (PIs) are substrates for P-gp. Saquinavir transport has been demonstrated in ABCB1-overexpressing cell lines13–16 and increased saquinavir brain accumulation was reported in mdr1a knockout mice.17 Saquinavir also inhibited P-gp function in human lymphocytes15 and insect cells.18 P-gp induction19 and inhibition19,20 by atazanavir have also been demonstrated in vitro. Because P-gp is expressed on lymphocyte plasma membranes, the cells targeted by HIV antiretrovirals, P-gp may limit the intracellular accumulation of PIs, rendering these drugs ineffectual and providing a sanctuary site for HIV.21
There is a high degree of interpatient variability in PI pharmacokinetic and pharmacodynamic parameters,22,23 and the occurrence of drug-related toxicities is difficult to foresee.24 In order to predict PI efficacy or toxicity, physicians monitor plasma concentrations25,26 that are assumed to correspond to pharmacologically relevant intracellular concentrations. A recent study by Colombo et al. demonstrated that saquinavir plasma and intralymphocytic concentrations correlated significantly (r = 0.80).27 However, there was considerable interindividual variability in the ratio between intracellular and plasma concentrations, with a coefficient of variation of 76%.27 This suggests that there is substantial interindividual variation in the cellular ‘permeability’ of saquinavir, which may be due to expression or functional differences in membrane transporters such as P-gp. Such variability in HIV antiretroviral pharmacokinetic and pharmacodynamic parameters may be influenced by polymorphisms in ABCB1 or other genes.28
Although the role of P-gp in PI pharmacokinetics and pharmacodynamics remains ambiguous, the effect of P-gp expression and function on antiretroviral response continues to be investigated. Conversely, few studies have explored the effect of PI exposure on lymphocyte P-gp in vivo. Studies have shown that oral administration of the antibiotic rifampicin up-regulates intestinal P-gp via the transcriptional factor pregnane X receptor (PXR) in a feedback loop, limiting systemic xenobiotic exposure.29 A similar phenomenon may occur when lymphocytes are exposed to PIs.
In the present study, we examined the effect of co-administration of the HIV PIs saquinavir and atazanavir on lymphocyte P-gp expression and function in healthy individuals. In addition, we determined if exposure to saquinavir or atazanavir in vitro resulted in a change in ABCB1 mRNA or P-gp expression in the CD4 cell line CEM, or if these drugs activate or inhibit P-gp function.
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Materials and methods |
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Chemicals and reagents
Histopaque, cyclosporin A and dimethyl sulphoxide (DMSO) were purchased from Sigma (St Louis, MO, USA). PBS, fetal bovine serum (FBS), penicillin–streptomycin and RPMI-1640 cell culture medium were purchased from the UCSF Cell Culture Facility (San Francisco, CA, USA). Calcein acetomethoxyester (calcein AM) was purchased from Molecular Probes (Eugene, OR, USA). Mouse IgG2a-allophycocyanin (APC) secondary antibody was purchased from Caltag Laboratories (Burlingame, CA, USA). The anti-human monoclonal P-gp antibody MRK16 was purchased from Kamiya Biomedical (Seattle, WA, USA). The RNeasy Mini Kit was purchased from Qiagen (Valencia, CA, USA). TRIzol reagent and Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA, USA). TaqMan buffer was purchased from the Genome Analysis Core (University of California, San Francisco, CA, USA). MgCl2 and AmpliTaq Gold were purchased from Applied Biosystems (Foster City, CA, USA) and dNTPs and M-MLV reverse transcriptase (RT) were purchased from Promega Corporation (Madison, WI, USA). Saquinavir mesylate and atazanavir sulphate were purchased from the University of California, San Francisco Medical Center pharmacy.
Quantification of PI effect on P-gp function
The human embryonic kidney cell line HEK293T was a gift from Dr Warner Greene (Gladstone Institute, San Francisco, CA, USA). Cell medium consisted of minimum essential medium supplemented with 10% FBS, 100 U/mL penicillin and 0.1 mg/mL streptomycin. HEK293T cells at 70% confluency were transfected with 10 µg of plasmid cDNA (ABCB1-pcDNA5/FRT or vector control) using 25 µL of Lipofectamine 2000 per T-25 flask. Cells were incubated at 37°C in a humidified 5% CO2 atmosphere for 24 h. Cells were harvested and counted, and 3 x 105 cells were incubated in 200 nM calcein AM ± 5 or 50 µM saquinavir or atazanavir or 5 µM cyclosporin A for 30 min at 37°C. Cells were collected by centrifugation, washed twice with ice-cold PBS and incubated with the anti-human P-gp antibody MRK16 (500 µg/mL) for 30 min at 4°C. Following two additional washes, cells were incubated with an APC-conjugated mouse IgG2a secondary antibody at a concentration of 1.2 µg/mL for 30 min at 4°C. Samples were washed twice and analysed on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Forward- and side-scatter signals were detected on a linear scale, and fluorescence was measured in channels 2 and 4 (FL-2, 
max 585 nm and FL-4, max 661 nm) on a logarithmic scale. Intracellular calcein fluorescence was evaluated in an MRK16+ P-gp-expressing subpopulation; at least 5000 events were collected per sample.
Incubation of CEM cells with HIV PIs
The human T-lymphoblast CEM cell line was obtained from ATCC (Manassas, VA, USA). Cell medium consisted of RPMI 1640 supplemented with 10% FBS, 100 U/mL penicillin and 0.1 mg/mL streptomycin. Cells were seeded at a density of 5 x 104 cells/mL in the presence of 5 or 10 µM saquinavir or atazanavir, or 0.1% DMSO (vehicle control), and maintained at 37°C in a humidified chamber in a 5% CO2 atmosphere. Cell medium was replaced after 48 h, and after 96 h cells were harvested for use in P-gp expression assays or for RNA isolation.
Eighteen HIV-seronegative volunteers (nine male, nine female) between the ages of 18 and 65 were recruited for the ASPIRE II (Atazanavir–Saquinavir Pharmacokinetic Research Endeavor) study coordinated by the Pacific Horizon Medical Group. Four subjects did not complete the study due to protocol non-compliance. The study consisted of three subsequent 11 day arms of ritonavir- or atazanavir-boosted saquinavir, separated by 10 day washout periods. In the first arm, subjects received 1000 mg of saquinavir and 100 mg of ritonavir twice daily; in the second, 1000 mg of saquinavir and 200 mg of atazanavir twice daily; and in the third, 1500 mg of saquinavir and 200 mg of atazanavir twice daily. For pharmacokinetic purposes, plasma was collected on day 11 of each arm; pharmacokinetic results have been published elsewhere.30 On day 1 of the first arm and day 11 of the third arm, blood samples were collected and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Histopaque. PBMCs were resuspended in cryopreservation medium (90% FBS, 10% DMSO) and stored in nitrogen vapour. The Pacific Horizon Medical Group Internal Review Board approved ASPIRE II, including the collection of lymphocytes for analysis of P-gp expression and function, and all subjects provided written informed consent prior to participation in this study. Analysis of ASPIRE II samples was approved by the University of California, San Francisco Committee on Human Research.
Quantification of lymphocyte P-gp expression
Cells were washed twice with PBS, then incubated in the P-gp antibody MRK16 at 167 µg/mL for 30 min at 4°C. Following two washes with ice-cold PBS, cells were incubated with an APC-conjugated mouse IgG2a secondary antibody at 0.75 µg/mL for 30 min at 4°C. Samples were then washed twice and analysed on a FACSCalibur flow cytometer. Forward- and side-scatter signals were detected on a linear scale, and fluorescence was measured in channel 4 on a logarithmic scale. A homogeneous lymphocyte population was selected for data collection based on cellular light-scattering attributes; at least 5000 events were collected per sample.
RNA isolation and quantification of lymphocyte ABCB1 expression
RNA was extracted from cells using the RNeasy Mini Kit (primary lymphocyte samples) or TRIzol reagent (CEM samples) following the manufacturer's instructions. RNA was quantified using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and reverse-transcribed by M-MLV RT according to the manufacturer's instructions, using random hexamers as primers. Gene expression was measured by quantitative real-time PCR (TaqMan) on a PRISM 7700 Sequence Detection System (Applied Biosystems). Each reaction contained 1 x TaqMan buffer, 5.5 mM MgCl2, 200 µM dNTP, 0.625 U of AmpliTaq Gold, 500 nM each primer and 200 nM probe in a final volume of 25 µL. Primers and probes were designed with the Primer Express 2.0 software (Applied Biosystems); sequences are available online [see Table S1, available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)]. Primers and probes were synthesized by Invitrogen and Integrated DNA Technologies (Coralville, IA, USA), respectively. PCR conditions were 12 min at 95°C, followed by 45 cycles of 15 s at 95°C and 1 min at 60°C. All samples were normalized to expression of the human control gene ß-glucuronidase (hGUS).
ABCB1 gene expression was normalized to hGUS and compared between baseline (first day of study arm 1) and treated (last day of study arm 3) samples. P-gp expression was compared between baseline and treated samples by considering the median fluorescence in FL-4. P-gp function was represented by the median intracellular fluorescence in FL-2. Statistical analyses of ABCB1 expression and P-gp expression and function in the inhibition and in vitro induction studies were performed using the non-parametric Kruskal–Wallis analysis of variance test, followed by the Dunn's multiple comparison test if significance was found in the Kruskal–Wallis test. ABCB1 expression and P-gp expression and function in the ASPIRE II primary lymphocyte samples were statistically analysed using the Wilcoxon matched pairs test. The level of significance for all statistical tests was P = 0.05. All statistical comparisons were performed using the Prism (GraphPad Software, San Diego, CA, USA) software package.
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Inhibition of P-gp function by saquinavir and atazanavir in vitro
The effects of saquinavir and atazanavir on P-gp function were investigated in HEK293T cells transiently expressing ABCB1. Flow cytometry analysis showed that the addition of a pharmacological concentration (5 µM) of saquinavir or atazanavir slightly increased intracellular calcein AM fluorescence compared with control cells, although this did not reach statistical significance (Figure 1). In the presence of 50 µM atazanavir, there was a significant increase in intracellular fluorescence (324 ± 12.4% of control, P < 0.01), indicating inhibition of P-gp function. Cyclosporin A, a P-gp inhibitor, also increased intracellular fluorescence (236 ± 73.8% of control, P < 0.05). A high concentration (50 µM) of saquinavir showed a trend towards increasing intracellular fluorescence, but this did not reach statistical significance. Both saquinavir and atazanavir demonstrated a trend towards inhibition of P-gp function.
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Effect of PI exposure on lymphocyte ABCB1 mRNA and P-gp surface expression in vitro
In order to determine whether saquinavir and atazanavir affected ABCB1 mRNA or P-gp levels in vitro, CEM cells were incubated with these drugs for 96 h. Quantitative RT–PCR showed a 2.0-fold increase (±1.58, P < 0.05) in ABCB1 mRNA expression in cells cultured in 5 µM saquinavir compared with vehicle control (Figure 2). Exposure to 10 µM atazanavir also increased ABCB1 mRNA expression (1.53 ± 0.54-fold, P < 0.05) compared with vehicle control. The small changes in ABCB1 mRNA levels in saquinavir- and atazanavir-treated CEM cells did not translate into changes in P-gp expression. Flow cytometry quantification of bound MRK16 antibody showed that P-gp expression levels were similar across all concentrations (data not shown).
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Effect of PI administration on lymphocyte ABCB1 mRNA and P-gp surface expression in vivo
The demographics of the ASPIRE II population are shown in Table 1. All subjects were healthy, HIV-seronegative, had no prior exposure to antiretrovirals and were not currently taking any inducers or inhibitors of P-gp or cytochrome P450 enzymes. The majority of the study population (12 of 14 subjects) was Caucasian. To ascertain whether atazanavir-boosted saquinavir affected ABCB1 mRNA lymphocyte expression in this population, a quantitative RT–PCR analysis was performed. ABCB1 expression was measured in lymphocyte samples obtained before and after treatment; data are expressed as a fold change from the pre-treatment level (Figure 3a). There was a 1.3 ± 0.4-fold increase in ABCB1 mRNA levels after PI exposure. Intersubject variability in response to PI treatment was substantial, with ABCB1 RNA levels increasing from 0.5–1.9-fold over baseline levels.
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The effect of PI administration on lymphocyte P-gp expression was examined through flow cytometric quantification of cell-surface protein. Fluorescence of the APC-conjugated P-gp-specific antibody was measured in lymphocyte samples collected before and after atazanavir-boosted saquinavir administration. The median fluorescence of the total lymphocyte population is shown as relative fluorescence units (RFU) (Figure 3b). No change in mean expression was detected after PI treatment, although P-gp expression increased
30% in two subjects. The effect of PI administration on the P-gp-positive subpopulation of lymphocytes was also not significant (Figure 3c). The median fluorescence of all subjects increased by an average of 21% (41.5 ± 3.38 RFU before treatment, compared with 50.2 ± 18.9 RFU after treatment), although this change does not reach statistical significance (P = 0.058). As with the analysis of the total lymphocyte population, there is a significant intersubject variability in the response to PI treatment, with some subjects showing a nearly 2-fold increase in lymphocyte P-gp levels. |
Discussion |
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The results of the present study indicate that the HIV PIs saquinavir and atazanavir interact with and affect expression of the xenobiotic transporter P-gp to a limited extent in vivo. P-gp transport of saquinavir has been previously demonstrated, both in cell culture systems31–33 and in animal studies.34 Several groups have also reported that saquinavir inhibits P-gp function.15,18 Atazanavir has also been shown to have both inhibitory19,20 and inductive effects.19 However, few studies have examined the effect of PIs on ABCB1/P-gp in vivo.
In the current study, 5 µM saquinavir and atazanavir exhibited a trend towards P-gp inhibition. These concentrations are similar to the peak plasma concentrations at steady state in patients receiving these drugs (Invirase and Reyataz package inserts; Roche and Bristol–Myers Squibb, respectively). At higher concentrations, saquinavir and atazanavir inhibited P-gp function to a greater extent. These findings may be of clinical importance if these drugs are administered to a patient who is also receiving xenobiotics that alter PI metabolism, as this may lead to increased plasma concentrations of the PIs.
The effect of PIs on ABCB1 expression may also be an important factor in patient response to HIV antiretrovirals, since an increase in lymphocyte P-gp expression may decrease the target cell drug concentration. In 2003, Chandler et al. found that a 3 day incubation with non-cytotoxic levels of saquinavir did not up-regulate lymphocyte P-gp in vitro,35 although in a subsequent study Dupuis et al. reported that prolonged exposure to saquinavir increased lymphocyte P-gp expression in vitro.36 Two studies from Perloff et al. demonstrated that a 4 day exposure to saquinavir or a 3 day exposure to atazanavir increased P-gp levels in an ABCB1-overexpressing cell line.19,37 It has been proposed that this P-gp up-regulation is caused by the nuclear receptor PXR, for which saquinavir is a weak ligand.38 PIs may act via this regulatory pathway to generate an increase in lymphocyte P-gp expression, since the presence of PXR has previously been observed in lymphocytes.39–41
In order to study the inductive properties of saquinavir and atazanavir, we cultured CEM lymphoblast cells in saquinavir and atazanavir for 4 days. Quantitative RT–PCR results showed that exposure to 5 µM saquinavir and 10 µM atazanavir increased ABCB1 mRNA expression compared with vehicle control by at most 2-fold. The relatively small magnitude of the overall change in CEM ABCB1 mRNA expression following exposure to therapeutic concentrations of these PIs suggests that these findings may be of little clinical relevance.
Observed changes in ABCB1 mRNA expression did not translate into P-gp cell surface expression (data not shown). This is in accordance with previous findings from Chandler et al.35 P-gp levels may not have changed after incubation in saquinavir and atazanavir because of the comparatively short period of cellular exposure to these drugs. Examination of P-gp expression during continuous exposure to PIs may provide more information about the potential for P-gp up-regulation in response to atazanavir or saquinavir treatment. It is also likely that the comparatively small changes in CEM ABCB1 mRNA levels are not large enough to significantly affect P-gp levels.
Lymphocytes obtained from ASPIRE II subjects were used to determine the effects of atazanavir-boosted saquinavir on ABCB1 expression and function in healthy individuals. A large interindividual variability was observed in baseline ABCB1 mRNA expression, as well as in the change in ABCB1 mRNA expression after atazanavir–saquinavir treatment. Overall, quantitative RT–PCR analysis suggested that ABCB1 expression increased slightly (1.2-fold) after administration of atazanavir and saquinavir.
In accordance with our in vitro results, this slight up-regulation did not translate into an increase in lymphocyte P-gp expression, either with respect to the total lymphocyte population or in only that subset which expresses P-gp. However, the overall mean of the median fluorescence intensities of the FL-4+ lymphocyte subpopulations from each subject showed a trend towards up-regulation—in effect, the amount of cell surface P-gp increased slightly in the lymphocytes that were positive for P-gp antibody staining. This small overall increase was primarily due to several outliers who showed significant increases in P-gp expression after saquinavir and atazanavir administration, which may indicate increased sensitivity to regulatory stimuli in a subset of individuals, possibly due to polymorphisms in the ABCB1 promoter region or in nuclear receptors such as PXR.
It should be noted that the effect of HIV infection on lymphocyte ABCB1 expression was not addressed in this study, since all study participants were HIV-negative. Previous studies have addressed this question, with differing results. Andreana et al. demonstrated that the proportion of lymphocytes expressing P-gp was higher in HIV-infected subjects than in uninfected controls, and that this proportion increased with disease progression, although the increase in P-gp expression did not correlate with increased function.42 However, Meaden et al. showed that lymphocyte P-gp expression was reduced in HIV-infected subjects compared with controls.43 The results of both studies have been reproduced by several groups.44–46 Our analyses cannot rule out the possibility that the effects of atazanavir–saquinavir on lymphocyte ABCB1 expression observed in the ASPIRE II population may differ in an HIV-infected population.
The high degree of interindividual variability that we observed in baseline lymphocyte ABCB1 expression can be attributed in large part to genetic and environmental factors. Genetic polymorphisms in ABCB1 have been identified through comprehensive SNP discovery efforts;47 studies are ongoing to correlate these polymorphisms with P-gp expression and function. Subjects are excluded from a clinical study if they are exposed to substances known to influence ABCB1/P-gp expression, thus controlling for environmental factors to the greatest practical extent. In a clinical setting, however, it is much more difficult to regulate a patient's environment, leading to variability in drug pharmacokinetics. Genetic factors can also affect drug pharmacokinetics and pharmacodynamics.
Previous studies have demonstrated considerable interpatient variability in saquinavir pharmacokinetics.23,48–50 A 2005 report examined ritonavir-boosted saquinavir pharmacokinetics in HIV-seropositive patients in Thailand and the UK in three separate studies and found an average coefficient of variation for saquinavir Cmin of 86%, with a number of subjects (ranging from 14% to 50%) having trough concentrations below the minimum effective concentration of 0.1 mg/L.51 Interestingly, study site was found to significantly correlate with saquinavir AUC, suggesting that ethnic factors influence saquinavir pharmacokinetics. These factors may be environmental or genetic, or a combination of both.51 It is possible that polymorphisms in genes in the saquinavir pharmacokinetic or pharmacodynamic pathways, including transporters such as P-gp, influence how a patient responds to saquinavir treatment. The avoidance of excessively low saquinavir plasma levels may increase virological suppression and immunological response in certain patients,52 although plasma levels are not the sole determinant of saquinavir efficacy.53 The use of pharmacogenetic markers to predict which patients may require higher dosages may considerably advance HIV treatment.
The findings from the present study indicate that atazanavir-boosted saquinavir exposure may up-regulate ABCB1 expression in some individuals. The large interindividual variability in the change in transporter expression after atazanavir–saquinavir treatment may be caused by genetic variation, either in the ABCB1 gene, its regulatory partners, or in other genes involved in the disposition of these drugs (e.g. uptake and/or other ABC transporters, metabolizing enzymes or plasma binding proteins). Although it is clear that a high degree of interindividual variability exists in both basal transporter expression and in drug response, the source of this variability is at present speculative. The identification of these sources of variability will allow clinicians to more efficiently optimize HIV antiretroviral therapy on an individual basis.
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None to declare.
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Supplementary data |
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Table S1 is available as Supplementary data at JAC Online (http://jac.oxfordjournals.org).
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Acknowledgements |
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We thank Debbie Lin for the purification and chemical analysis of saquinavir and atazanavir. This work was supported by NIH grant GM61390 and Roche.
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References |
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