Prevalence and type of drug-drug interactions involving ART in patients attending a specialist HIV outpatient clinic in Kampala, Uganda.

OBJECTIVES: Scale-up of HIV services in sub-Saharan Africa has rapidly increased, necessitating evaluation of medication safety in these settings. Drug-drug interactions (DDIs) involving antiretrovirals (ARVs) in sub-Saharan Africa are poorly characterized. We evaluated the prevalence and type of ARV DDIs in Ugandan outpatients and identified the patients most at risk. METHODS: A total of 2000 consecutive patients receiving ARVs at the Infectious Diseases Institute, Kampala were studied. The most recent prescription for each patient was screened for clinically significant DDIs using www.hiv-druginteractions.org. Univariable and multivariable logistic regression were used to identify risk factors for DDIs. A screening tool was developed using significant risk factors and tested in a further 500 patients. RESULTS: Clinically significant DDIs were observed in 374 (18.7%) patients, with a total of 514 DDIs observed. Only 0.2% of DDIs involved a contraindicated combination. Comedications commonly associated with DDIs were antibiotics (4.8% of 2000 patients), anthelmintics (2.2%) and antifungals (3.5%). Patient age, gender, CD4 count and weight did not affect risk of DDIs. In multivariable analysis, the patient factors that independently increased risk of DDIs were two or more comedications (P < 0.0001), a PI-containing ARV regimen (P < 0.0001), use of an anti-infective (P < 0.0001) and WHO clinical stage 3-4 (P = 0.04). A scoring system based on having at least two of these risk factors identified between 75% and 90% of DDIs in a validation cohort. CONCLUSIONS: Significant ARV DDIs occur at similar rates in resource-limited settings and developed countries; however, the comedications frequently causing DDIs differ. Development of tools that are relevant to particular settings should be a priority to assist with prevention and management of DDIs.

Therapeutic drug monitoring of atazanavir/ritonavir in pregnancy.

OBJECTIVES: Pregnant women experience physiological changes during pregnancy that can have a significant impact on antiretroviral pharmacokinetics. Ensuring optimal plasma concentrations of antiretrovirals is essential for maternal health and to minimize the risk of vertical transmission. Here we describe atazanavir/ritonavir (ATV/r) plasma concentrations in a cohort of pregnant women undergoing routine therapeutic drug monitoring (TDM). METHODS: Pregnant HIV-positive women received ATV/r as part of their routine pre-natal care. Demographic and clinical data were collected. ATV plasma concentrations ([ATV]) were determined in the first (T1), second (T2) and third (T3) trimesters and at postpartum (PP) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: From January 2007, 44 women (37 black African) were enrolled in the study. All received ATV/r at a dose of 300/100 mg once a day. Twenty-four had received antiretroviral therapy (ART) prior to pregnancy, and 20 initiated ATV/r in pregnancy. At the time nearest to delivery, 36 patients had undetectable plasma viral loads. [ATV] values were determined in 11 (T1), 25 (T2), 34 (T3) and 28 (PP) patients. [ATV] at 24 hours post-dose (C24) values significantly lower at T2/T3 relative to PP. CONCLUSIONS: This study was carried out in one of the larger cohorts of women undergoing TDM for ATV in pregnancy. Lower [ATV] values were seen in T2/T3 compared with T1/PP. However, [ATV] were not associated with a lack of virologic suppression at delivery. Nonetheless, careful monitoring of women in pregnancy is required, and dose adjustment of ATV to 400 mg may be an option.

Interactions between tenofovir and nevirapine in CD4+ T cells and monocyte-derived macrophages restrict their intracellular accumulation.

OBJECTIVES: There is no pharmacokinetic interaction between tenofovir and nevirapine, but a higher emergence rate of resistance mutations has been reported when these drugs are coadministered. We sought to examine if there is a potential intracellular interaction that may account for the emergence of resistant virus. METHODS: Primary CD4+ and CD14+ cells were isolated from healthy volunteer blood. Monocyte-derived macrophages were differentiated from CD14+ cells. Accumulation of radiolabelled tenofovir and nevirapine was then assessed in these cells. RESULTS: We show here that tenofovir and nevirapine immune cell intracellular concentrations are lower when coincubated in CD4+ cells and monocyte-derived macrophages, but not in CD14+ cells. CONCLUSIONS: These data indicate a potential intracellular drug-drug interaction between these drugs that warrants further investigation.

Improved oral bioavailability of lopinavir in melt-extruded tablet formulation reduces impact of third trimester on lopinavir plasma concentrations.

Lopinavir exposure was reduced during the third trimester in pregnant women receiving standard dosing of the soft-gel capsule (SGC; 400/100 mg twice daily [b.i.d.]). Pharmacokinetic data on the lopinavir tablet in pregnancy are limited. On the basis of the tablet's improved bioavailability, standard dosing (400/100 mg b.i.d.) may provide adequate lopinavir exposure in pregnancy without a need for dose adjustment. Here we compared the total and unbound lopinavir pharmacokinetics throughout pregnancy in the second and third trimesters in HIV-infected women receiving standard dosing of the lopinavir SGC or tablet. Postpartum sampling was also performed in patients continuing therapy postdelivery. Blood samples were collected at 0 to 12 h postdosing, and lopinavir concentrations were determined by high-pressure liquid chromatography-tandem mass spectrometry. Nineteen patients were included: 8 received the SGC (cohort 1) and 11 received the tablet (cohort 2). Total lopinavir exposures in the third trimester were lower than those in the second trimester (35 and 28% for cohorts 1 and 2, respectively) and postpartum (35% for cohort 2). In the third trimester, the area under the concentration-time curve (AUC) from 0 to 12 h (AUC(0-12)) and maximum concentration were ∼15% and 25% higher, respectively, for the lopinavir tablet than the SGC. One SGC patient had lopinavir concentrations of <1,000 ng/ml; all patients on the tablet had concentrations of >1,000 ng/ml. In cohort 2, the percentage of the AUC that was unbound was higher (nonsignificantly) in the second (1.28%) and third (1.18%) trimesters than postpartum (1.01%). Seventeen of 19 patients had an undetectable viral load at delivery. There were no HIV transmissions. Although lopinavir (tablet) exposures were reduced during the third trimester, the higher total and unbound concentrations achieved in women receiving the tablet than in women receiving the SGC suggest that the tablet's improved oral bioavailability may partly compensate for the reduction in lopinavir exposure during the later stages of pregnancy.

Therapeutic drug monitoring of lopinavir/ritonavir in pregnancy.

OBJECTIVES: The aim of the study was to determine total and unbound lopinavir (LPV) plasma concentrations in HIV-infected pregnant women receiving lopinavir/ritonavir (LPV/r tablet) undergoing therapeutic drug monitoring (TDM) during pregnancy and postpartum. METHODS: Women were enrolled in the study who were receiving the LPV/r tablet as part of their routine prenatal care. Demographic and clinical data were collected and LPV plasma (total) and ultrafiltrate (unbound) concentrations were determined in the first, second and third trimesters using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Postpartum sampling was performed where applicable. Antepartum and postpartum trough concentrations (C(trough) ) were compared independently [using analysis of variance (anova)] and on a longitudinal basis (using a paired t-test). RESULTS: Forty-six women were enrolled in the study (38 Black African). Forty women initiated LPV/r treatment in pregnancy. Median (range) gestation at initiation was 25 (15-36) weeks and median (range) baseline CD4 count and viral load were 346 (14-836) cells/µL and 8724 (<50-267408) HIV-1 RNA copies/mL, respectively. Forty women (87%) had LPV concentrations above the accepted minimum effective concentration for wild-type virus (MEC; 1000 ng/mL). Geometric mean (95% confidence interval [CI]) total LPV concentrations in the first/second [3525 (2823-4227) ng/mL; n=16] and third [3346 (2813-3880) ng/mL; n=43] trimesters were significantly lower relative to postpartum [5136 (3693-6579) ng/mL; n=12] (P=0.006). In a paired analysis (n=12), LPV concentrations were reduced in the third trimester [3657 (2851-4463) ng/mL] vs. postpartum (P=0.021). No significant differences were observed in the LPV fraction unbound (fu%). Conclusions The above target concentrations achieved in the majority of women and similarities in the fu% suggest standard dosing of the LPV/r tablet is appropriate during pregnancy. However, reduced LPV concentrations in the second/third trimesters and potentially compromised adherence highlight the need for TDM-guided dose adjustment in certain cases.

The impact of pharmacogenetics on HIV therapy.

The use of highly active antiretroviral therapy in the treatment of HIV infection has resulted in significant reductions in mortality and morbidity worldwide. However, there is considerable interindividual variability in patient outcomes in terms of drug disposition, drug efficacy and adverse events. The basis of these differences is multifactorial, but host genetics are believed to play a significant part. To date, most attempts to explain this variability have focused on isolated single nucleotide polymorphisms. The most exciting development to date is the discovery of human leukocyte antigen subtype B*5701 (HLA B*5701) as a strong predictor of the abacavir hypersensitivity reaction. There is a gradual move away from single candidate gene analyses towards a high throughput whole genome approach. These studies must be performed on well characterized cohorts and reported associations must be validated in independent, ethnically diverse populations.

Detection of ABCC2, CYP2B6 and CYP3A4 in human peripheral blood mononuclear cells using flow cytometry.

ABCC2 has a wide tissue distribution and can mediate the efflux of a number of therapeutic compounds from cells and contribute to potential treatment failure. Its diverse expression and ability to efflux a number of substrates imply a number of physiological and pharmacological roles. CYP2B6 and CYP3A4 are responsible for the metabolism of a number of therapeutic compounds. Reports on the expression of these proteins in various cells and tissues have been contradictory mainly due to differences in experimental approach and cell type studied. With the advances in commercially available antibodies we describe here a simplified technique for the detection of ABCC2, CYP2B6 and CYP3A4 in human peripheral blood mononuclear cells (PBMC) by flow cytometry. Results are expressed as mean increase in fluorescence compared to isotypically matched controls. Using these assays we confirmed the expression of these proteins in human PBMC. These methods are rapid and reproducible and have potential use for both in vitro and clinical applications.

Comparison of the induction profile for drug disposition proteins by typical nuclear receptor activators in human hepatic and intestinal cells.

BACKGROUND AND PURPOSE: Certain nuclear receptors (NRs) such as the constitutive androstane receptor (CAR), pregnane X receptor (PXR) and farnesoid X receptor (FXR) mediate induction of some cytochrome P450 enzymes and ABC transporters but conflicting reports exist. The purpose of this study was to assess the reasons for these discrepancies and use a standardized approach to compare activators of NRs. EXPERIMENTAL APPROACH: Dexamethasone, pregnenolone 16alpha-carbonitrile, rifampicin, phenobarbital and chenodeoxycholic acid were incubated with HepG2, Caco-2 and cryopreserved human hepatocytes prior to analysis of mRNA and protein for CYP2B6, CYP3A4, CYP3A5, ABCB1, ABCC1, ABCC2, PXR, CAR and FXR. KEY RESULTS: Dexamethasone significantly up-regulated PXR, CYP3A4 and ABCB1 expression in HepG2 and Caco-2 cells. As a result, including dexamethasone as a media supplement masked the induction of these genes by pregnenolone 16alpha-carbonitrile, which may explain discrepancies between previous reports. In the absence of dexamethasone, significant activator-dependent induction was observed in all cell types. Significant correlations were observed between the fold increase in mRNA and in protein, which were, for most instances, logarithmic. Changes in mRNA expression were greater in cell lines than primary cells but for most transcripts correlations were observed between fold increases in HepG2 and hepatocytes. CONCLUSIONS AND IMPLICATIONS: Clearly, no in vitro system can imitate the physiology of a hepatocyte or intestinal cell within an intact organ in vivo, but these data explain some of the discrepancies reported between laboratories and have important implications for study design.

A call for a consortium for optimal management of drug-drug interactions in patient care.

During clinical development of medicines, manufacturers are obliged to assess the risk of drug-drug interactions (DDIs) with their new drug. There is no doubt that product labels of drugs that are nowadays introduced to the market contain much more information on DDIs than in the past. Indeed, the drug label is often the first source for DDIs available to physicians and pharmacists. But how informative are the data presented in the drug labels?

The Effect of Gene Variants on Levonorgestrel Pharmacokinetics When Combined With Antiretroviral Therapy Containing Efavirenz or Nevirapine.

Reduced levonorgestrel concentrations from the levonorgestrel contraceptive implant was previously seen when given concomitantly with efavirenz. We sought to assess whether single nucleotide polymorphisms (SNPs) in genes involved in efavirenz and nevirapine metabolism were linked to these changes in levonorgestrel concentration. SNPs in CYP2B6, CYP2A6, NR1I2, and NR1I3 were analyzed. Associations of participant demographics and genotype with levonorgestrel pharmacokinetics were evaluated in HIV-positive women using the levonorgestrel implant plus efavirenz- or nevirapine-based antiretroviral therapy (ART), in comparison to ART-naïve women using multivariate linear regression. Efavirenz group: CYP2B6 516G>T was associated with lower levonorgestrel log10 Cmax and log10 AUC. CYP2B6 15582C>T was associated with lower log10 AUC. Nevirapine group: CYP2B6 516G>T was associated with higher log10 Cmax and lower log10 Cmin . Pharmacogenetic variations influenced subdermal levonorgestrel pharmacokinetics in HIV-positive women, indicating that the magnitude of the interaction with non-nucleoside reverse transcriptase inhibitors (NNRTIs) is influenced by host genetics.

Interactions between oral contraceptives and other drugs.

PIP: There is increasing evidence from various types of studies that commonly prescribed drugs can interact with OCs (oral contraceptives), leading to a decreased contraceptive efficacy. Additionally, contraceptive steroids influence the response to the other drug therapies as well. Antituberculous drugs enhance metabolism of the estrogenic component of OCs, thus reducing the contraceptive effect. Other types of drugs which have been shown to interfere with the contraceptive effect of OCs are: anticonvulsant, antibiotic, and analgesic drugs. The effect of OCs on anticoagulant, antihypertensive, antidiabetic, and antidepressant drugs is also outlined. Tables and graphs illustrate some of these disadvantegeous drug interactions.^ieng

Modulation of P-glycoprotein function in human lymphocytes and Caco-2 cell monolayers by HIV-1 protease inhibitors.

OBJECTIVES: To determine the effect of the protease inhibitors ritonavir, nelfinavir and indinavir on the P-glycoprotein (P-gp)-mediated transport of saquinavir in Caco-2 cell monolayers. To study the modulation of P-gp function in human lymphocytes by saquinavir, ritonavir, nelfinavir and indinavir. METHODS: We examined the effect of the protease inhibitors on P-gp function in human lymphocytes by using Rhodamine 123 (Rh 123; a fluorescent substrate of P-gp) by flow cytometry. Efflux of Rh 123 correlates with P-gp function and inhibition of P-gp results in dye retention. Verapamil, a P-gp modulator and inhibitor of active transport at 4 degrees C was used as a positive control. The transport of [14C]saquinavir (1 microM) across Caco-2 cell monolayers was investigated, alone and in the presence of verapamil and ketoconazole (500 microM) and the protease inhibitors at 100 microM. Caco-2 cells are an in vitro model of the intestinal epithelium that is widely used for the study of P-gp function. The transport of saquinavir was determined in both the apical to basolateral (AP-BL) and basolateral to apical (BL-AP) directions. RESULTS: Saquinavir and ritonavir (10 microM) markedly inhibited Rh 123 efflux with an increase in fluorescence intensity similar to that obtained with verapamil. A small but statistically significant increase in fluorescence intensity was observed with nelfinavir; however indinavir did not modulate Rh 123 efflux. In Caco-2 cells the apparent permeability coefficient for BL-AP efflux of saquinavir exceeded that for AP-BL efflux by a factor of 26: this is indicative of an active efflux pump. Known P-gp modulators caused a decrease in BL-AP efflux and an increase in AP-BL transport. The protease inhibitors displayed some P-gp modulation with ritonavir having the most potent effect. CONCLUSIONS: We have demonstrated that saquinavir is a substrate for P-gp and that ritonavir, nelfinavir and indinavir modulate P-gp function in both human lymphocytes and Caco-2 cells.

Saquinavir pharmacokinetics alone and in combination with nelfinavir in HIV-infected patients.

OBJECTIVE: To investigate the pharmacokinetics of saquinavir (SQV) hard gel when administered alone and in combination with nelfinavir (NLF) to HIV-positive patients. DESIGN: Six patients receiving triple therapy (dual nucleoside plus SQV 600 mg three times daily) were studied. On the first study day blood samples were drawn for assay of SQV. Prior to the second study day, patients received their usual medication plus NLF 750 mg three times daily for 2 days. METHODS: Blood samples were obtained at times 0, 1, 2, 4, 6 and 8 h after dosing on study days 1 and 2. Following centrifugation, separated plasma was heated at 58 degrees C for at least 30 min to inactivate HIV and stored at -80 degrees C until analysis using high performance liquid chromatography. RESULTS: The geometric mean Cmax and AUC0-8 h on the first study day were 253 ng/ml (range, < 25-1200 ng/ml) and 1106 ng/ml.h (range, < 100-3479 ng/ml.h), respectively, and on the second study day were 1204 ng/ml (range, 379-2755 ng/ml) and 5472 ng/ml.h (range, 1434-12,538 ng/ml.h), respectively. The geometric mean ratio for Cmax was 4.75 and for AUC0-8 h was 4.94. CONCLUSIONS: NLF increases the oral bioavailability of SQV (hard gel) approximately fivefold. For some patients the addition of NLF to SQV will increase the drug levels from subtherapeutic to the therapeutic range. In one of our patients the addition of NLF resulted in SQV levels that were much higher than previous work suggests are necessary for maximum antiviral effect. The variability in SQV concentrations both at baseline and following addition of NLF suggest that dosing may best be adjusted by individual therapeutic drug monitoring.

Differences in the intracellular accumulation of HIV protease inhibitors in vitro and the effect of active transport.

OBJECTIVES: To investigate the intracellular accumulation of HIV protease inhibitors (PI) and to assess the effect of active transport on this accumulation. METHODS: CEM cells were incubated with a PI for 18 h and the intracellular concentration determined using cell number and radioactivity. The effect of active transport was investigated using cells expressing P-glycoprotein (CEM(VBL)) and cells expressing multidrug resistance-associated protein 1 (MRP1; CEM(E1000)). Incubations were also carried out at 4 degrees C and in the presence of 2-deoxyglucose plus rotenone to examine the effect of inhibiting active transport. RESULTS: Nelfinavir (NFV) accumulated to the greatest extent (> 80-fold) followed by saquinavir (SQV; approximately 30-fold), ritonavir (RTV; 3-7-fold) and finally indinavir (IDV; extracellular equivalent to intracellular). In CEM(VBL) cells there was a significant reduction in the intracellular accumulation of NFV, SQV and RTV and in CEM(E1000) cells there was reduced accumulation of SQV and RTV. Inhibition of active transport processes caused a reduction in SQV and RTV accumulation but had no effect on IDV accumulation in all cell types. NFV accumulation was increased in CEM(VBL) cells as a result of inhibition of active transport. CONCLUSIONS: Marked differences can be detected in the intracellular accumulation of HIV PI drugs in vitro. Both P-glycoprotein and MRP1 may play a role in limiting the intracellular concentration of the PI and active influx mechanisms may contribute to drug accumulation.

Saquinavir pharmacokinetics alone and in combination with ritonavir in HIV-infected patients.

OBJECTIVE: The most important hepatic enzyme involved in the metabolism of protease inhibitors is cytochrome P450 3A4 (CYP3A4). Ritonavir (RIT) is a potent inhibitor of CYP3A4 and inhibits saquinavir (SQV) metabolism in healthy volunteers. In this study we investigated the kinetics of SQV when administered alone and in combination with RIT in HIV-infected patients. DESIGN: SQV pharmacokinetics were determined in seven patients who had advanced HIV disease. Steady-state SQV profiles were obtained on two occasions following treatment with SQV 600 mg three times daily alone and when administered with RIT 300 mg twice daily. METHODS: Blood samples were obtained at times 0, 1, 2, 4, 6 and 8 h post-dosing. Following centrifugation, separated plasma was heated at 58 degrees C for at least 30 min to inactivate HIV and stored at -80 degrees C until analysis using high performance liquid chromatography. RESULTS: For patients treated with SQV alone there was a 12-fold variability in the area under the SQV concentration-time curve (AUC0-8h) ranging from 293 to 3446 ng.h/ml. When combined with RIT there was a marked increase in the maximum plasma concentration of SQV [median (range), 146 (57-702) versus 4795 (1420-15810) ng/ml; approximately 95% confidence interval (CI), 2988-6819; P = 0.0006, Mann-Whitney U test]. The AUC0-8h for SQV was also significantly increased in the presence of RIT [median (range), 470 (29-3446) versus 27,458 (7357-108,001) ng.h/ml; approximately 95% CI, 16,628-35,111; P = 0.0006]. CONCLUSIONS: For some patients, administration of SQV 600 mg three times daily results in very low SQV plasma levels and possibly little antiviral effect. Combination of SQV with RIT results in a significant drug interaction mediated by enzyme inhibition which exposes patients to very high SQV concentrations and potential toxicity. If combination therapy with SQV plus RIT is considered then the dose of SQV should be greatly reduced.

The pharmacokinetics of combination therapy with nelfinavir plus nevirapine.

OBJECTIVE: To investigate the pharmacokinetics of nelfinavir (NFV) administered alone and in combination with nevirapine (NVP) to HIV-positive patients. DESIGN: Seven patients with advanced HIV disease received dual nucleoside analogues in addition to NFV (750 mg three times daily) and subsequently NVP (200 mg daily for 2 weeks followed by 200 mg twice daily) as salvage therapy. On the first study day (day 3), blood samples were taken for assay of NFV. The second study day followed the introduction of NVP for 3 weeks. METHODS: Blood samples were obtained at 0, 1, 2, 3, 4, 6 and 8 h after dosing on both study days. Separated plasma was heated to 58 degrees C for 30 min to inactivate HIV and stored at -80 degrees C until analysis by high performance liquid chromatography for both NFV and NVP. RESULTS: The geometric mean NFV area under the concentration-time curve to 8 h (AUC0-8h) was 23.4 microg x h/ml (range, 13.5-49.2) and 11.6 microg x h/ml (range, 6.6-23.2) on the first and second study days, respectively. The geometric mean ratio was 0.49 (95% confidence interval, 0.33-0.72; P = 0.016). This represented a 50% reduction in plasma NFV concentrations. Maximum and minimum concentrations were also reduced during NVP therapy (from 4.4 to 2.5 microg/ml and from 1.7 to 0.8 microg/ml, respectively). Time to maximum concentration was reduced from 4 to 2 h. NVP concentrations were determined with a maximum concentration of 5.4 microg/ml at 4 h. CONCLUSIONS: NVP is currently being used in combination therapy with protease inhibitors for antiretroviral-experienced patients in the setting of treatment failure. This study demonstrates that when patients are coadministered NVP there is a 50% reduction in the plasma AUC of NFV. Although the mean trough concentrations of NFV remained above the stated minimum effective concentration of 0.4 microg/ml, there is nevertheless concern that some patients will fall below this value when NVP is added to treatment regimens. In the absence of therapeutic drug monitoring we suggest that an increase in the standard NFV dosage of 750 mg three times daily will be required to ensure satisfactory NFV plasma concentrations, thereby maintaining antiviral efficacy.

Penetration of efavirenz into the male genital tract: drug concentrations and antiviral activity in semen and blood of HIV-1-infected men.

Efavirenz is a potent non-nucleoside reverse transcriptase inhibitor, licensed for the treatment of HIV-1. Data on sanctuary site penetration are limited. Therefore, we measured efavirenz concentrations in the blood and semen of 19 HIV-1-positive men and found concentrations in seminal plasma averaged 10% of those in blood plasma. Furthermore, seminal plasma viral loads were suppressed by 24 weeks of therapy in all patients. These data suggest that efavirenz-containing regimens have antiviral activity within the male genital tract.

The effect of zidovudine dose on the formation of intracellular phosphorylated metabolites.

OBJECTIVES: Zidovudine (ZDV) requires intracellular phosphorylation to ZDV triphosphate (ZDV-TP) prior to the inhibition of HIV replication. The effect of ZDV dose on the formation of intracellular phosphorylated metabolites may help define the optimum daily dose of ZDV, which is still unknown. DESIGN AND METHODS: The plasma and intracellular phosphorylated metabolite concentrations of ZDV were determined over a 12 h period following oral administration of 100 and 300 mg ZDV to 10 HIV-seropositive patients at steady state during two dosing regimens (i.e., 100 mg three times daily and 300 mg twice daily). The intracellular ZDV phosphates, ZDV monophosphate (ZDV-MP), ZDV diphosphate (ZDV-DP) and ZDV-TP were measured in peripheral blood mononuclear cells using a combination of high-performance liquid chromatography and radioimmunoassay. RESULTS: There was a greater than threefold increase in maximum plasma concentration (Cmax) following 300 mg ZDV when compared with 100 mg ZDV (mean +/- SD, 2.59 +/- 0.52 versus 0.70 +/- 0.14 mumol/l). The area under the concentration time curve (AUC0-12 h) was also significantly increased (4.59 +/- 0.79 versus 1.42 +/- 0.51 mumol/l x h) following 300 mg ZDV dose. For total intracellular ZDV phosphate metabolites the AUC0-12 h was doubled (7.64 +/- 3.67 versus 3.71 +/- 1.83 pmol/10(6) cells x h) in patients taking 300 mg ZDV compared with 100 mg. The AUC0-12 h for ZDV-MP was significantly increased at the higher dose (6.47 +/- 3.14 versus 2.77 +/- 1.70 pmol/10(6) cells x h), whereas the active moiety ZDV-TP was variable and not significantly different (0.42 +/- 0.42 versus 0.61 +/- 0.81 pmol/10(6) cells x h) following 100 and 300 mg ZDV. CONCLUSIONS: Administration of 100 mg ZDV orally produces significantly less of the potentially toxic metabolite, ZDV-MP, and comparative, although variable, concentrations of the active metabolite ZDV-TP when compared with 300 mg ZDV orally. This finding supports clinical data indicating the efficacy of low-dose (300 mg daily) ZDV. The measurement of intracellular phosphorylated metabolites advances our understanding of the clinical pharmacology of ZDV.

P-Glycoprotein and transporter MRP1 reduce HIV protease inhibitor uptake in CD4 cells: potential for accelerated viral drug resistance?

BACKGROUND: The multidrug transporters P-glycoprotein (P-gp) and MRP1 are functionally expressed in several subclasses of lymphocytes. HIV-1 protease inhibitors interact with both; consequently the transporters could reduce the local concentration of HIV-1 protease inhibitors and, thus, influence the selection of viral mutants. OBJECTIVES: To study the effect of the expression of P-gp and MRP1 on the transport and accumulation of HIV-1 protease inhibitors in human lymphocytes and to study the effects of specific P-gp and MRP1 inhibitors. METHODS: The initial rate and the steady-state intracellular accumulation of radiolabelled ritonavir, indinavir, saquinavir and nelfinavir was measured in three human lymphocyte cell lines: control CEM cells, CEM-MDR cells, which express 30-fold more P-gp than CEM cells, and CEM-MRP cells, which express fivefold more MRP1 protein than CEM cells. The effect of specific inhibitors of P-gp (GF 120918) and MRP1 (MK 571) was also examined. RESULTS: Compared with CEM cells, the initial rates of uptake and the steady-state intracellular concentrations of all protease inhibitors are significantly reduced in CEM-MDR cells. The intracellular concentrations of the protease inhibitors are increased upon co-administration with GF 120918, in some cases to levels approaching those in CEM cells. The intracellular concentrations of the protease inhibitors are also significantly reduced in CEM-MRP cells. Co-administration with MK -571 can partially overcome these effects. CONCLUSIONS: The overexpression of multidrug transporters significantly reduces the accumulation of protease inhibitors at this major site of virus replication, which, potentially, could accelerate the acquisition of viral resistance. Targeted inhibition of P-gp may represent an important strategy by which this problem can be overcome.

Interaction of sildenafil and indinavir when co-administered to HIV-positive patients.

OBJECTIVES: The prevalence of erectile dysfunction in HIV-infected men is estimated to be 33%. Sildenafil citrate (Viagra; Pfizer Ltd, Sandwich, Kent, UK) is the first oral drug for this condition. Since sildenafil and the protease inhibitors are both metabolized by, and act as inhibitors of cytochrome P450 3A4, we evaluated the pharmacokinetics of the combination sildenafil plus indinavir in HIV-infected patients. DESIGN AND METHODS: Six patients at steady state in treatment with indinavir participated in the study. On the first day blood samples for indinavir assay were drawn at times 0, 1, 2, 3, 4, 6 and 8 h after dosing. On the second study day patients received a single dose of 25 mg of sildenafil in addition to their routine morning medication. Blood samples were taken as described. Separated plasma was stored at -80 degrees C until analysis by high performance liquid chromatography. In a parallel study, the effect of indinavir, ritonavir, saquinavir and nelfinavir on the in vitro hepatic metabolism of sildenafil was assessed. RESULTS: The geometric mean area under the concentration curve for 0-8 h (AUC0-8h) and maximum plasma concentration (Cmax) for indinavir were 19.69 microg/ml h (range, 9.19-31.99 microg/ml h) and 7.02 microg/ml (range, 2.33-16.17 microg/ml), respectively, on the first study day. In the presence of sildenafil, the mean AUC0-8h and Cmax of indinavir were 22.37 microg/ml h [range, 10.08-37.25 microg/ml h; 95% confidence interval (CI) for difference between means, -15 to 13.25) and 9.11 microg/ml (range, 3.41-22.78 microg/ml; 95% CI, -13 to 6.37), respectively. The geometric mean AUC0-8h and Cmax for sildenafil were 1631 ng/ml h (range, 643-2970 ng/ml h) and 384 ng/ml (range, 209-766 ng/ml) respectively. The AUC for sildenafil was 4.4 times higher than data from historical controls given either 50 mg or 100 mg of sildenafil and dose normalized to 25 mg. Indinavir was a potent inhibitor of sildenafil hepatic metabolism in vitro [concentration producing 50% inhibition of control enzyme activity (IC50) = 0.39 +/- 0.17 microM, mean +/- SD]. CONCLUSIONS: Co-administration of sildenafil 25 mg did not significantly alter the plasma indinavir levels. However, plasma sildenafil AUC was markedly increased in the presence of indinavir compared with historical controls. From the in vitro data, the mechanism of increase is indinavir inhibition of the hepatic metabolism of sildenafil. The magnitude of this interaction suggests a lower starting dose of sildenafil may be more appropriate in this clinical setting.