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In vivo inhibition of human immunodeficiency virus (HIV) replication via combination antiretroviral therapy can be achieved with drugs targeting different HIV enzymes (i.e., reverse transcriptase and protease) or with drugs targeting the same viral enzyme (i.e., reverse transcriptase or protease). Although the combined use of HIV reverse transcriptase and protease inhibitors in HIV-infected individuals has been associated with significant virologic and clinical benefits (2, 3, 8, 15; British HIV Association Guidelines for the Treatment of HIV Disease with Antiretroviral Therapy [http://www.bhiva.org] and Department of Health and Human Services and Henry J. Kaiser Family Foundation Panel on Clinical Practices for the Treatment of HIV Infection, Guidelines for the Use of Antiretroviral Agents in HIV-Infected Adults and Adolescents [http://www.hivatis.org]), the high anti-HIV potency of the protease inhibitors provides a rationale for examining the clinical utility of dual protease inhibitor combinations.

The success of a dual protease inhibitor combination approach, however, may depend on potential drug-drug interactions. All of the currently available HIV protease inhibitors are metabolized in the liver and gastrointestinal tract, primarily by the cytochrome P450 system (1). In addition, all protease inhibitors are substrates for transport by the P-glycoprotein drug transport protein (10, 11). The actions of each protease inhibitor in a dual combination regimen on the pharmacokinetics of the partner protease inhibitor must be assessed to determine whether drug-drug interactions that affect plasma drug concentrations will occur and whether two specific protease inhibitors can be safely coadministered.

Amprenavir (Agenerase, formerly 141W94) is a potent HIV protease inhibitor that was originally synthesized using a structure-based drug design process (9). The mean 50% inhibitory concentration of amprenavir against 334 HIV clinical isolates is 29 nM (19). Amprenavir binds to proteins in normal human plasma or serum to the extent of ~90%, with the greatest degree of binding to ₁-acid-glycoprotein (AAG) (89%) and albumin (42%) (12). The metabolism of amprenavir, like that of the other approved HIV protease inhibitors, appears to be primarily dependent upon the 3A4 isozyme of the hepatic cytochrome P450 system (CYP3A4), based on in vitro and in vivo studies (6; J. Woolley, S. Studenberg, C. Boehlert, G. Bowers, A. Sinhababu, and P. Adams, Abstr. 37th Intersci. Conf. Antimicrob. Agents Chemother., abstr. A-60, 1997). These and other drug interaction studies have shown that amprenavir inhibits CYP3A4 to a degree comparable to that exhibited by indinavir and nelfinavir.

We report here the steady-state pharmacokinetics of amprenavir administered alone and as part of three dual protease inhibitor combinations with indinavir, nelfinavir, or saquinavir soft gel capsule (sgc) formulation in protease inhibitor-naïve, HIV-infected adults (Glaxo Wellcome protocol PROA2001). The pharmacokinetics of each partner protease inhibitor used in combination with amprenavir were also assessed and compared with historical monotherapy data for each drug as indicated: nelfinavir (B. Kerr, personal communication), indinavir (Merck & Co., complete prescribing information for indinavir [Crixivan]), and saquinavir (16). Preliminary data from this study have been previously presented (B. M. Sadler, J. Eron, J. Wakeford, G. Pagano, C. Rawls, J. McCrea, K. Mazina, and P. J. Deutsch, Abstr. 37th Intersci. Conf. Antimicrob. Agents Chemother., abstr. A-56, 1997; B. Sadler, C. Gillotin, G. E. Chittick, and W. T. Symonds, Abstr. 12th World AIDS Conf., abstr. 12389, 1998). This 3-week, phase I trial was part of a larger 48-week phase I-II study to explore the antiviral effect and evaluate the long-term safety and tolerability of amprenavir-containing dual protease inhibitor therapy (7). The pharmacokinetic interactions of ritonavir and amprenavir were studied separately (19).

Study population. The study entry criteria were per the work of Eron et al. (7). Briefly, subjects were enrolled in the study if they were 18 years of age and were HIV-seropositive adults with CD4⁺ cell counts of 200 cells/mm³ and plasma HIV RNA levels of >10,000 copies/ml. Subjects were excluded from the study if they had prior treatment with a protease inhibitor or received any antiretroviral therapy within 2 weeks prior to enrollment. Subjects with malabsorption, acute opportunistic infections, or standard laboratory values outside of prespecified ranges were excluded. Women of childbearing potential were required to have a negative serum human chorionic -gonadotropin test within 7 days of the start of dosing. Prior antiretroviral therapy (with drugs other than protease inhibitors) was permitted, but subjects were required to discontinue antiretroviral therapy 2 weeks prior to enrollment. All subjects provided written informed consent to participate in the study. All subjects were monitored for clinical adverse experiences and/or abnormal laboratory test findings throughout the study period, including the follow-up evaluation.

Study design and drug administration. A single-dose, phase IA interaction study of amprenavir and indinavir was conducted prior to the start of the 3-week, phase I multiple-dose study of the dual protease inhibitor combinations. The single-dose study was carried out to determine the pharmacokinetics of amprenavir and indinavir when administered in combination, to assess whether pharmacokinetic interactions exist between amprenavir and indinavir when administered as a single-dose combination, and to determine the dose of indinavir to be used in the later multiple-dose phases (phases I and II) of this study. Subjects were given a single oral dose of indinavir (800 mg) and amprenavir (800 mg) simultaneously, after an overnight fast of at least 8 h and 1.5 h before a meal.

Plasma sampling. Blood samples were collected on the first day of dosing to determine the single-dose indinavir and amprenavir pharmacokinetic parameters (phase IA) and at the week 2 study visit to determine steady-state pharmacokinetic parameters for amprenavir, indinavir, nelfinavir, and saquinavir. Blood samples were collected at predose and then at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, and 8 h postdose. For amprenavir, blood was collected in EDTA-containing tubes, and for indinavir, nelfinavir, and saquinavir sgc, blood was collected in sodium heparin tubes. All plasma samples were stored frozen at 20°C until analysis. Plasma sampling for AAG was conducted at weeks 1, 2, and 3.

Safety evaluations. Safety and tolerability were evaluated at predose; day 1; and weeks 1, 2, 3, and 4 and every 4 weeks thereafter. Evaluations were conducted by physical examination, vital signs, electrocardiogram, hematology, clinical chemistry, urinalysis, and monitoring for clinical adverse experiences.

Assays for protease inhibitors. Concentrations of amprenavir in plasma were determined using cross-validated isocratic reversed-phase chromatography with fluorescence detection as previously described (18) and an automated high-performance liquid chromatography method with detection by tandem mass spectrometry. Concentrations of indinavir, saquinavir, and nelfinavir in plasma were analyzed by specific, validated high-performance liquid chromatography assays (22, 23; document no. RD1998/01807/00, RD1998/00615/00, and RD1998/01806/00, Glaxo Wellcome Inc.) with UV detection conducted by the following laboratories: Oneida Research Services, Whitesboro, N.Y. (saquinavir); BAS Analytics, West Lafayette, Ind. (indinavir); and PPD-Pharmaco, Richmond, Va. (nelfinavir and M8-nelfinavir). Calibration curves for indinavir in plasma were linear for the concentration range of 5 to 500 ng/ml. The intraday precision ranged from 1.8 to 6.3%, the interday precision was 1.8 to 2.8%, and the interday accuracy ranged from 91.2 to 94.5%. For saquinavir, calibration curves were linear for the concentration range of 10 to 350 ng/ml. Interday precision was 8.62%, and the interday accuracy ranged from 99.88 to 106.38%. Intraday precision was 8.02%, and the intraday accuracy ranged from 96.66 to 116.18%. The calibration curves for nelfinavir were linear for the concentration range of 0.05 to 10 µg/ml. The intraday precision was 1.53 to 7.21%, and the intraday accuracy was 16%, while the interday precision was 2.32 to 5.43%, and the interassay accuracy was 9.75%.

Pharmacokinetic analyses. Model-independent pharmacokinetic parameters for single and multiple oral dosing were calculated using WinNonlin Pro, version 1.5 (Scientific Consulting, Inc., Cary, N.C.). The peak plasma concentration (C_max), the time to reach peak plasma concentration (T_max), the peak plasma concentration at steady state (C_max,ss), and the time to reach maximum plasma concentration (T_max,ss) were observed from the individual plasma concentration-time data. The minimum drug concentration at steady state (C_min,ss) was calculated as (C₀ + C_t)/2, where C₀ is the plasma concentration before the last dose and C_t is the plasma concentration of the last steady-state dosing interval. The area under the plasma concentration-time curve (AUC_0t) from the time of the predose sample to the time of the last sample was calculated using the linear trapezoidal rule. When necessary, AUC_0t was extrapolated to steady-state (AUC_ss) by adding C_t/_z[1  e_z^(  t)] to AUC_0t, where t is the time of the last plasma concentration sample during the steady-state dosing interval, _z is the elimination rate constant, and  is the length of the steady-state dosing interval. The apparent total clearance (CL/F) was calculated as dose/AUC_ss.

Statistical analyses. Only descriptive statistical analysis was performed for phase IA. The mean amprenavir AUC and mean indinavir AUC_{08 h} and C_max were compared with their respective historical control values. The 95% confidence intervals (CI) of the means were calculated and not considered different from their historical values if the mean used as the reference fell within the limit of the 95% CI. The single-dose historical pharmacokinetic data for indinavir monotherapy was from the work of Yeh et al. (24); that for amprenavir monotherapy, in which a single dose of 900 mg was administered, was from two studies (17, 18). Values obtained from the two amprenavir studies were recalculated to reflect the AUC_{08 h} only and normalized to an 800-mg dose for comparison purposes in this study.

Subject demographics and accountability. This study was conducted between January 1997 and October 1998 at three centers in the United States. A total of 34 HIV-infected subjects were enrolled in phase I and randomized to receive study medication. Of these 34 subjects, 12 (all from one center) participated in phase IA and all 12 were included in the single-dose pharmacokinetic analysis. Of the 33 subjects who initiated study treatment in phase I (one subject randomized to the amprenavir-alone group withdrew from the study before receiving the first dose), 31 were included in the multiple-dose pharmacokinetic analysis. One of the subjects not included in the analysis withdrew consent at week 3, and the other subject was lost to follow-up at week 2.

Single-dose amprenavir-indinavir pharmacokinetic interactions (phase IA). Coadministration of amprenavir and indinavir produced an increase in the mean amprenavir AUC_{08 h} relative to the historical amprenavir monotherapy data: 25.32 versus 21.49 µg · h/ml (17, 18) (see Table 1). This change in mean amprenavir AUC_{08 h} represents an increase of 18% over that observed for amprenavir alone (18). Coadministration of amprenavir and indinavir produced a decrease in both indinavir AUC_{08 h} (35%) and C_max (23%) compared with historical indinavir monotherapy data (24). Only the decrease in indinavir C_max was statistically different from indinavir control data (see Table 2).

Phase I safety and tolerability. No serious adverse events occurred during the study. A full description of the safety findings is given in the work of Eron et al. (7).

Effect of multiple-dose indinavir, nelfinavir, or saquinavir sgc on amprenavir pharmacokinetics. The steady-state pharmacokinetic values obtained for amprenavir in dual combination with indinavir, nelfinavir, or saquinavir sgc and amprenavir alone after multiple oral doses are presented in Table 1. The GLS mean ratios for AUC_ss (0.68 [90% CI, 0.51 to 0.91]) and C_max,ss (0.63 [90% CI, 0.46 to 0.86]) between the saquinavir sgc-amprenavir combination and amprenavir alone are significantly different, as are the GLS means for AUC_ss between the indinavir-amprenavir combination and amprenavir alone, which increased by 33% (90% CI, 1.02 to 1.73; ratio, 1.33). The GLS mean ratio for C_min,ss of the nelfinavir-amprenavir combination was also significantly increased by 2.89-fold (90% CI, 1.52 to 5.48) compared to the amprenavir-alone treatment group.

Effect of multiple-dose amprenavir on indinavir, nelfinavir, or saquinavir sgc pharmacokinetics. The steady-state pharmacokinetics of indinavir, nelfinavir, and saquinavir, estimated from subjects treated with each of these protease inhibitors in dual combination regimens with amprenavir, are presented in Table 2. Also given in the table are historical values of steady-state pharmacokinetic parameters for each of the protease inhibitors administered as monotherapy. Compared with indinavir-alone historical data, amprenavir was found to decrease the AUC_ss, C_max,ss, and C_min,ss of indinavir by 38, 22, and 27%, respectively, and to increase the CL/F by 72% (Table 2). The decreases in indinavir AUC_ss, C_max,ss, and C_min,ss and the increase in CL/F produced by coadministration with amprenavir were statistically different from historical values (indinavir alone), as indicated by historical reference values falling outside the 95% CI range of the means of these pharmacokinetic parameters for the indinavir and amprenavir combination. None of the pharmacokinetic parameter values for either saquinavir sgc or nelfinavir obtained from the historical studies fell outside the 95% CI range observed in this study.

AAG, treatment, and amprenavir pharmacokinetic parameters. Analysis of covariance revealed that, of the factors evaluated (AAG, age, albumin, bilirubin, HIV risk factor, race, treatment, and weight), only serum AAG concentrations and protease inhibitor coadministration treatment had a statistically significant effect on amprenavir steady-state pharmacokinetic parameters. AAG concentrations significantly influenced amprenavir AUC_ss (P = 0.004) and C_max,ss (P = 0.026). Treatment also had a statistically significant effect on amprenavir AUC_ss (P = 0.004) and C_max,ss (P = 0.015).