INTRODUCTION

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Amprenavir (USAN approved, VX-478, 141W94; Glaxo Wellcome Inc., Research Triangle Park, N.C.) is a new human immunodeficiency virus type 1 (HIV-1) protease inhibitor which has potent in vitro and in vivo activity for HIV (2). In vitro data indicate that amprenavir is extensively metabolized by the microsomal P450 enzyme, CYP3A4, to oxidized products (8, 28, 38). In humans, less than 2% of an oral dose of amprenavir appears in urine as unchanged drug (38).

The CYP3A4 isoenzyme is the major route of metabolism for many drugs, including all of the currently available protease inhibitors (9, 10, 19, 24). The rifamycin antibiotics rifampin and rifabutin are well-known inducers of this isoenzyme (16, 18, 23, 24, 31; J. A. Smith, T. C. Hardin, T. F. Patterson, M. G. Rinaldi, and J. R. Graybill, Abstr. 2nd Natl. Conf. Hum. Retrovir. Relat. Infect., abstr. 126, p. 77, 1995), although rifabutin is believed to cause substantially less isoenzyme induction than rifampin (16, 23). In addition, the multidrug transporter, P-glycoprotein contributes to elimination of the protease inhibitors, and rifampin increases the activity of this transporter (17, 20, 35, 37). The potential for an interaction between amprenavir and these rifamycin antibiotics is clinically important because HIV-infected patients may receive rifampin or rifabutin for the prevention and treatment of opportunistic infections caused by mycobacteria (4, 6). The prevalence of infection caused by Mycobacterium tuberculosis, including multi drug-resistant tuberculosis, has increased with the emergence of AIDS (6). This has made treatment difficult, because rifampin induces metabolism of the available HIV-1 protease inhibitors and reduces the mean area under the curve (AUC) for individual agents by 35 to 92% (4, 6). Guidelines from the Centers for Disease Control and Prevention recommend that rifampin not be used with most protease inhibitors, and if rifabutin is used in place of rifampin, that it be given at a reduced dose (6). This study was undertaken to determine if a pharmacokinetic interaction exists when amprenavir and rifabutin or rifampin are coadministered.

The erythromycin breath test (ERMBT) is a measure of hepatic CYP3A4 activity (14, 36; ERMBT assay product information, Metabolic Solutions Inc., Nashua, N.H.). The inclusion of the ERMBT in this study was intended to evaluate the following questions. (i) Is amprenavir an inhibitor of hepatic CYP3A4 in vivo? (ii) What are the effects of rifabutin and rifampin on CYP3A4 activity as measured by the ERMBT? (iii) Do the results of the ERMBT help explain the pharmacokinetics of amprenavir when administered alone and in combination with rifabutin and rifampin?

	MATERIALS AND METHODS

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Subjects. Nonsmoking men, aged 18 to 55 years, were eligible for this study, which was approved by the Committee on the Conduct of Human Research at Virginia Commonwealth University (VCU). Each subject gave written informed consent. A complete medical history; physical examination that included vital signs; and routine laboratory tests that included a 13-test chemistry screen, complete blood count with differential, urinalysis (dipstick), urine drug screen for illicit controlled substances, test for HIV antibiodies, and electrocardiogram were completed for each subject. Subjects were ineligible if they had a clinically significant abnormality at the screening evaluation, had donated blood within the past month, were taking concomitant medication(s) which could not be withheld for the duration of the study, or had a prior adverse reaction to rifabutin, rifampin, or another rifamycin antibiotic. Subjects were instructed to use a barrier method of contraception (condoms) while enrolled in the study and for a minimum of 1 month after administration of their last dose of study drugs. Additionally, subjects abstained from taking concomitant medications and from consuming alcohol from 48 h before each treatment day until discharge from the study center. The same restrictions were placed on consumption of grapefruit and grapefruit juice. Tea, coffee, chocolate, and other beverages and foods containing methyl xanthines were prohibited on each pharmacokinetic sampling day.

Experimental design and procedures. This was an open-label, parallel-group, three-period study conducted at the School of Pharmacy Center for Drug Studies, VCU/Medical College of Virginia Campus. Following the screening evaluation, the study consisted of a treatment phase of up to 5 weeks' duration and a follow-up evaluation comprising up to four separate visits over a 3-month period. The screening evaluation was scheduled within 14 days before administration of the first dose of study drug. Subjects eligible after screening were randomized to two dosing cohorts (dosing cohort 1 [DC1] and DC2) and received the following treatments.

(i) DC1. Subjects in DC1 were treated as follows: dosing days 1 to 4 (treatment 1), amprenavir (1,200 mg twice daily) for 31/2 days, followed by a 7-day washout period; dosing days 5 to 18 (treatment 2), rifabutin (300 mg every morning) for 14 days; dosing days 19 to 28 (treatment 3), amprenavir (1,200 mg twice daily) plus rifabutin (300 mg every morning) for 10 days.

(ii) DC2. Subjects in DC2 were treated as follows: dosing days 1 to 4 (treatment 1), amprenavir (1,200 mg twice daily) for 31/2 days, followed by a 7-day washout period; dosing days 5 to 18 (treatment 4), rifampin (600 mg every morning) for 14 days; dosing days 19 to 22 (treatment 5), amprenavir (1,200 mg twice daily) plus rifampin (600 mg every morning) for 4 days.

Administration and analysis of the ERMBT. The ERMBT (Metabolic Solutions, Inc.) was administered within 1 week before treatment period 1 (to establish baseline), at 2 h after the final dose of amprenavir (treatment 1), at 2 h after the morning dose of either rifampin or rifabutin at 7 and 14 days, at 2 h after the final dose of combined treatment with amprenavir and rifampin or rifabutin, and at the follow-up evaluation. For each ERMBT, subjects received an intravenous injection containing 3 µCi of [N-methyl-¹⁴C] erythromycin as a 1-min bolus infusion according to the manufacturer's directions. Twenty minutes later, subjects exhaled through a straw into a vial containing 20 ml of a 50:50 hyamine-ethanol solution containing thymolphthaline until there was a color change, from blue to clear, indicating the trapping of 2 mmol of CO₂.

Sample procurement and assay of plasma and urine samples for amprenavir, rifampin, and rifabutin and their respective metabolites. On dosing days 4 and 28 (DC1) or 22 (DC2), blood was obtained for determination of amprenavir concentrations in plasma on 15 separate occasions during the respective treatment periods: 0 (taken 5 min before dosing), 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, and 12 h after drug administration. On dosing days 18 and 28 (DC1) or 22 (DC2), blood samples to determine concentrations of rifabutin, rifampin, and their 25-O-desacetyl metabolites in plasma were drawn at the same interval as amprenavir blood samples, but with additional samples for amprenavir, rifampin, rifabutin, and metabolites at 16 and 24 h postdosing. Blood samples were collected by venipuncture or peripheral venous catheter. Each blood sample for amprenavir analysis was collected in a prelabeled 4-ml lavender-stoppered Vacutainer tube (containing freeze-dried K₂EDTA). Blood for analysis of the concentrations of rifabutin, rifampin, and their metabolites in plasma was drawn in 5-ml green-stoppered tubes containing freeze-dried sodium heparin. Ascorbic acid was added to prevent oxidation of rifampin and rifabutin. The exact time that each blood sample was drawn was recorded. Once separated, the plasma was stored in a polypropylene tube in an upright position at 20°C until shipment for analysis.

Pharmacokinetic analysis. The observed peak concentrations in plasma at steady state (C_max,ss) of amprenavir, rifabutin, and rifampin and the time to C_max,ss (T_max,ss) were obtained by inspection of the individual plasma concentration-time data. Individual estimates of the apparent terminal elimination rate constant (k) for each drug were obtained by log-linear regression of the terminal portions of the plasma concentration-time curves. Half-lives were calculated as 0.693/k. The steady-state AUC (AUC_ss) from time zero to the last quantifiable sample at 24 for rifampin and rifabutin or at 12 h for amprenavir was calculated by the linear trapezoidal method. The AUC from 0 h to infinity was calculated by adding C_last/k to AUC_ss. The apparent total clearance from plasma at steady state (CL/F) was calculated as dose/AUC. Similar formulae were used to determine the pharmacokinetic parameters for the metabolites (with the exception of CL/F). For each metabolite, the ratio of the metabolite AUC to that of the parent drug was also calculated based upon the AUC.

Statistical analysis. Data are presented as mean values ± standard deviations. Pharmacokinetic parameters other than T_max, were log transformed, and analysis of variance with treatment as the fixed effect and subject as the random effect was performed on the calculated parameters. The geometric least-squares (LS) mean ratio and its 90% CI were calculated for each pharmacokinetic parameter and for the results of the ERMBT. Two one-sided t tests (90% CI) were performed on each dosing cohort to compare treatments. Pearson's correlation coefficient was calculated to assess the relationship of ERMBT to pharmacokinetic parameters of amprenavir, rifabutin, and rifampin. Differences and associations were interpreted as statistically significant when P was 0.05.

	RESULTS

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Subject characteristics. Twenty-four healthy, HIV-seronegative, male subjects were enrolled in the study (19 Caucasian, 4 African-American, 1 Asian), with 12 subjects in each dosing cohort. The subjects' ages ranged from 18 to 49 years (mean, 27.3 years), and their weights ranged from 58.8 to 100 kg (mean, 75.9 kg). Demographic characteristics were similar between the two dosing cohorts.

Tolerability of study medications. All 24 subjects completed treatment period 1 (amprenavir alone). The most common adverse events that occurred during treatment with amprenavir alone in both cohorts were nausea, oral numbness, dizziness, diarrhea, and headache.

Pharmacokinetics. Twenty-four subjects were included in the pharmacokinetic analysis of amprenavir alone, 11 subjects for rifabutin alone and 11 for rifampin alone. There were 11 subjects included in the pharmacokinetic analysis of amprenavir and rifampin when given in combination, but there were only 6 subjects included in the pharmacokinetic analysis when amprenavir was given in combination with rifabutin.

Amprenavir. Table 1 provides the summary pharmacokinetic parameters, geometric LS mean ratio, and the 90% CI estimates for amprenavir pharmacokinetic parameters, alone and in combination with rifabutin and rifampin. There were no statistically significant differences in the pharmacokinetics of amprenavir when coadministered with rifabutin, but only 6 subjects were available for a full pharmacokinetic profile. In contrast, rifampin resulted in statistically significant decreases in AUC_ss, C_max,ss and minimum concentration in plasma (C_min,ss) (82, 70, and 92%, respectively) of amprenavir and a 5.45-fold increase in amprenavir CL/F. Figure 1 illustrates the effects of rifampin and rifabutin on the mean concentration-time profile of amprenavir.

View larger version (13K): [in this window] [in a new window]   FIG. 1.   Mean amprenavir concentrations in plasma following 1,200 mg administered alone (open diamond), following 2 weeks of rifampin (600 mg once daily) (open triangle), and following 2 weeks of rifabutin (300 mg once daily) (closed circle).

Rifabutin. Table 2 provides the summary pharmacokinetic data for rifabutin alone and when administered with amprenavir. There were statistically significant 2.93-, 2.19-, and 3.71-fold increases in rifabutin AUC_ss, C_max,ss and C_min,ss when coadministered with amprenavir. There was a 66% decrease in rifabutin CL/F. The median T_max,ss following administration of the combined treatment was 1.0 h longer than after administration of rifabutin alone. There was a 2.51-fold increase in the amount of rifabutin excreted in the urine as unchanged parent drug when it was administered with amprenavir. Figure 2 illustrates the effect of amprenavir on the mean concentration-time profile of rifabutin.

View larger version (14K): [in this window] [in a new window]   FIG. 2.   Mean rifabutin and 25-O-desacetylrifabutin concentrations in plasma following doses 300 mg once daily given for 2 weeks (closed squares and open squares, respectively) and following coadministration with amprenavir (1,200 mg) (closed triangles and open triangles, respectively).

25-O-Desacetylrifabutin. Table 2 provides the summary pharmacokinetic data for 25-O-desacetylrifabutin, when rifabutin was administered alone and in combination with amprenavir. When coadministered, the AUC_ss, C_max,ss and C_min,ss of 25-O-desacetylrifabutin were increased by 13.35-, 7.39-, and 32.9-fold, respectively, compared to rifabutin alone. There was a 4.27-fold increase in the AUC ratio (AUC of 25-O-desacetylrifabutin to that of rifabutin). There was a 23% decrease in CL_R when rifabutin was administered with amprenavir. The percent dose of rifabutin excreted in the urine as 25-O-desacetylrifabutin was 9.5-fold greater following the administration of rifabutin with amprenavir. Figure 2 illustrates the effect of amprenavir on the mean concentration-time profile of 25-O-desacetylrifabutin.

Rifampin. There were no significant differences in AUC_ss, C_max,ss and CL/F when rifampin was given alone and in combination with amprenavir (Table 3). There was a 22% decrease in CL_R and an 18% decrease in the amount of rifampin excreted in the urine as parent drug following coadministration with amprenavir.

25-O-Desacetylrifampin. There were no statistically significant differences in 25-O-desacetylrifampin pharmacokinetics when rifampin was administered alone or in combination with amprenavir (data not shown).

Erythromycin breath test. Amprenavir treatment reduced the LS mean ratio for the ERMBT to 17% of baseline for both dosing cohorts, and both rifampin and rifabutin significantly increased the ERMBT at 1 and 2 weeks of treatment. At follow-up, the ERMBT mean ratios had returned to baseline for both dosing cohorts. Figure 3 illustrates the ERMBT results for the rifampin group. Results for the rifabutin group were similar (data not shown), except for the combination treatment regimen of rifabutin plus amprenavir, in which the ERMBT result was significantly lower.

View larger version (21K): [in this window] [in a new window]   FIG. 3.   Individual ERMBT results at baseline; following administration of amprenavir (1,200 mg twice daily), administration of rifampin (600 mg once daily) for 7 and 14 days, and administration of the combination of amprenavir and rifampin; and at follow-up.

	DISCUSSION

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The pharmacokinetics of amprenavir, rifabutin, and rifampin when administered alone are in agreement with those reported in previous studies (1, 2, 5, 29). The effects of rifabutin and rifampin on amprenavir pharmacokinetics are consistent with observations for other protease inhibitors, indicating that amprenavir is a substrate for CYP3A4 (8, 28), rifabutin is a less potent inducer of metabolic clearance for protease inhibitors than is rifampin (4, 6, 10, 16, 23, 24), and rifabutin and its 25-O-desacetyl metabolite are metabolized by CYP3A4 (15, 32).

Rifabutin and amprenavir. Amprenavir significantly decreased clearance of rifabutin and 25-O-desacetylrifabutin. These results are similar to the effects of ritonavir(5) and other protease inhibitors (4, 6, 10, 24). on the pharmacokinetics of rifabutin. An assessment of the effect of rifabutin on the pharmacokinetics of amprenavir is confounded by the poor tolerability of the combination and the relatively few subjects who completed combination therapy. Although there is a 15% mean reduction in amprenavir AUC_ss, only six subjects were able to provide full pharmacokinetic data. However, even a true difference of this magnitude is unlikely to be clinically important.

Rifampin and amprenavir. The coadministration of amprenavir and rifampin resulted in significant changes in the pharmacokinetics of amprenavir, including an 82% decrease in the AUC_ss and an increase of greater than fivefold in amprenavir CL/F. This most likely reflects induction of hepatic and intestinal CYP3A4 by rifampin, and possibly enhancement of P-glycoprotein transport, resulting in an increase in clearance of amprenavir. Decreases in amprenavir concentrations in plasma of the magnitude observed in this study are of probable clinical relevance as trough concentrations are below the 95% inhibitory concentration for HIV isolates (2). Of the steady-state pharmacokinetic parameters for the HIV-1 protease inhibitors, C_min,ss has been shown to be the best predictor of antiviral response, as well as being associated with the development of resistance (7, 21, 30). Because rifampin markedly increases amprenavir's metabolism, coadministration of these drugs is contraindicated (4, 6).

ERMBT results. Amprenavir reduced hepatic CYP3A4 activity, as measured by the ERMBT, to 17% of baseline. Similar results were observed in our other studies with amprenavir (3, 25). Compared with the baseline, the mean ERMBT increased at 7 and 14 days of rifabutin and rifampin treatment by 181 and 187% and 164 and 156%, respectively. It appears that induction is not greater following 2 weeks of rifamycin therapy than following 1 week. These results are similar to the findings of a prior investigation with rifampin that revealed a mean 186% increase in the ERMBT (11). The effects of rifabutin on the ERMBT have not been previously reported.