INTRODUCTION

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Rifapentine is an orally administered rifamycin derivative that has antituberculous activity and that is similar to rifampin (11, 12, 16, 27). The MICs for sensitive strains are usually in the range of 0.03 to 0.12 mg/liter, and MICs for resistant strains are 8 mg/liter (15). Cross-resistance between rifapentine and rifampin is virtually complete (15). Cross-resistance between rifapentine and rifampin is virtually complete (15). Rifapentine has a longer elimination half-life than rifampin, allowing the possibility of less frequent (twice- or once-weekly) administration (17).

There have been no previous reports of the intrapulmonary concentrations or pharmacokinetics of rifapentine in humans. The purpose of this study was to compare the concentrations of rifapentine in plasma, alveolar cells (ACs), and epithelial lining fluid (ELF) of normal volunteers and to compare the drug's pharmacokinetics in these three compartments.

	MATERIALS AND METHODS

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Subjects. The protocol was approved by the Human Research Committee of the University of California, San Francisco. Written informed consent was obtained from each subject by an experienced research nurse. Subjects were required to be 18 to 45 years of age. If the subjects were female, they were required to be nonlactating and not pregnant and to agree to use adequate contraception (e.g., barrier methods or abstinence) during the study and for 2 weeks following completion of the study. Women using oral contraceptives were required to agree to use a barrier method in addition for 1 month following the study. Subjects who were lactating or pregnant, had a history of intolerance to rifamycin drugs or topical lidocaine, had clinically significant organ dysfunction, or were required to take chronic medications other than self-prescribed vitamins, birth control pills, or thyroid replacement therapy or who were smoking on a regular basis within 6 months of the study were excluded. Subjects who reported drug or alcohol dependence or who had psychiatric problems that would interfere with participation in the study were also excluded. Twenty subjects were women, and 10 subjects were men. The subjects' age, height, and weight (mean ± standard deviation [SD]) were 29.7 ± 6.2 years, 169 ± 9 cm, and 66.7 ± 13.8 kg, respectively. All subjects had normal renal function as measured by determination of the serum creatinine level (0.8 ± 0.1 mg/dl).

Study design. The investigation was prospective but not randomized or blinded. Thirty normal volunteers divided into six subgroups of five subjects each were assigned to undergo standardized bronchoscopy and bronchoalveolar lavage (BAL) (7, 10) at specified time intervals from 4 to 48 h following oral administration of a single dose of rifapentine. Rifapentine (600 mg) was administered by a research nurse in the Endoscopy Unit, followed by a timed bronchoscopy, as indicated in Table 1. All patients were observed for at least 30 min after taking the antibiotic for any signs of an allergic reaction. Bronchoscopy and BAL were performed at 4, 5, 7, 12, 24, and 48 h. Baseline data included the results of a medical history and physical examination; blood tests including a complete blood count with differential and a platelet count and alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total bilirubin, total protein, albumin, blood urea nitrogen, serum creatinine, and electrolyte level determinations; and urinalysis, including specific gravity, pH, albumin, glucose, ketone, and bilirubin level determinations, and microscopic examination of spun sediment. The postbronchoscopy assessment included a medical history and physical examination, a review of voluntarily described and observed adverse experiences, collection of a sample for antibiotic concentration determination, and a repeat of the laboratory testing of blood and urine. Major adverse reactions were defined as those that were fatal or life-threatening, resulted in permanent disability, required hospitalization, were the result of drug overdose, or suggested significant hazard to the subject.

Bronchoscopy and BAL. Bronchoscopy and BAL were performed in the Endoscopy Unit of the Moffitt-Long Hospitals. The blood pressure, respiratory rate, and heart rate of each subject were recorded before, at the completion of, and at approximately 1 h after bronchoscopy. Oxygenation was monitored by fingertip oximetry throughout the procedure. A 4% topical lidocaine gargle followed by a 4% topical lidocaine spray was used to prepare the subjects for the procedure. Four percent topical lidocaine was then applied to each side of the posterior pharynx, followed by the application of topical 1% lidocaine more distally. Systemic sedation was not used. A fiberoptic bronchoscope (Pentax FB-19H) was inserted into the right middle lobe. The instrument was in place for an average of 4.8 min (range, 3 to 9 min). Four 50-ml aliquots of normal saline were instilled into the right middle lobe, and each was immediately aspirated into a trap.

Specimen handling. All specimens were kept in ice until they were frozen. The blood was centrifuged, and the plasma was separated and then frozen until it was assayed.

Assay for rifapentine concentrations. The rifapentine concentration in plasma was measured by the laboratory at Hoechst Marion Roussel, Inc. (Kansas City, Mo.) by a high-pressure liquid chromatography method that was reported previously (18). Briefly, plasma proteins were precipitated with methanol containing internal standard (25-O-desacetyl rifampin). After centrifugation the supernatant was injected onto a 5-µm Primesphere C₁₈-HC column (2.0 by 150 mm; Phenomenex, Inc., Torrance, Calif.) preceded by a matching guard column. Analytes were eluted with a mobile phase consisting of 40% methanol, 25% acetonitrile, and 35% water containing 0.5% acetic acid. Drug peaks were detected at 480 nm. The validation statement provided by personal communication from an internal research and development report of 5 January 1996 (Marion Merrell Dow Inc., Kansas City, Mo.) stated the following: the assay was validated over the concentration range of 0.5 to 60 µg/ml. Two replicates of seven freshly prepared calibration standards (0, 0.5, 1.0, 15, 30, 45, and 60 µg/ml) and 30 replicates of four validation quality control sample pools (0.5, 1.0, 30, and 60 µg/ml) were assayed in each of three batches run over a period of 2 days. The intraday coefficients of variation ranged from 2.8 to 6.2% for rifapentine and 2.7 to 5.8% for 25-desacetyl rifapentine. The interday coefficients of variation ranged from 0.8 to 4.4% for rifapentine and 0.8 to 5.7% for 25-desacetyl rifapentine. The linearity of the assay (R²) ranged from 0.9993 to 0.9998.

Determination of ELF volume and concentration of antibiotics in ELF and ACs. The ELF volume was determined by the urea dilution method (29). The concentration of urea in serum was analyzed by the clinical laboratory at the University of California, San Francisco, by a coupled urease-glutamate dehydrogenase enzymatic method (32) modified by Boehringer Mannheim Corporation (Indianapolis, Ind.). Measurements were made at a fixed time interval that permitted automated analysis with a BM 747 analyzer (Boehringer Mannheim). The urea concentration in the BAL fluid supernatant was measured by a modified enzymatic assay (BUN kit UV-66; Sigma), and the results were read on a Spectronic 20D spectrophotometer (Milton Roy, Rochester, N.Y.). The proportions of the reagent to the specimen were changed from 3.0 ml/0.005 ml, as recommended by the manufacturer, to 2.5 ml/0.5 ml, as reported by Rennard et al. (29). Standard curves prepared in normal saline with concentrations ranging from 0.047 to 0.750 mg/dl were linear (r²  0.99). Controls with concentrations of 0.094 and 0.375 mg/dl were run with every standard curve used to assay the BAL fluid samples. If the values for the controls were not within 10% of the known value, the standard curve, controls, and BAL fluid sample assays were repeated.

Statistical analysis and modeling. Statistical analysis and database management were performed by using Prophet, version 6.0 (AbTech Corp., Charlottesville, Va.). Modeling was performed by using Prophet, version 4.1 (BBN, Inc., Cambridge, Mass.), on a Sun 10 Sparc Station (Sun Microsystems, Milpitas, Calif.). The linear regression analysis used the method of least-squares estimation. A P value of <0.05 was regarded as statistically significant. Prior to comparison of the data sets, tests for normality (Wilk-Shapiro test) and equality of variances (Levene's test) were performed. Parametric and nonparametric comparisons were performed by the Neuman-Keuls (all pairwise) or Kruskal-Wallis (unblocked data) and Friedman (blocked data) tests, respectively (34). A P value of <0.05 was regarded as statistically significant.

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	RESULTS

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None of the 30 subjects experienced a major adverse reaction. Systemic sedation was not required for any of the subjects. During the postbronchoscopy observation period, eight subjects (27%) experienced self-limited lightheadedness of unclear etiology, possibly a lidocaine effect. Transient shortness of breath, cough, fatigue, and headache were reported by one (3%), two (7%), four (13%), and one (3%) of the subjects, respectively. None of the subjects experienced postbronchoscopy fever. Fifteen of the 30 subjects (50%) experienced an elevation in the level of one of the liver enzymes, but the level returned to normal on retesting. Two of the subjects experienced borderline elevated (43 and 43 U/liter) concentrations of aspartate aminotransferase. One of the two subjects was retested, and the level returned to the normal range (<40 U/liter). Twelve of the subjects developed mildly abnormal total bilirubin concentrations (mean ± SD, 1.6 ± 0.3 mg/dl; range, 1.3 to 2.4 mg/dl). For all of these subjects the concentrations returned to normal on retesting.

Recovery of cells and ELF from BAL fluid. The number of cells recovered ranged from 8.52 × 10⁷ ± 2.67 × 10⁷ to 1.72 × 10⁸ ± 1.18 × 10⁸ cells/liter (Table 1), and the number of cells recovered was not significantly different for the six groups (P > 0.05). The most predominant cell types were monocytes/macrophages (range, 70.8% ± 21.7% to 89.9% ± 9.0%). The percentages of each cell type were compared among the six groups, and none of the percentages were significantly different (P > 0.05). The volumes of ELF ranged from 0.81 ± 0.48 to 1.21 ± 0.57 ml (Table 1) and were not significantly different among the six groups (P > 0.05).

Plasma rifapentine and 25-desacetyl rifapentine concentrations at time of bronchoscopy. The plasma rifapentine concentrations ranged from 4.5 ± 1.7 to 13.9 ± 3.6 µg/ml at 2 h following drug administration and from 4.1 ± 2.6 to 10.7 ± 4.0 µg/ml at 20 to 24 h following drug administration. At the time of bronchoscopy, the concentrations in plasma ranged from a low of 3.4 ± 3.2 µg/ml at 48 h to a high of 26.2 ± 6.1 µg/ml at 5 h. The corresponding concentrations of 25-desacetyl rifapentine were less at all time periods (Table 2). The plasma rifapentine concentrations determined at the time of bronchoscopy (at 4, 5, 7, 12, 24, and 48 h) declined biexponentially (r² = 0.98; log likelihood of the fit = 6.97; residual sum of squares = 3.59), with a t_1/2 of 18.3 h and an AUC_0- of 520 mg · h ml¹ (Fig. 1 and Table 3).

View larger version (12K): [in this window] [in a new window]   FIG. 1.   Plasma concentration-time curve derived from the means of the concentrations in the plasma of the six timed-bronchoscopy groups. See Table 2 for the SDs for each group.

Intrapulmonary rifapentine concentrations. AC rifapentine concentrations (mean ± SD) ranged from a low of 0.6 ± 1.0 µg/ml at 48 h to a high of 5.3 ± 2.5 µg/ml at 7 h and were lower than the corresponding concentrations in plasma at all time periods (Table 4). Intracellular 25-desacetyl rifapentine was detectable at very low concentrations at all time periods except at 7 h, when it was not detectable. The concentrations in ACs declined monoexponentially (r² = 0.95, log likelihood of the fit = 2.1, residual sum of squares = 0.71), with a t_1/2 of 13.0 h and an AUC_0- of 133 mg · h ml¹ (Fig. 2 and Table 4). The AUC_0- for ACs/AUC_0- for plasma ratio was 0.26. 

View larger version (13K): [in this window] [in a new window]   FIG. 2.   AC concentration-time curve derived from the means of the concentrations in the ACs of the six timed-bronchoscopy groups. See Table 4 for the SDs for each group.

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View larger version (14K): [in this window] [in a new window]   FIG. 3.   ELF concentration-time curve derived from the means of the concentrations in the ELF of the six timed-bronchoscopy groups. See Table 4 for the SDs for each group.

	DISCUSSION

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The observations presented here confirm our previous experiences (6, 7, 9, 10) and those of others (13, 14, 25, 26) that bronchoscopy and BAL for research purposes can safely be carried out with healthy volunteers.

The standardized technique that we use for bronchoscopy and BAL results in adequate and reliable recovery of ACs and ELF. The number of cells (approximately 100 × 10⁶ to 150 × 10⁶), the cell type (approximately 80 to 90% monocytes/macrophages), and the volume of ELF (approximately 1 ml) are similar to the values that we and others have reported previously (1, 2, 4, 7, 10, 26, 29, 33). Used with sensitive and specific drug assay techniques, this procedure permits an accurate estimation of the intrapulmonary drug concentrations in these compartments.

It is noteworthy that the C_max (26.2 µg/ml), T_max (5 h), t_1/2-LZ (18.3 h), and AUC (520 mg · h ml¹) values derived from this study were very similar to those reported in earlier studies with normal volunteers (19-21).

These data confirm that the long plasma t_1/2 of rifapentine also appears to result in a long intrapulmonary t_1/2, i.e., 13.0 h in ACs and 20.8 h in ELF. We believe that these estimates of t_1/2 are reasonably accurate. They would be improved by additional data obtained at intervals of greater than 48 h. While the drug concentrations and AUC values for ACs and ELF are substantially less than those for plasma, inspection of Fig. 2 and 3 reveals that the intrapulmonary drug concentrations (and the plasma drug concentrations in Fig. 1) resulting from the administration of a single 600-mg dose remain above the MIC breakpoint for Mycobacterium tuberculosis for 48 h. The data suggest a pharmacologic rationale for extended-interval dosing. Whether these pharmacokinetic estimates would be materially affected by the presence of pulmonary tuberculosis is unknown. The optimum dosing interval and the effect of multiple-dose administration on the intrapulmonary pharmacokinetics of rifapentine have not been determined. The relationship between the MIC of rifapentine for M. tuberculosis, dosing interval, and drug effectiveness requires further investigation. The reversible abnormalities in liver function that we observed in this study have been reported previously (17).

We have recently completed a steady-state study of intrapulmonary pyrazinamide concentrations (8). The t_1/2-LZ of pyrazinamide is also long and has been reported to be from 9 to 23 h (5, 22, 23, 28, 31). We found that pyrazinamide, unlike rifapentine, is highly concentrated in ELF. After the administration of five daily 1.0-g doses, the concentration in ELF/concentration in plasma ratio at 4 h was 22, whereas the concentration in ELF/concentration in plasma ratio was 0.2 for rifapentine in this study. The concentration in ACs/concentration in plasma ratio at 4 h was 0.6 for pyrazinamide, whereas it was 0.26 for rifapentine.

Determination of whether drugs such as pyrazinamide that achieve greater concentrations in the lungs and that therefore have greater inhibitory or killing ratios are more effective will require controlled trials. In general, high inhibitory or killing ratios are viewed as favorable in the treatment of infectious diseases. It is likely that the prolonged intrapulmonary rifapentine concentrations above the MIC for M. tuberculosis observed in these subjects are in part responsible for this drug's effectiveness for the treatment of pulmonary tuberculosis. The significance of the low (usually below 1.0 µg/ml) but detectable concentrations of rifapentine's main metabolite, 25-desacetyl rifapentine, in ACs and ELF is unknown. This was a single-dose study, and multiple doses may result in higher intrapulmonary concentrations than we observed in the subjects in the present study.