Introduction

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Rifapentine is a semisynthetic, rifamycin-class antibiotic (Arioli et al., 1981) under development for the treatment of pulmonary tuberculosis and for the prophylaxis of Mycobacterium tuberculosis and Mycobacterium avium complex infections. The antimicrobial spectrum of rifapentine is similar to that of its homologues, rifampin and rifabutin (Arioli et al., 1981; Dickinson and Mitchison, 1987; Heifets et al., 1990; Yates and Collins, 1982); however, rifapentine has demonstrated greater therapeutic efficacy in experimental mycobacterium infections, compared with rifampin (Arioli et al., 1981; Pattyn, 1987; Truffot-Pernot et al., 1983).

The structure of rifapentine is shown in fig. 1. Rifapentine differs from rifampin by the presence of a cyclopentyl ring instead of a methyl group at the piperazinyl moiety, which makes rifapentine more lipophilic. Pharmacokinetic studies of rats (Assandri et al., 1978) and healthy volunteers (Birmingham et al., 1978, Buniva et al., 1983) dosed with rifapentine indicate differences in the pharmacokinetic profiles between it and rifabutin. Peak serum concentrations obtained after oral or iv doses of rifapentine were comparable to those produced by rifampin, but the elimination half-lives of the two drugs were markedly different, with rifapentine persisting in serum about 4-5 times longer.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Structures of compounds either identified (standard exists) or characterized (MS data only) in mouse, rat, and monkey feces after iv or oral doses or 14C-rifapentine. All molecular weights are the monoisotope mass. The asterisk (*) denotes the position of the 14C label.

Previous studies in mice, rats, and monkeys have identified the pharmacokinetic profile of rifapentine and its metabolite, 25-desacetyl-rifapentine. In a study designed to assess disposition of radiolabeled-rifapentine in rats, Assandri and colleagues (Assandri et al., 1978) identified feces as the major route of elimination of intact rifapentine and metabolites. In a separate study (Assandri et al., 1984), the pharmacokinetics of ¹⁴C-rifapentine in rat, mouse, and rabbit were investigated. The mean bioavailability of rifapentine after a single oral dose of 3 mg/kg in rats was 84%. The primary route of elimination of radioactivity was through the feces (about 90%), with less than 10% of the radioactivity recovered in urine. The mean terminal elimination half-life of rifapentine was 14-21 hr in rats, 17-23 hr in mice, and only about 2 hr in rabbits. In these previous rifapentine studies, no attempts were made to determine mass balance or identify compounds.

The purpose of our studies was to determine the disposition and biotransformation of ¹⁴C-rifapentine in mice, bile duct-cannulated and uncannulated rats, and monkeys after either a single oral or iv dose. Mass balance studies included ¹⁴C analysis of urine, feces, bile, cage wash, carcasses, and cage air collected for up to 120 hr postdose. Metabolite profiling of ¹⁴C in urine, bile, and feces was conducted using HPLC ¹ with radioisotope detection. Mass spectra of selected radioactive chromatographic peaks were obtained. `

	Materials and Methods

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Chemicals and Dose Formulations. Standards of rifapentine, 25-desacetyl-rifapentine, and 3-formyl-rifapentine were synthesized at Hoechst Marion Roussel (Cincinnati, OH). The labeled drug was prepared with the imine carbon containing the ¹⁴C label (fig. 1). Unlabeled drug was coprecipitated with labeled drug to produce ¹⁴C-rifapentine and to limit the radioactivity exposure to less than 40 µCi/animal. The radiochemical purity was 98.41% and the chemical purity was 97.71%.

Animals. Mice. Two groups, each consisting of six male NMRI mice weighing between 25 and 30 g, received either a single iv or oral 10 mg/kg dose of ¹⁴C-rifapentine. The mice were housed in glass Roth-type cages in a light- and temperature-controlled room. The animals fasted overnight before dosing, then had free access to food and water after dosing. The iv dose was administered into the tail vein and the oral dose was delivered by gavage.

Rats. Three parallel groups of four male Wistar rats weighing 250-300 g were administered 10 mg/kg ¹⁴C-rifapentine either orally (two groups) or by iv (one group). The rats were housed in glass Roth-type cages in a light- and temperature-controlled room. The animals fasted overnight before dosing, then had free access to food and water after dosing. The iv dose was administered into the tail vein and the oral doses were delivered by gavage.

Monkeys. One group of four male Cynomolgus monkeys weighing 3.0-5.0 kg was administered one of three treatments: 10 mg/kg ¹⁴C-rifapentine administered orally or by iv, or 40 mg/kg ¹⁴C-rifapentine administered orally. Monkeys were housed in individual stainless steel cages designed for the separation and collection of urine and feces. The treatments were administered sequentially, with a 3-week washout period between treatments. Before each dose, the animals fasted overnight and until about 4 hr postdose. The iv dose was administered via the saphenous vein, and the oral doses were administered via intragastric intubation.

Sample Collection. The collection times for urine, bile (rats only), and fecal samples were intervals of 0-6, 6-12, 12-24, 24-48, 48-72, 72-96, and 96-120 hr after the dose was administered. The samples were collected in tared glass containers for mice and rats and in plastic containers for monkeys. Cage washes were conducted at the end of each urine-collection interval (mice and rats) or after each 24-hr fecal collection (monkeys). In addition, for mice and the uncannulated rats, cage air was scrubbed through a CO₂ trapping solution (5 M ethanolamine in 2-methoxyethanol, 3:7 v/v) to trap any ¹⁴C-labeled CO₂ at 12-hr intervals. At the end of the mass balance study, carcasses of mice and rats were analyzed for radioactivity content.

Fecal Sample Preparation for LC/UV/¹⁴C or LC/MS. Fecal samples were thawed and mixed with a methanol solution (1 µg/ml ascorbic acid) in a ratio of about 2 ml/g of fecal homogenate. Samples were centrifuged at 3500 rpm for 2 min after mixing; the supernatant was then transferred to a test tube. This extraction procedure was repeated two more times and the extracts were combined and dried in a vacuum centrifuge. The dry residue was reconstituted in a methanol/water solution, filtered, and injected onto the HPLC system.

Radioactivity Analysis. Concentrations of ¹⁴C in urine, bile, cage wash, carcass, and expired air were measured by direct liquid scintillation counting (LSC) with external standardization for quench correction. Weighed aliquots of urine, bile, and cage wash were transferred to vials, and 5-15 ml of Ultima Gold^® (Packard, Meriden, CT) scintillant added. After incubation of carcass with Soluene-350 (Packard), 10 ml of Hionic Fluor^® (Packard) was added as scintillant. The CO₂ trapping solution from expired air was aliquoted into LSC vials and 10-15 ml of Permafluor V^® (Packard) was added as scintillant. Activity of ¹⁴C in feces was determined after combustion of the dried samples in a Packard 307 oxidizer. The CO₂ generated by the combustion was trapped in 9 ml of Carbo-Sorb E^® (CO₂ absorber; Packard), then 11 ml of Permafluor was added as scintillant. Each sample was counted by liquid scintillation analysis on a Tri-Carb 2300TR Liquid Scintillation Analyzer (Packard). All samples were counted for at least 5 min or 100,000 counts.

Metabolite Characterization. HPLC radiochromatograms and mass spectra were obtained for samples with radioactivity greater than 5% of the total dose. The HPLC system used for metabolite profiling included a Hitachi AS-4000 autosampler with a Hitachi L-6200A pump (San Jose, CA), Hitachi L-4250 UV detector set at 480 nm, and an IN/US Systems RAM radioactivity detector. A Mac-Mod Zorbax RX-C8 column (250 × 4.6 mm, 5 µm; Chadds Ford, PA) with a gradient mobile phase was used to separate ¹⁴C-rifapentine-related compounds in urine, bile, and feces. Mobile phase A was 95% water/5% acetonitrile/0.1% heptafluorobutyric acid, and mobile phase B was 100% acetonitrile/0.1% heptafluorobutyric acid. A linear gradient was used to separate compounds in the extracts. Initially, 100% A flowed through the column for 1 min after the injection, followed by a linear ramp to 100% B in 35 min, where it remained for 10 more min. The flow rate was set to 1 ml/min and a 200 µl aliquot sample was injected onto the HPLC system. The LC/MS system used to analyze the fecal extracts and bile samples consisted of a Gilson Model 231 autosampler (Middleton, WI), HPLC pump (Michrom BioResources Inc., Auburn, CA), Waters Model 490-MS UV detector with the Programmable Detector set at 480 nm (Milford, MA), and a Finnigan MAT TSQ 710 triple quadrupole mass spectrometer (San Jose, CA). The column and the mobile phase were identical to those used for the LC/UV/¹⁴C profiling analyses. The mass spectrometer was set in the positive ion mode to scan from 400 to 1400 atomic mass units in 2 sec. The standard Finnigan electrospray ionization source was used to produce ions. The heated capillary temperature was 150°C and spray voltage set at 4500 volts. The 1 ml/min flow from the LC column was split 10:1 before entering the mass spectrometer.

Determination of Extraction Efficiency and Column Recovery of ¹⁴C-Labeled Components. To determine extraction efficiency, mouse, rat, and monkey fecal samples were accurately weighed and processed as described above. A small amount of methanol was used to redissolve the dry residue, and the liquid was transferred to a scintillation vial along with scintillant. The sample was counted using a liquid scintillation counter. The extraction efficiency was calculated by comparing the radioactivity of a processed sample with the theoretical maximum value, based upon combustion data.

	Results

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Extraction Efficiency and Column Recovery. The extraction efficiencies for mouse and rat fecal samples were relatively high, ranging from 89% to 93% (mean, 90%) in mice, and from 82% to 105% (mean, 93%) in rats. Since the extraction efficiencies were high, the assumption was that no significant metabolite was unaccounted for in fecal homogenates in mice or rats. In monkeys, the extraction efficiencies ranged from 67% to 71% (mean, 69%). Because the extraction efficiencies were relatively low in this species, predose monkey feces was spiked with ¹⁴C-rifapentine to determine if a difference between spiked and study samples was observed. The extraction efficiencies for the spiked predose fecal samples ranged from 78% to 88% (mean, 83%). Thus a matrix effect may account in part for the lower yield. The mean column recovery of duplicate measurements was 102% in mice, 102% in rats, and 103% in monkeys. No significant amount of radioactivity was retained on the column.

Mass Balance Studies. Mice. Mass balance data of excreted compounds after iv and oral administration of ¹⁴C-rifapentine in mice are summarized in table 1. After iv administration, radioactivity excreted in urine and feces during the 120-hr collection periods was 4.9% and 73.0%, respectively. The cage wash contained 3.7% of the dose, and 3.4% of the dose remained in the carcass. Expired air did not contain any radioactivity over the first 24 hr postdose. The mass balance averaged 86.2% of dose. Radioactivity recovery results with oral administration were quite similar to the iv dose. During the 120-hr postdose collection period, 4.5% and 75.5% of the oral dose was recovered in urine and feces, respectively. The mass balance averaged 88.5% of dose.

Rats. After iv administration of ¹⁴C-rifapentine to bile duct-cannulated rats, radioactivity excreted in urine, feces, and bile over the 96 hr postdose was 4.8%, 23.2%, and 30.2% of the dose, respectively; 35.8% remained in the carcass (table 1). The total recovery of radioactivity was 94.6%. Similar to the findings with mice, disposition results after oral administration of ¹⁴C-rifapentine to bile duct-cannulated rats were comparable to iv dosing results. During the 96-hr postdose collection period, 4.2%, 15.4%, and 21.5% of the oral dose was recovered in urine, feces, and bile, respectively. The amount of radioactivity in the carcass was 41.7% of the oral dose and the mass balance was 84.2%. After oral administration in uncannulated rats, radioactivity excreted in urine and feces was 4.4% and 100.2% of the dose, respectively, over 144 hr postdose; only 3.1% of the radioactive dose remained in the carcass and the mass balance was 108.4%. No radioactivity was detected in expired air in any of the treatment groups.

Monkeys. After iv administration of 10 mg/kg ¹⁴C-rifapentine, the radioactivity excreted in urine and feces over 120 hr postdose was 2.8% and 88.1% of the dose, respectively (table 1). After oral administration, the total recovery of radioactivity in urine and feces was 3.3% and 92.1% of the 10 mg/kg dose, respectively, and 4.5% and 85.1% of the 40 mg/kg dose, respectively. The mass balances after dosing of 10 mg/kg IV, 10 mg/kg oral, and 40 mg/kg oral were 91.8%, 95.8%, and 89.9% of dose respectively.

Metabolism. None of the mouse, rat, or monkey urine samples contained more than 5% of the radiolabeled dose of rifapentine. Therefore, urine samples were not analyzed except for the one that contained the most radioactivity (range, 0.1% to 0.7% of dose). Fecal samples containing radioactivity greater than 5% of dose were subjected to LC/UV/¹⁴C. For all three species, the oral and iv routes of rifapentine administration produced similar metabolite profiles in feces samples.

Mice. A summary of identified metabolites and their abundance in fecal samples is presented in table 2. Representative LC/¹⁴C chromatograms (fig. 2) resulting from injection of a mouse fecal extract contained 10 regions of significant radioactivity. The radiochromatograms were similar after either intravenous or oral dosing. One of the abundant regions of radioactivity (8-18% of dose) eluted form the column as a broad peak with retention time from 9 to 17 min. Occasionally, small peaks were observed on top of the broad peak. Unsuccessful attempts were made to resolve the broad peaks into individual components using other columns and mobile phase components and conditions. The entire region was summed for the purpose of calculating percentage of dose.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Radiochromatogram resulting from injection of a mouse sample (72-hr) fecal extract after the animal received a 10 mg/kg iv dose of 14C-rifapentine.

Rat feces. A summary of identified metabolites and their abundance in fecal samples is presented in table 2. A representative radiochromatogram from a rat fecal extract is shown in fig. 3. Radiochromatograms from rat fecal extracts were similar after either iv or oral dosing. For rat extracts, as for mouse extracts, approximately 10 regions of significant radioactivity were observed in the chromatograms. Four chromatographic peaks observed in the LC/¹⁴C chromatograms were assigned as 25-desacetyl-rifapentine, rifapentine, 3-formyl-25-desacetyl-rifapentine, and 3-formyl-rifapentine, respectively. The assignments were based upon a retention time match of chromatographic peaks from authentic standards to those from rat fecal extracts. The largest individual region consistently corresponded to rifapentine (3%-35% of dose). Generally, no significant new peaks were observed as time progressed postdose. Instead, a decrease in the intensity of the peaks that were already present at earlier times was found. In the feces, one of the significant chromatographic regions of radioactivity (1%-19% of dose) was a very broad region (8- to 10-min rise) in the background (about 7%- to 17-min retention time), with few distinct peaks within it. The presence of rifapentine in the rat fecal extract could be confirmed by LC/MS, based upon a comparison of the retention time and mass spectrum to an authentic standard. The other identified metabolites, based upon LC/UV/¹⁴C data, were not observed by LC/MS, probably because of the relatively poor MS response of rifapentine and analogs combined with their low concentration.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Representative 14C radiochromatogram of bile duct-cannulated rat fecal extract after iv administration of 14C-rifapentine.

Rat Bile. A summary of identified metabolites and their abundance in bile samples is presented in table 2. A representative radiochromatogram from a rat bile extract is shown in fig. 4. Radiochromatograms from rat bile extracts were similar after either iv or oral dosing. Three chromatographic peaks observed in the LC/¹⁴C chromatograms were assigned as 25-desacetyl-rifapentine, rifapentine, and 3-formyl-rifapentine, respectively, based upon a retention time match with authentic standards. A significant broad region of radioactivity was also present in the LC/¹⁴C chromatograms from 7 to 19 min and corresponded to 0%-2.6% of the dose. Four distinct peaks were observed from 13.5 to 18.7 min, with varying intensities of 0%-8.7% of dose. The presence of 25-desacetyl-rifapentine and rifapentine in the rat fecal extract was confirmed by LC/MS, based upon a comparison of the retention time and mass spectrum to an authentic standard. The other identified metabolites, based upon LC/UV/¹⁴C data, were not observed by LC/MS probably because of the relatively poor MS response of rifapentine and analogs combined with their low concentration. No mass spectra (unique from predose fecal extracts) were observed for any of the other radioactive compounds eluting from the LC column, presumably because of low concentrations and/or low ionization yields.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Representative 14C radiochromatogram of bile duct-cannulated rat bile extract after iv administration of 14C-rifapentine.

Monkeys. A representative LC/¹⁴C chromatogram for the 10 mg/kg iv dose in monkeys that was obtained 72 hr postdose is presented in fig. 5. Most radiochromatograms obtained after the iv and oral doses were similar, with seven regions of significant radioactivity observed. As with the other species, one of the abundant regions of radioactivity (12%-19% of the dose) eluted from the column as a peak with retention time from 6 to 15 min. Four peaks in each radiochromatogram were assigned as 25-desacetyl-rifapentine, rifapentine, 3-formyl-25-desacetyl-rifapentine, and 3-formyl-rifapentine. In general, rifapentine represented 17-29% of the dose in a sample, while 25-desacetyl-rifapentine represented 14-20% of the dose. These were the largest amounts of radioactivity eluting from the column.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Radiochromatogram resulting from injection of a monkey sample fecal extract after the animal received a 10 mg/kg iv dose of 14C-rifapentine.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   LC/MS (m/z labeled) and LC/UV (user trace) chromatograms resulting from injection of a monkey sample fecal extract after the animal received a 10 mg/kg iv dose of 14C-rifapentine. The m/z value corresponds to the (M+H)+ ion for the labeled analyte. User trace is UV wavelength 480 nm.

	Discussion

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The results of the rifapentine mass balance studies for mouse, rat, and monkey were strikingly similar. During the 120-hr collection period, less than 5% of radioactive drug was excreted in the mouse, rat, and monkey urine, indicating that renal excretion is a minor route of elimination of rifapentine in these species. The major route of elimination of radioactivity was into the feces, where more than 75% of the radioactive dose was recovered. In contrast to rifapentine, mass balance studies of ¹⁴C-rifabutin in rats and monkeys revealed that the amounts of radioactivity excreted in urine and feces after oral and iv doses were similar (about 44% of the dose each) (Battaglia et al., 1990, 1991).

Virtually no differences were found between the percentages of dose excreted in urine and feces after oral and iv administration of rifapentine in mouse, uncannulated rats, and monkeys. The relative amounts of radioactivity excreted into bile, feces, and urine after oral administration of rifapentine in bile duct-cannulated rats were also similar to that after iv administration. The ratio of radioactivity excreted in rat bile to feces was approximately 3:2 after both oral and iv dosing. These data support other reports that absorption of rifapentine is nearly complete (Weber et al., 1983).

Biliary excretion was a major route of elimination of radioactivity (approximately 51% of excreted radioactivity) after iv administration of rifapentine in bile duct-cannulated rats. Approximately 40% of the excreted radioactivity (about 23% of the dose) was recovered in feces after iv administration in bile duct-cannulated rats. This finding suggests that rifapentine may be cleared by another route of elimination, such as direct excretion through the intestinal wall. The role of gastrointestinal secretion in the clearance of rifapentine in rats is currently under investigation. The ratio of rifapentine to 25-desacetyl-rifapentine was approximately 10:1 in fecal samples of the cannulated rats, but for bile, the ratio was approximately 1:3. Since previous studies found no detectable amount of 25-desacetyl-rifapentine in circulating plasma (Weber et al., 1983), it appears that the 25-desacetyl metabolite is rapidly excreted into bile after being formed in the liver.

The total radioactivity recovered in urine and feces over 96 hr postdose in orally dosed uncannulated rats (about 104.6%) was much greater than the recovery in urine, bile, and feces found in orally dosed bile duct-cannulated rats (about 41.1%). The balance of the radioactivity was primarily found in the carcass (table 1). This difference was most likely due to changes in the physiology of the rats, stemming from the stress of cannulation and/or surgical trauma. Further investigation of dose recovery after bile duct cannulation in rats is planned.

In general, examination of urine samples using LC/UV/¹⁴C was not done in any of the species because the total and individual sample recoveries of radioactivity in urine was less than 5% and 0.7% of dose, respectively. Only the urine sample with the highest level of radioactivity in each species was examined using LC/¹⁴C. The radiochromatogram contained small peaks corresponding to rifapentine and its hydrolysis product, 3-formyl-rifapentine.

The radiochromatograms from rat fecal and bile samples after oral and iv administration of ¹⁴C-rifapentine were similar. By comparing the HPLC retention times of existing standards and/or mass spectral analysis of chromatographic peaks, components identified in the fecal and bile samples included rifapentine, 25-desacetyl-rifapentine, 3-formyl-rifapentine, and 3-formyl-25-desacetyl-rifapentine. The primary peak observed in fecal samples was rifapentine in all three species. Rifapentine is known to be unstable in solution and one major degradation product is 3-formyl-rifapentine. Similar to rifampin, the 3-formyl derivatives found in fecal samples are probably formed by nonenzymatic hydrolysis (Battaglia et al., 1990). Since these metabolites have not been observed in plasma, formation most likely occurs as the excreta resides in the gut.

In all three species, one region of radioactivity occurred as a broad rise in the background with few distinct peaks. The approximate retention time of this area was 9-17 min in mice (accounting for 8%-18% of dose), 7-17 min in rats (accounting for 1%-19% of dose), and 6-15 min in monkeys (accounting for approximately 12%-19% of dose). Attempts to resolve the broad peaks into individual components using other columns and mobile phase conditions were unsuccessful; therefore, structures could not be assigned. No unique mass spectra (from predose fecal extracts) were observed for any of the radioactive compounds eluting from the LC column other than rifapentine, 25-desacetyl-rifapentine, 3-formyl-rifapentine, or 3-formyl-25-desacetyl-rifapentine, presumably because of low concentrations and/or because the ionization efficiency was insufficient to yield discernible mass spectra.

A search of the published literature failed to identify any studies that evaluated the metabolism and excretion of rifampin in animals. The mass balance and metabolic profile of another rifamycin analog, rifabutin, has been reported in animals and humans (Battaglia et al., 1990, 1991; Utkin et al., 1997; Koudriakova et al., 1996). Parent drug accounted for only 8.5% of the radioactive rifabutin dose in urine of rats, and less than 0.5% of the dose in rabbits and monkeys (Battaglia et al., 1990). Most of the urinary radioactivity (more than 93%) after a single oral dose of ¹⁴C-rifabutin in rats, rabbits, and monkeys was constituted by unidentified peaks of polar compounds (Battaglia et al., 1990). The most abundant polar metabolite identified was N-isobutyl-4-hydroxy-piperidine. Lipophilic metabolites, which accounted for less than 20% of urinary radioactivity in rats and humans, included 25-O-desacetyl-rifabutin, 27-O-demethyl-rifabutin, and 31-hydroxy-rifabutin (Battaglia et al., 1990; Utkin et al., 1997; Koudriakova et al., 1996). Only small amounts of the 25-desacetyl derivative were found in the urine of rats (2.1% of the dose) (Battaglia et al., 1990). In bile duct-cannulated rats, approximately 24% of the rifabutin dose was excreted in bile, almost exclusively (98%) as metabolites (Koudriakova et al., 1996). These data suggest rifabutin is more extensively metabolized in animals than rifapentine is, with metabolic pathways that include oxidation.

In summary, the disposition and biotransformation of rifapentine in mouse, rat, and monkey are quite similar. Rifapentine is excreted primarily as intact drug in feces; less than 5% of rifapentine and its metabolites are excreted in the urine. The primary metabolite in bile and feces is 25-desacetyl-rifapentine, with smaller amounts of the degradation byproducts, 3-formyl-rifapentine and 3-formyl-25-desacetyl-rifapentine, formed in the gut.