Introduction

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Atevirdine mesylate (U-87201E) is a nonnucleoside inhibitor of HIV-1¹ reverse transcriptase (Romero et al., 1991; Romero, 1994a). Atevirdine mesylate is the first-generation member of the bisheteroarylpiperazine class of nonnucleoside inhibitors, which inhibits reverse transcriptase noncompetitively by binding near the catalytic site (Althaus et al., 1993). Atevirdine inhibits antiviral activity in a manner equivalent to that of AZT, but unlike AZT it is selective for reverse transcriptase over cellular polymerases. Atevirdine inhibits HIV-1 replication in infected peripheral blood mononuclear cells with a 50% inhibitory concentration of 1 nM and a cytotoxic-to-inhibitory ratio of approximately 10,000-fold (Romero, 1994a) and inhibits HIV-1 replication in H9 cells with a median effective concentration of 40 nM and is cytotoxic to 50% of the cells at >10 µM (Romero, 1994a). Atevirdine is also active in the SCID-hu mouse model of HIV-1 infection (Romero et al., 1991; Romero, 1994a). In addition, AZT-resistant and 2'-3'-dideoxyinosine-resistant clinical isolates are inhibited by atevirdine with a median inhibitory concentration of 0.74 µM (Campbell et al., 1993). Clinical studies with atevirdine mesylate have shown that the drug is well tolerated (Been-Tiktak et al., 1995; Reichman et al., 1995; Ward et al., 1992; Borleffts et al., 1992; Cox et al., 1992). Further clinical evaluation of atevirdine in monotherapy and in combination therapy is underway.

This article describes the absorption, distribution, metabolism, and excretion of atevirdine in the rat.

	Materials and Methods

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Chemicals. All chemicals used in this study were of analytical grade. Solvents were Burdick & Jackson high-purity grade (Burdick & Jackson, Muskegon, MI). Water was distilled and purified through a Milli-Q reagent system (Millipore Corp., Bedford, MA). Ultima Gold (Packard Instrument Co., Meriden, CT) was used for liquid scintillation counting (LSC). Carbo-sorb E (Packard) and Permafluor E⁺ (Packard) were used for combustion of samples. Flo-Scint II (Packard) was used as scintillant for flow-through detection. -Glucuronidase (from Helix pomatia, Type H-5) and Trizma buffer (0.2 M, pH 7.4) were obtained from Sigma Chemical Company (St. Louis, MO). Methanol-d₄ (99.96% D) was purchased from Cambridge Isotope Laboratories (Cambridge, MA).

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Structure of atevirdine mesylate.

Animals and Husbandry. Rats were obtained from Charles River Laboratories (Portage, MI). Male and female Sprague-Dawley rats were used for all phases of this study. In addition, male Long-Evans rats and full-term (18-19 days' gestation) pregnant Sprague-Dawley rats were used for the tissue-distribution phase. For the excretion portion of this study, male rats (9 weeks old, 225-273 g at study initiation) and female rats (13 weeks old, 217-250 g) were surgically implanted with superior vena cava cannulas and housed in Nalgene metabolism cages (Nalge Company, Rochester, NY). For the tissue-distribution phase, male and female Sprague-Dawley and male Long-Evans rats (5-6 weeks old, 107-137 g) and pregnant Sprague-Dawley rats (12 weeks old, 252-311 g) were housed in stainless steel cages with wire mesh floors. The rats were fed Certified Purina Rat Chow 5002 (Purina Mills Inc., St. Louis, MO) and water ad libitum, except during fasting periods. The animal rooms were environmentally controlled, with 12 hr light/dark cycle, a room temperature range of 68°F-76°F, and a relative humidity range of 40%-70%.

Instrumentation. Samples were oxidized in a Packard model 307 sample oxidizer equipped with a Packard Oximate 80 robotics system. A Packard Tri-Carb liquid scintillation analyzer model 1900CA or 1900TR was used for radioactivity counting. Feces were homogenized using a Stomacher blender (Tekmar Co., Cincinnati, OH). A Turbo Vap LV evaporator (Zymark Corp., Hopkinton, MA) was used to concentrate samples. A cryomicrotome PMV 450 (LKB Instruments, Stockholm, Sweden) or Jung Cryomacrocut (Leica Instruments GmbH, Nussloch, Germany) was used for sectioning carcasses. A Microcomputer Imaging Device system (Imaging Research Inc., St. Catharines, Ontario, Canada) was used for quantitation of the optical densities of the autoradiograms (Kodak SB-5 scientific imaging films; Eastman Kodak Co., Rochester, NY). ¹⁴C calibration standards from American Radiochemicals (St. Louis, MO) were used for quantitation of the optical densities.

Chromatography. The HPLC system consisted of a Perkin Elmer 410 quaternary pump (Perkin-Elmer Corp., Norwalk, CT), a Perkin Elmer ISS 200 sample processor, a Perkin Elmer LC-235C or LC-235 diode array detector, and a Radiomatic Model A-515 flow-through detector (Packard Instrument Co., Meriden, CT) containing a 500-µl liquid cell. Samples were analyzed on YMC 5µ-basic 4.5 mm i.d. x 25 cm HPLC columns (YMC, Inc., Wilmington, NC). A Foxy 200 fraction collector (Isco, Inc., Lincoln, NE) equipped with a diverter valve was used to collect fractions at 0.25- to 1-min intervals in a recycling mode.

Nuclear Magnetic Resonance Spectroscopy. NMR experiments were recorded on a Bruker AMX-500 instrument (Bruker, Inc., Billerica, MA) operating at 500.13 MHz for ¹H and 125.7 MHz for ¹³C. Samples were dissolved in approximately 300-400 µl of methanol-d₄, transferred to a 4 mm o.d. Teflon (DuPont, Wilmington, DE) NMR tube (Wilmad Glass Company, Buena, NJ), and the Teflon tube was inserted into a 5 mm o.d. glass NMR tube (Wilmad). Proton spectra were referenced to the solvent pentet at 3.30 ppm. All experiments were run without spinning of the sample. Spectra were recorded at either 300°K or 303°K. Resolution-enhancing Gaussian windows were applied to one-dimensional ¹H free induction decays prior to transformation. Where advantageous, presaturation suppression of the large hydroxyl peak at 4.8 ppm was utilized to obtain better signal to noise. Two-dimensional experiments included ¹H-¹H COSY, ¹H-¹³C HMQC (Bax et al., 1983) and ¹H-¹³C HMBC (Bax and Summers, 1986). COSY and HMBC experiments were run in the magnitude-mode (non-phase-sensitive) in F1. HMQC experiments were phase-sensitive, using the time-proportional phase incrementation method for quadrature detection in F1. HMQC experiments used globally optimized alternating-phase rectangular pulse decoupling (Shaka et al., 1985) of ¹³C during acquisition. Resolution-enhancing sine-bell or sine-bell squared windows were generally applied prior to transform, though line-broadening sine-bell squared windows shifted by /2 radians were applied in the proton dimension of the heteronuclear experiments to increase signal to noise.

Mass Spectrometry. PCI/LC/MS was performed on a Finnigan 4021 quadrupole mass spectrometer (Finnigan MAT, San Jose, CA) equipped with a thermal pneumatic nebulizer coupled to a momentum separator (Thermabeam^®; Extrel Corp., Pittsburgh, PA). The instrument was operated in the PCI mode using ammonia as reagent gas. CI gas pressure was optimized by the appearance of the m/z 52 ion [(NH₃)₂NH₄⁺]. Electron energy was set to 70 eV. The HPLC system consisted of a Perkin Elmer ISS 100 sample processor interfaced to a Perkin Elmer 410 quaternary pump with a Perkin Elmer SEC-4 solvent environment control and a Waters 490 MS UV detector (Waters, Milford, MA). Samples were analyzed at 0.5 ml/min with a 20-min linear gradient from 10% A/90% D to 60% A/40% D, followed by 20-min 60% A/40% D, then a 10-min linear gradient to 80% A/20% D (A, acetonitrile; D, 0.1 M ammonium acetate buffer, pH 4).

Study Design and Collection of Samples. Two separate studies were conducted: a single- and multiple-dose absorption, excretion, and metabolism study, and a single-dose tissue distribution study.

Single- and Multiple-Dose Absorption, Excretion, and Metabolism Study. Atevirdine mesylate and [¹⁴C]atevirdine mesylate were dissolved in 80% propylene glycol/20% water containing 3 µl methanesulfonic acid per milliliter to final concentrations of 5 and 50 mg/ml. Four male and four female rats received single oral radiolabeled doses of 10 mg/kg and 100 µCi/kg, followed by multiple oral nonradiolabeled doses of 20 mg/kg/day given as 10 mg/kg doses twice a day (multiple-dose regimen was started 24 hr after the radiolabeled dose; one dose in the morning and a second dose 8 hr later) for the next 8 days; then a second radiolabeled dose of 10 mg/kg and 100 µCi/kg was administered. In addition, four male and four female rats received the same dosing regimen at single radiolabeled doses of 100 mg/kg and 100 µCi/kg and multiple nonradiolabeled doses of 200 mg/kg/day (100 mg/kg doses given twice a day). Doses were administered by gastric gavage at a constant dosing volume of 3 ml/kg. Rats were fasted 17 hr before and 4 hr after radiolabeled dose administration. Urine and feces were collected and weighed at 0-12, 12-24, and at 24-hr intervals up to 192 and 120 hr after the first and second radiolabeled doses, respectively. Blood samples (75-400 µl aliquots) were drawn at 0, 1, 2, 4, 8, 12, 24, and 48 hr after each radiolabeled dose and at 0 and 4 hr after the morning nonradiolabeled dose. Aliquots of the samples were immediately analyzed for radioactivity; the remaining samples were stored at below 10°C.

Tissue Distribution Study. Atevirdine mesylate and [¹⁴C]atevirdine mesylate were dissolved in 10% propylene glycol/15% polyethylene glycol 400/45% glycerin/30% water (pH adjusted to 2.0 with 10% HCl) to a final concentration of 10 mg/ml. Twelve male and 9 female Sprague-Dawley rats, 8 male Long-Evans rats, and 3 pregnant Sprague-Dawley rats were administered a single 10 mg/kg and 200 µCi/kg oral dose of [¹⁴C]atevirdine mesylate. Rats were fasted overnight before dosing; food was returned 4 hr after dosing to animals that were to be euthanized later than 8 hr. Rats to be sacrificed later than 24 hr after dosing were fasted overnight prior to euthanasia. Male Sprague-Dawley rats were euthanized by CO₂ inhalation at 1, 4, 8, and 24 hr; female and pregnant Sprague-Dawley rats were euthanized at 1, 4, and 24 hr; and male Long-Evans rats were euthanized at 1, 8, 24, and 48 hr. The carcasses were immediately frozen in a dry ice/heptane bath. The frozen carcasses were embedded and frozen in 5% carboxymethylcellulose blocks and stored below 10°C until sectioned. The blocks were sagittally sectioned (20 µm) at approximately 20°C with a cryomicrotome. After freeze-drying at approximately 20°C for 2 to 3 days, representative sections were apposed to film along with the ¹⁴C calibration standards for 1 to 8 weeks. The optical densities of the autoradiograms were converted to µg-eq/g using ¹⁴C calibration standard values and the specific activity of the dose formulation.

Sample Preparation and Analysis. Duplicate 500-mg aliquots of urine and cagewash samples were mixed with 10 ml of Ultima Gold and analyzed by LSC. A single 25-mg aliquot of plasma was transferred into scintillation vials containing 5 ml of Ultima Gold. Duplicate 25-mg aliquots of blood were combusted and analyzed by LSC. Fecal samples were homogenized with 3-5 volumes of water, and triplicate 500-mg aliquots of fecal homogenate were combusted and analyzed by LSC. LSC of combusted samples was determined in 9 ml Carbo-sorb E and 10 ml Permafluor E⁺ scintillation cocktail. Radioactivity was measured by LSC with 10 min counting.

HPLC Analyses of Plasma for Atevirdine, N-Desethyl Atevirdine, and O-Desmethyl Atevirdine. A 25-µl aliquot of plasma was mixed with 50 µl internal standard solution in acetonitrile (10 µg/ml of U-90152S in acetonitrile) and 10 µl of methanol. The mixture was centrifuged at 14,000 rpm for 5 min at 4°C. A 75-µl aliquot of the supernatant was mixed with 75 µl of 0.1 M ammonium acetate buffer (pH 4), and a 75-µl portion was analyzed by HPLC with UV detection at 295 nm. The HPLC conditions consisted of isocratic elution at 1.0 ml/min with 38% acetonitrile/2% isopropyl alcohol/60% 0.1 M ammonium acetate (pH 4). Quantitation of atevirdine, N-desethyl atevirdine, and O-desmethyl atevirdine in plasma was determined using peak area ratios relative to the internal standard and linear regression parameters calculated from calibration curve standards prepared in blank rat plasma. The assay was linear from 0.0314 to 502 µM for atevirdine and from 0.0314 to 251 µM for N-desethyl atevirdine and O-desmethyl atevirdine. Inter- and intraday assay variability were acceptable with no more than 10% coefficient of variation. Recovery of parent drug and metabolites was >95%.

Pharmacokinetic Analysis. Pharmacokinetic parameters were determined by noncompartmental analyses of the concentration-time data for drug-related radioactivity, atevirdine, N-desethyl atevirdine, and O-desmethyl atevirdine. AUC was determined by the linear trapezoidal rule. t_max was the time at which maximum concentration (C_max) was achieved; both values were based on the highest observed concentrations. The percentage of atevirdine, N-desethyl atevirdine, and O-desethyl atevirdine in circulation was calculated from the respective AUCs relative to radiochemical AUC of plasma.

Metabolite Profiles. Urine and feces samples were profiled for parent drug and metabolites by HPLC with radiochemical detection. Urine samples were directly profiled; feces (2-g aliquots) were extracted with 90% acetonitrile/10% 0.1 ammonium acetate (pH 4) and concentrated prior to HPLC analysis. Extraction procedures were validated with a predose fecal sample fortified with [¹⁴C]atevirdine mesylate (95% recovery). Chromatographic analyses were performed using gradient elution at 1.0 ml/min with 10% A/90% D for 5 min, followed by a 35-min linear gradient to 60% a/40% D, then 60% A/40% D for 5 min (A, acetonitrile; D, 0.1 M ammonium acetate buffer, pH 4). The HPLC effluent was mixed with Flo-Scint II in a 1:3 ratio. Radioactivity was quantified using peak area integration.

Metabolite Isolation and Identification. MET-3 was isolated as follows: Urine (~60 ml of 0-12 hr urine from a male rats given 200 mg/kg/day) was lyophilized and the residue reconstituted in 5-6 ml of 10% acetonitrile/90% 0.1 M ammonium acetate buffer (pH 4). The concentrated urine (3.1 mg drug-related radioactivity) was purified by HPLC using the same chromatographic conditions as for the metabolite profiles to afford 0.72 mg of partially purified MET-3. Subsequent isocratic HPLC purification (0.5 ml/min, 10% acetonitrile/2% isopropyl alcohol/88% 0.1 M ammonium acetate [pH 4]) gave 0.2 mg of purified MET-3.

Enzyme Hydrolysis. A 250-µl aliquot of urine (0-12 hr from male and female rats given 200 mg/kg/day) was mixed with 250 µl of 0.1 M sodium acetate buffer (pH 5) and 100 µl of -glucuronidase solution (1,250 units/ml in 0.2% saline). The mixture was incubated at 37°C for 1 hr. The reaction was quenched by addition of 500 µl of acetonitrile, concentrated, and a 50-µl aliquot was analyzed by HPLC with radiochemical detection (chromatographic conditions were the same as for metabolite profiles). A control sample containing rat urine and sodium acetate buffer (pH 5) was carried out similarly.

Microsomal Metabolism. Liver microsomes from untreated rats were used for control activity. For selective induction of CYP3A, rats were treated with daily 100 mg/kg oral doses of PCN (100 mg/ml suspension in corn oil) for 3 days and euthanized 24 hr after last dose. Livers were perfused with cold saline and, if necessary, frozen in liquid nitrogen and stored at 80°C. Hepatic microsomes were prepared by differential centrifugation of liver homogenates (Imai and Sato, 1974; vanderHoeven and Coon, 1974). All tissue manipulations were conducted at 4°C. Liver tissue was homogenized in 4 volumes 1.15% KCl, 10 mM EDTA (pH 7.4), using a motor-driven Teflon mortar-glass pestle homogenizer. Cellular debris, nuclei, mitochondria and lysosomes were removed by centrifugation at 10,000 g for 20 min and the supernatant was centrifuged at 227,000g for 40 min. The resulting microsomal pellet was washed by homogenization in 100 mM sodium pyrophosphate (pH 7.4), 1 mM EDTA, and pelleted by centrifugation at 227,000g for 40 min. The microsomal pellet was finally homogenized in 0.25 M sucrose, 0.1 mM EDTA and stored at 80°C.

	Results

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Absorption and Excretion of Atevirdine. A comparison of the cumulative excretion of drug-related radioactivity in the single 10 mg/kg dose, single 100 mg/kg dose, multiple 20 mg/kg/day dose, and multiple 200 mg/kg/day dose is presented in fig. 2. In the four groups after oral administration, 12%-20% of the administered radioactivity was excreted in urine, and 76%-86% was recovered in feces. The majority of the radioactivity (>81%) was excreted within 48 hr. The overall quantitative pattern of excretion of radioactivity was similar for males and females in the four groups. Excretion of radioactivity was also similar for the two dose levels and for single and multiple doses.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Excretion of drug-related radioactivity in rats after oral administration of [14C]atevirdine mesylate

Distribution of Atevirdine. Concentrations of drug-related radioactivity in selected tissues and organs for the four groups of animals at 1 and 24 hr after dose are shown in table 1. Drug-related radioactivity distributed rapidly to most tissues and organs of albino and pigmented rats, with maximum concentrations achieved at 1 hr. The distribution patterns in male and female Sprague-Dawley rats were similar. Liver contained the highest ¹⁴C levels. ¹⁴C concentrations in brain were slightly lower than blood levels at 1 hr after dose but were significantly below blood levels at later time points. Most fetal tissues had ¹⁴C levels significantly above maternal blood levels. An autoradiogram of a pregnant Sprague-Dawley rat is shown in fig. 3. Drug-related radioactivity also distributed into basophilic melanin in the pigmented uveal tract and pigmented skin of Long-Evans rats and declined twofold at 24 hr.

View larger version (86K): [in this window] [in a new window]   Fig. 3.   Distribution of drug-related radioactivity in a pregnant Sprague-Dawley rat at 1 hr after a 10 mg/kg single dose of [14C]atevirdine mesylate.

Radioactivity, Atevirdine, N-Desethyl Atevirdine, and O-Desmethyl Atevirdine Levels in Plasma. Plasma concentration-time profiles of drug-related radioactivity, atevirdine, N-desethyl atevirdine, and O-desmethyl atevirdine are shown in fig. 4. Pharmacokinetic parameters are listed in table 2. In general, C_max was attained at the 1- or 4-hr time points. Concentrations of O-desmethyl atevirdine were sustained in female rats. Plasma concentrations of N-desethyl atevirdine increased at the 48-hr time point after multiple-dose administration, suggesting enterohepatic recirculation of this metabolite. Relative to AUC of drug-related radioactivity, atevirdine constituted 20% and 45% in male and female rats, respectively, given a 10 mg/kg single-dose and 49% and 74% in male and female rats, respectively, after a 100 mg/kg single-dose.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Plasma concentration-time profiles of drug-related radioactivity, atevirdine, N-desethyl atevirdine, and O-desmethyl atevirdine in male and female Sprague-Dawley rats after oral administration of [14C]atevirdine mesylate.

Metabolite Profiles of Urine and Feces. Results from the HPLC analyses of urine and feces samples for parent drug and metabolites are summarized in table 3. Representative chromatograms are shown in fig. 5. Less than 0.1% of the administered radioactivity was excreted in urine as unchanged parent drug. The major components in urine were MET-3 and MET-5. MET-3 was the major metabolite in male rats, whereas MET-5 was predominant in females. In feces of the 10 mg/kg single-dose and 20 mg/kg/day multiple-dose groups, <1.5% of the dose was unchanged atevirdine. Parent drug constituted 8.7% and 25% of the dose in the 100 mg/kg single-dose and 200 mg/kg/day multiple-dose groups, respectively. The increase in the amount of unchanged parent drug in feces at higher doses could be attributed to unabsorbed drug.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Representative HPLC radiochromatograms of rat urine and feces after a 20 mg/kg/day multiple dose of [14C]atevirdine mesylate and of microsomes from a male rat.

Microsomal Metabolism. The microsomal metabolite profile after incubation with [¹⁴C]atevirdine is shown in fig. 5. Three significant metabolites were observed (MET-8, MET-9, and MET-16), with MET-8 as the major metabolite. In addition MET-15 was observed as a minor component.

Metabolism of Atevirdine. The structures and proposed mechanism for formation of the metabolites discussed below are shown in fig. 6.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Metabolic pathway of atevirdine in the rat.

MET-3 (RT 14 min) and MET-6 (RT 23 min). Treatment of urine with -glucuronidase resulted in a decrease in MET-3 concomitant with an increase in MET-6, suggesting that MET-3 was a glucuronide or sulfate conjugate of MET-6. The UV _max at 316 nm indicated the presence of an indole ring. SIMS/MS of MET-3 (table 5) showed a protonated molecular ion at m/z 544, 164 amu higher than parent drug, and indicated loss of the ethyl chain and addition of a hydroxyl group followed by conjugation with glucuronic acid. The CI spectrum of MET-6 (table 5) showed a protonated molecular ion at m/z 368, 12 amu lower than atevirdine, indicating loss of the ethyl group and further hydroxylation. Structurally informative ions at m/z 452, 179, and 205 arising from cleavage of the piperazine-pyridine, carbonyl-piperazine, and carbonyl-indole linkages, respectively, indicated hydroxylation in the indole ring. The ¹H NMR spectrum (table 6) of MET-3 showed the presence of six aromatic protons, a methoxy group, a sugar moiety, and eight piperazine protons. Resonances for the ethyl chain were not observed, and the pyridine ring resonances were similar to those in N-desethyl atevirdine. In addition, the resonance for the indole H-6 proton was missing and the indole ring H-4 and H-7 protons had simplified from doublets to singlets. Assignments were confirmed with a 2D NMR homonuclear COSY experiment (table 7). These spectroscopic data identified MET-3 as N-desethyl 6-O-glucuronic acid atevirdine and MET-6 as N-desethyl 6-hydroxy atevirdine.

MET-4 (RT 16 min). The CI spectrum of MET-4 (table 5) showed a protonated molecular ion at m/z 338, 42 amu lower than parent drug, indicating loss of the methyl and ethyl groups. Cleavage of the carbonyl-indole linkage generated ions at m/z 207 and 134. These data were consistent with MET-4 as N-desethyl O-desmethyl atevirdine.

MET-5 (RT 20 min), MET-7 (RT 25 min), and MET-9 (RT 30 min). Treatment of urine with -glucuronidase resulted in hydrolysis of MET-5 and MET-7 to MET-9, a peak with similar retention time as O-desmethyl atevirdine, and incubation with sulfatase hydrolyzed MET-7 to MET-9. SIMS of MET-5 (table 5) showed a protonated molecular ion at m/z 542, 162 amu higher than parent drug, indicative of O-demethylation and conjugation with glucuronic acid, and key fragment ions indicated that the glucuronic acid was located in the indole ring. The CI spectra of MET-7 and MET-9 showed a protonated molecular ion at m/z 366 and similar fragmentation as MET-5 (table 5). The ¹H NMR spectrum (table 6) of MET-5 showed the presence of seven aromatic protons, an ethyl group, a sugar moiety, and eight piperazine protons. A resonance for the methoxy group was not observed. The chemical shifts for the pyridine H-4', H-5', and H-6' protons were comparable to those of O-desmethyl atevirdine. Resonances for the indole ring H-3 and H-7 were similar to those in atevirdine, while the indole ring H-4 and H-6 were shifted 0.2-0.3 ppm downfield, compared with those in atevirdine, consistent with the presence of a sugar moiety at the indole C-5. Proton assignments were confirmed with a 2D COSY NMR experiment (table 7). ¹³C NMR resonances are assigned in table 7. These spectroscopic data identified MET-5 as O-desmethyl 5-glucuronic acid atevirdine, MET-7 as O-desmethyl 5-sulfate atevirdine, and MET-9 as O-desmethyl atevirdine.

MET-8 (RT 28 min) and MET-10 (RT 22 min). MET-8 had similar retention time as N-desethyl atevirdine (U-89255). The CI spectrum of MET-8 (table 5) showed a protonated molecular ion at m/z 352, 28 amu lower than parent drug, indicating loss of the ethyl group. Fragmentation of the piperazine-pyridine (m/z 95 and 260), carbonyl-piperazine (m/z 179 and 176), and carbonyl-indole (m/z 207 and 148) linkages identified MET-8 as N-desethyl atevirdine. The CI spectrum (table 5) of MET-10 was similar to that of MET-8, indicating that MET-10 was a conjugate of N-desethyl atevirdine, probably a sulfamate.

MET-11 (RT 22 min) and MET-12 (RT 20 min). The CI spectra of MET-11 and MET-12 (table 5) gave protonated molecular ions at m/z 572 and 396, respectively, and indicated that MET-11 was the glucuronide conjugate of MET-12. Ions for MET-11 at m/z 123, 207, and 235 indicated the pyridine and piperazine rings were intact. Only the ion at m/z 123 was observed for MET-12. Both MET-11 and MET-12 showed an ion at m/z 164, corresponding to cleavage of the carbonyl-indole bond to 5-methoxy-hydroxy indole. These data established that MET-11 and MET-12 were hydroxylated at the indole ring and, in the case of MET-11, further conjugated with glucuronic acid.

MET-13 (RT 22 min). The CI spectrum of MET-13 (table 5) showed a protonated molecular ion at m/z 368, 12 amu lower than atevirdine, indicating loss of the ethyl group and addition of a hydroxyl moiety. Ions at m/z 111, 195, and 223 resulting from cleavage of the piperazine-pyridine, carbonyl-piperazine, and carbonyl-indole bonds, respectively, indicated N-deethylation and hydroxylation of the pyridine ring. Ions at m/z 260, 176, and 148 indicated that the indole and piperazine rings were intact. These data established MET-13 as N-desethyl atevirdine hydroxylated at the pyridine ring.

MET-14 (RT 23 min) and MET-15 (RT 23 min). The CI spectra of MET-14 and MET-15 (table 5) gave protonated molecular ions at m/z 572 and 396, respectively, indicating that MET-14 was the glucuronide conjugate of MET-15. The protonated molecular ion for MET-15 was 16 amu higher than atevirdine and indicated addition of a hydroxyl group. Ions at m/z 260 and 148 established that the indole and piperazine rings were intact, while ions at m/z 139, 223, and 251 indicated hydroxylation of the pyridine ring. These spectroscopic data identified MET-14 and MET-15 as atevirdine glucuronidated and hydroxylated at the indole ring, respectively.

MET-16 (RT 30 min). The CI spectrum of MET-16 (table 5) gave a protonated molecular ion at m/z 396, 16 amu higher than atevirdine, indicating addition of a hydroxyl group. Ions at m/z 123, 207, and 235 indicated the pyridine and piperazine rings were intact, while ions at m/z 164 and 192 suggested hydroxylation at the indole ring.

Atevirdine (RT 36 min). The CI spectrum of atevirdine showed a protonated molecular ion at m/z 380 and characteristic fragments (summarized in table 5).

	Discussion

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The results from this study demonstrate that atevirdine was rapidly absorbed, distributed to most tissues and organs, and metabolized extensively before excretion. Peak plasma values of atevirdine at the lower doses suggested rapid absorption. Absorption in the high-dose groups appeared to be prolonged, compared with that of the lower doses; however, this could be attributed to the limited time points in this study. In all dose groups, plasma concentrations of N-desethyl atevirdine and O-desmethyl atevirdine were lower than plasma concentrations of atevirdine. The increase in plasma concentrations of N-desethyl atevirdine at the 48-hr time point after multiple-dose administration suggested enterohepatic recirculation of this metabolite. Systemic exposure to atevirdine was significantly higher in female rats than in male rats. In addition, the ratio of AUC of atevirdine to AUC of drug-related radioactivity was approximately twofold higher in females than in males, while the ratio of AUC of N-desethyl atevirdine to AUC of drug-related radioactivity was nearly twofold lower in females than in males. In contrast, systemic exposure to O-desmethyl atevirdine and the ratio of AUC of O-desmethyl atevirdine to AUC of drug-related radioactivity were two- to fivefold higher in female rats than in male rats. These gender-differences are explained by the involvement of CYP3A in the biotransformation of atevirdine to N-desethyl atevirdine (Rosser et al., 1994). The observed gender differences in the biotransformation of atevirdine to O-desmethyl atevirdine suggest the involvement of a female-dominant cytochrome P450. The AUC of atevirdine increased more than proportionally with dose and indicated that the pharmacokinetics of atevirdine were nonlinear. In addition, substantial interanimal variability in the values of the pharmacokinetic parameters was observed. These findings are in agreement with results from clinical trials (Been-Tiktak et al., 1995; Reichman et al., 1995; Ward et al., 1992). The higher plasma atevirdine concentrations than the plasma metabolite concentrations from the present study differed from those in a previous clinical study (Been-Tiktak et al., 1995) in which serum metabolite concentrations typically exceeded the serum atevirdine concentrations. Thus the human appears to have a higher capacity to clear atevirdine via metabolism to N-desethyl atevirdine than does the rat.

Atevirdine and its metabolites were widely distributed to all tissues, with no gender differences in the distribution pattern. Atevirdine and its metabolites effectively crossed the blood-brain barrier. Brain concentrations of drug-related radioactivity were 50%-100% of blood concentrations in albino rats at 1 hr after dose, although penetration into the brain was transient. The results from this study differed from those in an earlier rat study in which atevirdine had a constant brain/plasma ratio of 0.4 (Anstadt et al., 1991); however, the present study quantified drug-related radioactivity, whereas the earlier study measured atevirdine. The ability of atevirdine to cross the blood-brain barrier was in contrast to that of delavirdine (Romero et al., 1993; Romero, 1994b), a structurally related reverse transcriptase inhibitor. In a mouse study, brain concentrations of delavirdine were <3% of plasma concentrations (Chang et al., 1997b). Likewise, brain concentrations of AZT in rats were 10% of the concurrent concentrations in plasma at 1 hr after a 10 mg/kg ip dose (Daluge et al., 1997). The greater lipophilicity of atevirdine (The Upjohn Company, 1993), as measured by the logarithm of the n-octanol-water partition coefficient, allows a relatively higher uptake of atevirdine into the brain, compared with delavirdine (The Upjohn Company, 1995) and AZT (Daluge et al., 1997; Tsuzuki et al., 1994; Zimmerman et al., 1987). The present study also showed that atevirdine and its metabolites crossed the placenta, with concentrations of drug-related radioactivity in fetal tissues typically higher than those in maternal tissues. Levels of radioactivity in fetal blood were one- to fourfold higher than those in maternal circulation. Levels of drug-related radioactivity in fetal brain were slightly lower than those in maternal brain at 1 hr after dose; however, levels in fetal brain remained quantifiable at the 24-hr time point. The maternal-fetal transfer observed in rats is in good agreement with results from an ex vivo human placenta study, in which atevirdine was found to have at least twice the clearance index of AZT and more than five times greater than that of ddI (Roberts et al., 1995). The high rate of maternal-fetal transfer and the high brain penetration suggest that atevirdine may have clinical utility for the treatment of fetuses of HIV-1-infected mothers and of HIV-1-associated brain disease.

Excretion was rapid, with most of the drug-related radioactivity excreted in feces. Less than 0.1% of the administered radioactivity was excreted in urine as unchanged parent drug, indicating that atevirdine was almost completely metabolized before elimination in urine. A gender difference in the urinary excretion of MET-3 and MET-5 was observed. MET-3, N-desethyl 6-O-glucuronic acid atevirdine, was the major metabolite in male rats, whereas MET-5, O-desmethyl 5-O-glucuronic acid atevirdine, was predominant in females. Likewise, a higher percentage of the radioactive dose was excreted in feces as MET-9, O-desmethyl atevirdine, in females, compared with males. These gender differences were consistent with the involvement of the male-dominant CYP3A in the N-deethylation of atevirdine and a female-dominant cytochrome P450 in the O-demethylation of atevirdine.

The metabolites of atevirdine were identified by LC/MS. The characteristic fragmentation for atevirdine and its metabolites observed by SIMS and CI/MS allowed assignment of hydroxylation or the glucuronic acid moiety on the indole ring at either the indole or pyridine ring, although the specific position of substitution was not determined for minor metabolites. The major metabolites, MET-3 and MET-5, were isolated and identified by NMR spectroscopy. Comparison of the proton resonances for the metabolites to those for parent drug facilitated assignment of the substitution position. Assignments of the proton resonances were confirmed with 2D NMR experiments. Because of the chromatographic properties of MET-1 and MET-2, these metabolites were not further characterized.

The microsomal metabolite profiles were consistent with the observed in vivo metabolism. Microsomal formation of N-desethyl atevirdine, O-desmethyl atevirdine, and hydroxy indole atevirdine was increased in male rats treated with PCN, suggesting the likely involvement of CYP3A in the formation of all three metabolites. However, since the ratio of metabolite formation changes from males to females and in males after PCN treatment, it seems likely that other P450 isoforms also contribute to the metabolism of atevirdine. Preliminary kinetic analysis for metabolite formation revealed apparent K_ms ranging from 10 to 22 µM and linear Eadie-Hofstee plots, indicating apparent single or closely related K_m processes (data not shown). Thus partial saturation of metabolic clearance might be expected as plasma levels of atevirdine exceed the measured K_ms. Correlation of sample-to-sample variation for metabolite formation among 23 human liver microsomal samples showed high correlations (r>0.96) of catalytic rates for the three metabolites, with a marker for CYP3A4 (testosterone 6-hydroxylation) and with each other (data not shown).

A proposed scheme for the metabolism of atevirdine is shown in fig. 6. The metabolism of atevirdine in the rat involved four pathways: First, N-dealkylation to MET-8, followed by conjugation with sulfate (MET-10) (alternatively, N-desethyl atevirdine is hydroxylated at the pyridine ring to MET-13 or at C-6 of the indole ring to MET-6, followed by conjugation with glucuronic acid to MET-3); second, O-demethylation of atevirdine to MET-9, followed by conjugation with glucuronic acid (MET-5) or sulfate (MET-7)O-demethylation of MET-8 or N-deethylation of MET-9 give MET-4; third, pyridine ring hydroxylation of atevirdine to MET-15 and subsequent conjugation with glucuronic acid to MET-14; fourth, indole ring hydroxylation of atevirdine to MET-12 and MET-16, and further glucuronidation to MET-11.

N-Dealkylation is one of the major pathways of biotransformation for both atevirdine and delavirdine (Chang et al., 1997a) and results in inactive metabolites (Romero et al., 1994). On the other hand, biological activity is retained upon O-demethylation of atevirdine (Romero et al., 1993). Despite the structural similarities between atevirdine and delavirdine, the metabolic hydroxylation occurs at different sites. Hydroxylation at the C-6 position of the indole ring was a major pathway of atevirdine in the rat. In contrast, the metabolism of the structurally similar delavirdine in the rat favored hydroxylation at the C-4' and C-6' positions of the pyridine ring (Chang et al., 1997a). Moreover, not only was hydroxylation of the indole ring a minor pathway for delavirdine, but it occurred at the C-4 position (Chang et al., 1997a). This illustrates that the position of metabolic hydroxylation is influenced by the type of substituents on the ring as predicted by theories of aromatic electrophilic substitution (Foye, 1981).

In summary, atevirdine was rapidly absorbed, widely distributed to most tissues and organs, and metabolized extensively before excretion. Its high degree of maternal-fetal transfer and its effectiveness in crossing the blood-brain barrier hold promise for the treatment of HIV-1 infection.