RESUMEN
P450 complex formation and the unusual pharmacokinetics of methylenedioxyphenyl HIV protease inhibitors were examined by in vitro studies using human and rat liver microsomes and by in vivo oral dosing studies. In vitro spectral studies indicated that the formation of a P450 complex having absorbance maxima at 425 and 456 nm was time and concentration dependent; 27-60% of the total P450 was complexed in dexamethasone-induced rat liver microsomes after a 30-min incubation with 100 microM HIV protease inhibitors. Methoxy substitution on the phenyl ring of the methylenedioxyphenyl moiety increased formation of the P450 complex, whereas chlorine substitution markedly decreased the P450 complexation. Kinetic studies on the P450 complex formation indicated that both methoxy and chlorine substitution affected the maximum complex formation rate (Vmax), while it had little effect on Km values (approximately 10 microM). This complexation in human liver microsomes was inhibited markedly by an anti-CYP3A1 antibody. Furthermore, the P450 complex formation resulted in a time-dependent loss of CYP3A-catalyzed marker activities (testosterone 2beta/6beta-hydroxylase) in both rat and human liver microsomes. Collectively, these results point to the involvement of CYP3A isoforms in P450 complexation by methylenedioxyphenyl HIV protease inhibitors. Additionally, after oral administration to rats, one of these HIV protease inhibitors (Compound I), which complexed P450 to the greatest extent, showed no elimination over a period of 500 min after administration of the highest dose. It is suggested that formation of a quasi-irreversible metabolite-CYP3A complex with methylenedioxyphenyl HIV protease inhibitors was responsible for the CYP3A-selective time-dependent loss of catalytic function and the unusual dose-dependent pharmacokinetics after oral administration.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/metabolismo , Inhibidores de la Proteasa del VIH/farmacología , Isoenzimas/metabolismo , Microsomas Hepáticos/efectos de los fármacos , Animales , Biotransformación , Catálisis , Cromatografía Líquida de Alta Presión , Citocromo P-450 CYP3A , Inhibidores de la Proteasa del VIH/metabolismo , Inhibidores de la Proteasa del VIH/farmacocinética , Semivida , Humanos , Hidroxilación , Masculino , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Indinavir, a potent and specific inhibitor of human immunodeficiency virus protease, is used for the treatment of AIDS. This study was designed to investigate the sex-related differences in kinetics and metabolism of indinavir in rats, dogs, and monkeys to support the toxicity studies. When given intravenously, indinavir was cleared rapidly in a polyphasic manner in all species. Indinavir exhibited significant differences in elimination kinetics among species. The rat had the highest plasma clearance (CLp; 41-89 ml/min/kg), and the dog had the lowest CLp (15-26 ml/min/kg), with the monkey exhibiting an intermediate value (36-39 ml/min/kg). Furthermore, marked sex-related differences in CLp were observed in rats and dogs, but not in monkeys. The CLp was 89 ml/min/kg for male rats and 41 ml/min/kg for female rats. In contrast to rats, female dogs cleared indinavir more rapidly than male dogs; the CLp was 26 ml/min/kg for female dogs and 15 ml/min/kg for male dogs. Consistent with the in vivo observations, hepatic microsomes from male rats had a substantially higher metabolizing activity toward indinavir than that from females, whereas liver microsomes from female dogs catalyzed the drug at a higher rate than that from male dogs. Qualitatively, in vitro metabolic profiles of indinavir were similar among species and between male and female animals. Studies with an anti-rat cytochrome P450 (CYP) 3A1 antibody pointed to the probable involvement of isoforms in the CYP3A subfamily in the oxidative metabolism of indinavir in both males and females of all species. The functional activity of CYP3A measured by the formation of testosterone 6beta-hydroxylation and immunoblot analysis of the level of CYP3A proteins strongly suggested that gender differences in the levels of CYP3A isoforms may contribute to the observed sex-related differences in indinavir metabolism in rats and dogs.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Inhibidores de la Proteasa del VIH/farmacocinética , Indinavir/farmacocinética , Animales , Área Bajo la Curva , Cromatografía Líquida de Alta Presión , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/metabolismo , Perros , Femenino , Inhibidores de la Proteasa del VIH/administración & dosificación , Humanos , Técnicas In Vitro , Indinavir/administración & dosificación , Inyecciones Intravenosas , Macaca mulatta , Masculino , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Ratas , Ratas Sprague-Dawley , Caracteres Sexuales , Especificidad de la EspecieRESUMEN
Indinavir, a potent and specific inhibitor of human immunodeficiency virus protease, is undergoing clinical investigation for the treatment of acquired immunodeficiency syndrome. The studies described herein were designed to characterize the absorption, distribution, metabolism, and excretion of the drug in rats, dogs, and monkeys. Indinavir exhibited marked species differences in elimination kinetics. The plasma clearance was in the rank order: rat (107 ml/min/kg) > monkey (36 ml/min/kg) > dog (16 ml/min/kg). Significant differences in the bioavailability of indinavir also were observed. When given orally as a solution in 0.05 M citric acid, the bioavailability varied significantly from 72% in the dog to 19% in the monkey, and 24% in the rat. These differences in bioavailability were attributed mainly to species differences in the magnitude of hepatic first-pass metabolism. The distribution of indinavir was studied only in rats, both intravenously and orally. Intravenously, indinavir was distributed widely throughout the body. Brain uptake studies showed that indinavir penetrated the blood-brain barrier, but that the penetration was limited. After oral administration, indinavir was distributed rapidly into and out of the lymphatic system. The rapid lymph transfer is of clinical relevance, because a primary clinical hallmark of acquired immunodeficiency syndrome is the depletion of CD4 lymphocytes. Biliary and urinary recovery studies revealed that metabolism was the major route of indinavir elimination in all species, and N-dealkylation, N-oxidation, and hydroxylation seemed to be the major pathways. Although limited to qualitative aspects, the metabolite profile obtained from in vitro microsomal studies generally reflected the in vivo oxidative metabolism of indinavir in all species studies. Results from the chemical and immunochemical inhibition studies indicated the possible involvement of isoforms of the CYP3A subfamily in the oxidative metabolism of indinavir in rats, dogs, and monkeys. This is consistent with our previous studies, which have shown that CYP3A4 is the isoform responsible for the oxidative metabolism of indinavir in human liver microsomes. Furthermore, the in vivo oxidative metabolism of indinavir in rats, dogs, and monkeys was qualitatively similar to that in humans. The high degree of similarity in the metabolite profiles of drug metabolism between animals and humans validates the use of these animal models for toxicity studies of indinavir. Attempts were made to quantitatively extrapolate in vitro metabolic data to in vivo metabolism. With the application of the well-stirred and parallel-tube models, the hepatic clearance and hepatic extraction ratio were calculated using the in vitro Vmax/Km values. In rats, the predicted hepatic clearance (31 ml/ min/kg) and hepatic extraction ratio (0.47) agreed well with the observed in vivo hepatic clearance (43 ml/min/kg) and hepatic extraction ratio (0.68). In addition, the hepatic clearance of indinavir was predicted reasonably well in dogs and monkeys. Based on the in vitro intrinsic clearance of human liver microsomes, a small but significant hepatic first-pass metabolism (ca. 25%) is expected in humans.
Asunto(s)
Inhibidores de la Proteasa del VIH/farmacocinética , Indinavir/farmacocinética , Animales , Área Bajo la Curva , Perros , Inhibidores de la Proteasa del VIH/metabolismo , Haplorrinos , Indinavir/metabolismo , Masculino , Microsomas Hepáticos/metabolismo , Ratas , Ratas Sprague-Dawley , Especificidad de la EspecieRESUMEN
A high-performance liquid chromatographic method coupled with liquid-liquid sample extraction and ultraviolet detection has been developed for the quantification of L-735,524 (I), a potent, highly selective and orally bioavailable inhibitor of recombinant human immunodeficiency virus (HIV) protease in rat, dog and monkey plasma. The present method is reproducible and reliable with limits of quantification of 25, 12.5 and 6.25 ng/ml, respectively for rat, dog and monkey plasma. The standard curve was linear over the range of 6.25-2000 ng/ml in the biological fluid. The mean coefficients of variation for concentration within the range of standard curve were 7.94, 6.91 and 4.52%, respectively, for intra-day analysis and 5.58, 9.27 and 5.45%, respectively, for inter-day analysis. The recoveries of I and L-707,943 (II), an analog of I used as the internal standard, from plasma samples were all over 88% through the extraction procedure. I and II are stable in mobile phase over a 48-h period while waiting for injection at ambient temperature and over a 144-h period in rat, dog and monkey plasma while stored at -20 degrees C. Aqueous solubility of I is pH dependent, 60 mg/ml at pH 3.5 and 0.3 mg/ml at pH 4.8. The analytic procedures described in this report have been successfully employed to quantify the concentration of I in rat, dog and monkey plasma and provide the kinetic information for toxicological and pharmacological studies.
Asunto(s)
Cromatografía Líquida de Alta Presión , Inhibidores de la Proteasa del VIH/sangre , Piridinas/sangre , Administración Oral , Animales , Perros , Inhibidores de la Proteasa del VIH/administración & dosificación , Inhibidores de la Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/farmacocinética , Humanos , Concentración de Iones de Hidrógeno , Indinavir , Inyecciones Intravenosas , Absorción Intestinal , Macaca mulatta , Masculino , Piridinas/administración & dosificación , Piridinas/química , Piridinas/farmacocinética , Ratas , Ratas Sprague-Dawley , Solubilidad , Soluciones , Espectrofotometría UltravioletaRESUMEN
L-735,524, a potent and specific inhibitor of human immunodeficiency virus protease, is currently under investigation for the treatment of acquired immunodeficiency syndrome. The aqueous solubility of L-735,524 was pH-dependent, > 100 mg/ml at pH below 3.5 and 0.03 mg/ml at pH 6. When L-735,524 was given orally as a suspension in 0.5% methocel (pH 6.5) at 10 mg/kg, the bioavailability was approximately 16% for both dogs and rats. When the same dose of the drug was administered in 0.05 M citric acid (pH 2.5), the bioavailability increased 4.5-fold in dogs (72%), but only slightly in rats (24%). The pH- and species-dependent differences in bioavailability observed in rats and dogs may be because of differences in the rate of gastric acid secretion and in the magnitude of hepatic first-pass effect. Gastric acid secretion is poor in dogs but substantial in rats. When L-735,524 was administered in 0.5% methocel, a large portion of the drug in dogs, but not in rats, remained undissolved, resulting in poor absorption in dogs. On the other hand, when L-735,524 was administered in citric acid, most of the drug would be in solution allowing for better absorption in dogs. The hypothesis of pH-dependent absorption was further supported by the findings that absorption was significantly increased in dogs after feeding, but substantially decreased in rats after pretreatment with famotidine, a potent H2-receptor antagonist. L-735,524 underwent an extensive first-pass metabolism in rats, but not in dogs.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Inhibidores de la Proteasa del VIH/farmacocinética , Piridinas/farmacocinética , Administración Oral , Animales , Citratos/química , Ácido Cítrico , Perros , Inhibidores de la Proteasa del VIH/sangre , Inhibidores de la Proteasa del VIH/química , Concentración de Iones de Hidrógeno , Indinavir , Absorción Intestinal , Hígado/metabolismo , Masculino , Metilcelulosa/farmacología , Piridinas/sangre , Piridinas/química , Ratas , Ratas Sprague-Dawley , Solubilidad , Especificidad de la EspecieRESUMEN
L-754,394 is a potent and specific inhibitor of the HIV-1 encoded protease that is essential for the maturation of the infectious virus. The drug exhibited dose-dependent kinetics in all species studied (rat, dog and monkey); the apparent clearance decreased when the dose was increased. However, the dose-dependency cannot be explained by Michaelis-Menten kinetics. L-754,394 in plasma declined log-linearly with time, but with an apparent half-life that increased with dose. The apparent terminal half-life of L-754,394 in rats increased from 20 min at 0.5 mg/kg i.v. to 118 min at 10 mg/kg i.v. Furthermore, L-754,394 exhibited time-dependent pharmacokinetics. After chronic i.v. doses for 7 days (1 mg/kg/dose/day), the apparent clearance of L-754,394 in rats decreased from 87 ml/min/kg after the first dose to 25 ml/min/kg after the last dose. Similar results were observed in dogs and monkeys. In vitro spectral studies indicated that approximately 40 to 60% of the content of cytochrome P-450 was inactivated when L-754,394 (10 microM) was incubated with rat, dog and monkey liver microsomes in the presence of NADPH. Little or no inactivation of cytochrome P-450 was observed when either NADPH or L-754,394 was omitted. In addition, L-754,394 selectively inhibited CYP 2C11-dependent testosterone 2 alpha- and 16 alpha-hydroxylase activity and CYP 3A1/2-dependent testosterone 6 beta-hydroxylase activity, but not CYP 2D1/2-dependent bufuralol 1'-hydroxylase activity nor CYP 1A2-dependent phenacetin O-deethylase activity in rat liver microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)