MK-639 (L-735,524) is a potent human immunodeficiency virus protease inhibitor under investigation in the treatment of acquired immunodeficiency syndrome. Five in vitro approaches have been used to ...identify the cytochrome P450 isoform(s) responsible for the human microsomal oxidative metabolism of MK-639. These approaches are: 1) chemical inhibition; 2) immunochemical inhibition; 3) metabolism by cDNA-expressed human cytochrome P450 enzymes; 4) a correlation analysis; and 5) competitive inhibition of marker activities. Ketoconazole and troleandomycin, both selective inhibitors for cytochrome P450 3A4 (CYP3A4), markedly inhibited the formation of all oxidative metabolites of MK-639; whereas other inhibitors (furafylline, sulfaphenazole, quinidine, S-mephenytoin, and diethyldithiocarbamate) had little effect on MK-639 metabolism. This suggested the involvement of CYP3A4 in MK-639 metabolism. Consistent with this, an anti-rat CYP3A1 rabbit polyclonal antibody, which shows a cross-reactive inhibition of CYP3A4-dependent testosterone 6beta-hydroxylation in human liver microsomes, completely inhibited MK-639 metabolism. Human recombinant CYP3A4 showed a high metabolic activity to form all MK-639 metabolites found in native human liver microsomes. In addition, the formation of individual MK-639 metabolites correlated well with each other and with testosterone 6beta-hydroxylation in 12 different human liver microsomes, whereas no correlation was observed between MK-639 metabolite formation and bufuralol 1'-hydroxylation (or tolbutamide methyl hydroxylation). Furthermore, MK-639 strongly inhibited testosterone 6beta-hydroxylation in a concentration-dependent manner. Kinetic analysis showed that MK-639 is a very potent competitive inhibitor for testosterone 6beta-hydroxylation, with a Ki value of approximately 0.5 mu M. Collectively, these results consistently indicate that CYP3A4 is the isoform responsible for the oxidative metabolism of MK-639 in human liver microsomes.
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.
CONTEXT Both attenuated heart rate recovery following exercise and the Duke
treadmill exercise score have been demonstrated to be independent predictors
of mortality, but their prognostic value ...relative to each other has not been
studied. OBJECTIVE To assess the associations among abnormal heart rate recovery, treadmill
exercise score, and death in patients referred specifically for exercise electrocardiography. DESIGN AND SETTING Prospective cohort study conducted in an academic medical center between
September 1990 and December 1997, with a median follow-up of 5.2 years. PATIENTS A total of 9454 consecutive patients (mean SD age, 53 11 years;
78% male) who underwent symptom-limited exercise electrocardiographic testing.
Exclusion criteria included age younger than 30 years, history of heart failure
or valvular disease, pacemaker implantation, and uninterpretable electrocardiograms. MAIN OUTCOME MEASURES All-cause mortality, as predicted by abnormal heart rate recovery, defined
as failure of heart rate to decrease by more than 12/min during the first
minute after peak exercise, and by treadmill exercise score, defined as (exercise
time) − (5 × maximum ST-segment deviation) − (4 ×
treadmill angina index). RESULTS Three hundred twelve deaths occurred in the cohort. Abnormal heart rate
recovery and intermediate- or high-risk treadmill exercise score were present
in 20% (n = 1852) and 21% (n = 1996) of patients, respectively. In univariate
analyses, death was predicted by both abnormal heart rate recovery (8% vs
2% in patients with normal heart rate recovery; hazard ratio HR, 4.16; 95%
confidence interval CI, 3.33-5.19; χ2 = 158; P<.001) and intermediate- or high-risk treadmill exercise score
(8% vs 2% in patients with low-risk scores; HR, 4.28; 95% CI, 3.43-5.35; χ2 = 164; P<.001). After adjusting for age,
sex, standard cardiovascular risk factors, medication use, and other potential
confounders, abnormal heart rate recovery remained predictive of death (among
the 8549 patients not taking β-blockers, adjusted HR, 2.13; 95% CI, 1.63-2.78; P<.001), as did intermediate- or high-risk treadmill
exercise score (adjusted HR, 1.49; 95% CI, 1.15-1.92; P = .002). There was no interaction between these 2 predictors. CONCLUSIONS In this cohort of patients referred specifically for exercise electrocardiography,
both abnormal heart rate recovery and treadmill exercise score were independent
predictors of mortality. Heart rate recovery appears to provide additional
prognostic information to the established treadmill exercise score and should
be considered for routine incorporation into exercise test interpretation.
Montelukast (L-706,631, MK-0476, SINGULAIR), a potent and selective leukotriene D4 (CysLT1) receptor antagonist, is currently under development for the treatment of asthma. In vitro studies were ...conducted using human liver microsomes to evaluate: 1) the difference in the metabolic kinetics of montelukast between adult and pediatric subjects; 2) the relative contribution of flavin-containing monooxygenase and cytochrome P450 (P450) to the sulfoxidation; and 3) the P450 isoforms responsible for montelukast oxidation. No statistically significant difference was observed in the in vitro kinetics for acyl glucuronidation and oxidative metabolism between the two age groups. Results from studies on heat inactivation of flavin-containing monooxygenase and immunochemical inhibition by an anti-rat NADPH P450 reductase antibody on montelukast oxidation indicated that all oxidative metabolism of montelukast-including diastereomeric sulfoxidations, as well as 21- and methyl-hydroxylations-are catalyzed exclusively by P450. Five in vitro approaches have been used to identify the P450 isoforms responsible for the human liver microsomal oxidation of montelukast. The experimental results consistently indicated that CYP3A4 catalyzes sulfoxidation and 21-hydroxylation, whereas CYP2C9 selectively mediates methyl-hydroxylation.
L-754,394, N-2(R)-hydroxy-1(S)-indanyl-5-2(S)-(1,1-dimethylethylaminocarbonyl )-4- (furo2,3-bpyridin-5-yl)methylpiperazin-1-yl-4(S)-hydroxy-2(R) - phenylmethylpentanamide, is a potent and specific ...inhibitor of the human immune deficiency virus (HIV) protease. The drug selectively inhibited human liver microsomal CYP 3A4-dependent testosterone 6 beta-hydroxylase and CYP 2D6-dependent bufuralol 1'-hydroxylase activities in a time- and concentration-dependent manner in the presence of an NADPH-generating system. L-754,394 was found to be a very potent inactivator of CYP 3A4. Thus, for testosterone 6 beta-hydroxylase, the inactivation kinetic constants, Kl and kinact, were 7.5 microM and 1.62 min-1, respectively, and the partition ratio (moles product formed per moles enzyme inactivated) was approximately 1.35. To a lesser extent, L-754,394 also was an inactivator of CYP 2D6, for which the corresponding values for Kl, kinact and partition ratio were 32 microM, 0.18 min-1 and 40, respectively. CYP 3A4 inactivation was reduced markedly by ketoconazole, a selective CYP 3A4 inhibitor. Similarly, CYP 2D6 inactivation also was prevented by quinidine, a specific competitive inhibitor of this isoform. However, exogenously added nucleophiles (GSH, semicarbazide and N-acetylcysteine) failed to protect against P-450 inactivation. These results suggest that the inactivation process likely is mediated by a reactive metabolite of L-754,394 that alkylates, and thereby destroys, the enzyme. Furthermore, this electrophilic intermediate may not be released into the medium before the inactivation event.
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.
L-754,394, a furanopyridine derivative, is an experimental HIV protease inhibitor. Previous studies from this laboratory have demonstrated that L-754,394 is cleared very rapidly in animals, and that ...this drug is a potent mechanism-based inactivator (suicide inhibitor) for CYP3A4 in human liver microsomes. Because L-754,394 is a high-clearance drug and an enzyme inactivator, it is expected that this drug will be subject to significant first-pass metabolism, and that the degree of enzyme inactivation will be dependent not only on the dose, but also on the route of administration. The purpose of this study is to examine the effects of dose and route of administration on the kinetics of L-754,394 using rats and dogs as animal models. In both rats and dogs, L-754,394 exhibited marked dose-dependent pharmacokinetics after i.v. and oral administration. Irrespective of i.v. or oral administration, the area under the plasma concentration-time curve from zero to infinity increased with dose in a greater than proportional manner. However, the magnitude of area under the plasma concentration-time curve from zero to infinity increase was much greater after oral dosing than after i.v. administration, indicating route-dependent pharmacokinetics. Data from in vitro and in vivo studies suggested that the dose- and route-dependent pharmacokinetics were due mainly to the inactivation (destruction) of the enzymes responsible for its own metabolism.
1. The in vitro metabolism of indinavir (CRIXIVAN, MK-0639, L-735,524), an HIV protease inhibitor, was evaluated using liver microsomes from cynomolgus monkey, rhesus monkey, chimpanzee and human. ...Indinavir exhibited marked species differences in metabolism. The overall rate of indinavir metabolism varied 4-fold among primates (84 pmol/min/mg protein in cynomolgus monkey versus 20.4 pmol/min/mg protein in human) and followed the rank order: cynomolgus monkey > rhesus monkey > chimpanzee > human. 2. The cis-(indan) hydroxylated metabolite of indinavir was formed only in cynomolgus and rhesus monkey livers, whereas trans-(indan) hydroxylation and N-dealkylation were observed as the major metabolites in all primates tested. Inhibition studies with P450-selective inhibitors (ketoconazole, quinine, quinidine) and monoclonal antibodies (against CYP2D6 or CYP3A4) indicated that a cytochrome P450 isoform of the CYP2D subfamily is involved in the formation of the unique cis-(indan) hydroxylated metabolite in monkey, whereas all other oxidative metabolites, including the trans-(indan) hydroxylated metabolite, are formed by CYP3A isoform(s). 3. The present study has demonstrated that monkeys were unique in their abilities to form the stereoselective metabolite and were not appropriate surrogates for the qualitative prediction of indinavir metabolism in human.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Previous studies in vitro have revealed that L-754,394, an HIV protease inhibitor, is a potent suicide inhibitor of cytochrome P-450 enzymes. The present report examines the effect of chronic ...treatment of L-754,394 on hepatic cytochrome P-450s in adult male rats. L-754,394 was administered orally once a day for 7 days and resulted in significant changes in marker activities. An unusual parabolic (ascending, then descending) profile was observed for testosterone 2beta-/6beta-(CYP 3A1/2-catalyzed) hydroxylase activities during the 7-day treatment with 20 mg/kg L-754,394. These activities, which were elevated 2-fold on day 2, returned to basal levels by day 8. In contrast, testosterone 2alpha-/16alpha-(CYP2C11-catalyzed) hydroxylase activities showed an opposite parabolic (descending, then ascending) profile during the same period, reducing to 40% of control activities on day 4, followed by a rebounding trend. Immunoquantitation of CYP 3A1/2 and 2C11 showed that the expressed protein levels were in parallel with the associated activities. Furthermore, mRNA levels of CYP 3A2 and CYP2C11 showed the same trends as the protein expression of the respective isoforms. These observations show that L-754,394 perturbs the relative abundance of P-450 isoforms in rat liver by affecting the regulation at a pretranslational step. This may further involve a disturbance of hormonal homeostasis. Although serum levels of testosterone did not show a marked change during treatment, thyroxine and triiodothyronine markedly decreased on days 2 and 4, and subsequently increased to basal levels.
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