Preclinical pharmacokinetics of MK-0974, an orally active calcitonin-gene related peptide (CGRP)-receptor antagonist, mechanism of dose dependency and species differences.

The underlying mechanism for low oral bioavailability of MK-0974, a potent calcitonin-gene related peptide (CGRP)-receptor antagonist, in monkeys and for species-dependent non-linear pharmacokinetics in monkeys and rats were investigated. In monkeys, MK-0974 displayed moderate clearance (14-20 ml min(-1) kg(-1)), while oral bioavailability was 6%. The pharmacokinetics of MK-0974 remained linear across 0.5-10 mg kg(-1) intravenous dose in monkeys, but the oral area under the plasma concentration-time curve (AUC) increase (5-30 mg kg(-1)) was 15-fold over dose-proportional. Based on a comparison of AUC following hepatic portal vein administration and cephalic vein infusion, MK-0974 exhibited a low-to-moderate hepatic extraction ratio (36%) in monkeys. Following oral dose of [14C]MK-0974 to monkeys, the hepatic portal AUC ratio of MK-0974 versus total radioactivity was 0.32, and the total radioactivity recovered in bile and urine was 45-83%. MK-0974 undergoes significant oxidative metabolism (cytochrome P450 (CYP) 3A) in monkey intestinal microsomes. In contrast, oral AUC of MK-0974 in rats was near dose-proportional (15-100 mg kg(-1)). Following oral administration of [14C]MK-0974 to rats, the hepatic portal AUC ratio of MK-0974 to total radioactivity (0.67) was higher than in monkeys. Additionally, the metabolic rate of MK-0974 was slower in rat than in monkey intestinal microsomes. Collectively, intestinal first-pass metabolism played a significant role in the low oral bioavailability in monkeys and contributed to the species-dependent non-linear oral pharmacokinetics in rats and monkeys of MK-0974.

Validation of a microdose probe drug cocktail for clinical drug interaction assessments for drug transporters and CYP3A.

A microdose cocktail containing midazolam, dabigatran etexilate, pitavastatin, rosuvastatin, and atorvastatin has been established to allow simultaneous assessment of a perpetrator impact on the most common drug metabolizing enzyme, cytochrome P450 (CYP)3A, and the major transporters organic anion-transporting polypeptides (OATP)1B, breast cancer resistance protein (BCRP), and MDR1 P-glycoprotein (P-gp). The clinical utility of these microdose cocktail probe substrates was qualified by conducting clinical drug interaction studies with three inhibitors with different in vitro inhibitory profiles (rifampin, itraconazole, and clarithromycin). Generally, the pharmacokinetic profiles of the probe substrates, in the absence and presence of the inhibitors, were comparable to their reported corresponding pharmacological doses, and/or in agreement with theoretical expectations. The exception was dabigatran, which resulted in an approximately twofold higher magnitude for microdose compared to conventional dosing, and, thus, can be used to flag a worst-case scenario for P-gp. Broader application of the microdose cocktail will facilitate a more comprehensive understanding of the roles of drug transporters in drug disposition and drug interactions.

Bioavailability of cyclosporine with concomitant rifampin administration is markedly less than predicted by hepatic enzyme induction.

The pharmacokinetics of cyclosporine was studied in six healthy volunteers after administration of the drug orally (10 mg/kg) and intravenously (3 mg/kg) with and without concomitant rifampin administration. Both blood and plasma (separated at 37 degrees C) samples were analyzed for cyclosporine concentration. For blood and plasma, respectively, clearances of cyclosporine were calculated to be 0.30 and 0.55 L/hr/kg, values for volume of distribution at steady state were 1.31 and 1.68 L/kg, and bioavailabilities were 27% and 33% during the pre-rifampin phase. Post-rifampin phase clearances of cyclosporine were 0.42 and 0.79 L/hr/kg, values for volume of distribution at steady state were 1.36 and 1.35 L/kg, and bioavailabilities were 10% and 9% for blood and plasma, respectively. Rifampin not only induces the hepatic metabolism of cyclosporine but also decreases its bioavailability to a greater extent than would be predicted by the increased metabolism. The decreased bioavailability most probably can be explained by an induction of intestinal cytochrome P450 enzymes, which appears to be markedly greater than the induction of hepatic metabolism.

Non-peptide glycoprotein IIb/IIIa inhibitors. 17. Design and synthesis of orally active, long-acting non-peptide fibrinogen receptor antagonists.

The synthesis and pharmacological evaluation of 5 (L-738, 167), a potent, selective non-peptide fibrinogen receptor antagonist is reported. Compound 5 inhibited the aggregation of human gel-filtered platelets with an IC50 value of 8 nM and was found to be > 33000-fold less effective at inhibiting the attachment of human endothelial cells to fibrinogen, fibronectin, and vitronectin than it was at inhibiting platelet aggregation. Ex vivo platelet aggregation was inhibited by > 85% 24 h after the oral administration of 5 to dogs at 100 micrograms/kg. The extended pharmacodynamic profile exhibited by 5 appears to be a consequence of its high-affinity binding to GPIIb/IIIa on circulating platelets and suggests that 5 is suitable for once-a-day dosing.

(+)-bufuralol 1'-hydroxylation activity in human and rhesus monkey intestine and liver.

(+)-Bufuralol 1'-hydroxylation, a commonly used marker of hepatic CYP2D6 activity, was investigated in human and rhesus monkey intestinal microsomes and compared with that in hepatic microsomes. The cumene hydroperoxide (CuOOH)-mediated metabolism of (+)-bufuralol suggested that at least two enzymes were responsible for bufuralol 1'-hydroxylation in both human and monkey intestinal microsomes. In contrast, the kinetics of the CuOOH-mediated metabolism in human and monkey livers were monophasic. The Km values for the higher affinity component of the intestinal enzyme(s) of both species were similar to, while the corresponding Vmax values were much lower than, those obtained with the livers. Bufuralol metabolism mediated by NADPH exhibited biphasic kinetics and was less efficient than that observed in the presence of CuOOH in both human and monkey intestines, in agreement with the observations in the livers. Inhibition of bufuralol hydroxylase activity in the intestine and liver preparations from the same species by known CYP2D6 inhibitors/substrates was qualitatively similar. Quinidine was the most potent inhibitor of (+)-bufuralol 1'-hydroxylation in all tissues studied. Western immunoblots using anti-CYP2D6 peptide antibody revealed a protein band in human and monkey intestinal microsomes of the same molecular weight as that observed in the liver preparations. The intestinal CYP2D protein content appeared to be much less than that of liver, and correlated with the (+)-bufuralol hydroxylase activity. Immunoinhibition studies indicated significant (up to 50%) inhibition of the CuOOH-mediated (+)-bufuralol metabolism in human and monkey intestines only by anti-CYP2D6, and not by anti-CYP2A6, or anti-CYP2E1. Inhibition of the bufuralol 1'-hydroxylase activity by anti-rat CYP3A1 was only slight (20%) in human, but marked (60-65%) in monkey intestinal microsomes. The hepatic metabolism of (+)-bufuralol in humans and monkeys was only inhibited (75%) by anti-CYP2D6, but not by anti-CYP3A1. Overall, the results suggest that (1) tissue and species differences exist in the catalysis of (+)-bufuralol 1'-hydroxylation, and (2) CYP2D6-related enzymes are partially or primarily responsible for the bufuralol hydroxylase activity in human and monkey intestines or monkey liver.

Transport and metabolism of cyclosporine in isolated rat hepatocytes. The effects of lipids.

The effects of lipids on the uptake and metabolism of cyclosporine (CyA) were investigated in isolated rat hepatocytes. In the absence of lipids, CyA was rapidly taken up (reaching apparent steady state within 5 min) and highly associated with the cells (more than 80%). The CyA uptake was concentration independent over the concentration range studied (0.6 to 11.2 micrograms/mL). Metabolism, however, was relatively slow and saturable. Except for cholesterol (at concentrations up to 15.5 mM), all lipids tested [oleic acid; low density lipoproteins (LDL); and high density lipoproteins (HDL)] reduced CyA cell uptake as well as its metabolism in a concentration-dependent manner. The effects of LDL were much more pronounced when compared to those of HDL and oleic acid. At an LDL concentration of 1 microM, drug uptake, indicated by the cell-associated concentration at steady state, was about 49% of the control value, while CyA metabolism was inhibited completely. Drug uptake of about 82 and 91% and CyA disappearance of 75 and 68% of the relevant control values were observed with HDL and oleic acid at concentrations of 10 microM and 0.7 mM, respectively. Apparently, lipids decreased CyA metabolism by reducing the concentration of CyA available for transport into the cells. These findings further support the suggestion of an important role for plasma lipids in the disposition of CyA.

Pharmacokinetics of orally and intravenously administered cyclosporine in pre-kidney transplant patients.

The pharmacokinetics of cyclosporine (CSA) and four metabolites were evaluated in eight hemodialysis subjects awaiting renal transplantation to compare metabolic patterns with those observed in post-transplant patients and normal volunteers. Each subject received a single 4-mg/kg intravenous and a single 10-mg/kg oral dose separated by a 1-week washout period. Blood samples were collected before and at .5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 14, and 24 hours after CSA dosing. Cyclosporine blood, plasma, and metabolite (M17, M1, M18, M21) levels were determined by high-pressure liquid chromatography. Mean (+/- standard deviation) CSA blood clearance was .47 +/- .15 L/hour/kg, steady-state volume of distribution (Vss) was 1.9 +/- .5 L/kg, and mean residence time (MRT) was 4.4 +/- 1.8 hours after intravenous dosing. With plasma, mean clearance was .70 +/- .31 L/hour/kg, Vss was 2.4 +/- 1.2 L/kg, and MRT was 3.7 +/- 2.2 hours. Cyclosporine bioavailability (F) averaged 24 +/- 11 and 24 +/- 15%, using blood and plasma, respectively. Values for clearance and Vss were approximately 30 to 100% greater than comparable estimates in healthy volunteers, but F and MRT were not altered to this extent. These changes might be explained on the basis of decreased protein binding in uremic patients. The area under the curve ratio for M17 and M1 to CSA increased an average of 1.7- and 3.9-fold, respectively, after oral dosing compared with intravenous administration, indicating increased conversion during first-pass metabolism.

Concurrent administration of the erythromycin breath test (EBT) and oral midazolam as in vivo probes for CYP3A activity.

Given the prominent role of CYP3A in the metabolism of drugs, it is important to identify whether new chemical entities will affect this enzyme system and produce clinically relevant drug interactions. This study evaluated concomitant administration of intravenous [14C N-methyl] erythromycin (3 microCi) (erythromycin breath test; EBT) and 2 mg oral midazolam as probes of systemic and of systemic plus presystemic CYP3A activity, respectively. Twelve males received the probes in a two-period crossover fashion: one period included the probes on two occasions, 5 days apart; in the second period, 200 mg ketoconazole was given orally 2 hours prior to the probes. The within-subject CV for EBT (%14CO2/h) and midazolam AUC0-last was 4.9% and 16.9%, respectively. Ketoconazole reduced %14CO2/h by 43% and increased midazolam AUC0-last by approximately fivefold. In a nonrandomized third period (N = 5), ketoconazole was given simultaneously with midazolam (no EBT); midazolam AUC0-last was similar whether ketoconazole was given 2 hours prior to or simultaneously with the midazolam. The low midazolam dose was generally well tolerated; mild sedation was occasionally seen. Concurrent administration of the EBT and oral midazolam is a sensitive and reproducible tool to screen new chemical entities for potentially important CYP3A interactions.

Simvastatin does not affect CYP3A activity, quantified by the erythromycin breath test and oral midazolam pharmacokinetics, in healthy male subjects.

Potential for inhibition of CYP3A activity by simvastatin, an HMG-CoA reductase inhibitor, was evaluated in 12 healthy male subjects who received placebo or 80 mg of simvastatin, the maximal recommended dose, once daily for 7 consecutive days. On day 7, an intravenous injection of 3 microCi [14C N-methyl]erythromycin for the erythromycin breath test (EBT) was coadministered with a 2 mg oral solution of midazolam. The values for percent 14C exhaled during the first hour (for EBT) and the pharmacokinetic parameters of midazolam (AUC, Cmax, t1/2) were not affected following multiple once-daily oral doses of simvastatin 80 mg. The 95% confidence interval was 0.97 to 1.18 for EBT and 0.99 to 1.23 for midazolam AUC. In addition, the total urinary recoveries of midazolam and its 1'-hydroxy metabolites (free plus conjugate) obtained from both treatments were not statistically different (p > 0.200). These data demonstrate that multiple dosing of simvastatin, at the highest recommended clinical dose, does not significantly alter the in vivo hepatic or intestinal CYP3A4/5 activity as measured by the commonly used EBT and oral midazolam probes.

Interactions between simvastatin and troglitazone or pioglitazone in healthy subjects.

Two randomized, two-period crossover studies were conducted to evaluate the effects of repeat oral dosing of troglitazone (Study I) and pioglitazone (Study II) on the pharmacokinetics of plasma HMG-CoA reductase inhibitors following multiple oral doses of simvastatin and of simvastatin on the plasma pharmacokinetics of troglitazone (Study I) in healthy subjects. In both studies, each subject received two treatments. Treatment A consisted of once-daily oral doses of troglitazone 400 mg (Study I) or pioglitazone 45 mg (Study II) for 24 days with coadministration of once-daily doses of simvastatin 40 mg (Study I) or 80 mg (Study II) on Days 15 through 24. Treatment B consisted of once-daily oral doses of simvastatin 40 mg (Study I) or 80 mg (Study II) for 10 days. In Study I, the area under the plasma concentration-time profiles (AUC) and maximum plasma concentrations (Cmax) of HMG-CoA reductase inhibitors in subjects who received both troglitazone and simvastatin were decreased modestly (by approximately 30% for Cmax and approximately 40% for AUC), but time to reach Cmax (tmax) did not change, as compared with those who received simvastatin alone. Simvastatin, administered orally as a 40 mg tablet daily for 10 days, did not affect the AUC or tmax (p > 0.5) but caused a small but clinically insignificant increase (approximately 25%) in Cmax for troglitazone. In Study II, pioglitazone, at the highest approved dose for clinical use, did not significantly alter any of the pharmacokinetic parameters (AUC, Cmax, and tmax) of simvastatin HMG-CoA reductase inhibitory activity. For all treatment regimens, side effects were mild and transient, suggesting that coadministration of simvastatin with either troglitazone or pioglitazone was well tolerated. The modest effect of troglitazone on simvastatin pharmacokinetics is in agreement with the suggestion that troglitazone is an inducer of CYP3A. The insignificant effect of simvastatin on troglitazone pharmacokinetics is consistent with the conclusion that simvastatin is not a significant inhibitor for drug-metabolizing enzymes. The lack of pharmacokinetic effect of pioglitazone on simvastatin supports the expectation that this combination may be used safely.

Immunopharmacodynamic studies of cyclosporine in patients awaiting renal transplantation.

The immunopharmacodynamics of cyclosporine were investigated in eight hemodialysis patients awaiting renal transplantation. Cyclosporine was administered orally (10 mg/kg) and intravenously (4 mg/kg), with both administrations separated by at least one week. Plasma samples were processed at 37 degrees C and analyzed for specific cyclosporine and its four major metabolites (AM1, AM1c, AM9, and AM4N) using high-performance liquid chromatography. In addition, the in vitro immunosuppressive activity of these serial plasma samples was estimated as a relative percentage inhibition of third party mitogenic lymphocyte proliferation stimulated with phytohemagglutinin. The relationships between concentration and effect of cyclosporine versus time were noted. These results suggest that unchanged cyclosporine concentrations in plasma correlate with mitogen-induced lymphocyte suppression yielding significant immunosuppressant activity of cyclosporine. Control studies with plasma from healthy volunteers spiked with cyclosporine in the concentration range of 0-10,000 ng/mL were developed. A sigmoidal Emax model was fitted to the effect versus plasma concentration data. The ratio of effect versus predicted effect were calculated for intravenous cyclosporine dosing. There was a good correlation between the observed and predicted inhibitory effect.

Analysis of metabolite kinetics by deconvolution and in vivo-in vitro correlations of metabolite formation rates: studies of fibrinogen receptor antagonist ester prodrugs.

The pharmacokinetics of L-767,679, a potent fibrinogen receptor antagonist, were characterized following administration of its ethyl ester prodrug to dogs and monkeys. Deconvolution analysis was performed to determine the rate and extent of (1) the formation of L-767,679 from the prodrug in the systemic circulation, (2) the composite input (systemic and presystemic) of L-767,679 to the general circulation after oral administration of the prodrug, (3) the oral input of the prodrug, and (4) the input of the presystemically formed L-767,679 following oral administration of the prodrug. The results indicated that there were species differences in the kinetics of the disposition of L-767,679 and its prodrug. In dogs, the prodrug was absorbed faster than it was converted to the active drug, and the presystemic formation of L-767,679 contributed to about one-half of the total input of L-767,679 following oral administration of the prodrug. In monkeys, the low input of L-767,679 following oral administration of the prodrug was not due to an inefficient formation of L-767,679 in the systemic circulation but rather to the low oral bioavailability of the prodrug. Virtually all of the total oral input of L-767,679 following administration of its prodrug to monkeys resulted from the presystemic metabolism of the prodrug. These results were consistent with the finding in monkeys that the ester prodrug underwent extensive transformation to metabolites other than L-767,679. In addition, the present study also demonstrated a correlation between in vivo formation rates of L-767,679 determined using deconvolution analysis following its ethyl, methyl, and isopropyl esters in dogs and the ethyl ester in monkeys and in vitro formation rates of L-767,679 obtained following incubations of the corresponding esters with dog and monkey liver microsomes. The results suggested that deconvolution and/or convolution analysis together with in vitro metabolism results could potentially be used to predict in vivo formation rates of other ester prodrugs of L-767,679 and also plasma concentrations of L-767,679 as a function of time, following administration of its prodrugs.

UGT2B17 genetic polymorphisms dramatically affect the pharmacokinetics of MK-7246 in healthy subjects in a first-in-human study.

MK-7246, an antagonist of the chemoattractant receptor on T helper type 2 (Th2) cells, is being developed for the treatment of respiratory diseases. In a first-in-human study, we investigated whether genetic polymorphisms contributed to the marked intersubject variability in the pharmacokinetics of MK-7246 and its glucuronide metabolite M3. Results from in vitro enzyme kinetic studies suggested that UGT2B17 is probably the major enzyme responsible for MK-7246 metabolism in both the liver and the intestine. As compared with those with the UGT2B17*1/*1 wild-type genotype, UGT2B17*2/*2 carriers, who possess no UGT2B17 protein, had 25- and 82-fold greater mean dose-normalized values of area under the plasma concentration-time curve (AUC) and peak concentration of MK-7246, respectively, and a 24-fold lower M3-to-MK-7246 AUC ratio. The apparent half-life of MK-7246 was not as variable between these two genotypes. Therefore, the highly variable pharmacokinetics of MK-7246 is attributable primarily to the impact of UGT2B17 genetic polymorphisms and extensive first-pass metabolism of MK-7246.

Hepatic metabolism of MK-0457, a potent aurora kinase inhibitor: interspecies comparison and role of human cytochrome P450 and flavin-containing monooxygenase.

MK-0457 (N-[4([4-(4-methylpiperazin-1-yl)-6-[(3-methyl-1H-pyrazol-5 -yl)amino]pyrimidin-2-yl]thio)phenyl]cyclopropanecarboxamide), an Aurora kinase inhibitor in development for the treatment of cancer, was evaluated for its in vitro metabolism in different species. This compound primarily underwent N-oxidation and N-demethylation in human, monkey, dog, and rat liver preparations. However, N-demethylation was less significant in dogs. The formation of minor metabolites varied with species, but all metabolites generated in human hepatocytes were observed in animals. Results of immunoinhibition, selective chemical inhibition, thermal inactivation, and metabolism by recombinant cytochromes P450 and flavin-containing monoogygenases (FMOs) strongly suggest that CYP3A4 and FMO3 comparably contributed to MK-0457 N-oxidation in human liver microsomes, where the reaction conformed to Michaelis-Menten kinetics. These studies indicate a major role of CYP2C8 in the N-demethylation reaction, whereas CYP3A4 only made a minor contribution. However, significant substrate inhibition was observed with MK-0457 N-demethylation at high substrate concentrations (>10 microM) in human liver microsomes relative to the anticipated therapeutic exposure. A multienzyme metabolic pathway such as this may mitigate the potential of drug interactions in clinical treatment with MK-0457.

Effects of fibrates on human organic anion-transporting polypeptide 1B1-, multidrug resistance protein 2- and P-glycoprotein-mediated transport.

The effects of different fibric acid derivatives (bezafibrate, clofibrate, clofibric acid, fenofibrate, fenofibric acid and gemfibrozil) on human organic anion transporting-polypeptide 1B1 (OATP2, OATP-C, SLC21A6), multidrug resistance protein 2 (MRP2/ABCC2) and MDR1-type P-glycoprotein (P-gp/ABCB1) were examined in vitro. Cyclosporin A (a known inhibitor of OATP1B1 and P-gp), MK-571 (a known inhibitor of MRP2) and cimetidine (an organic cation) were also tested. Bezafibrate, fenofibrate, fenofibric acid and gemfibrozil showed concentration-dependent inhibition of estradiol 17-beta-D-glucuronide uptake by OATP1B1-stably transfected HEK cells, whereas clofibrate and clofibric acid did not show any significant effects up to 100 microM. Inhibition kinetics of gemfibrozil, which exhibited the most significant inhibition on OATP1B1, was shown to be competitive with a Ki = 12.5 microM. None of the fibrates showed any significant inhibition of MRP2-mediated transport, which was evaluated by measuring the uptake of ethacrynic acid glutathione into MRP2-expressing Sf9 membrane vesicles. Only fenofibrate showed moderate P-gp inhibition as assessed by measuring cellular accumulation of vinblastine in a P-gp overexpressing cell-line. Cyclosporin A significantly inhibited OATP1B1 and P-gp, whereas only moderate inhibition was observed on MRP2. The rank order of inhibitory potency of MK-571 was determined as OATP1B1 (IC50: 0.3 microM) > MRP2 (4 microM) > P-gp (25 microM). Cimetidine did not show any effects on these transporters. In conclusion, neither MRP2- nor P-gp-mediated transport is inhibited significantly by the fibrates tested. Considering the plasma protein binding and IC50 values for OATP1B1, only gemfibrozil appeared to have a potential to cause drug-drug interactions by inhibiting OATP1B1 at clinically relevant concentrations.

Simple and micro high-performance liquid chromatographic method for simultaneous determination of p-aminohippuric acid and iothalamate in biological fluids.

A simple, rapid and micro high-performance liquid chromatographic method was developed for separate or simultaneous determination of p-aminohippuric acid and iothalamate in plasma and urine using p-aminobenzoic acid as an internal standard. The method involved deproteinizing samples with two volumes of acetonitrile followed by injection of 5 microliters of deproteinized supernatant onto a C18 reversed-phase column. The mobile phase contained 3.5% acetonitrile in 0.04% phosphoric acid and flowed at a rate of 1.5 ml/min. The column effluent was monitored by an ultraviolet detector at 254 nm. Retention times for p-aminohippuric acid, iothalamate and p-aminobenzoic acid were approximately 4.5, 6 and 8 min, respectively. This method requires as little as 5 microliters of sample and can be used to measure accurately down to 1 microgram/ml p-aminohippuric acid and 0.5 microgram/ml iothalamate in plasma samples. The coefficients of variation of the assay with or without the use of internal standard were generally low (below 7%). No interferences from endogenous substances or any drugs tested were found.

Simultaneous determination of ranitidine and its metabolites in human plasma and urine by high-performance liquid chromatography.

A sensitive high-performance liquid chromatographic method was developed for the simultaneous determination of ranitidine and its metabolites, ranitidine N-oxide, ranitidine S-oxide and desmethylranitidine, in human plasma and urine. For the plasma analysis, 1-ml plasma samples spiked with phenylpyramidol as the internal standard were extracted at basic pH with acetonitrile-ethyl acetate (3:2, v/v). After evaporation and reconstitution, the samples were chromatographed on a cation-exchange column, with a mobile phase of 0.1 M sodium acetate buffer (pH 5)-acetonitrile-tetrahydrofuran (56.5:36:7.5, v/v) and ultraviolet detection at 320 nm. The extraction recoveries were 99.8, 30.4, 74.2 and 80.2% and the detection limits were 5, 15, 10 and 4 ng/ml for ranitidine, ranitidine N-oxide, ranitidine S-oxide and desmethylranitidine, respectively. For the urine analysis, a simple deproteinization with an equal volume of acetonitrile was satisfactory for sample preparation. The applicability of this method for the pharmacokinetic study of ranitidine following oral administration was demonstrated.

Effects of low-density lipoprotein and ethinyl estradiol on cyclosporine metabolism in isolated rat liver perfusions.

The effects of low-density lipoprotein (LDL) on cyclosporine (CyA) metabolism were studied in the isolated perfused rat liver, in a recirculating mode, using Krebs-Ringer buffer in the absence (control perfusion) or presence of LDL (1 microM) (LDL perfusion). In the LDL perfusions, CyA concentrations at all sampling times were about 2-fold higher, whereas the biliary excretion of CyA and measured metabolites (AM1, AM9, AM1c, and AM4N) were all lower than those obtained with the control perfusions. At the end of the perfusion (3 hr), the percentage of total CyA remaining (liver, bile, and perfusate) was significantly higher (76 +/- 1.2% to 85 +/- 2.4%) and the percentage of dose metabolized to AM9 was lower (4.8 +/- 1.2% to 2.4 +/- 0.6%) in the LDL perfusions (N = 4). These results further suggest the inhibitory effects of LDL on CyA uptake, and, thereby, its metabolism as we observed previously in isolated rat hepatocyte studies. Because ethinyl estradiol (EE) is known to increase LDL receptors in rats, we investigated the possible involvement of LDL receptors in transporting CyA into liver cells using rats pretreated with EE (5 mg/kg/day sc for 5 days). The effects of LDL in maintaining CyA perfusate concentrations, and in decreasing biliary excretion of CyA and its metabolites in the EE-treated animals, were in the same direction as those noted in animals without EE, but the differences due to LDL were not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

HPLC assay for FK 506 and two metabolites in isolated rat hepatocytes and rat liver microsomes.

Despite the current use of a standard two-step enzyme immunoassay in the clinical monitoring of the immunosuppressant FK 506, the lack of specificity for the parent drug in this assay renders it unsuitable for drug metabolism studies. An HPLC assay has been developed for studying the metabolism of FK 506 in isolated hepatocytes and microsomal mixtures. This assay allows simultaneous measurement of the parent drug and two of its time dependent metabolites. Metabolism of this drug was studied in intact rat liver cells and rat liver microsomes. We have shown that the metabolites observed are products of phase 1 oxidation reactions. Correlation of the 6 beta-testosterone hydroxylase activity with the FK 506 metabolite (M1) initial formation rate is consistent with the belief that CYP 3A isozymes are involved in FK 506 metabolism in male rats.

Drug interactions with calcium channel blockers: possible involvement of metabolite-intermediate complexation with CYP3A.

The inhibitory effects of six commonly used calcium channel blockers on three major cytochrome P-450 activities were examined and characterized in human liver microsomes. All six compounds reversibly inhibited CYP2D6 (bufuralol 1'-hydroxylation) and CYP2C9 (tolbutamide methyl hydroxylation) activities. The IC(50) values for the inhibition of CYP2D6 and CYP2C9 for nicardipine were 3 to 9 microM, whereas those for all others ranged from 14 to >150 microM. Except for nifedipine, all calcium channel blockers showed increased inhibitory potency toward CYP3A activities (testosterone 6beta-hydroxylation and midazolam 1'-hydroxylation) after 30-min preincubation with NADPH. IC(50) values for the inhibition of testosterone 6beta-hydroxylase obtained in the NADPH-preincubation experiment for nicardipine (1 microM), verapamil (2 microM), and diltiazem (5 microM) were within 10-fold, whereas those for amlodipine (5 microM) and felodipine (13 microM) were >200-fold of their respective plasma concentrations reported after therapeutic doses. Similar results also were obtained based on midazolam 1'-hydroxylase activity. Unlike the observations with mibefradil, a potent irreversible inhibitor of CYP3A, the NADPH-dependent inhibition of CYP3A activity by nicardipine and verapamil was completely reversible on dialysis, whereas that by diltiazem was partially restored (80%). Additional experiments revealed that nicardipine, verapamil, and diltiazem formed cytochrome P-450-iron (II)-metabolite complex in both human liver microsomes and recombinant CYP3A4. Nicardipine yielded a higher extent of complex formation ( approximately 30% at 100 microM), and was a much faster-acting inhibitor (maximal inhibition rate constant approximately 2 min(-1)) as compared with verapamil and diltiazem. These present findings that the CYP3A inhibition caused by nicardipine, verapamil, and diltiazem is, at least in part, quasi-irreversible provide a rational basis for pharmacokinetically significant interactions reported when they were coadministered with agents that are cleared primarily by CYP3A-mediated pathways.