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.

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.

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.

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.

Disposition of a novel and potent alpha(v)beta3 antagonist in animals, and extrapolation to man.

1. The disposition of 3-[2-oxo-3-[3-(5,6,7,8-tetrahydro-[1,8]naphthyridin-2-yl) propyl]-imidazolidin-1-yl]-3(S)-(6-methoxy-pyridin-3-yl)propionic acid (compound A), a potent and selective alpha(v)beta(3) antagonist, was characterized in several animal species in support of its selection for preclinical safety studies and potential clinical development. 2. Compound A exhibited marked species differences in pharmacokinetics; the plasma clearances and bioavailabilities ranged from 33-47 ml min(-1) kg(-1) in rats and mice to 4-9 ml min(-1) kg(-1) in dogs and monkeys, and about 20% in rats to 70-80% in dogs and monkeys, respectively. Both the intravenous (i.v.) and oral kinetics of compound A were linear over the dose range studied in dogs (0.1-5 mg kg(-1) i.v. and 0.25-20 mg kg(-1) orally [p.o.]) and rats (1-30 mg kg(-1) i.v. and 4-160 mg kg(-1) p.o.). 3. Compound A was eliminated substantially by urinary excretion; the urinary recovery of the unchanged drug was 67% in rhesus, 48% in dogs and about 30% in rats. In these animal species, biotransformation was modest. 4. Following i.v. administration of [(14)C]-compound A to rats, the radioactivity rapidly distributed to all tissues investigated, with high levels of the radioactivity detected in liver, kidney and intestine soon after the drug administration. The radioactivity declined rapidly, with less than 1% of the i.v. dose remaining at 30-h post-dose. 5. Compound A was moderately bound to plasma proteins, with unbound fractions of 26, 20, 14 and 5% for rats, dogs, monkeys and humans, respectively. It was bound primarily to human alpha(1)-acid glycoprotein (about 85% binding at 0.1% concentration), as compared with human albumin (< 50% binding at 4% concentration). 6. Using simple allometry, compound A was predicted to exhibit relatively low clearance (1-3 ml min(-1) kg(-1)) and low volume of distribution (0.1-0.3 l kg(-1)) in humans. Based on the predicted values, compound A was projected to exhibit a favourable oral pharmacokinetic profile in humans, with good bioavailability (50-80%). These predicted values provided a basis for compound selection for further development.

Renal elimination of a novel and potent alphavbeta3 integrin antagonist in animals.

Compound A (3-{2-oxo-3-[3-(5,6,7,8-tetrahydro-[1,8]napthyridin-2-yl)propyl]-imidazolidin-1-yl}-3(S)-(6-methoxy-pyridin-3-yl)propionic acid), a hydrophilic zwitter-ion, is a potent and selective alphavbeta3 integrin antagonist currently under clinical development for the treatment of osteoporosis. The mechanism of renal excretion of compound A was investigated using a combination of in vivo and in vitro approaches. In rats, renal excretion of compound A involved tubular secretion; ratios between renal clearance, corrected for unbound fraction in plasma (CLr,u) and glomerular filtration rate (GFR) were greater than unity (2-5). The tubular secretion of compound A was saturable at high plasma levels (> 26 microM), and was inhibited significantly, although modestly (about twofold) by relatively high plasma concentrations of the organic anion PAH (160 microM) and the cation cimetidine (about 400 microM), but not by the P-gp inhibitor quinidine (about 50 microM). However, compound A (about 100 microM) had a minimal effect on CLr/GFRs for cimetidine and PAH. In rhesus monkeys, renal elimination of compound A also involved tubular secretion, with a CLr,u/GFR ratio of about 30. The renal secretion of compound A was not affected by either cimetidine (about 120 microM) or PAH (about 80 microM). Similarly, compound A (about 40 microM) had a minimal effect on the renal tubular secretion of both cimetidine and PAH. At the doses studied, neither rat nor monkey plasma protein binding of compound A, cimetidine or PAH was affected in the presence of each other. In vitro transport studies showed that compound A was not a substrate for P-gp in the Caco-2, human MDR1 and mouse mdr1a transfected LLC-PK1 cell lines. In an uptake study using rOAT1 and rOAT3 transfected HEK cell lines, compound A was shown to be a substrate for rat OAT3 (Km= 15 microM), but not rat OAT1. The results suggest that the tubular secretion of compound A is not mediated by P-gp, but rather is mediated, at least in part, via the organic anion transporter OAT3, the renal transporter shown to be capable of transporting both the organic anion PAH and the organic cation cimetidine. Although there is a possibility for pharmacokinetic interactions between compound A and substrates or inhibitors of OAT3, at the renal excretion level, the magnitude of interaction would likely be modest in humans at clinically relevant doses.

Stereoselective hepatic disposition of a diastereomeric pair of alphavbeta3 antagonists in rat.

1. The study investigated mechanisms underlying the stereoselective hepatic disposition observed in rats of a zwitterionic diastereomeric pair ((3S)-3-[(3R or 3S)-2-oxo-3-[3-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propyl]pyrrolidin-1-yl]-3-quinolin-3-ylpropanoic acid) with different lipophilicities. 2. In a recirculating isolated rat liver system, the more hydrophilic diastereomer II possessed biliary clearance, CLb, and bile-to-liver concentration ratio higher (about 10-30-fold) than the lipophilic zwitterion I, whereas both I and II exhibited comparably high concentration ratios between liver and perfusate. Although MK-571, a known multidrug resistance protein (MRP) inhibitor, significantly inhibited the CLb of both compounds, it did not inhibit their canalicular transport, as evident by unchanged concentration ratios between bile and liver of either I or II. 3. Following an intravenous infusion of I or II to Sprague-Dawley rats, the biliary clearance calculated either based on plasma (CL(b,p)) or liver concentration (CL(b,l)), of II was much higher than that of I (about 5-50-fold). In rats lacking multidrug resistance protein 2 (Mrp2) (Eisai hyperbilirubinemic rat, EHBR), the biliary excretion rate and CL(b,p) of II were also higher than the corresponding values for I. However, both CL(b,p) or CL(b,l) of either I or II were not reduced in EHBR, as compared with control SD rats. 4. In the in vitro rat canalicular membrane vesicle study, I and II exhibited no differences in their inhibitory effect on the Mrp2 mediated ATP-dependent [3H]DNP-SG initial uptake (no inhibition at 10 microM and only about 40% inhibition at 100 microM). 5. Collectively, these results suggested that (1) the difference in the hepatic disposition between the two isomers was due primarily to the difference in their transport mechanism across the canalicular membrane and (2) Mrp2 did not play a major role in the observed differences in the biliary excretion of the diastereomers I and II in rats.

Differences in the absorption, metabolism and biliary excretion of a diastereomeric pair of alphavbeta3-antagonists in rat: limited role of P-glycoprotein.

1. The study investigated mechanisms underlying the pharmacokinetic differences of two zwitterionic diastereomers ((3S)-3-[(3R or 3S)-2-oxo-3-[3-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propyl]pyrrolidin-1-yl]-3-quinolin-3-ylpropanoic acid) with different lipophilicities using a combination of in vivo and in vitro approaches. 2. In rat, both isomers possessed comparable plasma clearances (CL). However, the more lipophilic diastereomer I exhibited a higher metabolic clearance (>2-fold higher than II), whereas the hydrophilic zwitterion II exhibited a higher biliary clearance (approximately 5-fold higher than I). Following oral administration, the bioavailability (F) of I (17%) was much higher than that of II (1%). 3. Consistent with these in vivo observations and the expectation based on their lipophilicity differences, the metabolism in rat liver microsomes was faster and the permeability in Caco-2 and LLC-PK1 cells and in situ rat intestinal loop was better for I than for II. 4. Only the absorption of the more lipophilic diastereomer I was subjected to an efflux system in the Caco-2 and in situ rat intestinal loop models. I was a good substrate for P-glycoprotein (P-gp) in both the human MDR1 and mouse mdr1a transfected cell lines, and in the wild-type mdr1a (-/-) mouse when compared with the P-gp-deficient mdr1a (-/-) mouse. Concomitant administration of I with verapamil in rat caused significant increases in oral AUC, F and Cmax of I without affecting its CL, further supporting the effect of P-gp in limiting the intestinal absorption of I in vivo in this animal model. 5. Since the findings that the lipophilic diastereomer I, but not II, was a good P-gp substrate were not in line with the observations that I was excreted to bile much slower than II and that I was absorbed better than II, the results suggested that P-gp played a minor role to the observed differences in the biliary excretion and intestinal absorption of the diastereomers I and II in rat.

An extended point-area deconvolution approach for assessing drug input rates.

PURPOSE: To describe an extended point-area deconvolution approach for evaluating drug input rates based on the application of piecewise cubic polynomial functions. METHODS: Both the nonimpulse response data and the impulse reference data were independently represented by the piecewise cubic polynomials to obtain interpolations, numerical integration, and reduced step size for the staircase input rates. A moving average algorithm was employed to compute the input rate estimates. The method was illustrated using data from preclinical and human studies. Simulations were used to examine the effects of data noise. RESULTS: In all cases examined, the piecewise cubic interpolation functions combined with the moving average algorithm yielded estimates that were reasonable and acceptable. Compared to the standard point-area approach based on the trapezoidal rule, the present method resulted in estimates that were closer to the expected values. CONCLUSIONS: The point-area deconvolution analysis is one of the preferred approaches in assessing pharmacokinetic and biopharmaceutic data when it is undesirable to assume the functional forms of the input processes. The present method provides improved performance and greater flexibility of this approach.

beta-Oxidation of simvastatin in mouse liver preparations.

All current 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors [simvastatin (SV), lovastatin (LV), atorvastatin, pravastatin, fluvastatin, and cerivastatin] are believed to undergo an atypical beta-oxidation of the dihydroxy heptanoic or heptanoic acid side chain. Metabolites, which are shortened by two- and/or four-carbon units consistent with beta-oxidation products, have been reported exclusively in rodents following LV and SV administration and across species (rodents, dogs, and humans) following the other statins. In this study, in vitro formation of a beta-oxidation product of simvastatin hydroxy acid (SVA) and its intermediates in mouse livers is described. Incubation of SVA with mouse liver preparations fortified with CoASH and ATP led to formation of SV and two major products (P1 and P2). Based on mass spectrometry (MS), tandem mass spectrometry, and/or NMR spectral characteristics, P1 was an alpha,beta-unsaturated metabolite, formed by dehydration of the D,D-dihydroxy heptanoic acid side chain, whereas P2 was probably the L,D-dihydroxy acid isomer of SVA, formed by stereospecific hydration of P1. When NAD(+) was also included in the incubation mixture, there were two additional metabolites with the MS and/or NMR characteristics consistent with a two-carbon shortened product (P3) and its dehydrated derivative (P4). In a complete incubation system with all cofactors (ATP, CoASH, NAD(+), and NADPH) present, there was an additional product with MS spectra and liquid chromatography retention time identical to the beta-oxidized, unsubstituted pentanoic acid metabolite (P5) detected in rats and mice following simvastatin administration. The involvement of CoASH and NAD(+) and the presence of the four metabolic intermediates suggest that SVA (and presumably the other statins) is a substrate for the beta-oxidation enzyme complex in mice. Additionally, the present finding of CoASH-dependent formation of SV substantiates a mechanism proposed previously for the in vivo lactonization of statin hydroxy acids.

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.

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.

Interspecies comparison and role of human cytochrome P450 and flavin-containing monooxygenase in hepatic metabolism of L-775,606, a potent 5-HT(1D) receptor agonist.

1. Quantitative species differences and human liver enzymes involved in the metabolism of L-775,606, a potent and selective 5-HT1D receptor agonist developed for the acute treatment of migraine headache, have been investigated in vitro. 2. In human, monkey, dog and rat liver microsomes, formation of the hydroxylated M1 and the N-dealkylated M2 was mediated by enzyme(s) of high-affinity (apparent Km approximately 1-6 microM), and that of the two N-oxide isomers (M3) was catalysed by those of low affinity (apparent Km approximately 50-110 microM). In dog, M3 constituted a major pathway (approximately 40%), whereas in all other species it was a minor metabolite (< 5%). 3. In human liver microsomes, a marked inhibition (> or =80%) of M1 and M2 formation was observed by SKF525-A, troleandomycin, ketoconazole and anti-CYP3A antibodies, whereas the inhibition was modest (approximately 20-40%) with quercetin. Of seven cDNA-expressed human P450 tested, only CYP3A4 and CYP2C8 were capable of oxidizing L-775,606, resulting primarily in M1 and M2. However, CYP3A4 possessed much higher affinity (> or = 20-fold) and much higher intrinsic activity (> 100-fold) than CYP2C8. 4. In contrast, N-oxidation was not inhibited by any inhibitors of P450 tested, but rather was reduced significantly by heat treatment and methimazole, and was increased substantially with an incubation pH>7.4. Human flavin-containing monooxygenase form 3 (FMO3) catalysed exclusively the N-oxidation to M3, with apparent Km and optimum pH comparable with those observed in human liver microsomes. 5. These results demonstrated quantitative interspecies differences in the metabolism of L-775,606. In human, metabolism of L-775,606 to the principal metabolites, M1 and M2, was mediated primarily by CYP3A4 with minimal contribution from CYP2C8, whereas the minor N-oxidative pathway was catalysed mainly by FMO3.

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.

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.

In vitro and in vivo studies on the metabolism of tirofiban.

Tirofiban hydrochloride [L-tyrosine-N-(butylsulfonyl)-O-[4-(4-piperidinebutyl)] monohydrochloride, is a potent and specific fibrinogen receptor antagonist. Radiolabeled tirofiban was synthesized with either (3)H-label incorporated into the phenyl ring of the tyrosinyl residue or (14)C-label in the butane sulfonyl moiety. Neither human liver microsomes nor liver slices metabolized [(14)C]tirofiban. However, male rat liver microsomes converted a limited amount of the substrate to a more polar metabolite (I) and a relatively less polar metabolite (II). The formation of I was sex dependent and resulted from an O-dealkylation reaction catalyzed by CYP3A2. Metabolite II was identified as a 2-piperidone analog of tirofiban. There was no evidence for Phase II biotransformation of tirofiban by microsomes fortified with uridine-5'-diphospho-alpha-D-glucuronic acid. After a 1 mg/kg i.v. dose of [(14)C]tirofiban, recoveries of radioactivity in rat urine and bile were 23 and 73%, respectively. Metabolite I and unchanged tirofiban represented 70 and 30% of the urinary radioactivity, respectively. Tirofiban represented >90% of the biliary radioactivity. At least three minor biliary metabolites represented the remainder of the radioactivity. One of them was identified as I. Another was identified as II. When dogs received 1 mg/kg i.v. of [(3)H]tirofiban, most of the radioactivity was recovered in the feces as unchanged tirofiban. The plasma half-life of tirofiban was short in both rats and dogs, and tirofiban was not concentrated in tissues other than those of the vasculature and excretory organs.

Metabolic interactions between mibefradil and HMG-CoA reductase inhibitors: an in vitro investigation with human liver preparations.

AIMS: To determine the effects of mibefradil on the nletabolism in human liver microsomal preparations of the HMG-CoA reductase inhibitors simvastatin, lovastatin, atorvastatin, cerivastatin and fluvastatin. METHODS: Metabolism of the above five statins (0.5, 5 or 10 microM), as well as of specific CYP3A4/5 and CYP2C8/9 marker substrates, was examined in human liver microsomal preparations in the presence and absence of mibefradil (0.1-50 microM). RESULTS: Mibefradil inhibited, in a concentration-dependent fashion, the metabolism of the four statins (simvastatin, lovastatin, atorvastatin and cerivastatin) known to be substrates for CYP3A. The potency of inhibition was such that the IC50 values (<1 microM) for inhibition of all of the CYP3A substrates fell within the therapeutic plasma concentrations of mibefradil, and was comparable with that of ketoconazole. However, the inhibition by mibefradil, unlike that of ketoconazole, was at least in part mechanism-based. Based on the kinetics of its inhibition of hepatic testosterone 6beta-hydroxylase activity, mibefradil was judged to be a powerful mechanism-based inhibitor of CYP3A4/5, with values for Kinactivation, Ki and partition ratio (moles of mibefradil metabolized per moles of enzyme inactivated) of 0.4 min(-1), 2.3 microM and 1.7, respectively. In contrast to the results with substrates of CYP3A, metabolism of fluvastatin, a substrate of CYP2C8/9, and the hydroxylation of tolbutamide, a functional probe for CYP2C8/9, were not inhibited by mibefradil. CONCLUSION: Mibefradil, at therapeutically relevant concentrations, strongly suppressed the metabolism in human liver microsomes of simvastatin, lovastatin, atorvastatin and cerivastatin through its inhibitory effects on CYP3A4/5, while the effects of mibefradil on fluvastatin, a substrate for CYP2C8/9, were minimal in this system. Since mibefradil is a potent mechanism-based inhibitor of CYP3A4/5, it is anticipated that clinically significant drug-drug interactions will likely ensue when mibefradil is coadministered with agents which are cleared primarily by CYP3A-mediated pathways.

In vitro and in vivo evaluations of intestinal barriers for the zwitterion L-767,679 and its carboxyl ester prodrug L-775,318. Roles of efflux and metabolism.

The barriers to oral delivery of the hydrophilic zwitterion L-767, 679 (I) and its carboxyl ester prodrug L-775,318 (II) were examined. In the Caco-2 cell model, transport of II, but not I, was strongly oriented in the secretory direction. The basal-to-apical transport of II displayed saturable kinetics and was markedly inhibited by verapamil and quinidine, known P-glycoprotein inhibitors. In Caco-2 cells, metabolism of I was not observed, whereas hydrolysis of II was modest (

Effects of pentobarbital on pharmacokinetics and pharmacodynamics of a potent fibrinogen receptor antagonist, L-734,217, in dogs.

Effects of pentobarbital on pharmacokinetics and pharmacodynamics of L-734,217, a potent fibrinogen receptor antagonist, were studied in male dogs. L-734,217 was given intravenously at 0.01 mg kg-1, in a cross-over fashion, to conscious dogs or to dogs anesthetized with pentobarbital. Plasma concentrations of L-734,217 were measured using a radioimmunoassay and inhibitory effects on ex vivo platelet aggregation induced by ADP or collagen were determined. In pentobarbital-treated dogs, L-734,217 plasma concentrations during the first 3 h collection period were significantly higher than those in the control animals. Corresponding to the increased plasma levels, the mean ex vivo inhibitory effects on ADP- or collagen-induced platelet aggregation in dogs under anesthesia appeared greater than in those without the anesthetic treatment. Pharmacokinetic analysis revealed a modest, but significant (up to 40%) elevation in the area under the plasma concentration-time curve during 6 h of the drug administration, and a reduction in L-734,217 plasma clearance and volumes of distribution, in the anesthetized dogs. Analysis of pharmacodynamic data indicated that the EC50 and the Hill coefficient of the platelet aggregation response-plasma concentration curve were not altered by pentobarbital treatment. The results are in agreement with the findings that the administration of pentobarbital alone (in the absence of L-734,217) did not affect appreciably the ex vivo platelet aggregatory responses. In a separate group of dogs, L-734,217 was found to be metabolically stable, and was eliminated unchanged renally (64 +/- 4%) and hepatically (32 +/- 6%). In addition, L-734,217 did not bind substantially to canine plasma proteins or blood cellular components. It is possible that alterations of regional hemodynamics, reportedly mediated by pentobarbital, contributed to changes observed in the present study. That is, alterations occurred in L-734,217 elimination and distribution processes which resulted in an increase in drug plasma levels. Since pentobarbital anesthesia influenced only the pharmacokinetics, and not the pharmacodynamics, of L-734,217, the apparent increases in the inhibition of platelet aggregation responses observed following L-734,217 administration to the anesthetized dogs were probably sequential effects of the pharmacokinetic interactions.