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.

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.

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 (

In vitro metabolism of simvastatin in humans [SBT]identification of metabolizing enzymes and effect of the drug on hepatic P450s.

Simvastatin (SV) is a lactone prodrug used for the treatment of hypercholesterolemia. Upon incubation of SV with liver microsomal preparations from human donors, four major metabolic products were formed (3'-hydroxy SV, 6'-exomethylene SV, 3',5'-dihydrodiol SV, and the active hydroxy acid, SVA), together with several minor unidentified metabolites. The 3',5'-dihydrodiol SV, a new metabolite, was inactive as an inhibitor of HMG-CoA reductase. Kinetic studies of SV metabolism in human liver microsomes suggested that the major NADPH-dependent metabolites (3'-hydroxy SV, 6'-exomethylene SV, and 3',5'-dihydrodiol SV) were formed with relatively high intrinsic clearances, consistent with the extensive metabolism of SV observed in vivo. Based on four different in vitro approaches, namely 1) correlation analysis, 2) chemical inhibition, 3) immunoinhibition, and 4) metabolism by recombinant human P450, it is concluded that CYP3A is the major enzyme subfamily responsible for the metabolism of SV by human liver microsomes. Both CYP3A4 and CYP3A5 were capable of catalyzing the formation of 3',5'-dihydrodiol, 3'-hydroxy, and 6'-exomethylene metabolites. However, CYP3A4 exhibited higher affinity (> 3 fold) for SV than CYP3A5. Also, the studies indicated that CYP2D6, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP1A2, and CYP2E1 did not play significant roles in the metabolism of SV in vitro. Over the concentration range of 0-40 microM, SV inhibited the activity of CYP3A, but not the activities of CYP2C8/9, CYP2C19, or CYP2D6 in human liver microsomes. The inhibition of hepatic midazolam 1'-hydroxylase, a CYP3A marker activity, by SV was competitive with a Ki value of approximately 10 microM. SV was > 30-fold less potent than ketoconazole and itraconazole as an inhibitor of CYP3A. Under the same conditions, SVA, the hydrophilic hydroxy acid form of SV, did not inhibit CYP3A, CYP2C8/9, CYP2C19, or CYP2D6 activities. The results suggested that the in vivo inhibitory effects of SV on the metabolism of CYP3A substrates likely would be less than those of ketoconazole and itraconazole at their respective therapeutic concentrations. In addition, metabolic activities mediated by the other P450 enzymes tested are unlikely to be affected by SV.

In vitro and in vivo evaluations of the metabolism, pharmacokinetics, and bioavailability of ester prodrugs of L-767,679, a potent fibrinogen receptor antagonist: an approach for the selection of a prodrug candidate.

The present study demonstrates the utility of an in vitro-in vivo correlative approach in the selection of an optimum prodrug candidate of L-767,679 (N-([7-(piperazin-1-yl)-3,4-dihydro-1(1H)-isoquinolinone-2-yl]acetyl)-3(S)-(ethynyl)-beta-alanine), a potent fibrinogen receptor antagonist. As an initial screening step, a comparative in vitro hepatic metabolism study was conducted for L-767,679 and a series of aliphatic and aromatic ester prodrugs in dogs, monkeys, and humans. In all species, the active acid L-767,679, but not the ester prodrugs, was resistant to metabolism. Only the methyl, ethyl, and isopropyl esters were converted exclusively to the active acid in liver microsomal preparations from dogs and humans, and thus were selected for further studies. In the preparations from monkeys, all of the esters investigated were metabolized efficiently to both the active acid and several other products. The absolute formation rates of L-767,679 from the esters followed the rank order: methyl approximately ethyl > isopropyl in all species, and in humans > dogs for the three esters. The three ester prodrugs did not undergo appreciable hydrolysis in blood or upon incubation with intestinal S9 from any of the studied species. In vivo evaluation of the previous three aliphatic esters in dogs and monkeys supported the in vitro findings. L-767,679 was metabolically stable in both dogs and monkeys. After intravenous administration of the prodrugs to either species, the extent of acid formation was higher in dogs than in monkeys. In addition, the extent of L-767,679 formed from these prodrugs followed the rank order: methyl approximately ethyl > isopropyl. Similar results were obtained after oral dosing of the prodrugs, such that the bioavailability of L-767,679 was higher in dogs than in monkeys, and the bioavailability was higher after the ethyl ester than after the isopropyl prodrug in both species. In either species, both ethyl and isopropyl ester prodrugs were better absorbed than L-767,679. Overall, the results suggested that the bioavailability of the active acid after administration of an ester prodrug was dictated primarily by two factors, viz.:1) the relative rates of ester hydrolysis versus competing metabolic reactions and 2) the absolute rates of ester hydrolysis. In the case of L-767,679 prodrugs, absorption was not a limiting factor. Consequently, the bioavailability of L-767,679 after oral administration of the ester prodrugs would likely be greater in humans than in dogs, and in humans would be higher with the ethyl ester than with the isopropyl ester. On this basis, the ethyl ester was considered as a promising candidate for clinical evaluation as a fibrinogen receptor antagonist prodrug.

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.

Disposition of L-738,167, a potent and long-acting fibrinogen receptor antagonist, in dogs. Dose-dependent pharmacokinetics.

L-738,167 is a potent and long-acting fibrinogen receptor antagonist and may be useful for treatment of chronic thrombotic occlusive disorders. The purposes of this study were to characterize the metabolism and disposition of L-738,167, and to investigate factors affecting its pharmacokinetic behaviors in dogs, one of the animal models used in pharmacological and toxicological studies. In vitro and in vivo experiments indicated that L-738,167 was not metabolized to any appreciable extent in dogs. Biliary excretion was found to be the major route (approximately 75%) of drug elimination. Following 1 and 3 micrograms/kg iv doses, blood pharmacokinetics of L-738,167 were linear. Total blood clearance (CLB) was much lower than hepatic blood flow, and the apparent volume of distribution at steady-state (Vdss,B) was comparable with blood volume. Blood pharmacokinetics in the dose range of 3-250 micrograms/kg were dose-dependent; both CLB and Vdss,B for L-738,167 increased markedly with increasing doses. However, the terminal half-life (t1/2) was dose-independent, with a mean value of approximately 4 days. L-738,167 was found to bind negligibly to dog plasma proteins. Determinations of whole blood (WB), platelet-rich plasma, and platelet-poor plasma concentrations after several intravenous doses of [3H]L-738,167 revealed significant concentration-dependent binding of the compound to platelets. Kinetic analysis of the platelet binding indicated that L-738,167 was bound to dog platelets with high affinity (apparent Kd approximately 1 nM platelet-poor plasma concentration) and relatively low capacity (approximately 70 nM WB concentration). Findings are consistent with the binding kinetics of L-738,167 to glycoprotein IIb/IIIa (GP IIb/IIIa) receptor, supporting that GP IIb/IIIa was the primary binding component on the platelets. It was concluded that the dose-dependent pharmacokinetics of L-738,167 were the consequence of the concentration-dependent drug-platelet binding. Due to this extensive platelet binding, L-738,167, when given in therapeutic doses or lower, resided primarily in the vascular compartment-the site of pharmacological action. At doses exceeding the receptor binding capacity, the excess amount or the unbound drug was eliminated rapidly. In all cases, the equally long t1/2 of L-738,167 was also a consequence of the high-affinity binding to platelets, in good agreement with its prolonged pharmacodynamic profile.

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.

Species and organ differences in first-pass metabolism of the ester prodrug L-751,164 in dogs and monkeys. In vivo and in vitro studies.

The pharmacokinetics and bioavailability of L-751,164, an ethyl ester prodrug of a potent fibrinogen receptor antagonist, L-742,998, were studied in beagle dogs and rhesus monkeys. In both species, L-751,164 exhibited high clearance. After an intravenous dose, L-751,164 was converted to the parent L-742,998 to the extent of approximately 20% in dogs and 90% in monkeys. After oral administration of the prodrug, however, the bioavailability, measured either as the prodrug or as the active parent, was < 5% in both species. Several experiments were conducted subsequently to investigate possible causes for the observed similarities in the low oral bioavailability of the prodrug between species despite its differences in the in vivo conversion. In vitro metabolism studies using dog liver subcellular fractions indicated extensive metabolism of L-751,164 to metabolites other than L-742,998. Kinetically, L-742,998 formation accounted only for approximately 25% of the prodrug disappearance. In contrast, monkey liver preparations converted L-751,164 exclusively and rapidly to L-742,998. Good agreement between the in vitro hepatic metabolism and the in vivo observations suggests that liver was the major eliminating organ after intravenous administration of the prodrug in both species. In dogs, this suggestion was further supported by low bioavailability of the prodrug (20%) and the parent (below detection limit) after intraportal administration of the prodrug. In vitro metabolism of L-751,164 using intestinal S9 fractions revealed substantial metabolism in monkeys, but not in dogs. Several NADPH-dependent metabolites were observed with monkey intestinal preparation, with the parent L-742,998 being the minor product (approximately 25-30%). Furthermore, L-751,164 was shown, by means of an in vitro Caco-2 cell, and in situ rat intestinal loop models, to be highly permeable to intestinal barriers. Collectively, these results suggest that the apparent species differences in the prodrug conversion observed in vivo likely were due to species differences in the hepatic metabolism of the prodrug. In both species, the high first-pass metabolism of the prodrug, and the extensive conversion of the prodrug to metabolic products other than the parent contributed, at least in part, to the low bioavailability of the prodrug and active parent, respectively, obtained after an oral dose of the prodrug. The latter process was species-dependent, involving primarily the hepatic first-pass elimination in dogs and the intestinal first-pass metabolism in monkeys.

Comparative studies of drug-metabolizing enzymes in dog, monkey, and human small intestines, and in Caco-2 cells.

Drug-metabolizing enzymes were studied in subcellular fractions of dog, monkey, and human small intestines, and in the human adenocarcinoma cell line Caco-2, a commonly used in vitro absorption model. Immunoblot analysis indicated the presence of enzymes related to cytochrome P450 (CYP) 1A1/CYP1A2, CYP2D6, CYP3A, and carboxylesterases (ESs) in human and monkey intestines, and of CYP3A and ES in dog intestines. Catalytically, human and monkey intestines exhibited significant and comparable testosterone 6 beta-hydroxylase, (+)-bufuralol 1'-hydroxylase, and ES activities. In contrast, dog intestine possessed moderate testosterone 6 beta-hydroxylase, much lower ES, and undetectable bufuralol hydroxylase activities. In addition, low tolbutamide methylhydroxylase activity was observed in human and monkey intestines, but not in dog intestines. Of the phase I enzymes investigated, only ES was detected immunologically and functionally in Caco-2 cells. With respect to phase II enzymes, human and monkey intestines contained relatively high intestinal glucuronyltransferase, N-acetyltransferase (NAT), sulfotransferase, and glutathione S-transferase activities. Except for NAT, all phase II enzymes studied were detectable in dog intestines. In Caco-2 cells, acetaminophen sulfation activity was below the limit of detection, whereas all other conjugating activities were evident. Studies of enzyme kinetics and inhibition by known inhibitors of testosterone 6 beta-hydroxylase activity, the major intestinal mono-oxygenase in all species, revealed some similarities between the responsible enzymes. Comparative studies with human liver microsomes suggested the possible involvement of CYP3A enzymes in the intestinal catalysis of testosterone 6 beta-hydroxylation similar to those observed with human hepatic CYP3A. Further studies on ESs, however, revealed multiplicity and species and/or tissue differences in the microsomal and cytosolic enzymes. Based on kinetic studies, monkey intestines and Caco-2 cells possessed NAT activities, with properties similar to those in human intestine and liver. Overall, the results demonstrated that both the preparations of small intestines and Caco-2 cells exhibited significant drug-metabolizing enzyme activities, although several differences were noted between the intestinal enzymes in the animals or in the Caco-2 cells and those found in humans.

Nutritional influences on plutonium absorption from the gastrointestinal tract of the rat.

Rats that were fasted and/or fed diets consisting of whole milk, dry milk, milk supplement, orange juice, low calcium, or low vitamin D were gavaged with 238Pu nitrate to measure plutonium absorption. All dietary variations resulted in increases in both absorption and retention. The increases ranged between 2 and 20 times the absorption values obtained for rats fed commercial chow. Sucklings of dams fed a calcium-deficient diet also exhibited increased 238Pu absorption. The results indicate that nutrition is an important factor influencing gastrointestinal absorption of plutonium.

The effect of X rays, DTPA, and aspirin on the absorption of plutonium from the gastrointestinal tract of rats.

To measure the effect of radiation on plutonium transport, rats that were exposed to 250-kVp X rays were given 238Pu 3 days afterwards by either gavage or injection into a ligated segment of the duodenum. In a second group of experiments, rats were either injected intraduodenally with 238Pu-DTPA or administered the chelate intravenously and the 238Pu by gavage. In a third experiment, rats that had been gavaged with 200 or 400 mg/kg/day of aspirin for 2 days were injected intragastrically with 238Pu nitrate. Results of the first experiment showed a dose-dependent increase in 238Pu absorption between 800 and 1500 rad of lower-body X irradiation. Intravenous or intraduodenal injections of DTPA caused a marked increase in 238Pu absorption but resulted in decreased plutonium deposition in the skeleton and liver. Retention of 238Pu in the skeleton of rats given aspirin was double that of controls, but the effect on plutonium absorption was less marked than that of DTPA.

Can information on the gastrointestinal absorption of actinide elements by neonatal rats, guinea pigs, dogs and swine be extrapolated to man?

Neonatal mammals that acquire passive immunity by absorbing immunoglobulins from mother's milk (colostrum), as well as species that develop immunity prenatally, absorb macromolecules from the gut. This permeability of the GI tract to large molecules also serves as a route of entry for actinides in quantities greatly in excess of adult absorption. Administration of 238Pu nitrate to piglets results in absorption that is 1000 times the adult value but neonatal dogs, guinea pigs and rats absorb only 100 times more 238Pu than adults. Neonatal pigs and rats retain as much as 60% of the dose in the gut mucosa for several days but dogs and guinea pigs retain much less. We have also found this difference in GI tract 238Pu retention to occur after inhalation, indicating that the gut is an important route of entry for actinides that are inhaled by neonates. Although retention of different gavaged actinides by suckling rats is within the same range (1-3%) we found retention by piglets to range between 35% for 233U and about 2% for 237Np, 244Cm and 241Am nitrate. Piglets that were gavaged with 238Pu nitrate retained 19% in the liver and carcass a week later. After one year, half of that still remained. Neonatal rats, on the other hand, retained 3% initially and only 0.1% at one year. While the GI tract of the human infant is probably not nearly as permeable to macromolecules as that of the swine, it is known to be more permeable to macromolecules than that of the adult human. If actinide absorption is also increased in the child, the retention of these elements would probably be more like that in swine than in rats, because of the greater similarity between man and swine in skeletal maturation, in retention of actinides in liver and their longer life span. These studies suggest a need for further information on neonatal absorption of actinides in a large animal species that has a developing GI function more similar to that of humans.

Further studies on the absorption of actinide elements from the gastrointestinal tract of neonatal animals.

Plutonium retention was measured after intragastric administration to neonatal rats, dogs and swine. At 1 week after administration, substantially more of the actinide remained in swine and dogs than in rats. The quantity of 238Pu absorbed by piglets was markedly influenced by such factors as compound solubility, mass of plutonium administered, oxidation state of the actinide, and age of the animal at gavage. Cortisone treatment reduced absorption, but was less effective in piglets than in neonatal rats. Measurements of 238Pu transport from ligated segments of the neonatal swine intestine indicated highest absorption from the duodenum, where the actinide was shown, autoradiographically, to be deposited in the epithelial region; in the ileum, deposition was predominantly in the lacteal region. Absorption of actinides by neonatal swine decreased in the order of 233U greater than 238Pu greater than 237Np greater than 244Cm greater than 241Am. Measurements at 1 yr after gavage showed a much higher retention by swine than by rats.