Influence of beta-adrenoceptor antagonists on the pharmacokinetics of rizatriptan, a 5-HT1B/1D agonist: differential effects of propranolol, nadolol and metoprolol.

AIMS: Patients with migraine may receive the 5-HT1B/1D agonist, rizatriptan (5 or 10 mg), to control acute attacks. Patients with frequent attacks may also receive propranolol or other beta-adrenoceptor antagonists for migraine prophylaxis. The present studies investigated the potential for pharmacokinetic or pharmacodynamic interaction between beta-adrenoceptor blockers and rizatriptan. METHODS: Four double-blind, placebo-controlled, randomized crossover investigations were performed in a total of 51 healthy subjects. A single 10 mg dose of rizatriptan was administered after 7 days' administration of propranolol (60 and 120 mg twice daily), nadolol (80 mg twice daily), metoprolol (100 mg twice daily) or placebo. Rizatriptan pharmacokinetics were assessed. In vitro incubations of rizatriptan and sumatriptan with various beta-adrenoceptor blockers were performed in human S9 fraction. Production of the indole-acetic acid-MAO-A metabolite of each triptan was measured. RESULTS: Administration of rizatriptan during propranolol treatment (120 mg twice daily for 7.5 days) increased the AUC(0, infinity) for rizatriptan by approximately 67% and the Cmax by approximately 75%. A reduction in the dose of propranolol (60 mg twice daily) and/or the incorporation of a delay (1 or 2 h) between propranolol and rizatriptan administration did not produce a statistically significant change in the effect of propranolol on rizatriptan pharmacokinetics. Administration of rizatriptan together with nadolol (80 mg twice daily) or metoprolol (100 mg twice daily) for 7 days did not significantly alter the pharmacokinetics of rizatriptan. No untoward adverse experiences attributable to the pharmacokinetic interaction between propranolol and rizatriptan were observed, and no subjects developed serious clinical, laboratory, or other significant adverse experiences during coadministration of rizatriptan with any of the beta-adrenoceptor blockers. In vitro incubations showed that propranolol, but not other beta-adrenoceptor blockers significantly inhibited the production of the indole-acetic acid metabolite of rizatriptan and sumatriptan. CONCLUSIONS: These results suggest that propranolol increases plasma concentrations of rizatriptan by inhibiting monoamine oxidase-A. When prescribing rizatriptan to migraine patients receiving propranolol for prophylaxis, the 5 mg dose of rizatriptan is recommended. Administration with other beta-adrenoceptor blockers does not require consideration of a dose adjustment.

Metabolites of caspofungin acetate, a potent antifungal agent, in human plasma and urine.

Caspofungin acetate (MK-0991) is a semisynthetic pneumocandin derivative being developed as a parenteral antifungal agent with broad-spectrum activity against systemic infections such as those caused by Candida and Aspergillus species. Following a 1-h i.v. infusion of 70 mg of [(3)H]MK-0991 to healthy subjects, excretion of drug-related material was very slow, such that 41 and 35% of the dosed radioactivity was recovered in urine and feces, respectively, over 27 days. Plasma and urine samples collected around 24 h postdose contained predominantly unchanged MK-0991, together with trace amounts of a peptide hydrolysis product, M0, a linear peptide. However, at later sampling times, M0 proved to be the major circulating component, whereas corresponding urine specimens contained mainly the hydrolytic metabolites M1 and M2, together with M0 and unchanged MK-0991, whose cumulative urinary excretion over the first 16 days postdose represented 13, 71, 1, and 9%, respectively, of the urinary radioactivity. The major metabolite, M2, was highly polar and extremely unstable under acidic conditions when it was converted to a less polar product identified as N-acetyl-4(S)-hydroxy-4-(4-hydroxyphenyl)-L-threonine gamma-lactone. Derivatization of M2 in aqueous media led to its identification as the corresponding gamma-hydroxy acid, N-acetyl-4(S)-hydroxy-4-(4-hydroxyphenyl)-L-threonine. Metabolite M1, which was extremely polar, eluting from HPLC column just after the void volume, was identified by chemical derivatization as des-acetyl-M2. Thus, the major urinary and plasma metabolites of MK-0991 resulted from peptide hydrolysis and/or N-acetylation.

Metabolism of A dopamine D(4)-selective antagonist in rat, monkey, and humans: formation of A novel mercapturic acid adduct.

3-([4-(4-Chlorophenyl)piperazin-1-yl]-methyl)-1H-pyrrolo-2, 3-beta-pyridine (L-745,870) is a dopamine D(4) selective antagonist that has been studied as a potential treatment for schizophrenia, with the expectation that it would not exhibit the extrapyramidal side effects often observed with the use of classical antipsychotic agents. The metabolism of L-745,870 in vivo was investigated in the rat, rhesus monkey, and human using liquid chromatography-tandem mass spectrometry and/or NMR techniques in conjunction with radiochemical detection. In all three species, two major metabolic pathways were identified, namely N-dealkylation at the substituted piperazine moiety and the formation of a novel mercapturic acid adduct. It is proposed that the latter biotransformation process involves the formation of an electrophilic imine methide intermediate, analogous to that produced from 3-methyl indole. This report appears to represent the first example of metabolic activation of a 3-alkyl-7-azaindole nucleus.

Biotransformation of lovastatin. V. Species differences in in vivo metabolite profiles of mouse, rat, dog, and human.

Lovastatin is a prodrug lactone whose open-chain 3,5-dihydroxy acid is a potent, competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-limiting enzyme in cholesterol biosynthesis. The compound undergoes extensive and complex metabolism in animals and humans, with the metabolites excreted predominantly in bile. Radiochromatograms of bile from three human subjects and of bile and liver homogenates from mouse, rat, and dog displayed obvious species differences. Biotransformation of lovastatin occurred by three distinct routes, namely hydrolysis of the lactone ring to yield the pharmacologically active dihydroxy acid, cytochrome P-450-mediated oxidation of the fused-ring system, and beta-oxidation of the dihydroxy acid side chain. The first two reactions occurred in all four species, but the last was observed in mouse and rat only. The P-450 reactions, hydroxylation and a novel dehydrogenation reaction, yielded a 6'-hydroxylated metabolite of the dihydroxy acid and a 6'-exomethylene derivative as major and minor metabolites, respectively, in the bile of rat and dog. Human bile, which contained predominantly polar metabolites, yielded these metabolites in similar proportions only after mild hydrolysis at pH 5.0. In mouse and rat an atypical beta-oxidation of the dihydroxy acid side chain occurred to give a pentanoic acid derivative that was observed in liver homogenates. This metabolite was subsequently conjugated with taurine and excreted in the bile. From these studies, cytochrome P-450 oxidation is the primary route of phase I metabolism for lovastatin in human and dog, but beta-oxidation plays a major metabolic role in rodents.

In vivo and in vitro metabolism studies on a class III antiarrhythmic agent.

The metabolism of L-691,121 (I), a class III antiarrhythmic agent, was studied in vivo in rats and dogs and in vitro by using liver S9 or slices from these species and humans. After oral doses of [14C]I to rats (5 mg/kg) and dogs (1 mg/kg), urinary recoveries of label were, respectively, 6% and 28%. Biliary excretion (0-24 hr) accounted for 68% of a 5 mg/kg, po dose in rats and 19% of a 10 mg/kg dose, po in dogs. Metabolites were identified by application of FAB/MS, NMR, and diode-array UV spectroscopy. The major dog metabolites were the secondary alcohol (II) produced by carbonyl reduction and its glucuronide conjugate (III). It was estimated that II and III represented 24 and 36%, respectively, of the dog biliary radioactivity. After a 50 mg/kg dose of I, II represented approximately 50% of the dog urinary label. A minor metabolite (IV) in dog urine was produced by reduction and loss of N-substitution. There were species differences in that, relative to dogs, II represented a much smaller fraction of the excreted dose in rats and there was no evidence for excretion of III in rats. N-Dealkylated I (V) was excreted, along with IV in rat bile. Dog liver slices and S9 fractions were most efficient (relative to human and rat liver tissues) at reducing I to II. Metabolic reduction of I to II was highly stereoselective and yielded the (-)-antipode as determined by chiral chromatography.

Biotransformation of lovastatin. IV. Identification of cytochrome P450 3A proteins as the major enzymes responsible for the oxidative metabolism of lovastatin in rat and human liver microsomes.

Previous studies from our laboratories have shown that the metabolism of the cholesterol-lowering drug lovastatin by rat and human liver microsomes occurs primarily at the 6'-position, giving 6' beta-hydroxy- and 6'-exomethylene-lovastatin and that these oxidations are catalyzed by cytochrome P450-dependent monooxygenases. In the present study, the specific cytochrome P450 form involved in lovastatin oxidation was identified through immunoinhibition studies. Among several antibodies prepared against various cytochrome P450s, only anti-rat P450 3A IgG inhibited lovastatin metabolism in liver microsomes from untreated, phenobarbital-treated, and pregnenolone-16 alpha-carbonitrile-treated rats. Lovastatin metabolism at the 6'-position was markedly inhibited (6' beta-hydroxy, greater than 95%; 6'-exomethylene, 70-80%) by this antibody whereas the effect of anti-rat P450 3A on the 3"-hydroxylation was variable depending on the source of the microsomes. With human liver microsomes, both anti-rat P450 3A and anti-human P450 3A inhibited lovastatin metabolism. Correlation between lovastatin oxidation and the P450 3A content in human liver microsomes (measured by immunoblot analysis) was excellent (r2 = 0.97). In addition, preincubation of human liver microsomes with troleandomycin and NADPH inhibited metabolism by 60%. These results clearly indicate that cytochrome P450 3A enzymes are primarily responsible for the metabolism of lovastatin in rat and human liver microsomes.

HMG-CoA reductase inhibitor-induced myopathy in the rat: cyclosporine A interaction and mechanism studies.

Recent clinical evidence indicates a potential for skeletal muscle toxicity after therapy with HMG-CoA reductase inhibitors (HMGRIs) in man. Although the incidence of drug-induced skeletal muscle toxicity is very low (0.1-0.2%) with monotherapy, it may increase following concomitant drug therapy with the immunosuppressant, cyclosporine A (CsA), and possibly with certain other hypolipidemic agents. In the Sprague-Dawley rat, very high, pharmacologically comparable dosages (150-1200 mg/kg/day) of structurally similar HMGRIs (lovastatin, simvastatin, pravastatin and L-647, 318) produced dose-related increases in the incidence and severity of skeletal muscle degeneration. Physical signs included inappetence, decreased activity, loss of body weight, localized alopecia and mortality. To evaluate the interaction between HMGRIs and CsA, a rat model of CsA-induced cholestasis was developed. In this 2-week model, the skeletal muscle toxicity of the HMGRIs was clearly potentiated by CsA (10 mg/kg/day). Doses of HMGRIs which did not produce skeletal muscle toxicity when given alone caused between 75 and 100% incidence of myopathy (very slight to marked skeletal muscle degeneration) when CsA was coadministered. Typical light microscopic changes included myofiber necrosis with interstitial edema and inflammatory infiltration in areas of acute injury. Histochemical characterization of the muscle lesion indicated that type 2B fibers (primarily glycolytic white fibers) were most sensitive to this toxicity but that, with prolonged administration, all fiber types were ultimately affected. Results of pharmacokinetic studies in rats treated with various HMGRIs +/- CsA indicated that coadministration of CsA alters the disposition of these compounds, resulting in increased systemic exposure (e.g., increased area under the plasma drug concentration vs. time curve-AUC) and consequent (up to 13-fold) increases in skeletal muscle drug levels. Evaluation of the potential interaction between the HMGRI, lovastatin and CsA at the level of hepatic microsomal metabolism indicated that CsA did not inhibit the metabolism of lovastatin in isolated microsomes from female rats. In light of the above findings, it appears that HMGRI-induced myopathy is a class effect in the rat, which is potentiated by CsA as the result of altered clearance and resultant increased tissue exposure. Cholestasis associated with CsA and HMGRIs may form the basis for decreased elimination and the resultant elevated systemic exposure. Furthermore, this toxicity is muscle fiber-selective and may be associated with impaired skeletal muscle energy metabolism.

Metabolism of antiparkinson agent dopazinol by rat liver microsomes.

Metabolism of dopazinol (DZ) by liver microsomes from control and phenobarbital- and 3-methylcholanthrene-treated rats has been investigated. Liver microsomes from control and treated rats metabolized DZ to N-despropyl-DZ (39-53% of total metabolites); 8-hydroxy-DZ, a catechol metabolite (32-39%); and 5- or 6-hydroxy-DZ (12-20%). The last metabolite was identified as its dehydration product 5,6-dehydro-DZ. N-Dealkylation was favored only slightly over catechol formation (ratio = 1.2) by liver microsomes from control and phenobarbital-treated rats, whereas with liver microsomes from 3-methylcholanthrene-treated rats, N-dealkylation predominated (ratio = 1.7). Liver microsomes from control rats metabolized DZ at a rate of 0.86 nmol/nmol cytochrome P-450/min. Pretreatment of rats with phenobarbital or 3-methylcholanthrene stimulated rates of metabolism by 2.4- and 3-fold, respectively. Metabolism of DZ was inhibited by SKF 525-A, methimazole, and thiobenzamide. SKF 525-A completely inhibited metabolism of DZ, while methimazole and thiobenzamide, two alternate substrates of the microsomal flavin-containing monooxygenase (MFMO) inhibited N-dealkylation only. These results indicated that while the cytochrome P-450-dependent monooxygenase is the primary enzyme system in DZ oxidation, the MFMO also catalyzes the N-dealkylation reaction. The catechol metabolite was converted to isomeric O-methylated derivatives in approximately 1:1 ratio by purified catechol-O-methyl transferase or 105,000g liver cytosol. The late eluting isomer was 8-methoxy-DZ.

In vitro and in vivo biotransformation of simvastatin, an inhibitor of HMG CoA reductase.

Simvastatin (SV), an analog of lovastatin, is the lactone form of 1', 2', 6', 7', 8', 8a'-hexahydro-3,5-dihydroxy-2', 6'-dimethyl-8' (2", 2"-dimethyl-1"-oxobutoxy)-1'-naphthalene-heptanoic acid (SVA) which lowers plasma cholesterol by inhibiting 3-hydroxy-3-methylglutaryl-CoA reductase. SV but not its corresponding hydroxy acid form SVA underwent microsomal metabolism. Major in vitro metabolites were 6'-OH-SV (I) and 3"-OH-SV (III) formed by allylic and aliphatic hydroxylation, respectively, and 6'-exomethylene-SV (IV) formed by dehydrogenation. In rats, dogs, and humans, biliary excretion is the major route of elimination. Biliary metabolites (as both hydroxy acids and lactones) also included 6'-CH2OH-SV (V) and 6'-COOH-SV (VI) in both of which the 6'-chiral center had been inverted. High levels of esterase in rodent plasma favored the formation of SVA from SV. The formation of 1', 2', 6', 7', 8', 8a'-hexahydro-2', 6'-dimethyl-8'-(2",2"-dimethyl-1-oxobutoxy)-1'-naphthalene-pentano ic acid (VII) only in rodents represented a species difference in the metabolism of SV. It is proposed that VII is formed by beta-oxidation pathways of fatty acid intermediary metabolism. Several metabolites resulting from microsomal oxidation (after subsequent conversion from lactones to hydroxy acids) are effective inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase and may contribute to the cholesterol lowering effect of SV. Qualitatively, the metabolism of SV closely resembles that of lovastatin.

Biotransformation of lovastatin. I. Structure elucidation of in vitro and in vivo metabolites in the rat and mouse.

Structures of in vitro microsomal and in vivo metabolites of lovastatin, a new cholesterol-lowering drug, were elucidated with the combined application of HPLC, UV, fast atom bombardment-MS, and NMR spectroscopy. Liver microsomes from rats and mice catalyzed the biotransformation of lovastatin, primarily at the 6'-position of the molecule, to form 6'-hydroxy-lovastatin and a novel 6'-exomethylene derivative. Hydroxylation at the 6'-position occurred stereoselectively, giving 6'-beta-hydroxy-lovastatin. Stereoselective hydroxylation at the 3"-position of the methylbutyryl side chain and hydrolysis of the lactone group to the corresponding hydroxy acid were the other two pathways of microsomal metabolism. 3'-Hydroxy-iso-delta 4',5'-lovastatin was isolated, but is not believed to be a direct metabolite since 6'-beta-hydroxy-lovastatin rearranges to this compound under mildly acidic conditions. The major metabolites excreted in bile of rats treated with the hydroxy acid form of the drug were identified as the 3'-hydroxy analog and a taurine conjugate of a beta-oxidation product of lovastatin. The pentanoic acid derivative of lovastatin, formed by beta-oxidation of the heptanoic acid moiety, was a major metabolite in livers of mice dosed with the hydroxy acid form of lovastatin. The microsomal metabolites, in their hydroxy acid forms, were active inhibitors of HMG-CoA reductase. The relative enzyme inhibitory activities of hydroxy acid forms of lovastatin, 6'-beta-hydroxy-, 6'-exomethylene-, and 3"-hydroxy-lovastatin were 1, 0.6, 0.5, and 0.15, respectively.

Biotransformation of lovastatin. II. In vitro metabolism by rat and mouse liver microsomes and involvement of cytochrome P-450 in dehydrogenation of lovastatin.

Metabolism of lovastatin, a new cholesterol-lowering drug, by liver microsomes from rats and mice was investigated. Liver microsomes from rats catalyzed biotransformation of lovastatin at a rate of 3 nmol/mg of protein/min, whereas the rate of metabolism was 37% higher with liver microsomes from mice. The profiles of metabolites were similar, but the relative abundance of individual metabolites was species dependent. Hydroxylation at the 6'-position was the principal pathway of lovastatin biotransformation, whereas hydroxylation at the 3"-position of the side chain was a minor pathway. In both species the 6'-beta-hydroxy-lovastatin accounted for half of the total metabolism. Liver microsomes from rats produced 2- to 4-fold higher amounts of the other three metabolites, namely, 6'-exomethylene-, 3"-hydroxy-, and the hydroxy acid form, than mouse liver microsomes. The conversion of lovastatin to the novel 6'-exomethylene metabolite was catalyzed by cytochrome P-450 since it required microsomes and NADPH and was inhibited by SKF-525A, metyrapone, and 2,4,-dichloro-6-phenylphenoxyethylamine (DPEA). Furthermore, neither 6'-beta-hydroxy-lovastatin nor the 6'-hydroxymethyl analogs could be demonstrated to be intermediates in the formation of the 6'-exomethylene metabolite. The hydroxy acid form of lovastatin was not a substrate for liver microsomes from either species.

Regioselectivity and stereoselectivity in the metabolism of HMG-CoA reductase inhibitors.

Biotransformation of three analogs of simvastatin, L-672,201, L-157,012 and L-672,220, by rat liver microsomes has been examined. These compounds differ from each other at the 6' position of the hexahydronaphthalene system. When 6'-substituents were in the alpha configuration, rat liver microsomes catalysed biotransformation primarily at the 6' position. Hydroxylation was stereoselective giving 6' beta-hydroxy derivatives as major metabolites. In contrast, when the 6'-substituent had a beta-configuration, metabolism at this site was blocked. Rates of metabolism (nmols/mg protein/min) also indicated that 6' beta-derivatives were poorer substrates than their 6' alpha-counterparts. The results indicate that cytochrome P-450 exhibits a high degree of regio- and stereoselectivity in the metabolism of HMG-CoA reductase inhibitors.

Biotransformation of lovastatin--III. Effect of cimetidine and famotidine on in vitro metabolism of lovastatin by rat and human liver microsomes.

The effects of the H2-receptor antagonists, cimetidine and famotidine, on the microsomal metabolism of [14C]lovastatin were investigated. Liver microsomes were prepared from control, phenobarbital- and 3-methylcholanthrene-pretreated rats and humans (male and female). Concentration-dependent inhibition of the metabolism of lovastatin (0.1 mM) was observed with cimetidine (0.1 to 1.0 mM). In contrast, famotidine at a similar concentration was a very weak inhibitor. The formation of 6'beta-hydroxy-lovastatin, the major microsomal metabolite of lovastatin, was similarly inhibited. The results suggest that in vivo metabolic interaction with concomitantly administered lovastatin is less likely with famotidine than with cimetidine. Phenobarbital pretreatment produced 58% stimulation in overall metabolism, whereas 3-methylcholanthrene pretreatment had no effect relative to control rats (5.4 nmol/mg protein/min). Liver microsomes from phenobarbital-pretreated rats produced 67% more of the 6'beta-hydroxy-lovastatin but 63-66% less of the 3''-hydroxy and 6'-exomethylene metabolites. Liver microsomes from 3-methylcholanthrene-treated rats also produced less 3"-hydroxy-lovastatin (49%) but similar quantities of the other two metabolites. 6'beta-Hydroxy-lovastatin was a major metabolite with human liver microsomes. Interestingly with these microsomes, hydroxylation at the 3''-position of the molecule was a negligible pathway and hydrolysis to the hydroxy acid form was not observed. The formation of 6'-exomethylene-lovastatin was also catalyzed by human liver microsomes (0.5 to 0.8 nmol/mg protein/min).