The potential for CYP2D6 inhibition screening using a novel scintillation proximity assay-based approach.

High throughput inhibition screens for human cytochrome P450s (CYPs) are being used in preclinical drug metabolism to support drug discovery programs. The versatility of scintillation proximity assay (SPA) technology has enabled the development of a homogeneous high throughput assay for cytochrome P450 2D6 (CYP2D6) inhibition screen using [O-methyl-(14)C]dextromethorphan as substrate. The basis of the assay was the trapping of the O-demethylation product, [(14)C]HCHO, on SPA beads. Enzyme kinetics parameters V(max) and apparent K(m), determined using pooled human liver microsomes and microsomes from baculovirus cells coexpressing human CYP2D6 and NADPH-cytochrome P450 reductase, were 245 pmol [(14)C]HCHO/min/mg protein and 11 microM, and 27 pmol [(14)C]HCHO/min/pmol and 1.6 microM, respectively. In incubations containing either pooled microsomes or recombinant CYP2D6, [(14)C]dextromethorphan O-demethylase activity was inhibited in the presence of quinidine (IC(50) = 1.0 microM and 20 nM, respectively). By comparison, inhibitors selective for other CYP isoforms were relatively weak (IC(50) > 25 microM). In agreement, a selective CYP2D6 inhibitory monoclonal antibody caused greater than 90% inhibition of [(14)C]dextromethorphan O-demethylase activity in human liver microsomes, whereas CYP2C9/19- and CYP3A4/5-selective antibodies elicited a minimal inhibitory effect. SPA-based [(14)C]dextromethorphan O-demethylase activity was also shown to correlate (r(2) = 0.6) with dextromethorphan O-demethylase measured by high-performance liquid chromatography in a bank of human liver microsomes (N = 15 different organ donors). In a series of known CYP2D6 inhibitors/substrates, the SPA-based assay resolved potent inhibitors (IC(50) < 2 microM) from weak inhibitors (IC(50) >or= 20 microM). It is concluded that the SPA-based assay described herein is suitable for CYP2D6 inhibition screening using either native human liver microsomes or cDNA-expressed CYP2D6.

Role of human liver cytochrome P4503A in the metabolism of etoricoxib, a novel cyclooxygenase-2 selective inhibitor.

Etoricoxib, a potent and selective cyclooxygenase-2 inhibitor, was shown to be metabolized via 6'-methylhydroxylation (M2 formation) when incubated with NADPH-fortified human liver microsomes. In agreement with in vivo data, 1'-N'-oxidation was a relatively minor pathway. Over the etoricoxib concentration range studied (1-1300 microM), the rate of hydroxylation conformed to saturable Michaelis-Menten kinetics (apparent K(m) = 186 +/- 84.3 microM; V(max) = 0.76 +/- 0.45 nmol/min/mg of protein; mean +/- S.D., n = 3 livers) and yielded a V(max)/K(m) ratio of 2.4 to 7.3 microl/min/mg. This in vitro V(max)/K(m) ratio was scaled, with respect to yield of liver microsomal protein and liver weight, to obtain estimates of M2 formation clearance (3.1-9.7 ml/min/kg of b.wt.) that agreed favorably with in vivo results (8.3 ml/min/kg of b.wt.) following i.v. administration of [(14)C]etoricoxib to healthy male subjects. Cytochrome P450 (P450) reaction phenotyping studies-using P450 form selective chemical inhibitors, immunoinhibitory antibodies, recombinant P450s, and correlation analysis with microsomes prepared from a bank of human livers-revealed that the 6'-methyl hydroxylation of etoricoxib was catalyzed largely (approximately 60%) by member(s) of the CYP3A subfamily. By comparison, CYP2C9 (approximately 10%), CYP2D6 (approximately 10%), CYP1A2 (approximately 10%), and possibly CYP2C19 played an ancillary role. Moreover, etoricoxib (0.1-100 microM) was found to be a relatively weak inhibitor (IC(50) > 100 microM) of multiple P450s (CYP1A2, CYP2D6, CYP3A, CYP2E1, CYP2C9, and CYP2C19) in human liver microsomes.

Rizatriptan, a novel 5-HT1B/1D agonist for migraine: single- and multiple-dose tolerability and pharmacokinetics in healthy subjects.

Rizatriptan is a novel 5-HT1D/1B agonist for relief of migraine headache. The pharmacokinetics, metabolite profiles, and tolerability of rizatriptan were examined in a multiple-dose study in healthy subjects. Rizatriptan (N = 24) (or placebo, N = 12) was administered as a single 10 mg dose, followed 48 hours later by administration of one 10 mg dose every 2 hours for three doses on 4 consecutive days, corresponding to the maximum daily dose for a migraine attack. The AUC of rizatriptan and its active N-monodesmethyl metabolite after three 10 mg doses was approximately threefold greater than the plasma concentrations following a single 10 mg dose. Metabolite profiles were similar after single and multiple doses. Adverse events during rizatriptan were mild and transient; similar events occurred during placebo, with a somewhat reduced incidence. Diastolic blood pressure tended to increase compared with placebo (approximately 5 mmHg), particularly on the first multiple-dose day (p < .01 vs. placebo). In conclusion, rizatriptan is well tolerated by healthy subjects during multiple-dose administration, with no unexpected accumulation of drug in plasma.

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.

Metabolism of MK-499, a class III antiarrhythmic agent, in rats and dogs.

MK-499 [(+)-N-[1'-(6-cyano-1, 2, 3, 4-tetrahydro-2(R)-naphthalenyl)-3, 4-dihydro-4(R)-hydroxyspiro(2H-1-benzopyran-2, 4'-piperidin)-6-yl]methanesulfonamide] monohydrochloride is an investigational class III antiarrhythmic agent for treatment of malignant ventricular tachyarrhythmias. The disposition of [3H]MK-499 and [14C]MK-499 was studied in rats and dogs after oral and iv administration. MK-499 was concentrated in organs of excretion and the heart. In the rat, urinary radioactivity elimination values after iv (0.5 mg/kg) and oral (6.25 mg/kg) doses were 21 +/- 3% and 10 +/- 2%, respectively. Corresponding fecal recoveries were 68 +/- 6% and 78 +/- 7%. Similar results were found after corresponding doses of [14C]MK-499. In dogs, urine and feces accounted for 16 +/- 3% and 75 +/- 4% of recovered radioactivity after a [3H]MK-499 iv dose (0.1 mg/kg). Corresponding recoveries after an oral dose (1 mg/kg) were 12 +/- 2% and 76 +/- 3%. Biliary (0-24 hr) excretion accounted for 39 +/- 5% and 41 +/- 18% of [3H] and [14C] oral doses in rats, respectively. Dogs excreted 34% of [3H] oral dose in (0-24 hr) bile. The data indicated that a substantial amount of MK-499 was absorbed by rats and dogs. MK-499, metabolite I (formed by loss of N-substitution), and metabolite II (an acid formed by metabolic scission across the benzopyran ring) each represented 30% of rat urinary label. Rat bile contained MK-499 (10%), II (20%), and IV (10%), which was formed by carbon-4 hydroxylation of the tetralin ring. Additionally, rat bile included glutathione (V) and N-acetyl-1-cysteine (VI) conjugates of a ring-opened metabolite. Metabolite III, a positional isomer of IV, was excreted in rat urine. The major labeled species excreted in dog bile were unchanged MK-499 and its glucuronide (VII), which, respectively, represented 50% and 30% of the biliary radioactivity. MK-499 and a small amount of I represented dog urinary radioactivity. The bioavailability of MK-499 was high in dogs (100%) but low in rats (17%). This difference was probably due to the more extensive presystemic metabolism of MK-499 in rats.

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.

Identification of covalent adducts to protein sulfur nucleophiles by alkaline permethylation.

We recently reported on the identification of metabolites of the hepatotoxin bromobenzene covalently bound to rat liver protein sulfur nucleophiles (D. E. Slaughter and R. P. Hanzlik, Chem. Res. Toxicol. 4, 349-359 (1991). Central to that study was our development of a method called alkaline permethylation which converts protein-S adducts of xenobiotic electrophiles to stable extractable thioanisole derivatives. We report here on substantial improvements to our original alkaline permethylation method which should greatly expand its potential utility. Specifically, we have developed significantly milder reaction conditions, eliminated side reactions, improved the amount of and consistency of thioanisole yields from various mercapturic acid model compounds, and increased the overall sensitivity of the method at least 50-fold. Using the procedure described herein it is routinely possible to generate, detect, and identify by GC/MS as little as 2 pmol of a thioanisole derivative. This method is potentially quite general and should prove useful for studies in the toxicology of reactive metabolites, for industrial hygiene and biomonitoring, and for agrichemical residue analysis.

Identification of epoxide- and quinone-derived bromobenzene adducts to protein sulfur nucleophiles.

Bromobenzene (BB) hepatotoxicity is widely attributed to the alkylation of cellular proteins by chemically reactive metabolites, particularly BB-3,4-oxide. This laboratory recently reported the first conclusive evidence that BB epoxides actually do alkylate proteins; i.e., acid hydrolysates of hepatic proteins from phenobarbital-(PB-) induced BB-treated rats contain S-(o-, S-(m-, and S-(p-bromophenyl)cysteine [Weller, P.E., and Hanzlik, R.P. (1991) Chem. Res. Toxicol. 4, 17-20]. However, these three compounds account for less than 0.5% of total protein covalent binding. Bromoquinone metabolites of BB are also suspected of alkylating proteins. To search for such adducts to protein cysteinyl or methionyl residues, we heated hepatic proteins from PB-induced BB-treated rats with a two-phase mixture of 16 N KOH and CH3I ("alkaline permethylation"). Under these conditions S-alkylated residues are cleaved via elimination and the phenoxide and thiophenoxide groups on the fragments are methylated. Product analysis by 14C HPLC and GC/MS revealed o-, m-, and p-bromothioanisoles in amounts comparable to the content of S-(bromophenyl)cysteines found by acid hydrolysis (para much greater than meta, ortho). This, too, clearly implicates protein-SH alkylation by BB-2,3- and 3,4-oxides. In addition, 2,3-dimethoxy-5-bromothioanisole and another unidentified isomer were observed. However, by far the major adduct (5-6% of total covalent binding) was 2,5-dimethoxythioanisole (i.e., a debrominated adduct). When BB-d5 was administered, the latter contained mostly 3 deuterium atoms/mol. These latter results clearly show that alkylation of protein sulfur nucleophiles in vivo by quinone metabolites is 10-15 times more extensive than their alkylation by BB epoxides. After BB-d5 was administered, the bromothioanisoles and dimethoxybromothioanisoles contained 4 and 2 deuterium atoms/mol, respectively. A weighted average calculation of deuterium retention across the six major sulfur adducts agreed well with 3H/14C retention ratios determined earlier for total liver protein covalent binding of dual-labeled [3H/14C]BB, indicating that the overall pattern of BB metabolite binding to all protein nucleophiles may closely parallel that seen here specifically for protein sulfhydryl groups. The identification of a variety of specific BB-derived adducts to protein now affords the opportunity to investigate their relative contributions to the toxicity of bromobenzene.