Metoprolol oxidation by rat liver microsomes: Inhibition by debrisoquine and other drugs

doi:10.1016/0006-2952(86)90186-3

Biochemical Pharmacology

Volume 35, Issue 16, 15 August 1986, Pages 2757-2761

https://doi.org/10.1016/0006-2952(86)90186-3 Get rights and content

Abstract

The oxidative metabolism of metoprolol has been shown to display genetic polymorphism of the debrisoquine-type. The use of in vitro inhibition studies has been proposed as a means of denning whether one or more forms of cytochrome P-450 are involved in the rnonogenically-controlled metabolism of two substrates. We have, therefore, tested the ability of debrisoquine and other substrates to inhibit the oxidation of metoprolol by rat liver microsomes. Debrisoquine and guanoxan were potent competitive inhibitors of the α-hydroxylation and O-desmethylation of metoprolol as well as its metabolism by all routes (measured by substrate disappearance). Cimetidine and ranitidine, drugs which are known to impair the clearance of metoprolol in man, showed an inhibitory action comparable to that of debrisoquine in rat liver microsomes. Antipyrine, a compound whose metabolism is not impaired in poor metabolisers of debrisoquine, was found to be only a weak inhibitor of the metabolism of metoprolol. These findings suggest that the oxidation of metoprolol is linked closely to that of debrisoquine, cimetidine and ranitidine but not to that of antipyrine in the rat.

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In this paper, we validated the depletion assay by comparing Km and the maximum metabolic intrinsic clearance (CLint) obtained from the substrate depletion assay with relevant data reported in the metabolite formation assay. For this purpose, we employed propranolol and metoprolol (a β-adrenoceptor antagonist) that are subject to multiple and/or sequential biotransformations,14−17 and nisoldipine (a calcium channel blocker), which shows a more complex biotransformation.18,19 Furthermore, we investigated the usefulness of the depletion assay for predicting nonlinearities of metoprolol, timolol, and propranolol, which exhibited a marginal, moderate, and large nonlinear pharmacokinetics after oral administrations in rats, respectively.
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Although glucuronidation of propranolol has been identified as one of the metabolic pathways in humans,17 this biotransformation appears to be a minor pathway in rats because no glucuronide was detected in preliminary incubations using freshly isolated rat hepatocytes (data not shown). For metoprolol, two main metabolic pathways, α-hydroxylation and O-desmethylation, have been shown to be inhibited by debrisoquine in a competitive manner,31 and these results and our data regarding the inhibition of metoprolol metabolism by quinine are consistent. Although CYP2D was the major isoform responsible for the overall metabolic pathways of metoprolol and propranolol, oral application of propranolol yielded a notable strain difference.
The purpose of this study was to clarify the pharmacokinetics of CYP2D6 substrates in female DA and Wistar rats, which are regarded as animal models of poor metabolizers and extensive metabolizers, respectively. In vivo pharmacokinetic and in vitro metabolic studies were conducted using metoprolol and propranolol, which show substantial and marginal polymorphisms in humans, respectively. After oral administration, the areas under the plasma concentration curves (AUC) for metoprolol and propranolol in DA rats were ca. 5- and 35-fold higher, respectively, than those in Wistar rats. There were no strain differences for serum protein binding or metabolism inhibition by quinine between the two compounds. Using a substrate depletion assay, the intrinsic clearances estimated for the two strains differed by 7.2-fold for metoprolol and 4.5-fold for propranolol. The discrepancy between the in vitro and in vivo profiles observed for propranolol, but not metoprolol, would be due to nonlinearity between the normalized AUC and the oral doses in DA rats, being associated with lower K_m values. The larger strain difference in the AUCs of propranolol was proved by the in vitro kinetic parameters, implying that DA rats do not always reflect the polymorphic profiles in humans.
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A new form of cytochrome P450 (P450 DUT2) was purified from untreated male dog liver microsomes. The final preparation (a specific content of 19.1 nmol P450/mg protein) showed a single band with an apparent monomeric molecular weight of 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but was further separated into two apoproteins (P450 DUT2_a and P450 DUT2_b) by reverse-phase HPLC. Both proteins had identical NH₂-terminal amino acid sequences, but the first three amino acids of P450 DUT2_b were truncated in P450 DUT2_a. Purified P450 DUT2 showed 5 to 18 times higher catalytic activities than did hepatic microsomes toward desipramine, metoprolol, and dextromethorphan. These activities in dog liver microsomes were strongly inhibited by anti-P450 DUT2-IgG. A 1.7-kilobase pair cDNA (cDUT2) encoding a male dog liver P450 of 500 amino acid residues (molecular weight 56,400) was isolated and sequenced. The first 35 NH₂-terminal amino acid sequence of P450 DUT2_b coincided with the deduced amino acid sequence of cDUT2 at 2-36. The deduced total amino acid sequence of cDUT2 shared high similarity with the reported 2D forms (with 2D6, 74.6%; 2D14, 75.4%; 2D1, 65.4%; and 2D9, 63.6%). Moreover, the expressed P450 DUTS in COS-7 cells had catalytic activities similar to those of purified P450 DUT2. Therefore, this paper is the first report about dog CYP2D. Furthermore, Northern and Western blot analyses indicated that the expressed levels of mRNA and protein were almost equal between male and female dogs. Western blot analysis suggested that P450 DUT2 is a constitutive and major (approximately 20% of the total P450) form, indicating that the 2D subfamily P450 in dog Liver is quite unique from CYP2D members of other species.
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Enantio- and diastereoselective aspects of oxidative metabolism of metoprolol (1) were examined in the presence of rat liver and human liver microsomes using a pseudoracemate of 1, made up of equal molar $(2R)- 1 - d_{0}$ and $(2S)- 1 - d_{2}$ , as substrate. Both O-demethylation and α-hydroxylation showed only slight enantioselectivity, $2R 2S$ ratios being 1.18 and 0.93 for these pathways in rat liver microsomes and 1.09 and 0.92 in human liver microsomes. In the presence of the rat liver microsomal fraction, α-hydroxylation yielded predominantly the 1′R-hydroxy product, $1′ R 1′S ratio > 12$ , regardless of the stereochemistry of the side chain. In humans (extensive metabolizers) administered a single 50 mg oral dose of pseudoracemic metoprolol tartrate, urinary α-hydroxymetoprolol (2) accounted for 9.3 ± 2.4% of the dose, $2R 2S ratio of 0.85 ± 0.14$ , and the carboxylic acid metabolite 4, accounted for 52.7 ± 6.8% of the dose, $2R 2S$ ratio 1.15 ± 0.09. The data suggested that preferential O-demethylation of the (2R)-enantiomer of 1 could contribute to the 2S > 2R plasma ratio of metoprolol enantiomers observed in this population.
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Metoprolol oxidation by rat liver microsomes: Inhibition by debrisoquine and other drugs

Abstract

Biochem. Pharmac.

Lancet

J. biol. Chem.

J. Chromatogr.

Biochem. Pharmac.

Life Sci.

Biochem. Pharmac.

Biochem. Pharmac.

Biochem. Pharmac.

Biochem. Pharmac.

Biochem. Pharmac.

Life Sci.

N. Engl. J. Med.

Br. J. clin. Pharmac.

Br. J. clin. Pharmac.

Molec. Pharmac.

Clin. Pharmac. Ther.

Br. Med. J.

Clin. Pharmac. Ther.