Regio- and stereoselective oxidation of propranolol enantiomers by human CYP2D6, cynomolgus monkey CYP2D17 and marmoset CYP2D19
Graphical abstract
HPLC chromatograms showing the oxidation of PL enantiomers by primates recombinant enzymes.
Introduction
Since various human tissues and recombinant enzymes have become available, the metabolic profiles of drug candidates can be predicted fairly accurately in experiments in vitro. However, toxic and pharmacokinetic profiles of drug candidates should still be evaluated in vivo using experimental animals and the data obtained extrapolated to humans. In this context, monkeys such as cynomolgus monkeys, rhesus monkeys and marmosets are superior to non-primates such as rodents, rabbits, and dogs as experimental animal species. Cynomolgus monkeys are rather big (5–10 kg in body weight) as compared with marmosets (200–300 g), which affects handling and feeding. Though cynomolgus monkeys and rhesus monkeys have been used extensively in research into drug metabolism and toxicology, relatively little data has been obtained from marmosets.
Drug metabolism is divided into phase I reactions consisting of oxidation, reduction and hydrolysis and phase II reactions consisting of various kinds of conjugation. The oxidation catalyzed by cytochrome P450s (CYPs) makes up about 80% of phase I reactions [1]. CYPs compose a superfamily of hemethiolate enzymes, and over 10,000 CYPs from animals, birds, fish, plants, microorganisms etc. are known to exist [2]. Major isoenzymes of CYP1, 2 and 3 are responsible for drug metabolism in humans, namely CYP1A1/2, -2A6, -2B6, 2C8, -2C9, -2C19, -2D6, -2E1 and -3A4/5 [3]. CYP2D6 is clinically important because it contributes as the major enzyme to the oxidation of 15% of clinically prescribed medicines [4], though it accounts for only about 2% of all hepatic CYPs [5]. CYP2D6 shows extensive genetic polymorphism, and some 80 allelic variants have been reported to date [6], resulting in variation in drug-response phenotypes such as poor, intermediate, extensive and super-extensive metabolizers [4]. Cynomolgus monkeys and marmosets also have many CYPs [2] including CYP2D enzymes; CYP2D17 for cynomolgus monkeys [7] and CYP2D19 [8] and CYP2D30 [9] for marmosets.
Propranolol (PL) is a classical adrenoceptor blocking agent used clinically to treat arrhythmia and hypertension. PL has an asymmetric carbon atom in its side-chain, yielding the enantiomers R-PL and S-PL. Though S-PL has much more pharmacological activity as a β-blocker than R-PL [10], PL is given as a racemate. PL undergoes extensive metabolism in humans as shown in Fig. 1. For example, it is oxidized at the aromatic 4- and 5-positions mainly by CYP2D6 yielding 4-hydroxypropranolol (4-OH-PL) and 5-OH-PL, respectively, whereas the oxidation of the PL side-chain is mainly catalyzed by CYP1A2 giving N-desisopropylpropranolol (NDP) [11]. The oxidative metabolites as well as the parental compound are the substrates for UDP-glucuronosyltransferases and sulfotransferases [12], [13]. PL is thus a useful substrate to study species differences in the regio- and stereoselective metabolism by CYP and conjugation enzymes.
Recently, we examined the oxidation of PL enantiomers by microsomal fractions from cynomolgus monkey and marmoset livers, and compared it with that by a human liver microsomal fraction [14]. As a result, we obtained experimental evidence that CYP2D enzymes are involved not only in the ring hydroxylation at the 4- and 5-positions but also in the side-chain N-desisopropylation in the monkey liver microsomal fractions [14]. In the present study, we expressed cynomolgus monkey CYP2D17 and marmoset CYP2D19 as well as human CYP2D6 in yeast cells, and compared the oxidation of PL enantiomers by yeast cell microsomal fractions among monkeys and humans.
Section snippets
Materials
PL enantiomers as hydrochlorides were obtained from Sigma–Aldrich (St. Louis, MO); 4-OH-PL and 5-OH-PL as hydrochlorides from C/D/N Isotopes Inc. (Quebec, Canada); NDP as a hydrochloride from AstraZenaca (Cheshire, England); and 4-hydroxybunitrolol (4-OH-BTL) as a hydrochloride from Nippon Boehringer Ingelheim Co. (Hyogo, Japan). The RNeasy Mini kit, QIA shredder, and MiniElute Gel Extraction kit were purchased from Qiagen (Heiden, Germany). The RNA PCR kit v3.0, DNA ligation kit v2.1, Taq DNA
Expression of CYP2D enzymes in yeast cells
Yeast cell microsomal fractions expressing CYP2D6 and CYP2D17 yielded typical reduced carbon monoxide (CO)-difference spectra, while the CYP2D19 fraction showed a spectrum having a low Soret peak at 450 nm (Fig. 2). The amounts of the recombinant enzymes were 39.1 ± 11.5, 63.3 ± 18.8 and 4.38 ± 0.83 pmol/mg protein for CYP2D6, CYP2D17 and CYP2D19, respectively. The concentration of CYP2D19 was significantly lower than that of CYP2D6 or CYP2D17. Western blot analysis using the polyclonal antibody
Discussion
We previously examined the oxidative metabolism of PL enantiomers by liver microsomal fractions from cynomolgus monkeys and marmosets, and found that the kinetic profiles were considerably different between the two monkey species [14]. However, we obtained some lines of experimental evidence that not only PL aromatic ring hydroxylation at the 4- and 5-positions but also side-chain N-desalkylation was mediated by monkey CYP2D enzymes, because the formation of the three metabolites from PL
Conflict of interest
The author declares that there are no conflicts of interest.
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The marmoset cytochrome P450 superfamily: Sequence/phylogenetic analyses, genomic structure, and catalytic function
2020, Biochemical PharmacologyCitation Excerpt :Similarly, cynomolgus macaque P450 2D6 and 2D8 also catalyze dextromethorphan O-demethylation and bufuralol 1′-hydroxylation [58]. The enantio-selectivities of propranolol oxidation by P450 2D6 enzymes are similar for humans, cynomolgus macaques, and marmosets [59]. Both human and marmoset liver microsomes catalyze R-metoprolol O-demethylation, but those of cynomolgus macaques and rats effectively catalyze S-metoprolol O-demethylation and R-metoprolol α-hydroxylation.
Characterization of marmoset CYP2B6: CDNA cloning, protein expression and enzymatic functions
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