Regio- and stereoselective oxidation of propranolol enantiomers by human CYP2D6, cynomolgus monkey CYP2D17 and marmoset CYP2D19

doi:10.1016/j.cbi.2010.12.014

Chemico-Biological Interactions

Volume 189, Issue 3, 1 February 2011, Pages 146-152

https://doi.org/10.1016/j.cbi.2010.12.014 Get rights and content

Abstract

Toxic and pharmacokinetic profiles of drug candidates are evaluated in vivo often using monkeys as experimental animals, and the data obtained are extrapolated to humans. Well understanding physiological properties, including drug-metabolizing enzymes, of monkeys should increase the accuracy of the extrapolation. The present study was performed to compare regio- and stereoselectivity in the oxidation of propranolol (PL), a chiral substrate, by cytochrome P450 2D (CYP2D) enzymes among humans, cynomolgus monkeys and marmosets. Complimentary DNAs encoding human CYP2D6, cynomolgus monkey CYP2D17 and marmoset CYP2D19 were cloned, and their proteins expressed in a yeast cell expression system. The regio- and stereoselective oxidation of PL enantiomers by yeast cell microsomal fractions were compared. In terms of efficiency of expression in the system, the holo-proteins ranked CYP2D6 ≒ CYP2D17 ≫ CYP2D19. This may be caused by the bulky side chain of the amino acid residue at position 119 (leucine for CYP2D19 vs. valine for CYP2D6 and CYP2D17), which can disturb the incorporation of the heme moiety into the active-site cavity. PL enantiomers were oxidized by all of the enzymes mainly into 4-hydroxyproranolol (4-OH-PL), followed by 5-OH-PL and N-desisopropylpropranolol (NDP). In the kinetic analysis, apparent K_m values were commonly in the μM range and substrate enantioselectivity of R-PL < S-PL was observed in both K_m and V_max values for the formation of the three metabolites from PL enantiomers. The activity to produce NDP tended to be higher for the monkey enzymes, particularly CYP2D17, than for the human enzyme. These results indicate that in the oxidation of PL enantiomers by CYP2D enzymes, stereoselectivity is similar but regioselectivity is different between humans and monkeys.

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.

References (22)

D.C. Mankowski et al.
Molecular cloning, expression, and characterization of CYP2D17 from cynomolgus monkey liver
Arch. Biochem. Biophys.
(1999)
T. Igarashi et al.
Marmoset liver cytochrome P450s: study for expression and molecular cloning of their cDNAs
Arch. Biochem. Biophys.
(1997)
H. Hichiya et al.
Cloning and functional expression of a novel marmoset cytochrome P450 2D enzyme, CYP2D30: comparison with the known marmoset CYP2D19
Biochem. Pharmacol.
(2004)
B. Silber et al.
Stereoselective disposition and glucuronidation of propranolol in human
J. Pharm. Sci.
(1982)
T. Shimizudani et al.
Comparative study of the oxidation of propranolol enantiomers in hepatic and small intestinal microsomes from cynomolgus and marmoset monkeys
Chemico-Biol. Interact.
(2010)
S. Narimatsu et al.
Molecular cloning and functional analysis of cytochrome P450 1A2 from Japanese monkey liver: comparison with marmoset cytochrome P450 1A2
Chemico-Biol. Interact.
(2005)
T. Omura et al.
The carbon monoxide-binding pigment of liver microsomes I. Evidence for its hemoprotein nature
J. Biol. Chem.
(1964)
O.H. Lowry et al.
Protein measurement with the Folin phenol reagent
J. Biol. Chem.
(1951)
T. Omura et al.
The carbon monoxide-binding pigment of liver microsomes II. Solubilization, purification and properties
J. Biol. Chem.
(1964)
W.E. Evans et al.
Pharmacogenomics: translating functional genomics into rational therapeutics
Science
(1999)

The home page of Dr. David Nelson;...

Cited by (18)

Polymorphic cytochromes P450 in non-human primates
2022, Advances in Pharmacology
Citation Excerpt :
Similarly, cynomolgus CYP2D6 and CYP2D8 catalyze dextromethorphan O-demethylation and bufuralol 1′-hydroxylation (Uno, Matsushita, et al., 2010). The enantioselectivity of propranolol oxidation by CYP2D6 enzymes is similar in humans, cynomolgus macaques, and marmosets (Narimatsu et al., 2011). R-metoprolol O-demethylation is catalyzed in human and marmoset liver microsomes, whereas S-metoprolol O-demethylation and R-metoprolol α-hydroxylation are effectively catalyzed in cynomolgus liver microsomes (Table 2).
Cynomolgus macaques (Macaca fascicularis, an Old World monkey) are widely used in drug development because of their genetic and physiological similarities to humans, and this trend has continued with the use of common marmosets (Callithrix jacchus, a New World monkey). Information on the major drug-metabolizing cytochrome P450 (CYP, P450) enzymes of these primate species indicates that multiple forms of their P450 enzymes have generally similar substrate selectivities to those of human P450 enzymes; however, some differences in isoform, activity, and substrate specificity account for limited species differences in drug oxidative metabolism. This review provides information on the P450 enzymes of cynomolgus macaques and marmosets, including cDNA, tissue expression, substrate specificity, and genetic variants, along with age differences and induction. Typical examples of important P450s to be considered in drug metabolism studies include cynomolgus CYP2C19, which is expressed abundantly in liver and metabolizes numerous drugs. Moreover, genetic variants of cynomolgus CYP2C19 affect the individual pharmacokinetic data of drugs such as R-warfarin. These findings provide a foundation for understanding each P450 enzyme and the individual pharmacokinetic and toxicological results in cynomolgus macaques and marmosets as preclinical models. In addition, the effects of induction on some drug clearances mediated by P450 enzymes are also described. In summary, this review describes genetic and acquired individual differences in cynomolgus and marmoset P450 enzymes involved in drug oxidation that may be associated with pharmacological and/or toxicological effects.
Two dimensional chromatography mass spectrometry: Quantitation of chiral shifts in metabolism of propranolol in bioanalysis
2020, Journal of Chromatography A
Citation Excerpt :
In vivo concentrations of (S)−5-HOPL and (S)-7-HOPL in analysed urine samples are often below LOQ or even non-detectable (Fig. 6). In literature, in vitro formation of 7-HOPL by rhCYP2D6 often is not reported [7, 28]. Our experiments clarify, that recombinant human rhCYP2D6 as well as rhCYP1A2 possess the ability to catalyse the formation of (R)−5-HOPL, (R)−4-HOPL and (R)−7-HOPL in vitro (Fig. 7).
In this study a heart-cutting 2D-LC method was successfully developed and optimized in order to discriminate and quantitate (S)-propranolol, (R)-propranolol, and its hydroxy metabolites, namely the isomeric (S)-4′‑hydroxy propranolol, (R)-4′‑hydroxy propranolol, (S)-5′‑hydroxy propranolol, (R)-5′‑hydroxy propranolol, (S)-7′-hydroxy propranolol, and (R)-7′‑hydroxy propranolol in one chromatographic run. Thereby, experiments investigating chiral discrimination in ring hydroxylation of propranolol were made feasible.
Analysis of human urine samples after administration of a single oral dose of 40 mg of propranolol clearly revealed considerable chiral shifts in propranolol and its 4′-, 5′-, and 7′-hydroxy metabolites. Furthermore, the excretion rates of the individual (S)- and (R)-enantiomers were continuously monitored over 24 h post administration.
Studies were performed utilizing a 2D-LC system hyphenated to a triple quadrupole mass spectrometer. The chromatographic system was endued with a reversed phase column (phenyl-hexyl) in first dimension and a teicoplanin based chiral column in second dimension. The method was basically validated and successfully evaluated as robust. Calibration was performed achieving accuracy between 80% and 120%. Maximal excretion rates of (S)-propranolol, (R)-propranolol, (S)-4′‑hydroxy propranolol, (R)-4′‑hydroxy propranolol, (S)-5′‑hydroxy propranolol, (R)-5′‑hydroxy propranolol, and (R)-7′‑hydroxy propranolol were 237 ng/min, 281 ng/min, 4 ng/min, 4 ng/min, 1 ng/min, 9 ng/min, and 3 ng/min, respectively.
The marmoset cytochrome P450 superfamily: Sequence/phylogenetic analyses, genomic structure, and catalytic function
2020, Biochemical Pharmacology
Citation 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.
The common marmoset (Callithrix jacchus) is a New World monkey that has attracted much attention as a potentially useful primate model for preclinical testing. A total of 36 marmoset cytochrome P450 (P450) isoforms in the P450 1–51 subfamilies have been identified and characterized by the application of genome analysis and molecular functional characterization. In this mini-review, we provide an overview of the genomic structures, sequence identities, and substrate selectivities of marmoset P450s compared with those of human P450s. Based on the sequence identity, phylogeny, and genomic organization of marmoset P450s, orthologous relationships were established between human and marmoset P450s. Twenty-four members of the marmoset P450 1A, 2A, 2B, 2C, 2D, 2E, 3A, 4A, and 4F subfamilies shared high degrees of homology in terms of cDNA (>89%) and amino acid sequences (>85%) with the corresponding human P450s; P450 2C76 was among the exceptions. Phylogenetic analysis using amino acid sequences revealed that marmoset P450s in the P450 1–51 families were located in the same clades as their human and macaque P450 homologs. This finding underlines the evolutionary closeness of marmoset P450s to their human and macaque homologs. Most marmoset P450 1–4 enzymes catalyzed the typical drug-metabolizing reactions of the corresponding human P450 homologs, except for some differences of P450 2A6 and 2B6. Consequently, it appears that the substrate specificities of enzymes in the P450 1–4 families are generally similar in marmosets and humans. The information presented here supports a better understanding of the functional characteristics of marmoset P450s and their similarities and differences with human P450s. It is hoped that this mini-review will facilitate the successful use of marmosets as primate models in drug metabolism and pharmacokinetic studies.
Utility of non-human primates in drug development: Comparison of non-human primate and human drug-metabolizing cytochrome P450 enzymes
2016, Biochemical Pharmacology
Cynomolgus monkeys (Macaca fascicularis, an Old World Monkey) have been widely used as a non-human primate model in preclinical studies because of their genetic and physiological similarity to humans. This trend has been followed by common marmoset (Callithrix jacchus, a New World Monkey). However, drug-metabolism properties in these non-human primates have not been fully understood due to limited information on cytochrome P450 (P450) enzymes, major drug-metabolizing enzymes in humans. Multiple forms of cynomolgus monkey P450 enzymes have been identified and characterized in comparison to those of humans, including a cynomolgus monkey specific form, P450 2C76. Similarly, marmoset P450 1A/B, 2A, 2C, 2D, and 4F enzymes were recently identified and characterized to understand drug metabolism properties. In this research update, updates for marmoset, cynomolgus monkey, and human P450 cDNAs are provided. Marmoset and cynomolgus monkey P450 enzymes showed high sequence homology to their human counterparts and generally had similar substrate recognition functionality to human P450 enzymes; however, they also possibly contribute to limited specific differences in drug oxidative metabolism partly due to small differences in amino acid residues. These findings provide a foundation for successful use of non-human primates as preclinical models and will help to further understand molecular mechanisms of human P450 function. In addition to the P450 enzymes, flavin-containing monooxygenases, another monooxygenase family, in these non-human primates have been found to be involved in the oxidation of a variety of compounds associated with pharmacological and/or toxicological effects in humans and are also described.
Characterization of marmoset CYP2B6: CDNA cloning, protein expression and enzymatic functions
2013, Biochemical Pharmacology
Citation Excerpt :
In marmosets, cDNA nucleotide sequences and deduced amino acid sequences for eight drug-metabolizing type CYP enzymes [CYP1A2 (accession number D86475), CYP2C8 (AB242600), CYP2D19 (D29822), CYP2D30 (AY082602), CYP2E1 (D86477), CYP3A4 (D31921), CYP3A5 (EF589801) and CYP3A90 (EF589800)] have been registered in GenBank to date. Previous studies have characterized the enzymatic functions of marmoset CYP1A2 [6,7], CYP2C8 [8], CYP2D19 [9–11] and CYP2D30 [10] expressed in various heterologus expression systems, but no reliable experimental data have been published on the enzymatic functions of recombinant marmoset CYP2E1 or CYP3A enzymes so far. In the present study, we focused on CYP2B enzymes in marmoset livers, because only very little information has been reported on the marmoset CYP2B family.
The common marmoset is a promising species for evaluating the safety of drug candidates. To further understand the capacity for drug metabolism in marmosets, a cDNA encoding a CYP2B enzyme was cloned from the total RNA fraction of marmoset liver by 3′- and 5′-RACE methods. Nucleotide and deduced amino acid sequences showed 90.8 and 86.2% identity, respectively, with human CYP2B6. The marmoset CYP2B6 (marCYP2B6) protein was expressed in insect cells, and its enzymatic properties were compared with those of human (humCYP2B6) and cynomolgus monkey (cynCYP2B6) orthologs in liver and insect cell microsomes. Enzymatic functions were examined for the oxidation of 7-ethoxy-4-(trifluoromethyl)coumarin (7-ETC), bupropion (BUP) and efavirenz (EFV). The kinetic profiles for the oxidation of the three substrates by liver microsomal fractions were similar between humans and cynomolgus monkeys (biphasic for 7-ETC and monophasic for BUP and EFV), but that of marmosets was unique (monophasic for 7-ETC and biphasic for BUP and EFV). Recombinant enzymes, humCYP2B6 and cynCYP2B6, also yielded similar kinetic profiles for the oxidation of the three substrates, whereas marCYP2B6 showed activity only for 7-ETC hydroxylation. In silico docking simulations suggested that two amino acid residues, Val-114 and Leu-367, affect the activity of marCYP2B6. In fact, a marCYP2B6 mutant with substitutions V114I and L367V exhibited BUP hydroxylase activity that was 4-fold higher than that of humCYP2B6, while its EFV 8-hydroxylase activity was only 10% that of the human enzyme. These results indicate that the amino acids at positions 114 and 367 affect the enzymatic capacity of marmoset CYP2B6.
Advances and challenges in the pharmacokinetics and bioanalysis of chiral drugs
2022, Chirality

View all citing articles on Scopus

View full text

Regio- and stereoselective oxidation of propranolol enantiomers by human CYP2D6, cynomolgus monkey CYP2D17 and marmoset CYP2D19

Abstract

Graphical abstract

Introduction

Section snippets

Materials

Expression of CYP2D enzymes in yeast cells

Discussion

Conflict of interest

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

Biochem. Pharmacol.

J. Pharm. Sci.

Chemico-Biol. Interact.

Chemico-Biol. Interact.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Pharmacogenomics: translating functional genomics into rational therapeutics

Science