Elsevier

Biochemical Pharmacology

Volume 73, Issue 6, 15 March 2007, Pages 880-890
Biochemical Pharmacology

Diclofenac hydroxylation in monkeys: Efficiency, regioselectivity, and response to inhibitors

https://doi.org/10.1016/j.bcp.2006.11.016Get rights and content

Abstract

The catalytic efficiency, regioselectivity, and response to chemical inhibitors of diclofenac (DF) hydroxylation in three Old World monkey liver microsomes (rhesus, cynomolgus, and African green monkey) are different from those determined with human liver microsomes. In contrast to the high affinity–high capacity (low Km–high Vmax) characteristics of DF 4′-hydroxylation in humans, this reaction proceeded in all monkey species with catalytic efficiencies >20-fold lower. However, DF 5-hydroxylation, a negligible reaction in human liver microsomes, was kinetically favored in monkeys mainly due to the increased Vmax values. Chemical inhibitors (reversible or mechanism-based) selective to human CYP3A4 and CYP2C9 failed to differentiate monkey orthologs involved in DF hydroxylation. Immunoinhibition studies with monoclonal antibodies against human CYPs revealed the major contribution of CYP2C and CYP3A to 4′- and to 5-hydroxylation, respectively, in rhesus and cynomolgus liver microsomes. However, in African green monkeys, in addition to CYP2C, CYP3A also appeared to be involved in 4′-hydroxylation. Further studies with recombinant rhesus and African green monkey CYP2C and CYP3A enzymes (rhesus CYP2C75, 2C74, and 3A64; African green monkey CYP2C9agm and CYP3A4agm) confirmed the major role of CYP enzymes of these two subfamilies in DF 4′- and 5-hydroxylation. Clearly, while monkey CYP2C and 3A enzymes retain the same substrate selectivity towards DF hydroxylation as their human orthologs, their altered catalytic efficiency and response to chemical inhibitors may indicate different structural features of active sites as opposed to human orthologs.

Introduction

Cytochrome P450 2C9 (CYP2C9) accounts for approximately 18% of the CYP protein content in human liver microsomes and catalyzes approximately 20% of the CYP-mediated metabolic reactions of drugs currently on the market [1], [2]. It is also genetically polymorphic with a number of variants showing reduced catalytic activity [3]. As a result, drug interactions associated with CYP2C9 and altered pharmacokinetics in CYP2C9 polymorphic subjects have been an important theme in both academic fields and the pharmaceutical industry. It is well known that the success of these two types of studies relies heavily on the selection of the probe substrates. Selectivity is the most important criterion for in vitro studies, while clinical relevance and safety have to be considered for in vivo studies. Diclofenac (DF), a non-steroidal anti-inflammatory drug, is considered a preferred and acceptable in vitro and in vivo probe [4].

DF can be oxidized at both the 4′- and 5-positions (Fig. 1), but it is selectively metabolized to 4′-hydroxyl DF (4′-OHD) by CYP2C9 with a high catalytic efficiency. Interestingly, the other members of the human CYP2C subfamily, CYP2C8, 2C18, and 2C19, also are able to catalyze DF hydroxylation, but they lead to simultaneous formation of 4′-OHD and 5-hydroxy DF (5-OHD) with a much lower efficiency. Studies of the CYP2C9 active site employing DF and its derivatives [5], homology modeling [6], and site-directed mutagenesis [7] suggest that DF is strictly positioned in the CYP2C9 active site via its COO functionality in favor of position 4′, which is not chemically preferred. Thus, DF 4′-hydroxylase activity has been measured in many in vitro and in vivo drug interaction and genotyping studies to indicate the function of human CYP2C9.

Ethical constraints and cost-effective considerations have led to rigorous endeavors in searching for animal models to predict the potentials of drug interactions in humans. While mice and rats have been widely used for this purpose, differences among species in many physiological and biochemical functions quite often make it difficult to extrapolate rodent data to humans. Hence, nonhuman primates, which are genetically closer to humans than are rodents, are considered to be better species in this regard. Rhesus monkeys (Macaca mulatta) and cynomologus monkeys (Macaca fascicularis) are commonly used throughout the pharmaceutical industry as preclinical safety species. They have successfully mimicked drug interactions by inhibitors or inducers verified in humans, although mainly for CYP3A [8], [9], [10], [11], [12]. Recently, African green monkeys (Cercopithecus aethiops) have been reported to be a good model for studying induction of brain and liver CYP2E1 by nicotine [13], [14] and CYP2B6 by Phenobarbital [15]. However, similar cases with monkey CYP2C enzymes are lacking. In contrast to human livers where CYP2C9 accounts for the major component of CYP2C members [16], a recent report [17] describes the identification of a novel major member CYP2C76 in cynomolgus monkey liver. This new CYP2C member was also detected in rhesus monkey liver but not in humans or the great apes. Although its functional characteristics are unknown, the emergence of this new CYP2C member may underscore the importance of a better understanding of substrate selectivity of monkey CYP2C enzymes, if a drug interaction study involving monkey CYP2C enzymes is investigated.

Although DF has been used to characterize heterologously expressed monkey CYP2C enzymes [18], its suitability as a probe for monkey CYP2C enzymes orthologous to human CYP2C9 has not been adequately characterized. Instead, 5-hydroxylation catalyzed by monkey CYP3A has drawn more attention, presumably due to bioactivation and quinidine stimulation concerning this pathway [19], [20]. Involvement of CYP2C enzymes in DF 4′-hydroxylation was deduced mainly based on cross-inhibition by antibodies raised against human or rat CYP2C enzymes [17], [21], [22]. Little effort has been reported comparing this reaction in humans and monkeys in terms of the substrate specificity, kinetic properties, and responses to inhibitors. When a discrepancy was observed between in vitro and in vivo rifampin induction of monkey DF 4′-hydroxylase activity, the suitability of DF as a CYP2C probe substrate was questioned [23]. In this report, we disclose our efforts in evaluating DF as a probe substrate for monkey CYP2C and 3A with the aid of monoclonal antibodies against human CYP3A and 2C enzymes, and recombinant human (CYP2C9 and 3A4), rhesus (CYP2C74, 2C75, and 3A64), and African green monkey (CYP2C9agm and 3A4agm) CYPs. We also investigated whether common human CYP2C9 and 3A4 inhibitors were effective in inhibiting respective monkey orthologs. The results of these studies indicate that rhesus and cynomolgus monkeys are closer to humans than African green monkeys in the regioselectivity of DF hydroxylation, but kinetic parameters and response to chemical inhibitors are significantly different between humans and all monkey species.

Section snippets

Chemicals and biological materials

DF, 4′-OHD, sulfaphenazole (SLF), ketoconazole (KTZ), and tienilic acid (TA) were purchased from Sigma Chemicals (St. Louis, MO). The procedures for in-house preparations of human CYP2C9 and CYP3A4 and rhesus CYP3A64 have been described elsewhere [24], [25]. Rhesus CYP2C74 (GenBank accession no. AY635462) and CYP2C75 (GenBank accession no. AY635463), and African green monkey CYP2C9agm (GenBank accession no. DQ022201) and CYP3A4agm (GenBank accession no. DQ022197) have been cloned and expressed

Catalytic profiles of DF hydroxylation in human, rhesus, cynomolgus, and African green monkey liver microsomes

LC–MS/MS analysis of extracts of all liver microsomal incubations containing DF (250 μM) and NADPH revealed the presence of two major oxidative metabolites with an increase of the protonated molecular weights (MH+) by 16 Da. They were confirmed to be 4′- and 5-hydroxy DF by comparison of retention times and product spectra with authentic standards (data not shown). Since these two metabolites possess similar UV characteristics [5], their regioselective formation in different species is

Discussion

The present study demonstrated that monkeys differed functionally from humans in DF hydroxylation, as reflected in catalytic efficiency, regioselectivity, and responses to chemical inhibitors. Relative to humans, the overall catalytic efficiency (Vmax/Km) of DF oxidation in monkey liver microsomes of all three species was lower in catalyzing DF oxidation (>10-fold). While 4′-hydroxylation was similarly favored over 5-hydroxylation in rhesus and cynomolgus monkeys, the relative level of

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