Characterization of the Rhesus Monkey CYP3A64 Enzyme: Species Comparisons of CYP3A Substrate Specificity and Kinetics Using Baculovirus-Expressed Recombinant Enzymes

Carr, Brian A.; Norcross, Ryan; Fang, Yulin; Lü, Ping; Rodrigues, A. David; Shou, Magang; Rushmore, Tom; Booth-Genthe, Catherine

doi:10.1124/dmd.106.009977

Cited by 53 publications

(59 citation statements)

References 35 publications

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“…As a consequence, the modifications of CL int (the ratio of V max to K m ) paralleled those of V max . The absolute values of CL int of the recombinant enzymes were very similar to those previously obtained by Carr et al (2006) (3.3 and 0.1 ml/min per pmol CYP for rCYP3A2 and rCYP3A1, respectively), thus confirming the greater catalytic efficiency of CYP3A2.…”

Section: Resultssupporting

confidence: 74%

Differential Effect of Liver Cirrhosis on the Pregnane X Receptor–Mediated Induction of CYP3A1 and 3A2 in the Rat

et al. 2014

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Conflicting results have been obtained by clinical studies investigating the effect of liver cirrhosis on enzyme induction. Because ethical concerns do not give consent for methodologically rigorous studies in humans, we addressed this question by examining the effect of the prototypical inducer dexamethasone (DEX) on the pregnane X receptor (PXR)-mediated induction of CYP3A1 and 3A2 in a validated animal model of liver cirrhosis obtained by exposure of rats to carbon tetrachloride. For this purpose, we assessed mRNA levels, protein expressions, and enzymatic activities of both CYP3A enzymes, as well as mRNA and protein expressions of PXR in rat populations rigorously stratified according to the severity of liver insufficiency. Constitutive mRNA and protein expressions of CYP3A1 and CYP3A2 and their basal enzyme activities were not affected by liver dysfunction. DEX treatment markedly increased steadystate mRNA level, protein content, and enzymatic activity of CYP3A1 in healthy and cirrhotic rats, irrespective of the degree of liver dysfunction. On the contrary, the inducing effect of DEX on gene and protein expressions and enzyme activity of CYP3A2 was preserved in moderate liver insufficiency, whereas it was greatly curtailed when liver insufficiency became severe. mRNA and protein expressions of PXR were neither reduced by liver dysfunction nor increased by DEX treatment. These results indicate that even the inducibility of cytochrome P450 isoforms under the transcriptional control of the same nuclear receptor may be differentially affected by cirrhosis and may partly explain why conflicting results were obtained by human studies.

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Section: Resultssupporting

confidence: 74%

Differential Effect of Liver Cirrhosis on the Pregnane X Receptor–Mediated Induction of CYP3A1 and 3A2 in the Rat

et al. 2014

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“…After consulting with the P450 Nomenclature Committee (http://drnelson.utmem.edu/ cytochromeP450), in this article, we designate cynomolgus CYP3A8 and rhesus CYP3A64, both orthologous to human CYP3A4, as CYP3A4, and rhesus CYP3A66, orthologous to human CYP3A5, as CYP3A5. Rhesus CYP3A4 possesses metabolic capabilities and induction properties similar to human CYP3A4 (Carr et al, 2006). Cynomolgus CYP3A4 and CYP3A5 exhibited testosterone 6␤-hydroxylation (Uno et al, 2007), similar to their human P450 counterparts, indicating similar drug-metabolizing properties of macaque and human CYP3A.…”

Section: Introductionmentioning

confidence: 85%

“…Several groups, including ours, have identified cDNAs highly homologous to human CYP3A4 or CYP3A5 cDNA in cynomolgus and rhesus macaques (Komori et al, 1992;Carr et al, 2006;Uno et al, 2007). Between these macaque species and humans, sequence identity of CYP3A4 and CYP3A5 is approximately 95 and 94% in cDNA and 93 and 91% in amino acid sequence, respectively.…”

Section: Introductionmentioning

confidence: 99%

“…This interindividual variability in CYP3A5 protein expression is highly correlated with CYP3A5*3, a defective allele, because the aberrant splicing imposed by the mutant allele gives rise to a nonfunctional protein (Kuehl et al, 2001). Identification and characterization of such genetic variants are essential for understanding drugmetabolizing properties of CYP3A4 and CYP3A5 enzymes.Several groups, including ours, have identified cDNAs highly homologous to human CYP3A4 or CYP3A5 cDNA in cynomolgus and rhesus macaques (Komori et al, 1992;Carr et al, 2006;Uno et al, 2007). Between these macaque species and humans, sequence identity of CYP3A4 and CYP3A5 is approximately 95 and 94% in cDNA and 93 and 91% in amino acid sequence, respectively.…”

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confidence: 99%

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Genetic Variants ofCYP3A4andCYP3A5in Cynomolgus and Rhesus Macaques

Uno¹,

Matsushita²,

Osada³

et al. 2009

Drug Metab Dispos

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ABSTRACT:Cynomolgus and rhesus macaques are frequently used in preclinical trials due to their close evolutionary relationships to humans. We conducted an initial screening for genetic variants in cynomolgus and rhesus macaque genes orthologous to human CYP3A4 and CYP3A5. Genetic screening of 78 Indochinese and Indonesian cynomolgus macaques and 34 Chinese rhesus macaques revealed a combined total of 42 CYP3A4 genetic variants, including 12 nonsynonymous variants, and 34 CYP3A5 genetic variants, including nine nonsynonymous variants. Four of these nonsynonymous variants were located at substrate recognition sites or the hemebinding region, domains essential for protein function, including c.886G>A (V296M) and c.1310G>A (S437N) in CYP3A4 and c.1437C>G (N479K) and c.1310G>C (T437S) in CYP3A5. The mutant proteins of these genetic variants were expressed in Escherichia coli and purified. Metabolic activity of these proteins measured using midazolam and nifedipine as substrates showed that none of these protein variants substantially influences the drugmetabolizing capacity of CYP3A4 or CYP3A5 protein. In Indonesian cynomolgus macaques, we also found IVS3؉1delG in CYP3A4 and c.625A>T in CYP3A5, with which an intact protein cannot be produced due to a frameshift generated. Screening additional genomes revealed that two of 239 animals and three of 258 animals were heterozygous for IVS3؉1delG of CYP3A4 and c.625A>T of CYP3A5, respectively. Some genetic variants were unevenly distributed between Indochinese and Indonesian cynomolgus macaques and between cynomolgus and rhesus macaques. Information on genetic diversity of macaque CYP3A4 and CYP3A5 presented here could be useful for successful drug metabolism studies conducted in macaques.Cynomolgus (Macaca fascicularis) and rhesus (Macaca mulatta) macaques have been used to predict the metabolic fate of drugs in humans due to their evolutionary closeness to humans. As with humans, macaques have a diverse genetic background as evidenced by numerous genetic polymorphisms that have been reported Hernandez et al., 2007;Street et al., 2007). In cynomolgus macaques, interanimal differences have been noted in drug metabolism by in vivo analysis using dextromethorphan and S-mephenytoin as probe substrates (Jacqz et al., 1988), which could be, in some part, attributable to genetic variability of drug-metabolizing enzymes, because genetic variants of cytochromes P450 (P450s) such as CYP2C76 have been identified (Uno et al., 2009).Human CYP3As are considered the major drug-metabolizing cytochrome P450 (P450) subfamily, comprised of CYP3A4, CYP3A5, CYP3A7, and CYP3A43 (Gellner et al., 2001). In humans, CYP3As account for more than half of the total P450 content in human liver (Thummel and Wilkinson, 1998) and metabolize more than half of all prescription drugs, such as nifedipine, midazolam, and testosterone (Thummel and Wilkinson, 1998;Evans and Relling, 1999). Numerous interindividual differences in drug-metabolizing capability mediated by CYP3A4 and CYP3A5 have been reported in hum...

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“…After addition of NADPH to the enzyme solutions at a 1 mM final concentration and preincubation at 37°C for 5 min, the reaction was started by adding MDZ at a 200 nM final concentration (total volume of the reaction mixture: 900 l). The MDZ concentration in reaction mixtures (200 nM) was thought to be considerably lower than the K m values of CYP3A2 and CYP2C11 for MDZ metabolism (Carr et al, 2006;Chovan et al, 2007). At 0, 4, 8, and 15 min, the reaction was stopped by adding 150 l of the reaction mixture to 75 l of acetonitrile containing 200 nM bufuralol as the internal standard (IS).…”

Section: Methodsmentioning

confidence: 99%

Quantitative Prediction of Mechanism-Based Inhibition Caused by Mibefradil in Rats

Sekiguchi¹,

Kato²,

Takada³

et al. 2011

Drug Metab Dispos

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ABSTRACT:It was previously demonstrated that mibefradil, which shows mechanism-based inhibition in humans, also caused drug-drug interactions (DDIs) with midazolam (MDZ) in rats. In this study, we aimed to quantitatively predict the DDIs observed in rats using a physiologically based pharmacokinetic (PBPK) model from in vitro inactivation parameters. For more precise predictions, contribution ratios of cytochrome P450 (P450) isozymes involved in MDZ metabolism and inactivation parameters of mibefradil against each isozyme were incorporated in the predictive model. The evaluation of metabolic rate using recombinant P450s suggested that CYP3A2 and CYP2C11 contributed to 89 and 11% of MDZ metabolism, respectively. Inactivation studies of mibefradil against the two isozymes showed that the maximal inactivation rate constants (k inact ) were considerable in both isozymes (0.231-0.565 min ؊1 ), whereas the inhibitor concentration producing half the k inact (K I, app ) of CYP3A2 (0.263-0.410 M) was a good deal lower than that for CYP2C11 (6.82-11.4 M). As a result of predicting the DDIs using the PBPK model, predicted increases in areas under the concentration-time curve of MDZ with coadministration of mibefradil (284 and 510% at 6 and 12 mg/kg mibefradil, respectively) closely corresponded to the observed values (226 and 545%, respectively). From those results, it was thought that the construction of a predictive model for DDIs using the PBPK model in detail would enable us to quantitatively predict in vivo DDIs from in vitro data. This approach to predict DDIs on the basis of the contributing isozymes would be important for predicting clinical DDIs of drugs metabolized by multiple enzymes.

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Characterization of the Rhesus Monkey CYP3A64 Enzyme: Species Comparisons of CYP3A Substrate Specificity and Kinetics Using Baculovirus-Expressed Recombinant Enzymes

Cited by 53 publications

References 35 publications

Differential Effect of Liver Cirrhosis on the Pregnane X Receptor–Mediated Induction of CYP3A1 and 3A2 in the Rat

Differential Effect of Liver Cirrhosis on the Pregnane X Receptor–Mediated Induction of CYP3A1 and 3A2 in the Rat

Genetic Variants ofCYP3A4andCYP3A5in Cynomolgus and Rhesus Macaques

Quantitative Prediction of Mechanism-Based Inhibition Caused by Mibefradil in Rats

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