Utility of non-human primates in drug development: Comparison of non-human primate and human drug-metabolizing cytochrome P450 enzymes

Biochemical Pharmacology xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm

Research update

Utility of non-human primates in drug development: Comparison of non-human primate and human drug-metabolizing cytochrome P450 enzymes Yasuhiro Uno a, Shotaro Uehara b, Hiroshi Yamazaki b,⇑ a b

Shin Nippon Biomedical Laboratories, Ltd., Kainan, Wakayama 642-0017, Japan Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan

a r t i c l e

i n f o

Article history: Received 13 May 2016 Accepted 14 June 2016 Available online xxxx Keywords: Common marmoset Cynomolgus monkey P450 2C9 P450 2C19 Warfarin

a b s t r a c t Cynomolgus monkeys (Macaca fascicularis, an Old World Monkey) have been widely used as a nonhuman 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 nonhuman 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. Ó 2016 Elsevier Inc. All rights reserved.

Contents 1. 2. 3. 4. 5.

Updated information for marmoset, cynomolgus monkey, and human cytochrome P450 cDNAs . . . . . . . . . . . . . . . . . . . . . . . . . Stereoselective warfarin elimination mediated by different P450 enzymes in marmosets, cynomolgus monkeys, and humans . Limited different roles of P450 enzymes in drug oxidations in marmosets, cynomolgus monkeys, and humans . . . . . . . . . . . . . Age-related pharmacokinetic changes and other monooxygenases in humans and cynomolgus monkeys . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

00 00 00 00 00 00 00

1. Updated information for marmoset, cynomolgus monkey, and human cytochrome P450 cDNAs ⇑ Corresponding

author at: Showa Pharmaceutical University, Higashi-tamagawa Gakuen, Machida, Tokyo 194-8543, Japan. E-mail address: [email protected] (H. Yamazaki).

3-3165

Species differences for drug metabolism are an important issue for drug development. The Old World primate cynomolgus monkeys (Macaca fascicularis) and New World primate common

http://dx.doi.org/10.1016/j.bcp.2016.06.008 0006-2952/Ó 2016 Elsevier Inc. All rights reserved.

Please cite this article in press as: Y. Uno et al., Utility of non-human primates in drug development: Comparison of non-human primate and human drugmetabolizing cytochrome P450 enzymes, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.06.008

2

Y. Uno et al. / Biochemical Pharmacology xxx (2016) xxx–xxx

Table 1 Homologous P450 cDNAs in humans, cynomolgus monkeys, and marmosets. P450 subfamily

1A 1B 1D 2A

2B 2C

2D 2E 2G 2J 3A

4A 4F

Marmoset

Cynomolgus monkey

Human

P450

GenBank accession no.

P450

GenBank accession no.

P450

GenBank accession no.

1A1 1A2 1B1

KJ922553 NM_001204434 KX231947

2A6

KJ922555

2B6 2C8 2C18 2C19 2C58 2C76 2D6(19) 2D8 2E1

KJ922556 NM_001204437 KJ922558 KJ922561 KJ922559 KJ922560 NM_001204438 KJ922562 NM_001267751

NM_000499 NM_000761 NM_000104 N/A NM_000762 NM_000764 NM_000766 NM_000767 NM_000770 NM_000771 NM_000772 NM_000769

KX231948 NM_001204440 NM_001204442 NM_001204443

NM_001319482 NM_001319483 KX231946 NM_001319506 NM_001285268 NM_001285348 NM_001284028 NM_001287637 NM_001283763 NM_001287625 NM_001319509 NM_001283290 NM_001284789 NM_001283966 NM_001284773 NM_001319495 NM_001283163 DQ074794 NM_001284534 NM_001319511 NM_001319505

1A1 1A2 1B1 [1D1P] 2A6 [2A7] 2A13 2B6 2C8 2C9 [2C18] 2C19

2J2 3A4(21) 3A5 3A90

1A1 1A2 1B1 1D1 2A23 2A24 2A26 2B6 2C8(20) 2C9(43) 2C18 2C19(75) 2C76 2D6(17) 2D8(44) 2E1 2G2 2J2 3A4(8) 3A5 3A43

4A11 4F2 4F3(3B) 4F71(11) 4F12

KX231949 KX231950 KX231951 KX231952 KX231953

4A11 4F2(45) 4F3v2 4F11 4F12

NM_001283882 NM_001319492 NM_001283131 NM_001319493 NM_001283264

2D6 [2D8P] 2E1 [2G2P] 2J2 3A4 3A5 3A7 3A43 4A11 4F2 4F3 4F11 4F12

NM_000106 N/A NM_000773 N/A NM_000775 NM_017460 NM_000777 NM_000765 NM_022820 NM_000778 NM_001082 NM_001199208 NM_021187 NM_023944

Human P450 enzymes whose sequences are highly similar to P450 enzymes identified in marmosets or cynomolgus monkeys are taken from Uno et al. [2] and Shimizu et al. [8] and updated. For P450 enzymes that have been renamed, the original names are indicated in parentheses. Human genes in brackets indicate pseudogenes or those considered as nonfunctional genes. N/A, not available.

marmosets (Callithrix jacchus) are commonly used and potentially useful for preclinical studies, due to their close evolutionary relationship to humans, to overcome some species difference problems. One of the classical species differences is seen in thalidomide, which causes a teratogen in humans or non-human primates [1], but not in rodents. Multiple forms of cynomolgus monkey cytochrome P450 (P450) enzymes (major xenobiotic-metabolizing enzymes), including a cynomolgus monkey specific form (P450 2C76), were previously summarized by Uno et al. [2] in comparison to human P450s. Although characterization of P450 enzymes has been partly summarized for cynomolgus monkeys as important experimental animal models [3–7], more current information has recently become available involving marmoset P450 enzymes. Marmoset P450 1A/B, 2A/B/C/D/E/J, 3A, and 4A/F enzymes recently identified and/or characterized [8,9] are listed together in Table 1 that provide better understanding of drug metabolism properties from the updated information on marmoset, cynomolgus monkey, and human P450 cDNAs. It should be noted that marmoset CYP4F3B and CYP4F11, respectively, have been renamed to be CYP4F3 and CYP4F71 by the P450 nomenclature committee (http://drnelson. uthsc.edu/CytochromeP450.html). In general, marmoset or cynomolgus monkey P450 enzymes showed high sequence homology to their human counterparts as summarized in Table 2. Substrate specificity of cynomolgus monkey P450 enzymes has been partly clarified in Table 3. These findings suggest that marmoset and cynomolgus monkey P450 enzymes likely have similar substrate specificity to human P450 enzymes with slightly different characteristics as mentioned below. For example, it should be noted that cynomolgus monkey P450 3A enzymes also showed similar substrate selectivity to human P450 3A4 and 3A5 enzymes, but cynomolgus monkey P450 3A enzymes exhibited wider substrate selectivity toward human P450 2D substrates [10], resulting in high P450 2Drelated drug clearance in cynomolgus monkeys.

In humans, P450 2C enzymes are secondary essential drugmetabolizing enzymes for prescribed drugs [11]. Cynomolgus monkey P450 2C9 (formerly P450 2C43, Tables 1 and 2) and P450 2C19 (formerly P450 2C75) exhibit high amino acid sequence identity to human P450 2C9 and P450 2C19 [2]. On the other hand, cynomolgus monkey P450 2C76, which has no ortholog in humans, shows lower amino acid sequence identity to any human P450 2C forms [12]. Because a broad evaluation of potential substrates for cynomolgus monkey P450 2C enzymes was not conducted, to obtain the comprehensive information of substrate selectivity for major cynomolgus monkey P450 2C enzymes, 89 commercially available drugs were investigated [13–16]. Cynomolgus monkey P450 2C19 metabolized 34 of the 89 compounds, including representative human P450 2C19 substrates such as diazepam and omeprazole and human P450 2C9 substrates such as diclofenac, warfarin, and flurbiprofen [15]. Cynomolgus monkey P450 2C9 metabolized 20 of the 89 compounds, and 17 of those 20 drugs have been reported as substrates or inhibitors of human P450 2C9 and P450 2C19, but also metabolized efavirenz which is a marker substrate for P450 2B6 in humans [13]. Cynomolgus monkey P450 2C76 metabolized 19 of the 89 compounds with a wide variety of diverse structures from small to large molecules and formed a unique nifedipine metabolite which was not reported in humans [14]. These findings collectively suggest that cynomolgus monkey P450 2C enzymes have similar substrate recognition functionality as human P450 2C forms, but there would be possibly limited specific differences in drug oxidative metabolism between the two species. 2. Stereoselective warfarin elimination mediated by different P450 enzymes in marmosets, cynomolgus monkeys, and humans In a definitive pharmacokinetic study [17], caffeine, S-warfarin, omeprazole, metoprolol, and midazolam (in combination) have

Please cite this article in press as: Y. Uno et al., Utility of non-human primates in drug development: Comparison of non-human primate and human drugmetabolizing cytochrome P450 enzymes, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.06.008

Y. Uno et al. / Biochemical Pharmacology xxx (2016) xxx–xxx Table 2 Similarities of amino acid sequences of marmoset and cynomolgus monkey P450s in comparison with human P450s. Marmoset

Cynomolgus monkey

Human

P450

Homology to human P450

P450

Homology to human P450

P450

1A1 1A2 1B1 – 2A6

90 88 92 – 89 86 93 – – – – – – 86 88 93 86 86 – – 87 79 78 78 68 68 79 69 91 85 89 – 91 90 89 – 82 88 78 74 88 93 93 89 87

1A1 1A2 1B1 1D1 2A23

94 93 94 91a 92 89 94 95 93 94 93 91 93 91 92 96 93 91 93 92 – – – – 70 71 72 72 93 91 94 93b 95 94 91 97 – – – – 95 94 95 91 92

1A1 1A2 1B1 [1D1P] 2A6 [2A7] 2A13 2A6 [2A7] 2A13 2A6 [2A7] 2A13 2B6 2C8 [2C18] 2C9 2C19 2C9 2C19 2C8 2C9 [2C18] 2C19 2C8 2C9 [2C18] 2C19 2D6 2D6 2E1 [2G2P] 2J2 3A4 3A5 3A43 3A4 3A5 3A7 3A43 4A11 4F2 4F3B 4F11 4F12

– – – – – – 2B6 2C8 2C18 2C19 – – 2C58

2C76

2D6(19) 2D8 2E1 – 2J2 3A4(21) 3A5 – 3A90

4A11 4F2 4F3 4F71 4F12

2A24

2A26

2B6 2C8(20) 2C18 2C9(43) 2C19(75) – – – – 2C76

2D6(17) 2D8(44) 2E1 2G2 2J2 3A4(8) 3A5 3A43 – – – – 4A11 4F2(45) 4F3v2 4F11 4F12

a Amino acid sequences of cynomolgus monkey P450 1D1 were compared to amino acid sequence inferred from the P450 1D1 pseudogene sequence. b Amino acid sequences of cynomolgus monkey P450 2G2 were compared to amino acid sequence inferred from the P450 2G2 pseudogene sequence.

been reported as probe substrates of human P450 1A2, 2C9, 2C19, 2D6, and 3A, respectively. Human P450 cocktail probe metabolic clearance previously described [17] was compared to those of the same cassette dosing in cynomolgus monkeys [18] and marmosets [19] recently. Based on the pharmacokinetics of cynomolgus monkeys and marmosets, simplified human physiologically based pharmacokinetic models for the probe substrates [20,21] could successfully estimate the corresponding human pharmacokinetics. Cynomolgus monkey P450 2C19, highly homologous to human P450 2C19, catalyzed R-warfarin 7-hydroxylation [22,23] (Table 3). In vivo pharmacokinetics of racemic warfarin and its metabolites at a dose of 1.0 mg/kg were studied after oral and intravenous administrations to fasted male cynomolgus monkeys (n = 11, from Indochina, 4–8 years of age, 3.5–7.4 kg of body weight) [24], which had been genotyped for P450 2C19 p.[(Phe7Leu; Ser254Leu; Ile469Thr)] [25]. Kinetic parameters for S-warfarin determined after chiral

3

separation were not different among homozygous mutant, heterozygous mutant, or wild type groups; however, elimination half-lives and area under the curves of R-warfarin in the homozygous mutant group were 9- and 8-times greater than those values in the wild type group after oral administration, respectively. Clearance of R-warfarin from plasma in the homozygous mutant group was 7-fold that of the wild-type group after intravenous administration. The 7-hydroxy- and 6-hydroxywafarin formations from R/S-warfarin were in the order of wild type, heterozygous mutant, then homozygous mutant groups. Inter-animal variations of warfarin metabolism, especially R-warfarin clearance, in cynomolgus monkeys are associated with P450 2C19 genotypes p.[(Phe100Asn; Ala103Val; Ile112Leu)]. On the other hand, significant faster in vivo S-warfarin clearance than R-warfarin after administration of racemic warfarin to marmosets was consistent with reported warfarin stereoselectivity in humans [26]. Newly identified marmoset P450 2C19, highly homologous to human P450 2C19, catalyzed S-warfarin 7-hydroxylation [27]. Similarly, in vivo pharmacokinetics of racemic warfarin and its metabolites at a dose of 1.0 mg/kg were studied after oral and intravenous administrations to fasted male marmosets (n = 6, >2 years of age, 0.3 kg of body weight), which had been genotyped for P450 2C19 p.[(Phe7Leu; Ser254Leu; Ile469Thr)] [28]. Although mean plasma concentrations of R-warfarin in marmosets determined after chiral separation were similar between the homozygous mutant and wild-type groups up to 24 h after the intravenous and oral administrations of racemic warfarin, S-warfarin clearances from plasma were significantly faster in the three wild-type marmosets compared to the three homozygous mutant marmosets. These marmoset P450 2C19 polymorphisms influenced metabolic activities of S-warfarin 7-hydroxylation in 18 marmoset livers in vitro because the homozygotes and heterozygotes showed significantly reduced catalytic activities in liver microsomes toward S-warfarin 7-hydroxylation compared to the wild-type group [28]. Because inter-individual variability of P450 2C-dependent drug clearances in cynomolgus monkeys and marmosets is partly accounted for by polymorphic P450 2C19 variants, similar to humans, genotyping of drug-metabolizing enzyme genes would be beneficial before and after drug metabolism testing and evaluations in cynomolgus monkeys and marmosets. In the future, it would be of use to investigate warfarin-target protein vitamin K epoxide reductase levels in monkeys in association with polymorphic P450 2C enzymes in this updates, with respect to human clinical problem of modulating the dosage of warfarin. To our best knowledge, we observed a variety of monkey P450 2C variants which had not completely matched to corresponding human P450 variants extensively reported so far. However, we could image somehow altered catalytic activities of monkey P450 variants having amino acid substations found inside the substrate recognition sites. We experienced fast eliminations of P450 2D-dependent metoprolol and P450 3A-dependent midazolam in limited numbers of cynomolgus monkeys [18] and marmosets [19]. It should be noted that understanding of polymorphic monkey P450 variants in the future would be important for using monkeys in drug development and identification of such P450 2D/3A variants that lead to adverse drug reactions.

3. Limited different roles of P450 enzymes in drug oxidations in marmosets, cynomolgus monkeys, and humans In vivo caffeine elimination rates in plasma were similar among marmosets, cynomolgus monkeys, and humans [18,19]; however, main caffeine metabolite formations were 7-N-demethylation (theophylline formation) in cynomolgus monkeys [29] and

Please cite this article in press as: Y. Uno et al., Utility of non-human primates in drug development: Comparison of non-human primate and human drugmetabolizing cytochrome P450 enzymes, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.06.008

4

Y. Uno et al. / Biochemical Pharmacology xxx (2016) xxx–xxx

Table 3 Typical substrates for marmoset, cynomolgus monkey, and human P450 enzymes. Marmoset 1A1 [47] 1A2 [47] 1B1 [47]

2A6 [48]

Cynomolgus monkey 7-Ethoxycoumarin 7-Ethoxyresorufin 7-Ethoxycoumarin Phenacetin 7-Ethoxyresorufin b-Estradiol 7-Ethoxycoumarin Phenacetin

1A1 [47,54] 1A2 [47,54] 1B1 [47] 1D1 [54] 2A23 [48]

2A24 [48] 2A26 [48] 2B6 [49]

7-Ethoxycoumarin

2B6 [49]

2C8 [27]

Paclitaxel Tolbutamide S-Warfarin Tolbutamide Flurbiprofen Omeprazole Flurbiprofen Tolbutamide 7-Ethoxycoumarin

2C8(20) [16,55]

2C19 [27]

2C58 [27]

2C9(43) [13,55] 2C19(75) [15,27,55]

2C76 [27]

Tolbutamide

2C76 [14,55,56]

2D6(19) [50,51]

2D6(17) [51,57]

3A4(21) [19]

Bufuralol Dextromethorphan Metoprolol Propranolol Bufuralol Dextromethorphan Metoprolol Chlorzoxazone Astemizole Terfenadine Midazolam

3A5 3A90 [19]

Midazolam Midazolam

3A5 [10,68]

4A11 4F2 [33]

Arachidonic acid Lauric acid Leukotriene B4

4F3 [33]

2D8 [50]

Human 7-Ethoxycoumarin 7-Ethoxyresorufin 7-Ethoxycoumarin Phenacetin 7-Ethoxyresorufin b-Estradiol 7-Ethoxyresorufin Coumarin 7-Ethoxycoumarin Phenacetin Coumarin Coumarin 7-Ethoxycoumarin Bupropion Efavirenz 7-Ethoxycoumarin Paclitaxel Amodiaquine Efavirenz S-Mephenytoin R-Warfarin S-Warfarin Diclofenac Flurbiprofen Tolbutamide Omeprazole Pitavastatin Tolbutamide Nifedipine Bupropion Bufuralol Dextromethorphan Propranolol

1A1 [47,54,60] 1A2 [47,54,60] 1B1 [47,60]

2A6 [48,60] 2A13 [48]

Coumarin Phenacetin 7-Ethoxycoumarin

2B6 [49,60]

Bupropion Efavirenz 7-Ethoxycoumarin Paclitaxel Amodiaquine Tolbutamide S-Warfarin Diclofenac Tolbutamide Flurbiprofen Omeprazole S-Mephenytoin

2C8 [27,60,66]

2C9 [27,60]

2C19 [27,60]

2D6 [50,51,57,60]

Bufuralol Dextromethorphan Metoprolol Propranolol

2E1 [58,60] 2J2 [53]

Chlorzoxazone Astemizole Terfenadine Midazolam Nifedipine Testosterone Alprazolam Midazolam Nifedipine Testosterone

2D8(44) [57]

Bufuralol Dextromethorphan

2E1 [58] 2J2 [53]

4A11 [59]

Chlorzoxazone Astemizole Terfenadine Midazolam Nifedipine Testosterone Alprazolam Midazolam Nifedipine Testosterone Alprazolam Arachidonic acid

4F2(45) [59]

Arachidonic acid

4F2 [62–64]

Leukotriene B4

4F3v2 [59]

Arachidonic acid

4F3B [63,65]

4F71 [33]

Leukotriene B4

4F11 [59]

Arachidonic acid

4F11 [65]

4F12 [33]

Leukotriene B4 Ebastine

4F12 [33]

Ebastine

4F12 [33,64,67]

2E1 [52] 2J2 [53]

3A4(8) [10,68]

C-8-hydroxylation [30] in marmosets (Fig. 1), mainly mediated by cynomolgus monkey liver microsomal P450 2C9 (an experimental Vmax/Km value of 1.7 mM min
1) and marmoset P450 3A4 (Vmax/Km of 2.6 mM min
1), respectively. Because human P450 1A2 enzymes efficiently catalyzed caffeine 3-N-demethylation (Vmax/Km of 1.4 mM min
1), roles of P450 enzymes of humans, cynomolgus monkeys, and marmosets in caffeine oxidative metabolism were different, implying some functional characteristics different in terms of substrate specificities and catalytic activities. Interspecies differences regarding caffeine oxidation exist, but the predominant

7-Ethoxycoumarin 7-Ethoxyresorufin 7-Ethoxycoumarin Phenacetin 7-Ethoxyresorufin b-Estradiol

3A4 [10,60,68]

3A5 [10,60]

4A11 [61,62]

Arachidonic acid Lauric acid Arachidonic acid Leukotriene B4 Arachidonic acid Leukotriene B4 Arachidonic acid Leukotriene B4 Arachidonic acid Leukotriene B4 Ebastine Astemizole Terfenadine

C-8-hydroxylation of caffeine in rodents and rabbits have been shown to be broadly similar [31]. Although efavirenz is known as a probe substrate for human P450 2B6, efavirenz was oxidized in a limited manner by cynomolgus monkey P450 2B6 but efficiently by cynomolgus monkey P450 2C9 [13] (Table 3). Liquid chromatography-mass spectrometry analysis revealed that both cynomolgus monkey P450 2C9 and human P450 2B6 formed the same 8-hydroxy- and 8,14dihydroxy-efavirenz [13]. An anti-histaminic drug ebastine has been recognized as a human P450 2J2 probe, but also partly

Please cite this article in press as: Y. Uno et al., Utility of non-human primates in drug development: Comparison of non-human primate and human drugmetabolizing cytochrome P450 enzymes, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.06.008

nmol/min/mg microsomal protein

Y. Uno et al. / Biochemical Pharmacology xxx (2016) xxx–xxx

0.10

0.05

0 Marmoset Monkey Human Liver microsomes

Fig. 1. Caffeine oxidation activities of liver microsomes form marmosets, cynomolgus monkeys, and humans. Caffeine 1-N- (open), 3-N- (shaded), and 7-N- (closed) demethylation and C-8-hydroxylation (hatched) activities are taken from Utoh et al. [29] and Uehara et al. [30].

metabolized by human P450 4F12 [32]. On the other hand, both marmoset P450 4F12 and cynomolgus monkey P450 4F12, expressed in small intestines and livers, more efficiently metabolized ebastine oxidation than marmoset and monkey P450 2J2 did [33]. These findings indicated that some marmoset or cynomolgus monkey P450 enzymes had apparently different functional characteristics to those of humans. Accumulation of such information of cynomolgus monkeys will lead to profound understanding of drug metabolism in cynomolgus monkeys and humans. 4. Age-related pharmacokinetic changes and other monooxygenases in humans and cynomolgus monkeys Among a variety of factors for inter-individual differences, aging is one of many important and determinant factors in pharmacokinetics and drug responses (pharmacodynamics) that may cause adverse drug reactions. The age-related alterations of pharmacokinetics have been well summarized in humans [34–36]. During drug development, it is important to predict how the pharmacokinetics of drug candidates will change in older patients to reduce related risks. Cynomolgus monkeys (aged 16 years) may be a suitable animal model to predict the age-related alterations of pharmacokinetics and physiological parameters in humans, because age-related alterations of physiological parameters or reduced hepatic clearances of some human P450 substrates in cynomolgus monkeys are in agreement with clinical observations in humans [18,37], as briefly indicated in Table 4. At least, cynomolgus monkey P450 2C19- and P450 3A-mediated drug

Table 4 Age-related pharmacokinetic changes in humans and cynomolgus monkeys. Drug

Property

Reported human clearance

Monkey in vivo, n = 3 or 6 (in vitro, n = 55)

Antipyrine

Marker of hepatic metabolic activity and total body water Hepatic blood flowlimited and protein bound Renally eliminated Human P450 2C19 substrate Human P450 2D6 substrate Human P450 3A4 substrate

;, t1/2"

;, t1/2"

;

;, t1/2"

; ;

;, t1/2" ? (;)

?

? (;, by P450 3A)

;

; (?)

Diphenhydramine

Ofloxacin Omeprazole Metoprolol Midazolam

Taken from Koyanagi et al. [18,37]. t1/2, a half-life.

5

clearances showed similar age-related decreases in hepatic clearance values to those of humans (Table 4). Age-related pharmacokinetic changes in animal models would bring up an important aspect of drug development for elderly patients. In the future, a pharmacokinetic model for child would be one of the challenging studies with non-human primates. Flavin-containing monooxygenases (FMOs), another monooxygenase family, are also important drug oxygenation enzymes catalyzing a wide variety of nitrogen-, sulfur-, or phosphorouscontaining xenobiotics. Drug oxygenations mediated by human FMO3 are linked to their pharmacological and toxicological actions [38]. Cynomolgus monkey FMO1-5 were highly identical to human FMO1-5 and were closely clustered with human FMO1-5 in a phylogeny, respectively [39]. Molecular and functional characterizations of FMOs in cynomolgus monkeys [39–41] and marmosets [9] were also reported. In addition to the P450 enzymes, FMO enzymes in these non–human primates have been found to be similarly involved in the oxidation of a variety of compounds associated with pharmacological and/or toxicological effects in humans. 5. Conclusion Research has elucidated human liver microsomal P450 isoform contents [42], their relatively broad but selective substrate specificities [43,44], P450 induction and inhibition [45], P450mediated drug interactions [46], and genetic polymorphisms of P450 enzymes associated with pharmacological and/or toxicological actions. Marmoset and cynomolgus monkey P450 enzymes (Table 1) showed high sequence homology to their human counterparts (Table 2) and generally had similar substrate recognition functionality to human P450 enzymes (Table 3). On the other hand, they also possibly contribute to limited specific differences in drug oxidative metabolism owing to small differences in amino acid residues. These current findings collectively suggest that marmoset and cynomolgus monkey P450 enzymes most likely had similar substrate specificity to human P450 enzymes with slightly different characteristics. These findings also provide a foundation for successful use of non-human primates as preclinical drug metabolism models and will help to further understand molecular mechanisms of human P450 function. Inter-individual variability of P450-dependent drug clearances in marmosets and cynomolgus monkeys is partly accounted for by polymorphic P450 variants and aging (Table 4), similar to humans. Genotyping of drugmetabolizing P450 enzyme genes in non-human primates would be beneficial before and after drug metabolism testing and evaluations in marmosets or cynomolgus monkeys. These findings on inter-individual variability and substrate specificity of marmoset or cynomolgus monkey P450 enzymes should help to understand apparent species differences in drug metabolism and disposition among marmosets, cynomolgus monkeys, and humans and to extrapolate the preclinical study data obtained using marmosets or cynomolgus monkeys to humans. Acknowledgments We thank Drs. Masahiro Utoh, Erika Sasaki, Takashi Inoue, Norie Murayama, Makiko Shimizu, and Sakura Ishii for their support in experiments and Mr. Lance Bell for advice on English writing. This work was supported in part by ‘‘Construction of System for Spread of Primate Model Animals” under the Strategic Research Program for Brain Sciences of Japan Agency for Medical Research and Development, the Japan Society for the Promotion of Science Grants-inAid for Scientific Research 26460206 (H.Y.) and 15K18934 (S.U.), and partly supported by the MEXT (Ministry of Education, Science, Sports and Culture of Japan)-Supported Program for the Strategic Research Foundation at Private Universities, 2013–2018.

Please cite this article in press as: Y. Uno et al., Utility of non-human primates in drug development: Comparison of non-human primate and human drugmetabolizing cytochrome P450 enzymes, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.06.008

6

Y. Uno et al. / Biochemical Pharmacology xxx (2016) xxx–xxx

References [1] D.E. Poswillo, W.J. Hamilton, D. Sopher, The marmoset as an animal model for teratological research, Nature 239 (1972) 460–462. [2] Y. Uno, K. Iwasaki, H. Yamazaki, D.R. Nelson, Macaque cytochromes P450: nomenclature, transcript, gene, genomic structure, and function, Drug Metab. Rev. 43 (2011) 346–361. [3] R. Ise, S. Uehara, H. Akiyama, S. Kondo, K. Iwasaki, R. Nagata, H. Nobumasa, H. Yamazaki, Y. Uno, A newly developed DNA microarray is useful to assess induction of cytochromes P450 in cynomolgus monkey, Drug Metab. Pharmacokinet. 26 (2011) 228–235. [4] C. Emoto, K. Iwasaki, R. Koizumi, M. Utoh, N. Murayama, Y. Uno, H. Yamazaki, Species difference between cynomolgus monkeys and humans on cytochromes P450 2D and 3A-dependent drug oxidation activities in liver microsomes, J. Health Sci. 57 (2011) 164–170. [5] C. Emoto, N. Yoda, Y. Uno, K. Iwasaki, K. Umehara, E. Kashiyama, H. Yamazaki, Comparison of P450 enzymes between cynomolgus monkeys and humans: P450 identities, protein contents, kinetic parameters, and potential for inhibitory profiles, Curr. Drug Metab. 14 (2013) 239–252. [6] Y. Uno, H. Fujino, K. Iwasaki, M. Utoh, Macaque CYP2C76 encodes cytochrome P450 enzyme not orthologous to any human isozymes, Curr. Drug Metab. 11 (2010) 142–152. [7] K. Iwasaki, Y. Uno, Cynomolgus monkey CYPs: a comparison with human CYPs, Xenobiotica 39 (2009) 578–581. [8] M. Shimizu, S. Iwano, Y. Uno, S. Uehara, T. Inoue, N. Murayama, J. Onodera, E. Sasaki, H. Yamazaki, Qualitative de novo analysis of full length cDNA and quantitative analysis of gene expression for common marmoset (Callithrix jacchus) transcriptomes using parallel long-read technology and short-read sequencing, PLoS One 9 (2014) e100936. [9] S. Uehara, Y. Uno, T. Inoue, N. Murayama, M. Shimizu, E. Sasaki, H. Yamazaki, Activation and deactivation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) by cytochrome P450 enzymes and flavin-containing monooxygenases in common marmosets (Callithrix jacchus), Drug Metab. Dispos. 43 (2015) 735– 742. [10] K. Iwasaki, N. Murayama, R. Koizumi, Y. Uno, H. Yamazaki, Comparison of cytochrome P450 3A enzymes in cynomolgus monkeys and humans, Drug Metab. Pharmacokinet. 25 (2010) 388–391. [11] S.P. Rendic, F.P. Guengerich, Survey of human oxidoreductases and cytochrome P450 enzymes involved in the metabolism of chemicals, Chem. Res. Toxicol. 28 (2015) 38–42. [12] Y. Uno, K. Matsuno, N. Murayama, C. Nakamura, H. Yamazaki, Metabolism of P450 probe substrates by cynomolgus monkey CYP2C76, Basic Clin. Pharmacol. Toxicol. 109 (2011) 315–318. [13] S. Hosaka, N. Murayama, M. Satsukawa, S. Uehara, M. Shimizu, K. Iwasaki, S. Iwano, Y. Uno, H. Yamazaki, Comprehensive evaluation for substrate selectivity of cynomolgus monkey cytochrome P450 2C9, a new efavirenz oxidase, Drug Metab. Dispos. 43 (2015) 1119–1122. [14] S. Hosaka, N. Murayama, M. Satsukawa, M. Shimizu, S. Uehara, H. Fujino, K. Iwasaki, S. Iwano, Y. Uno, H. Yamazaki, Evaluation of 89 compounds for identification of substrates for cynomolgus monkey cytochrome P450 2C76, a new bupropion/nifedipine oxidase, Drug Metab. Dispos. 43 (2015) 27–33. [15] S. Hosaka, N. Murayama, M. Satsukawa, S. Uehara, M. Shimizu, K. Iwasaki, S. Iwano, Y. Uno, H. Yamazaki, Similar substrate specificity of cynomolgus monkey cytochrome P450 2C19 to reported human P450 2C counterpart enzymes by evaluation of 89 drug clearances, Biopharm. Drug Dispos. 36 (2015) 636–643. [16] S. Hosaka, N. Murayama, M. Satsukawa, S. Uehara, M. Shimizu, K. Iwasaki, S. Iwano, Y. Uno, H. Yamazaki, Identification of putative substrates for cynomolgus monkey cytochrome P450 2C8 by substrate depletion assays with 22 human P450 substrates and inhibitors, Biopharm. Drug Dispos. (2016), http://dx.doi.org/10.1002/bdd.1998 (in press). [17] S. Turpault, W. Brian, H.R. Van, A. Santoni, F. Poitiers, Y. Donazzolo, X. Boulenc, Pharmacokinetic assessment of a five-probe cocktail for CYPs 1A2, 2C9, 2C19, 2D6 and 3A, Br. J. Clin. Pharmacol. 68 (2009) 928–935. [18] T. Koyanagi, Y. Nakanishi, N. Murayama, Y. Yamaura, K. Ikeda, K. Yano, S. Uehara, M. Utoh, S. Kim, Y. Uno, H. Yamazaki, Age-related changes of hepatic clearances of cytochrome P450 probes, midazolam and R-/S-warfarin in combination with caffeine, omeprazole, and metoprolol in cynomolgus monkeys using in vitro-in vivo correlation, Xenobiotica 45 (2015) 312–321. [19] S. Uehara, T. Inoue, M. Utoh, A. Toda, M. Shimizu, Y. Uno, E. Sasaki, H. Yamazaki, Simultaneous pharmacokinetics evaluation of human cytochrome P450 probes, caffeine, warfarin, omeprazole, metoprolol, and midazolam, in common marmosets (Callithrix jacchus), Xenobiotica 46 (2016) 163–168. [20] S. Shida, M. Utoh, N. Murayama, M. Shimizu, Y. Uno, H. Yamazaki, Human plasma concentrations of cytochrome P450 probes extrapolated from pharmacokinetics in cynomolgus monkeys using physiologically based pharmacokinetic modeling, Xenobiotica 45 (2015) 881–886. [21] M. Utoh, H. Suemizu, M. Mitsui, M. Kawao, A. Toda, S. Uehara, Y. Uno, M. Shimizu, E. Sasaki, H. Yamazaki, Human plasma concentrations of cytochrome P450 probe cocktails extrapolated from pharmacokinetics in mice transplanted with human hepatocytes and from pharmacokinetics in common marmosets using physiologically based pharmacokinetic modeling, Xenobiotica (2016), http://dx.doi.org/10.3109/00498254.2016.1147102 (in press).

[22] Y. Hosoi, Y. Uno, N. Murayama, H. Fujino, M. Shukuya, K. Iwasaki, M. Shimizu, M. Utoh, H. Yamazaki, Monkey liver cytochrome P450 2C19 is involved in Rand S-warfarin 7-hydroxylation, Biochem. Pharmacol. 84 (2012) 1691–1695. [23] Y. Uno, A. Matsushita, M. Shukuya, Y. Matsumoto, N. Murayama, H. Yamazaki, CYP2C19 polymorphisms account for inter-individual variability of drug metabolism in cynomolgus macaques, Biochem. Pharmacol. 91 (2014) 242– 248. [24] M. Utoh, T. Yoshikawa, Y. Hayashi, M. Shimizu, K. Iwasaki, Y. Uno, H. Yamazaki, Slow R-warfarin 7-hydroxylation mediated by P450 2C19 genetic variants in cynomolgus monkeys in vivo, Biochem. Pharmacol. 95 (2015) 110–114. [25] Y. Uno, H. Yamazaki, Development of a genotyping tool for a functionally relevant CYP2C19 allele (Phe100Asn, Ala103Val, Ile112Leu) in cynomolgus macaques, J. Vet. Med. Sci. 78 (2016) 147–148. [26] R.S. Obach, F. Lombardo, N.J. Waters, Trend analysis of a database of intravenous pharmacokinetic parameters in humans for 670 drug compounds, Drug Metab. Dispos. 36 (2008) 1385–1405. [27] S. Uehara, Y. Uno, S. Inoue, M. Kawano, M. Shimizu, A. Toda, M. Utoh, E. Sasaki, H. Yamazaki, Novel marmoset cytochrome P450 2C19 in livers efficiently metabolizes human P450 2C9 and 2C19 substrates, S-warfarin, tolbutamide, flurbiprofen, and omeprazole, Drug Metab. Dispos. 43 (2015) 1408–1416. [28] S. Uehara, Y. Uno, T. Inoue, M. Kawano, M. Shimizu, A. Toda, M. Utoh, E. Sasaki, H. Yamazaki, Individual differences in metabolic clearance of S-warfarin efficiently mediated by polymorphic marmoset cytochrome P450 2C19 in livers, Drug Metab. Dispos. (2016), http://dx.doi.org/10.1124/dmd.116.070383 (in press). [29] M. Utoh, N. Murayama, Y. Uno, Y. Onose, S. Hosaka, H. Fujino, M. Shimizu, K. Iwasaki, H. Yamazaki, Monkey liver cytochrome P450 2C9 is involved in caffeine 7-N-demethylation to form theophylline, Xenobiotica 43 (2013) 1037–1042. [30] S. Uehara, Y. Uno, T. Inoue, T. Suzuki, M. Utoh, E. Sasaki, H. Yamazaki, Caffeine 7-N-demethylation and C-8-oxidation mediated by liver microsomal cytochrome P450 enzymes in common marmosets, Xenobiotica 46 (2016) 573–578. [31] F. Berthou, B. Guillois, C. Riche, Y. Dreano, E. Jacqz-Aigrain, P.H. Beaune, Interspecies variations in caffeine metabolism related to cytochrome P4501A enzymes, Xenobiotica 22 (1992) 671–680. [32] T. Hashizume, S. Imaoka, M. Mise, Y. Terauchi, T. Fujii, H. Miyazaki, T. Kamataki, Y. Funae, Involvement of CYP2J2 and CYP4F12 in the metabolism of ebastine in human intestinal microsomes, J. Pharmacol. Exp. Ther. 300 (2002) 298–304. [33] S. Uehara, Y. Uno, Y. Yuki, T. Inoue, E. Sasaki, H. Yamazaki, A new marmoset P450 4F12 enzyme expressed in small intestines and livers efficiently metabolizes an anti-histaminic drug ebastine, Drug Metab. Dispos. 44 (2016) 833–841. [34] U. Klotz, Pharmacokinetics and drug metabolism in the elderly, Drug Metab. Rev. 41 (2009) 67–76. [35] A.J. McLachlan, L.G. Pont, Drug metabolism in older people – a key consideration in achieving optimal outcomes with medicines, J. Gerontol. A Biol. Sci. Med. Sci. 67 (2012) 175–180. [36] A. Corsonello, C. Pedone, R.A. Incalzi, Age-related pharmacokinetic and pharmacodynamic changes and related risk of adverse drug reactions, Curr. Med. Chem. 17 (2010) 571–584. [37] T. Koyanagi, K. Yamaura, K. Yano, S. Kim, H. Yamazaki, Age-related pharmacokinetic changes of acetaminophen, antipyrine, diazepam, diphenhydramine, and ofloxacin in male cynomolgus monkeys and beagle dogs, Xenobiotica 44 (2014) 893–901. [38] H. Yamazaki, M. Shimizu, Survey of variants of human flavin-containing monooxygenase 3 (FMO3) and their drug oxidation activities, Biochem. Pharmacol. 85 (2013) 1588–1593. [39] Y. Uno, M. Shimizu, H. Yamazaki, Molecular and functional characterization of flavin-containing monooxygenases in cynomolgus macaque, Biochem. Pharmacol. 85 (2013) 1837–1847. [40] M. Yamazaki, M. Shimizu, Y. Uno, H. Yamazaki, Drug oxygenation activities mediated by liver microsomal flavin-containing monooxygenases 1 and 3 in humans, monkeys, rats, and minipigs, Biochem. Pharmacol. 90 (2014) 159– 165. [41] T. Taniguchi-Takizawa, M. Shimizu, T. Kume, H. Yamazaki, Benzydamine Noxygenation as an index for flavin-containing monooxygenase activity and benzydamine N-demethylation by cytochrome P450 enzymes in liver microsomes from rats, dogs, monkeys, and humans, Drug Metab. Pharmacokinet. 30 (2015) 64–69. [42] T. Shimada, H. Yamazaki, M. Mimura, Y. Inui, F.P. Guengerich, Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians, J. Pharmacol. Exp. Ther. 270 (1994) 414–423. [43] F.P. Guengerich, S. Rendic, Update information on drug metabolism systems? 2009, Part I, Curr. Drug Metab. 11 (2010) 1–3. [44] S. Rendic, F.P. Guengerich, Update information on drug metabolism systems2009, Part II: summary of information on the effects of diseases and environmental factors on human cytochrome P450 (CYP) enzymes and transporters, Curr. Drug Metab. 11 (2010) 4–84. [45] T. Niwa, N. Murayama, H. Yamazaki, Stereoselectivity of human cytochrome P450 in metabolic and inhibitory activities, Curr. Drug Metab. 12 (2011) 549– 569.

Please cite this article in press as: Y. Uno et al., Utility of non-human primates in drug development: Comparison of non-human primate and human drugmetabolizing cytochrome P450 enzymes, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.06.008

Y. Uno et al. / Biochemical Pharmacology xxx (2016) xxx–xxx [46] A. Hisaka, Y. Ohno, T. Yamamoto, H. Suzuki, Prediction of pharmacokinetic drug-drug interaction caused by changes in cytochrome P450 activity using in vivo information, Pharmacol. Ther. 125 (2010) 230–248. [47] S. Uehara, Y. Uno, T. Inoue, E. Sasaki, H. Yamazaki, Molecular cloning, tissue distribution, and functional characterization of marmoset cytochrome P450 1A1, 1A2, and 1B1, Drug Metab. Dispos. 46 (2016) 8–15. [48] S. Uehara, Y. Uno, T. Inoue, E. Sasaki, H. Yamazaki, Substrate selectivities and catalytic activities of marmoset liver cytochrome P450 2A6 differed from those of human P450 2A6, Drug Metab. Dispos. 43 (2015) 969–976. [49] K. Mayumi, N. Hanioka, K. Masuda, A. Koeda, S. Naito, A. Miyata, S. Narimatsu, Characterization of marmoset CYP2B6: cDNA cloning, protein expression and enzymatic functions, Biochem. Pharmacol. 85 (2013) 1182–1194. [50] S. Uehara, Y. Uno, Y. Hagihira, N. Murayama, M. Shimizu, T. Inoue, E. Sasaki, H. Yamazaki, Marmoset cytochrome P450 2D8 in livers and small intestines metabolizes typical human P450 2D6 substrates, metoprolol, bufuralol, and dextromethorphan, Xenobiotica 45 (2015) 757–765. [51] S. Narimatsu, T. Nakata, T. Shimizudani, K. Nagaoka, H. Nakura, K. Masuda, T. Katsu, A. Koeda, S. Naito, S. Yamano, A. Miyata, N. Hanioka, Regio- and stereoselective oxidation of propranolol enantiomers by human CYP2D6, cynomolgus monkey CYP2D17 and marmoset CYP2D19, Chem. Biol. Interact. 189 (2011) 146–152. [52] T.G. Schulz, R. Thiel, D.S. Davies, R.J. Edwards, Identification of CYP2E1 in marmoset monkey, Biochim. Biophys. Acta 1382 (1998) 287–294. [53] S. Uehara, Y. Uno, T. Inoue, E. Okamoto, E. Sasaki, H. Yamazaki, Marmoset cytochrome P450 2J2 mainly expressed in small intestines and livers effectively metabolizes human P450 2J2 probe substrates, astemizole and terfenadine, Xenobiotica (2016), http://dx.doi.org/10.3109/00498254.2016. 1146366 (in press). [54] Y. Uno, S. Uehara, N. Murayama, H. Yamazaki, CYP1D1, pseudogenized in human, is expressed and encodes a functional drug-metabolizing enzyme in cynomolgus monkey, Biochem. Pharmacol. 81 (2011) 442–450. [55] Y. Uno, S. Uehara, S. Kohara, K. Iwasaki, R. Nagata, K. Fukuzaki, M. Utoh, N. Murayama, H. Yamazaki, Newly identified CYP2C93 is a functional enzyme in rhesus monkey, but not in cynomolgus monkey, PLoS One 6 (2011) e16923. [56] Y. Uno, T. Kumano, G. Kito, R. Nagata, T. Kamataki, H. Fujino, CYP2C76mediated species difference in drug metabolism: a comparison of pitavastatin metabolism between monkeys and humans, Xenobiotica 37 (2007) 30–43. [57] Y. Uno, S. Uehara, S. Kohara, N. Murayama, H. Yamazaki, Cynomolgus monkey CYP2D44 newly identified in liver, metabolizes bufuralol, and dextromethorphan, Drug Metab. Dispos. 38 (2010) 1486–1492.

7

[58] N. Hanioka, M. Yamamoto, H. Iwabu, H. Jinno, T. Tanaka-Kagawa, S. Naito, T. Shimizu, K. Masuda, T. Katsu, S. Narimatsu, Functional characterization of human and cynomolgus monkey cytochrome P450 2E1 enzymes, Life Sci. 81 (2007) 1436–1445. [59] Y. Uno, K. Matsuno, C. Nakamura, M. Utoh, H. Yamazaki, Cynomolgus macaque CYP4 isoforms are functional, metabolizing arachidonic acid, J. Vet. Med. Sci. 73 (2011) 487–490. [60] H. Yamazaki, M. Nakamura, T. Komatsu, K. Ohyama, N. Hatanaka, S. Asahi, N. Shimada, F.P. Guengerich, T. Shimada, M. Nakajima, T. Yokoi, Roles of NADPHP450 reductase and apo- and holo-cytochrome b5 on xenobiotic oxidations catalyzed by 12 recombinant human cytochrome P450s expressed in membranes of Escherichia coli, Protein Express. Purif. 24 (2002) 329–337. [61] H. Kawashima, T. Naganuma, E. Kusunose, T. Kono, R. Yasumoto, K. Sugimura, T. Kishimoto, Human fatty acid x-hydroxylase, CYP4A11: determination of complete genomic sequence and characterization of purified recombinant protein, Arch. Biochem. Biophys. 378 (2000) 333–339. [62] P.K. Powell, I. Wolf, R.Y. Jin, J.M. Lasker, Metabolism of arachidonic acid to 20-hydroxy-5,8,11,14- eicosatetraenoic acid by P450 enzymes in human liver: Involvement of CYP4F2 and CYP4A11, J. Pharmacol. Exp. Ther. 285 (1998) 1327–1336. [63] V. Hirani, A. Yarovoy, A. Kozeska, R.P. Magnusson, J.M. Lasker, Expression of CYP4F2 in human liver and kidney: assessment using targeted peptide antibodies, Arch. Biochem. Biophys. 478 (2008) 59–68. [64] J. Eksterowicz, D.A. Rock, B.M. Rock, L.C. Wienkers, R.S. Foti, Characterization of the active site properties of CYP4F12, Drug Metab. Dispos. 42 (2014) 1698– 1707. [65] A. Kalsotra, C.M. Turman, Y. Kikuta, H.W. Strobel, Expression and characterization of human cytochrome P450 4F11: putative role in the metabolism of therapeutic drugs and eicosanoids, Toxicol. Appl. Pharmacol. 199 (2004) 295–304. [66] X.Q. Li, A. Bjorkman, T.B. Andersson, M. Ridderstrom, C.M. Masimirembwa, Amodiaquine clearance and its metabolism to N-desethylamodiaquine is mediated by CYP2C8: a new high affinity and turnover enzyme-specific probe substrate, J. Pharmacol. Exp. Ther. 300 (2002) 399–407. [67] T. Hashizume, S. Imaoka, T. Hiroi, Y. Terauchi, T. Fujii, H. Miyazaki, T. Kamataki, Y. Funae, CDNA cloning and expression of a novel cytochrome p450 (cyp4f12) from human small intestine, Biochem. Biophys. Res. Commun. 280 (2001) 1135–1141. [68] T. Ohtsuka, T. Yoshikawa, K. Kozakai, Y. Tsuneto, Y. Uno, M. Utoh, H. Yamazaki, T. Kume, Alprazolam as an in vivo probe for studying induction of CYP3A in cynomolgus monkeys, Drug Metab. Dispos. 38 (2010) 1806–1813.

Please cite this article in press as: Y. Uno et al., Utility of non-human primates in drug development: Comparison of non-human primate and human drugmetabolizing cytochrome P450 enzymes, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.06.008