Simultaneous pharmacokinetics evaluation of human cytochrome P450 probes,caffeine, warfarin,omeprazole, metoprolol and midazolam,in common marmosets (Callithrix jacchus)

1.?Pharmacokinetics of human cytochrome P450 probes (caffeine, racemic warfarin, omeprazole, metoprolol and midazolam) composite, after single intravenous and oral administrations at doses of 0.20 and 1.0?mg?kg^?1, respectively, to four male common marmosets were investigated.

2.?The plasma concentrations of caffeine and warfarin decreased slowly in a monophasic manner but those of omeprazole, metoprolol and midazolam decreased extensively after intravenous and oral administrations, in a manner that approximated those as reported for pharmacokinetics in humans.

3.?Bioavailabilities were ～100% for caffeine and warfarin, but <25% for omeprazole and metoprolol. Bioavailability of midazolam was 4% in marmosets, presumably because of contribution of marmoset P450 3A4 expressed in small intestine and liver, with a high catalytic efficiency for midazolam 1′-hydroxylation as evident in the recombinant system.

4.?These results suggest that common marmosets, despite their rapid clearance of some human P450 probe substrates, could be an experimental model for humans and that marmoset P450s have functional characteristics that differ from those of human and/or cynomolgus monkey P450s in some aspects, indicating their importance in modeling in P450-dependent drug metabolism studies in marmosets and of further studies.     

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

1.?The pharmacokinetic data of cytochrome P450 probes in humans can be extrapolated from corresponding data in cynomolgus monkeys, dogs and minipigs using simplified physiologically based pharmacokinetic (PBPK) modeling. In this study, the modeling methodology was further adapted to estimate human plasma concentrations of P450 probes based on data from mice transplanted with human hepatocytes or based on data from marmosets.

2.?Using known species allometric scaling factors, the observed plasma concentrations of caffeine, warfarin, omeprazole, metoprolol, and midazolam in chimeric TK-NOG mice with humanized liver were scaled to human oral monitoring equivalents. Using the same approach, the previously reported pharmacokinetics of the five P450 probes in marmosets were also scaled to reported equivalents in humans using in vitro metabolic clearance data.

3.?Human plasma concentration profiles of the five P450 probes estimated by simplified human PBPK models based on the observed pharmacokinetics in mice with humanized liver and on the reported pharmacokinetics in marmosets were consistent with previously published pharmacokinetic data in Caucasians.

4.?These results suggest that mice with humanized liver and/or marmosets could be suitable pharmacokinetic models for humans during research into new drugs, especially when used in combination with simple PBPK models.     

Human plasma concentrations of five cytochrome P450 probes extrapolated from pharmacokinetics in dogs and minipigs using physiologically based pharmacokinetic modeling

1. The pharmacokinetics of cytochrome P450 probes in humans can be extrapolated from corresponding data in cynomolgus monkeys using simplified physiologically based pharmacokinetic (PBPK) modeling. In the current study, despite some species difference in drug clearances, this modeling methodology was adapted to estimate human plasma concentrations of P450 probes based on data from commonly used medium-sized experimental animals, namely dogs and minipigs.

2. Using known species allometric scaling factors and in vitro metabolic clearance data, the observed plasma concentrations of slowly eliminated caffeine and warfarin and rapidly eliminated omeprazole, metoprolol and midazolam in two young dogs were scaled to human oral monitoring equivalents. Using the same approach, the previously reported pharmacokinetics of the five P450 probes in minipigs was also scaled to human monitoring equivalents.

3. The human plasma concentration profiles of the five P450 probes estimated by the simplified human PBPK models based on observed/reported pharmacokinetics in dogs/minipigs were consistent with previously published pharmacokinetic data in humans.

4. These results suggest that dogs and minipigs, in addition to monkeys, could be suitable models for humans during research into new drugs, especially when used in combination with simple PBPK models.

     

Human plasma concentrations of cytochrome P450 probes extrapolated from pharmacokinetics in cynomolgus monkeys using physiologically based pharmacokinetic modeling

Abstract

1.?Cynomolgus monkeys are widely used in preclinical studies as non-human primate species. Pharmacokinetics of human cytochrome P450 probes determined in cynomolgus monkeys after single oral or intravenous administrations were extrapolated to give human plasma concentrations.

2.?Plasma concentrations of slowly eliminated caffeine and R-/S-warfarin and rapidly eliminated omeprazole and midazolam previously observed in cynomolgus monkeys were scaled to human oral biomonitoring equivalents using known species allometric scaling factors and in vitro metabolic clearance data with a simple physiologically based pharmacokinetic (PBPK) model. Results of the simplified human PBPK models were consistent with reported experimental PK data in humans or with values simulated by a fully constructed population-based simulator (Simcyp).

3.?Oral administrations of metoprolol and dextromethorphan (human P450 2D probes) in monkeys reportedly yielded plasma concentrations similar to their quantitative detection limits. Consequently, ratios of in vitro hepatic intrinsic clearances of metoprolol and dextromethorphan determined in monkeys and humans were used with simplified PBPK models to extrapolate intravenous PK in monkeys to oral PK in humans.

4.?These results suggest that cynomolgus monkeys, despite their rapid clearance of some human P450 substrates, could be a suitable model for humans, especially when used in conjunction with simple PBPK models.     

Caffeine 7-N-demethylation and C-8-oxidation mediated by liver microsomal cytochrome P450 enzymes in common marmosets

1.?3-N-Demethylation of caffeine (1,3,7-trimethylxanthine) is mediated by human cytochrome P450 1A2, whereas 7-N-demethylation and C-8-hydroxylation are reportedly catalyzed by monkey P450 2C9 and rat P450 1A2, respectively.

2.?Roles of marmoset P450 enzymes in caffeine oxidation were investigated using nine marmoset liver microsomes and 14 recombinantly expressed marmoset P450 enzymes.

3.?Predominant caffeine 7-N-demethylation and C-8-hydroxylation activities in marmoset liver microsomes were moderately (r?=?0.78, p?<?0.05) and highly (r?=?0.82, p?<?0.01) correlated with midazolam 1′-hydroxylation activities, respectively, while the former was not strongly affected by ketoconazole or α-naphthoflavone.

4.?Caffeine C-8-hydroxylation in liver microsomes was inhibited by ketoconazole and activated by α-naphthoflavone, suggesting main involvements of P450 3As.

5.?Recombinant marmoset P450 3As had high V_max/K_m values for C-8-hydroxylation, comparable to K_m values for marmoset liver microsomes. Marmoset P450 1As efficiently mediated caffeine 3-N-demethylation and C-8-hydroxylation with apparently lower K_m values than those of liver microsomes.

6.?These results collectively suggest highly active marmoset P450 3A enzymes toward caffeine 8-hydorxylaiton and involvement of multiple P450 isoforms including P450 1A in caffeine 7-N- and 3-N-demethylations in marmoset livers. Marmoset P450s have slightly different properties to human or monkey P450s regarding caffeine metabolic pathways.     

Effect of kanglaite on rat cytochrome P450

Abstract

Context: Kanglaite (KLT) is an oily substance extracted from Coix lacryma-jobi Linn. (Cramineae) and has been proved to significantly improve the life span and quality of life of patients, when combined with chemotherapy, radiotherapy, or surgery.

Objective: The purpose of this study was to find out whether KLT influences the effect on rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2B6, CYP2C9, CYP2C19, and CYP3A4) by using cocktail probe drugs in vivo.

Materials and methods: A cocktail solution at a dose of 5?mL/kg, which contained phenacetin (20?mg/kg), bupropion (20?mg/kg), tolbutamide (5?mg/kg), omeprazole (20?mg/kg), and midazolam (10?mg/kg), was given as oral administration to rats treated with 7?d intraperitoneal injection of KLT. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC-MS/MS. The corresponding pharmacokinetic parameters were calculated by the software of DAS 2.0 (SPPS Inc., Chicago, IL).

Results: In the experiment, there was a statistically significant difference in the t_1/2, C_max, AUC_(0–∞), and CL for phenacetin, bupropion, tolbutamide, omeprazole, and midazolam. Our study showed that treatment with multiple doses of KLT had induction effect on rat CYP1A2, while CYP2B6, CYP2C9, CYP2C19, and CYP3A4 enzyme activities had been inhibited after multiple doses of KLT treatment.

Conclusions: KLT can either induce or inhibit activities of CYP. Therefore, caution is needed when KLT is co-administration with some CYP substrates in clinic, which may result in herb–drug interactions.     

Suitable albumin concentrations for enhanced drug oxidation activities mediated by human liver microsomal cytochrome P450 2C9 and other forms predicted with unbound fractions and partition/distribution coefficients of model substrates

1. Albumin has reportedly enhanced cytochrome P450 (P450)-mediated drug oxidation rates in human liver microsomes. Consequently, measurements of clearances and fractions metabolized could vary depending on the experimental albumin concentrations used.

2. In this study, the oxidation rates of diclofenac and warfarin by human liver microsomes were significantly enhanced in the presence of 0.10% (w/v) bovine serum albumin, whereas those of tolbutamide and phenytoin required 1.0% and 2.0% of albumin for significant enhancement. Values of the fractions metabolized by P450 2C9 for four substrates did not markedly change in the presence of albumin at the above-mentioned concentrations.

3. The oxidation rates of bupropion, omeprazole, chlorzoxazone and phenacetin in human liver microsomes were reportedly enhanced by 0.5%, 1%, 2% and 2% of albumin, respectively. Analysis of reported intrinsic clearance values and suitable albumin concentrations for the currently analyzed substrates and the reported substrates revealed an inverse correlation, with warfarin as an outlier.

4. Suitable albumin concentrations were multivariately correlated with physicochemical properties, that is, the plasma unbound fractions, octanol–water partition coefficient and acid dissociation constant (r?=?0.98, p<.0001, n?=?10). Therefore, multiple physicochemical properties may be determinants of suitable albumin concentrations for substrate oxidations in human liver microsomes.

     

Progesterone hydroxylation by cytochromes P450 2C and 3A enzymes in marmoset liver microsomes

1.?Common marmosets (Callithrix jacchus) are potentially useful nonhuman primate models for preclinical drug metabolism studies. However, the roles of marmoset cytochrome P450 (P450) isoforms in the oxidation of endobiotic progesterone have not been fully investigated. In this study, the roles of marmoset P450 isoforms in progesterone hydroxylation were extensively determined.

2.?The activities of liver microsomes from individual marmosets with respect to progesterone 21/17α- and 16α/6β-hydroxylation were significantly correlated with those for flurbiprofen 4-hydroxylation and midazolam 1′-hydroxylation, respectively, as similar correlations have been found in humans. Anti-P450 2?C and 3?A antibodies suppressed progesterone 21/17α- and 16α/6β-hydroxylation, respectively, in marmoset liver microsomes.

3.?Recombinant marmoset P450 2C58 and 2C19 catalyzed progesterone to form 21-hydroxyprogesterone and 16α-hydroxyprogesterone, respectively, as major products with high maximum velocity/K_m values of 0.53 and 0.089?mL/min/nmol, respectively. Recombinant marmoset P450 3A4/90 oxidized progesterone to form 6β-hydroxyprogesterone as a major product with homotropic cooperativity (>1 of Hill coefficients).

4.?These results indicate that the overall activities and roles of liver microsomal P450 enzymes in marmoset livers are similar to those in humans, especially for progesterone 21/17α- and 16α/6β-hydroxylation by marmoset P450 2?C and 3?A enzymes, respectively, suggesting important roles for these P450 enzymes in the metabolism of endobiotics in marmosets.     

Association with polymorphic marmoset cytochrome P450 2C19 of in vivo hepatic clearances of chirally separated R-omeprazole and S-warfarin using individual marmoset physiologically based pharmacokinetic models

1. Simulated clearances of R-warfarin and efavirenz were recently reported for individual cynomolgus monkeys genotyped for cytochrome P450 2C19 and 2C9, respectively. To expand and verify this modeling procedure, simulations of R/S-omeprazole and R/S-warfarin clearances after oral administrations in individual marmosets were performed using individual simplified physiologically based pharmacokinetic (PBPK) modeling consisting of gut, liver and central compartments.

2. Pharmacokinetics of R/S-omeprazole were chirally determined using the previously reported plasma microsamples in this study. The areas under the plasma concentration/time curves (AUC) of R-omeprazole and S-warfarin, but not S-omeprazole and R-warfarin, after oral administrations in the P450 2C19 homozygous mutant group were significantly higher than those in the wild-type group. These modeled hepatic intrinsic clearances were also significantly associated with the marmoset P450 2C19 genotypes. Other parameter values, e.g. absorption rate constants or systemic circulation volumes, were not likely determining factors.

3. The reported individual AUC values measured in 4–6 marmosets after oral R-omeprazole and S-warfarin administrations were significantly correlated with the AUC values predicted using the PBPK models after virtual administrations.

4. This study indicates that clearances of R-omeprazole, S-warfarin and related medicines associated with polymorphic P450 2C19 in individual marmosets can be simulated using simplified individual PBPK models.     

Drug interactions of diclofenac and its oxidative metabolite with human liver microsomal cytochrome P450 1A2-dependent drug oxidation

Abstract

1.?The purpose of this study was to investigate the inhibitory effects of diclofenac on human cytochrome P450 1A2-, 2C19- and 3A4-mediated drug oxidations and to evaluate the drug interaction potential of diclofenac and 4′-hydroxydiclofenac.

2.?Diclofenac was converted to 4′-hydroxydiclofenac by recombinantly expressed human P450 1A2 with K_m and V_max values of 33?µM and 0.20?min^?1, respectively. Diclofenac and 4′-hydroxydiclofenac suppressed flurbiprofen 4′-hydroxylation by P450 2C9 strongly and moderately, respectively; however, they did not affect P450 2C19-dependent S-mephenytoin hydroxylation or P450 3A4-dependent midazolam hydroxylation.

3.?Although the caffeine 3-N-demethylation activity of liver microsomal P450 1A2 was inhibited by simultaneous incubation with diclofenac, the riluzole N-hydroxylation activities of recombinant P450 1A2 and human liver microsomes were inhibited after preincubation with diclofenac or 4′-hydroxydiclofenac for 20?min in the presence of NADPH. Using the inhibition constant (37?µM) of diclofenac on caffeine 3-N-demethylation and the reported 95th percentiles of maximum plasma concentration (10.5?µM) after an oral dose of diclofenac, the in vivo estimated increase in area under the plasma concentration–time curve was 29%.

4.?These results suggest that diclofenac could inhibit drug clearance to a clinically important degree that depends on P450 1A2. Clinically relevant drug interactions in vivo with diclofenac are likely to be invoked via human P450 1A2 function in addition to those caused by the effect of diclofenac on P450 2C9.     

Omeprazole and the cytochrome P450 system

Enzymes of the cytochrome P450 superfamily play a key role in xenobiotic metabolism. Their properties and significance are discussed with particular reference to interactions with the H⁺, K⁺-ATPase blocker, omeprazole. Such interactions include both inhibitory (subfamily 2C) and inducing effects (subfamily 1A). Delayed metabolic elimination of diazepam, warfarin, carbamazepin and phenytoin is probably due to omeprazole competition for the concerned isoform of subfamily 2C; however, these effects are modest to negligible in magnitude and, for phenytoin, not consistently reproducible. Also, induction of subfamily 1A is only minor as assessed from the resultant changes in N-3-demethylation of caffeine, a reaction specific to this subfamily. Concerns about a possible activation of procarcinogens that might arise from subfamily 1A induction appear ill-founded given the fact that cruciferous vegetables such as broccoli and Brussels sprouts are potent inducers, but rather seem to lower the incidence of certain types of cancer. Likewise, the idea that the toxicity of acetaminophen might increase upon subfamily 1A induction appears far-fetched, mainly because much stronger inducers of subfamily 1A (cigarette smoke and charcoaled beef) are unable to alter acetaminophen metabolism.     

Assessment of multiple cytochrome P450 activities in metabolically inactivated human liver microsomes and roles of P450 2C isoforms in reaction phenotyping studies

The fraction of substrate metabolized (f_m) can be used to estimate drug interactions and can be determined by comparison of the intrinsic clearances (CL_int) of victim drugs obtained from inhibited and uninhibited hepatic enzymes_. Commercially available human liver microsomes were recently developed in which one cytochrome P450 (P450) isoform is selectively inactivated. These inactivated liver microsomes were used to evaluate the roles of P450 2C isoforms in the depletion and oxidation of probe substrates. Determination of CL_int with sets of control and P450 2C9‐inactivated liver microsomes yielded f_{m,P450 2C9} values of 0.69–1.0 for celecoxib, diclofenac and warfarin. Apparent minor contributions of P450 1A2/2C8/3A4 were seen in depletion assays, yielding ~1 for the sum of the f_m values. Selectively inactivated liver microsomes were thereby shown to be potentially useful for determining the in vitro f_m values for major P450 2C9 contributions to substrate oxidations. Metabolite formations from diclofenac and warfarin were suppressed by 62–84% by the replacement of control liver microsomes with P450 2C9‐inactivated liver microsomes. R‐, S‐ and racemic omeprazole and troglitazone oxidation activities by liver microsomes at multiple substrate concentrations were suppressed by 26–36% and 22–50%, respectively, when P450 2C19‐ and 2C8‐inactivated liver microsomes were used in place of control liver microsomes. This study provides important information to help elucidate the different roles of P450 isoforms in metabolite formation at different substrate concentrations. The data obtained allow the fractions metabolized to be calculated for victim drugs.     

Simultaneous and comprehensive in vivo analysis of cytochrome P450 activity by using a cocktail approach in rats

A cocktail approach can detect the activities of multiple cytochrome P450 (CYP) isoforms following the administration of multiple CYP‐specific substrates in a single experiment. This study aimed to develop a simultaneous and comprehensive in vivo analysis of CYP activity in rats. The rats received an oral administration of losartan (10 mg/kg) and omeprazole (40 mg/kg). Caffeine (1 mg/kg), dextromethorphan (10 mg/kg) and midazolam (10 mg/kg) were administered 15 min later. In the drug‐interaction phase, the rats were treated orally with dexamethasone (80 mg/kg) 24 h before, or with ketoconazole (10 mg/kg), fluvoxamine (100 mg kg) or fluconazole (10 mg/kg) 1 h before the administration of cocktail drugs. The concentrations of the drugs and their metabolites were determined by LC/MS/MS. Plasma concentrations of five CYP substrates and their metabolites were simultaneously evaluated after the oral drug administration. Fluvoxamine and fluconazole significantly increased the C_max and AUC of caffeine, and the AUC of omeprazole and midazolam. Dexamethasone significantly increased C_max and AUC of losartan, while it decreased the C_max of midazolam. Ketoconazole showed no significant effect on the pharmacokinetic parameters of the tested drugs. In conclusion, a cocktail approach was developed for simultaneous and comprehensive analysis of the activities of multiple CYP isoforms in rats. In this approach, the effects of inhibitors and an inducer of various CYP isoforms were examined. Although further studies are necessary to predict the effects in humans, this approach may be expected to serve as a convenient method for detecting drug–drug interactions in rats. Copyright © 2014 John Wiley & Sons, Ltd.     

R-warfarin clearances from plasma associated with polymorphic cytochrome P450 2C19 and simulated by individual physiologically based pharmacokinetic models for 11 cynomolgus monkeys

1.?Cynomolgus monkey cytochrome P450 2C19 (formerly known as P450 2C75), homologous to human P450 2C19, has been identified as R-warfarin 7-hydroxylase. In this study, simulations of R-warfarin clearance in individual cynomolgus monkeys genotyped for P450 2C19 p.[(Phe100Asn; Ala103Val; Ile112Leu)] were performed using individual simplified physiologically based pharmacokinetic (PBPK) modeling.

2.?Pharmacokinetic parameters and absorption rate constants, volumes of the systemic circulation, and hepatic intrinsic clearances for individual PBPK models were estimated for eleven cynomolgus monkeys.

3.?One-way ANOVA revealed significant effects of the genotype (p?R-warfarin among the homozygous mutant, heterozygous mutant, and wild-type groups. R-Warfarin clearances in individual cynomolgus monkeys genotyped for P450 2C19 were simulated by simplified PBPK modeling. The modeled hepatic intrinsic clearances were significantly associated with the P450 2C19 genotypes. The liver microsomal elimination rates of R-warfarin for individual animals after in vivo administration showed significant reductions associated with the genotype (p?4.?This study provides important information to help simulate clearances of R-warfarin and related medicines associated with polymorphic P450 2C19 in individual cynomolgus monkeys, thereby facilitating calculation of the fraction of hepatic clearance.     

Interaction profile of armodafinil with medications metabolized by cytochrome P450 enzymes 1A2, 3A4 and 2C19 in healthy subjects

BACKGROUND AND OBJECTIVE: Armodafinil, a wakefulness-promoting agent, is the pure R-enantiomer of racemic modafinil. The objective of this article is to summarize the results of three clinical drug-interaction studies assessing the potential for drug interactions of armodafinil with agents metabolized by cytochrome P450 (CYP) enzymes 1A2, 3A4 and 2C19. Study 1 evaluated the potential for armodafinil to induce the activity of CYP1A2 using oral caffeine as the probe substrate. Study 2 evaluated the potential for armodafinil to induce gastrointestinal and hepatic CYP3A4 activity using intravenous and oral midazolam as the probe substrate. Study 3 evaluated the potential for armodafinil to inhibit the activity of CYP2C19 using oral omeprazole as the probe substrate. METHODS: Healthy men and nonpregnant women aged 18-45 years with a body mass index of 相似文献   

Effects of Alismatis rhizome on rat cytochrome P450 enzymes

Context: Alismatis rhizome (RA) (Water Plantain Family, also called “Zexie” in Chinese), one of the commonly used components of traditional Chinese medicines, is derived from the dried rhizomes of Alisma orientalis (Sam.) Juzep. (Alismataceae).

Objective: This study explores the RA influences on rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C9, CYP2E1 and CYP3A4) by using cocktail probe drugs in vivo.

Materials and methods: A cocktail solution at a dose of 5?mL/kg, which contained phenacetin (20?mg/kg), tolbutamide (5?mg/kg), chlorzoxazone (20?mg/kg) and midazolam (10?mg/kg), was orally administration to rats treated twice daily with RA (10, 20 and 40?g/kg) for consecutive 14?days. Blood samples (0.2?mL) were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC-MS/MS. The corresponding pharmacokinetic parameters were calculated by the software of DAS 2.0 (Wenzhou Medical College, Zhejiang, China).

Results: In the experiment, there was a statistically significant difference in the t_1/2, C_max, AUC_(0-∞) and CL for phenacetin and midazolam, while there was no statistical pharmacokinetics difference for tolbutamide and chlorzoxazone. Our study showed that treatment with multiple doses of RA had an inductive effect on rat CYP1A2 and an inhibitory effect on rat CYP3A4 enzyme activity. However, RA has no inductive or inhibitory effect on the activities of CYP2C9 and CYP2E1.

Conclusions: Caution is needed when RA is co-administration with some CYP1A2 or CYP3A4 substrates in clinic, because it may result in treatment failure and herb–drug interactions.     

Terfenadine t-butyl hydroxylation catalyzed by human and marmoset cytochrome P450 3A and 4F enzymes in livers and small intestines

1.?Roles of human cytochrome P450 (P450) 3A4 in oxidation of an antihistaminic drug terfenadine have been previously investigated in association with terfenadine–ketoconazole interaction. Several antihistamine drugs have been recently identified as substrates for multiple P450 enzymes. In this study, overall roles of P450 3A4, 2J2, and 4F12 enzymes in terfenadine t-butyl hydroxylation were investigated in small intestines and livers from humans, marmosets, and/or cynomolgus monkeys.

2.?Human liver microsomes and liver and small intestine microsomes from marmosets and cynomolgus monkeys effectively mediated terfenadine t-butyl hydroxylation. Ketoconazole and N-hydroxy-N′-(4-butyl-2-methylphenyl)-formamidine (a P450 4A/F inhibitor) almost completely and moderately inhibited these activities, respectively, in human liver microsomes; however, these chemicals did not show substantially suppression in marmoset liver. Anti-human P450 3A and 4F antibodies showed the roughly supportive inhibitory effects.

3.?Recombinant P450 3A4/90 and 4F12 showed high terfenadine t-butyl hydroxylation activities with substrate inhibition constants of 84–144?μM (under 26–76?μM of K_m values), in similar manners to liver and intestine microsomes.

4.?These results suggest that human and marmoset P450 3A4/90 and 4F12 in livers or small intestines played important roles in terfenadine t-butyl hydroxylation. Marmosets could be a model for humans during first pass extraction of terfenadine and related substrates.     

Monkey liver cytochrome P450 2C9 is involved in caffeine 7-N-demethylation to form theophylline

Abstract

1. Caffeine (1,3,7-trimethylxanthine) is a phenotyping substrate for human cytochrome P450 1A2. 3-N-Demethylation of caffeine is the main human metabolic pathway, whereas monkeys extensively mediate the 7-N-demethylation of caffeine to form pharmacological active theophylline.

2. Roles of monkey P450 enzymes in theophylline formation from caffeine were investigated using individual monkey liver microsomes and 14 recombinantly expressed monkey P450 enzymes, and the results were compared with those for human P450 enzymes.

3. Caffeine 7-N-demethylation activity in microsomes from 20 monkey livers was not strongly inhibited by α-naphthoflavone, quinidine or ketoconazole, and was roughly correlated with diclofenac 4′-hydroxylation activities. Monkey P450 2C9 had the highest activity for caffeine 7-N-demethylation. Kinetic analysis revealed that monkey P450 2C9 had a high V_max/K_m value for caffeine 7-N-demethylation, comparable to low K_m value for monkey liver microsomes. Caffeine could dock favorably with monkey P450 2C9 modeled for 7-N-demethylation and with human P450 1A2 for 3-N-demethylation.

4. The primary metabolite theophylline was oxidized to 8-hydroxytheophylline in similar ways by liver microsomes and by recombinant P450s in both humans and monkeys.

5. These results collectively suggest a high activity for monkey liver P450 2C9 toward caffeine 7-N-demethylation, whereas, in humans, P450 1A2-mediated caffeine 3-N-demethylation is dominant.     

Marmoset cytochrome P450 2J2 mainly expressed in small intestines and livers effectively metabolizes human P450 2J2 probe substrates,astemizole and terfenadine

1.?Common marmoset (Callithrix jacchus), a New World Monkey, has potential to be a useful animal model in preclinical studies. However, drug metabolizing properties have not been fully understood due to insufficient information on cytochrome P450 (P450), major drug metabolizing enzymes.

2.?Marmoset P450 2J2 cDNA was isolated from marmoset livers. The deduced amino acid sequence showed a high-sequence identity (91%) with cynomolgus monkey and human P450 2J2 enzymes. A phylogenetic tree revealed that marmoset P450 2J2 was evolutionarily closer to cynomolgus monkey and human P450 2J2 enzymes, than P450 2J forms in pigs, rabbits, rats or mice.

3.?Marmoset P450 2J2 mRNA was abundantly expressed in the small intestine and liver, and to a lesser extent in the brain, lung and kidney. Immunoblot analysis also showed expression of marmoset P450 2J2 protein in the small intestine and liver.

4.?Enzyme assays using marmoset P450 2J2 protein heterologously expressed in Escherichia coli indicated that marmoset P450 2J2 effectively catalyzed astemizole O-demethylation and terfenadine t-butyl hydroxylation, similar to human and cynomolgus monkey P450 2J2 enzymes.

5.?These results suggest the functional characteristics of P450 2J2 enzymes are similar among marmosets, cynomolgus monkeys and humans.