1.?Although the New World non-human primate, the common marmoset (Callithrix jacchus), is a potentially useful animal model, comprehensive understanding of drug metabolizing enzymes is insufficient.
2.?A cDNA encoding a novel cytochrome P450 (P450) 2D8 was identified in marmosets. The amino acid sequence deduced from P450 2D8 cDNA showed a high sequence identity (83–86%) with other primate P450 2Ds. Phylogenetic analysis showed that marmoset P450 2D8 was closely clustered with human P450 2D6, unlike P450 2Ds of miniature pig, dog, rabbit, guinea pig, mouse or rat.
3.?Marmoset P450 2D8 mRNA was predominantly expressed in the liver and small intestine among the tissues types analyzed, whereas marmoset P450 2D6 mRNA was expressed predominantly in the liver where P450 2D protein was detected by immunoblotting.
4.?By metabolic assays using marmoset P450 2D8 protein heterologously expressed in Escherichia coli, although P450 2D8 exhibits lower catalytic efficiency compared to marmoset and human P450 2D6 enzymes, P450 2D8 mediated O-demethylations of metoprolol and dextromethorphan and bufuralol 1′-hydroxylation.
5.?These results suggest that marmoset P450 2D8 (also expressed in the extrahepatic tissues) has potential roles in drug metabolism in a similar manner to those of human and marmoset P450 2D6.
Cynomolgus monkeys are widely used to predict human pharmacokinetic and/or toxic profiles in the drug developmental stage. Characterization of cynomolgus monkey P450s such as the mRNA expression level, substrate specificity, and inhibitor selectivity were conducted to provide helpful information in designing monkey in vivo studies and monkey-to-human extrapolation.
The expression levels of 12 monkey P450 mRNAs, which are considered to be important P450 subfamilies in drug metabolism, were investigated in the liver, small intestine (duodenum, jejunum, and ileum), and colon of individual monkeys.
iIn vitro activities and intrinsic clearance values were determined in monkey intestinal and liver microsomes (MIM and MLM, respectively) using nine typical oxidative reactions for human P450s. Paclitaxel 6α-hydroxylation, diclofenac 4′-hydroxylation, and S-mephenytoin 4′-hydroxylation showed low activities in MIM and MLM.
IC50 values of eight selective inhibitors of human P450s were determined in MIM and MLM. Inhibitory effects of furafylline and sulfaphenazole were weak in monkeys on phenacetin O-deethylation and diclofenac 4′-hydroxylation, respectively.
These results show profiles of monkey P450s in both the intestine and liver in detail and contribute to a better understanding of the species difference in substrate specificity and inhibitor selectivity between cynomolgus monkeys and humans.
The roles of human cytochrome P450 (P450 or CYP) 2A6 in the oxidation of flavanone [(2R)- and (2S)-enantiomers] and flavone were studied in human liver microsomes and recombinant human P450 enzymes.
CYP2A6 was highly active in oxidizing flavanone to form flavone, 2′-hydroxy-, 4′-, and 6-hydroxyflavanones and in oxidizing flavone to form mono- and di-hydroxylated products, such as mono-hydroxy flavones M6, M7, and M11 and di-hydroxy flavones M3, M4, and M5.
Liver microsomes prepared from human sample HH2, defective in coumarin 7-hydroxylation activity, were very inefficient in forming 2′-hydroxyflavanone from flavanone and a mono-hydroxylated product, M6, from flavone. Coumarin and anti-CYP2A6 antibodies strongly inhibited the formation of these metabolites in microsomes prepared from liver samples HH47 and 54, which were active in coumarin oxidation activities.
Molecular docking analysis showed that the C2′-position of (2R)-flavanone (3.8 Å) was closer to the iron center of CYP2A6 than the C6-position (10 Å), while distances from C2′ and C6 of (2S)-flavanone to the CYP2A6 were 6.91 Å and 5.42 Å, respectively.
These results suggest that CYP2A6 catalyzes site-specific oxidation of (racemic) flavanone and also flavone in human liver microsomes. CYP1A2 and CYP2B6 were also found to play significant roles in some of the oxidations of these flavonoids by human liver microsomes.
The evaluation of drug-mediated cytochrome P450 (P450) induction using human hepatocytes is important for predicting drug interactions. In this study, we prepared hepatocytes from chimeric mice with humanised livers (Hu-Liver mice) and evaluated the expression and inducibility of P450s in these hepatocytes.
Up to 95% of the Hu-Liver cells stained positive for human leukocyte antigen and the mean viability exceeded 85% (n?=?10). Monolayer-cultured Hu-Liver cells displayed a similar morphology to cultures of the corresponding human hepatocytes used as transplantation donors.
The mRNA expression levels in Hu-Liver cells of 16 P450 forms belonging to P450 subfamilies 1–4 correlated well with the expression levels of the same enzymes in human hepatocytes. The variations in individual P450 mRNA levels between Hu-Liver cells and the corresponding human hepatocytes were within five-fold for 13 P450 forms.
The production of 6β-hydroxytestosterone in Hu-Liver cells was significantly increased (p?<?.05) following treatment with the CYP3A inducer, rifampicin.
Hu-Liver cells have characteristics similar to those of human hepatocytes in terms of mRNA expression levels and the inducibility of the various P450 forms. Thus, Hu-Liver cells can potentially be used for in vitro drug-mediated induction assays of human hepatic P450s.
A 1,2,4-oxadiazole ring-containing compound DS-8500a was developed as a novel G protein-coupled receptor 119 agonist. In vivo metabolic fates of [14C]DS-8500a differently radiolabeled in the benzene ring or benzamide side carbon in rats were investigated.
Differences in mass balances were observed, primarily because after the oxadiazole ring-opening and subsequent ring-cleavage small-molecule metabolites containing the benzene side were excreted in the urine, while those containing the benzamide side were excreted in the bile.
DS-8500a was detected at trace levels in urine and bile, demonstrating extensive metabolism prior to urinary/biliary excretion. At least 16 metabolite structures were proposed in plasma, urine, and bile samples from rats treated with [14C]DS-8500a.
Formation of a ring-opened metabolite (reduced DS-8500a) in hepatocytes of humans, monkeys, and rats was confirmed; however, it was not affected by typical inhibitors of cytochrome P450s, aldehyde oxidases, or carboxylesterases in human hepatocytes. Extensive formation of the ring-opened metabolite was observed in human liver microsomes fortified with an NADPH-generating system under anaerobic conditions.
These results suggest an in vivo unique reductive metabolism of DS-8500a is mediated by human non-cytochrome P450 enzymes.
Glutathione S-transferase (GST) is a family of enzymes important for conjugation with glutathione of endogenous and exogenous compounds. Human GSTM1 null allele is associated with toxicity and cancers. Cynomolgus and rhesus macaques have molecular and enzymatic similarities of GSTs to humans; however, genetic variants have not been investigated. In macaques, instead of pseudogenized GSTM1, GSTM5 is a predominant GSTM isoform.
In this study, re-sequencing of GSTM5 in 64 cynomolgus and 31 rhesus macaques found 6 non-synonymous variants, and 1 variant (IVS5?+?1) causing exon skip. Of these 7 variants, 3 and 1 were found only in Indochinese and Indonesian cynomolgus macaques, respectively.
Cynomolgus GSTM5-mediated styrene 7,8-oxide and trans-stilbene oxide conjugation activities correlated with GSTM protein levels immunochemically quantified in cynomolgus liver samples. Using recombinant GSTM5 proteins, 4 of the 6 non-synonymous variants including E29Q, L96R, M166V and S201N showed substantially lower metabolic activities. Moreover, a homozygote for E29Q and heterozygotes for S201N or IVS5?+?1 showed significantly lower conjugation activities in liver cytosolic fractions as compared with wild-type animals.
Therefore, the present results suggest that inter-animal variability of GST-dependent drug metabolism is at least partly accounted for by GSTM5 variants in cynomolgus and rhesus macaques as pre-clinical animal models.
The objective is to evaluate methoxsalen as an in vitro phenotyping tool in comparison to ABT as a nonspecific inactivator of P450 mediated metabolism.
The reversible inhibition of methoxsalen and ABT against the P450, FMO, AO, MAO-A and -B, enzymes were evaluated using standard marker probe reactions. The time-dependent inhibition of P450 enzymes was evaluated in human liver microsomes. CES1 activities were determined by monitoring the depletion of known substrate, the clopidogrel. The metabolism of P450 substrates in the presence and absence of methoxsalen or ABT was evaluated in human liver microsomes.
Methoxsalen is a direct inhibitor and inhibited the activities (>90%) of all enzymes at a concentration of 300?µM except for CYP2C9. Methoxsalen is also a potent time-dependent inhibitor of all P450 enzymes except for CYP2C19 (moderate) at a concentration of 300?µM. Methoxsalen inhibited the metabolism of P450 substrates in the pre-incubation mode. ABT is a potent TDI of several P450 except for CYP2C19 (47%) and CYP2C9 (27%).
The results indicate that methoxsalen is a potent pan P450 inhibitor than ABT and can be a better tool in distinguishing P450 mediated metabolism form non-P450 metabolism in human liver microsomes.
The absorption, distribution, metabolism, and excretion of fasiglifam were investigated in rats, dogs, and humans.
The absolute oral bioavailability of fasiglifam was high in all species (>76.0%).
After oral administration of [14C]fasiglifam, the administered radioactivity was quantitatively recovered and the major route of excretion of radioactivity was via feces in all species.
Fasiglifam was a major component in the plasma and feces in all species. Its oxidative metabolite (M-I) was observed as a minor metabolite in rat and human plasma (<10% of plasma radioactivity). In human plasma, hydroxylated fasiglifam (T-1676427), the glucuronide of fasiglifam (fasiglifam-G), and the glucuronide of M-I were detected as additional minor metabolites (<2% of plasma radioactivity). None of these metabolites were specific to humans. Fasiglifam-G was the major component in the rat and dog bile.
In vitro cytochrome P450 (CYP) and uridine diphosphate glucuronosyltransferase (UGT) reaction phenotyping indicated that oxidation (to form M-I and T-1676427) and glucuronidation of fasiglifam are mainly mediated by CYP3A4/5 and UGT1A3, respectively.
Fasiglifam and fasiglifam-G are substrates of BCRP and Mrp2/MRP2, respectively.
Glucuronidation of fasiglifam-G was found to be the predominant elimination pathway of fasiglifam in all species tested, including humans.
Characteristics of twelve cytochromes P450 (CYPs) from cynomolgus monkeys were compared with those of human CYPs that play an important role in drug metabolism.
Eleven members of CYP1A, CYP2A, CYP2C, CYP2D, CYP2E, and CYP3A subfamilies from cynomolgus monkeys exhibited a high degree of homologies (more than 90%) in cDNA and amino acid sequences with corresponding human CYPs, and catalysed typical reactions of corresponding human CYPs.
One member of the cynomolgus monkey CYP2C subfamily, CYP2C76, exhibited a lower homology (around 70%) in amino acid sequences with other cynomolgus monkey and human CYP2C subfamilies. CYP2C76 catalysed typical CYP2C substrates with low activities, and has not been found in humans.
CYPs identified in cynomolgus monkeys were similar to CYP1A1, CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5 in humans.
These results indicate that cynomolgus monkeys express CYPs similar to human CYPs that are important in drug metabolism.
The disposition and metabolism of lemborexant, a novel dual orexin receptor antagonist currently under development as a therapeutic agent for insomnia disorder, were evaluated after a single oral administration of [14C]lemborexant in Sprague-Dawley rats (10?mg/kg) and cynomolgus monkeys (3?mg/kg).
In both species, [14C]lemborexant was rapidly absorbed: radioactivity concentration in blood peaked at 0.83–1.8?h, and decreased with elimination half-life of 110?h. The radioactivity administered was excreted primarily into faeces, with relatively little excreted into urine.
Lemborexant was not detected in bile, urine or faeces, indicating that lemborexant administered orally was completely absorbed from the gastrointestinal tract and that the main elimination pathway was metabolism in both species.
In rats, lemborexant was found to be minor in plasma (≤5.2% of total radioactivity), and M9 (hydroxylated form) was the major circulating metabolite. In monkeys, the major circulating components were lemborexant, M4 (N-oxide metabolite), M13 (di-oxidised form), M14 (di-oxidised form) and M16 (glucuronide of mono-oxidised form).
In both species, lemborexant was metabolised to various metabolites by multiple pathways, the primary of which was oxidation of the dimethylpyrimidine or fluorophenyl moiety.
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.
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.
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.
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.
The metabolism of deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in liver microsomes, liver cytosol and plasma from male Sprague–Dawley rats aged 15, 21 and 90 days and from adult humans.
DLM and CPM were metabolised by rat hepatic microsomal cytochrome P450 (CYP) enzymes and to a lesser extent by microsomal and cytosolic carboxylesterase (CES) enzymes, whereas TPM was metabolised to a greater extent by CES enzymes.
In human liver, DLM and TPM were mainly metabolised by CES enzymes, whereas CPM was metabolised by CYP and CES enzymes.
The metabolism of pyrethroids by cytosolic CES enzymes contributes to the overall hepatic clearance of these compounds.
DLM, CPM and TPM were metabolised by rat, but not human, plasma CES enzymes.
This study demonstrates that the ability of male rats to metabolise DLM, CPM and TPM by hepatic CYP and CES enzymes and plasma CES enzymes increases with age. In all instances, apparent intrinsic clearance values were lower in 15 than in 90?day old rats. As pyrethroid-induced neurotoxicity is due to the parent compound, these results suggest that DLM, CPM and TPM may be more neurotoxic to juvenile than to adult rats.
The metabolism of the pyrethroids deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in human expressed cytochrome P450 (CYP) and carboxylesterase (CES) enzymes.
DLM, CPM and TPM were metabolised by human CYP2B6 and CYP2C19, with the highest apparent intrinsic clearance (CLint) values for pyrethroid metabolism being observed with CYP2C19. Other CYP enzymes contributing to the metabolism of one or more of the three pyrethroids were CYP1A2, CYP2C8, CYP2C9*1, CYP2D6*1, CYP3A4 and CYP3A5. None of the pyrethroids were metabolised by CYP2A6, CYP2E1, CYP3A7 or CYP4A11.
DLM, CPM and TPM were metabolised by both human CES1 and CES2 enzymes.
Apparent CLint values for pyrethroid metabolism by CYP and CES enzymes were scaled to per gram of adult human liver using abundance values for microsomal CYP enzymes and for CES enzymes in liver microsomes and cytosol. TPM had the highest and CPM the lowest apparent CLint values for total metabolism (CYP and CES enzymes) per gram of adult human liver.
Due to their higher abundance, all three pyrethroids were extensively metabolised by CES enzymes in adult human liver, with CYP enzymes only accounting for 2%, 10% and 1% of total metabolism for DLM, CPM and TPM, respectively.