The cynomolgus monkey is an animal species widely used to study drug metabolism because of its evolutionary closeness to humans. However, drug-metabolizing enzyme activities have not been compared in various parts of the liver and small intestine in cynomolgus monkeys. In this study, therefore, drug-metabolizing enzyme activities were analyzed in the liver (the five lobes) and small intestine (six sections from the duodenum to the distal ileum). 7-Ethoxyresorufin O-deethylation, coumarin 7-hydroxylation, paclitaxel 6α-hydroxylation, diclofenac 4'-hydroxylation, tolbutamide methylhydroxylation, S-mephenytoin 4'-hydroxylation, bufuralol 1'-hydroxylation, chlorzoxazone 6-hydroxylation, midazolam 1'-hydroxylation, and testosterone 6β-, 16α-, 16β-, and 2α-hydroxylation were used as the probe reactions for this investigation. In liver, all probe reactions were detected and enzyme activity levels were similar in all lobes, whereas, in the small intestine, all enzyme activities were detected (except for coumarin 7-hydroxylase and testosterone 16α-hydroxylase activity), but from jejunum to ileum there was a decrease in the level of enzyme activity. This includes midazolam 1'-hydroxylation and testosterone 6β-hydroxylation, which are catalyzed by cynomolgus monkey cytochrome P450 (CYP) 3A4/5, orthologs of human CYP3A4/5, which are important drug-metabolizing enzymes. The data presented in this study are expected to facilitate the use of cynomolgus monkeys in drug metabolism studies.
"After centrifugation at 1500g for 10 min, the supernatant or extract was analyzed by reverse-phase high-performance liquid chromatography with a fluorescence or UV detector. Metabolic assays using diclofenac and testosterone as substrates were carried out as described previously (Nakanishi et al., 2011). To estimate a correlation between drugmetabolizing enzyme activities and P450 amounts, linear regression analysis was performed using Origin7.5J "
[Show abstract][Hide abstract] ABSTRACT: The cynomolgus monkey is widely used as a primate model in preclinical studies because of its evolutionary closeness to humans. Despite their importance in drug metabolism, the content of each cytochrome P450 (P450) enzyme has not been systematically determined in cynomolgus monkey livers. In this study, liver microsomes of 27 cynomolgus monkeys were analyzed by immunoblotting using selective P450 antibodies. The specificity of each antibody was confirmed by analyzing the cross-reactivity against 19 CYP1-3 subfamily enzymes using recombinant proteins. CYP2A, CYP2B6, CYP2C9/19, CYP2C76, CYP2D, CYP2E, CYP3A4, and CYP3A5 were detected in all 27 animals. In contrast, CYP1A, CYP1D, and CYP2J were below detectable levels in all liver samples. The average content of each P450 showed that among the P450s analyzed CYP3A (3A4 and 3A5) was the most abundant (40% of total immunoquantified P450), followed by CYP2A (25%), CYP2C (14%), CYP2B6 (13%), CYP2E1 (11%), and CYP2D (3%). No apparent sex differences were found for any P450. Interanimal variations ranged from 2.6-fold (CYP3A) to 11-fold (CYP2C9/19), and most P450s (CYP2A, CYP2D, CYP2E, CYP3A4, and CYP3A5) varied 3- to 4-fold. To examine the correlations of P450 content with enzyme activities, metabolic assays were performed in 27 cynomolgus monkey livers using 7-ethoxyresorufin, coumarin, pentoxyresorufin, flurbiprofen, bufuralol, dextromethorphan, and midazolam. CYP2D and CYP3A4 contents were significantly correlated with typical reactions of human CYP2D (bufuralol 1'-hydroxylation and dextromethorphan O-deethylation) and CYP3A (midazolam 1'-hydroxylation and 4-hydroxylation). The results presented in this study provide useful information for drug metabolism studies using cynomolgus monkeys.
Journal of Pharmacology and Experimental Therapeutics 08/2011; 339(2):654-61. DOI:10.1124/jpet.111.185009 · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cynomolgus macaques, frequently used in drug metabolism studies, are bred mainly in the countries of Asia; however, comparative studies of drug metabolism between cynomolgus macaques bred in these countries have not been conducted. In this study, hepatic gene expression profiles of cynomolgus macaques bred in Cambodia (mfCAM), China (mfCHN), and Indonesia (mfIDN) were analyzed. Microarray analysis revealed that expression of most hepatic genes, including drug-metabolizing enzyme genes, was not substantially different between mfCAM, mfCHN, and mfIDN; only 1.1% and 3.0% of all the gene probes detected differential expression (>2.5-fold) in mfCAM compared with mfCHN and mfIDN, respectively. Quantitative polymerase chain reaction showed that the expression levels of 14 cytochromes P450 (P450s) important for drug metabolism did not differ (>2.5-fold) in mfCAM, mfCHN, and mfIDN, validating the microarray data. In contrast, expression of CYP2B6 and CYP3A4 differed (>2.5-fold, p < 0.05) between cynomolgus (mfCAM, mfCHN, or mfIDN) and rhesus macaques, indicating greater differences in expression of P450 genes between the two lineages. Moreover, metabolic activities measured using 14 P450 substrates did not differ substantially (<1.5-fold) between mfCAM and mfCHN. These results suggest that gene expression profiles, including drug-metabolizing enzyme genes such as P450 genes, are similar in mfCAM, mfCHN, and mfIDN.
Drug Metabolism and Pharmacokinetics 12/2011; 27(3):307-16. DOI:10.2133/dmpk.DMPK-11-RG-133 · 2.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nonsteroidal anti-inflammatory drugs (NSAIDs) can cause serious gastrointestinal injury including jejunal/ileal mucosal ulceration, bleeding, and even perforation, in susceptible patients. The underlying mechanisms are largely unknown, but they are distinct from those related to gastric injury. Based on recent insights from experimental models, including genetics and pharmacology in rodents typically exposed to diclofenac, indomethacin, or naproxen, we propose a multiple-hit pathogenesis of NSAID enteropathy. The multiple-hits start with an initial pharmacokinetic determinant caused by vectorial hepatobiliary excretion and delivery of glucuronide NSAID or oxidative metabolite conjugates to the distal small intestinal lumen, where bacterial β-glucuronidase produces critical aglycones. The released aglycones are then taken up by enterocytes and further metabolized by intestinal cytochrome P450s to potentially reactive intermediates. The "first hit" is caused by the NSAID and/or oxidative metabolites that induce severe endoplasmic reticulum stress or mitochondrial stress and lead to cell death. The "second hit" is created by the significant subsequent inflammatory response that would follow such a first-hit injury. Based on these putative mechanisms, strategies have been developed to protect the enterocytes from being exposed to the parent NSAID and/or oxidative metabolites. Among these, a novel strategy already demonstrated in a murine model is the selective disruption of bacteria-specific β-glucuronidases with a novel small molecule inhibitor that does not harm the bacteria and that alleviates NSAID-induced enteropathy. Such mechanism-based strategies require further investigation, but provide potential avenues for the alleviation of the GI toxicity caused by multiple NSAID hits.
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