Predictions of CYPMediated Drug-Drug Interactions Using Cryopreserved Human Hepatocytes

consistent with an underestimation of in vitro inhibition potency in this system. In conclusion, the HHSHP system proved to be a simple, accurate predictor of DDIs for 3 major CYPs and superior to the protein-free approach

Interpatient heterogeneity in expression of CYP3A4 and CYP3A5 in small bowel: Lack of prediction by the erythromycin breath test

The CYP3A subfamily of cytochromes P450 metabolize many medications and environmental contaminants. CYP3A4 and, in 25% of patients, CYP3A5 seem to be the major CYP3A genes expressed in adult liver. Hepatic levels of CYP3A4 can be estimated by the erythromycin breath test and vary at least 10-fold among patients. CYP3A4 has also been shown to be present in small bowel where it is responsible for significant "first-pass" metabolism of orally administered substrates. However, it is not known whether there is significant interindividual variability in the intestinal expression of CYP3A4, or whether the liver and intestinal catalytic activities of CYP3A4 correlate within an individual. It is also not known whether CYP3A5 is expressed in the small intestine. To address these questions, we administered the erythromycin breath test to 20 patients and obtained biopsies from their small bowel. There was a 6-fold variation in CYP3A catalytic activity (midazolam hydroxylation), an 11-fold variation in CYP3A4 protein content, and an 8-fold variation in CYP3A4 mRNA content in intestinal biopsies. There was an excellent correlation between intestinal CYP3A4 protein level and catalytic activity (r = 0.86; p = 0.0001); however, neither parameter significantly correlated with hepatic CYP3A4 activity as measured by the erythromycin breath test result (r = 0.27; p = 0.24 and r = 0.33; p = 0.15, respectively). We also found that CYP3A5 protein was readily detectable in biopsies from 14 (70%) of the patients, indicating that CYP3A5 is commonly expressed in human small intestine

Cyp3A gene expression in human gut epithelium

CYP3A4, a major Phase I xenobiotic metabolizing enzyme present in liver, is also present in human small bowel epithelium where it appears to catalyse significant 'first pass' metabolism of some drugs. To determine whether CYP3A4 or the related enzymes CYP3A3, CYP3A5, and CYP3A7 are present in other regions of the digestive tract, we used CYP3A-specific antibodies to examine histological sections and epithelial microsomes obtained from a human organ donor. CYP3A-related proteins were detected in epithelia throughout the digestive tract and in gastric parietal cells, in pericentral hepatocytes, and in ductular cells of the pancreas. Immunoblot analysis suggested that the major CYP3A protein present in liver, jejunum, colon, and pancreas was CYP3A4 or CYP3A3, whereas CYP3A5 was the major protein present in stomach. Both CYP3A4 and CYP3A5 mRNA were detectable in all regions of the digestive tract using the polymerase chain reaction (PCR); however, only CYP3A4 could be detected by Northern blot analysis. CYP3A7 mRNA was consistently detected only in the liver by PCR and CYP3A3 mRNA was not detected in any of the tissues. We conclude that CYP3A4 and CYP3A5 are present throughout the human digestive tract and that differences in the expression of these enzymes may account for inter-organ differences in the metabolism of CYP3 A substrates

Acute Human Lethal Toxicity of Agricultural Pesticides: A Prospective Cohort Study

In a prospective cohort study of patients presenting with pesticide self-poisoning, Andrew Dawson and colleagues investigate the relative human toxicity of agricultural pesticides and contrast it with WHO toxicity classifications, which are based on toxicity in rats

The erythromycin breath test selectively measures P450IIIA in patients with severe liver disease

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/109969/1/cptclpt199217.pd

Flavin-containing monooxygenase-mediated Noxidation of the M1-muscarinic agonist xanomeline. Drug Metab Dispos 27

This paper is available online at http://www.dmd.org ABSTRACT: The involvement of flavin-containing monooxygenases (FMOs) in the formation of xanomeline N-oxide was examined in various human and rat tissues. Expressed FMOs formed xanomeline Noxide at a significantly greater rate than did expressed cytochromes P-450. Consistent with the involvement of FMO in the formation of xanomeline N-oxide in human liver, human kidney, rat liver, and rat kidney microsomes, this biotransformation was sensitive to heat treatment, increased at pH 8.3, and inhibited by methimazole. The latter two characteristics were effected to a lesser extent in human kidney, rat liver, and rat kidney microsomes than were observed in human liver microsomes, suggesting the involvement of a different FMO family member in this reaction in these tissues. As additional proof of the involvement of FMO in the formation of xanomeline N-oxide, the formation of this metabolite by a characterized human liver microsomal bank correlated with FMO activity. The FMO forming xanomeline N-oxide by human kidney microsomes exhibited a 20-fold lower K M (average K M ‫؍‬ 5.5 M) than that observed by the FMO present in human liver microsomes (average K M of 107 M). The involvement of an FMO in the formation of xanomeline N-oxide in rat lung could not be unequivocally demonstrated. These data and those in the literature suggest that the increased prevalence of N-oxidized metabolites of xanomeline after s.c. dosing as compared with oral dosing may be due to differences in the affinity of various FMO family members for xanomeline or to differences in exposure to xanomeline that these enzymes receive under different dosing regimens

Identification of the human cytochromes p450 responsible for in vitro formation of R- and S-norfhoxetine. Journal of pharmacology and experimental therapeutics

ABSTRACT The formation of R-and S-norfluoxetine was analyzed in vitro in human liver microsomes. Low apparent K m values for R-norfluoxetine formation of Յ8 M and S-norfluoxetine of Ͻ0.2 M were determined. R-Norfluoxetine formation rates in a characterized microsomal bank correlated with the catalytic activities for cytochrome P450 (CYP) 2D6, CYP2C9, and CYP2C8. Expressed CYP2C9, CYP2C19, and CYP2D6 formed R-norfluoxetine following incubation with 1 M Rfluoxetine and exhibited apparent K m values of 9.7, 8.5, and 1.8 M, respectively. Multivariate correlation analysis identified CYP2C9 and CYP2D6 as significant regressors with R-norfluoxetine formation. Antibodies to the CYP2C subfamily and CYP2D6 each exhibited moderate inhibition of Rnorfluoxetine formation. Therefore, CYP2D6 and CYP2C9 contribute to this biotransformation. At pharmacological concentrations of S-fluoxetine, S-norfluoxetine formation rates in the bank of microsomes were found to correlate only with CYP2D6 catalytic activity and only expressed CYP2D6 was found to be capable of forming S-norfluoxetine. Thus, it would appear that both CYP2D6 and CYP2C9 contribute to the formation of R-norfluoxetine, whereas only CYP2D6 is responsible for the conversion to S-norfluoxetine. Since the enantiomers of fluoxetine and norfluoxetine are inhibitors of CYP2D6, upon chronic dosing, the CYP2D6-mediated metabolism of the fluoxetine enantiomers would likely be inhibited, resulting in R-norfluoxetine formation being mediated by CYP2C9 and S-norfluoxetine formation being mediated by multiple high K m enzymes. Fluoxetine, a racemic mixture of R-and S-fluoxetine, is a selective serotonin reuptake inhibitor currently marketed for the treatment of depression and other disorders. R-fluoxetine was a drug candidate in development for use in psychiatric illness. The major route of metabolism of the enantiomers of fluoxetine is N-demethylation It has long been recognized that the enantiomers of fluoxetine and norfluoxetine are inhibitors of CYP2D6-mediated reactions. S-Fluoxetine and S-norfluoxetine are approximately 5-fold more potent in their ability to inhibit CYP2D6-mediated reactions than R-fluoxetine and R-norfluoxetine (K i values of 0.22, 0.31, 1.38, and 1.48 M, respectively) There have been a few studies in humans that have examined the clearance of the enantiomers of fluoxetine following both single and multiple doses of racemic fluoxetine. In a study examining the pharmacokinetics of a single dose of racemic fluoxetine a major role for CYP2D6 in S-fluoxetine metabolism was proposed, for S-fluoxetine clearance was 12-fold slower in poor metabolizers (PMs) of CYP2D6-mediated reactions than that observed in extensive metabolizers (EMs