How important is intestinal cytochrome P450 3A metabolism?

Contains fulltext : 80456.pdf (publisher's version ) (Closed access)Cytochrome P450 3A (CYP3A) enzymes metabolize a wide variety of xenobiotics including many drugs. Because CYP3A is localized in both the liver and intestine, it can make a major contribution to the presystemic elimination of substrate drugs after oral administration ('first-pass metabolism'). However, assessments of the relative importance of intestinal versus hepatic CYP3A-mediated first-pass metabolism have been difficult to make and are subject to extensive discussion. To assess systematically the relative contributions of the intestine and liver to first-pass metabolism in vivo, Cyp3a knockout mice expressing human CYP3A4 in the liver or intestine have recently been generated. Analysis of these mice, together with previous observations in humans, substantiates that intestinal CYP3A4 can operate independently of the liver as a highly efficient metabolic barrier during the uptake of various drugs from the intestine. We expect that the insights obtained with these mouse models will contribute to the development of better oral drugs and treatment regimens

Topological role of cytochrome P450 2D6 active site residues

Recent reports have identified Phe120, Asp301, Thr309, and Glu216 as important residues in cytochrome P450 2D6 (CYP2D6) substrate binding and catalysis. Complementary homology models have located these amino acids within the binding pocket of CYP2D6 and in the present study we have used aryldiazenes to test these models and gain further insight in the role these amino acids have in maintaining the integrity of the active site cavity. When Phe120 was replaced to alanine, there was a significant increase in probe migration to pyrrole nitrogens C and D, in agreement with homology models which have located the phenyl side-chain of Phe120 above these two pyrrole rings. No changes in topology were observed with the D301Q mutant, supporting claims that in this mutant the electrostatic interactions with the B/C-loop are largely maintained and the loop retains its native orientation. The T309V mutation resulted in significant topological alteration suggesting that, in addition to its potential role in dioxygen activation, Thr309 plays an important structural role within the active site crevice. Replacement of Ile106 with Glu, engineered to cause electrostatic repulsion with Glu216, had a profound topological effect in the higher region within the active site cavity and impaired the catalytic activity towards CYP2D6 probe substrates. © 2006 Elsevier Inc. All rights reserved

Intestinal cytochrome P450 3A plays an important role in the regulation of detoxifying systems in the liver.

Contains fulltext : 80111.pdf (publisher's version ) (Closed access)CYP3A4 is an important xenobiotic metabolizing enzyme. We previously found that CYP2C55 is highly up-regulated in Cyp3a(-/-) mice. Here, we have further investigated the mechanism of regulation of CYP2C55 and other detoxifying systems in Cyp3a(-/-) mice. Induction studies with prototypical inducers demonstrated an important role for the nuclear receptors PXR and CAR in the up-regulation of CYP2C55. Subsequent diet-switch experiments revealed that food-derived xenobiotics are primarily responsible for the increased induction of CYP2C55, as well as of several other primary detoxifying systems in Cyp3a(-/-) mice. Our data suggest that CYP3A normally metabolizes food-derived activators of PXR and/or CAR, explaining the increased levels of such activators in Cyp3a(-/-) mice and subsequent up-regulation of a range of detoxifying systems. Interestingly, our studies with tissue-specific CYP3A4 transgenic Cyp3a(-/-) mice revealed that not only hepatic but also intestinal expression of CYP3A4 could reduce the hepatic expression of detoxifying systems to near wild-type levels. Apparently, intestinal CYP3A4 can limit the hepatic exposure to food-derived activators of nuclear receptors, thereby regulating the expression of a range of detoxifying systems in the liver. This broad biological effect further emphasizes the importance of intestinal CYP3A activity and could have profound implications for the prediction of drug exposure

Molecular modeling-guided site-directed mutagenesis of cytochrome P450 2D6

Cytochrome P450 (CYP) 2D6 is one of the most important drug metabolizing enzymes and the rationalization and prediction of potential CYP2D6 substrates is therefore advantageous in the discovery and development of new drugs. Experimentally, the active site of CYP2D6 can be probed by site directed mutagenesis studies. Such studies can be designed from structural models of enzyme-substrate complexes. Modeling approaches can subsequently be used to rationalize the observed effect of mutations on metabolism and inhibition. The current paper will present the construction, refinement and validation of the CYP2D6 homology model used in our laboratory for the prediction and rationalisation of CYP2D6 substrate metabolism and CYP2D6-ligand interactions. The model could explain reported site-directed mutagenesis data (for example, mutation of E216 and D301). Furthermore, based on the model, new CYP2D6 mutants were constructed and studied in our lab, and also for these mutants a rationalization of experimentally observed characteristics could be achieved (I106E, F120A, T309V, F483A). CYP2D6-substrate interaction fingerprint analysis of docked substrates in our homology model suggests that several other active site residues are probably interacting with ligands as well, opening the way for further mutagenesis studies. Our homology model was found to agree with most of the details of the recently solved substrate-free CYP2D6 crystal structure [Rowland et al. J. Biol. Chem. 2006, 281, 7614-7622]. Structural differences between the homology model and crystal structure were the same differences observed between substrate-free and substrate-bound structures of other CYPs, suggesting that these conformational changes are required upon substrate binding. The CYP2D6 crystal structure further validates our homology modeling approach and shows that computational chemistry is a useful and valuable tool to provide models for substrate-bound complexes of CYPs which give insight into CYP-ligand interactions. This information is essential for successful pre-experimental virtual screening, as well as accurate hypothesis generation for in vitro studies in drug discovery and development. © 2007 Bentham Science Publishers Ltd

Evidence of CYP3A Allosterism In Vivo: Analysis of Interaction Between Fluconazole and Midazolam

The allosteric effect of fluconazole (effector) on the formation of 1’-hydroxymidazolam (1’-OH-MDZ) and 4-hydroxymidazolam (4-OH-MDZ) from the CYP3A4/5 substrate, midazolam (MDZ), was examined in healthy volunteers. Following pre-treatment of fluconazole, AUC(4-OH)/AUC(MDZ) increased 35–62%, while AUC(1’-OH)/AUC(MDZ) decreased 5–37%; AUC(1’-OH)/AUC(4-OH) ratio decreased 46–58% by fluconazole and had no association with CYP3A5 genotype. 1’-OH-MDZ formation in vitro was more susceptible than 4-OH-MDZ formation to inhibition by fluconazole. Fluconazole decreased the intrinsic formation clearance ratio of 1’-OH-MDZ/4-OH-MDZ to an extent that was quantitatively comparable to in vivo observations. The elimination clearance of midazolam metabolites appeared unaffected by fluconazole. This study demonstrated that fluconazole alters midazolam product formation both in vivo and in vitro in a manner consistent with an allosteric interaction. The 1'-OH-MDZ/4-OH-MDZ ratio may serve as a biomarker of such interactions between midazolam, CYP3A4/5 and other putative effectors

Effect of ABCC2 (MRP2) Transport Function on Erythromycin Metabolism

The macrolide antiobiotic erythromycin undergoes extensive hepatic metabolism and is commonly used as a probe for CYP3A4 activity. Using a transporter screen, erythromycin was identified as a substrate for the transporter ABCC2 (MRP2) and its murine ortholog, Abcc2. Since these proteins are highly expressed on the biliary surface of hepatocytes, we hypothesized that impaired Abcc2 function may influence the rate of hepatobiliary excretion and thereby enhance erythromycin metabolism. Using Abcc2-knockout mice, we found that erythromycin metabolism was significantly increased, whereas murine Cyp3a protein expression and microsomal Cyp3a activity were not affected by Abcc2 deficiency. Next, in a cohort of 108 human subjects, we observed that homozygosity for a common reduced-function variant in ABCC2 (rs717620) was also linked with increased erythromycin metabolism, but was not correlated with the clearance of midazolam. These results suggest that impaired ABCC2 function can alter erythromycin metabolism independently of changes in CYP3A4 activity

A Perspective on Efflux Transport Proteins in the Liver

Detailed knowledge regarding the influence of hepatic transport proteins on drug disposition has advanced at a rapid pace over the past decade. Efflux transport proteins located in the basolateral and apical (canalicular) membranes of hepatocytes play an important role in the hepatic elimination of many endogenous and exogenous compounds, including drugs and metabolites. This review focuses on the role of these efflux transporters in hepatic drug excretion. The impact of these proteins as underlying factors for disease is highlighted, and the importance of hepatic efflux proteins in the efficacy and toxicity of drugs is discussed. In addition, a brief overview of methodology to evaluate the function of hepatic efflux transport proteins is provided. Current challenges in predicting the impact of altered efflux protein function on systemic, intestinal and hepatocyte exposure to drugs and metabolites are highlighted