ABSTRACT:The human cytochromes P450 (P450) CYP3A contribute to the biotransformation of 50% of oxidatively metabolized drugs. The predominant hepatic form is CYP3A4, but recent evidence indicates that CYP3A5 contributes more significantly to the total liver CYP3A than was originally thought. CYP3A7 is the major fetal form and is rarely expressed in adults. To compare the metabolic capabilities of CYP3A forms for 10 substrates, incubations were performed using a consistent molar ratio (1:7:9) of recombinant CYP3A, P450 reductase, and cytochrome b5. A wide range of substrate concentrations was examined to determine the best fit to kinetic models for metabolite formation. In general, K m or S 50 values for the substrates were 3 to 4 times lower for CYP3A4 than for CYP3A5 or CYP3A7. For a more direct comparison of these P450 forms, clearance to the metabolites was determined as a linear relationship of rate of metabolite formation for the lowest substrate concentrations examined. The clearance for 1-hydroxy midazolam formation at low substrate concentrations was similar for CYP3A4 and CYP3A5. For CYP3A5 versus CYP3A4, clearance values at low substrate concentrations were 2 to 20 times lower for the other biotransformations. The clearance values for CYP3A7-catalyzed metabolite formation at low substrate concentrations were substantially lower than for CYP3A4 or CYP3A5, except for clarithromycin, 4-OH triazolam, and N-desmethyl diltiazem (CYP3A5 Ϸ CYP3A7). The CYP3A forms demonstrated regioselective differences in some of the biotransformations. These results demonstrate an equal or reduced metabolic capability for CYP3A5 compared with CYP3A4 and a significantly lower capability for CYP3A7.

Platelet aggregation and activation are serious concerns for patients who undergo percutaneous coronary intervention and stent placement. The underlying mechanism of platelet aggregation is mediated through two G protein-coupled P2 receptors, P2Y 1 and P2Y 12 (Gachet, 2001). P2Y 1 activation leads to a transient aggregation, whereas P2Y 12 activation maintains a sustained aggregation. To reduce platelet aggregation, the development of P2Y 12 -selective inhibitors has yielded the thienopyridine prodrugs, which include ticlopidine, clopidogrel (structures available in Farid et al., 2008), and prasugrel ( Fig. 1), a novel thienopyridine currently in clinical development.Although the active metabolites for prasugrel and clopidogrel have equipotency at the P2Y 12 receptor in vitro (Sugidachi et al., 2007), p.o. administered prasugrel is 10 and 100 times more effective on an equal-dose basis in inhibiting platelet aggregation than clopidogrel and ticlopidine, respectively (Niitsu et al., 2005). Clopidogrel and prasugrel differ markedly in the biotransformation pathways leading to their activation. Prasugrel (Fig. 1) has a single dominant metabolic pathway leading to the active metabolite (Farid et al., 2007a). However, clopidogrel has two competing metabolic pathways for the parent compound, with the major pathway leading to the formation of an inactive metabolite, clopidogrel carboxylic acid derivative (Caplain et al., 1999). The clopidogrel carboxylic acid derivative is formed through ester hydrolysis by the human carboxylesterase (hCE) 1 (Tang et al., 2006). The minor pathway in clopidogrel metabolism yielding the active metabolite requires two sequential steps of cytochrome P450 (P450) biotransformation (Kurihara et al., 2005), whereas prasugrel bioactivation requires the hydrolysis of the ester and then oxidation of the formed thiolactone, R-95913 (Farid et al., 2007a) (Fig. 1), to form the active metabolite of prasugrel. The oxidation of R-95913 has been shown to be mediated by several P450 enzymes but primarily by CYP3A and CYP2B6 (Rehmel et al., 2006).The carboxylesterases are a multigene family that hydrolyze compounds containing an ester, amide, or thioester linkage. Carboxylesterases are broadly expressed throughout the body with two major Article, publication date, and citation information can be found at

ABSTRACT:The carboxylesterases (CESs) are a family of serine hydrolases that hydrolyze compounds containing an ester, amide, or thioester. In humans, two dominant forms, CES1 and CES2, are highly expressed in organs of first-pass metabolism and play an important role in xenobiotic metabolism. The current study was conducted to better understand species-related differences in substrate selectivity and tissue expression of these enzymes. To elucidate potential similarities and differences among these enzymes, a series of 4-nitrophenyl esters and a series of gemcitabine prodrugs were evaluated using enzyme kinetics as substrates of expressed and purified CESs from beagle dog, cynomolgus monkey, and human genes. For the substrates examined, human and monkey CES2 more efficiently catalyzed hydrolysis compared with CES1, whereas CES1 was the more efficient enzyme in dog. Quantitative real-time polymerase chain reaction and Western blot analyses indicate that the pattern of CES tissue expression in monkey is similar to that of human, but the CES expression in dog is unique, with no detectable expression of CES in the intestine. Loperamide, a selective human CES2 inhibitor, was also found to be a CES2-selective inhibitor in both dog and monkey. This is the first study to examine substrate specificity among dog, human, and monkey CESs.

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