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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ABSTRACT:A liquid chromatography-tandem mass spectrometry method was developed to chromatographically separate the four stereoisomers of the active metabolite of prasugrel, R-138727, in human plasma after derivatization with bromomethoxyacetophenone to stabilize the molecule. This technique was designed to determine the relative contribution of each stereoisomer, based on statistical analyses of each stereoisomer's chromatographic peak areas. The methodology was validated and used for the analysis of clinical samples in which R-138727 had been derivatized at the time of blood collection. This technique can be useful to determine the ratios of stereoisomers in biological samples (e.g., plasma) especially in situations in which authentic standards of each individual stereoisomer are scarce or unavailable. In humans, the metabolic formation of R-138727 from prasugrel was found to be stereoselective, where 84% of R-138727 was present as RS and RR, the two most pharmacologically potent isomers, whereas the SR and SS enantiomers accounted for ϳ16%. The ratios of the R-138727 stereoisomers were consistent among subjects, regardless of the dose or time of sample collection or whether the blood was sampled after the first dose or after 4 weeks of therapy.Prasugrel, a novel thienopyridine antiplatelet compound, is a prodrug that is rapidly hydrolyzed in vivo to the pharmacologically inactive thiolactone R-95913, which is then metabolized by various cytochrome P450 enzymes to the pharmacologically active R-138727 ( Fig. 1) (Rehmel et al., 2006;. The pharmacological action of prasugrel is the result of R-138727 binding irreversibly to the P2Y 12 platelet adenosine diphosphate receptor, thus inhibiting platelet activation and aggregation (Jakubowski et al., 2006). R-138727 possesses two chiral centers, and it has been reported that the four stereoisomers possess differing antiplatelet activity, with the two stereoisomers with the R-configuration at the thiol group (R-125689 and R-125690) being the most potent (Hasegawa et al., 2005).Xenobiotics that contain multiple chiral centers (stereocenters) pose a challenging dimension to the overall drug development process. The chemistry and pharmacological significance of multiple stereocenters have been reviewed (Testa et al., 1993;Tomaszewski and Rumore, 1994;Reist et al., 1995;Van Miert, 2003). A compound with two chiral centers can exist in four stereoisomeric forms: RR, SS, RS, and SR. Enantiomers are stereoisomers that are mirror images (RR and SS are enantiomers as are SR and RS). Diastereomers are stereoisomers but are not mirror images, e.g., RR is a diastereomer of both RS and SR; likewise, SS is a diastereomer of both RS and SR. Such molecules pose a significant challenge for the bioanalytical chemist when analyzing plasma samples for pharmacokinetic analyses (Marzo and Heftman, 2002). Typically, "pure" standards of these stereoisomers are not available; therefore, the molecule is quantified as a single peak (achiral assay) or as doublets (RR/SS and RS/SR) using reverse-p...

A series of highly potent and specific fibrinogen receptor antagonists have been discovered and optimized through structural modification of the novel amidinoindole and benzofuran compounds, I and II. Systematic linker optimization afforded the amidinobenzofuran-containing inhibitor 29, which displayed an IC50 value of 250 nM in platelet aggregation assays. Attempts to enhance activity by modification of the beta-position of the beta-alanyl carboxylate group of 29 had only a modest effect on inhibitory activity in aggregation assays. Analogues prepared to enhance the activity by conformational restriction were also found to be equally or less potent. In contrast, modification at the alpha-position of the beta-alanyl carboxylate group resulted in the identification of extremely potent and novel amidinobenzofuran-containing derivatives 46-49. Reexamination of 5,6-bicyclic aromatic nucleus led to the further identification of amidinoindole- and amidinoindazole-containing derivatives 53-55. These analogues, 46-49 and 53-55, exhibited potent in vitro activity with IC50 values of 25-65 nM in platelet aggregation assays and an IC50 value of 2 nM in fibrinogen binding assays and demonstrated a selectivity of > 50,000-fold for GPIIb-IIIa versus the most closely related integrin, the vitronectin receptor, alpha v beta 3.