ABSTRACT:The intrinsic clearances (CL int ) of 50 neutral and basic marketed drugs were determined in fresh human hepatocytes and the data used to predict human in vivo hepatic metabolic clearance (CL met ). A statistically significant correlation between scaled CL met and actual CL met was observed (r 2 ‫؍‬ 0.48, p < 0.05), and for 73% of the drugs studied, scaled clearances were within 2-fold of the actual clearance. These data have shown that CL int data generated in human hepatocytes can be used to provide estimates of human hepatic CL met for both phase I and phase II processes. In addition, the utility of commercial and in-house cryopreserved hepatocytes was assessed by comparing with data derived from fresh cells. A set of 14 drugs metabolized by the major human cytochromes P450 (P450s) (CYP1A2, 2C9, 2C19, 2D6, and 3A4) and uridine diphosphate glucuronosyltransferases (UGT1A1, 1A4, 1A9, and 2B7) have been used to characterize the activity of freshly isolated and cryopreserved human and dog hepatocytes. The cryopreserved human and dog cells retained on average 94% and 81%, respectively, of the CL int determined in fresh cells. Cryopreserved hepatocytes retain their full activity for more than 1 year in liquid N 2 and are thus a flexible resource of hepatocytes for in vitro assays. In summary, this laboratory has successfully cryopreserved human and dog hepatocytes as assessed by the turnover of prototypic P450 and UGT substrates, and both fresh and cryopreserved human hepatocytes may be used for the prediction of human hepatic CL met .

The glucuronidation of a number of commonly used hepatic uridine diphosphate glucuronosyltransferase drug substrates has been studied in human tissue microsomes. Prediction of in vivo hepatic drug glucuronidation from liver microsomal data yielded a consistent 10-fold underprediction. Consideration of protein binding was observed to be pivotal when predicting in vivo glucuronidation for acid substrates. Studies using human intestinal microsomes demonstrated the majority of drugs to be extensively glucuronidated such that the intrinsic clearance (CL int ) of ethinylestradiol (CL int ϭ 1.3 l/min/mg) was twice that obtained using human liver microsomes (CL int ϭ 0.7 l/min/ mg). The potential extrahepatic in vivo glucuronidation was calculated for a range of drug substrates from human microsomal data. These results indicate the contribution of intestinal drug glucuronidation to systemic drug clearance to be much less than either hepatic or renal glucuronidation. Therefore, data obtained with intestinal microsomes may be misleading in the assessment of the contribution of this organ to systemic glucuronidation. The use of hepatocytes to assess metabolic stability for drugs predominantly metabolized by glucuronidation was also investigated. Metabolic clearances for a range of drugs obtained using fresh preparations of human hepatocytes predicted accurately hepatic clearance reported in vivo. The use of cryopreserved hepatocytes as an in vitro tool to predict in vivo metabolism was also assessed with an excellent correlation obtained for a number of extensively glucuronidated drugs (R 2 ϭ 0.80, p Ͻ 0.001).

ABSTRACT:The wealth of information that has emerged in recent years detailing the substrate specificity of hepatic transporters necessitates an investigation into their potential role in drug elimination. Therefore, an assay in which the loss of parent compound from the incubation medium into hepatocytes ("media loss" assay) was developed to assess the impact of hepatic uptake on unbound drug intrinsic clearance in vivo (CL int ub in vivo ). Studies using conventional hepatocyte incubations for a subset of 36 AstraZeneca new chemical entities (NCEs) resulted in a poor projection of CL int ub in vivo (r 2 ‫؍‬ 0.25, p ‫؍‬ 0.002, average fold error ‫؍‬ 57). This significant underestimation of CL int ub in vivo suggested that metabolism was not the dominant clearance mechanism for the majority of compounds examined. However, CL int ub in vivo was described well for this dataset using an initial compound "disappearance" CL int obtained from media loss assays (r 2 ‫؍‬ 0.72, p ‫؍‬ 6.3 ؋ 10 ؊11 , average fold error ‫؍‬ 3). Subsequent studies, using this method for the same 36 NCEs, suggested that the active uptake into human hepatocytes was generally slower (3-fold on average) than that observed with rat hepatocytes. The accurate prediction of human CL int ub in vivo (within 4-fold) for the marketed drug transporter substrates montelukast, bosentan, atorvastatin, and pravastatin confirmed further the utility of this assay. This work has described a simple method, amenable for use within a drug discovery setting, for predicting the in vivo clearance of drugs with significant hepatic uptake. Prentis et al. (1988) highlighted the importance of drug metabolism and pharmacokinetics (DMPK) in reducing the attrition of candidate drugs in early clinical trials. This has subsequently led to a realignment of DMPK within the drug discovery process and increased use of a plethora of high-throughput screens early in lead optimization (Riley and Grime, 2004).Arguably one of the most critical tasks within DMPK is the accurate prediction of in vivo clearance from in vitro data (Riley, 2001). Although the theory behind this process was published almost 30 years ago (Rane et al., 1977), the potential impact on drug discovery was not fully appreciated until the review by Houston (1994). Subsequently, hepatic microsomes and hepatocytes prepared from both preclinical species and humans have been used to predict in vivo clearance successfully (Obach, 1999;Soars et al., 2002;Ito and Houston, 2004;McGinnity et al., 2004;Ito and Houston, 2005;Riley et al., 2005). However, recent studies with hepatocytes have shown a significant underprediction of in vivo clearance for a distinct set of drugs, which has been attributed in some cases to hepatic uptake Soars et al., 2007).During the last decade a rapid increase has been seen in the number of publications in which researchers have investigated the role of hepatic uptake in drug clearance (Mizuno et al., 2003;Shitara et al., 2006). Perhaps the most important superfamily of enzymes for the hepatic up...

This article is available online at http://dmd.aspetjournals.org ABSTRACT:Traditionally, the Michaelis-Menten equation has been used to determine kinetic parameters for in vitro glucuronidation assays. Recently, estradiol-3-glucuronide formation was shown to exhibit non-Michaelis-Menten kinetics consistent with autoactivation. A concern with the observation of nontraditional kinetics is that they may result as an artifact of the incubation conditions. To examine this concern, the formation of estradiol-3-glucuronide was investigated using human liver microsomes prepared by two different methods, a range of assay conditions, and activation by alamethecin, sonication, or Brij 58 (polyoxyethylene monocetyl ether). Interestingly, holding the other assay components constant, estradiol-3-glucuronide formation was up to 2.5-fold greater using microsomes prepared in phosphate buffer compared with those prepared in sucrose. Incubations activated by alamethecin consistently exhibited the highest rates of estradiol glucuronidation versus the other activators. Furthermore, estradiol-3-glucuronidation exhibited autoactivation kinetics in all of the conditions investigated (n ‫؍‬ 1.2-1.7). Nontraditional kinetics were also observed when other UGT1A1 substrates such as ethinylestradiol, buprenorphine, and anthraflavic acid were studied with both human liver microsomes and recombinant UGT1A1. Naphthol, propofol, morphine, and androstanediol were used as probe UGT substrates selective for UGT1A6, UGT1A9, UGT2B7, and UGT2B15, respectively. Of these substrates, only androstanediol exhibited nontraditional kinetics using human liver microsomes. In conclusion, the Hill and/or Michaelis-Menten equations should be used to fit kinetic data to obtain an accurate assessment of in vitro glucuronidation.

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