ABSTRACT:The aim of this study was to evaluate a unified method for predicting human in vivo intrinsic clearance (CL int, in vivo ) and hepatic clearance (CL h ) from in vitro data in hepatocytes and microsomes by applying the unbound fraction in blood (fu b ) and in vitro incubations (fu inc ). Human CL int, in vivo was projected using in vitro data together with biological scaling factors and compared with the unbound intrinsic clearance (CL int, ub, in vivo ) estimated from clinical data using liver models with and without the various fu terms. For incubations conducted with fetal calf serum (n ‫؍‬ 14), the observed CL int, in vivo was modeled well assuming fu inc and fu b were equivalent. CL int, ub, in vivo was predicted best using both fu b Existing methods for the prediction of drug clearance in humans involve the use of in vitro human metabolic stability (intrinsic clearance, CL int ) data (Iwatsubo et al., 1997), consideration of preclinical animal data (Boxenbaum, 1982), or a combination of these approaches (Lave et al., 1997a;Naritomi et al., 2001). In vitro drug metabolism kinetic parameters can provide an estimate of in vivo CL int via "scaling" with established biological scaling factors (SFs) e.g., hepatocellularity for isolated hepatocytes, or a SF for microsomes based on incomplete microsomal recovery from human liver tissue using the cytochrome P450 (P450) content in homogenate and microsomes (Houston, 1984). CL int may subsequently be used to provide an estimate of hepatic clearance (CL h ) using several liver models (Houston, 1984;Ito and Houston, 2004).To date, the more extensive analyses of human clearance predictions have concentrated on P450 substrates, and data have therefore been generated in human liver microsomes (Iwatsubo et al., 1997;Obach, 1999;Naritomi et al., 2001). In general, these studies have been less comprehensive in the range of approaches investigated, with only occasional attention given to chemical class (Obach et al., 1997). Interestingly, these reports have also assessed the ability to predict human CL h rather than the more fundamental parameter, CL int , as advocated initially (Houston, 1984;Ito and Houston, 2004). Some controversy also still exists over use of fu inc , with some laboratories having suggested that fu inc and fu b may cancel, negating their inclusion in liver models (Obach et al., 1997). Recent reports have challenged this assumption (Obach, 1999;Austin et al., 2002) and perhaps suggest that consideration of CL h rather than CL int, in vivo may desensitize such analyses, particularly to errors associated with higher enzyme activities (Ito and Houston, 2004).The aim of this study was to investigate direct in vitro-in vivo scaling of human in vitro data generated in hepatocytes and microsomes for predicting human clearance in vivo by applying recently described models for estimating fu inc (Austin et al., 2002(Austin et al., , 2005. To provide a more mechanistic insight, in vitro human CL int data were compiled from recent in-house and published stud...

P450 cytochromes (P450) catalyze many types of oxidative reactions, including the conversion of olefinic substrates to epoxides by oxygen insertion. In some instances epoxidation leads to the formation of products of physiological importance from naturally occurring substrates, such as arachidonic acid, and to the toxicity, carcinogenicity, or teratogenicity of foreign compounds, including drugs. In the present mechanistic study, the rates of oxidation of model olefins were determined with N-terminal-truncated P450s 2B4 and 2E1 and their respective mutants in which the threonine believed to facilitate proton delivery to the active site was replaced by alanine. Styrene epoxidation, cyclohexene epoxidation and hydroxylation to give 1-cyclohexene-3-ol, and cis-or trans-butene epoxidation (without isomerization) and hydroxylation to give 2-butene-1-ol were all significantly decreased by the 2B4 T302A mutation. Reduced proton delivery in this mutant is believed to interfere with the activation of dioxygen to the oxenoid species, as shown earlier by decreased hydroxylation of several substrates and enhanced aldehyde deformylation via a presumed peroxo intermediate. Of particular interest, however, the T303A mutation of P450 2E1 resulted in enhanced epoxidation of all of the model olefins along with decreased allylic hydroxylation of cyclohexene and butene. These results and a comparison of the ratios of the rates of epoxidation and hydroxylation support the concept that two different species with electrophilic properties, hydroperoxo-iron (FeO 2 H) 3؉ and oxenoid-iron (FeO) 3؉ , can effect olefin epoxidation. The ability of cytochrome P450 to use several different active oxidants generated from molecular oxygen may help account for the broad reaction specificity and variety of products formed by this versatile catalyst.

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 .

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