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Translational and ADME Sciences Leadership Group Induction Working Group (IWG) presents an analysis on the time course for cytochrome P450 induction in primary human hepatocytes. Induction of CYP1A2, CYP2B6, and CYP3A4 was evaluated by seven IWG laboratories after incubation with prototypical inducers (omeprazole, phenobarbital, rifampicin, or efavirenz) for 6-72 hours. The effect of incubation duration and model-fitting approaches on induction parameters (Emax and EC50) and drug-drug interaction (DDI) risk assessment was determined. Despite variability in induction response across hepatocyte donors, the following recommendations are proposed: 1) 48 hours should be the primary time point for in vitro assessment of induction based on mRNA level or activity, with no further benefit from 72 hours; 2) when using mRNA, 24-hour incubations provide reliable assessment of induction and DDI risk; 3) if validated using prototypical inducers (>10-fold induction), 12-hour incubations may provide an estimate of induction potential, including characterization as negative if

11β-Hydroxysteroid dehydrogenase 1 (11β-HSD1) is distributed mainly in the human liver, with no detectable levels in the intestine or kidney, based on a newly developed proteomic approach. 11β-HSD1 is mostly membrane-bound and retained in the liver microsomal fraction. Interindividual variability of 11β-HSD1 is relatively low, with about a 3-fold difference. A significant correlation was not observed between various demographic variables (ethnicity, gender, age, weight, smoking, and alcohol use) and 11β-HSD1 protein expression or activity based on data from 31 donors. PF-915275 has been identified as a selective 11β-HSD1 inhibitor with minimal effects on carbonyl reductase 1 and major cytochrome P450 enzymes. 11β-HSD1 has been shown, for the first time, to be involved in doxorubicin metabolism, accounting for approximately 30% of doxorubicinol formation in human hepatocytes.

Fraction unbound (fu) of liver tissue, hepatocytes, and other cell types is an essential parameter used to estimate unbound liver drug concentration and intracellular free drug concentration. fu,liver and fu,cell are frequently measured in multiple species and cell types in drug discovery and development for various applications. A comparison study of 12 matrices for fu,liver and fu,cell of hepatocytes in five different species (mouse, rat, dog, monkey, and human), as well as fu,cell of Huh7 and human embryonic kidney 293 cell lines, was conducted for 22 structurally diverse compounds with the equilibrium dialysis method. Using an average bioequivalence approach, our results show that the average difference in binding to liver tissue, hepatocytes, or different cell types was within 2-fold of that of the rat fu,liver Therefore, we recommend using rat fu,liver as a surrogate for liver binding in other species and cell types in drug discovery. This strategy offers the potential to simplify binding studies and reduce cost, thereby enabling a more effective and practical determination of fu for liver tissues, hepatocytes, and other cell types. In addition, fu under hepatocyte stability incubation conditions should not be confused with fu,cell, as one is a diluted fu and the other is an undiluted fu Cell density also plays a critical role in the accurate measurement of fu,cell.

The purpose of this study was to determine whether functional purinergic P2 receptors are present in trabecular meshwork cells. The human trabecular cell line HTM-3 and cultured bovine trabecular cells were used to assess the effects of P2 agonists on intracellular Ca(2+) levels, extracellular signal-regulated kinase (ERK1/2) activation, and P2Y receptor expression. ATP, UTP, ADP, and 2-methyl-thio-adenosine triphosphate (2-MeS-ATP) each produced a concentration-dependent increase in intracellular Ca(2+) in bovine trabecular cells and the HTM-3 cell line. The addition of UDP did not produce any detectable rise in intracellular Ca(2+). Pretreatment with the P2Y(1) receptor antagonist 2'-deoxy-N(6)-methyladenosine-3',5'-diphosphate (MRS-2179) blocked the ADP- and 2-MeS-ATP-induced rise in intracellular Ca(2+). However, the ATP- or UTP-induced rise in intracellular Ca(2+) was not inhibited by MRS-2179 pretreatment. The addition of ADP, 2-MeS-ATP, ATP, or UTP were also found to activate the ERK1/2 signaling pathway. This activation of ERK1/2 was blocked by pretreatment with the mitogen-activated protein kinase kinase inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene (U-0126) or the protein kinase C inhibitor chelerythrine chloride, but not by MRS-2179. Analysis of mRNA from HTM-3 cells by reverse transcription-polymerase chain reaction revealed the expression of P2Y(1), P2Y(4), and P2Y(11) receptor subtypes. These data demonstrate that multiple P2Y receptors are present in trabecular cells. Our results are consistent with the idea that the mobilization of intracellular Ca(2+)results from the activation of P2Y(1) and P2Y(4) receptors, whereas the activation of the ERK1/2 pathway results from the activation of P2Y(4) receptors alone. However, a role for the P2Y(11) receptors in mobilization of Ca(2+), or activation of the ERK1/2 pathway, cannot be discounted.