Because drug-induced liver injury (DILI) remains a major reason for late-stage drug attrition, predictive assays are needed that can be deployed throughout the drug discovery process. Clinical DILI can be predicted with a sensitivity of ~50% and a false positive (FP) rate of ~5% using 24-h cultures of sandwich-cultured primary human hepatocytes and imaging of four cell injury endpoints (Xu et al., 2008). We hypothesized that long-term drug dosing in a functionally stable model of primary hepatocytes (micropatterned cocultures [MPCCs]) could provide for increased predictivity over short-term dosing paradigms. We used MPCCs with either primary human or rat hepatocytes to understand possible species differences along with standard endpoints (glutathione levels, ATP levels, albumin, and urea secretion) to test 45 drugs either known or not known to cause clinical DILI. Human MPCCs correctly detected 23 of 35 compounds known to cause DILI (65.7% sensitivity), with a FP rate of 10% for the 10 negative compounds tested. Rat MPCCs correctly detected 17 of 35 DILI compounds (48.6% sensitivity) and had a higher FP rate than human MPCCs (20 vs. 10%). For an additional 19 drugs with the most DILI concern, human MPCCs displayed a sensitivity of 100% when at least two hepatocyte donors were used for testing. Furthermore, MPCCs were able to detect relative clinical toxicities of structural drug analogs. In conclusion, MPCCs showed superiority over conventional short-term cultures for predictions of clinical DILI, and human MPCCs were more predictive for human liabilities than their rat counterparts.

Elevated levels of proinflammatory cytokines associated with infection and inflammation can modulate cytochrome P450 enzymes, leading to potential disease-drug interactions and altered small-molecule drug disposition. We established a human-derived hepatocyte-Kupffer cell (Hep:KC) coculture model to assess the indirect cytokine impact on hepatocytes through stimulation of KC-mediated cytokine release and compared this model with hepatocytes alone. Characterization of Hep:KC cocultures showed an inflammation response after treatment with lipopolysaccharide and interleukin (IL)-6 (indicated by secretion of various cytokines). Additionally, IL-6 exposure upregulated acutephase proteins (C-reactive protein, alpha-1-acid glycoprotein, and serum amyloid A2) and downregulated CYP3A4. Compared with hepatocytes alone, Hep:KC cocultures showed enhanced IL-1b-mediated effects but less impact from both IL-2 and IL-23. Hep:KC cocultures treated with IL-1b exhibited a higher release of proinflammatory cytokines, an increased upregulation of acute-phase proteins, and a larger extent of metabolic enzyme and transporter suppression. IC 50 values for IL-1b-mediated CYP3A4 suppression were lower in Hep:KC cocultures (98.0-144 pg/ml) compared with hepatocytes alone (IC 50 > 5000 pg/ml). Cytochrome suppression was preventable by blocking IL-1b interaction with IL-1R1 using an antagonist cytokine or an anti-IL-1b antibody. Unlike IL-1b, IL-6-mediated effects were comparable between hepatocyte monocultures and Hep:KC cocultures. IL-2 and IL-23 caused a negligible inflammation response and a minimal inhibition of CYP3A4. In both hepatocyte monocultures and Hep:KC cocultures, IL-2RB and IL-23R were undetectable, whereas IL-6R and IL-1R1 levels were higher in Hep:KC cocultures. In summary, compared with hepatocyte monocultures, the Hep:KC coculture system is a more robust in vitro model for studying the impact of proinflammatory cytokines on metabolic enzymes.

Binding of cocaine to the dopamine transporter (DAT) protein blocks synaptic dopamine clearance, triggering the psychoactive effects associated with the drug; the discrete drug-protein interactions, however, remain poorly understood. A longstanding postulate holds that cocaine inhibits DAT-mediated dopamine transport via competition with dopamine for formation of an ionic bond with the DAT transmembrane aspartic acid residue D79. In the present study, DAT mutations of this residue were generated and assayed for translocation of radiolabeled dopamine and binding of radiolabeled DAT inhibitors under identical conditions. When feasible, dopamine uptake inhibition potency and apparent binding affinity K i values were determined for structurally diverse DAT inhibitors. The glutamic acid substitution mutant (D79E) displayed values indistinguishable from wild-type DAT in both assays for the charge-neutral cocaine analog 8-oxa-norcocaine, a finding not supportive of the D79 "salt bridge" ligand-docking model. In addressing whether the D79 side chain contributes to the DAT binding sites of other portions of the cocaine pharmacophore, only inhibitors with modifications of the tropane ring C-3 substituent, i.e., benztropine and its analogs, displayed a substantially altered dopamine uptake inhibition potency as a function of the D79E mutation. A single conservative amino acid substitution thus differentiated structural requirements for benztropine function relative to those for all other classical DAT inhibitors. Distinguishing the precise mechanism of action of this DAT inhibitor with relatively low abuse liability from that of cocaine may be attainable using DAT mutagenesis and other structure-function studies, opening the door to rational design of therapeutic agents for cocaine abuse.Millions of individuals worldwide are addicted to cocaine, a public health crisis that also carries a substantial burden to society in the form of medical expenses, lost earnings, and increased crime associated with psychostimulant abuse. Although therapeutics including buprenorphine and methadone are clinically accessible to treat opiate abuse and addiction, no such Food and Drug Administration-approved medication is available for the treatment of cocaine addiction. Pharmacological and behavioral studies have established that the euphoric and addictive effects of cocaine are initiated by its binding to the dopamine transporter (DAT) protein, blocking clearance of dopamine from central nervous system synapses and thereby prolonging dopaminergic neurotransmission in brain areas associated with reward (Ritz et al., 1987). The DAT is therefore a logical target for development of medications for cocaine abuse. Elucidating the relationship between the DAT and its substrates and inhibitors is essential, especially regarding cocaine points of contact with the DAT protein relevant to its inhibition of dopamine uptake.Because dopamine and all classical DAT inhibitors possess a protonated nitrogen atom, a longstanding model for cocaine

Laboratory animal models are the industry standard for preclinical risk assessment of drug candidates. Thus, it is important that these species possess profiles of drug metabolites that are similar to those anticipated in human, since metabolites also could be responsible for biologic activities or unanticipated toxicity. Under most circumstances, preclinical species reflect human in vivo metabolites well; however, there have been several notable exceptions, and understanding and predicting these exceptions with an in vitro system would be very useful. Human micropatterned cocultured (MPCC) hepatocytes have been shown to recapitulate human in vivo qualitative metabolic profiles, but the same demonstration has not been performed yet for laboratory animal species. In this study, we investigated several compounds that are known to produce human-unique metabolites through CYP2C9, UGT1A4, aldehyde oxidase (AO), or N-acetyltransferase that were poorly covered or not detected at all in the selected preclinical species. To perform our investigation we used 24-well MPCC hepatocyte plates having three individual human donors and a single donor each of monkey, dog, and rat to study drug metabolism at four time points per species. Through the use of the multispecies MPCC hepatocyte system, the metabolite profiles of the selected compounds in human donors effectively captured the qualitative in vivo metabolite profile with respect to the human metabolite of interest. Human-unique metabolites that were not detected in vivo in certain preclinical species (normally dog and rat) were also not generated in the corresponding species in vitro, confirming that the MPCC hepatocytes can provide an assessment of preclinical species metabolism. From these results, we conclude that multispecies MPCC hepatocyte plates could be used as an effective in vitro tool for preclinical understanding of species metabolism relative to humans and aid in the choice of appropriate preclinical models.

Primary hepatocytes display functional and structural instability in standard monoculture systems. We have previously developed a model in which primary hepatocytes are organized in domains of empirically optimized dimensions and surrounded by murine embryonic fibroblasts (HepatoPac™). Here, we assess the long-term phenotype of freshly isolated and cryopreserved rat hepatocytes in a 96-well HepatoPac format. The viability, cell polarity (actin microfilaments, bile canaliculi), and functions (albumin, urea, Phase I/II enzymes, transporters) of fresh and cryopreserved rat hepatocytes were retained in HepatoPac at similar levels for at least 4 weeks as opposed to rapidly declining over 5 days in collagen/Matrigel™ sandwich cultures. Pulse or continuous exposure of rat HepatoPac to GW-7647, a selective agonist of PPARα, caused reproducible induction of CYP4A1 and 3-hydroxy-3-methylglutaryl-CoA synthase over 4 weeks. In conclusion, rat HepatoPac in a 96-well format can be used for chronic dosing of highly functional hepatocytes and assessment of perturbed hepatocellular pathways.