Simplifying the Execution of HepatoPac MetID Experiments: Metabolite Profile and Intrinsic Clearance Comparisons.

The HepatoPac micropatterned coculture (MPCC) hepatocyte system has been shown to be an effective tool to investigate the qualitative human and preclinical species' metabolite profiles of new drug candidates. However, additional improvements to the overall study conditions and execution, layout, and human-donor count could be made. To that end, we have evaluated several ways to increase the amount of data one can generate per MPCC plate and how to more efficiently execute a MPCC study for the purpose of metabolite generation. Herein, we compare a set of compounds using single- and 10-donor pooled human MPCC hepatocytes. Intrinsic clearance and mean metabolic activities assessed by diverse enzyme markers were comparable between the single- and 10-donor pool. We have confirmed that the generated metabolite profiles were indistinguishable between the single- and 10-donor pool and also that rat MPCC can be performed at 400 µl media volume, which greatly simplifies study execution. Additional tips for successful study execution are also described. SIGNIFICANCE STATEMENT: When using the HepatoPac micropatterned coculture (MPCC) system, sometimes simple experimental condition variables or problematic plate designs can hamper productive study execution. We evaluated conditions to increase the amount of data one can generate per MPCC plate and, perhaps more importantly, execute that study more efficiently with less likelihood of error. We describe some of our key learnings, provide an examination of enzyme activity levels and clearance values, and provide some recommendations to simplify the execution of a HepatoPac experiment.

Discovery and Characterization of (R)-6-Neopentyl-2-(pyridin-2-ylmethoxy)-6,7-dihydropyrimido[2,1-c][1,4]oxazin-4(9H)-one (PF-06462894), an Alkyne-Lacking Metabotropic Glutamate Receptor 5 Negative Allosteric Modulator Profiled in both Rat and Nonhuman Primates.

We previously observed a cutaneous type IV immune response in nonhuman primates (NHP) with the mGlu5 negative allosteric modulator (NAM) 7. To determine if this adverse event was chemotype- or mechanism-based, we evaluated a distinct series of mGlu5 NAMs. Increasing the sp3 character of high-throughput screening hit 40 afforded a novel morpholinopyrimidone mGlu5 NAM series. Its prototype, (R)-6-neopentyl-2-(pyridin-2-ylmethoxy)-6,7-dihydropyrimido[2,1-c][1,4]oxazin-4(9H)-one (PF-06462894, 8), possessed favorable properties and a predicted low clinical dose (2 mg twice daily). Compound 8 did not show any evidence of immune activation in a mouse drug allergy model. Additionally, plasma samples from toxicology studies confirmed that 8 did not form any reactive metabolites. However, 8 caused the identical microscopic skin lesions in NHPs found with 7, albeit with lower severity. Holistically, this work supports the hypothesis that this unique toxicity may be mechanism-based although additional work is required to confirm this and determine clinical relevance.

Application of a Micropatterned Cocultured Hepatocyte System To Predict Preclinical and Human-Specific Drug Metabolism.

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.