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    • Similar to GPR A activation of G

    2022-08-15

    Similar to GPR109A, activation of G-protein-coupled receptor 81 (GPR81, also called HCAR1 (hydroxycarboxylic Glucose Uptake Fluorometric Assay Kit receptor 1)) by lactate suppresses lipolysis (Fig. 1), suggesting GPR81 to be a potential drug target for treating T2DM (Boyd et al., 1974, Cai et al., 2008, Gold et al., 1963, Houghton et al., 1971, Liu et al., 2009). GPR81 is primarily expressed in adipose tissue with no evidence of expression in epidermal Langerhans cells (Ge et al., 2008, Wise et al., 2003), indicating that a GPR81 agonist would not confer the flush associated with GPR109A. Indeed, selective GPR81 agonists indicating separation between lipolysis suppression and flush have recently been reported (Sakurai et al., 2014). GPR81 is 52% identical to GPR109A at the amino-acid level and belongs to the same subfamily of receptors as GPR109A and GPR109B (Ahmed et al., 2009, Blad et al., 2011). The high degree of receptor sequence identity suggests that compounds may have dual activity, and GPR109A agonism needs to be monitored during development.Partial agonism of GPR109A has been shown to be a way to avoid flush (Lai et al., 2008), but anti-lipolytic effects still seem to be transient. An in vitro-in vivo correlation (IVIVC) between receptor potency and in vivo effect is fundamental to efficiently design new compounds in a chemical series by in vitro screening (Yamaguchi et al., 2013, Yu et al., 2006). A sufficiently good IVIVC confers reductions in cost and number of in vivo experiments. When dealing with multiple-target in vivo pharmacology where the targets are studied one at a time in vitro, the relationship between in vitro and in vivo effects may become difficult to discern. To some degree, this may be overcome by complex in vitro or ex vivo methods that increase confidence in the in vivo effect of the compounds before proceeding to in vivo experiments, but at the expense of being more resource-intensive. In this study, we explore different mathematical models for describing the IVIVC of a preclinical dataset of 12 different dual GPR109A/GPR81 agonists. The objective is to establish a predictive model of in vivo lipolysis suppression in the rat based on in vitro potency data. First, a nonlinear mixed effects pharmacokinetic and pharmacodynamic (PKPD) model was applied to a longitudinal data set of rat in vivo lipolysis (Fig. 2, path 1). From that model, in vivo EC50 values of NEFA inhibition were derived. These estimates subsequently served as the targets for a number of different in vitro-in vivo prediction models. Either primary adipocyte lipolysis potency data (Fig. 2, path 2), or in vitro cell assay potency data for GPR81 (Fig. 2, path 3) or GPR109A (Fig. 2, path 4) were used to fit simple linear regression models of in vivo EC50. The in vitro potency data were acquired from overexpressing cell systems, i.e., cells that artificially express high levels of either GPR81 or GPR109A. The assay works by measuring decreases in cAMP, which occurs when GPR81 or GPR109A become activated and inhibits the production of cAMP via their effect on adenylyl cyclase (see Fig. 1). We also compared three simple methods of combining the in vitro cell assay data. Current biological understanding suggests that GPR81 and GPR109A function as metabolic sensors activated by different endogenous mediators, and that activation of either GPR81 or GPR109A is likely sufficient to deactivate the cAMP pathway (Cai et al., 2008, Ahmed, 2011). Based on this, a logical-circuit OR-model was our primary candidate (Fig. 2, path 5a). To challenge this model we also considered a logical-circuit AND-model (Fig. 2, path 5b), and a multivariate linear regression model (Fig. 2, path 5c). We demonstrate that combining the in vitro data from the two cell assays improves prediction of in vivo effects for all three models, to the same level or better than using primary adipocyte data. Among these models the OR-model is most suitable for future in vitro screening due to its mechanistically inspired structure. Not only does this model provide adequate predictions of in vivo EC50 but it also predicts the potential for a compound to selectively target GPR81. Finally, as an illustration of how to apply the OR-model in practice, we used it to predict in vivo potencies for 1651 other compounds whose potencies so far only have been determined in the two in vitro cell assays.