The elucidation of species-specific receptor pharmacology: a case study using subtype selective para- and meta-carborane estrogen receptor agonists.

Estrogen receptors are essential pharmacological targets for treating hormonal disorders and estrogen-dependent malignancies. Selective activation of estrogen receptor (ER) ß is hypothesized to provide therapeutic benefit with reduced risk of unwanted estrogenic side-effects associated with ERα activity. However, activating ERß without activating α is challenging due to the high sequence and structural homology between the receptor subtypes. We assessed the impact of structural modifications to the parent compound OSU-ERß-12 on receptor subtype binding selectivity using cell-free binding assays. Functional selectivity was evaluated by transactivation in HEK-293 cells overexpressing human or murine estrogen receptors. In vivo selectivity was examined through the uterotrophic effects of the analogs after oral administration in estrogen-naïve female mice. Furthermore, we evaluated the in vivo pharmacokinetics of the analogs following single dose IV and oral administration. Regarding selectivity, a single compound exhibited greater functional selectivity than OSU-ERß-12 for human ERß. However, like others in the meta-carborane series, its poor in vivo pharmacokinetics limit its suitability for further development. Surprisingly, and at odds with their pharmacokinetic and in vitro human activity data, most analogs potently induced uterotrophic effects in estrogen-naïve female mice. Further investigation of activity in HEK293 cells expressing murine estrogen receptors revealed species-specific differences in the ER-subtype selectivity of these analogs. Our findings highlight species-specific receptor pharmacology and the challenges it poses to characterizing developmental therapeutics in preclinical species. Significance Statement This study investigates para- and meta-substituted carborane analogs targeting estrogen receptors, revealing the greater selectivity of carborane analogs for human ERß compared to the mouse homolog. These findings shed light on the intricacies of using preclinical species in drug development to predict human pharmacology. The report also provides insights for the refinement and optimization of carborane analogs as potential therapeutic agents for estrogen-related disease states.

Decoupling FcRn and tumor contributions to elevated immune checkpoint inhibitor clearance in cancer cachexia.

High baseline clearance of immune checkpoint inhibitors (ICIs), independent of dose or systemic exposure, is associated with cachexia and poor outcomes in cancer patients. Mechanisms linking ICI clearance, cachexia and ICI therapy failure are unknown. Here, we evaluate in four murine models and across multiple antibodies whether altered baseline catabolic clearance of administered antibody requires a tumor and/or cachexia and whether medical reversal of cachexia phenotype can alleviate altered clearance. Key findings include mild cachexia phenotype and lack of elevated pembrolizumab clearance in the MC38 tumor-bearing model. We also observed severe cachexia and decreased, instead of increased, baseline pembrolizumab clearance in the tumor-free cisplatin-induced cachexia model. Liver Fcgrt expression correlated with altered baseline catabolic clearance, though elevated clearance was still observed with antibodies having no (human IgA) or reduced (human H310Q IgG1) FcRn binding. We conclude cachexia phenotype coincides with altered antibody clearance, though tumor presence is neither sufficient nor necessary for altered clearance in immunocompetent mice. Magnitude and direction of clearance alteration correlated with hepatic Fcgrt, suggesting changes in FcRn expression and/or recycling function may be partially responsible, though factors beyond FcRn also contribute to altered clearance in cachexia.

Murine cancer cachexia models replicate elevated catabolic pembrolizumab clearance in humans.

BACKGROUND: Monoclonal antibody (mAb) immune checkpoint inhibitor (ICI) therapies have dramatically impacted oncology this past decade. However, only about one-third of patients respond to treatment, and biomarkers to predict responders are lacking. Recent ICI clinical pharmacology data demonstrate high baseline drug clearance (CL0) significantly associates with shorter overall survival, independent of ICI exposure, in patients receiving ICI mAb therapies. This suggests CL0 may predict outcomes from ICI therapy, and cachectic signalling may link elevated CL0 and poor response. Our aim was to determine if mouse models of cancer cachexia will be useful for studying these phenomena and their underlying mechanisms. METHODS: We evaluated pembrolizumab CL in the C26 and Lewis lung carcinoma mouse models of cancer cachexia. A single treatment of vehicle or pembrolizumab, at a dose of 2 or 10 mg/kg, was administered intravenously by tail vein injection. Pembrolizumab was quantified by an ELISA in serial plasma samples, and FcRn gene (Fcgrt) expression was assessed in liver using real-time quantitative reverse transcription PCR. Non-compartmental and mixed-effects pharmacokinetics analyses were performed. RESULTS: We observed higher pembrolizumab CL0 and decreased Fcgrt expression in whole liver tissue from tumour-bearing vs. tumour-free mice. In multivariate analysis, presence of tumour, total murine IgG, muscle weight and Fcgrt expression were significant covariates on CL, and total murine IgG was a significant covariate on V1 and Q. CONCLUSIONS: These data demonstrate increases in catabolic clearance of monoclonal antibodies observed in humans can be replicated in cachectic mice, in which Fcgrt expression is also reduced. Notably, FcRn activity is essential for proper antigen presentation and antitumour immunity, which may permit the study of cachexia's impact on FcRn-mediated clearance and efficacy of ICI therapies.