8041 Eneboparatide, A Potent Stimulator Of Urinary Calcium Reabsorption, Induces Prolonged Renal Cortex Retention And PTH1 Receptor Signaling
Authors: Ravel Guillaume , Datta Rakesh , Milano Stéphane , Ovize Michel , Allas Soraya , Aouadi Myriam , Culler Michael D 
Published: October 2024

Abstract
Disclosure: G. Ravel: Employee; Self; Amolyt Pharma. R. Datta: Consulting Fee; Self; Amolyt Pharma. S. Milano: Employee; Self; Amolyt Pharma. M. Ovize: Employee; Self; Amolyt Pharma. S. Allas: Employee; Self; Amolyt Pharma. M. Aouadi: None. M.D. Culler: Employee; Self; Amolyt Pharma.
Natural parathyroid hormone (PTH) replacement therapy for chronic hypoparathyroidism (cHP) is not ideal due to both short circulating half-life and brief receptor signaling, making the effect too transient. Following agonist binding to the PTH 1 receptor (PTH1R), the magnitude and duration of the receptor signal, and consequently the biological response, appears to depend on the affinity of the ligand for a specific receptor conformation termed R0, which determines the duration of ligand binding. Eneboparatide (Enebo) is a hybrid analog of PTH and PTH-related peptide designed to strongly bind to the R0 conformation while having a short circulating half-life similar to that of PTH. When tested in cHP patients, Enebo eliminated the need for calcium (Ca) (88% patients) and Vitamin D (92% patients) supplementation, while maintaining serum Ca levels over a full 24 hours. Urinary calcium (uCa) was rapidly decreased and was normalized in 92% of patients who were hypercalciuric prior to treatment. To explore the mechanism of the potent effect of Enebo on uCa, a binding assay specific for the R0 conformation of the PTH1R confirmed the high affinity of Enebo versus natural PTH1-34, with IC50 = 1.5nM and 30.9nM, respectively. Postulating that the high-affinity binding of Enebo to R0 should result in longer tissue retention in vivo, we examined the tissue levels of PTH1-34 and Enebo in male, 200-300g rats using quantitative whole-body autoradiography (QWBA) following subcutaneous injection of 3H-labeled Enebo and PTH1-34. Tissue levels of the compounds were normalized based on the concentrations observed in cardiac blood. The highest level observed for both compounds was in the renal cortex, the major site of active PTH-induced Ca reabsorption, with peak concentrations observed 30 minutes post-injection. Six hours after injection, 96.3% of PTH1-34 had disappeared from the renal cortex, as compared with only 61.4% of Enebo, despite both compounds having similar circulating half-lives. With respect to activating the PTH1R, both compounds showed a similar increase in cAMP in human embryonic kidney (HEK293) cells transfected with the PTH1R and the Green Downward cADDis cAMP biosensor, with EC50 of 0.32 and 0.48nM for Enebo and PTH1-34, respectively. To mimic the effect of rapid elimination in vivo, the ligands were added to the cells, washed-off after 15 minutes, and cAMP production followed over the next 16 hours. After washout, the cAMP signal induced by PTH1-34 rapidly returned to baseline in less than 60 minutes, whereas the cAMP signal induced by Enebo gradually decreased and was still evident after 16 hours. Collectively, these data confirm high affinity of Enebo for the R0 conformation of the PTH1R, as well as prolonged receptor signaling and retention by the renal cortex, clearly differentiating Enebo from PTH1-34 and providing strong rationale for the efficacy of Enebo in reducing uCa in cHP patients.
Presentation: 6/3/2024