Sustained elevation of parathyroid hormone (PTH) is catabolic to cortical bone, as evidenced by deterioration in bone structure (cortical porosity), and is a major factor for increased fracture risk in chronic kidney disease (CKD). Etelcalcetide (AMG 416), a novel peptide agonist of the calcium-sensing receptor, reduces PTH levels in subtotal nephrectomized (Nx) rats and in hemodialysis patients with secondary hyperparathyroidism (SHPT) in clinical studies; however, effects of etelcalcetide on bone have not been determined. In a rat model of established SHPT with renal osteodystrophy, etelcalcetide or vehicle was administered by subcutaneous (s.c.) injection to subtotal Nx rats with elevated PTH (>750pg/mL) once per day for 6weeks. Sham-operated rats receiving vehicle (s.c.) served as non-SHPT controls. Prior to treatment, significant increases in serum creatinine (2-fold), blood urea nitrogen (BUN, 3-fold), PTH (5-fold), fibroblast growth factor-23 (FGF23; 13-fold) and osteocalcin (12-fold) were observed in SHPT rats compared to non-SHPT controls. Elevations in serum creatinine and BUN were unaffected by treatment with vehicle or etelcalcetide. In contrast, etelcalcetide significantly decreased PTH, FGF23 and osteocalcin, whereas vehicle treatment did not. Cortical bone porosity increased and bone strength decreased in vehicle-treated SHPT rats compared to non-SHPT controls. Cortical bone structure improved and energy to failure was significantly greater in SHPT rats treated with etelcalcetide compared to vehicle. Mineralization lag time and marrow fibrosis were significantly reduced by etelcalcetide. In conclusion, etelcalcetide reduced bone turnover, attenuated mineralization defect and marrow fibrosis, and preserved cortical bone structure and bone strength by lowering PTH in subtotal Nx rats with established SHPT.

Etelcalcetide, a novel peptide agonist of the calcium-sensing receptor, prevents vascular calcification in a rat model of renal insufficiency with secondary hyperparathyroidism. Vascular calcification occurs frequently in patients with chronic kidney disease (CKD) and is a consequence of impaired mineral homeostasis and secondary hyperparathyroidism (SHPT). Etelcalcetide substantially lowers parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF23) levels in SHPT patients on hemodialysis. This study compared the effects of etelcalcetide and paricalcitol on vascular calcification in rats with adenine-induced CKD and SHPT. Uremia and SHPT were induced in male Wistar rats fed a diet supplemented with 0.75% adenine for 4 weeks. Rats were injected with vehicle, etelcalcetide, or paricalcitol for 4 weeks from the beginning of adenine diet. Rats fed an adenine-free diet were included as nonuremic controls. Similar reductions in plasma PTH and parathyroid chief cell proliferation were observed in both etelcalcetide- and paricalcitol-treated rats. Serum calcium and phosphorus were significantly lower in etelcalcetide-treated uremic rats and was unchanged in paricalcitol-treated rats. Both serum FGF23 and aortic calcium content were significantly lower in etelcalcetide-treated uremic rats compared with either vehicle- or paricalcitol-treated uremic rats. The degree of aortic calcium content for etelcalcetide-treated rats was similar to that in nonuremic controls and corroborated findings of lack of histologic aortic mineralization in those groups. In conclusion, etelcalcetide and paricalcitol similarly attenuated progression of SHPT in an adenine rat model of CKD. However, etelcalcetide differentially prevented vascular calcification, at least in part, due to reductions in serum FGF23, calcium, and phosphorus levels.

Etelcalcetide is a novel d-amino acid peptide that functions as an allosteric activator of the calcium-sensing receptor and is being developed as an intravenous calcimimetic for the treatment of secondary hyperparathyroidism in patients with chronic kidney disease on hemodialysis. To support clinical development and marketing authorization, a comprehensive nonclinical safety package was generated. Primary adverse effects included hypocalcemia, tremoring, and convulsions. Other adverse effects were considered sequelae of stress associated with hypocalcemia. Cardiovascular safety evaluations in the dog revealed an anticipated prolongation of the corrected QT interval that was related to reductions in serum calcium. Etelcalcetide did not affect the human ether-a-go-go gene ion channel current. Etelcalcetide was mutagenic in some strains of Salmonella, however, based on the negative results in 2 in vitro and 2 in vivo mammalian genotoxicity assays, including a 28-day Muta mouse study, etelcalcetide is considered nongenotoxic. Further support for a lack of genotoxicity was provided due to the fact that etelcalcetide was not carcinogenic in a 6-month transgenic rasH2 mouse model or a 2-year study in rats. There were no effects on fertility, embryo-fetal development, and prenatal and postnatal development. All of the adverse effects observed in both rat and dog were considered directly or secondarily related to the pharmacologic activity of etelcalcetide and the expected sequelae associated with dose-related reductions in serum calcium due to suppression of parathyroid hormone secretion. These nonclinical data indicate no safety signal of concern for human risk beyond that associated with hypocalcemia and associated QT prolongation.

AMG 416 is a novel D-amino acid-containing peptide agonist of the calcium-sensing receptor (CaSR) that is being evaluated for the treatment of secondary hyperparathyroidismin chronic kidney disease patients receiving hemodialysis. The principal amino acid residues and their location in the CaSR that accommodate AMG 416 binding and mode of action have not previously been reported. Herein we establish the importance of a pair of cysteine residues, one from AMG 416 and the other from the CaSR at position 482 (Cys482), and correlate the degree of disulfide bond formation between these residues with the pharmacological activity of AMG 416. KP-2067, a form of the CaSR agonist peptide, was included to establish the role of cysteine in vivo and in disulfide exchange. Studies conducted with AMG 416 in pigs showed a complete lack of pharmacodynamic effect and provided a foundation for determining the peptide agonist interaction site within the human CaSR. Inactivity of AMG 416 on the pig CaSR resulted from a naturally occurring mutation encoding tyrosine for cysteine (Cys) at position 482 in the pig CaSR. Replacing Cys482 in the human CaSR with serine or tyrosine ablated AMG 416 activity. Decidedly, a single substitution of cysteine for tyrosine at position 482 in the native pig CaSR provided a complete gain of activity by the peptide agonist. Direct evidence for this disulfide bond formation between the peptide and receptor was demonstrated using a mass spectrometry assay. The extent of disulfide bond formation was found to correlate with the extent of receptor activation. Notwithstanding the covalent basis of this disulfide bond, the observed in vivo pharmacology of AMG 416 showed readily reversible pharmacodynamics.

A novel peptide, AMG 416 (formerly KAI-4169, and with a United States Adopted Name: velcalcetide), has been identified that acts as an agonist of the calcium-sensing receptor (CaSR). This article summarizes the in vitro and in vivo characterization of AMG 416 activity and the potential clinical utility of this novel compound. AMG 416 activates the human CaSR in vitro, acting by a mechanism distinct from that of cinacalcet, the only approved calcimimetic, since it can activate the CaSR both in the presence or the absence of physiologic levels of extracellular calcium. Administration of AMG 416 in vivo into either normal or renally compromised rats results in dose-dependent reductions in parathyroid hormone (PTH) levels and corresponding decreases in serum calcium, regardless of the baseline level of PTH. Treatment of 5/6 nephrectomized rats with AMG 416 resulted in dramatic improvements in their metabolic profile, including lower PTH and serum creatinine levels, reduced amounts of vascular calcification, attenuated parathyroid hyperplasia, and greater expression of the parathyroid gland regulators CaSR, vitamin D receptor, and FGF23 receptor compared with vehicle-treated animals. No drug accumulation was observed under this dosing regimen, and the terminal half-life of AMG 416 was estimated to be 2-4.5 hours. As a long-acting CaSR agonist, AMG 416 is an innovative new therapy for the treatment of hemodialysis patients with secondary hyperparathyroidism.