SAT-402 Investigating Analogues of Parathyroid Hormone (PTH) and PTH-Related Peptide (PTHrP) to Improve Anabolic: Catabolic Response Ratios Using UMR106-01 Osteocytic Cells

Abstract

Osteoporosis, the most common bone disease in humans, is characterised by decreased bone mass and increased fracture risk1. Osteoporosis development is associated with an imbalance of bone resorption and deposition mediated by all three bone cell types; osteoblasts, osteocytes and osteoclasts2. Current treatments for osteoporosis either prevent further bone resorption (Bisphosphonates and anti-RANKL) or increase bone deposition through anabolism (Teriparatide and Abaloparatide)3. The two anabolic treatments are truncated analogues of endogenous peptides; parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP), respectively. Both are administered intermittently, act on PTH1R receptors on osteoblasts and osteocytes leading to an increase in bone mineral density and bone strength. However, both treatments also have side-effects of hypercalcemia and cortical bone porosity caused by osteoclast stimulation. We therefore compared an array of PTH and PTHrP analogues for potential stimulation of catabolic side effects and desired anabolic effects. Screening assays used are UMR106-01 osteocytic cells that have high levels of PTH1R to measure mRNAs involved in anabolic responses (suppression of WNT inhibitors SOST and Dkk1) and catabolic responses (stimulation of RANKL/OPG, IL6). Peptides were also analysed by real-time cellular impedance assays (xCELLigence) to measure unbiased receptor stimulation. xCELLigence assays showed PTH1-34, PTHrP1-34 and Abaloparatide had the highest potencies (3.7 nM,1.4 nM,1.7nM respectively) while Tyr1PTH1-34 and PTH2-34 had significantly decreased potencies (64nM and 130nM), the β-arrestin biased agonist, D-Trp12Tyr34PTH7–34, had no effect. PTH1-34 potently inhibited SOST (IC50 0.29nM), and catabolic genes (RANK/OPG EC50 0.5nM, IL6 EC50 2.5nM). PTHrP1-34 provided higher potencies for anabolic (inhibited SOST IC50 0.08nM) and catabolic (RANKL/OPG EC50 0.3nM, IL6 EC50 1nM) genes. Altering the first amino acid to Tyrosine; Tyr1PTH1-34 caused potent anabolic responses (SOST IC50 0.97nM) yet showed decreased potency for catabolic responses (RANKL/OPG EC50 20nM, IL6 EC50 > 100nM). Removing the first amino acid of PTH to PTH2-34 drastically decreases the effectiveness of the peptide (OPG, SOST and RANKL IC50 – no effect, IL6 EC50 > 100nM). These results indicate the importance of the N-terminal amino acid for PTH affinity and efficacy and suggest that Tyr1PTH1-34 may offer the best combination of bone stimulation without causing hypercalcemia.References 1 A.Gupta, L. March. (2016) Treating osteoporosis, Aust Prescr 2 Chen X, et al. (2018) Osteoblast–osteoclast interactions. Connect. Tissue Res 3 Tu KN, et al. (2018) Osteoporosis: A review of treatment options. P T