Therapeutic Potentials of Dimeric Cys25PTH(1–34) Peptide for Osteoporosis and Fracture Healing of the Bones- Buy One, Get One Free

Abstract

Objective: The extraordinarily high bone densities identified in the hypoparathyroidism patients originating from PTH R25C mutation suggested the possibility that this modified protein has unique biologic effects and contributes to the gain of bone volume. Interestingly, western blot of cell lysates stably transfected with PTH R25C construct revealed that Cys25PTH(1–84) formed a dimer, presumably due to disulfide bonding of the cysteine residues. This study aims to study the characteristics of Cys25PTH(1–34) dimeric peptide (Dimer) both in vitro and in vivo for potential therapeutic application of Dimer as a novel anabolic agent. Methods: Identity of the chemically synthesized Dimer was confirmed by its molecular weight and purity using MS and HPLC, respectively. Basic characteristics, for example, ability to bind to PTHR and cAMP production were investigated using a variety of cells. In addition, the ligand-receptor internalization was investigated using TMR tagged Dimer. In vivo characteristics, such as calcemic and phosphatemic responses, pharmacokinetics and pharmacodynamics, were assessed in CD1 mice. The osteoanabolic effects of Dimer were assessed using a fracture-healing model, a calvarial-injection model and an OVX mouse model. Results:In vivo study showed that Dimer has similar calcemic and phosphatemic responses to PTH(1–34; WT). In cell assays, Dimer showed a similar cAMP production but slightly lower binding affinity compared to WT. Dimer-receptor complex was internalized into the cells. Surprisingly, Dimer showed a potent anabolic effect in the fracture-healing model in mice measured as the callus volume fraction by microCT. We also observed a comparable anabolic effect of Dimer in calvarial-injection model and OVX model. Conclusions: Dimeric Cys25PTH(1–84) peptide might play a substantial role in the high bone mass in hypoparathyroidism patients, originating from the PTH R25C mutation. This may be translated into the development of potential therapeutic modality for the treatment of osteoporosis and fracture healing using Dimer.

Enhancing Bone Regeneration and Osseointegration using rhPTH(1-34) and DimericR25CPTH(1-34) in an Osteoporotic Beagle Model

“…This biological difference is thought to be due to dimeric R25C PTH(1-34) exhibiting a more preferential binding affinity for the RG versus R0 PTH1R conformation, despite having a diminished affinity for either conformation. (Lee and Lee, 2022; Park et al, 2021) Additionally, the potency of cAMP production in cells was lower for dimeric R25CPTH compared to monomeric R25CPTH, consistent with its lower PTH1R-binding affinity. (Noh et al, 2024) One of the potential clinical advantages of dimeric R25CPTH(1-34) is its partial agonistic effect in pharmacodynamics.…”

“…Although the capacity of R25C PTH(1-34) to bind to the PTH1R and stimulate cAMP production was slightly lower in the human osteoblast-derived SaOS-2 cell line ( Lee and Lee, 2022 ), yet it showed comparable anabolic activity in a mouse model ( Bae et al, 2016 ). Furthermore, the dimeric formation of the R25C PTH (1-84) peptide, presumably through disulfide bonding of the cysteine residues, has implied that dimeric R25C PTH(1-34) might partake in unique biological actions, which potentiate its clinical applications ( Park et al, 2021 ).…”

Enhancing Bone Regeneration and Osseointegration using rhPTH(1-34) and Dimeric R25CPTH(1-34) in an Osteoporotic Beagle Model

“…Although the capacity of R25C PTH(1-34) to bind to the PTH1R and stimulate cAMP production was slightly lower in the human osteoblast-derived SaOS-2 cell line (Lee and Lee, 2022 ), yet it showed comparable anabolic activity in a mouse model (Bae et al, 2016 ). Furthermore, the dimeric formation of the R25C PTH (1-84) peptide, presumably through disulfide bonding of the cysteine residues, has implied that dimeric R25C PTH(1-34) might partake in unique biological actions, which potentiate its clinical applications (Park et al, 2021 ).…”