Repurposing of Treprostinil and Cinacalcet to Facilitate Hematopoietic Cell Transplantation
Authors: Prchal-Murphy Michaela , Zehenter Julia , Fischer Marlene , Strunk Dirk , Farr Alex , Freissmuth Michael , Zebedin-Brandl Eva Maria 
Published: December 2024

Background
Hematopoietic stem and progenitor cell (HSPC) transplantation (HCT) has significantly advanced over the past decades and is now a standard treatment for various life-threatening conditions. HCT involves harvesting CD34+ HSPCs from sources such as bone marrow (BM), peripheral blood (PB), or umbilical cord blood (CB). However, this process can be physically demanding for stem cell donors, and both PB and BM transplants pose a risk of graft-versus-host disease (GvHD) due to the presence of T cells in the transplant. CB offers advantages with lower risks for donors and a reduced incidence of GvHD, but it is limited by the small number of HSPCs available in a single CB unit.
Despite these challenges, HCT remains the most successful somatic cell therapy and, due to its well-established medical use, does not require regulatory approval.
This exemption, however, does not apply to primed HSPCs, which must comply with regulatory standards for safety, efficacy, and good manufacturing practices, like traditional small molecules or therapeutic proteins.
Repurposing already clinically approved drugs is beneficial as it leverages existing safety and efficacy data, significantly reducing the time and costs required for drug development.
In this study, we utilized two approved drugs, treprostinil and cinacalcet, which target distinct signaling pathways in HSPCs, conducting a comprehensive analysis of their single and combined effects. We defined an optimal treatment regimen that enhances the efficacy of all HCTs, regardless of HSPC source, thereby addressing several limitations in current treatments.
Methods
Lineage-negative (lin-) HSPCs were isolated from murine BM, and CD34+ cells were obtained from the CB of healthy donors. The effects of treprostinil and cinacalcet, both individually and in combination, were assessed by analyzing the migration, adhesion, and differentiation of HSPCs in vitro. In vivo, NSG or NSG-S mice were (xeno)transplanted, and homing and engraftment were evaluated using flow cytometry in limiting cell number conditions. Long-term reconstitution capacity was determined through replating assays and secondary transplantation in animals.
Results
The experiments provided four major insights: (i) treprostinil was more effective than cinacalcet in stimulating the migration of human HSPCs. (ii) cinacalcet was superior in promoting adhesion. (iii) treprostinil and cinacalcet were mutually antagonistic when used together. (iv) a transient rise in cAMP levels, induced by priming with treprostinil/forskolin, was sufficient to promote the subsequent differentiation of early progenitors (CFU-GEMMs) but impaired the outgrowth of the erythroid lineage (BFU-Es).
These insights allowed us to exploit the unique properties of each agent to define an optimal drug regimen for HCT. This regimen involved the sequential priming of murine or human HSPCs with treprostinil and forskolin, followed by 10 days treating recipient animals with cinacalcet. This approach resulted in accelerated recovery of peripheral blood cells and 100% survival of transplanted mice. Moreover, the long-term reconstitution capacity of transplanted HSPCs was elevated.
Summary
Our findings suggest that the sequential use of treprostinil and cinacalcet as the first “rapid engraftment regimen” can enhance the efficacy of all HCTs, regardless of the HSPC source, and potentially rejuvenate cord blood hematopoietic cell transplantation. This regimen boosts HSPC potency, improves bone marrow reconstitution, and enhances homing and engraftment properties both in vitro and in vivo.
It offers two key benefits - i) enabling successful transplants with sub-threshold HSPC collections and ii) reducing the number of cells needed from donors, thereby minimizing the risks and discomfort associated with harvesting.
These advantages increase the pool of willing donors and broaden the use of HCT in various clinical settings. Future studies will investigate the safety and mechanisms of this regimen, potentially transforming HCT by making it safer and more accessible for patients and donors.