Everolimus and Cyclosporine in Combination with 2Gy TBI as Pre- and Posttransplant Immunosuppression in a Canine Transplantation Model.
Authors: Lange Sandra , Rathsack Susanne , Wacke Rainer , Altmann Simone , Weirich Volker , Vogel Heike , Drewelow Bernd , Freund Mathias , Junghanss Christian 
Published: October 2019

Abstract
BACKGROUND: Everolimus (RAD) is a potent immunosuppressant used for the prevention of acute allograft rejection in renal and cardiac transplantation. RAD in combination with cyclosporine A (CSA) demonstrates comparable efficacy to mycophenolate mofetil (MMF) and azathioprine, respectively, whereas it has a more feasible side effect profile. Studies that investigate RAD in hematopoietic stem cell transplantation (HSCT) are lacking. The aim of the present study was to evaluate the efficacy and safety of RAD in combination with CSA in a nonmyeloablative HSCT model.
METHODS: Dogs were given conditioning with 2Gy total body irradiation (TBI) before allogeneic HSCT (d0). Dog leukocyte antigen-identical littermate marrow was infused i.v. at a median of 3.6x10E8 TNC/kg. Three dogs received unmodified marrow (group 1) whereas three other dogs were given sensitized marrow from a donor that had rejected the graft of a prior HSCT from the sibling that served as recipient in the current experiments (group 2). All dogs received an immunosuppression consisting of 15mg/kg CSA BID PO (days -1 to 35) and 0.25mg BID PO (days 0 to 27) initially. Pharmacokinetics of RAD and CSA at day 5 were used for doses adaptation to reach targeted trough level of 3 8ng/ml for RAD and C0 and C2 level of 300 500ng/ml and 1500 2000ng/ml for CSA, respectively. Hematotoxicity, blood lipids, and creatinine were monitored.
RESULTS: All dogs engrafted following HSCT and first hematopoietic mixed chimerism was detectable by day 7. Dogs in group 1 engrafted with a median maximum granulocyte chimerism of 82% (54–95%) at day 28. Median maximum PBMC chimerism was 52% (26–62%) at day 34. One dog rejected the graft after 10 weeks, 1 dog died from infection at week 9 but was a mixed hematopoietic chimera at the time of death (28% granulocytes, 36% PBMC), and 1 dog continues to be mixed chimeric through week 27 (0% granulocytes, 16% PBMC). Two of the 3 dogs in group 2 developed full donor chimerism among granulocytes and PBMC and remain stable full hematopoietic chimeras through weeks 15 an 26. The other dog rejected the graft after 10 weeks. Median RAD trough level at day 5 was 7.2ng/ml (2.3–12.0ng/ml). Synergism between RAD and CSA caused enhancement in CSA C0, C2, and AUC values by 112, 106 and 45%, respectively, compared to CSA levels in CSA/MMF treated dogs. CSA dose reduction was initiated in 3/6 dogs based on pharmacokinetics. Within all dogs a transient decrease in platelet and leukocyte counts with median nadirs of 0x10E9/l (0–21x10E9/l) at day 11 and 1.6x10E9/l (0.7–3.2x10E9/l) at day 8 was observed. Creatinine, cholesterol, triglyceride levels remained within normal limits.
CONCLUSION: Immunosuppression with RAD/CSA as pre- and posttransplant conditioning is feasible in the canine HSCT model. RAD was well tolerated and allowed dose reduction of CSA. Using RAD and pre-sensitized bone marrow, full donor chimerism was achievable. Therefore, our data indicate that the combination of RAD/CSA might be used as alternative for CSA/MMF in the 2Gy nonmyeloablative HSCT setting.