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FTY720 compares with FK 506 as rescue therapy in rat heterotopic cardiac transplantation
FTY720 Compares With FK 506 as Rescue Therapy in Rat Heterotopic Cardiac Transplantation M. Xu, J. Pirenne, S. Antoniou, B. Gunson, M. D’silva, and P. McMaster
T
HE immunosuppressive agent FTY720 (2-amino-2-[2(4-octylphenyl)ethyl]-1,3-propanediol hydrochloride) has been reported to possess unique immunosuppressive mechanisms distinct from those of FK 506 and cyclosporine (CyA).1–3 Our previous study has also demonstrated that FTY720 possesses marked anti-rejection effect when used as induction therapy.4 The aim of this study was to determine whether FTY720 or FK 506 used as rescue therapy can reverse ongoing acute rejection in rat heterotopic cardiac transplantation. MATERIALS AND METHODS Inbred DA and Lewis rats were used as donors and recipients, respectively. Abdominal heterotopic cardiac transplantation was performed using the modified technique described by Ono and Lindsey.5 Transplanted hearts that did not beat strongly immediately after reperfusion and for 2 days’ posttransplant were excluded from this study. Graft survival time was designated from the day of transplantation to 1 day before rejection.
Immunosuppressive Agents FTY720 (synthesized in powder form by Taito Co Ltd, Tokyo, and Yoshitomi Pharmaceutical Ind, Ltd, Osaka, Japan) and FK 506 (Fujisawa Pharmaceuticals GmbH, Munich, Germany) was dissolved in normal saline and administered orally by gavage.
Experimental Design The dose and duration of treatment for each experimental group are shown in Table 1. Group 1 was given nothing as control. Group 2 (early treatment rejection) was given FTY720 5 mg/kg 1 day before and on the day of grafting. Group 3 was given FTY720 5 mg/kg on postoperative days 2 and 3. Group 4 was given FK 506 1 mg/kg from day 2 to day 6. Group 5 (late treatment rejection) was given FTY720 5 mg/kg on days 4 and 5. Group 6 was given FK 506 1 mg/kg from days 4 to 6.
Statistical significance of differences between medians was analyzed using nonparametric tests (Mann-Whitney U test). A P-value of ,.05 was considered to reflect a significant difference.
RESULTS
The median survival time of abdominal heterotopic heart transplant is shown in Table 1. In Group 1, without immunosuppression, the median survival time was 5 days. The median survivals in the early rejection Groups 3 and 4 were 8 and 11 days, respectively. There were significant statistical differences in median survival time between Group 1 (control) and Groups 3 and 4. The median survivals in late rejection Groups 5 and 6 were 7 and 6 days, respectively. There was significant statistical difference in median survival time between Group 1 and Group 5, but there was no significant statistical difference in median survival between Group 6 and Group 1 (P 5 .076). DISCUSSION
FTY720 was reported to have a potential immunosuppressive property. In our MHC mismatched rat strain combination, FTY720 significantly prolonged graft survival from 5 (control) to 13 days. It was shown that FTY720 is an effective pretransplant drug as a short induction treatment. When FTY720 was given at early stage (day 2 and day 3) and late stage (day 4 and day 5), the median survivals in Group 3 and 5 were prolonged to 8 and 7 days, respectively. FTY720 significantly prolonged graft survival (P 5 0.009, From Liver Unit, Queen Elizabeth Hospital, University of Birmingham, Edgbaston, Birmingham, UK. Address reprint requests to Paul McMaster, MD, Director and Professor of Liver Unit, Queen Elizabeth Hospital, University of Birmingham, Edgbaston, Birmingham B15 2TH, UK.
Table 1. FTY720 and FK 506 as Rescue Therapy in Rat Heterotopic Heart Transplantation Group
1 (n 2 (n 3 (n 4 (n 5 (n 6 (n
5 5 5 5 5 5
5) 6) 5) 5) 5) 5)
Treatment
Median/Range (d)
Control (none) FTY720, 5 mg/kg, days 21, 0 FTY720, 5 mg/kg, days 2, 3 FK 506, 1 mg/kg, days 2– 6 FTY720, 5 mg/kg, days 4, 5 FK 506, 1 mg/kg, days 4 – 6
5 (4 –5) 13 (11–16) 8 (8 –9) 11 (10 –16) 7 (7– 8) 6 (5–26)
P-value
.006, .009, .009, .009, .076,
— vs Group vs Group vs Group vs Group vs Group
1 1 1 1 1
© 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/98/$19.00 PII S0041-1345(98)00596-X
Transplantation Proceedings, 30, 2221–2222 (1998)
2221
2222
Group 3 vs Group 1; P 5 .009, Group 5 vs Group 1) and delayed ongoing rejection. Similarly, when FK 506 was given at early stage (1 mg/kg from day, 2 to day 6), the median survival in Group 4 was prolonged to 11 days and delayed ongoing rejection, but FK 406 given 1 mg/kg from day 4 to day 5 did not prolong graft survival (p 5 .076). With regard to the immunosuppressive mechanism of FTY720, the induction of apoptosis in lymphocytes was suggested to be the inhibitory effect of FTY720. In vitro, rat spleen cells incubated with FTY720 demonstrated features of apoptosis, such as the absence of surface microvilli, chromatin condensation, and the formation of apoptotic bodies by means of electron microscopy and genemic DNA fragmentation by agarose gel electrophoresis.3 Our study also showed lymphopenia in transplant and nontransplant rats after the administration of two doses of FTY720 5 mg/kg (data not shown). The phenomenon of lymphopenia reflects immunosuppressive activity of FTY720. Further study showed FTY720 selectively decreased the peripheral mature T cells and did not affect B cells, bone marrow cells, thymocytes, and polymorphonuclear cells in peripheral blood. Other in vitro studies showed FTY720 did not affect IL-2 production from mitogen-stimulated T cells.2,6,7 In conclusion, our study has shown that FTY720 used as induction therapy and rescue therapy significantly extended allograft survival in rat heterotopic cardiac transplantation.
XU, PIRENNE, ANTONIOU ET AL
FTY720 given both at early and late stage was proven to slow ongoing rejection; FK 506 given at early stage was proven to slow ongoing rejection, but not at late stage in this model.
ACKNOWLEDGMENT We would like to thank Seiichi Suzuki, MD (Department of Experimental Surgery and Bioengineering, National Children’s Medical Research Center, Taishido, Setagaya-ku, Tokyo 154, Japan) for his generous supply of FTY720.
REFERENCES 1. Adachi K, Kohara T, Nakano N, et al: Bioorg Med Chem 5:853, 1995 2. Chiba K, Hoshino Y, Suzuki C, et al: Transplant Proc 28:1056, 1996 3. Suzuki S, Enosawa S, Kakefuda T, et al: Transplantation 61:200, 1996 4. Xu M, Antoniou E, Afford S, et al: Transplant Proc 29(7): 2964, 1997 5. Ono K, Lindsey ES: J Thorac Cardiovasc Surg 57:225, 1969 6. Hoshino Y, Suzuki C, Ohtsuki M, et al: Transplant Proc 28:1060, 1996 7. Kawaguchi T, Hoshino Y, Rahman F, et al: Transplant Proc 28:1062, 1996
T
HE immunosuppressive agent FTY720 (2-amino-2-[2(4-octylphenyl)ethyl]-1,3-propanediol hydrochloride) has been reported to possess unique immunosuppressive mechanisms distinct from those of FK 506 and cyclosporine (CyA).1–3 Our previous study has also demonstrated that FTY720 possesses marked anti-rejection effect when used as induction therapy.4 The aim of this study was to determine whether FTY720 or FK 506 used as rescue therapy can reverse ongoing acute rejection in rat heterotopic cardiac transplantation. MATERIALS AND METHODS Inbred DA and Lewis rats were used as donors and recipients, respectively. Abdominal heterotopic cardiac transplantation was performed using the modified technique described by Ono and Lindsey.5 Transplanted hearts that did not beat strongly immediately after reperfusion and for 2 days’ posttransplant were excluded from this study. Graft survival time was designated from the day of transplantation to 1 day before rejection.
Immunosuppressive Agents FTY720 (synthesized in powder form by Taito Co Ltd, Tokyo, and Yoshitomi Pharmaceutical Ind, Ltd, Osaka, Japan) and FK 506 (Fujisawa Pharmaceuticals GmbH, Munich, Germany) was dissolved in normal saline and administered orally by gavage.
Experimental Design The dose and duration of treatment for each experimental group are shown in Table 1. Group 1 was given nothing as control. Group 2 (early treatment rejection) was given FTY720 5 mg/kg 1 day before and on the day of grafting. Group 3 was given FTY720 5 mg/kg on postoperative days 2 and 3. Group 4 was given FK 506 1 mg/kg from day 2 to day 6. Group 5 (late treatment rejection) was given FTY720 5 mg/kg on days 4 and 5. Group 6 was given FK 506 1 mg/kg from days 4 to 6.
Statistical significance of differences between medians was analyzed using nonparametric tests (Mann-Whitney U test). A P-value of ,.05 was considered to reflect a significant difference.
RESULTS
The median survival time of abdominal heterotopic heart transplant is shown in Table 1. In Group 1, without immunosuppression, the median survival time was 5 days. The median survivals in the early rejection Groups 3 and 4 were 8 and 11 days, respectively. There were significant statistical differences in median survival time between Group 1 (control) and Groups 3 and 4. The median survivals in late rejection Groups 5 and 6 were 7 and 6 days, respectively. There was significant statistical difference in median survival time between Group 1 and Group 5, but there was no significant statistical difference in median survival between Group 6 and Group 1 (P 5 .076). DISCUSSION
FTY720 was reported to have a potential immunosuppressive property. In our MHC mismatched rat strain combination, FTY720 significantly prolonged graft survival from 5 (control) to 13 days. It was shown that FTY720 is an effective pretransplant drug as a short induction treatment. When FTY720 was given at early stage (day 2 and day 3) and late stage (day 4 and day 5), the median survivals in Group 3 and 5 were prolonged to 8 and 7 days, respectively. FTY720 significantly prolonged graft survival (P 5 0.009, From Liver Unit, Queen Elizabeth Hospital, University of Birmingham, Edgbaston, Birmingham, UK. Address reprint requests to Paul McMaster, MD, Director and Professor of Liver Unit, Queen Elizabeth Hospital, University of Birmingham, Edgbaston, Birmingham B15 2TH, UK.
Table 1. FTY720 and FK 506 as Rescue Therapy in Rat Heterotopic Heart Transplantation Group
1 (n 2 (n 3 (n 4 (n 5 (n 6 (n
5 5 5 5 5 5
5) 6) 5) 5) 5) 5)
Treatment
Median/Range (d)
Control (none) FTY720, 5 mg/kg, days 21, 0 FTY720, 5 mg/kg, days 2, 3 FK 506, 1 mg/kg, days 2– 6 FTY720, 5 mg/kg, days 4, 5 FK 506, 1 mg/kg, days 4 – 6
5 (4 –5) 13 (11–16) 8 (8 –9) 11 (10 –16) 7 (7– 8) 6 (5–26)
P-value
.006, .009, .009, .009, .076,
— vs Group vs Group vs Group vs Group vs Group
1 1 1 1 1
© 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/98/$19.00 PII S0041-1345(98)00596-X
Transplantation Proceedings, 30, 2221–2222 (1998)
2221
2222
Group 3 vs Group 1; P 5 .009, Group 5 vs Group 1) and delayed ongoing rejection. Similarly, when FK 506 was given at early stage (1 mg/kg from day, 2 to day 6), the median survival in Group 4 was prolonged to 11 days and delayed ongoing rejection, but FK 406 given 1 mg/kg from day 4 to day 5 did not prolong graft survival (p 5 .076). With regard to the immunosuppressive mechanism of FTY720, the induction of apoptosis in lymphocytes was suggested to be the inhibitory effect of FTY720. In vitro, rat spleen cells incubated with FTY720 demonstrated features of apoptosis, such as the absence of surface microvilli, chromatin condensation, and the formation of apoptotic bodies by means of electron microscopy and genemic DNA fragmentation by agarose gel electrophoresis.3 Our study also showed lymphopenia in transplant and nontransplant rats after the administration of two doses of FTY720 5 mg/kg (data not shown). The phenomenon of lymphopenia reflects immunosuppressive activity of FTY720. Further study showed FTY720 selectively decreased the peripheral mature T cells and did not affect B cells, bone marrow cells, thymocytes, and polymorphonuclear cells in peripheral blood. Other in vitro studies showed FTY720 did not affect IL-2 production from mitogen-stimulated T cells.2,6,7 In conclusion, our study has shown that FTY720 used as induction therapy and rescue therapy significantly extended allograft survival in rat heterotopic cardiac transplantation.
XU, PIRENNE, ANTONIOU ET AL
FTY720 given both at early and late stage was proven to slow ongoing rejection; FK 506 given at early stage was proven to slow ongoing rejection, but not at late stage in this model.
ACKNOWLEDGMENT We would like to thank Seiichi Suzuki, MD (Department of Experimental Surgery and Bioengineering, National Children’s Medical Research Center, Taishido, Setagaya-ku, Tokyo 154, Japan) for his generous supply of FTY720.
REFERENCES 1. Adachi K, Kohara T, Nakano N, et al: Bioorg Med Chem 5:853, 1995 2. Chiba K, Hoshino Y, Suzuki C, et al: Transplant Proc 28:1056, 1996 3. Suzuki S, Enosawa S, Kakefuda T, et al: Transplantation 61:200, 1996 4. Xu M, Antoniou E, Afford S, et al: Transplant Proc 29(7): 2964, 1997 5. Ono K, Lindsey ES: J Thorac Cardiovasc Surg 57:225, 1969 6. Hoshino Y, Suzuki C, Ohtsuki M, et al: Transplant Proc 28:1060, 1996 7. Kawaguchi T, Hoshino Y, Rahman F, et al: Transplant Proc 28:1062, 1996