Assessment of the in vivo immunosuppressive activity of the major cyclosporine metabolite by leukemia allograft rejection.
AM1 (M17) is the major metabolite of cyclosporine found in the blood of human transplant recipients, and trough levels of this derivative exceed those of the parent compound approximately two-fold. Studies performed in vitro indicate that AM1 retains only 10-20% of the biological activity of the parent compound, but very little is known about its in vivo immunosuppressive effects. We therefore developed a rapid and sensitive method, based on the rejection of allogeneic L1210 (H-2d) leukemia cells by C57BL/6 (H-2b) mice, to assess the immunosuppressive activity of AM1 in vivo. Rejection of the leukemia allograft was determined by analyzing the spleens from mice injected intravenously with 10(5) L1210 cells for the presence of H-2Kd-positive cells by flow cytometry using an FITC-conjugated monoclonal anti-H-2Kd antibody. Nonimmunosuppressed mice rejected the allogeneic cells and survived indefinitely. Spleens from these mice were virtually free of H-2Kd-positive cells (0.51 +/- 0.21%) by day 7. In contrast, C57BL/6 mice treated with 10 mg/kg/day s.c. of CsA all died from the L1210 challenge (mean survival time of 9 +/- 1 days). Spleens from mice treated in this manner contained 11.02 +/- 3.31% H-2Kd-positive cells on day 7. There was a direct correlation between the dose of CsA administered (7.5-50 mg/kg/day) and the percentage of H-2Kd-positive cells in the spleen. We then compared the immunosuppressive activity of AM1 and CsA in this model. AM1 was purified from the urine of CsA-treated renal allograft recipients by a combination of preparative adsorption-desorption chromatography and preparative elution high-performance liquid chromatography. AM1 at a dose of 10 mg/kg/day exhibited no demonstrable immunosuppressive effect, and trough levels of AM1 on day 7 were only 36 +/- 4 ng/ml. Increasing the dose of AM1 to 50 mg/kg/day resulted in only 1.05 +/- 0.16% H-2Kd-positive cells in the spleens (P = NS) and a mean trough level of 221 +/- 27 ng/ml. In contrast, mice treated with 50 mg/kg/day of CsA exhibited 17.7 +/- 2.9% H-2Kd-positive cells in their spleens and a mean trough CsA level of 3036 +/- 277 ng/ml. The half-life of a single subcutaneous dose of 10 mg/kg of AM1 (4.6 hr) was significantly shorter than that of CsA (9.7 hr) in mice. Compared with CsA, the lack of immunosuppressive effect of AM1 in vivo therefore appears to be due to a combination of decreased immunosuppressive activity and increased rate of clearance in mice.
Freed
B M
BM
Department of Surgery, Albany Medical College, NY 12208.
Bennett
J A
JA
Rosano
T G
TG
Brooks
C A
CA
Cramer
S M
SM
Lempert
N
N
D000818:Animals; D016572:Cyclosporine / Q000097:blood / Q000378:metabolism* / Q000493:pharmacokinetics / Q000494:pharmacology; D005260:Female; D005434:Flow Cytometry; D006084:Graft Rejection / Q000187:drug effects / Q000276:immunology*; D006207:Half-Life; D007166:Immunosuppressive Agents / Q000378:metabolism*; D016219:Immunotherapy, Adoptive; D007939:Leukemia L1210 / Q000473:pathology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D014184:Transplantation, Homologous
D007166:Immunosuppressive Agents; D016572:Cyclosporine