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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.

We have evaluated the effects of CsA, methylprednisolone (MP), 6-mercaptopurine (6-MP), and FK506 on T cell-dependent and T cell-independent immunoglobulin production. FK506 and 6-MP were potent inhibitors of IgG and IgM production by PWM-stimulated peripheral blood mononuclear cells, which depend on the presence of T cells. CsA was less effective in this system and MP actually enhanced IgG and IgM production. In order to assess the direct effects of these various immunosuppressive agents on B cells, we utilized human B cell lines representing different stages of B cell differentiation. The B cell lines CESS and SKW6.4 exhibit increased production of IgG and IgM, respectively, in response to interleukin-6. These cells represent activated, but not fully differentiated, B cells. CsA inhibited IL-6-induced IgG production by CESS cells by 64% at 100 ng/ml and 6-MP inhibited this response by 82% at 250 ng/ml. Neither CsA nor 6-MP effectively inhibited IL-6-induced IgM production by SKW6.4 cells. MP at 250 ng/ml inhibited IL-6-induced IgG production by 89%, but enhanced IL-6-induced IgM production more than two-fold. FK506 did not inhibit IL-6-induced IgG or IgM production, suggesting that it has no direct effect on the ability of B cells to respond to this differentiation factor. These experiments clearly demonstrate that CsA, MP and 6-MP have direct inhibitory effects on the response of human B cells to IL-6. In contrast, FK506 has no direct effect on these B cells lines, but is more potent than the other agents at inhibiting T cell-dependent immunoglobulin production.

The in vitro biological activity of cyclosporine (CsA) and four of its metabolites (M1, M8, M17, and M21) was determined. M1, M17, and M21 are primary metabolites, while M8 is a secondary metabolite derived from either M1 or M17. The order of inhibitory activity in production assays was phytohemagglutinin (PHA), concanavalin A (ConA), mixed lymphocyte culture (MLC), and interleukin-2 (IL-2) CsA greater than M17 greater than M1 greater than M21 much greater than M8. In the PHA assay, CsA was significantly more inhibitory than M17, but in Con A and MLC assays, the inhibitory activity of M17 approached that of CsA. More importantly, M17 and M1 inhibited the production of IL-2 in the MLC to the same extent as CsA. M21 was significantly less inhibitory than either M17 or M1, and M8 appeared to be largely devoid of biological activity. These experiments demonstrate that single hydroxylations of amino acids 1 (M17) and 9 (M1) do not significantly affect the ability of the molecule to block IL-2 production, but hydroxylation of both amino acids renders the molecule virtually inactive. In addition, the presence of the N-methyl group on amino acid 4 appears to be very important, since removal of this group (M21) greatly diminishes the immunosuppressive activity.

Vascular endothelial cells (VEC) are clearly immunogenic and express antigens unique to vascular endothelial cells. The observation that peripheral blood monocytes also express this VEC antigen system made prospective testing feasible. Preformed antibody to the VEC/monocyte antigen system of the donor usually leads to graft rejection in HLA-identical combinations, but antibody directed against donor monocytes exclusively (monocyte-specific antigens) appears to be benign. Clearly the VEC antigen system is an important immunogen in HLA-identical living-related donor combinations--and, in addition, this antigen system may be equally important in the non-HLA-identical combinations. The identification of antibody directed against the VEC/monocyte antigens of the donor is frequently masked by the concurrent development of anti-HLA antibody. Preliminary family segregation studies support the concept of genetic linkage between the VEC/monocyte antigen system and the major histocompatibility complex. A group of 153 consecutive, prospective monocyte crossmatches performed have yielded approximately a 7% incidence of positive monocyte crossmatches, with traditional crossmatches being negative. This frequency of patients sensitized to the VEC/monocyte antigens of the donor could conceivably account for up to 70% of the observed early graft loss in living-related donors. We think a positive monocyte crossmatch to the donor in the presence of positive reactivity to concordant VEC and monocytes remains a contraindication to transplantation.

Cyclosporine levels by radioimmunoassay (RIA) and high-performance liquid chromatography (HPLC) were monitored in serial blood samples (n = 177) from 11 renal allograft recipients. HPLC analysis revealed three primary metabolites of CsA (M17, M1, and M21) in peak and trough blood samples; M17 was the preponderant metabolite. In 4 patients on whom serial metabolite assays were performed, M17 was found in the blood at 86-2004 ng/ml; M1 and M21 were found at up to 100 ng/ml. The immunosuppressive properties of purified metabolites M1, M17, M21, and M8 (which was not detected in the blood) were compared with CsA. M17--and, to a lesser extent, M1 and M21--were found to inhibit the in vitro response of human mononuclear cells in the mixed leukocyte culture and in mitogen (phytohemagglutinin [PHA], concanavalin A [Con A], and pokeweed mitogen [PWM]) assays at 1000 ng/ml. M8 exhibited no in vitro inhibitory activity. M17 was further tested at 10-1000 ng/ml in PHA and mixed lymphocyte culture (MLC) assays. M17 had considerably less inhibitory activity (12-43%) than CsA (18-70%) in the PHA assay. However, in MLC experiments M17 blocked the proliferative response by 39-72% at 100-800 ng/ml, which approached the degree of inhibition exhibited by CsA (63-87%). In 34 of 37 (92%) patient blood samples, the level of metabolite M17 was found to exceed the parent drug level and could not be measured accurately by RIA. The observed in vitro immunosuppressive activity of metabolites (particularly M17) and their presence in the blood of renal allograft recipients suggest a possible role for these metabolites in the immunopharmacology of CsA.