Blood distribution of rapamycin.

Rapamycin (RAPA) is a potent new immunosuppressive drug. Although blood concentration monitoring of RAPA is being performed in preclinical and clinical trials, little is known regarding the blood distribution of the drug. Such information would have an impact on the medium used for analysis of the drug. The distribution of RAPA was investigated by spiking human whole blood having an initial temperature of either 4 degrees C or 22 degrees C with a constant amount of 3H-RAPA and increasing amounts of RAPA to a final concentration of 5-100 micrograms/L. The drug concentration spans the range seen when immunosuppressive doses of the drug are administered. This was followed by incubation of the blood at 37 degrees C for 0 to 60 min before separation of cells. The dpm in the resulting plasma and RBC fractions was determined by scintillation counting. The plasma to formed blood elements and plasma to whole blood ratios were 0.05 +/- 0.051 and 0.09 +/- 0.016, respectively (mean +/- SD, n = 50). The distribution did not exhibit any temperature or concentration dependence. The proportion of the drug among cellular components was as follows (mean % distribution +/- SD); RBC 94.5 +/- 4.9%; plasma 3.1 +/- 2.5%; lymphocytes 1.01 +/- 1.02%; and granulocytes 1.0 +/- 0.88%. The free or unbound fraction of RAPA over the plasma concentration range of 5-100 micrograms/L as determined by ultracentrifugation was 2.5 +/- 0.2%. The drug was found to be associated primarily with nonlipoprotein fractions in plasma. The results suggest from an analytical perspective that whole blood as compared with plasma would be the most suitable medium for analysis due to the higher concentrations found in the former.

Efficacy of rapamycin, RS-61443 and cyclophosphamide in the prolongation of survival of discordant pig to rabbit cardiac xenografts.

OBJECTIVE: To evaluate the efficacy of two new immunosuppressive drugs, RS-61443 and rapamycin, alone and in combination with cyclophosphamide in the prolongation of heterotopic cardiac xenograft survival in a discordant neonatal pig to rabbit model. DESIGN: Animals were randomly assigned to receive rapamycin intravenously (1.0 mg/kg/day) or RS-61443 subcutaneously (160 mg/kg/day) commencing five days before surgery until rejection occurred. Cyclophosphamide, 20 mg/kg/day, was administered from one day prior to surgery. Combination of the above drugs (rapamycin 1.0 mg/kg/day and RS-61443 80 mg/kg/day) with and without treatment with cyclophosphamide was also evaluated. Trough blood concentrations of rapamycin and mycophenolic acid as well as rabbit antiporcine endothelial cell antibodies (immunoglobulin [Ig]M) were measured every second day from the commencement of drug therapy. RESULTS: The mean +/- SD survival time in control animals was 7.98 +/- 13.85 h (n = 11), with the majority of animals (n = 6) rejecting their grafts in less than 1 h. Rapamycin (11.5 +/- 9.5 h), RS-61443 (9.8 +/- 6.1 h), cyclophosphamide (17.9 +/- 16 h) alone, or the three drugs in combination (16.6 +/- 9.1 h) resulted in a trend to increase graft survival, which did not reach significance. In contrast, rapamycin and RS-61443 in combination (24.3 +/- 11.9 h) significantly (P < 0.05) prolonged graft survival. No significant (P > 0.05) decrease in rabbit antiporcine endothelial cell IgM concentrations was observed in all drug-treated groups; however, considerable variation was noted within each group. No relationship was found between trough concentrations of rapamycin and mycophenolic acid and xenograft survival or with a decrease in antibody concentrations. CONCLUSION: The data suggest that treatment modalities, in addition to the above drugs, is required for prolonged survival in cardiac xenografts in the discordant animal model used here.

Quality assurance program for cyclosporin G (OG37-325).

Cyclosporine-G (CsG) (OG37-325) an analogue of cyclosporine-A (CsA) is presently undergoing clinical trials. As therapeutic monitoring of CsG was an integral part of these evaluations, the performance of laboratories measuring the drug was assessed through an external quality assurance program, which included all North American centers participating in the trials. The assays used by participating centers were high-performance liquid chromatography (HPLC) (n = 3), radioimmunoassay (RIA) (n = 5), and fluorescence polarization immunoassay (FPIA) (n = 24). The latter two assays, developed for measurement of CsA, were adapted for measurement of CsG. The FPIA was the most precise, with the largest proportion of laboratories reporting coefficients of variation (CNs) of < 10%. RIA was the least accurate method, with approximately 74% of results differing by > 20% from target values. FPIA and RIA methods exhibited mean recoveries of CsG of 112 and 129%, respectively. The ranking of the specificity of the assays from measurement of parent drug was HPLC > RIA > FPIA. FPIA and RIA produced values that were, on average, approximately 60% higher than those reported by HPLC in pooled whole blood specimens obtained from renal transplant patients. It is recommended that if CsG is approved for routine clinical use, then assays developed specifically for measurement of the drug be used.

Distribution of cyclosporin G (NVa2 cyclosporin) in blood and plasma.

We report here on the distribution of cyclosporin G (CsG), an analog of cyclosporin A, in whole blood. CsG has a temperature-dependent distribution between erythrocytes (RBCs) and plasma. After 30 min at 37 degrees C, the plasma/whole blood and plasma/RBC ratios were relatively constant (0.7-0.8) up to CsG at 1000 micrograms/L. At 5000 micrograms/L, this ratio increased to 1.0-1.1 for plasma/whole blood and 1.7 for plasma/RBC. The primary CsG metabolites GM1 and GM9 were sequestered within RBCs to a greater extent than was the parent drug. In whole blood, approximately 2% of CsG was bound to granulocytes, 6% to lymphocytes, and 50-55% to RBC, and 35-40% was found in the plasma fraction. The free fraction of the drug as determined by ultracentrifugation was 5-6% and 13-17% of total drug at 37 and 4 degrees C, respectively. In plasma the drug was primarily associated with high-density lipoprotein (50-60%) and to a lesser degree with low-density (20-30%) and very-low-density (10%) lipoproteins.