Cyclosporine blood level monitoring. Cross-reactivity of anti-cyclosporine A monoclonal with its sulphate metabolite: an in vitro study.

Cyclosporine is metabolized in the liver by the Cytochrom P450 system. Many of the metabolites have been identified and thoroughly studied. However the sulphate metabolite is the least known. The metabolite is hydrophilic and thus it is not easily detectable by HPLC and it is not available in pure form for analysis. We have isolated the metabolite from transplant patients receiving CYA as part of their immunosuppressive therapy. The amino acid sequence was determined and the molecule was synthesised in our laboratories. We have tested the molecule in vitro using cell culture to determine its activity. 1/2 ml of blood+1/2 ml of RPMI was incubated with the following concentration of either CSA or CSS: 0, 25, 50, 100, 250, 500, 750, 1000, 1250, 1500, 2000 ng/ml for 2 and 1/2h at 37 degrees C with 5% CO2. The blood was then stimulated with Ionomycine and PMA (phorpol 12 myristate 13-acetate) for additional 2h. Supernatant was collected and assayed for the concentration of the following cytokines: IL-1a, IL-2, Interferon (IFNa), IL-6, IL-12, TNFa. The cells were used to evaluate the expression of cell activation marker CD69. Blood was assayed for CYA concentration using Abbott TDx assay. Results the level of the metabolite was actually higher than the parent compound indicating that the monoclonal antibodies detected the S form preferentially. The results with other CYA monoclonal was also similar. These results suggest that blood level monitoring of CYA many not be accurate as all the monoclonal antibodies currently used cross react with the metabolites.