The binding of cyclosporin A to human plasma: an in vitro microdialysis study.

PURPOSE

The human plasma binding of cyclosporin A was studied in vitro using the technique of microdialysis. The effect of temperature on the overall binding interaction between cyclosporin A and human plasma was also investigated.

METHODS

Flow-through loop-type microdialysis probes were constructed from fused silica tubing and regenerated cellulose tubing with a MWCO of 13000 daltons. Probes were perfused with phosphate buffer (0.5 microliters/min) and the concentration of 3H-cyclosporin A in the well-mixed medium (plasma or buffer) was 1200 ng/ml. Relative recoveries of cyclosporin A from plasma or buffer were determined for each probe by separate experiments to measure the solute gain or loss with reference to the perfusate.

RESULTS

Recoveries determined by loss were significantly greater than those determined by gain and in each case temperature dependent, with higher recoveries at higher temperatures. The plasma free fraction of cyclosporin A calculated from the recovery data and the perfusate to plasma concentration ratios was dependent on temperature in a log-linear fashion. Mean +/- s.d. plasma free fractions expressed in percent were 33.5 +/- 4.6, 17.9 +/- 3.6, 6.2 +/- 0.8, 3.0 +/- 0.6, and 1.5 +/- 0.2 at temperatures of 4, 10, 20, 30, and 37 degrees C, respectively. Assuming that the enthalpy of binding is constant over the temperature range studied and pseudo-first order conditions exist, the binding reaction at these temperatures was spontaneous, endothermic (delta H = 74.0 kJ/mole), and entropically driven (delta S = 0.274 kJ/mole/deg).

CONCLUSIONS

These results show that the free fraction of cyclosporin A in human plasma is dependent on temperature with the fraction unbound decreasing with temperature in the range of 4 to 37 degrees C. The thermodynamic parameters for the binding of cyclosporin A to plasma components indicate that the reaction is a spontaneous endothermic reaction that is mainly entropy driven, similar to the partitioning of lipophilic molecules from an aqueous to a hydrophobic phase. Moreover, these results show that microdialysis is a feasible method to determine the binding interactions between plasma and cyclosporin A, which indicates the method may be suitable for other difficult binding studies where the solutes have nonspecific binding to separation devices.