Cyclosporin a has low potency as a calcineurin inhibitor in cells expressing high levels of p-glycoprotein

Cyclosporin A (CsA) is a widely-used immunosuppressant drug whose therapeutic and toxic actions are mediated through inhibition of calcineurin (CN), a calcium- and calmodulin-dependent phosphatase. Inhibition of CN by CsA requires drug binding to its protein cofactor in the inhibition, cyclophilin. Because cyclophilin is a high affinity target for CsA it is expected that this protein can act as a reservoir for the drug in the cell and may be able to inhibit cellular efflux of CsA. P-glycoprotein (P-gp) is known to increase the rate of CsA efflux from CsA loaded cells but it is not clear if the P-gp drug efflux pump can compete effectively with cyclophilin at therapeutically relevant concentrations of CsA. To test the hypothesis that increased expression of Pgp confers protection against CsA-dependent inhibition of CN phosphatase activity, KB-V cells expressing varying levels of P-gp were analyzed to determine the potency of CsA as a CN inhibitor. When intact cells were treated with CsA, a positive correlation was observed between P-gp expression and resistance to CsA-dependent inhibition of CN: the IC₅₀ is approximately 20-fold higher in the multidrug resistant epidermal carcinoma cell line, KB-V, which expresses P-gp at a high level than in the parental, KB, cell line expressing very low levels of P-gp. The resistance displayed by KB-V cells is abrogated by co-administration of the P-gp inhibitor verapamil, whereas verapamil has no effect on CsA potency in control KB cells. In cell lysates from KB-V cells with different amounts of P-gp CsA exhibits equivalent potency, indicating that the difference in sensitivity to CsA among the cell types requires maintenance of cell integrity. These observations support the view that resistance to CN inhibition by CsA occurs in cells with moderately elevated P-gp activity. Therefore, P-gp activity appears to be an important determinant of CsA cellular specificity for both therapeutic and toxic effects.