*Purpose: Little is known about the pharmacokinetics of intracellular tacrolimus, particularly in T lymphocytes and monocytes. We demonstrated a significantly lower intracellular tacrolimus concentration in CD3⁺ (TAC_[CD3]) compared with CD14⁺ cells (TAC_[CD14]). Our objective was to investigate the role of 2 important proteins involved in intracellular tacrolimus distribution, namely FK-binding protein 12 (FKBP12) and P-glycoprotein (P-gp) to explain these differences in intra-cellular tacrolimus concentrations.

*Methods: Rhodamine (Rh), a substrate of P-gp (like tacrolimus), was used to determine the P-gp activity in isolated, pure CD3⁺ T lymphocytes and CD14⁺ monocytes obtained from healthy volunteers. FKBP12 and P-gp expression was semi-quantified by Western blot and flow cytometry comparing between T lymphocytes and monocytes. To determine the effect of P-gp on intracellular tacrolimus concentration, verapamil, a P-gp inhibitor, was added to kidney transplant recipient’s blood before the cell isolation process and the intracellular tacrolimus measurement. Results were compared with the same blood samples not treated with verapamil.

*Results: T lymphocytes showed a significantly lower percentage of Rh-positive cells after 2 hours incubation at 37^oC (61±14%) and 25^oC (80±9%) compared with 4^oC (94±4%) (p<0.001). Adding verapamil completely negated this temperature effect, suggesting that tacrolimus is pumped out of T lymphocytes if not processed at 4^oC or in the absence of a P-gp inhibitor. In contrast, monocytes did not show any difference of the percentage of Rh-positive cells regardless of temperature or the addition of verapamil (98-99%). Flow cytometric analysis revealed a significantly higher expression of P-gp on T lymphocytes than monocytes (mean fluorescence intensity 793(606-863) vs 664(466-718);p=0.012) and a lower intensity of FKBP12 in T lymphocytes than monocytes (182(70-232) vs 595(332-851);p=0.012). Western blot confirmed that T lymphocytes had higher P-gp and lower FKBP band density than monocytes. By adding verapamil to patient samples, TAC_[CD3] was 53-100% higher than samples from the same patients in the absence of verapamil.

*Conclusions: The higher activity of P-gp and the lower concentration of FKBP-12 explain the lower TAC_[CD3] compared with TAC_[CD14]. A substantial amount of tacrolimus is actively pumped out from T lymphocytes during the cell isolation process if P-gp is not properly inhibited. Verapamil blocks this process and gives reliable intracellular tacrolimus T lymphocyte concentrations.

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