Veicolazione dell’immunosoppressore Tacrolimus tramite eritrociti

Tacrolimus (FK506, Prograf) is a potent immunosuppressive macrolide lactone, produced by Streptomyces tsukubaensis, recently approved for prophylaxis of rejection in recipients of liver, kidney and pancreas transplants. Tacrolimus immunosuppressive activity is due to its ability of decreasing the formation of cytotoxic lymphocytes, primarily responsible for graft rejection. FK506 suppresses T-cell activation by inhibiting the phosphatase activity of calcineurin and this results in the disruption of T cell signaling pathways leading to the production of some activating interleukins (e.g. IL-2 and IL-4). Despite its therapeutic properties, Tacrolimus exhibits some toxicity including nephrotoxicity, neurotoxicity and glucose intolerance. At the molecular level the effect of Tacrolimus is mediated by the binding to a cytosolic protein (FKBP-12), which is responsible for the intracellular accumulation of the compound. FK506 in blood is mainly associated with erythrocytes (about 95%) followed by plasma (about 5%) and lymphocytes (less than 0.5%). The high RBC fraction is due to the presence into erythrocytes of two types of immunophilins that bind the drug with a very high affinity: FKBP-12, a 12-kDa cytosolic protein with a peptidyl-prolyl cis-trans isomerase activity, and FKBP-13, a 13-kDa membrane-associated protein. Our strategy intends to increase the amount of Tacrolimus carried by erythrocytes, in order to use them as a potential drug delivery system, and restrain possible toxic effects. Since RBC binding capacity for Tacrolimus is limited by the intracellular amount of immunophilins, we tried to increase the intra-erythrocytic concentration of FKBP-12. To this end we manufactured a recombinant form of human FKBP12 to be loaded into RBCs by means of a procedure of hypotonic dialysis and isotonic resealing. The FKBP12 cDNA was cloned into the pET-45b expression vector, fused to the N-terminal His-Tag. The recombinant protein induced in the BL21(DE3) E. Coli strain, was purified to homogeneity through a Ni sepharose High-Performance affinity chromatography, under native conditions. The ability of purified FKBP12 to bind the FK506 ligand was first assessed by in vitro binding studies. Then we encapsulated the recombinant FKBP12 into RBCs and we tested the capacity of such engineered erythrocytes of catching FK506. Experiments to assess the red cell capability of releasing Tacrolimus into the external medium were finally performed.