Declining intracellular T-lymphocyte concentration of cyclosporine a precedes acute rejection in kidney transplant recipients.

BACKGROUND: We investigated cyclosporine A (CsA) concentrations at the site of action, inside T-lymphocytes, to evaluate its applicability as a new supplementary therapeutic drug monitoring method after renal transplantation. METHOD: In this prospective single-center study, 20 kidney transplant recipients, mean age 54 (range 21-74) years, on CsA-based immunosuppression were included within 2 weeks posttransplant and followed for 3 months. Nine patients also had one full 12-hour pharmacokinetic profile performed. T-lymphocytes were isolated from 7 ml whole blood using Prepacyte and intracellular CsA concentrations were determined using a validated liquid chromatography double mass spectrometry method. RESULTS: Seven patients (35%) experienced acute rejections (all biopsy verified) during the first three months posttransplantation. Intracellular CsA concentrations tended to decline 1 week prior to acute rejection and the decrease was significant (-27.1+/-14.6%, P=0.014) three days before the rejection episodes were recognized clinically. In addition, the intracellular CsA area under the curve 0-12 measured during stable phase was 182% higher in the rejection-free patients (P=0.004). There was no difference between patients experiencing rejection and the rejection-free patients with respect to CsA C2-levels, dose (mg/kg), human leukocyte antigen mismatch, donor age, recipient age, or ABCB1 genotyping. CONCLUSION: Intracellular CsA T-lymphocyte concentrations declined significantly 3 days prior to a rejection episode and there was a general lower intracellular exposure of CsA in recipients experiencing rejection. Intracellular measurement of CsA therefore seems to have a potential to further improve individualization of therapeutic drug monitoring. Larger studies are needed to elucidate the role for intracellular T-lymphocyte measurements in ordinary clinical care, for both CsA and other immunosuppressive drugs.

Determination of ciclosporin A and its six main metabolites in isolated T-lymphocytes and whole blood using liquid chromatography-tandem mass spectrometry.

A specific and sensitive method for determination of intracellular ciclosporin A (CsA) and its six main metabolites AM1, AM9, AM1c, AM1c9, AM19 and AM4N, in isolated T-lymphocytes and whole blood is described. T-lymphocytes were separated from whole blood using Prepacyte. The analytes were extracted and purified from isolated lymphocytes and whole blood by protein precipitation followed by solid-phase extraction (SPE). The analytes and the internal standard, ciclosporin C (CsC), were separated on a reversed phase C8 column (30 mm x 2.1mm, 3 microm) with a 10 mm x 2 mm, 5 microm Drop-In Guard Cartridge, using gradient elution chromatography and tandem ion trap mass spectrometry detection. The method has been validated in accordance with FDA guidelines and showed linear range from 0.25 to 500 ng/mL for CsA, 0.5 to 500 ng/mL for AM1, AM9 and AM19, 1 to 500 ng/mL for AM4N, AM1c and AM1c9 in intracellular matrix, and 2.5 to 3000 ng/mL for all analytes in whole blood. The applicability of the method is shown on patient samples.