Strategies for the optimization of immunotherapy in kidney transplantation

The success of kidney transplantation is dependent upon a life-long maintenance therapy with immunosuppressants. Immunosuppressants are narrow therapeutic index drugs that exhibit high intra and inter-individual variability in their pharmacokinetics (PK) and pharmacodynamics (PD). Immunosuppressant doses are adjusted by their concentrations, a practice referred to as \u27therapeutic drug monitoring\u27. This dissertation aimed to devise novel strategies/methods to improve the monitoring of immunosuppressive agents and to compare their PK/PD between diabetic (D) and non-diabetic (ND) kidney transplant recipients. A liquid chromatography tandem mass spectrometric assay was developed and validated to analyze the concentrations of mycophenolic acid (MPA) in saliva over a 12-hour dosing interval from 29 stable kidney transplant recipients. The MPA saliva concentrations were significantly associated with its total (r=0.322, p\u3c0.001) or unbound (r=0.435, p\u3c0.001 ) plasma concentrations indicating that salivary MPA concentrations may prove to be a non-invasive method for its monitoring. Pharmacokinetic characteristics of tacrolimus (TAC) and ciclosporin (CsA) were compared between D and ND patients. Tacrolimus maximum concentration (Cmax), time to maximum concentration (tmax) and the area under the concentration time curves (AUC0-12) were comparable between D and ND patients. In contrast, absorption of CsA was delayed (t max D=128.4 vs. ND=93 min, p=0.009], AUC0-12 was lower and the unbound fraction was higher (D=1.20% vs. ND=0.72%, p=0.066) in diabetic patients. The mechanism of differential effect of diabetes on the pharmacokinetics of CsA and TAC requires further characterization. Pharmacodynamic properties of CsA and TAC were assessed using intracellular cytokine staining for IL-2, TNF-alpha and IFN-gamma in mitogen stimulated CD3+ T-lymphocytes. A strong correlation was observed between the expression of IL-2 with total CsA or TAC concentrations. The degree of immunosuppression was also compared between D (n=14) and ND (n=15) patients before dose and 2-hours after dose. B-cell activity was also determined by measuring fluorescence of surface markers CD54 (ICAM-1), CD86 (B7.2) and CD95 (Fas antigen) in pokeweed mitogen stimulated CD19 cells. Expression of intracellular IL-2 in CD3 cells was significantly lower in diabetics while CD95 in B-cells were significantly lower in diabetics both at resting and stimulated states. These results indicate that diabetics require lower concentrations of immunosuppressants than non-diabetics