| The key problem for limb, skin or other composite tissues transplantation is to control posttransplantation rejection, which encumbers long-term functional survival of transplanted organ and tissues. The first female facial composite tissue allotransplantation was performed in 2005 by Henri Monder (AH-HP) Hospital, France. This procedure, as well as other composite tissue transplants, offers the potential for correcting untreatable large tissue defects. In April 2006, a male facial composite tissue allotransplantation was performed successfully in xijing hospital, Fourth Military Medical University. Full thickness skin of the nose and upper lip, mimetic muscle and skin were reconstructed in the surgery. However, comparing with solid organ transplantation, skin has higher antigenicity and it is easier to have rejection in the composite tissue allograft, and concerns remain regarding obligatory chronic immunosuppression and long-term functional recovery. Degree and frequency of rejection correlated with long-term survival and functional recovery of the transplanted organ or tissues ,and dose of immunosuppressant correlated with the degree and frequency of rejection, so it is necessary for the recipient'custom-made clothes'to perform personalized medicine. Till now, there was no report about pharmacokinetics of tacrolimus (FK506) and mycophenolate mofetil in facial composite tissue allograft recipient. Pharmacokinetic of tacrolimus and mycophenolate mofetil would be studied to issue a guide to personalized medicine in the face allograft recipient and prevent post-transplantation rejection and toxic effects of immunosuppressants.AIM: To performe personalized medicine in the facial composite tissue allograft recipient and to assure a safety and efficacy therapeutic time window of immunosuppressants, based on the pharmacokinetic characteristics of tacrolimus and mycophenolate mofetil in the recipient.METHODS:The study includes three parts.Part 1: To establish analysis methods to determine tacrolimus and mycophenolic acid in human blood. First, a micro-particle enzyme immunoassay (MEIA) assay with high specificity, high precision and sensibility was established for determining concentrations of tacrolimus in whole blood samples. Precision, accuracy, linearity and LLOQ are selected to evaluate whether it could be used in the study of the pharmacokinetics of tacrolimus in human. Procedure: 150uL of untested or standard samples with different concentrations was added into the labeled tubes, and then 150μL of precipitating agent (saturated methanol and dimethyl carbinol solution of zinc sulphate) was added into the above tubes. The above tubes were vortexed and mixed and 200μL of clear supernatant was used to determine tacrolimus concentration in the whole blood. MODE 1 and(lower,medium,high concentration ) quality control (QC) was used to measure tacrolimus concentration in the whole blood. Secondly, an economic HPLC analysis with high specificity, high precision and sensibility was established for determining concentrations of mycophenolic acid in plasma samples. The chromatography column was an Irregular C18 column (250 mm×4.6 mm i.d., 5μm) coupled to a C18 guard column (4 mm×3.0 mm i.d.). The column temperature was maintained at 25°C. The mobile phase was composed of methanol/10 mM potassium dihydrogen phosphate buffer (3:2, v/v, 5 mM tetrabutylammonium bromide). The flow rate was 1 mL/min and the injection volume was 100μL. Precision, accuracy, linearity, LLOQ, extraction recovery and stability were selected to evaluate whether it could be used in the study of the pharmacokinetics of mycophenolic acid in human plasma samples.Part 2: To study pharmacokinetic characteristics of tacrolimus and mycophenolic acid in recipient and investigate the absorption, metabolism, and excretion of tacrolimus and mycophenolic acid in the recipient. Tacrolimus pharmacokinetic study: Whole blood samples (2 mL) were drawn and collected in EDTA-containing tubes before (C0), and 0.25 h, 0.5 h, 0.75 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 4 h, 6 h, 8 h and 12 h after oral drug administration to otherwise fasting (2 h before and 1 h after administration) patients. The samples were stored at 4°C until analyzed by the MEIA developed above. Mycophenolate mofetil pharmacokinetic study: Mycophenolic acid is main active metabolite of mycophenolate mofetil in human body. Plasma samples (2 mL) were drawn and collected in heparin-containing tubes before (C0), and 0.25 h, 0.5 h, 1 h, 1.5 h, 2 h,2.5 h,3.5 h, 5.5 h, 7.5 h, and 11.5h after oral drug administration to otherwise fasting (2 h before and 1 h after administration) patients. The plasma samples was frozen and stored at -20°C until analyzed by the HPLC developed above. The pharmacokinetic parameters were calculated by DAS2.0 statistic analysis software. Peak concentrations (Cmax) and time to peak concentration (Tmax) were taken directly from the observed data.Part 3: To perform personalized medicine of immunosuppressants in facial composite tissue allograft recipient based on clinical diagnosis, laboratory test, vital signs and pharmacokinetic characteristics of tacrolimus and mycophenolate mofetil in the recipient. A safe and effective therapeutic window of tacrolimus was optimized to prevent transplant rejection and toxic effects of immunodepressants.RESULTS:1. Tacrolimus was detected by EMIA in the study. The limit of quantification (LOQ) for tacrolimus was 3 ng·mL-1; the linear range was 3～30 ng·mL-1. The intra-plate precision (RSD) was within 12.7% and accuracy was during 94.3% to 100.8%. Inter-plate precision (RSD) were within 11.1% and accuracy was during 92.2% to 101.7%.2. Mycophenolic acid (MPA) was detected by HPLC in the study. Good selectivity was observed and there was no significant interference from endogenous substances observed at the retention time 7.45 min of MPA. The limit of quantification (LOQ) for MPA was 0.5 mg·L-1; the linear range was 0.5?32 mg·L-1. The intra-run RSD and inter-run RSD of MPA in this linear range was within 10% and accuracy was during 94.8% to 109.0%. The recovery of MPA was between 79.72～84.89%. The results of stability experiments showed that accuracy value of MPA was within 4.6%.3. Steady-state pharmacokinetic profiles of tacrolimus in facial composite tissue allograft recipient were as follows: Tmax was 1.5 h, Cmax was 51.4 ng·mL-1, T1/2 is13.4 h, AUC0~12 was 600.58 ng·mL-1·h-1.4. Steady-state pharmacokinetic profiles of MPA in facial composite tissue allograft recipient were as follows: Tmax was 0.5 h, Cmax was12.42 mg·L-1, T1/2 was 0.51 h, AUC0~11.5 was 13.31 mg·L-1·h-1.5. Immunosuppressive therapy consisted of tacrolimus, mycophenolate mofetil, prednisone and Zenapax was adopted in the facial composite tissue allograft. Drip infusion of methylprednisone and tacrolimus injections were administered on the day of transplantation and continued for a few days post-operatively in case of hyperacute rejection. Methylprednisone was given intravenously for the first 1~5 days,tapering from 250 mg per day to 130 mg per day, and then prednisone was given orally, tapering from 80mg per day at day 6 to 25 mg per day. Tacrolimus was given at an initial high dose for the first 1~3 days and then tapering to 13 mg per day after transplantation. Dose of tacrolimus was optimized according to tacrolimus's pharmacokinetic characteristic and clinical diagnosis during different post-transplant period. Mycophenolate mofetil was introduced at a dose of 1000 mg per day orally at day 2 and increased to 1500 mg per day. Zenapax was coadministred with other immunosuppressants after transplantation. 6. During the 1st month after transplantation, the therapeutic window of tacrolimus is 20.12±4.77 ng·mL-1; during the 2nd to 3rd month, the therapeutic window of tacrolimus is 20.17±4.02 ng·mL-1; during the 4th to 6th month, the therapeutic window of tacrolimus is 22.82±7.10 ng·mL-1; during the 7th to 12th month, the therapeutic window of tacrolimus is27.88±5.68 ng·mL-1. The results of clinical diagnosis, laboratory test and biopsy showed the therapeutic window is safe and effective for the first face transplantation recipient in China with less transplant rejection and toxic action.CONCLUSIONS:1. The EMIA method with high sensitivity, accuracy and wonderful specificity was established to determine tacrolimus in human blood. The HPLC analysis method with high sensitivity, accuracy, wonderful specificity, stability and recovery was developed to determine MPA in human plasma samples.2. Tacrolimus's pharmacokinetic model was developed in the study. Tmax was 1.5 h and T1/2 was 13.4 h, which were similar to the pharmacokinetic parameters of common people. Tacrolimus was eliminated slowly in the recipient and personalized medicine was designed by the T1/2.3. Mycophenolate mofetil's pharmacokinetic model was developed in the study. Tmax was 1.5 h and T1/2 was 13.4 h, which were similar to the pharmacokinetic parameters of common people. Mycophenolate mofetil was eliminated rapidly in the recipient and personalized medicine was designed by the therapeutic index of Mycophenolate mofetil 4. A personalized dose regimen was optimized for the first human face allograft recipient in China. Partial functional recovery of transplanted facial composite tissue has been observed. Morphologically, there was no red swelling, blood clot or ulcerate in the smooth and glossy transplanted skin; functionally, functional reaction of transplanted composite tissue has recovered partially to outside stimulation. |
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