The calcineurin inhibitor tacrolimus is an immunosuppressive drug that has a narrow therapeutic index with high interindividual variations in its pharmacokinetics, which makes it difficult to establish an empirical dosage regimen in organ transplant recipients (1 2 CYP3A5 genotype and tacrolimus pharmacokinetics (3, 4 CYP3A5*1 , cytochrome P450 (CYP) 3A5 expressers, exhibited the lower tacrolimus dose-normalized concentration (C/D ratio) and required a higher dose to reach the same blood concentration than the recipients with homozygous CYP3A5*3 allele, CYP3A5 nonexpressers (5, 6 7
As tacrolimus is metabolized by both intestinal and hepatic CYP3A5 enzymes, the combined contribution of CYP3A5 expressions in native intestine and liver allograft should be considered for the pharmacokinetics of tacrolimus in liver transplant recipients (4, 6 CYP3A5 genotypes of both the recipients’ native intestine themselves and donors’ liver allograft, recipient -donor combinational effect of CYP3A5 genotype, could be essential to the marked inter-individual variation in postoperative tacrolimus pharmacokinetics.
However, the recipient -donor combinational effect of CYP3A5 genotype on tacrolimus dose and blood C/D ratio in living donor liver transplantation (LDLT) remains to be elucidated. Living donor liver transplantation is becoming increasingly important strategy in reducing the waiting time mortality in liver failure patients (8, 9 10 CYP3A5 genotypes of both native intestine and the graft liver. To our knowledge, however, this time trend on tacrolimus pharmacokinetics has not been clearly demonstrated. The previous studies were limited to relatively large graft size from deceased donor liver transplantation (DDLT) (11, 12 13 14
Based on this background, we hypothesized that tacrolimus clearance would change over time by recipient -donor combinational effect of CYP3A5 genotypes in patients after LDLT and aimed to evaluate longitudinal effect of recipient -donor combinational CYP 3A5 genotypes on blood tacrolimus C/D ratio in Korean adult LDLT recipients.
RESULTS
Characteristics of the Study Participants
A total of 58 de novo ethnically Korean adult LDLT recipients administering tacrolimus were stratified into four study groups according to the CYP3A5 genotype of recipients and donors: CYP3A5 expresser recipient grafted from CYP3A5 expresser donor (RE DE , n=10); CYP3A5 expresser recipient grafted from CYP3A5 nonexpresser donor (RE DN , n=13); CYP3A5 nonexpresser recipient grafted from CYP3A5 expresser donor (RN DE , n=8); and CYP3A5 nonexpresser recipient grafted from CYP3A5 nonexpresser donor (RN DN , n=27). The demographic and baseline clinical characteristics of the study participants were compatible among 4 genotype groups (all P >0.05), except for recipient ’s age (P <0.05) (Table 1 ). RE DN were the youngest (44.5±8.8 years), whereas RN DN were the oldest (52.6±7.0 years) (P =0.049). The mean length of hospital stay for transplantation was 27±18 days, ranging from 13 to 127 days. The most prevalent primary indication of transplantation was hepatitis B viral cirrhosis, which accounted for 75.0% of all cases, followed by alcoholic cirrhosis (8.3%), hepatitis C viral cirrhosis (6.7%), biliary cirrhosis (5.0%), fulminant hepatitis (3.3%), and cryptogenic cirrhosis (1.7%). Incidental hepatocellular carcinoma in addition to the primary disease was present in 29 of the recipients (49.2%). Six patients died during the study (at 1 and 8 months and 1.5, 2, 3, and 4 years postoperation). Child-Pugh scores consisted of 13.6% of grade A (5–6), 22.0% of grade B (7–9), and 64.4% of grade C (10–15). A total of 23 cases of acute cellular rejection (ACR) were observed during the study (19 cases within the first 4 weeks, whereas the others occurred at 0.5, 0.7, 1, and 3.6 years, respectively). Severity of ACR based on the rejection activity index (RAI) (15
TABLE 1: Demographic and baseline characteristics of study participants
Genotype Frequencies
CYP3A5 genotype frequencies for recipients and donors of liver grafts are shown in Table 2 . As a feature of liver transplantation, there were cases in which the CYP3A5 genotype of the recipient was different from that of donor; however, there was no difference in CYP3A5 genotype frequencies between recipients and donors (P =0.610). The allele frequency of the CYP3A5 *3 variant of the whole recipient and donor was 79.7%. The observed CYP3A5 genotype frequency distributions for recipients and donors were consistent with Hardy-Weinberg equilibrium (HWE) (all P >0.05).
TABLE 2: Genotype frequencies of CYP3A5
Time Trend of the C/D Ratio Stratified by Recipient -Donor Combinational CYP3A5 Genotype
Figure 1 presents tacrolimus C/D ratio over 48 months posttransplantation stratified by four recipient -donor combinational CYP3A5 genetic groups. The C/D ratios for all 4 groups tended to decline, although with different degree, during the first 6 months posttransplantation, and remained fairly stable thereafter. Approximately after 4 months posttransplantation, C/D ratios of CYP3A5 expresser liver (donor expresser) were lower than those of nonexpresser regardless of recipients’ genotype (RE DE , RN DE < RE DN , RN DN ). Given the same donor genotype, C/D ratios of CYP3A5 expresser intestine (recipient expresser) were lower than those of nonexpresser (RE DE < RN DE given the same DE and RE DN < RN DN given the same DN ). However, during the early period until about 4 months posttransplantation, C/D ratios of CYP3A5 expresser intestine were lower than those of nonexpresser independent of donors’ genotype (RE DE , RE DN < RN DE , RN DN ). The overall C/D ratio during the entire study period was nearly twofold greater in both intestinal and hepatic nonexpressers (RN DN ) than both expressers (RE DE ).
FIGURE 1: Tacrolimus dose-normalized concentration (C/D ratio, ng/mL per mg) stratified by the four donor-recipient combinational CYP3A5 genotype groups over 48 months after transplantation. RE DE , CYP3A5 expresser recipient grafted from CYP3A5 expresser donor; RE DN , CYP3A5 expresser recipient grafted from CYP3A5 nonexpresser donor; RN DE , CYP3A5 nonexpresser recipient grafted from CYP3A5 expresser donor; RN DN , CYP3A5 nonexpresser recipient grafted from CYP3A5 nonexpresser donor.
Because C/D ratio of RN DE changed dramatically over time, we further analyzed the difference between RN DE and RE DN over time as illustrated in Figure 2 . As the 95% confidence band (shaded area) suggested (i.e., zero excluded), C/D ratio between the two groups was significantly different until approximately 1 month of transplantation, but the difference was diminished thereafter. This motivated performing separate analyses for comparison of C/D ratios among 4 genotype groups by two different periods, until 1 month and after 1 month.
FIGURE 2: Differences in log-transformed tacrolimus dose-normalized concentration (C/D ratio, mg/mL per mg) between RN DE and RE DN over 48 months after transplantation. The C/D ratios were logarithmically transformed as the distribution was skewed to the right. RN DE , CYP3A5 nonexpresser recipient grafted from CYP3A5 expresser donor; RE DN , CYP3A5 expresser recipient grafted from CYP3A5 nonexpresser donor.
Effects of CYP3A5 Genotype on the Tacrolimus C/D Ratio
Table 3 presents the results for fixed effects in the final model for examining the association between log-transformed tacrolimus C/D ratio and the recipient -donor combinational CYP3A5 genotype groups after adjusting for covariates. After adjustment of the covariates, CYP3A5 genotypes remained as the most important factor affecting tacrolimus C/D ratio. The nonexpression of CYP3A5 was significantly associated with higher tacrolimus C/D ratio. For example, both intestinal and hepatic nonexpressers, RN DN , was associated with an increase of 0.89 and 0.8 in log-transformed tacrolimus C/D ratio compared with both expressers, RE DE , until and after 1 month, respectively (P <0.001). If either intestine or liver was CYP3A5 nonexpresser (RN DE or RE DN ), C/D ratio increased by a range of ~0.5 when compared with both expressers (RE DE ) (P ≤0.01), whereas it decreased by approximately 0.3 in comparison with both nonexpressers (RN DN ) (all P <0.01 except P =0.178 for RN DE vs. RN DN until 1 month). Among covariates in the model, after adjusting for the genotypes, albumin was negatively associated with the log-transformed tacrolimus C/D ratio during the entire study period (P <0.001). Time after transplantation is an important variable in the changing tacrolimus C/D ratio.
TABLE 3: Results for fixed effects in the mixed-effects model for examining the association between log-transformed tacrolimus C/D ratio and the recipient -donor combinational CYP3A5 genotype groups
DISCUSSION
To the best of our knowledge, this prospective study is the first study to examine time-dependent effect of combinational CYP3A5 genotype in graft liver and native intestine on the C/D ratio of tacrolimus in adult LDLT recipients. Our major finding was that recipients’ native intestinal CYP3A5 genotype played more important role than donors’ hepatic genotype in early stage after transplantation, and the roles would be gradually changed to be switched in later stage. Thus, C/D ratio of RN DE was significantly higher than that of RE DN shortly after transplantation but decreased rapidly during the first month of posttransplantation so that there was no difference between the two groups after 1 month. In addition, as expected, our study confirmed that C/D ratio was consistently the greatest in RN DN , whereas it was the lowest in RE DE throughout the entire study period.
As hepatic metabolism by CYP3A enzymes is considered a major eliminating process of tacrolimus, liver regeneration from the ischemia reperfusion injury as well as enlarging the liver mass after transplantation in LDLT may have influence on the total clearance of tacrolimus. In LDLT, tacrolimus metabolism is reduced during the liver regeneration period (16 17, 18 19 N DE and RE DN groups was not distinctive after the first month of transplantation, indicating the recovery of hepatic metabolism along with the regeneration of partial liver allograft takes about 1 month. Meanwhile, this recovery time in DDLT can be conjectured to be shorter because of receipt of a full-sized liver graft instead of partial grafted liver such as in LDLT (11, 12
Donor’s CYP3A5 genotype not recipient ’s exhibited genetic effect on tacrolimus pharmacokinetics in the studies of DDLT subjects (11, 12, 20 21 recipient ’s CYP3A5 genotype (22, 23 CYP3A5 genotype had minimal influence on tacrolimus metabolism similar to what was previously reported (24 13 CYP3A5 genotype combination of the donor and recipient for the first 35 days after LDLT. They reported a gradual decline of the C/D ratio, no hepatic influence in intestinal nonexpressers, and significant hepatic influence in intestinal expressers; however, they observed no difference between RN DE and RE DN and no prolonged difference between RE DE and RN DE after 1 month. This may be attributable to the characteristics of their study population, which was composed of both pediatric and adult transplant recipients. As previously described for DDLT, it may be difficult to detect the influence of intestinal metabolism in pediatric LDLT patients receiving relatively larger graft size than adults. Fukudo et al. (14
Tacrolimus is extensively distributed to red blood cells and bound to plasma protein, and the physiological status of the liver transplanted is expected to influence the pharmacokinetics of tacrolimus. Thus, covariates that have been known to affect tacrolimus disposition, such as time since transplantation, hematocrit, albumin, total bilirubin, alanine transaminase (ALT), body weight, and graft-to-recipient body weight ratio (GRWR) as well as demographics (1 CYP3A5 genetic variants with the pharmacokinetics of tacrolimus over time in the current study. The overall C/D ratio, which had stabilized at 6 months posttransplantation, gradually increased until 2 to 3 years after transplantation (Fig. 1 ). This trend of increasing C/D ratio might be explained mostly by less intensive and frequent sampling of the tacrolimus trough blood levels because of prolonged or delayed outpatient follow-ups, poor adherence to tacrolimus treatment and blood sampling, and relatively low dose of tacrolimus as well as inevitable gradual deterioration of liver function over time.
The allele frequency of the CYP3A5 *3 variant was 79.7% in our study, which is consistent with the reported frequencies of 76.5% to 81.4% for this variant in the Korean population (21, 25–27 CYP3A5 allele was speculated to be the wild type (*1) when the CYP3A5 6986G variant was not detected, as the CYP3A5 *6 allele is not expressed to any significant degree among the Korean population (26, 27 CYP3A5 to hepatic drug metabolism have been reported (4, 6
In conclusion, CYP3A5 genotypes of both recipient and donor were independently important factors influencing interindividual pharmacokinetic variability of tacrolimus. The recipient -donor combinational genetic effect on tacrolimus C/D ratio changed over time since transplantation. To individualize tacrolimus therapy, however, quantitative guidelines should be developed for tacrolimus dosage regimens according to CYP3A5 genotype of donor and recipient pairs considering time after transplantation in adult LDLT.
MATERIALS AND METHODS
Subjects
Between July 9, 2004, and August 7, 2006, a total of 103 adult patients received LDLT at a tertiary hospital affiliated with a university in Seoul, South Korea. Fifty-nine of these patients were recruited into this prospective 4-year follow-up study. One patient was excluded because of severe hepatic impairment, which was never resolved. Inclusion criteria were as follows: (1) patients on tacrolimus-based immunosuppressive therapy, (2) corresponding donor participation, and (3) patients older than 18 years. Exclusion criteria were patients with (1) multiorgan transplantation, (2) retransplantation, and (3) administration of any investigational or immunosuppressive agent not included as part of the study regimen during the study period. The study protocol was approved by the institutional review board (C-0505-148-005). All procedures were performed in accordance with the recommendations of the Declaration of Helsinki on Biomedical Research Involving Human Subjects, as well as the International Conference on the Harmonization of the Technical Requirements for the Registration of Pharmaceuticals for Human Use–Good Clinical Practice guidelines. All participants provided written informed consent before enrollment in the study.
Genotype Identification
Genomic deoxyribonucleic acid (DNA) was extracted from peripheral whole-blood samples of each subject using a Qiagen DNA extraction kit (Qiagen, Hilden, Germany). The presence of the CYP3A5*3 allele (6986A>G, rs 776746) was identified by mismatch polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis, as reported previously (25
Immunosuppression Protocol
Our institutional immunosuppression protocol, a triple or double regimen including tacrolimus and corticosteroids with or without mycophenolate mofetil (MMF), was applied to all study participants. The initial tacrolimus dosage was 0.03 mg/kg for the triple regimen or 0.05 mg/kg for the double regimen, administered twice daily at 10:00 and 22:00 on an empty stomach starting on the postoperational day (POD) 1. The following doses were adjusted based on the measured blood concentration to achieve the following target trough concentrations: 8 to 13 ng/mL until 2 weeks; 7 to 10 ng/mL between 2 weeks and 3 months; 5 to 7 ng/mL between 3 and 6 months; approximately 5 ng/mL thereafter for the triple regimen: 13–17, 8–13, and 5–10 ng/mL; and approximately 5 ng/mL during the respective periods for the double regimen. Clinical staff adjusting tacrolimus dosage were blinded to the individual patient’s genotyping results. Methylprednisolone was administered intravenously at a dose of 500 mg before and after reperfusion, then tapered gradually, switched to oral prednisolone at 20 mg on POD 7, and then tapered to discontinuation approximately 6 months after transplantation. Mycophenolate mofetil was administered at 0.5 g with white blood cell (WBC) count of greater than 3500/L or 0.25 g with WBC count of 2500 to 3500/L twice a day with tacrolimus. Patients did not receive any other medications known to interact significantly with tacrolimus. Prophylactic fluconazole of 50 mg daily was allowed because of its small amount and application to all participants.
Data Collection and Therapeutic Drug Monitoring
Tacrolimus C/D ratio was calculated as tacrolimus trough blood concentration in nanograms per milliliter divided by a dose administered before blood sampling in milligrams. Tacrolimus trough concentrations from the routine TDM were adopted to calculate the C/D ratio. Blood samples for tacrolimus routine TDM were collected daily at about 09:00 in the morning, beginning the day after the first dose and continuing until the day of discharge. Subsequent samples were obtained at each outpatient visit. Whole-blood tacrolimus concentrations were determined by microparticle enzyme immunoassay using an IMx analyzer and tacrolimus II reagent (Abbott Laboratories Diagnostic Division, Abbott Park, IL). Clinical laboratory test markers were measured simultaneously.
The written and electronic medical records of each subject were reviewed for abstraction of basic demographic information, concomitant medications, and laboratory test results associated with tacrolimus concentration with corresponding dose and body weight, hematocrit, albumin, total bilirubin, ALT, and pathologic data. The amount of methylprednisolone was converted to prednisolone by multiplying by 1.25.
Statistics
We performed an exploratory analysis to examine the time trends of C/D ratio changes for the four groups stratified by the CYP3A5 genotype of recipients and donors. To further examine the differences in the time trends of C/D ratio between RN DE and RE DN , cluster bootstrap analysis was performed with 5000 bootstraps, and the median and 95% confidence band were constructed for the difference. The distribution of C/D ratio was skewed to the right, and therefore, logarithmically transformed C/D ratio values were used in all analyses to reduce the skewness of the distribution.
The exploratory analyses motivated performing separate analyses for two periods, from the first day until 1 month and thereafter. The differences in C/D ratio among the four groups were examined separately in these two periods, using mixed effects models to take into account the correlation due to repeated measurements. The following covariates were selected a priori based on their potential effects on C/D ratio and adjusted in the analyses: age, body weight at each tacrolimus measurement, GRWR, hematocrit, albumin, total bilirubin, and ALT. To model linear and nonlinear time trends, we included a continuous time variable as well as its quadratic function in the models. The GRWR values for two subjects were missing, which were imputed using the median of GRWR values from 56 subjects.
Hardy-Weinberg equilibrium was tested using Pearson χ2 test for HWE in the R package genetics and our own R program. Data were presented as means, SDs, or ranges.
All analyses were performed using STATA 11.0 (Stata Corp., College Station, TX) and the statistical programming language R version 2.10.0 (28
ACKNOWLEDGMENT
The authors thank Jiyung Jun for the help in laboratory genotyping.
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