Effect of Tacrolimus Metabolism Rate on Renal Func-tion Among Hispanic Kidney Transplant Recipients

Tacrolimus (TAC) is important immunosuppressant given for graft survival after renal transplant...


Introduction
Tacrolimus (TAC), a Calcineurin Inhibitor (CNI), is the immunosuppressive drug of choice after renal transplantation because it has reduced acute rejection and improved renal function and graft survival [1,2]. However, many factors make management of tacrolimus complicated: its narrow therapeutic index, the large inter and intra individual variations in its pharmacokinetics characteristics, pharmaceutical interactions and polymorphisms in genes encoding TAC metabolizing enzymes [3,4]. A narrow therapeutic window and proven association between drug concentration and effect requires to perform Therapeutic Drug Monitoring (TDM) of tacrolimus routinely [5]. Subtherapeutic level increases the risk of acute rejection and development of de novo donor specific antibodies (dnDSA) [6] whereas supratherapeutic level increases the risk of adverse effects such as nephrotoxicity, neurotoxicity, infection, diabetes and malignancies. Therefore, it is necessary to keep a balance between efficacy and toxicity of the tacrolimus [7,8].
However, although blood levels fall within the therapeutic range, the actual efficacy or adverse effect may be different due to variability of interindividual susceptibility in drug response [9].
Therefore, ultimately, we need the personalizing CNI therapy based on understanding the factors that determine drug disposition in individual patients [3]. Also, it is clear reliable predictive marker is necessary to estimate efficacy and side effect of tacrolimus to protect graft for tacrolimus-treated kidney recipient. From this, it has been investigated about tool to optimize the individual doses of tacrolimus to achieve therapeutic targets and parameter for categorize high-risk patients identified by high metabolite rate to develop tacrolimus side effects [15]. The previous several studies have been explored the C/D ratio (TAC trough concentration/daily TAC dose) which define tacrolimus metabolization as a potential predictor of nephrotoxicity and acute rejection, or even higher mortality independently for tacrolimus-treated patients after transplantation [15][16][17][18]. Even though ethnicity is one of the factor for interindividual variability of drug metabolism and response [19]. there have been no studies based on specific ethnic variability about tacrolimus metabolization rate influencing clinical outcome.

Objective
We aim to determine whether the rate of the metabolism of TAC affects the renal function after renal transplantation and evaluate whether the C/D ratio can be reliable clinical predictor to estimate risk on allograft loss after kidney transplantation among Hispanic patients.

Tacrolimus Metabolism Rate
Once all data was collected, tacrolimus metabolism rates were calculated by dividing the TAC trough Concentration (C) by the corresponding daily TAC dose (D), as published using the following formula [16]. C/D ratio (ng/mL x 1/mg) = blood TAC trough level (ng/mL)/daily TAC dose (mg) TAC metabolism rate was determined at discharge time and at month 1, 3, 6, 9 and 12 after renal transplantation. The mean C/D ratio of the six periods used for patient categorization. We decided the C/D ratio thresholds based its mean value (1.93 ng/mL x 1/mg) because it showed symmetric distribution. Since the mean value of the C/D ratio in this study was higher than the mean value (1.29ng/mLx1/mg) shown in the previous study, we decided that the C/D ratio (between 1.05 and 1.55), which previously defined the intermediate group, was also included as the fast metabolizer group. We used cut-off C/D ratio of 1.55 ng/mL x 1/mg, so patient with a TAC C/D ratio ≤1.55ng/mL x 1/mg were defined as fast metabolizer and TAC C/D ratio >1.55 ng/ mL x 1/mg were categorized as slow metabolizer.

Statistical analyses
The demographic and clinical characteristics of the patients are presented as mean, Standard Deviation (SD), and percentage.
The Chi-squared test for categorial variables and the independent ttest for continuous variables were used to compare the 2 different TAC metabolism groups. P value < 0.05 was considered statistically

Study patients
A total of 303 self-reported Hispanic patients who had renal transplantation were recruited into this study. From these patients, 165 kidney allograft recipients were fulfilled our study inclusion criteria and they were categorized in two groups with different TAC metabolism rates. Ninety-two recipients (55.8 %) were classified as slow metabolizers and 73 recipients (44.2%) were categorized as fast metabolizers.

Study patients' demographic and clinical characteristics
Baseline patient characteristics for slow and fast metabolizers are shown in Table 1. The recipient's height showed a statistically correlation with the metabolism rate. Fast metabolizers were taller than slow metabolizers (p<0.001). Except for height, there were no significant differences in demographic features and clinical characteristics between metabolizer groups ( Table 1). The mean (± SD) age for slow metabolizers and fast metabolizers was 54.9 ± 9.9 years and 52.7 ± 12.0 years. Fifty-five (59.8%) of slow metabolizers and 52 (71.2%) of fast metabolizers were male. Majority of donor type of slow and fast metabolizers was the decreased donor (94.6% vs 91.8%). The proportion of patients diagnosed with diabetes as cause of ESRD was 54.3% and 42.5% in slow and fast metabolizers, respectively. Note: Results are presented as mean ± standard deviation (SD) ap-value is from the independent t-test b p-value is from the Chisquared test

Tacrolimus Daily Dose, Trough Level and Metabolism Rate
At 1, 3, 6, 9, and 12 months, the mean daily dose for TAC was significantly higher in fast metabolizers than slow metabolizers (Table 2). Fast metabolizers required more than twice of the TAC maintenance dose compared to slow metabolizers for entire followup period. The mean TAC trough levels of slow metabolizers were significantly higher than those of faster metabolizers at 1 month and 6 months after renal transplantation (1month: 7.0 ± 2.3 vs 6.0 ± 2.0 ng/mL, p = 0.003; 6 months: 6.7 ± 2.4 vs 5.9 ± 1.8 ng/mL, p = 0.012). At all points, there were no differences of blood pressures between two groups ( Table 2). As shown in Figure 1, the C/D ratio had symmetric distribution. The mean C/D ration values was 1.93 ± 1.068 ng/mL x 1/mg (range 0.35 to 5.74).
It is demonstrated that specific alleles of CYP 3A4/5 and ABCB1 were associated with TAC rapid metabolization and contributed a lower estimated glomerular filtration rate [22]. The expressor patients with CYP3A5*1/*1 or *1/*3 genotype require the higher tacrolimus dose to get target level due to the high level of metabolism in intestine and hepatic line, while the non-expressor patients with CYP3A5*3/*3 need lower dose due to slow metabolism rate [7,23,24]. In other words, fast metabolizers, characterized by a high tacrolimus requirement to reach the target trough level, has shown the small C/D ratio and presented CYP 3A5 *1/*1 expressor, while slow metabolizers need small dose to achieve targeted concentration, characterized as CYP 3A5 *3/*3 nonexpresser recipients exhibiting the large C/D ratio [4,18,20,21,25]. According to one report, which has shown ethnic variability in the frequencies of polymorphism in gene encoding for drug metabolizing enzyme, Hispanic population in the US has the largest frequency in CYP3A5*3 (0.245-0.867) and a higher prevalence of CYP2C9*3, CYP2C19*4, and UGT2B7 IVS1+985 A>G compared to that in non-Hispanics [26]. More than 80% of Caucasians are non-expressors i.e. the CYP3A5*3 [7]. In our study, patients who were categorized as slow metabolizers (55.8 %) were more than fast metabolizer (44.2%).
The mean C/D ratio values also higher than that of previous study (1.93 mg/mL x 1/mg vs, 1.29ng/mL x 1/mg) [16]. This result reflected majority of Hispanics have CYP3A5*3 genotype and can be categorized the slow metabolizers with low dose of tacrolimus requirement and high C/D ratio in our study.
There have been several studies related to the relationship between TAC level and allograft outcome. It has been suggested that higher tacrolimus trough-level variability was related with inferior renal graft outcome [25].The mechanism of action by which CNI increases nephrotoxicity can be explained as follows: vasoconstriction of afferent and efferent glomerular arterioles, and reduction of renal blood flow and GFR by decreased formation of prostaglandin E2 and inhibiting COX-2 inhibition Nitric Oxide (NO) synthesis [27][28][29].Based on the evidence of nephrotoxicity correlated to the tacrolimus blood levels, some studies emphasized TAC minimizing protocols and presented that low exposure TAC have been shown to decrease premature allograft loss [11,29].
Studies on minimizing TAC exposure have shown a correlation between mean trough levels and graft survival [11,30,31]. For example. one largest randomized clinical trial with 1645 renal recipients showed that reduced exposure to tacrolimus based regimen (trough levels 3-7ng/ml) provided better estimated GFR and allograft survival [11] whereas the usual therapeutic trough level is range from 5-20mg/L and for preventing toxicity the usual range is 5-15mg/L [13]. However, when low exposure to TAC is too much, it may contribute to de novo DSA formation so it should be tailored to the degree of risk the patient has for preventing TACassociated nephrotoxicity [32].
Although tacrolimus TDM is essential for targeting, there is the shortcoming of TDM that toxicity can occur even within the therapeutic range. Therefore, new approach about personalized use of tacrolimus considering intra-patient variability has been required. A new concept, C/D ratio, might be able to help identify patients at risk for TAC toxicity and can be used to individualize and optimize tacrolimus therapy contributing to improved graft outcomes. In several published studies, the C/D ratio can reflect tacrolimus metabolism rate and the fast metabolizer has been associated with relatively higher risk of impaired kidney functions [15,17,19,21,33]. loss, so TAC minimization is possible, while minimization is not recommended for patients with low C/D ratios [15][16][17][18].
However, there are influencing factors on C/D ratio such as CYP3A5 gene polymorphism, race, hepatic function, age, time after transplantation, hematocrit, and drug or food that induce/inhibit CYP 3A and/or ABCB1 activity [35]. As a result of this, it is necessary to evaluate whether the C/D ratio can be reliable clinical predictor to estimate risk on allograft loss. Gijsen  We conducted retrospective single center study and didn't control some confounders including drug interactions, comorbid diseases and donor characteristics. However, we investigated one strategy to personalize immunosuppressant management with certain study population having specific genetic characteristics.
To our knowledge, this is the first study of Hispanic population which examines associations between patients' tacrolimus metabolism rate and clinical outcomes (renal function) after renal transplantation. We showed non consistent association between C/D ratio and graft function compared to previous studies. This new understanding would help to improve predictions of the impact of C/D ratio. Prospective, controlled and multicenter trials are needed to determine whether C/D ratio can be powerful predictor of the graft outcome.

Conclusion
Our finding shows that there are significant association between C/D ratio and estimated GFR in tacrolimus-treated Hispanic patients after transplantation. We suggest that slow metabolizers with C/D ratio above 1.55 should be categorized into high risk group developing deteriorated renal function after transplantation. We propose the use of C/D ratio as alternative methods for assessing tacrolimus exposure and we expect this method can be useful tool to personalize tacrolimus therapy and to ensure the safety of the patient by identifying those patients who are at increased risk of developing CNI nephrotoxicity.

Conflicts of Interest
All authors declare that they have no conflict of interest.