Brief Communication
Kidney Transplant Recipients Carrying the CYP3A4
22
Allelic Variant Have Reduced Tacrolimus Clearance
and Often Reach Supratherapeutic Tacrolimus
Concentrations
N. Pallet
1,2,3,4,*, A.-S. Jannot
3,4,5, M. El Bahri
1,
I. Etienne
6, M. Buchler
7, B. H. de Ligny
8,
G. Choukroun
9, C. Colosio
10, A. Thierry
11,
C. Vigneau
12, B. Moulin
13, Y. Le Meur
14,
A.-E. Heng
15, J.-F. Subra
16, C. Legendre
17,
P. Beaune
1,3,4, C. Alberti
18, M. A. Loriot
1,3,4and E. Thervet
2,3,41
Clinical Chemistry, Hoˆpital Europe´en Georges
Pompidou, Assistance Publique Hoˆpitaux de Paris, Paris, France
2
Department of Nephrology, Hoˆpital Europe´en Georges Pompidou, Assistance Publique Hoˆpitaux de Paris, Paris, France
3
Paris Descartes University, Paris, France
4
Sorbonne Paris Cite´, INSERM UMRS, 1147, Paris, France
5
Department of Medical Informatics and Public Health, Hoˆpital Europe´en Georges Pompidou, Assistance Publique Hoˆpitaux de Paris, Paris, France
6
Department of Nephrology, CHU Rouen, Rouen, France
7
Department of Nephrology, CHU Tours, Tours, France
8
Department of Nephrology, CHU Caen, Caen, France
9
Department of Nephrology, CHU Amiens, Amiens, France
10
Department of Nephrology, CHU Reims, Reims, France
11
Department of Nephrology, CHU Poitiers, Poitiers, France
12
Department of Nephrology, CHU Rennes, Rennes, France
13
Department of Nephrology, CHU Strasbourg, Strasbourg, France
14
Department of Nephrology, CHU Brest, Brest, France
15
Department of Nephrology, CHU Clermont-Ferrand, Clermont-Ferrand, France
16
Department of Nephrology, CHU Angers, Angers, France
17
Department of Nephrology, Necker Hospital, Assistance Publique Hoˆpitaux de Paris, Paris, France
18
Clinical Investigation Center, Assistance Publique-Hoˆpitaux de Paris, Hoˆpital Robert Debre´, Paris, France
Corresponding author: Nicolas Pallet, npallet@yahoo.fr
CYP3A422 is an allelic variant of the cytochrome P450 3A4 associated with a decreased activity. Carriers of this polymorphism may require reduced tacrolimus
(Tac) doses to reach the target residual concentrations (Co). We tested this hypothesis in a population of kidney transplant recipients extracted from a multi-center, prospective and randomized study. Among the 186 kidney transplant recipients included, 9.3% (18 patients) were heterozygous for the CYP3A422 geno-type and none were homozygous (allele frequency of 4.8%). Ten days after transplantation (3 days after starting treatment with Tac), 11% of the CYP3A422 carriers were within the target range of Tac Co (10– 15 ng/mL), whereas among the CYP3A41/1 carriers, 40% were within the target range (p¼ 0.02, OR ¼ 0.19 [0.03; 0.69]). The mean Tac Co at day 10 in the CYP3A41/22 group was 23.5 ng/mL (16.6–30.9) com-pared with 15.1 ng/mL (14–16.3) in the CYP3A41/1 group, p< 0.001. The Tac Co/dose significantly de-pended on the CYP3A4 genotype during the follow-up (random effects model, p< 0.001) with the correspond-ing equivalent dose for patients heterozygous for CYP3A422 being 0.67 [0.54; 0.84] times the dose for CYP3A41/1 carriers. In conclusion, the CYP3A422 allelic variant is associated with a significantly altered Tac metabolism and carriers of this polymorphism often reach supratherapeutic concentrations.
Abbreviations: Co, residual concentration; CYP, cyto-chrome; Tac, tacrolimus
Received 15 September 2014, revised and accepted for publication 08 October 2014
Introduction
Personalized medicine aims at tailoring the prescription of drugs to patients based on the knowledge of genetic polymorphisms eventually leading to functional variations. Generally based on genetic and/or phenotypic testing, this approach is particularly relevant for drugs with a narrow therapeutic window and a great inter-individual variability, such as immunosuppressive agents. A large body of evidence supports that prior knowledge of variations within genes encoding tacrolimus (Tac) drug metabolizing en-zymes and transporters, by means of pharmacogenetic testing, could help to better predict the target drug concentration, thereby, avoiding potentially harmful over-or underexposure (1,2).
and the American Society of Transplant Surgeons doi: 10.1111/ajt.13059
Tac is metabolized, and degraded, by the cytochrome P450 (CYP) system, mainly the CYP3A subfamily. The two isoforms CYP3A4 and 3A5 are implicated in the hepatic and intestinal metabolism of Tac, respectively, with CYP3A5 being more efficient in oxidative metabolism of Tac than CYP3A4 (3–5). Genetic polymorphisms of CYP3A5 are associated with variations in Tac pharmacokinetic profiles. Specifically, the g.6986A>G (rs776746) variant in the intron 3 of the CYP3A5 gene, encoding the CYP3A53 allele, promotes an RNA splicing defect leading to the lack of expression of the enzyme (6,7). Consequently, nonex-pressers of this enzyme (CYP3A53/3) have a higher bioavailability and exposure of Tac because of an increased intestinal absorption. Conversely, individuals carrying at least one functional CYP3A51 allele do express CYP3A5, and require higher Tac doses to reach target concentra-tions (5,8). This led to the concept that the knowledge of the CYP3A5 expresser/nonexpresser status prior to the intro-duction of Tac based-therapy could help to adapt the dosage of the drug, and, nowadays, routine testing of CYP3A53 prior to transplantation is implemented in clinical chemistry departments (9,10).
Tac is also metabolized in the liver by CYP3A4, and the inter-individual variations of activity of this enzyme are remark-able (11,12). Recently, a functional polymorphism has been described in the intron 6 of the gene encoding CYP3A4 (rs35599367, g.15389C>T), which is associated with a lower expression level of CYP3A4 mRNA and protein, leading to decrease in global enzyme activity (13). This so-called CYP3A422 allele may impact Tac clearance in kidney and heart transplant recipients (14–16).
The Tactique study was initially undertaken to test whether Tac residual blood concentration (Co) reached the target concentration (10–15 ng/mL) 10 days posttransplantation (3 days after starting treatment with Tac) more rapidly when the dosages of Tac were adapted to the CYP3A5 genotype (10). In the present study, we took advantage of this large, multicenter, prospective and randomized study to determine the influence of CYP3A422 on Tac metabolism during the first 3 months after kidney transplantation. To do this, we analyzed the association between CY3A422 genotype and the initial primary outcome of this trial, that is the proportion of patient reaching the target concentration 10 days after transplan-tation. We also tested the association between CYP3A4 genotype and the Co out of Tac daily dose ratio (Tac Co/ dose) over time.
Patients and Methods
Study design
The Tactique study was conducted to evaluate whether adaption of Tac dosing according to the CYP3A5 genotype would allow earlier achievement of target blood concentrations of Tac in kidney transplant recipients. The design of the study is detailed in (10). Briefly, from April 2006 to
October 2007, a total of 280 patients from 12 sites underwent randomization for the study. The patients were randomly assigned at day 7 posttrans-plantation to receive Tac at either a fixed dosage of 0.2 mg/kg/day (control group) or a dosage determined by their genotype: CYP3A5 expressers (i.e. carriers of the CYP3A51 allele) received 0.3 mg/kg/day, whereas CYP3A5 nonexpressers (CYP3A53/3 genotype) received 0.15 mg/kg/day (adapted-dose group). The first measurement of Co was performed after the intake of six doses (¼day 10 after transplantation), after which physicians could modify the daily dose in order to achieve a prespecified target range of Co (10–15 ng/mL). The primary efficacy end point was the proportion of patients for whom Tac Co was within target range (10–15 ng/mL) at day 10.
All patients received a biological induction (17.8% basiliximab, 82.2% rabbit thymocyte antiglobulin), 3 g of mycophenolate mofetil (Cell-Cept; Roche, Basel, Switzerland) daily for 15 days (tapered to 2 g/day) and a tapered corticosteroid regimen. The study was approved by the Institutional Review Board at each participating center, and written informed consent was obtained from all patients. The study was carried out in compliance with the provisions of the Declaration of Helsinki and the Good Clinical Practice guidelines. The clinical and research activities being reported are consistent with the Principles of the Declaration of Istanbul as outlined in the Declaration of Istanbul on Organ Trafficking and Transplant Tourism.
For the retrospective study presented here, we included all patients included in this trial having a Tac dosage at day 10 after transplantation. In addition, the patients included were nonexpressers for CYP3A5 (CYP3A53/3) because the vast majority (19 out of 21) of the CYP3A422 carriers were also CYP3A53/3 carriers (see below). The CYP3A422 carriers were distributed in the randomization groups as follows: 64% of CYP3A422 individuals were in the control group, and 50% of the CYP3A41 individuals were in the control group. The median 95% confidence interval (CI) in the control group (fixed Tac starting dose, 0.2 mg/kg/day) was for CYP3A422: 0.20 (0.18–0.20) mg/kg/day, and for CYP3A41: 0.20 (0.18–0.20) mg/kg/day. The median 95% CI in the dosage determined by genotype group (adapted Tac starting dose, 0.15 mg/kg/day for CYP3A53/3 or 0.30 mg/kg/day for CYP3A51), was for CYP3A422: 0.15 (0.14–0.16) mg/kg/day, and CYP3A41: 0.15 (0.14–0.15) mg/kg/day
CYP3A422 genotyping
DNA was extracted from peripheral blood leukocytes using the Blood DNA kit (Qiagen, Courtaboeuf, France) in accordance with standard protocols. Genotyping of the CYP3A422 allele was performed using TaqMan Assay Reagents for allelic discrimination (Applied Biosystems, Courtaboeuf, France) with a 7900HT Applied Biosystems thermal cycler.
Statistical analyses
We first tested the Hardy-Weinberg equilibrium for CYP3A4 genotype using a chi-square test comparing observed genotypic distribution and expected genotypic distribution under Hardy-Weinberg equilibrium.
We tested linkage disequilibrium between CYP3A4 and CYP3A5 by testing the association between the genotypes at the two loci using a Fisher exact test.
Demographic and baseline characteristics were summarized as counts and percentages for categorical variables, and continuous variables as medians with inter-quartiles ranges (IQR). Demographic and baseline characteristics were compared between CYP3A4 expressers and nonexpressers using Fisher exact test for categorical data, and using the Wilcoxon test for continuous variables. All tests were two tailed, and a p-value<0.05 was considered statistically significant.
For the primary outcome, we compared the proportion of patients within the target Tac Co range (10–15 ng/mL) 10 days after transplantation in both groups (CYP3A4 expressers and nonexpressers) by testing statistical significance between the two groups using a Fisher exact test. We estimated the corresponding odds ratio (OR) and its associated CI using contingency tables and by computing the proportions of patients within the target Co in both groups.
We tested the association between CYP3A4 genotype and Tac Co/dose (secondary outcome) using mixed linear regression models with CYP3A4 as independent variable and Tac Co/dose as dependent variables and with patients as random effect. We compared models including only random intercept and both random intercept and random time slope using an analysis of variance.
We plotted the relationship between CYP3A4 genotypes and Tac Co, Tac Co/dose and Tac dose for the different time points (median values and 95% CI).
All analyses were performed using R 3.1.0 software (package nlme for mixed linear models and ggplot2 for graphs; www.r-project.org/)
Results
Distribution and characteristics of the CYP3A422 genotypes
DNA samples were available for a total of 272 patients. Among these 272 patients, 251 patients were identified as WT homozygotes (CYP3A41/1), 21 were heterozygous (CYP3A41/22), and none were mutant homozygote (CYP3A422/22) (Table 1), therefore there was no depar-ture from Hardy-Weinberg equilibrium (p-value for chi-square test¼ 0.44).
The frequency of the CYP3A422 allele in the entire cohort including all CYP3A5 genotypes was 3.9%, which is in accordance with previously published data (14). The vast majority (19 out of 21, 90%) of the CYP3A422 carriers were nonexpressers for the CYP3A5 (CYP3A53/3). Of note, CYP3A4 and CYP3A5 were not in linkage disequilibri-um (p¼ 0.57, Fisher exact test for genotypes).
Because only two patients were CYP3A422 carriers and CYP3A5 expressers, we restricted our analysis to the CYP3A53/3 (nonexpressers) patients in order to have a
homogenous population in terms of CYP3A5 expression. Among the 217 nonexpressers for the CYP3A5, 186 (86%) had a dosage of Tac available at day 10. Consequently, the study population was made of 186 patients: 18 CYP3A41/
22 carriers and 168 CYP3A41/1 carriers (frequency of
4.8% for CYP3A422 allele in the study population). The characteristics and demographic data of this population of CYP3A53/3 carriers according to the CYP3A422 genotype are detailed on the Table 2. The two groups of patients were well balanced regarding the recipients and donors demographic and clinical characteristics. Notably, the initial daily dose of Tac (day 7 posttransplantation) was similar between CYP3A41/1 carriers and CYP3A41/22 carriers (median value of 0.16 mg/kg/day vs. 0.18 mg/kg/ day, respectively p¼ 0.13). All patients for whom ethnical origin was available (137, 74%) were Caucasian, and the genotypic distribution of CYP3A4 did not depend on the availability of this information (p¼ 0.57, Fisher exact test).
Table 1: Frequency and distribution of CYP3A4 alleles
Genotype CYP3A41/1 (normal metabolizer) CYP3A41/22 (slow
metabolizer) CYP3A422/22 Total CYP3A51/1 (expresser) 12 0 0 12 CYP3A51/3 (expresser) 41 2 0 43 CYP3A53/3 (nonexpresser) 198 19 0 217 Total 251 (92.2) 21 (7.7%) 0 272
Table 2: Demographic and baseline characteristics (median and interquartile ranges or number and proportion) of the CYP3A53/3 carriers according to the CYP3A422 status
CYP3A41/1 (168)
CYP3A41/22
(18) p
Recipient age (years) 48 (38–57) 49 (1–57) 0.82 Male recipient (n, %) 105 (62%) 15 (83%) 0.60 Weight (kg) 69 (61–80) 70 (65–77) 0.36 Cause of end-stage renal disease 0.09 Chronic glomerulonephritis 52 (31%) 4 (21%) – Polycystic kidney disease 30 (18%) 6 (35%) – Lupus 0 (0%) 1 (5%) – Uropathy 22 (13%) 2 (10%) – Diabetes 5 (3%) 2 (10%) – Unknown 15 (9%) 1 (5%) – Nephroangiosclerosis 14 (8%) 1 (5%) – Hereditary nephropathy 9 (5%) 1 (5%) – Other 18 (10%) 0 (0%) – Cause of donor death 0.30 Trauma 52 (31%) 5 (28%) – Cerebrovascular accident 75 (45%) 8 (44%) – Others 38 (23%) 5 (28%) – Cold ischemia time (hours) 15 (9–17) 16 (12–19) 0.68 Previous transplantation (n, %) 7 (4.5%) 2 (11%) 0.2 Tacrolimus dose at day 7 0.16 (0.15–0.20) 0.18 (0.16–0.21) 0.13 Creatinine level at day 7 148 (107–260) 217 (140–394) 0.14
Impact of the CYP3A422 genotype on residual Tac concentrations 10 days after transplantation
To evaluate the impact of the CYP3A422 allele on Tac Co, we measured the proportion of patients reaching the target Tac Co range (10–15 ng/mL) between carriers of CY-P3A41/22 and CYP3A41/1 genotypes 10 days after transplantation, which correspond to the first measure-ment of Tac Co after the intake of six doses, and which was the primary end point of the Tactique study. Only 11% of the CYP3A41/22 carriers were within the target range of Tac Co (10–15 ng/mL) at this time point, whereas among the CYP3A41/1 carriers, 40% were within the target range (p¼ 0.02, Fisher exact test, OR ¼ 0.19 [0.03; 0.69]) (Figure 1). The mean Co at day 10 in the CYP3A41/22 group was 23.5 ng/mL (16.6–30.9) compared with 15.1 ng/ mL (14–16.3) in the CYP3A41/1 group (Figure 2A), p< 0.001, using a Wilcoxon test.
Impact of the CYP3A422 allele on Tac Co/dose over time
The Tac Co/dose distribution was skewed and therefore we log-transformed it to obtain a Gaussian distribution. We tested the association between the log-ratio of the Tac Co/ dose and CYP3A4 genotype using a linear mixed effects model. We obtained a better fit (p< 0.0001, analysis of variance) for the linear model with both random intercept and random time slope than for the model with only random intercept. In the linear mixed effect model with fixed effects for CYP3A4 genotype and time, and random intercept and random time slope for patient effects, the log-ratio of the Tac Co/dose depended significantly on the genotype of CYP3A4 during the follow-up (p< 0.001) (Figure 2B). The regression coefficient for the fixed effect CYP3A4 was 0.67 [0.54; 0.84] indicating that the corresponding equivalent dose for patients heterozygous for CYP3A422 was 0.67 [0.54; 0.84] times the dose for patients homozygous for CYP3A41/1 (Figure 2C).
Figure 1: Proportion of patients within the prespecified Tac Co target range 10 days after transplantation. Plot representing the proportion 95% CI of patient reaching the target concentration at days 10, 14, 30, 60 and 90 after transplantation according CYP3A4 genotypes (CYP3A41/1 carriers: triangles with dotted line, CYP3A41/22: diamond with plain line). The statistical significance between the two groups has been tested using a Fisher exact test.p¼ 0.02. Prespecified Tac Co target range is between 10 and 15 ng/mL.
Figure 2: Impact of the CYP3A422 allele on Tac Co and daily dose over time. Plots representing mean 95% CI of Tac Co (A), log-ratio of Tac Co/dose (B) and Tac dose (C) at days 10, 14, 30, 60 and 90 after transplantation according to CYP3A4 genotypes (CYP3A41/1 carriers: triangles with dotted line, CYP3A41/22: diamond with plain line).p¼ 0.02, Wilcoxon test.
Discussion
In this study, we have demonstrated that a recently described polymorphism in the gene encoding the CYP3A4, and which leads to a decreased expression of the protein in the liver, is associated with a slower Tac metabolism and higher systemic exposition in the early posttransplant period, and that the patients carrying this gene variant may require 30% less Tac dose than patients with the CYP3A41/1 genotype. Importantly, the CY-P3A422 allele is almost exclusively found in the patients of our study who do not express the CYP3A5 in the intestine (CYP3A53/3). This indicates that among the patients who do not express the CYP3A5, and who may require less Tac doses than patients carrying one CYP3A51 allele, a sub group of patients (CYP3A422 carriers, 5–10%) may still require reduced Tac dose to reach the target Tac Co. Therefore, combining CYP3A4 and CYP3A5 genotypes may have an increased significance compared with the geno-types considered alone, and that a group classification might lead to a better prediction of the optimal Tac starting dose.
Our study is in accordance with the results recently published in heart and kidney transplant recipients, which demonstrated that the great majority of the patients carrying a CYP3A422 are CYP3A5 nonexpressers, and that the overall mean daily dose requirement to reach target Tac concentrations was in average 30% lower in these patients than in those carrying the CYP3A41/1 geno-type (14–16). These results are consistent with the fact that this variant is associated with a reduction of mRNA production and global activity of the enzyme in the liver, and constitutes the first relevant CYP3A4 gene variant with clinical impact.
Some caution should be paid in interpreting the role of CYP3A422 based on the present analysis because, inherent to its design, the kidney transplant recipients included in the trial were highly selected, without extended criteria donors, and their immunological risk of rejection was low. Even if the immunosuppressive regimen was the standard (corticosteroids, mycophenolate mofetil and Tac), the target concentrations of Tac are higher than at the present time, given that the current strategy aims at reducing calcineurin inhibitor exposure. Finally, due to the relatively low number of CYP3A422 carriers, some potential cofounders, like variations in absorption profiles among kidney transplant recipients, could limit the assess-ment of the impact of the genotype in Tac doses and concentrations.
In conclusion, the CYP3A422 allele is associated with Tac overexposure in the early posttransplant period, and patients carrying this variant allele may require lower Tac dose than the others. Multiple genotype-based dosage adjustment taking into account CYP3A5 and CYP3A4 allelic variants might help to optimize initial Tac doses more
accurately than CYP3A5 alone. Our study provides argu-ments for implementation of the CYP3A422 status in genotype-based guidelines on initial Tac dose adjustments, which, initially based on the CYP3A5 status only, recom-mend doubling the dose in CYP3A5 expressers (0.15 mg/ kg/day compared 0.075 mg/kg/day in the nonexpressers). Based on our findings, we propose that the CYP3A5 nonexpressers (CYP3A53/3 genotype), who are slow CYP3A4 metabolizers (carriers of CYP3A422 allele) could receive 30% less Tac dose, namely 0.05 mg/kg/day compared to CYP3A4 rapid metabolizers for who 0.075 mg/kg/day could be prescribed. Of note, this proposition is based on Co that are slightly higher than those currently recommended, and need to be evaluated in prospective trials. In addition, these recommendations cannot be extended to the very infrequent CYP3A422 carriers who express CYP3A5 (only two cases in our cohort).
Disclosure
The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.
References
1. Elens L, Hesselink DA, van Schaik RH, van Gelder T. Pharmacogenetics in kidney transplantation: Recent updates and potential clinical applications. Mol Diagn Ther 2012; 16: 331–345.
2. Hesselink DA, Bouamar R, van Gelder T. The pharmacogenetics of calcineurin inhibitor-related nephrotoxicity. Ther Drug Monit 2010; 32: 387–393.
3. Kamdem LK, Streit F, Zanger UM, et al. Contribution of CYP3A5 to the in vitro hepatic clearance of tacrolimus. Clin Chem 2005; 51: 1374–1381.
4. Lampen A, Christians U, Guengerich FP, et al. Metabolism of the immunosuppressant tacrolimus in the small intestine: Cytochrome P450, drug interactions, and interindividual variability. Drug Metab Dispos 1995; 23: 1315–1324.
5. Staatz CE, Goodman LK, Tett SE. Effect of CYP3A and ABCB1 single nucleotide polymorphisms on the pharmacokinetics and pharmacodynamics of calcineurin inhibitors: Part II. Clin Pharma-cokinet 2010; 49: 207–221.
6. Kuehl P, Zhang J, Lin Y, et al. Sequence diversity in CYP3A promoters and characterization of the genetic basis of polymorphic CYP3A5 expression. Nat Genet 2001; 27: 383–391.
7. Lee SJ, Usmani KA, Chanas B, et al. Genetic findings and functional studies of human CYP3A5 single nucleotide poly-morphisms in different ethnic groups. Pharmacogenetics 2003; 13: 461–472.
8. Staatz CE, Goodman LK, Tett SE. Effect of CYP3A and ABCB1 single nucleotide polymorphisms on the pharmacokinetics and pharmacodynamics of calcineurin inhibitors: Part I. Clin Pharma-cokinet 2010; 49: 141–175.
9. Elens L, Bouamar R, Shuker N, Hesselink DA, van Gelder T, van Schaik RH. Clinical implementation of pharmacogenetics in kidney
transplantation: Calcineurin inhibitors in the starting blocks. Br J Clin Pharmacol 2014; 77: 715–728.
10. Thervet E, Loriot MA, Barbier S, et al. Optimization of initial tacrolimus dose using pharmacogenetic testing. Clin Pharmacol Ther 2010; 87: 721–726.
11. Shimada T, Yamazaki H, Mimura M, Inui Y, Guengerich FP. Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: Studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther 1994; 270: 414–423. 12. Westlind A, Lofberg L, Tindberg N, Andersson TB,
Ingelman-Sundberg M. Interindividual differences in hepatic expression of CYP3 A4: Relationship to genetic polymorphism in the 50-upstream regulatory region. Biochem Biophys Res Commun 1999; 259: 201–205.
13. Wang D, Guo Y, Wrighton SA, Cooke GE, Sadee W. Intronic polymorphism in CYP3A4 affects hepatic expression and response to statin drugs. Pharmacogenomics J 2011; 11: 274–286. 14. Elens L, Bouamar R, Hesselink DA, et al. A new functional CYP3A4
intron 6 polymorphism significantly affects tacrolimus pharmaco-kinetics in kidney transplant recipients. Clin Chem 2011; 57: 1574– 1583.
15. Elens L, Capron A, van Schaik RH, et al. Impact of CYP3A422 allele on tacrolimus pharmacokinetics in early period after renal transplantation: Toward updated genotype-based dosage guide-lines. Ther Drug Monit 2013; 35: 608–616.
16. Gijsen VM, van Schaik RH, Elens L, et al. CYP3A422 and CYP3A combined genotypes both correlate with tacrolimus disposition in pediatric heart transplant recipients. Pharmacogenomics 2013; 14: 1027–1036.
