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Effect of CYP3A4*22, POR*28, and PPARA rs4253728 on sirolimus in vitro metabolism and trough concentrations in kidney transplant recipients.

Sirolimus (SRL) [7] is an inhibitor of the mammalian target of rapamycin used as a second-line immunosuppressive drug in solid organ transplantation. It is a substrate of intestinal and hepatic cytochrome P450 3A (CYP3A) (1) and of the P-glycoprotein (P-gp) (2, 3), which both contribute to its first-pass elimination and consequently to its low and variable bioavailability. There is currently no evidence supporting an influence of polymorphisms of the gene ABCB1 [ATP-binding cassette, sub-family B (MDR/TAP), member 1] [8] (the P-gp-encoding gene) (4) on SRL pharmacokinetics and clinical outcomes in kidney transplant patients. On the other hand, controversial results have been published concerning the impact on SRL exposure of polymorphisms of the gene CYP3A (cytochrome P450, family 3, subfamily A) (5-7). The CYP3A5*3 allele (rs776746 A>G), which results in a truncated enzyme (8), has been associated with higher SRL blood concentrations in kidney transplant recipients. However, this effect would be of concern only for patients on calcineurin inhibitor (CNI)-free regimens and was not consistently found across studies (5-7, 9-11).

Recently, a newly discovered polymorphism in CYP3A4 (cytochrome P450,

family 3, subfamily A, polypeptide 4) intron 6 (rs35599367 C>T; CYP3A4*22), associated with decreased mRNA hepatic expression and enzymatic activity, was found to be significantly associated with increased cyclosporine and tacrolimus doseadjusted concentrations in kidney transplant recipients (12,13). The variant allele also resulted in a higher risk of decreasing glomerular function and worse creatinine clearance in cyclosporine-treated patients (14).

A single-nucleotide polymorphism (SNP) in the POR [P450 (cytochrome) oxidoreductase] gene (rs1057868 C>T; POR*28 allele) was also recently shown to result in higher tacrolimus dose requirements in CYP3A5 (cytochrome P450, family 3, subfamily 5) nonexpressers (15). Consistently, this SNP was associated with a 1.6-fold increase in CYP3A activity as measured by the midazolam phenotyping test in 2 independent population datasets (16).

Finally, a comprehensive study associating in vitro and clinical data identified an SNP in PPARA (peroxisome proliferator-activated receptor alpha) (rs4253728) as another candidate to explain the variability in CYP3A4 activity (17).

Altogether, these results suggest that pharmacogenetic testing might allow the classification of individuals according to their CYP3A metabolic capability. None of these newly discovered genetic variations have been studied with regard to their impact on SRL metabolism, exposure, and adverse events.

Therefore, our aim was to assess the individual and combined impact of CYP3A4*22, CYP3A5*3, POR*28, and PPARA-rs4253728 alleles on (a) SRL in vitro hepatic metabolism, (b) SRL trough concentrations ([C.sub.0]), and (c) SRL adverse events in kidney transplant recipients.

Material and Methods

IN VITRO EXPERIMENTS

SRL (100 [micro]g/L) was incubated with individually prepared human liver microsomes (HLM) (n = 31) (donor characteristics are given in Table 1 in the Data Supplement that accompanies the online version of this report at http://www.clinchem.org/content/vol59/ issue 12) and pooled HLM as previously described (18). Briefly, the incubation mixture consisted of 0.1 mol/L Tris buffer, pH 7.4, 10 mmol/L Mg[Cl.sub.2], 2 mmol/LNADPH, HLM (0.1 mg protein/mL), and SRL prepared in acetonitrile/water (25/1.25 [micro]L) (1.25% acetonitrile in final incubation medium). Incubations (100 iL) were performed at 37 [degrees]C after a 5-min pre-incubation of the reagents. The reaction was initiated by adding NADPH. After 10 min, the reaction was stopped using ice-cold acetonitrile (100 [micro]L). The remaining concentration of SRL in the incubation mixture was determined by using liquid chromatographytandem mass spectrometry (LC-MS/MS) as described previously (18).

PATIENT POPULATION

One hundred and thirteen adult kidney transplant patients (80 males and 33 females) switched from a CNI to SRL at least 3 months after transplantation [median (25th-75th percentiles) 3.8 (1.2-9.3) years; minimum, 0.3, maximum, 25.9 years] were included in this retrospective study. Sixty-five percent of the patients were switched from cyclosporine and 35% from tacrolimus. At the time of the switch to SRL, the mean (SD) patient age was 52 (14) years. This study complied with the Declaration of Helsinki and was approved by an ethics committee, and written informed consent was obtained from each patient enrolled. None of the patients discontinued SRL within the first 6 months post-conversion for uncontrolled adverse events. SRL [C.sub.0] determination was chosen as a marker of exposure because of the excellent correlation between [C.sub.0] and interdose area under the concentration curve previously reported ([r.sup.2] = 0.83-0.95) (19-21). All patients had a [C.sub.0] determination once a week until they obtained the target value and then every month. For each patient, clinical data were recorded [date of transplantation and date of SRL introduction, age, sex, presence and date of occurrence of cutaneous adverse effects (AEs), lymphedemas, and infections]. Infections were defined as severe infections at any location that required hospitalization. Cutaneous AEs were defined as cutaneous lesions, folliculites, aphthous stomatitis, macula-papular and squamous lesions, and acne. Edemas and pneumonitis were also collected from the clinical files. Additionally, the doses of all immunosuppressants, hemoglobin concentrations, plasma total cholesterol, plasma triglyceride concentrations, and the dose and type of statins were retrospectively collected from patient charts at 1, 3, and 6 months after the initiation of SRL. Dose reduction was defined as reduction of SRL dose occurring in any context at 3 or 6 months compared to the dose at 1 month postswitch to SRL. SRL dose changes occurring within the period were not recorded. No period earlier than 1 month post-SRL introduction was considered, to take into account the time required to reach steady-state. Statin administration was categorized in 4 strata: no statin (statins = 0), low dose (statins = 1; 10mg/day of pravastatin, simvastatin, or atorvastatin), middle dose (statins = 2; 20, or 30 mg/day of pravastatin and 20-40 mg/day of simvastatin or atorvastatin), and high dose (statins = 3; 40 mg/day of pravastatin and 50-80 mg/ day of simvastatin or atorvastatin). Patient demographics and laboratory test values were previously reported (22).

IDENTIFICATION OF GENOTYPES

Microsomes and patients were genotyped for CYP3A5 rs776746 A>G (CYP3A5*3), CYP3A4 rs35599367 C>T (CYP3A4*22), POR rs1057868 C>T (POR*28), and PPARA rs4253728 G>A by use of Taqman[R] allelic discrimination assays on an ABI PRISM 7000 sequence detection system (Applied Biosystems). Assays were ordered from Applied-Biosystems as custom (CYP3A5 assay ID, AHD0879) or inventoried TaqMan SNP genotyp ing assays (POR assay, C_8890131_30; CYP3A4 assay, C_59013445_10; PPARA assay, C_31052401_10).

SRL LC-MS/MS ASSAY

SRL was determined in blood and in vitro samples by using turbulent-flow chromatography-tandem mass spectrometry using a validated, previously described method (18).

STATISTICAL ANALYSES

Statistical analyses were performed using R software version 2.13.1 (R foundation for statistical computing, http://www.r-project.org). Conformity of genotyping data with Hardy-Weinberg equilibrium was verified with the Fisher's exact test in the "SNPassoc" package. The Kruskal-Wallis or Mann-Whitney tests were used to compare the SRL in vitro disappearance rate between genotype groups. Genetic models were chosen on the basis of genotype frequencies and the literature. The normality of the distribution of continuous phenotypes was assessed by both visual examination of the probability density function and the Shapiro-Wilk test. SRL [C.sub.0] concentrations, SRL dose, and SRL [C.sub.0]/ dose were log-transformed before analysis to obtain normal distributions.

In a first step, associations between nongenetic covariates and phenotypes were investigated using a backward stepwise process. Intermediate models including the significant covariates were selected based on the Akaike criterion. Then, SNPs were included in these adjusted models and a backward selection based on the Bayesian information criterion was performed to obtain the final models.

We investigated the effects of genotypes on SRL dose, [C.sub.0] and [C.sub.0]/dose, and hemoglobin, cholesterol, and triglyceride plasma concentrations using linear mixed-effect models ("lme4" package), which allowed correlations between observations per patient. The effects of genotypes on infections, cutaneous AEs, edema, pneumonitis, and dose reduction were investigated by using logistic regression with only the first event taken into account. SRL exposure was studied in these analyses as the area under the curve of all the available [C.sub.0]/dose data in the follow-up period [cumulative exposure = equivalent to (mean [C.sub.0]/dose) X follow-up duration]. The Benjamini-Hochberg false discovery rate was applied to the final models (23) to account for multiple testing. All statistical analyses were 2-sided except in vitro comparisons. P < 0.05 was considered statistically significant.

Results

IN VITRO DATA

Microsomes carrying the CYP3A4*22 allele (n = 3 heterozygotes and 1 homozygote) metabolized SRL at significantly lower rates than noncarriers (n = 27) [65.5 (55.2-80.7) vs 86.1 (53.4-105.2) pmol/mg/min; P = 0.0411]. In contrast, no significant effect of POR*28 (P = 0.4606) or PPARA rs4253728 (P = 0.2766) was observed (Fig. 1).

GENOTYPE DISTRIBUTION

Patient genotypes are described in online Supplemental Table 2. Genotyping results were in HardyWeinberg equilibrium. An important linkage disequilibrium was found between rs35599367 (CYP3A4*22) and rs776746 (CYP3A5*3) (D' = 0.78, [r.sup.2] = 0.58). Patients carrying the CYP3A4*22 defective allele (n = 11) were more frequently carriers of the inactive CYP3A5*3 (n = 9/11) than the CYP3A5*1 allele (n = 2/11). According to genotype frequencies and to the literature, all the genotypes were studied using a dominant genetic model.

IMPACT OF GENETIC VARIATIONS ON SRL EXPOSURE

Effects of genotypes. No significant association was found between CYP3A4 and CYP3A5 genotypes and SRL dose, [C.sub.0], or [C.sub.0]/dose (Table 1). After correction for multiple testing, only the POR*28 allele (CT/TT vs CC) was associated with a significant decrease in SRL log transformed [C.sub.0] values over the follow-up period [[beta] = -0.15 (0.05); P = 0.0197] but it had no effect on SRL log-transformed dose or [C.sub.0]/dose (Table 1). The evolution of SRL [C.sub.0], [C.sub.0]/dose, and dose in carriers and non-carriers of POR*28 is depicted in Fig. 2. When combined with the CYP3A genotypes, this influence of the POR*28 polymorphism concerned only carriers of CYP3A4*1 (CYP3A4*1/POR*28 vs CYP3A4*1/POR*1 alleles; P = 0.0150). No significant differences were observed for patients carrying other allele combinations compared to the CYP3A4*1/POR*1 or CYP3A5*1/POR*1 combinations chosen as references (data not shown). No effect of the POR*28 allele was observed on log-transformed SRL [C.sub.0]/dose (Table 1), regardless of the combination of genotypes (Fig. 3).

Effect of predicted CYP3A metabolic status. The effect of combined CYP3A genotypes on log-transformed [C.sub.0] and [C.sub.0]/dose was investigated at each period. Patients were classified on the basis of their expected metabolic status. Poor metabolizers were defined as CYP3A4*22 and homozygous CYP3A5*3 carriers (n = 9); intermediate metabolizers as CYP3A4*22 and CYP3A5*1 carriers (n = 2) or homozygous CYP3A4*1 and homozygous CYP3A5*3 carriers (n = 83); and extensive metabolizers as homozygous CYP3A4*1 and CYP3A5*1 carriers (n = 19). No significant association between the metabolic status and log-transformed [C.sub.0] or [C.sub.0]/dose was found (see online Supplemental Fig. 1).

IMPACT OF GENETIC VARIATIONS ON SRL ADVERSE EVENTS

Table 2 shows the nongenetic covariates selected in the intermediate model of each phenotype. After adjustment for nongenetic covariates and correction for false discovery finding, none of the SNPs tested showed a significant association with phenotypes (Table 3).

Moreover, no association was found between SNPs and phenotypes using crude analysis (i.e., without adjustment on nongenetic covariates) (data not shown).

Discussion

In this study we investigated whether genetic variations in CYP3A and 2 associated regulators (POR and PPARA) contributed to the variability of SRL disposition or adverse effects in renal transplant patients. The effect of gene polymorphisms on SRL metabolism was investigated in vitro using human liver microsomes, and potential associations with SRL blood concentrations and AEs were explored in parallel in 113 kidney transplant recipients.

In vitro, only the newly discovered CYP3A4 intron 6 C>T SNP (CYP3A4*22) was identified as a significant determinant of the variability in SRL metabolism. In contrast, only the POR*28 allele influenced SRL [C.sub.0] concentrations in kidney transplant patients. None of the genotypes tested were associated with SRL AEs in our patient population.

The CYP3A4*22 allele was previously found to decrease CYP3A4 mRNA hepatic expression (24) and to significantly reduce the CYP3A4-catalyzed hydroxylation of testosterone in liver microsomes. In addition, this allele resulted in higher cyclosporine and tacrolimus dose-adjusted [C.sub.0] in kidney transplant recipients (12,13). Here the CYP3A4*22 allele was associated in vitro with a significant decrease of SRL metabolic clearance by human liver microsomes, but this effect did not translate into a difference in SRL blood exposure in patients. Given the limited effect observed in vitro (approximately 20% lower metabolic rates in microsomes carrying the CYP3A4*22 allele), it is likely that other environmental or genetic sources of variability have tempered the effect of this genotype in vivo. It is noteworthy that, based on our in vitro results, our population sample set had a sufficient size to detect the expected effect of the CYP3A4*22 allele; a power calculation indicated a power of 0.92 [[micro]1 = 80.8 (13.3), n1 = 102, [micro]2 = 66.7 (13.1), n2 = 11].

We did not find any association between the CYP3A5*1/*3 genotype and SRL blood concentrations. We previously provided in vitro evidence that CYP3A5 genotype had no influence on SRL metabolism by HLM (18). We indeed observed similar intrinsic clearance ([CL.sub.int]) for SRL hydroxylation, demethylation, and depletion between HLM carrying a CYP3A5*1 (active) allele and HLM not expressing CYP3A5. However, previous reports on such an association in patients are controversial. Two studies found an effect of the CYP3A5*1 allele on SRL exposure. However, it was specifically in patients without CNI cotreatment (5, 6), but this was not confirmed in a third study (7). In the study we report here, in which the number of patients was relatively large compared to those in previous studies, the results are not in favor of this effect either, despite the fact that patients received no CNI cotreatment, which may have concealed the genetic effect. When the CYP3A metabolic status (integrating the CYP3A4 and 3A5 genotypes) was taken into account, as previously done for tacrolimus (13), once again no significant association was observed with SRL exposure or adverse effects. Taken together, these results suggest that CYP3A genetic variability has a very limited impact on SRL concentrations in kidney transplant recipients.

The POR gene modulates the activity of CYP3A via an electron transfer from NADPH to microsomal P450 enzymes (25) and is thus another candidate to explain the variability in CYP3A activity. The POR*28 allele, associated with an amino acid substitution, was found to increase CYP3A in vivo activity, as assessed by the midazolam metabolic ratio in 2 population datasets (16). Although this allele had no effect on SRL in vitro metabolic clearance, we observed here a significant, although weak, decrease of SRL [C.sub.0] in carriers of the POR*28 allele (-0.15 p,g/L for carriers of at least 1 T allele). Subgroup analysis showed that this effect concerned only the patients carrying a CYP3A4*1 functional allele, which seems logical because decreased POR activity may not show up if CYP3A4 activity is also decreased. However, the POR*28 allele had no significant effect on SRL [C.sub.0]/dose or dose, whether in the overall study population or in subgroups. It is thus likely that the clinical relevance of this finding is limited.

De Jonge et al. (15) showed that the POR*28 allele (CT/TT vs CC) was associated with a decrease in tacrolimus [C.sub.0] in the first 3 days after tacrolimus initiation and then with a significant increase in tacrolimus daily dose to achieve similar [C.sub.0] compared to POR*28 noncarriers. These results concerned only CYP3A5 ex pressors. The authors provided no clear explanation for this finding, but again it is likely that a functional CYP3A is required for the influence of the POR genotype to become apparent. In our study the POR*28 allele had no significant influence on SRL concentrations or dose regardless of the CYP3A5 genotype, but we previously demonstrated using recombinant enzymes that CYP3A5 activity toward SRL is much more limited than that of CYP3A4 (CLint = 0.64 vs 2.36 [micro]L/pmol P450 per min) (18). Accordingly, as mentioned above, the CYP3A5*1 allele had no influence on SRL concentrations in this study.

Another potential source of SRL pharmacokinetic variability was the rs4253728G>A polymorphism in PPARA. Klein et al. showed that homozygous carriers of the rs4253728 expressed significantly less PPAR-a protein in the liver (17). This nuclear receptor is believed to influence CYP3A4 activity by (a) indirect regulation of CYP3A4 gene transcription through PXR, another nuclear receptor; (b) its anti-inflammatory properties, resulting in the inhibition of the down-regulation of CYP3A4 by inflammation; or (c) a direct activation of CYP3A4 gene transcription. In any case, based on the metabolism of atorvastatin in vitro and in vivo, Klein et al. concluded that the rs4253728 SNP influenced CYP3A4 gene expression and its activity (17). Here, we found no effect of this SNP on SRL metabolism in vitro using HLM or in kidney transplant recipients. Since this study is the first to investigate the effect of this SNP on SRL, further investigations are required.

The inclusion of in vitro data was useful to assess the genetic determinants of SRL hepatic metabolism, but this part of the work has limitations because non-genetic factors may influence HLM enzyme activity. We found no correlation between HLM donor age and SRL metabolism (Spearman correlation test, P = 0.6125), and the rate of SRL metabolism did not differ between HLM derived from women and men (MannWhitney test, P = 0.5058). Notably, other donor characteristics not recorded in the present study such as donor medication before the surgery might influence CYP3A activity and be potential confounding factors.

We found no significant association between SRL AEs and CYP3A4*22, CYP3A5*3, POR*28, or PPARArs4253728 SNPs, although as previously observed in the same group of patients (22), most of these AEs were influenced by SRL exposure (edemas, cutaneous ad verse events, and cholesterol and hemoglobin concentrations). No association with dose reduction, which could indirectly reflect the occurrence of an adverse event, was found either. The absence of significant influences of the investigated SNPs on SRL exposure (except for the weak effect of POR*28) probably account for their lack of influence on AEs. Other AEs (triglyceride concentrations, infection, and pneumonitis) appeared to be independent from patient exposure to SRL and were not associated with the genetic variations studied here.

In conclusion, we found that the CYP3A4*22 allele was associated with a moderate decrease in SRL hepatic metabolism in vitro but did not contribute significantly to the pharmacokinetic variability of SRL in renal transplant patients. On the other hand, the POR*28 allele was associated with a significant decrease in SRL [C.sub.0] in kidney transplant recipients, but this effect did not show up in vitro, and given its limited extent it is likely that it is not clinically relevant. Contrary to what was recently found for tacrolimus and cyclosporine, these 2 recently described genetic variations do not seem to contribute substantially to the pharmacokinetic variability of SRL in transplant patients and would not be useful for dose individualization.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, oranalysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.

Role of Sponsor: No sponsor was declared.

Acknowledgments: We are grateful toJ.H. Comte forhis contribution to laboratory analyses and K. Poole for manuscript editing.

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Jean-Baptiste Woillard, [1,2,3] Nassim Kamar, [4,5,6] Sandra Coste, [2] Lionel Rostaing, [4,5,6] Pierre Marquet, [1,2,3] and Nicolas Picard [1,2,3] *

[1] INSERM, UMR S-850, Limoges, France; [2] Univ Limoges, Limoges, France; [3] CHU Limoges, Department of Pharmacology and Toxicology, Limoges, France; [4] CHU Toulouse, Department of Nephrology-Dialysis and Multi-Organ Transplantation, Toulouse, France; [5] INSERM, U563, IFR-BMT, CHU Purpan, Toulouse, France; [6] Univ Paul Sabatier, Toulouse, France

[7] Nonstandard abbreviations: SRL, sirolimus; CYP3A, cytochrome P450 3A; P-gp, P-glycoprotein; CNI, calcineurin inhibitor; SNP, single-nucleotide polymorphism; POR: P450 oxydo-reductase; [C.sub.0], trough concentrations; AE, adverse effects; HLM, human liver microsomes; LC-MS/MS, liquid chromatography-tandem mass spectrometry; CLint, intrinsic clearance.

[8] Human genes: ABCB1, ATP-binding cassette, sub-family B (MDR/TAP), member 1; CYP3A, cytochrome P450, family 3, subfamily A; CYP3A4, cytochrome P450, family 3, subfamily A, polypeptide 4; POR, P450 (cytochrome) oxidoreductase; CYP3A5, cytochrome P450, family 3, subfamily 5; PPARA, peroxisome proliferatoractivated receptor alpha.

* Address correspondence to this author at: INSERM UMR-S850, 2 rue du Dr. Marcland, 87025 Limoges, France. Fax +33(0)555435936; e-mail nicolas. picard@unilim.fr.

Received February 8, 2013; accepted August 1, 2013.

Previously published online at DOI: 10.1373/clinchem.2013.204990

Table 1. Linear mixed effect regression analyses of SNP effects on
exposure phenotypes.

Variable            Category      Phenotype               Mean
                                                       difference

CYP3A4*22           CT/TT vs CC   log [C.sub.0]/dose     0.0812
rs35599367                        log dose               0.0004
                                  log [C.sub.0]          0.0343
CYP3A5*3 rs776746   GG vs GA      log [C.sub.0]/dose    -0.0186
                                  log dose               0.0595
                                  log [C.sub.0]          0.0853
POR*28 rs1057868    CT/TT vs CC   log [C.sub.0]/dose    -0.2002
                                  log dose               0.0639
                                  log [C.sub.0]         -0.1503
PPARA rs4253728     GG vs GA/AA   log [C.sub.0]/dose    -0.2155
                                  log dose               0.2148
                                  log [C.sub.0]          0.0249

Variable            Category      Phenotype              SD     P (a)

CYP3A4*22           CT/TT vs CC   log [C.sub.0]/dose   0.1602   0.8810
rs35599367                        log dose             0.1335   0.9976
                                  log [C.sub.0]        0.0932   0.8278
CYP3A5*3 rs776746   GG vs GA      log [C.sub.0]/dose   0.1235   0.8810
                                  log dose             0.1036   0.9976
                                  log [C.sub.0]        0.0692   0.6605
POR*28 rs1057868    CT/TT vs CC   log [C.sub.0]/dose   0.0940   0.1419
                                  log dose             0.0798   0.9976
                                  log [C.sub.0]        0.0524   0.0197
PPARA rs4253728     GG vs GA/AA   log [C.sub.0]/dose   0.1972   0.8305
                                  log dose             0.1642   0.7744
                                  log [C.sub.0]        0.1142   0.8278

(a) P values were subjected to the Benjamini/Hochberg correction to
account for the analysis of 3 phenotypes (log [C.sub.0]/dose, log
dose, and log [C.sub.0]).

Table 2. Intermediate models with significant nongenetic covariates
for each phenotype used for adjustment of SNP analysis.

Phenotype (case/total)      Covariates                Category

Infection (32/113)          Sex                       M vs F
Pneumonitis (4/113)         Age                       Per year increase
                            Time between              Per year increase
                              transplantation
                              and switch
Cutaneous adverse           SRL cumulative            Per unit increase
  events (41/113)             concentrations
Edemas (20/113)             Age                       Per year increase
                            Time between              Per year increase
                              transplantation
                              and switch
                            SRL cumulative exposure   Per unit increase
Dose reduction (37/113)     SRL cumulative exposure   Per unit increase
                            Statin                    High dosing vs no

Phenotype                   Covariates                Category

Hemoglobin concentrations   SRL [C.sub.0]/dose        Per unit increase
                            Time between              Per unit increase
                              transplantation
                              and switch
Total cholesterol plasma    SRL [C.sub.0]/dose        Per unit increase
  concentrations            Corticosteroids intake    Yes vs no
                            Statin                    High dosing vs no
Triglyceride plasma         Time between              Per unit increase
  concentrations              transplantation
                              and switch
                            Corticosteroids intake    Yes vs no
                            Statin                    High dosing vs no

Phenotype (case/total)      Covariates                   Odds ratio
                                                          (95% CI)

Infection (32/113)          Sex                       0.47 (0.20-1.13)
Pneumonitis (4/113)         Age                        1.19(1.04-1.46)
                            Time between              1.02 (1.00-1.04)
                              transplantation
                              and switch
Cutaneous adverse           SRL cumulative            1.16 (1.02-1.35)
  events (41/113)             concentrations
Edemas (20/113)             Age                       1.07 (1.02-1.14)
                            Time between              1.00 (0.99-1.01)
                              transplantation
                              and switch
                            SRL cumulative exposure   1.31 (1.12-1.60)
Dose reduction (37/113)     SRL cumulative exposure   1.21 (1.05-1.45)
                            Statin                    7.10 (2.09-26.82)

Phenotype                   Covariates                     P (SD)

Hemoglobin concentrations   SRL [C.sub.0]/dose          0.060 (0.039)
                            Time between               0.0022 (0.0014)
                              transplantation
                              and switch
Total cholesterol plasma    SRL [C.sub.0]/dose         -0.039 (0.027)
  concentrations            Corticosteroids intake      0.697 (0.225)
                            Statin                      0.495 (0.228)
Triglyceride plasma         Time between               -0.002 (0.001)
  concentrations              transplantation
                              and switch
                            Corticosteroids intake      0.373 (0.225)
                            Statin                      0.585 (0.214)

Phenotype (case/total)      Covariates                P

Infection (32/113)          Sex                       0.0892
Pneumonitis (4/113)         Age                       0.0388
                            Time between              0.0225
                              transplantation
                              and switch
Cutaneous adverse           SRL cumulative            0.0261
  events (41/113)             concentrations
Edemas (20/113)             Age                       0.00444
                            Time between              0.08960
                              transplantation
                              and switch
                            SRL cumulative exposure   0.00253
Dose reduction (37/113)     SRL cumulative exposure   0.01669
                            Statin                    0.00233

Phenotype                   Covariates                P

Hemoglobin concentrations   SRL [C.sub.0]/dose        0.1291
                            Time between              0.133
                              transplantation
                              and switch
Total cholesterol plasma    SRL [C.sub.0]/dose        0.161
  concentrations            Corticosteroids intake    0.002
                            Statin                    0.032
Triglyceride plasma         Time between              0.082
  concentrations              transplantation
                              and switch
                            Corticosteroids intake    0.101
                            Statin                    0.007

(a) SRL cumulative exposure = equivalent to (mean [C.sub.0]-dose) x
follow-up duration.

Table 3. Univariate analysis of SNPs using the model adjusted for
covariates selected at the first step.

Phenotype                           Variable   Category

Total cholesterol plasma            CYP3A4     CT/TT vs CC
concentration (g/L) adjusted on
[C.sub.0]/dose SRL,
corticosteroids, and statin
comedication

                                    CYP3A5     GG vs AG
                                    POR        CT/TT vs CC
                                    PPARA      GG vs GA/AA

Triglyceride plasma concentration   CYP3A4     CT/TT vs CC
(g/L) adjusted on time between
transplantation and switch to
SRL, corticosteroids, and statin
comedication

                                    CYP3A5     GG vs AG
                                    POR        CT/TT vs CC
                                    PPARA      GG vs GA/AA

Hemoglobin concentration (g/dL)     CYP3A4     CT/TT vs CC
adjusted on [C.sub.0]/dose SRL,
time between transplantation and
switch to SRL

                                    CYP3A5     GG vs AG
                                    POR        CT/TT vs CC
                                    PPARA      GG vs GA/AA

Phenotype                           Variable   Category

Cutaneous adverse events adjusted   CYP3A4     CT/TT vs CC
on SRL cumulative exposure (b)

                                    CYP3A5     GA vs GG
                                    POR        CT/TT vs CC
                                    PPARA      GA/AA vs GG

Pneumonitis adjusted on age and     CYP3A4     CT/TT vs CC
time between transplantation and
switch to SRL

                                    CYP3A5     GA vs GG
                                    POR        CT/TT vs CC
                                    PPARA      GA/AA vs GG

Infections adjusted on sex          CYP3A4     CT/TT vs CC
                                    CYP3A5     GA vs GG
                                    POR        CT/TT vs CC
                                    PPARA      GA/AA vs GG

Edemas adjusted on age, time        CYP3A4     CT/TT vs CC
between transplantation, and
switch to SRL and SRL cumulative
exposure

                                    CYP3A5     GA vs GG
                                    POR        CT/TT vs CC
                                    PPARA      GA/AA vs GG

Dose reduction adjusted on statin   CYP3A4     CT/TT vs CC
comedication and SRL cumulative
exposure

                                    CYP3A5     GA vs GG
                                    POR        CT/TT vs CC
                                    PPARA      GA/AA vs GG

Phenotype                             [beta]         SD         P
                                    coefficient

Total cholesterol plasma              -0.1088      0.2848     1.0000
concentration (g/L) adjusted on
[C.sub.0]/dose SRL,
corticosteroids, and statin
comedication

                                     -0.26338      0.22741    0.5352
                                       0.1291      0.1773     0.9126
                                       0.0352      0.1737     0.8395

Triglyceride plasma concentration     -0.3273      0.2719     1.0000
(g/L) adjusted on time between
transplantation and switch to
SRL, corticosteroids, and statin
comedication

                                      -0.2048      0.2250     0.5352
                                       0.1917      0.1668     0.9126
                                       0.2041      0.17059    0.8395

Hemoglobin concentration (g/dL)        -0.291       0.312     1.0000
adjusted on [C.sub.0]/dose SRL,
time between transplantation and
switch to SRL

                                       -0.529       0.280     0.4822
                                      -0.0213       0.203     0.9126
                                       0.1211      0.1814     0.8395

Phenotype                           Odds ratio     95% CI     P (a)

Cutaneous adverse events adjusted      1.40       0.33-5.30   1.0000
on SRL cumulative exposure (b)

                                       0.49       0.14-1.49   0.5352
                                       0.57       0.24-1.28   0.9126
                                       0.46       0.19-1.05   0.5651

Pneumonitis adjusted on age and         NAc          NA         NA
time between transplantation and
switch to SRL

                                       0.42       0.02-4.82   0.5352
                                       0.72       0.03-8.06   0.9126
                                       2.25       0.22-51.6   0.8395

Infections adjusted on sex             0.41       0.06-1.81   1.0000
                                       0.59       0.16-1.81   0.5352
                                       1.26       0.55-2.94   0.9126
                                       0.81       0.35-1.86   0.8395

Edemas adjusted on age, time           1.00       0.03-3.97   1.0000
between transplantation, and
switch to SRL and SRL cumulative
exposure

                                       0.61       0.11-2.62   0.5352
                                       0.84       0.23-2.91   0.9126
                                       0.62       0.16-2.25   0.8395

Dose reduction adjusted on statin      5.12       1.10-28.8   0.3536
comedication and SRL cumulative
exposure

                                       2.16       0.70-6.73   0.5352
                                       0.60       0.24-1.46   0.9126
                                       0.63       0.26-1.55   0.8395

(a) P values indicated are those corrected by the Benjamini-Hochberg
correction for 8 phenotypes.

(b) SRL cumulative exposure = equivalent to (mean [C.sub.0]/dose) x
follow-up duration.

(c) NA, not applicable.
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Title Annotation:Drug Monitoring and Toxicology
Author:Woillard, Jean-Baptiste; Kamar, Nassim; Coste, Sandra; Rostaing, Lionel; Marquet, Pierre; Picard, Ni
Publication:Clinical Chemistry
Article Type:Report
Date:Dec 1, 2013
Words:5765
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