Title Significant association between CYP 3 A 5 polymorphism and blood concentration of tacrolimus in patients with connective tissue diseases

Objectives. To assess the association between the blood level of tacrolimus (TAC) in patients with connective tissue disease (CTD) and CYP3A5 polymorphism that affects TAC level in patients of organ transplantation. Methods. 72 patients with CTD were genotyped for rs776746 in CYP3A5. The blood trough level of TAC after taking 3mg/day was retrospectively obtained for each patient. Results. Allele A of rs776746 showed a significant association with a decreasing serum level of TAC (p=0.0038). One copy of the A allele decreased the level of TAC on average by 21.2%. Conclusions. Rs776746 is associated with levels of TAC level in a dose-dependent manner in patients with CTD. Tacrolimus and CYP3A5 polymorphism Introduction Tacrolimus (FK506, TAC) is a calcineurin inhibitor isolated from Streptomyces tsukubaensis (1) and one of the many types of powerful immunosuppressants that are frequently used for solid organ transplantation to prevent organ rejection(2). TAC is also used for patients with connective tissue disease (CTD) including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), polymyositis (PM) and dermatomyositis (DM) to control disease activity(3, 4). TAC is metabolized mainly by cytochrome P450 (CYP) 3A in the liver and intestine(5). Because TAC level is highly variable among patients, to predict TAC level to achieve therapeutic effect is a big challenge. Previous studies revealed that the variation of TAC level is largely attributable to different expressions of CYP3A in patients of organ transplantation. Patients carrying CYP3A5*3 determined by the G allele of rs776746 were shown to have high TAC level than patients with the A allele(5, 6). Although TAC is a substrate for P-glycoprotein encoded by the ABCB1 gene, effects of polymorphisms in ABCB1 on TAC level is inconclusive(6, 7). Genetic studies have only been performed recruiting patients with organ transplantation to date. When TAC is given to patients with CTD, the dosage is around 3mg/day (3, 4) which is much lower than that given to patients of organ transplantation. For example, patients with renal transplantation receive 0.3mg/kg/day around the transplantation and 0.12mg/kg/day as maintainance (8). In addition, though recipients of renal transplantation take TAC twice daily(8), patients with CTD only take a single dose of TAC per day. The effect of CYP3A5 on TAC levels with low TAC exposure has not been studied so far. Furthermore, chronic, systemic and autoimmune inflammatory process in CTD may influence the metabolism and level of TAC. Thus, whether the associations between polymorphisms of CYP3A5 and TAC levels can be observed in patients with CTD remained unclear. Evidence of the association between CYP3A5 and TAC levels in patients with CTD would enable us to develop a predictive model of TAC levels in these patients. Here, we performed an association study to address this point. Materials and Methods This study was designed in accordance with the Helsinki Declaration. This study was approved by the ethics committee of Kyoto University Graduate School and Faculty of Medicine. Written informed consent was obtained from all the participants. Study Subjects A total of 72 subjects with CTD who were prescribed to take a single dose of TAC every day in the evening at the Department of Rheumatology and Clinical Immunology, Kyoto University Graduate School of Medicine from December in 2005 to December in 2012 were enrolled in this study. Patients took PROGRAF® (Astellas) capsules containing 1mg or 0.5mg tacrolimus. CTD patients Tacrolimus and CYP3A5 polymorphism fulfilled criteria for each disease, namely, American College of Rheumatology (ACR) criteria for RA in 1987(9) or ACR/EULAR criteria in 2010(10), criteria for SLE(11), PM and DM(12) and for Polyarteritis Nodosa and microscopic polyangiitis(13). Clinical Information Information of age, sex, weight, serum creatinine and date of prescription, dosage, and serum level of TAC were obtained from clinical charts retrospectively by the system previously described(14). Information of prescription and dosage of corticosteroid was also obtained. TAC level data was obtained at least one week after prescription was initiated or changed. The data of concurrent use of cyclosporine, bosentan or antibiotics was excluded. eGFR was inferred by serum creatinine, age, and sex. TAC level The serum trough TAC level with 3mg TAC (around 12 hours after taking TAC) was used. TAC levels were quantified by two measurements according to measuring time; namely, microparticle enzyme immunoassay (MEIA, IMxTM-TACRO II, Abott) until May 2009 and chemiluminescent immunoassay (CLIA, ARCHITECT_TACRO, Abott) from May 2009. These measurements can quantify even low TAC levels (1.5~ and 0.5~ng/ml, respectively). When TAC levels were available for both measurements in a patient, data of CLIA with lower measuring limit of TAC levels was used. 63 and 9 patients were quantified by CLIA and MEIA, respectively (hereafter termed as CLIA group and MEIA group, respectively). When multiple TAC levels with 3mg TAC were available, the mean of the levels was adopted. Calculations were performed based on logarithm of TAC levels to avoid negative level by linear regression model and excess influence of extreme data. Selection of SNPs and genotyping Rs776746, whose G allele determined CYP3A5*3, was selected based on previous studies. Genotyping was performed by the Taqman assay (Applied Biosystems). Statistical Analysis The association was analyzed between serum level of TAC in the patients and genotypes of rs776746, age, sex, weight, eGFR, whether the patients were affected with RA or with SLE by single or multiple linear regression analysis in each measurement separately. The overall associations were estimated by meta-analysis using inverse-variance method. P-value less than 0.05 was regarded as significant. These analyses were performed by R statistical software.