Tacrolimus, a calcineurin inhibitor, is a basic component of immunosuppressive therapy in transplant patients, with a narrow therapeutic range. Its pharmacokinetic characteristics vary greatly among individuals, and dosage regimens are adjusted according to whole-blood trough tacrolimus concentrations (C0). Achieving therapeutic C0 is of critical importance, especially during the period after immediately transplantation. Identification of the parameters predictive of the optimal tacrolimus dosage is of clinical interest for fast determination of an adequate therapeutic range.
Cytochrome P450 (CYP) proteins are classified into families and subfamilies on the basis of the percentage of amino-acid sequences similarities (1). Members of the CYP3A subfamily are implicated in the metabolism of structurally diverse endobiotics, drugs, and protoxic or procarcinogenic molecules (2). The CYP3A members are the most abundant CYPs in human liver and small intestine (3). Substantial interindividual differences in CYP3A expression contribute greatly to variation in the oral bioavailability and systemic clearance of CYP3A substrates (4). Human CYP3A activities reflect the heterogeneous expression of at least three CYP3A members—CYP3A4, CYP3A5, and CYP3A7—which are adjacent to each other on chromosome band 7q21 (5). Analysis of human liver CYP3A5 cDNA revealed that only individuals with at least one CYP3A5*1 allele (A at position 6986) produce high levels of full-length CYP3A5 mRNA and express CYP3A5 (6). Those with the CYP3A5*3 allele (G at position 6986) display sequence variability in intron 3 that creates a cryptic splice site; this CYP3A5*3 allele encodes an aberrantly spliced mRNA with a premature stop codon. For most whites and African Americans with the CYP3A5*1 allele, CYP3A5 accounts for at least 50% of the total CYP3A content (6). Because tacrolimus is also a substrate for the CYP3A5 gene, this polymorphism may explain the large interindividual variations in the pharmacokinetic characteristics of tacrolimus. We therefore investigated the effect of CYP3A5 genotype on tacrolimus doses and concentration-to-dose ratio (C0) in renal transplant recipients.
PATIENTS AND METHODS
All renal transplant recipients who underwent transplantation in our center between 1998 and 2001, and who were treated with tacrolimus, were invited to participate in this retrospective study. All patients gave written informed consent. Eighty kidney transplant recipients (40 men and 40 women) who were given a tacrolimus-based immunosuppressive treatment, were recruited for the investigation. Mean recipient age was 40.9±11.3 years, and mean recipient body weight was 62.7±13.7 kg. Seventy-two patients were white, 7 were African American, and one patient was Indian. For all patients, the initial dosage of tacrolimus was 0.2 mg/kg per day. This dose was subsequently adapted to blood tacrolimus concentrations. During the first 3 months, the targeted C0 was 10 to 15 ng/mL. In addition to tacrolimus, most patients received a purine inhibitor, consisting of either mycophenolate mofetil (n=50) or azathioprine (n=26), and steroids, given at a standard dose regimen of 500 mg of intravenous methylprednisolone at the time of surgery, 125 mg intravenously the following day, and then 20 mg of prednisone daily. Oral prednisone was then progressively tapered to 5 mg daily at 3 months after transplantation.
All patients had a clinical and laboratory assessment 1 month after tacrolimus initiation. Clinical evaluation included body weight measurement and evaluation of the effects of all concomitant medications, with possible CYP3A and/or P-glycoprotein (P-gp) interaction. We determined the incidence of delayed graft function and biopsy-proven acute rejection episodes. The daily dose of tacrolimus (mg) was recorded and its weight-adjusted dosage calculated (mg/kg/day). Tacrolimus blood C0 (ng/mL) measured 12 hr after the previous dose, were assayed using the semiautomated microparticle enzyme immunoassay (Abbott, Rungis, France). To reduce intra- individual variability, tacrolimus blood C0 was calculated as the mean of three consecutives measurements. The blood concentration measured was dose-normalized using the concentration:dose ratio obtained by dividing the tacrolimus C0 by the corresponding 24-hr dose on an mg/kg basis. The information thus obtained was the tacrolimus dose needed to obtain a given trough level.
Identification of CYP3A5 Genotype
For genotype identification, genomic DNA was extracted from EDTA-treated blood, using the Qiagen Kit (Courtabeauf, France).
PCR amplification was performed as described previously (7) using the forward 5′-CCTGCCTTCAATTTTCACT-3′ and reverse 5′-GGTCCAAACAGGGAAGAG G T-3′ primers. A single mismatch was introduced into the penultimate position of the reverse primer (see unlined G in the sequence of the reverse primer), which is predicted to result in the creation of an Rsa I restriction site when A is present at the adjacent position 6986 of the genomic sequence (CYP3A5*1 allele). PCR was performed in a final volume of 25 μl in a reaction mix consisting of 0.25 μM of each primer, 0.1 mM of each dNTP, 0.2 to 0.5 μg of genomic DNA, 0.25 U of Taq polymerase (Promega, Madison, WI) in 50 mM Tris-HCl (pH 9.1), 3.5 mM MgCl2, 16 mM ammonium sulphate, and 0.15 mg/mL bovine serum albumin. Amplification involved 35 cycles of 94°C for 1 min, 61°C for 1 min, and 70°C for 1 min followed by final extension at 72°C for 7 min. A product of 196 bp was obtained. For restriction digest of the PCR product, a 20-μl aliquot was incubated overnight at 37°C with 3 U of Rsa I (New England Biolabs). Digestion products were applied to a 10% polyacrylamide gel. Gels were stained with ethidium bromide for 15 min and band patterns visualized using a ultraviolet transilluminator.
Patient genotypes were used as categorical independent variables for analysis of continuous clinical and pharmacologic variables. For analysis of the daily dose of tacrolimus, blood tacrolimus level and tacrolimus concentration:dose ratio analysis, groups were compared using nonparametric statistical tests. To compare two groups, we used the Mann-Whitney U test, and to compare several groups, the Kruskal-Wallis test. P values less than 0.05 were considered statistically significant.
Frequency of CYP3A5 Genotypes in Renal Transplant Patients
We determined the frequency of the different CYP3A5 genotypes in our population. We found that the frequency of the homozygotes for the *1 allelic variant (CYP3A5*1/*1) was 5% (4/80); the frequency of heterozygotes allele (*1/*3) was 11% (9/80), and the frequency of *3 homozygotes was 84% (67/80). We found no correlation between clinical outcome (delayed graft function and acute rejection episode) and the CYP3A5 genotype.
Tacrolimus Dose Requirements
One month after tacrolimus initiation, the tacrolimus doses required ranged from 0.029 to 0.364 mg/kg per day (median, 0.168), thus confirming the large interindividual variations in tacrolimus pharmacokinetics (Fig. 1). The mean tacrolimus C0 was 11.8±2.8 ng/mL.
Also at 1 month, there was no difference between the C0 of the patients with different CYP3A5 genotypes. As shown in Figure 2, the CYP3A5*1/*1 genotype was significantly associated with the tacrolimus daily dose after 1 month of tacrolimus treatment with a gene-dose effect (P =0.05, Kruskal-Wallis test). The mean dose required to obtain the targeted C0 was significantly lower in patients with the CYP3A5*3/*3 genotype than in those with the CYP3A5*1/*1 genotype (0.16±0.07, 0.20±0.06, and 0.25±0.06 mg/kg/day, respectively; P =0.05). The average concentration:dose ratio, indicating the exposure to tacrolimus, correlated with the CYP3A5 genotype. The mean concentration:dose ratios were 44, 61, and 92 for CYP3A5*1/*1, CYP3A5*1/*3, and CYP3A5*3/*3 variants, respectively (P =0.01, Kruskal-Wallis test).
Our results confirmed the large interindividual variations in tacrolimus pharmacokinetics because a 1- to 12-fold range of doses was needed to achieve therapeutic concentrations 1 month after tacrolimus introduction. A significant proportion of these variations in the bioavailability of tacrolimus after oral administration can be attributed to the variability of CYP3A5 expression because of its genetic polymorphism. Because patients with the CYP3A5*1/*1 genotype were more likely to have a high rate of hepatic and intestinal metabolism, they required a higher daily dose to achieve adequate blood tacrolimus levels. These findings are in agreement with those of MacPhee et al. (8), who observed that a single nucleotide polymorphism in the CYP3AP1 pseudogene (A/G44) correlated with the tacrolimus dose requirement. However, even though this polymorphism is associated with hepatic CYP3A5 activity, this is a result of linkage disequilibrium within the CYP3A locus, whereas screening for the A6986G polymorphism is directly predictive of CYP3A5 expression (6). Interestingly, all but one patient with the CYP3A5*1/*1 genotype were African American, and this allele is indeed more common in African Americans. The higher frequency of the CYP3A5*1/*1 genotype may therefore at least partly explain the worse outcome after renal transplantation observed in this population if the tacrolimus dose is not correctly adjusted from the beginning of the treatment.
We and others have reported the importance for tacrolimus dose requirement of another genetic polymorphism, that of P-gp, the product of the multidrug-resistance (MDR1) gene (9). Several single nucleotide polymorphisms of the MDR1 have been recently described (10), with a correlation with the in vivo activity of P-gp. However, in one study, this correlation was weak (8). Determination of both MDR1 and CYP3A5 polymorphisms would probably improve the positive predictive value of a pharmacogenomic approach to the estimation of tacrolimus requirements.
Characterization of the CYP3A5 genotype is a reliable predictor of the optimal dose of tacrolimus in renal transplant recipients. It can easily be done before transplantation and helps to determine the initial daily dose needed by individual patients to obtain adequate immunosuppression without increasing the risk of nephrotoxicity. We are currently investigating this hypothesis.
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