Dennis A. Hesselink

Genetic polymorphisms of the CYP3A4, CYP3A5, and MDR‐1 genes and pharmacokinetics of the calcineurin inhibitors cyclosporine and tacrolimus

The calcineurin inhibitors cyclosporine (INN, ciclosporin) and tacrolimus have a narrow therapeutic index and show considerable interindividual variability in their pharmacokinetics. The low oral bioavailability of calcineurin inhibitors is thought to result from the actions of the metabolizing enzymes cytochrome P450 (CYP) 3A4 and CYP3A5 and the multidrug efflux pump P‐glycoprotein, encoded by MDR‐1.

Cyclosporine interacts with mycophenolic acid by inhibiting the multidrug resistance-associated protein 2

In mycophenolate mofetil (MMF)‐treated organ transplant recipients, lower mycophenolic acid (MPA) plasma concentrations have been found in cyclosporine (CsA) compared with tacrolimus (Tac)‐based immunosuppressive regimens. We previously demonstrated that CsA decreases exposure to MPA and increases exposure to its metabolite MPA‐glucuronide (MPAG), possibly by interfering with the biliary excretion of MPAG. To elucidate the role of the multidrug resistance‐associated protein (Mrp)‐2 in the interaction between MMF and CsA, we treated three groups of 10 Mrp2‐deficient rats (TR− rat) for 6 days with either vehicle, CsA (8 mg/kg) or Tac (4 mg/kg) by oral gavage. Hereafter, co‐administration with MMF (20 mg/kg) was started in all groups and continued through day 14. The 24‐h MPA/MPAG area under the concentration‐time curve (AUC) was determined after single (day 7) and multiple MMF doses (day 14). On both study days, there were no significant differences in the mean MPA and MPAG AUC between CsA and Tac‐treated animals. We conclude that the pharmacokinetics of MMF are comparable in Mrp2‐deficient rats receiving either CsA or Tac as co‐medication. This finding suggests that CsA‐mediated inhibition of the biliary excretion of MPAG by the Mrp2 transporter is the mechanism responsible for the interaction between CsA and MMF.

A New Functional CYP3A4 Intron 6 Polymorphism Significantly Affects Tacrolimus Pharmacokinetics in Kidney Transplant Recipients

BACKGROUND Tacrolimus (Tac) is a potent immunosuppressant with considerable toxicity. Tac pharmacokinetics varies between individuals and thus complicates its use in preventing rejection after kidney transplantation. This variability might be caused by genetic polymorphisms in metabolizing enzymes. METHODS We used TaqMan analyses to evaluate the impact of a newly discovered CYP3A4 (cytochrome P450, family 3, subfamily A, polypeptide 4) single-nucleotide polymorphism (SNP) (rs35599367C>T; CYP3A4*22) on Tac pharmacokinetics in 185 renal transplant recipients who participated in an international randomized controlled clinical trial (fixed-dose, concentration-controlled study). RESULTS The overall mean daily-dose requirement to reach the same predose Tac blood concentration was 33% lower for carriers of the T variant allele than for rs35599367CC patients (95% CI, -46% to -20%; P = 0.018). When combined with the *3 genotype of the CYP3A5 (cytochrome P450, family 3, subfamily A, polypeptide 5) gene, the rs35599367C>T SNP was also associated with a risk of supratherapeutic Tac concentrations (>15 μg/L) during the first 3 days after surgery, with an odds ratio of 8.7 for carriers of the CYP3A4 T allele plus CYP3A5*3/*3 (P = 0.027) and 4.2 for the CYP3A4 CC homozygotes plus CYP3A5*3/*3 (P = 0.002), compared with CYP3A4 CC homozygotes having 1 or 2 CYP3A5*1 alleles. The overall increase in the Tac dose-adjusted trough blood concentration was +179% for carriers of the CYP3A4 T allele with CYP3A5*3/*3 (P < 0.001), +101% for CYP3A4 CC homozygotes with CYP3A5*3/*3 (P < 0.001), and +64% for CYP3A4 T allele carriers with CYP3A5*1 (P = 0.020),compared with CYP3A4 CC homozygotes with CYP3A5*1. CONCLUSIONS The CYP3A4 rs35599367C>T polymorphism is associated with a significantly altered Tac metabolism and therefore increases the risk of supratherapeutic Tac concentrations early after transplantation. Analysis of this CYP3A4*22 SNP may help in identifying patients at risk of Tac overexposure.

Population pharmacokinetics of cyclosporine in kidney and heart transplant recipients and the influence of ethnicity and genetic polymorphisms in the MDR-1, CYP3A4, and CYP3A5 genes

Our objective was to determine the relationship between single nucleotide polymorphisms (SNPs) in the multidrug resistance 1 (MDR‐1) gene and the cytochrome P450 (CYP) genes CYP3A4 and CYP3A5 and the pharmacokinetics of cyclosporine (INN, ciclosporin).

CYP3A5 genotype is not associated with a higher risk of acute rejection in tacrolimus-treated renal transplant recipients

Objective Patients expressing the tacrolimus-metabolizing enzyme, cytochrome P450 (CYP) 3A5, require more tacrolimus to reach target concentrations. We studied the influence of the CYP3A5*3 allele, which results in the absence of CYP3A5 protein, on tacrolimus dose and exposure, as well as the incidence of biopsy-proven acute rejection (BPAR) after renal transplantation. Methods A total of 136 patients participating in a prospective, randomized-controlled clinical trial with the primary aim of comparing the efficacy of a fixed-dose versus a concentration-controlled mycophenolate mofetil immunosuppressive regimen, were genotyped for CYP3A5*3. The patients described herein, participated in a pharmacogenetic substudy and were all treated with mycophenolate mofetil, corticosteroids and tacrolimus. Tacrolimus predose concentrations (C0) were measured on day 3 and 10, and month 1, 3, 6 and 12. Results Compared with CYP3A5*3/*3 individuals (n=110), patients carrying at least one CYP3A5*1 (wild-type) allele (CYP3A5 expressers; n=26) had a lower tacrolimus C0 on day 3 only (16.6 versus 12.3 ng/ml, respectively), whereas dose-corrected tacrolimus C0 were significantly lower in the latter group at all time points. After day 3, the overall daily tacrolimus dose was 68% higher in CYP3A5 expressers (P<0.001). The incidence of BPAR was comparable between CYP3A5 expressers and nonexpressers (8 versus 16%, respectively; P=0.36). Conclusion We conclude that patients expressing CYP3A5 need more tacrolimus to reach target concentrations and have a lower tacrolimus exposure shortly after transplantation. This delay in reaching target concentrations, however, did not result in an increased incidence of early BPAR and therefore, genotyping for CYP3A5 is unlikely to improve short-term transplantation outcome.

The pharmacogenetics of calcineurin inhibitors: One step closer toward individualized immunosuppression?

The immunosuppressive drugs cyclosporin (CsA) and tacrolimus (Tac) are widely used to prevent acute rejection following solid-organ transplantation. However, the clinical use of these agents is complicated by their many side effects, a narrow therapeutic index and highly variable pharmacokinetics. The variability in CsA and Tac disposition has been attributed to interindividual differences in the expression of the metabolizing enzymes cytochrome P450 (CYP) 3A4 and 3A5, and in the expression of the drug transporter P-glycoprotein (encoded by the ABCB1 gene, formerly known as the multidrug resistance 1 gene). Variation in the expression of these genes could in turn be explained by several recently-identified single nucleotide polymorphisms (SNPs). Determination of these SNPs in (future) transplant recipients has the potential to identify individuals who are at risk of under-immunosuppression or the development of adverse drug reactions. Ultimately, genotyping for CYP3A and ABCB1 may lead to further individualization of immunosuppressive drug therapy for the transplanted patient.

The new CYP3A4 intron 6 C>T polymorphism (CYP3A4*22) is associated with an increased risk of delayed graft function and worse renal function in cyclosporine-treated kidney transplant patients.

Objective Cyclosporine A (CsA) is a substrate of cytochrome P450 3A4 (CYP3A4). Recently, a newly discovered intron 6 single-nucleotide polymorphism in CYP3A4 (rs35599367 C>T), defining the CYP3A4*22 allele, has been linked to reduced hepatic expression and activity of CYP3A4. In the present study, the clinical impact of this single-nucleotide polymorphism was investigated in a cohort of patients receiving a CsA-based immunosuppressive regimen. Materials and methods A total of 172 de-novo kidney transplant recipients, receiving CsA/mycophenolate mofetil as immunosuppressive therapy and participating in the Fixed-Dose Concentration Controlled study, were genotyped for the new CYP3A4*22 allele. CsA C0 and/or C2 levels were measured on days 3 and 10 and in months 1, 3, 6, and 12 after transplantation. Plasma creatinine concentrations, delayed graft function (DGF), and biopsy-proven acute rejection were recorded. Results The CYP3A4*22 allele was significantly associated with a higher risk of DGF compared with the CYP3A4*1/*1 patients after adjustment for known risk factors [odds ratio (OR)=6.34, confidence interval (CI95%: 1.38–29.3), P=0.015]. Mixed-model analysis demonstrated that the overall creatinine clearance was 20% lower in CYP3A4*22 allele carriers compared with CYP3A4*1/*1 patients [CI95% (−33.1 to −7.2%), P=0.002]. For ABCB1 3435C>T, T-variant carriers had a decreased risk of developing DGF compared with CC patients [CT: OR=0.30, CI95% (0.11–0.77), P=0.011; TT: OR=0.18, CI95% (0.05–0.67), P=0.011]. Conclusion CYP3A4*22 constitutes a risk factor for DGF and worse creatinine clearance in patients receiving CsA-based immunosuppressive therapy. Therefore, pretransplant genotyping for the CYP3A4*22 allele might help clinicians to identify patients at risk of DGF and poor renal function when treated with CsA.

Genetic and nongenetic determinants of between-patient variability in the pharmacokinetics of mycophenolic acid

c p c ( u i g t a m p Mycophenolate mofetil (MMF) (CellCept; offmann-La Roche, Basel, Switzerland) and mycopheolate sodium (Myfortic; Novartis Pharmaceuticals Corp, asel, Switzerland) are prodrugs of the immunosuppresive compound mycophenolic acid (MPA). MMF was pproved by the US Food and Drug Administration in 995, after the publication of the results of 3 phase III, andomized, controlled, double-blind, multicenter trials erformed in de novo kidney transplant recipients. hese so-called pivotal trials demonstrated that combinaion therapy consisting of MMF, cyclosporine (INN, iclosporin), and corticosteroids, with or without antithyocyte globulin induction treatment, significantly reuced the incidence of acute rejection compared with the ombination of cyclosporine, corticosteroids, and either lacebo or azathioprine. Since then, MMF has also roved effective in combination with other drugs, such as acrolimus and sirolimus, as well as after other forms of olid organ transplantation. As a result, there has been a ise in the use of MMF during the past decade. In 2002, 9% of kidney transplant recipients received MMF before ospital discharge. At present, MMF has largely replaced zathioprine as the antimetabolite of choice after renal ransplantation. A similar trend has occurred for heart and iver transplantation. In addition, MMF is being increasngly used in the prevention of graft-versus-host disease fter hematopoietic stem cell transplantation and is curently under investigation in several autoimmune disor-

The pharmacogenetics of calcineurin inhibitor-related nephrotoxicity.

Chronic calcineurin inhibitor (CNI)-induced nephrotoxicity is associated with prolonged use of cyclosporine and tacrolimus and has been observed after all types of transplantation, as well as during treatment of autoimmune disease. Extensive alterations in the renal architecture including glomerular sclerosis, tubular atrophy and interstitial fibrosis may lead to end-stage renal failure. Increasing evidence shows that pharmacogenetic factors explain part of the between-patient differences in susceptibility to developing CNI-induced nephrotoxicity. In this paper this evidence is reviewed, with special emphasis on the role of genetic factors influencing metabolism and transportation of CNIs in both acceptor and donor.

Impact of POR*28 on the pharmacokinetics of tacrolimus and cyclosporine A in renal transplant patients

Background: The P450 oxidoreductase (POR)*28 variant allele has been associated with altered cytochrome P450 3A enzyme activities. Both CYP3A5 and CYP3A4 are involved in the metabolism of calcineurin inhibitors and recent data show that POR*28 may explain part of the variability observed in tacrolimus (Tac) pharmacokinetics. The aim of this study was to investigate the impact of the POR*28 allele on Tac and cyclosporine A (CsA) immunosuppressive therapies. Methods: Kidney transplant recipients receiving either Tac (n = 184) or CsA (n = 174), participating in a prospective multicenter trial, were genotyped for POR*28, CYP3A4*22, and CYP3A5*3. Results: CYP3A5 expressers that were carriers of at least 1 POR*28 allele had a 16.9% decrease in dose-adjusted predose concentrations when compared CYP3A5 expressers that carried the POR*1/*1 genotype (P = 0.03), indicating an increased CYP3A5 activity for POR*28 carriers. In CYP3A5, nonexpressers carrying 2 POR*28 alleles, a 24.1% (confidence interval95% = −39.4% to −4.9%; P = 0.02) decrease in dose-adjusted predose concentrations was observed for Tac, suggesting higher CYP3A4 activity. For CsA, POR*28/*28 patients not expressing CYP3A5 and not carrying the CYP3A4*22 decrease-of-function allele showed 15% lower CsA dose-adjusted predose concentrations (P = 0.01), indicating also increased CYP3A4 activity. In both cohorts (ie, Tac and CsA), the POR*28 allele was neither associated with the incidence of delayed graft function nor with biopsy-proven acute rejection. These results were further confirmed in 2 independent cohorts. Conclusions: Our results show that the POR*28 allele is associated with increased in vivo CYP3A5 activity for Tac in CYP3A5 expressers, whereas POR*28 homozygosity was associated with a significant higher CYP3A4 activity in CYP3A5 nonexpressers for both Tac and CsA.