Tacrolimus and 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors: An interaction study in CYP3A5 non-expressors, renal transplant recipients.

OBJECTIVES: Atherosclerosis is a significant factor affecting long-term outcome in renal transplant recipients. Studies have been conducted to determine the pharmacogenomic pathways involved in statin efficacy, efficiency, and adverse effect likelihood. However, little is known about the influence of statins on tacrolimus kinetics. The aim of this study was to investigate possible pharmacological interactions between tacrolimus and statins in CYP3A5 non-expressors, renal transplant recipients.
MATERIALS AND METHODS: Twenty-four patients, treated with tacrolimus (n=24), methylprednisolone (n=24), and mycophenolate mofetil (n=19)/azathioprine (n=1)/everolimus (n=4), participated in the study. After an observation time of 112±36 days, statins, namely, atorvastatin (n=12), simvastatin (n=8), pravastatin (n=2), or fluvastatin (n=2), were administered for additional 101±34 days. DNA was extracted from whole blood sample and polymerase chain reaction followed by restriction fragment length polymorphism analysis was used for CYP3A5 genotyping. Student's t-test and Mann-Whitney test were used to test the significance of difference in variables that passed or did not pass Kolmogorov's normality test, respectively.
RESULTS: No statistically significant difference was observed in tacrolimus daily dose, concentration, concentration/dose ratio, and volume of distribution before and during the administration of statins. Statistically significant decrease in serum cholesterol was observed after initiation of statins. Renal and hepatic function remained unchanged and no skeletal muscle abnormalities were reported.
CONCLUSIONS: The results of this study show that tacrolimus and statins do not interact in terms of efficacy, efficiency, and adverse effect likelihood. No significant clinical interaction or effect was observed, even with the use of atorvastatin or simvastatin, which are metabolized by CYP3A4 such as tacrolimus.

Introduction

As a result of advances in the immunosuppressive treatment in renal transplant recipients, the survival of both the graft and the recipient has been prolonged. At present, the long-term survival seems to be affected by adverse effects of the immunosuppressive regimen. Tacrolimus remains the major immunosuppressant drug, and some of its common adverse effects include arterial hypertension, hyperlipidemia, and hyperglycemia. Thus, atherosclerosis has become a significant factor in patient survival after renal transplantation. Statins, also known as 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, are the most commonly used agents for the treatment of hyperlipidemia.[1] Two potentially serious adverse effects can be seen during statin use; these include liver dysfunction and skeletal muscle abnormalities.[2-5]

Although a number of studies have been conducted in an effort to determine the pharmacogenomic pathways involved in statin efficacy, efficiency, and adverse effect likelihood, little is known about the influence of statins in tacrolimus kinetics in renal transplant recipients.[6] Till date, the only proved interaction between statins and tacrolimus has been reported with the use of simvastatin in tacrolimus-treated rats.[7] Genetic variance in the metabolizing enzymes or drug transporters/receptors as well as co-administered therapeutic agents seem to affect the relationship between dose and concentration as well as between concentration and pharmacological effect of various drugs.[3] Discovering genetic allelic variants and other factors that may influence the impact of statins on tacrolimus kinetics may lead to better dose individualization.

Liver stands as the major clearance site for statins and tacrolimus. Both these drug agents are mainly metabolized by cytochrome P450 (CYP) metabolizing enzymes.[36] Tacrolimus is metabolized by two main enzymes of the CYP3A family: CYP3A5 and CYP3A4. CYP3A5 has a more catalytic role for tacrolimus biotransformation than CYP3A4 and when expressed, it may account for 50% of the total hepatic CYP3A protein.[8-11] Although statins are encountered as a single class of drugs, each one shows a distinct pharmacokinetic profile and is metabolized to a varied degree by CYP3A enzymatic system. Lipophilic statins such as lovastatin, simvastatin, and atorvastatin are metabolized by CYP3A4, whereas fluvastatin is mainly metabolized by CYP2C9.[2-51213] Simvastatin is metabolized by CYP2C8, and pravastatin, a hydrophilic statin, is excreted mostly unchanged.[2351213]

Taking into account the pharmacokinetic properties of statins and tacrolimus, physicians can potentially avoid several drug interactions and consequent side effects without compromising therapeutic benefits.[212] Although the interaction between cyclosporine and statins has been extensively described, potential interactions between tacrolimus and statins are still being studied. The aim of the present study was to investigate the pharmacokinetics of tacrolimus in statin-treated, CYP3A5 non-expressors, renal transplant recipients as well as to elucidate the impact of statin therapy in the renal and hepatic function in these patients.

Materials and Methods

Patient Population

The participants in this study included 24 (15 male and 9 female) renal transplant recipients who attended the Outpatient Clinic of Nephrology at our institution and were treated with triple immunosuppression therapy consisting of tacrolimus (24 patients), methylprednisolone (24 patients), and mycophenolatemofetil (19 patients) or azathioprine (1 patient) or everolimus (4 patients). The co-administered statin was atorvastatin in 12 patients, simvastatin in 8 patients, pravastatin in 2 patients and fluvastatin in the remaining 2 patients.

All patients had received the kidney transplant 213±57 days ago. After an observation time of 112±36 days, statin treatment was started and administered for an additional time of 101±34 days. Triple immunosuppression regimen was monitored, along with patient's body weight, co-administered drug agents, tacrolimus levels 12 hours post dose, and blood chemistry during the study time. Tacrolimus dose-adjusted concentration (concentration/dose ratio) and tacrolimus volume of distribution (dose/concentration ratio) were calculated.

Concomitant medication was allowed only if it had been started 3-4 weeks before the initiation of the study, especially if the administrated drugs influenced the kinetics of tacrolimus or statin. Adverse effects were registered during the study period and were evaluated by the investigators.

The protocol was approved by the Ethics Committee of our institution and informed consent was obtained from all subjects.

CYP3A5 genotyping

Five milliliter blood samples were collected by direct venipuncture from each patient and were drawn in a vacutainer tube containing ethylene diaminetetraacetic acid (EDTA). DNA was extracted from the 200-μl EDTA-treated whole blood sample by using QIAamp DNA Blood kit (QIACEN GmbH). Polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) analysis was used for CYP3A5 genotyping. PCR primers for CYP3A5 were designed to amplify a 293-bp fragment of CYP3A5 (forward primer: 5’-CATCAGTTAGTAGACAGATGA-3’ and reverse primer: 5’-GGTCCAAACAGGGAAGAAATA-3’).[10] PCR conditions were 1 min at 94°C, 40 cycles of 1 min at 94°C, 1 min at 55°C, 1 min at 72°C, and, finally, 7 min at 72°C.

Enzymatic digestion of PCR amplification products from blood samples of all patients was performed using the SspI endonuclease (New England BioLabs Inc.). The digestion products were separated using 3.5% agarose/Tris-borate EDTA gel electrophoresis and ethidium bromide staining [Figure 1]. CYP3A5*1/*1 genotype gives 148-bp, 125-bp, and 20-bp bands; CYP3A5*3/*3 genotype gives 168-bp and 125-bp bands; and CYP3A5*1/*3 genotype gives 168-bp, 148-bp, 125-bp and 20-bp.[10]

Figure 1

RFLP for the CYP3A5. Lane M, base pair marker (250-bp DNA ladder); lanes 1-11 SspI-digested PCR products from 11 PCR products (each PCR product refers to one patient). CYP3A5*3/*3 genotype gives 168-bp and 125-bp bands (lanes 1-11). CYP3A5*1/*1 and CYP3A5*1/*3 genotypes are not seen in this picture. Analysis was done on a 3.5% agarose/Tris-borate-EDTA gel

Data Analysis

Tacrolimus daily dose, concentration, dose-adjusted concentration (concentration/dose ratio), and volume of distribution (dose/concentration ratio) were used to estimate tacrolimus kinetics without and during concomitant administration of statins. Lipid-lowering effect of statins was evaluated with the use of serum total cholesterol. Serum creatinine and aspartate aminotransferase and alanine aminotransferase were used to estimate renal and hepatic function, respectively. Creatinine kinase level was examined for the evaluation of skeletal muscle abnormalities incidence.

Statistical Analysis

The Prism 4 (GraphPad Software, Inc.) statistical software package was employed for all statistical calculations and analyses. Quantitative variables were expressed as mean±SD. All statistical tests performed were two-sided and the threshold of statistical significance was set at 5% (a=0.05).

Kolmogorov's normality test was used to determine whether continuous data should be treated as originating from normal distribution. Student's t-test was used for testing the significance of difference of variables that passed the normality test. Mann-Whitney test was used for continuous variables that did not pass the normality test.

Results

The characteristics of the study population are presented in Table 1.

Table 1

Characteristics of the patient population

Tacrolimus Pharmacokinetics

Tacrolimus pharmacokinetics before and after the initiation of administration of statin are presented in Table 2. No statistically significant difference was observed in tacrolimus pharmacokinetic parameters (daily dose, concentration, dose-adjusted concentration, and volume of distribution) in our patient population after the initiation of statin treatment.

Table 2

Tacrolimus pharmacokinetics in patients before and after the initiation of administration of statin (data presented as mean ± SD, n = 24)

Lipid-Lowering Effect of Statins

Statistically significant decrease in cholesterol was observed in statin-treated patients (P < 0.001), in patients receiving atorvastatin (P < 0.001), and in patients receiving simvastatin (P = 0.001). The lipid-lowering effect of statins is presented in Table 3.

Table 3

The lipid-lowering effect of statins, their impact on renal and hepatic function, and incidence of skeletal muscle abnormalities (data presented as mean ± SD, n = 24)

Renal and Hepatic Function

Renal and hepatic function remained unchanged in statin-treated patients as well as in the subgroups treated with atorvastatin or simvastatin. No statistically significant difference was reported in serum creatinine, aspartate aminotransferase, and alanine aminotransferase after the initiation of statin treatment (P=0.61, P=0.41, and P=0.56, respectively). The impact of statins on renal and hepatic function is described in Table 3.

Incidence of Skeletal Muscle Abnormalities with the use of Statin

No skeletal muscle abnormalities were reported during the study period. No statistically significant elevation (P=0.55) was found in creatinine kinase level after the initiation of statin treatment [Table 3].

Discussion

The use of statins in transplant recipients has been restrained by adverse effects. Tacrolimus is thought to be a safer treatment option than cyclosporine in patients receiving a combination of calcineurin inhibitor and statin.[71415] Although studies propose that cyclosporine and tacrolimus are CYP3A4 inhibitors/substrates and that their co-administration with statins may increase the risk of myopathy and rhabdomyolysis, a significant increase in the systemic exposure to atorvastatin and its metabolite was reported only during cyclosporine treatment.[2314] Patients, treated with cyclosporine, experienced a several-fold higher systemic exposure to all statins compared to those that did not receive it.[5] Cyclosporine increases the risk of statin myotoxicity, with fluvastatin showing the smallest risk among all statins.[1316] The administration of cyclosporine, but not tacrolimus, in atorvastatin-treated healthy volunteers was reported to lead to a profound decrease in hepatic and intestinal P-glycoprotein and increase in the intestinal CYP3A4 enzyme.[14]

In the present study, only CYP3A5 non-expressors renal transplant recipients participated. CYP3A5 non-expressors are individuals who are homozygotes for CYP3A5*3, whereas individuals carrying at least one CYP3A5*1 allele express CYP3A5 protein.[891117-20] Healthy individuals who are CYP3A5*3 carriers have been shown to have elevated plasma area under the curve (AUC) while being treated with simvastatin.[6] Tacrolimus is metabolized by CYP3A4 and CYP3A5 enzymes. We studied CYP3A5 non-expressors to ensure that tacrolimus metabolism is mainly performed via CYP3A4.

The different pharmacokinetics of each statin should always be taken into account in order to predict potential drug interactions.[3] For this reason, four different statins (atorvastatin, simvastatin, pravastatin, and fluvastatin) were examined. Atorvastatin and simvastatin have been marked as inhibitors of CYP3A4 and multidrug resistance 1 (MDR-1), whereas fluvastatin inhibits only CYP3A4.[5] Pravastatin inhibits neither CYP3A4 nor MDR-1 action.[5] The concentration of fluvastatin is less varied than that of other statins when co-administered with potent inhibitors or inducers of CYP family.[13] Except for the CYP family, oral bioavailability of statins is influenced by a drug transporter in the small intestine – P-glycoprotein.[2] The only interaction between statins and tacrolimus has been reported with the use of simvastatin in tacrolimus-treated rats and this action was similar with that observed for cyclosporine.[7] In our study, no statistically significant difference was detected in the pharmacokinetic parameters of tacrolimus before and after the initiation of statin treatment.

Pharmacokinetic and pharmacodynamic studies of pravastatin in pediatric and adolescent cardiac transplant population receiving a triple immunosuppression treatment have revealed a statistically significant decrease in serum total cholesterol and low-density lipoprotein cholesterol without any clinically significant increase in serum alanine aminotransferase, creatinine kinase, or creatinine level.[21] In our study, no statistically significant difference was observed after the initiation of statin treatment in serum creatinine, aspartate aminotransferase, and alanine aminotransferase. Statistically significant lowering action was marked in serum cholesterol. Both findings strengthen the safe and effective role of statins in the management of hyperlipidemia and, consequently, in the prevention of atherosclerosis in renal transplant recipients.

The principal causes of the interaction between statins and co-administered drugs are the inhibition of CYP metabolizing enzymes and the transporters responsible for hepatic uptake and efflux.[5] Deciphering these interactions is of great importance in order to ensure the efficacy, tolerability, and safety of the treatment schedule, especially in patients taking drugs that are well known CYP3A4 substrates and/or inhibitors.[322] Our results empower the belief that tacrolimus and concomitant administrated statin do not seem to interact in terms of efficacy, efficiency, and side effect likelihood. No significant clinical interaction or effect was observed, even for atorvastatin and simvastatin, which are metabolized by CYP3A4 such as tacrolimus.