Effect of CYP3A5 Genotypes on the Pharmacokinetics of Carbamazepine when used as Monotherapy or Co-Administered with Phenytoin, Phenobarbital or Valproic Acid in Thai Patients

- Purpose: To determine the effects of CYP3A5 polymorphisms on carbamazepine (CBZ) pharmacokinetic parameters when CBZ is used either as monotherapy or co-administered with phenytoin (PHT), phenobarbital (PB) or valproic acid (VPA). Methods: Retrospective data were collected from an electronic database and medical records. Blood samples were obtained and drug concentrations analyzed as a part of routine therapeutic drug monitoring (TDM). Screening for wild-type CYP3A5*1 and CYP3A5*3 single nucleotide polymorphism (rs776746) by allelic discrimination assay using real-time polymerase chain reaction technique (real-time PCR) was performed. Pharmacokinetic parameters of CBZ; clearance and dose-adjusted CBZ levels in patients with different genotypes were calculated and compared. Results: Of the 70 patients assessed, 8 (11%) patients were homozygous CYP3A5*1/*1 , 28 (40%) patients were heterozygous CYP3A5*1/*3 , and 34 (49%) patients were homozygous CYP3A5*3/*3 . The CBZ clearance and dose-adjusted CBZ levels did not significantly differ between patients with CYP3A5*1 and CYP3A5*3 alleles when CBZ was used as monotherapy. For patients who used CBZ in combination with an enzyme-inducing antiepileptic drug (AED: PHT or PB), individuals carrying the CYP3A5*1 allele (CYP3A5 expressers) showed a trend of having higher CBZ clearance and lower dose-adjusted CBZ level as compared to individuals carrying the CYP3A5*3 allele, even though no statistical significance was recorded. Nevertheless, it was observed that AEDs significantly increased CBZ clearance only in patients carrying the active CYP3A5*1 allele. Conclusions: When CBZ was used in combination with enzyme-inducing AED, CYP3A5 expressers yielded a trend toward greater susceptibility to change in CBZ clearance and showed lower dose-adjusted CBZ levels compared to CYP3A5 non-expressers. The dosage regimen should be adjusted accordingly to gain a better clinical outcome.


INTRODUCTION
Carbamazepine (CBZ) is a first-line antiepileptic drug for partial and generalized tonic-clonic seizures [1][2][3][4][5]. CBZ is commonly used as monotherapy or co-administered with other antiepileptic drugs (AED), such as phenytoin (PHT), phenobarbital (PB) and valproic acid (VPA). Additionally, it is commonly used for other neurological diseases, for instance, pain relief in trigeminal neuralgia or bipolar disorder [5][6][7][8]. The serum concentration of CBZ reported to be within the accepted therapeutic range is 4-12 mg/L when the drug is used for the treatment of seizures; accordingly, the range for psychiatric disorders and trigeminal neuralgia is assumed to be the same [9]. Ninety-nine percent of CBZ is metabolized by the liver; CYP3A4 and CYP3A5 are the most prominent enzymes that converted CBZ to CBZ-10, 11-epoxide (CBZ-E) in the major pathway of CBZ metabolism [8][9][10][11] (Figure  1).
Studies about the clearance of CBZ are important for therapeutic drug monitoring. Among several factors that have been reported as influencing the elimination of CBZ, age, body weight, surface area, dose of CBZ, and comedication with PHT, PB, or VPA [8,9,[12][13][14], CYP3A5 polymorphism have more recently received greater focus. Seo et al. reported CBZ clearance in patients with CYP3A5*3/*3 to be 8% significantly higher than that in patient with CYP3A5*1/*1 or CYP3A5*1/*3 [15].
However, Park et al. reported the mean doseadjusted CBZ level in patients with CYP3A5*3/*3 to be 31% significantly higher than that in patients with CYP3A5*1/*1 or CYP3A5*1/*3 (p = 0.032). Consequently, CBZ clearance was reported to be 29% significantly lower in patients with CYP3A5*3/*3 compared to the rate in patients with CYP3A5*1/*1 or CYP3A5*1/*3 (p = 0.004) [16]. The effects of CYP3A5* 3 on CBZ pharmacokinetics (PK) when co-medicated with other AEDs reported to have a pharmacokinetic interaction with CBZ have never been clearly defined. Knowledge about the effect of CYP3A5 polymorphism on CBZ pharmacokinetics either when used alone or used in combination with other AEDs may be useful in therapeutic plans to avoid serum drug concentration-related adverse effects and inefficacy.
The purpose of this study was to determine the effects of CYP3A5 polymorphism on CBZ pharmacokinetic parameters when CBZ was used either as monotherapy or co-administered with PHT, PB or VPA.

Study Design
A retrospective study design was used. The protocol was approved by The Institutional Review Board/Independent Ethics Committee, Prasat Neurological Institute (Bangkok, Thailand).

Patients
During February 2010 -September 2010, patients at the epilepsy outpatient clinic of Prasat Neurological Institute, aged over 13 years old who used CBZ as monotherapy or coadministered with PHT, PB or VPA and whose therapeutic drug monitoring data (TDM) had been recorded and were available were included in this study. CBZ and VPA took the form of controlledrelease tablets (Tegretol CR or Zeptol CR and Depakine Chrono, respectively) while the rest took prompt release dosage forms. Patients were on a stable dosage regimen for at least 6 weeks; hence, a steady state was assumed and the pharmacokinetic interaction, if any, was assumed to be complete. We excluded pregnant women, patients with hepatic disease or renal disease. Additionally, patients who were concurrently using drugs which may interact with CBZ pharmacokinetics (except PHT, PB and VPA) were excluded, namely verapamil, diltiazem, haloperidol, theophylline, ticlopidine, cimetidine, omeprazole, trazodone, fluoxetine, risperidone, clarithromycin, erythromycin, rifampicin, isoniazid, isotretrinion, gemfibrozil and metronidazole [14][15][16]. Grapefruit consumers, if any, were excluded. All subjects gave written informed consent.

Blood Sampling and Assay
Blood samples were obtained as a part of routine TDM. All blood samples were drawn in the morning before drug intake (trough level). Total CBZ, PHT, PB and VPA concentrations in serum were determined using an immuno-turbidimetry assay method with an automated analyzer (Synchron LX ® Systems, Beckman Coulter Inc., Fullerton, California, USA). The assay method has been routinely validated according to company guidance. The analytical range for CBZ level was 2.0-20.0 mg/L, while the precision specification was 0.6 mg/L or 5.0%. The analytical range for PHT level was 2.5-40.0 mg/L, while the precision specification was 0.5 mg/L or 4.0%. The analytical range for PB level was 5.0-80.0 mg/L, while the precision specification was 1.0 mg/L or 4.0%. The analytical range for VPA level was 10.0-150.0 mg/L, while the precision specification was 3.6 mg/L or 6.0%.

Genotyping
Genomic DNA was extracted from peripheral whole blood using QIAamp ® DNA Blood Mini kit (Qiagen, Valencia, California, USA).   F is the bioavailability, S is the salt fraction for CBZ = 1,  is the dosing interval (hour or day), and Css ave is the measured serum drug concentration at steady state (mg/L). Because CBZ absolute bioavailability is unknown, apparent clearance (CL/F) was estimated [8,9].

DATA ANALYSIS
PK parameters were calculated using Microsoft Excel and statistical analyses were determined using SPSS version 17.0 (SPSS Co., Ltd., Bangkok Thailand). The level of significance was set at p ≤ 0.05. Continuous variables were determined for normality of the distribution using the Kolmogorov-Smirnov test and determined for homogeneity of variance using Levene's test. Statistical comparisons of CBZ clearance and dose-adjusted CBZ levels between patients with CYP3A5*1 and CYP3A5*3 were performed using independent t-test or Mann-Whitney U test, and between patients with CBZ monotherapy or coadministered with PHT, PB or VPA were performed using one-way ANOVA or Kruskal-Wallis test. All p-values were corrected for multiple testing.

RESULTS
Seventy patients who used CBZ as monotherapy or co-administered with PHT, PB or VPA and met the inclusion-exclusion criteria were included in this study. Their demographic data and plasma levels of related drugs were collected from the electronic database and medical records at the epilepsy outpatient clinic of Prasat Neurological Institute.

Frequencies of CYP3A5
The allele frequency of CYP3A5*1 was 31% and CYP3A5*3 was 69%. Allelic frequencies of CYP3A5 genotypes did not significantly differ from those predicted by Hardy-Weinberg Equilibrium.

Demographic Data
Of the 70 patients, 67 were diagnosed as suffering epilepsy and 3 neuropathic pain. Among these, 36 patients used CBZ as monotherapy, 7 patients used CBZ in combination with PHT, 11 patients used CBZ in combination with PB and 16 patients used CBZ in combination with VPA. The patient's gender, age, body weight and BMI did not significantly differ among the three different CYP3A5 genotype groups, as shown in Table 1.

Effect of CYP3A5 Polymorphism on CBZ Pharmacokinetics CBZ Monotherapy
Thirty six patients who used CBZ as monotherapy were categorized into a CYP3A5 expressor group and a CYP3A5 non-expressor group. The CYP3A5 expressor group comprised the genotypes CYP3A5*1/*1 and CYP3A5*1/*3. The CYP3A5 non-expressor group consisted of the CYP3A5*3/*3 genotype. There were no statistically significant differences in patient's demographic characteristics (i.e., age, body weight, BMI, and male gender) and pharmacokinetic parameters of CBZ between the two groups. The details are shown in Table 2.

CBZ used in combination with other AEDs CBZ in combination with enzyme-inducing AED group (CBZ+PHT or CBZ+PB)
Comparisons of CBZ PK parameters between CYP3A5 expressor group and CYP3A5 nonexpressor group among 18 patients who used CBZ in combination with enzyme-inducing AED revealed that the CBZ CL/F (L/kg/hr) in the CYP3A5 expressor group showed a higher trend than that observed in the CYP3A5 non-expressor group, but did not reach a statistically significantly different level. The details are shown in Table 3.

CBZ+VPA
Comparisons of CBZ PK parameters between CYP3A5 expressor group and CYP3A5 nonexpressor group among the 16 patients who used CBZ in combination with VPA showed no statistically significantly difference in either CBZ PK parameters as demonstrated in Table 3. Tables 4A and 4B show comparisons of PK parameters of CBZ within the same genotype group between patients who used CBZ as monotherapy with those who used CBZ in combined treatment with a different second AED, i.e. PHT, PB and VPA.

CYP3A5 Expressor Group
CBZ dose (mg/day, mg/kg/day) and CBZ level (mg/L) did not significantly differ among the three groups categorized based on different second AED used in combination with CBZ; however, when the level was standardized with the dose, dose-adjusted CBZ level (mcg/L/mg) and CBZ CL/F (L/hr, and L/kg/hr) significantly differed between the CBZ monotherapy group and CBZ combined with second AED group. The details are shown in Table 4A.
Post hoc comparisons of the CBZ PK parameters between CBZ monotherapy, CBZ + enzyme-inducing AEDs and CBZ+VPA within the CYP3A5 expressor group revealed that the mean CBZ CL/F (L/kg/hr) in patients who used CBZ + enzyme-inducing AEDs (PHT, PB) was significantly higher than the mean CBZ CL/F in patients who used CBZ as monotherapy (43.28%, p=0.013). Accordingly, the mean dose-adjusted CBZ level (mcg/L/mg) in patients who used CBZ + enzyme-inducing AEDs was significantly lower than that obtained in patients who used CBZ as monotherapy (78.10%, p=0.010), while the mean CBZ CL/F and the mean dose-adjusted CBZ level (mcg/L/mg) in patients who used CBZ + VPA did not significantly differ statistically from patients who used CBZ as monotherapy.  Table 4B. The mean dose-adjusted CBZ level (mcg/L/mg) in CBZ+ enzyme-inducing AEDs group was 20.83% lower than the mean in the monotherapy group; however, this figure did not reach a statistically significant level. The mean of CBZ CL/F (L/kg/hr) in patients who used CBZ+ enzyme-inducing AEDs was 16.47% higher than the mean of CBZ CL/F in patients who used CBZ as monotherapy -but this also did not statistically differ significantly. At the same time, the mean of CBZ CL/F in patients who used CBZ+VPA was 18.39% higher than the mean of CBZ CL/F in patients who used CBZ monotherapy, and showed no statistically significant difference.

DISCUSSION
The observed allelic frequency of CYP3A5*3 among the 70 patients participating in this study was 69%. This frequency was similar to previous studies in the Thai population (66%) and in most other Asian populations, including Chinese, Indian, Malaysian and Japanese (78%, 59%, 61% and 77% respectively). However, this frequency of CYP3A5*3 allele observed in our study was lower than that reported for Caucasians (92%) and higher than that recorded for African-Americans (48%). Other CYP3A5 coding variants have been described, but occur at relatively low allele frequencies [17][18][19][20][21][22]. In addition to CYP3A5, CYP3A4, as the most abundant isoform of the CYP3A family may play an important role in the metabolism of CBZ. The CYP3A4 gene is also very polymorphic; therefore, its genetic polymorphisms may influence the levels of CBZ obtained. However, unlike the CYP3A5 gene, there is no evidence of a 'null' allele for CYP3A4 [23]. Nonetheless, the over 30 SNPs identified in the CYP3A4 gene [24] may contribute to the marked inter-individual variation in CYP3A4-catalysed metabolism. However, since the frequencies of different CYP3A4 alleles are very low, the impact may not be distinct. In vitro data show that CYP3A5 exhibited metabolic activity comparable to that of CYP3A4 toward CBZ epoxidation [11]. The CYP3A5*3 causes alternative splicing and protein truncation, resulting in an absence of protein activity, whereas individuals with at least one CYP3A5*1 allele express the CYP3A5 isoenzyme. Because CYP3A5 may represent up to 50% of the total CYP3A protein in individuals polymorphically expressing CYP3A5, it may have a major role in the variation of CYP3A-mediated drug metabolism [22][23]. A previous study by Seo et al. of 144 Japanese epileptic patients reported that patients with CYP3A5*3/*3 exhibited CBZ clearance 8% higher than patients not carrying CYP3A5*3/*3 [15]. This result conflicted with the result from the study by Park et al. of 35 Korean epileptic patients, who reported that the CBZ clearance in patients with homozygous CYP3A5*3/*3 was 29% lower than that observed in patients with at least one CYP3A5*1 allele [16]. Seo et al. [15] recruited patients who used CBZ as either monotherapy or concurrently with the potent inducer of CYP3A, i.e. PHT and PB, into their study which may confound the effect of CYP3A5 genotypes on CBZ pharmacokinetics. The reason that CBZ clearance was found to be higher in the CYP3A5*3/*3 group might be due to the number of patients who used CBZ concurrently with the potent inducer being also higher in that group. Park et al. included only patients who used CBZ as monotherapy with their results indicating that CYP3A5*3/*3 would result in lower CBZ clearance.   In this study, in order to avoid the confounding effect of the enzyme-inducing factor, the effects of CYP3A5 polymorphism on CBZ pharmacokinetic parameters were determined by grouping the patients based on the AEDs used, i.e., CBZ monotherapy, CBZ+PHT or CBZ+PB, CBZ+VPA. Since a previous study on population pharmacokinetics by Vucicevic et al [14] reported that smoking had no effect on CBZ clearance, smokers were not excluded from this study.
Comparisons of CBZ PK parameters between groups of different genotypes among the 36 patients who used CBZ as monotherapy, either categorized patients into three groups as CYP3A5*1/*1, CYP3A5*1/*3 and CYP3A5*3/*3 or categorized patients into two groups as CYP3A5 expressors and CYP3A5 non-expressors. CBZ level and CBZ CL/F showed no significant difference between groups of different genotypes (comparisons of the categorized patients into three groups are not shown). These results conflict with the results reported by Park et al., who reported that the mean of dose-adjusted CBZ level in CYP3A5 expressors (9.94±3.38 mcg/L/mg) was significantly lower (p=0.032) than the mean of dose-adjusted CBZ level in CYP3A5 non-expressors (13.07±4.46 mcg/L/mg), while the mean of CBZ CL/F in CYP3A5 expressors (0.056±0.017 L/kg/hr) was significantly higher (p=0.004) than the mean of CBZ CL/F in CYP3A5 non-expressors (0.040±0.014 L/kg/hr) [16]. In our study, the mean of dose-adjusted CBZ level in CYP3A5 expressors was 11.06±3.92 mcg/L/mg, while the mean of CBZ level-to-dose ratio in CYP3A5 nonexpressors was 10.61±3.65 mcg/L/mg, meaning they were nearly equal and were not statistically significantly different (p=0.727). At the same time, the mean of CBZ CL/F in CYP3A5 expressors was 0.076±0.033 L/kg/hr while the mean of CBZ CL/F in CYP3A5 non-expressors was 0.071±0.019 L/kg/hr -6.58% lower but not statistically significantly different (p=0.552).
The results also demonstrated that the enzyme-inducing effect of PB and PHT on CBZ clearance was stronger than the effect caused by polymorphism of the CYP3A5 genotype.
Even though the CYP3A5 is not the only enzyme for CBZ metabolism, the drug-drug interaction that inhibits or induces this enzyme may cause significant alteration of CBZ clearance because it is a prominent enzyme in the major pathway of CBZ metabolism. PB and PHT are the common potent inducer of the CYP450 enzymes including CYP3A5. These AEDs will increase the clearance of CBZ due to enhanced metabolism [8][9][10][11][25][26]. Previous studies reported the increment of CBZ clearance to be within the range of 16-44% when concurrently used with PB, while the increment was 42-45 % in patients who used CBZ in combination with PHT as compared to patients who used CBZ monotherapy [13][14][27][28]. Consistent with this study, especially in CYP3A5 expressors who used CBZ+ enzyme-inducing AEDs, whose CBZ clearance was 43.28% higher when compared to that obtained in patients using CBZ monotherapy, while in CYP3A5 non-expressors CBZ clearance increased less (by 16.47%). However, the effect of AEDs on the induction of CYP3A5 has not been directly investigated. There are conflicting results on the effect of VPA on CBZ clearance: increase, decrease, or no change [7,[13][14][25][26][27][28]. In this study, the CBZ clearance in CYP3A5 expressors who used CBZ + VPA was quite similar to patients who used CBZ monotherapy, while in CYP3A5 non-expressors the CBZ clearance in patients who used CBZ + VPA was 18.39% higher than patients who used CBZ monotherapy. VPA which has a higher proteinbinding capacity than CBZ could increase CBZ clearance by replacing protein-bound CBZ and changing CBZ to a free form that would be eliminated faster [13,29]. The inability to measure CBZ-epoxide and CBZ-diol which are the major metabolites of CBZ and the too low sample size are the limitations of this study.

CONCLUSIONS
The CYP3A5 genotype did not substantially affect the pharmacokinetics of CBZ especially when the drug was used as monotherapy. However, in patients who used CBZ in combination with enzyme-inducing AED (PHT or PB), individuals carrying CYP3A5*1 allele yielded a trend toward being more susceptible to increments in CBZ clearance and showed lower dose-adjusted CBZ level as compared to individuals carrying CYP3A5*3.
Genotype identifying might be an advantage for predicting individual dosage requirements for targeted therapeutic levels, especially for those who have used CBZ in combination with enzymeinducing AED. Thus, better clinical outcome can be achieved.

ACKNOWLEDGEMENT
This study was supported by a grant from the 90 th anniversary of Chulalongkorn University fund (Ratchadaphiseksomphot Endowment Fund).