Effects of cytochrome P450 oxidoreductase genotypes on the pharmacokinetics of amlodipine in healthy Korean subjects

Abstract Background The aim of this study was to investigate the effects of P450 oxidoreductase (POR) genetic polymorphisms on the pharmacokinetic parameters of amlodipine. Methods After a single 10‐mg dose of amlodipine administration, 25 healthy male subjects completed genotyping for 12 single nucleotide polymorphisms (SNPs) of the POR genes, cytochrome P450 (CYP)3A4 g.25343G>A (CYP3A4*1G), and CYP3A5 g.12083G>A (CYP3A5*3). Stratified analysis and in silico analysis to predict the possible effects of given variants on splicing were performed. Results The maximum blood concentration (Cmax) of amlodipine in carriers of g.57332T>C and g.56551G>A SNPs of the POR gene was statistically significantly different. In addition, T‐allele carriers of g.57332T>C had a 21% higher Cmax than those with the CC genotype (p = .007). Subjects who carried the wild‐type g.56551G>A allele also had a 1.12‐fold significantly higher Cmax than subjects with mutant‐type homozygous carriers (p = .033). In stratified analyses, g.57332T>C was significantly associated with a 1.3‐fold increase in Cmax value in T‐allele carriers compared with subjects with the CC genotype in CYP3A4 and CYP3A5 expressers. POR g.57332T>C increased the score above the threshold in both ESEfinder 3.0 and HSF 3.1. Conclusion This study identified a novel SNP of the POR gene, which affected amlodipine metabolism and may reduce interindividual variation in responses to amlodipine.

CYP3A is involved in 40%-50% of the oxidative biotransformation of current therapeutic agents (Evans & Relling, 1999). Because it is located in the intestinal mucosa and liver tissue, it is associated with drug metabolism ability after oral drug administration (Wilkinson, 1996). Of the four types of CYP3A, CYP3A4 and CYP3A5 are the most abundant in the liver and intestine in that order, whereas CYP3A7 and CYP3A43 are undetectable or expressed at very low levels in the adult liver (Burk et al., 2002;Gellner et al., 2001;Nelson et al., 1996;Westlind et al., 2001). CYP3A has large interindividual variability of 5-to 20-fold in catalyzing drug metabolism, which are the substrates of CYP3A (Evans & Relling, 1999;Wilkinson, 1996). However, CYP3A polymorphism alone is not sufficient to account for the interindividual variation in CYP3A metabolic activity (Ingelman-Sundberg, Sim, Gomez, & Rodriguez-Antona, 2007;Özdemir et al., 2000).
Even though POR polymorphisms have important effects on interindividual variation in CYP activity, few studies have investigated these polymorphisms and their effects on amlodipine pharmacokinetics (PKs). Thus, the aim of this study was to investigate the effects of POR gene polymorphisms on the PK parameters of amlodipine.

| Study populations
The study population was included from 50 healthy male volunteers who had participated in a bioequivalence study of a 10-mg dose of amlodipine (Kim et al., 2013). Among these volunteers, 25 healthy men participated in this study after providing additional written consent for genotyping. Eligible subjects were men between the ages of 20 and 50 years, who were within 20% of their ideal body weight with no congenital abnormality or chronic disease. Exclusion criteria were as follows: (a) use of prescription drugs or herbal medications within 2 weeks or use of nonprescription drugs within 1 week before the study, which had the potential to interact with amlodipine; and (b) use of drugs that induce or inhibit drug-metabolizing enzymes within 1 month before the study, which had the potential to interact with study medications. Vital signs monitoring, physical examination, and routine laboratory tests were performed before the start of the study.

| Clinical study
The PK data of the study population were obtained from a previous single-dose study of amlodipine (Kim et al., 2013). Subjects took a 10-mg tablet of amlodipine orally with 240-mL water at 8 a.m. after an overnight fast for 10 hr. Venous blood samples were collected into ethylenediaminetetraacetic acid-containing tubes by an indwelling catheter inserted into the forearm at 0 (predose) and 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 16, 24, and 48 hr after dosing. Blood samples for genotyping were also collected, and genotyping was performed after the end of the study.

| Editorial policies and ethical considerations
The study protocol was approved by the Ethics Committee of the Institutional Review Board (IRB No. 2012-4-0283). Informed consent was obtained from all patients before study participation.

| PK analysis
PK parameters were calculated using actual sampling times. Maximum blood concentration (C max ) and time to maximum concentration (T max ) were determined by searching the observed data. The area under the plasma concentration-time curve from time zero to the time of the last concentration (AUC last ) was calculated using the linear trapezoidal rule. The AUC from time zero to infinity (AUC inf ) was the sum of AUC last and C last / k e , where C last is the last quantifiable concentration and k e is the terminal elimination rate constant; the half-life was 0.693/ k e . Plasma concentrations during the terminal phase were fitted to a log linear line by the least squares method to obtain the k e . PK parameters were analyzed by a noncompartmental method using WinNonlin5.3 (Pharsight Corporation).

| In silico analyses
To predict the possible effects of given variants on splicing, different computational tools were used. Netgene2 and Splice Site Prediction by Neural Network (NNSPLICE) were used for splice site predictions (Brunak, Engelbrecht, & Knudsen, 1991;Reese, Eeckman, Kulp, & Haussler, 1997). Alternations of the splicing factor-binding site pattern caused by the given mutation were evaluated by using Exonic Splicing Enhancer (ESE) finder 3.0 and Human Splicing Finder (HSF) 3.1 (Cartegni, 2003;Desmet et al., 2009). We used the default threshold values, and a score for a given sequence was considered to be potentially significant if it was above the threshold values.

| Statistical analysis
All PK data were expressed as the mean ± standard deviation (SD). Hardy-Weinberg equilibrium (HWE) was tested using the chi-square test. Differences in PK parameters among the genotype groups were evaluated using the Mann-Whitney rank sum test for two-group comparisons. Stratified analyses were conducted to investigate the effects of POR gene polymorphisms on amlodipine PKs using CYP3A expressers. p values < .05 were considered statistically significant. Statistical analyses were performed using SPSS 20.0 (International Business Machines Corp.).

| RESULTS
The mean age, weight, and height of the subjects were 26.8 ± 5.9 years, 67.9 ± 8.2 kg, and 174.2 ± 4.8 cm, respectively. The mean PK parameter values were as follows: C max : 6.09 ± 1.06 ng/ml, T max : 6.32 ± 0.75 hr, half-life: 40.75 ± 7.29 hr, AUC last : 257.45 ± 54.99 hr·μg/mL, AUC inf : 267.80 ± 59.71 hr·μg/mL, ke: 0.02 ± 0.00 hr -1 , oral clearance (CL/F): 39.25 ± 9.35 L/h, and volume of distribution (Vd/F): 2,278.51 ± 560.01 L. All SNPs were in accordance with HWE. The MAFs of g.57332T>C and g.56551G>A were 0.125 and 0.24, respectively. Table 1 described the association between polymorphisms of POR genes and PK parameter values. The locations of the 12 selected SNPs in the POR gene were the intron region (n = 8), 5'-untranslated region (UTR; n = 1), 3'-UTR (n = 1), missense region (n = 1), and synonymous region (n = 1). Of the 12 POR SNPs, g.57332T>C and g.56551G>A were significantly associated with the C max of amlodipine. T-allele carriers of g.57332T>C had a 21% higher C max than those with the CC genotype (p = .007). Subjects who carried the wild-type allele of g.56551G>A also showed a 1.12-fold significantly higher C max than subjects with mutant-type homozygous carriers (p = .033). The mean (±SD) plasma concentration-time profiles of amlodipine after oral administration according to genotypes of g.57332T>C and g.56551G>A are shown in Figure 1. There was no statistically significant difference between CYP3A polymorphisms and PK parameters of amlodipine. Stratified analyses were performed for the association between PK parameter values and POR polymorphisms in CYP3A expressers (Table 2). POR SNPs that were statistically significant in univariate analysis were included. POR g.57332T>C was significantly associated with the 1.3-fold increase in C max value in T-allele carriers compared with subjects with the CC genotype in CYP3A4 and CYP3A5 expressers.
Analysis of two SNPs (g.57332T>C and g.56551G>A) with Netgene2 and NNSPLICE showed a significant association with the C max of amlodipine, and did not show the presence of an altered splicing donor or acceptor. However, the results generated by ESEfinder 3.0 indicated that the g.57332T>C increased the score of a SRSF1 (IgM-BRCA1) from −0.873 to 2.232 (threshold: 1.867), creating a new high score-enhancing motif. HSF 3.1, with a different scoring algorithm from that of ESEfinder 3.0, also showed that this mutation led to the formation of two SF2/ASF (IgM-BRCA1)-enhancing motifs (CTCCCCG and CCCCGCT); the scores were 75.31 and 73.31, respectively (threshold: 70.51) (Supplement Table S1).

| DISCUSSION
The main result of this study was that g.57332T>C and g.56551G>A of the POR genes showed statistically significant differences in the C max of amlodipine. After adjusting for CYP3A effects, g.57332T>C remained a significant factor for amlodipine PKs.
POR is a gene that gives electrons to CYP450 enzymes (Masters, 2005). The structure of the POR gene consists of an NADPH-docking site and FAD in one lobe and FMN and P450-interacting domain in the other lobe (Wang et al., 1997). The electron transfer proceeds from NADPH to FAD, followed by FMN, and finally to P450. In a study that examined the effects of 35 POR variants on CYP enzymes, it was revealed that the POR variants are involved in the activity of the CYP enzymes, although the effects vary depending on the variants .
The association between POR polymorphisms and CYP3A4 metabolic activities using different substrates including midazolam, testosterone, erythromycin, and quinidine was assessed (Agrawal et al., 2010). This study revealed that the impact of POR polymorphisms on CYP3A4 activity was substrate specific, possibly due to the substrate-induced conformational changes in CYP3A4. In the case of the most common variant, c.1508C<T (POR*28), the polymorphism reduced the CYP3A4 activity to 61%-77% of wild-type with testosterone and midazolam while it had similar activity to wild-type with quinidine and erythromycin. Our study failed to show significant differences in any PK parameters of amlodipine according to genotypes of POR*28. Instead, novel POR variants, g.57332T>C and g.56551G>A, which affected C max value of amlodipine, were identified. In stratified analyses to rule out the effects of CYP3A polymorphisms and find POR variant effects, g.57332T>C remained significant SNP in both CYP3A4 and CYP3A5 expressers. In particular, the difference in C max values according to the g.57332T>C genotypes in expressers increased (20.7% vs. around 33%). The SNPs of g.57332T>C and g.56551G>A are located in intronic regions, which are thought to not be involved in protein production. However, intronic regions have the potential to affect mRNA splicing and alter protein expression or activity; thus, analyzing these regions may be useful (Pagani & Baralle, 2004;Raponi & Baralle, 2010). To evaluate the splicing effects of these two SNPs, we employed various computational tools and found that g.57332T>C affected the splicing of -POR. In addition, it has been revealed that intronic POR variants also affected CYP activity. An in vitro study evaluating the effect of POR polymorphisms on CYP activity showed that three intronic SNPs were associated with alteration of various CYP functions (Gomes et al., 2009). The intronic SNP g.18557G>A decreased CYP3A4 activity, whereas g.25676C>T and g.30986G>A increased microsomal activities of CYP1A2, 2C8, 2C19, and 3A4 and of CYP2C19 and 3A4, respectively. Therefore, g.57332T>C may be a candidate SNP that affects the amlodipine absorption rate.
CYP3A4 and CYP3A5 are major enzymes of CYP3A family (Zhu et al., 2014). CYP3A5 g.12083G>A (CYP3A5*3), which was analyzed in this study, is the most common CYP3A5 polymorphic form and causes a truncated CYP3A5 enzyme and loss of CYP3A5 activity (Kim et al., 2007;Kuehl et al., 2001;Park et al., 2006;Park et al., 2009). However, in an amlodipine PK study, subjects with CYP3A5*3/*3 had a lower plasma concentration of amlodipine than subjects who carried CYP3A5*1 . In terms of CYP3A4, g.25343G>A (CYP3A4*1G) has a high frequency in Asian populations (Fukushima-Uesaka et al., 2004;Zhou et al., 2011). In a study that demonstrated the impact of g.25343G>A (CYP3A4*1G) polymorphism on fentanyl metabolism, plasma concentration in the *1G/*1G group was significantly higher than that in the *1/*1G and *1/*1 groups, indicating loss-of-function mutation (Zhou et al., 2011). In contrast, a clinical study using clopidogrel showed similar PK profiles between wild-type homozygotes and mutant allele carriers (Danielak et al., 2017). As described above, the effects of CYP3A4 and CYP3A5 polymorphisms on drug PKs are not consistent. In this study, no significant effects of CYP3A4 and CYP3A5 polymorphisms including g.12083G>A (CYP3A5*3) and g.25343G>A (CYP3A4*1G) on the PK parameters of amlodipine were observed.
Only the C max among PK parameters was statistically significant; however, in applying these results to clinical settings, we need to recognize that failure to achieve statistical significance does not necessarily mean clinical insignificance. With respect to g.57332T>C in CYP3A4 and CYP3A5 expressers, the difference in AUC inf and CL/F was more than 25% according to genotypes, but statistical significance was not found. This was possibly due to the small sample size, resulting in an underpowered study. There were some limitations in this study. The sample size was too small to obtain statistically significant results. Our study population only comprised males, so it was impossible to analyze gender differences. Multiple testing correction was not performed to avoid the possible loss of true positives.
In conclusion, to the best of our knowledge, this is the first study to evaluate the association between POR gene polymorphisms and amlodipine PKs in a Korean population. The identified novel SNP of the POR gene, which was shown to affect amlodipine metabolism, may be useful for reducing interindividual variation in responses to amlodipine. However, these results should be interpreted with caution due to the risk of false-positive results. Additional studies are needed to verify the results of this study.