Influence of CYP2C9 Genetic Polymorphisms on the Pharmacokinetics of Losartan and Its Active Metabolite E-3174: A Systematic Review and Meta-Analysis

This study aimed to investigate the influence of CYP2C9 genetic polymorphisms on the pharmacokinetics of losartan and its active metabolite, E-3174, through a systematic review and meta-analysis. Eight studies published before March 2021 were included in this study. We used PubMed, the Cochrane Library, EMBASE, and Web of Science, based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The data analysis was conducted through Review Manager (RevMan), version 5.3, and R software. We found that healthy volunteers with CYP2C9*2 or *3 carriers had higher area under the curve (AUC0-∞) of losartan (mean difference (MD) 0.17 μg·h/mL; 95% confidence intervals (CI): 0.04, 0.29) and lower AUC0-∞ of E-3174 (MD −0.35 μg·h/mL; 95% CI: −0.62, −0.08) than those with CYP2C9*1/*1. Subjects with CYP2C9*2 or *3 carriers showed lower maximum concentration (Cmax) of E-3174 than those with CYP2C9*1/*1 (MD −0.13 μg/mL; 95% CI: −0.17, −0.09). For half-life, subjects with CYP2C9*2 or *3 carriers had longer half-lives of losartan and E-3174 than those with CYP2C9*1/*1 (MD 0.47 h; 95% CI: 0.32, 0.61 and MD 0.68 h; 95% CI: 0.44, 0.92, respectively). This meta-analysis suggests that the pharmacokinetics of losartan and E-3174 are associated with the CYP2C9 polymorphisms


Introduction
Losartan is an angiotensin II receptor blocker (ARB) that is widely used for hypertension, heart failure, and diabetic nephropathy. It blocks the angiotensin II type 1 (AT1) receptor. It is absorbed from the gastrointestinal tract after oral administration and undergoes substantial first-pass metabolism, resulting in a systematic bioavailability of approximately 33%. It is metabolized to an active carboxylic acid metabolite E-3174, which has up to 40 times greater pharmacological activity than losartan [1,2].
Safe and effective drug therapy requires an understanding of a drug's pharmacokinetic, pharmacodynamic, and pharmacogenomic interrelationships [6]. Drug response and adverse events can be predicted using pharmacokinetic parameters [7]. Genetic polymorphisms can affect drug concentrations and effectiveness. However, the association between CYP2C9 gene polymorphisms and losartan pharmacokinetic parameters has been inconsistent, possibly due to small sample sizes [8][9][10][11]. This study aimed to investigate the effects of the CYP2C9 polymorphisms on the pharmacokinetic characteristics of losartan and E-3174 through systematic review and meta-analysis.

Methods
The paper was conducted based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [12].

Search Strategy
Two investigators independently conducted a systemic search for all studies published before 4 March 2021, using PubMed, Cochrane Library, EMBASE, and Web of Science. The following search terms were included: (losartan OR (losartan potassium) OR Cozaar) AND ((CYP2C9*) OR (cytochrome p450 2C9) OR (cytochrome p 450 2C9)) AND (polymorph* OR variant* OR mutation* OR genotyp* OR phenotyp* OR haplotyp* OR allele* OR SNP* OR pharmacogen*).

Selection Criteria and Data Extraction
Studies were selected if they (1) compared the pharmacokinetic characteristics of subjects with CYP2C9*2 or *3 alleles to those with CYP2C9*1/*1 after oral administration of losartan; (2) included healthy adults who received a single dose of 50 mg losartan; and (3) were randomized, controlled trials (RCT) or cohort studies.
Studies were excluded if they (1) were editorials, notes, abstracts, reviews, news, letters, posters, or comments; (2) were in vitro or animal studies; (3) did not include blood sample data; (4) had concomitant medications with losartan; or (5) were unable to extract outcome data. If there were overlapping data, only the most recent and comprehensive data were included in the meta-analysis.
The following parameters were extracted independently by two investigators: name of the first author, year of publication, nation, studied polymorphisms, age, number of subjects, body mass index (BMI), genotyping methods, and quantitative methods. The AUC 0-∞ (primary outcome), C max , and half-life (secondary outcomes) of losartan and E-3174 were also extracted from each study.
Study quality was assessed by the Newcastle-Ottawa scale (NOS) tool [13]. The NOS tool assessment is based on three primary domains, including selection of subjects (0-4 points), comparability of study groups (0-2 points), and determination of outcomes of interest (0-3 points).
We performed a subgroup analysis by ethnicity and conducted a sensitivity analysis, using sequential omission of each study, to validate the robustness of the results. Begg's rank correlation test and Egger's regression test were used to detect publication bias. Statistical analyses were performed using Review Manager (RevMan) version 5.3 (The Cochrane Collaboration, Copenhagen, Denmark) and R software (version 4.0.5; R Foundation for Statistical Computing, Vienna, Austria). A p-value < 0.05 was considered statistically significant.

Sensitivity Analysis
To assess the stability of the results, a sensitivity analysis was performed by sequentially excluding each study. Sensitivity analysis of AUC 0-∞ of losartan and E-3174 showed similar results to the main analysis; MD between CYP2C9*2 or *3 carriers and CYP2C9*1/*1 ranged from 0.09 to 0.22 for AUC 0-∞ of losartan and −0.54 to −0.25 for AUC 0-∞ of E-3174, respectively. When the study by Cabaleiro et al. was eliminated, the heterogeneity of losartan AUC 0-∞ decreased from 64% to 11% [16].

Sensitivity Analysis
To assess the stability of the results, a sensitivity analysis was performed by sequentially excluding each study. Sensitivity analysis of AUC0-∞ of losartan and E-3174 showed similar results to the main analysis; MD between CYP2C9*2 or *3 carriers and CYP2C9*1/*1 ranged from 0.09 to 0.22 for AUC0-∞ of losartan and −0.54 to −0.25 for AUC0-∞ of E-3174, respectively. When the study by Cabaleiro et al. was eliminated, the heterogeneity of losartan AUC0-∞ decreased from 64% to 11% [16].

Discussion
This is the first meta-analysis to evaluate the effect of CYP2C9 gene polymorphisms on the pharmacokinetic properties of losartan. The results showed that CYP2C9*2 or *3 carriers had a higher AUC0-∞ of losartan and lower AUC0-∞ of E-3174 than those with CYP2C9*1/*1. There was no significant difference in the Cmax of losartan between subjects with CYP2C9*2 or *3 and CYP2C9*1/*1, whereas the subjects with CYP2C9*2 or *3 carriers had lower Cmax of E-3174 than those with CYP2C9*1/*1. The half-lives of both losartan and E-3174 were longer in CYP2C9*2 or *3 carriers than CYP2C9*1/*1. Similar results were obtained in the comparison between CYP2C9*1/*1 and CYP2C9*3 carriers. There was an ethnic difference in the effects of CYP2C9 gene polymorphisms on the AUC0-∞ values of losartan and E-3174 between Asians and Caucasians.

Discussion
This is the first meta-analysis to evaluate the effect of CYP2C9 gene polymorphisms on the pharmacokinetic properties of losartan. The results showed that CYP2C9*2 or *3 carriers had a higher AUC 0-∞ of losartan and lower AUC 0-∞ of E-3174 than those with CYP2C9*1/*1. There was no significant difference in the C max of losartan between subjects with CYP2C9*2 or *3 and CYP2C9*1/*1, whereas the subjects with CYP2C9*2 or *3 carriers had lower C max of E-3174 than those with CYP2C9*1/*1. The half-lives of both losartan and E-3174 were longer in CYP2C9*2 or *3 carriers than CYP2C9*1/*1. Similar results were obtained in the comparison between CYP2C9*1/*1 and CYP2C9*3 carriers. There was an ethnic difference in the effects of CYP2C9 gene polymorphisms on the AUC 0-∞ values of losartan and E-3174 between Asians and Caucasians.
There are several studies on the effect of CYP2C9 inhibitors, such as fluconazole, bucolome, and ticlopidine, on the pharmacokinetic parameters of losartan. An in vitro study revealed that the C max and AUC 0-∞ of E-3174 significantly decreased to 30% and 47% by fluconazole, respectively, compared to the control group [22]. Kobayashi et al. reported that concomitant use of losartan and bucolome resulted in the significant decrease of AUC 0-∞ of E-3174 and increase of AUC 0-∞ of losartan in healthy male volunteers [23]. There was also a report that ticlopidine increased the AUC 0-∞ of losartan in rats [24]. The aforementioned studies indicate that the CYP2C9 enzyme was a significant metabolizer of losartan.

Discussion
This is the first meta-analysis to evaluate the effect of CYP2C9 gene polymorphisms on the pharmacokinetic properties of losartan. The results showed that CYP2C9*2 or *3 carriers had a higher AUC 0-∞ of losartan and lower AUC 0-∞ of E-3174 than those with CYP2C9*1/*1. There was no significant difference in the C max of losartan between subjects with CYP2C9*2 or *3 and CYP2C9*1/*1, whereas the subjects with CYP2C9*2 or *3 carriers had lower C max of E-3174 than those with CYP2C9*1/*1. The half-lives of both losartan and E-3174 were longer in CYP2C9*2 or *3 carriers than CYP2C9*1/*1. Similar results were obtained in the comparison between CYP2C9*1/*1 and CYP2C9*3 carriers. There was an ethnic difference in the effects of CYP2C9 gene polymorphisms on the AUC 0-∞ values of losartan and E-3174 between Asians and Caucasians.
There are several studies on the effect of CYP2C9 inhibitors, such as fluconazole, bucolome, and ticlopidine, on the pharmacokinetic parameters of losartan. An in vitro study revealed that the C max and AUC 0-∞ of E-3174 significantly decreased to 30% and 47% by fluconazole, respectively, compared to the control group [22]. Kobayashi et al. reported that concomitant use of losartan and bucolome resulted in the significant decrease of AUC 0-∞ of E-3174 and increase of AUC 0-∞ of losartan in healthy male volunteers [23]. There was also a report that ticlopidine increased the AUC 0-∞ of losartan in rats [24]. The aforementioned studies indicate that the CYP2C9 enzyme was a significant metabolizer of losartan.
The pharmacokinetic properties of losartan can affect its response. One study found that the CYP2C9*3 variant reduces losartan metabolism and its hypotensive effect after oral administration of losartan [25]. In this study, the C max of E-3174 in the CYP2C9*1/*3 group was 30% lower than that in the CYP2C9*1/*1 group. Subjects with CYP2C9*1/*3 had a less hypotensive effect in the diastolic blood pressure (DBP) at 10 h and 12 h post-dosing than those with CYP2C9*1/*1. The CYP2C9*1/*1 group had a more significant decrease in the systolic blood pressure (SBP) from 1 h to 12 h than the CYP2C9*1/*3 group.
CYP2C9 polymorphism may differently affect the ARB drug responses. Losartan has lower efficiency in CYP2C9*2 or *3 carriers, despite an increased concentration of losartan. The low efficiency in CYP2C9*2 or *3 carriers was attributed to the decreased AUC 0-∞ of E-3174, which has more potent activity than losartan. In contrast, in the case of irbesartan, the CYP2C9*1/*3 genotype carriers showed both a higher concentration of irbesartan and greater DBP responses compared to the CYP2C9*1/*1 genotype carriers; this was possibly because the metabolite has no pharmacological activity [26]. Chen et al. also showed that subjects with the CYP2C9*1/*3 genotype had significantly higher plasma irbesartan concentrations compared with those with the CYP2C9*1/*1 genotype [27].
We further investigated the effect of the complex heterozygous genotype of CYP2C9*2 and CYP2C9*3 on losartan pharmacokinetics or pharmacodynamics. There was only one study to examine the pharmacokinetic difference between patients with both CYP2C9*2 and CYP2C9*3 alleles and those with CYP2C9*1/*1 [11]. Subjects with the complex heterozygous genotype of CYP2C9*2 and CYP2C9*3 had a significantly higher C max and AUC of E-3174 than those with CYP2C9*1/*1 (179 vs. 603 nmol/L and 2134 vs. 4346 nmol/L·h, respectively). However, in the case of losartan, the C max and AUC did not show a statistically significant difference between CYP2C9*2/*3 and CYP2C9*1/*1 (635 vs. 675 nmol/L and 1697 vs. 2006 nmol/L·h, respectively).
Polymorphisms of CYP2C9 should be considered in the case of antihypertensive drug polytherapy, because other hypertensive agents, such as carvedilol, torsemide, and indapamide, are also known to be CYP2C9 substrates [31]. According to Pan et al., CYP2C9 variants decreased the intrinsic clearance of carvedilol [32]. For torsemide, it was shown that CYP2C9*3 resulted in lower oral clearance and metabolite formation clearance [33]. In the study of Wang et al., patients with homozygous variants of CYP2C9 rs4918758, which showed lowered CYP2C9 activity, had higher C max and AUC and lower clearance of indapamide [34]. As CYP2C9 is involved in the metabolism of several antihypertensive agents, CYP2C9 loss-of-function alleles may increase the parent drug level. There are some limitations to this meta-analysis that should be considered when interpreting the results. First, the limited number of studies may lead to low statistical power in detecting differences or heterogeneity. However, according to Herbison et al., meta-analyses with as few as four or five studies could produce robust results that are consistent with long-term results [35]. Second, some potential confounder variables, which could be associated with pharmacokinetics (e.g., kidney and liver functions), could not be adjusted due to a lack of information from individual studies. Third, only healthy volunteers were involved in this study, indicating that the results may not be applicable to patients. Fourth, other CYP2C9 genotypes, such as CYP2C9*13, were not included in this meta-analysis because of low frequencies. Fifth, we could not conduct a meta-analysis comparing CYP2C9*2 carriers with CYP2C9*1/*1 carriers due to a lack of studies.
Despite these drawbacks, this study is the first systematic review and meta-analysis to evaluate the association between CYP2C9 genotypes and pharmacokinetic characteristics of losartan and its active metabolite. By combining the results of several studies, this study suggests that CYP2C9*2 or *3 alleles may be significantly associated with the pharmacokinetics of losartan and its active metabolite.
In conclusion, we found that CYP2C9*2 or *3 carriers showed higher AUC 0-∞ of losartan and lower AUC 0-∞ of E-3174 compared to those with CYP2C9*1/*1. As altered pharmacokinetics can affect the therapeutic responses of losartan, genotyping CYP2C9 may be useful in understanding individual pharmacokinetic and pharmacodynamic differences. Funding: This study did not receive any funding.
Institutional Review Board Statement: Ethical review and approval were waived for this study, due to the nature of the review article.
Informed Consent Statement: Patient consent was waived, due to the nature of the review article.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest:
The authors declare no conflict of interest.