Correlation between CYP2C9 polymorphisms and office blood pressure levels in patients treated with irbesartan and valsartan

Introduction
According to the World Health Organization (WHO) [1], approximately 50% of adults with hypertension remain undiagnosed. By 2030, approximately 40% of the adult population in the United States is projected to have some form of cardiovascular disease, including hypertension [2]. In this regard, the WHO has identified a 25% reduction in the prevalence of hypertension by 2025 as a global priority [3]. High blood pressure is the leading cause of disability-adjusted life years (DALYs) lost [4] and is responsible for a significant proportion of cardiovascular events [5] and premature mortality [6] worldwide. Despite the obvious importance of prevention in limiting the growth in the prevalence of hypertension and associated comorbid conditions, most clinical guidelines continue to focus primarily on the treatment of the disease [7, 8]. One of the promising areas for increasing the effectiveness of pharmacotherapy for hypertension in patients who regularly take antihypertensive drugs in accordance with current clinical guidelines but do not achieve target blood pressure levels is the introduction of personalized medicine principles. Historically, the idea of ​​the need for an individual approach to treatment has been developing in medicine for centuries, but only in recent decades has the approach to individualization of therapy been based on objective molecular genetic data. The modern concept of personalized medicine involves the use of scientifically based methods for selecting the most effective and safe pharmacotherapy taking into account the individual characteristics of the patient, including genetic markers that affect the pharmacokinetics and pharmacodynamics of drugs. The completion of the Human Genome Project, the active development and clinical implementation of molecular genetic technologies, as well as the accumulation of knowledge about the molecular mechanisms of drug action have contributed to the transition from an empirical to an evidence-based approach in choosing therapy. Given the growing demands for the effectiveness and safety of medical care, the use of pharmacogenetic data is becoming increasingly important in the treatment of chronic diseases, including arterial hypertension [9].
Single nucleotide polymorphisms (SNPs) of cytochrome P450 (CYP) enzymes, key participants in the first phase of biotransformation of many drugs, are among the most important genetic determinants of interindividual variability in drug response. Currently, more than 700 allelic variants of CYP enzymes have been described in various populations, many of which significantly affect their catalytic activity and pharmacokinetic parameters of the corresponding drugs. The highly genetically polymorphic CYP2C9 enzyme is involved in the metabolism of approximately 15–18% of all clinically used drugs, including warfarin, glimepiride, diclofenac, carvedilol, torasemide, and angiotensin II receptor blockers (particularly losartan). The most studied variants that differ from the wild-type CYP2C9*1 allele are CYP2C9*2 (Arg144Cys) and CYP2C9*3 (Ile359Leu), although a significantly larger number of allelic variants are currently known. The CYP2C9*2 allele is caused by the substitution of cytosine for thymine at position 430 of the nucleotide sequence, which results in the substitution of arginine for cysteine ​​at position 144 of the amino acid chain and the formation of an enzyme with reduced metabolic activity. The CYP2C9*3 allele is characterized by the substitution of adenine for cytosine at position 1075, which causes the substitution of isoleucine for leucine at amino acid 359, also associated with a decrease in the functional activity of the enzyme [10–12].

Objective
To study the pharmacodynamic parameters of the effectiveness of therapy with angiotensin II receptor blockers as monotherapy and as part of combination drugs in patients with hypertension depending on the genetic characteristics of patients – Arg144Cys, Ile359Leu polymorphisms of the CYP2C9 gene.

Materials and methods
The study included 179 patients with newly diagnosed stage 1–2 hypertension (AH) living in the Moscow region. The study sample was dominated by women — 141 patients (78.8%); there were 38 men (21.2%). The patients' age ranged from 32 to 69 years; the mean age was 58.2±6.4 years, the median age was 60 years (interquartile range: 57–63 years). Inclusion criteria: established diagnosis of stage 1–2 hypertension, age from 18 to 74 years, presence of signed informed voluntary consent to participate in the study [13].
The study was approved by the local ethics committee of the I.M. Sechenov First Moscow State Medical University of the Ministry of Health of the Russian Federation (Sechenov University) (protocol No. 05-21 dated March 10, 2021). The study design was an open, randomized, controlled clinical trial. The clinical and instrumental examination program included collecting complaints and anamnesis (including the presence of risk factors and comorbidities), physical examination, biochemical blood test, office measurement of blood pressure (BP), electrocardiography (ECG) to exclude patients with rhythm disturbances and structural heart pathology, and echocardiography. BP was measured on both arms using the Korotkov method after the patient had rested for 10 minutes in a sitting position. The average value of three consecutive measurements taken at one-minute intervals was used for analysis.
For genotyping, venous blood was collected in 4 ml VACUETTE® vacuum tubes (Greiner Bio-One, Austria) (size 13×75 mm) containing K2-EDTA as an anticoagulant. The tubes were gently inverted at least 10 times to ensure uniform mixing of blood with the anticoagulant, then the samples were frozen and stored at -20°C until DNA extraction. Genetic polymorphisms were analyzed by real-time polymerase chain reaction (RT-PCR) using a CFX96 Touch Real-Time PCR Detection System and CFX Manager software (Bio-Rad, USA), as well as commercial reagent kits. Genotyping of CYP2C9*2 (Arg144Cys) and CYP2C9*3 (Ile359Leu) alleles was performed using the RealBest-Genetics Warfarin kit (cat. No. D-3827, Vector-Best, Russia), based on PCR followed by analysis of amplicon melting curves. Statistical analysis and data visualization were performed in the R software environment version 4.2.3 (R Foundation for Statistical Computing, Vienna, Austria). Descriptive characteristics of categorical variables are presented as absolute numbers and relative frequencies. Fisher's exact test was used to compare groups by categorical characteristics. The assessment of the compliance of the genotype distribution with the Hardy-Weinberg principle was carried out using the likelihood ratio criterion [13].

Results
All patients included in the study had not previously received regular antihypertensive therapy. After randomization using the simple random allocation method (envelope method), the participants were divided into two groups receiving therapy with irbesartan or valsartan. Angiotensin II receptor blockers (ARBs) - irbesartan and valsartan - were used as monotherapy or in combination with hydrochlorothiazide for three months. In the irbesartan group, 83 patients received the drug at a dose of 150 mg once a day; of these, 32 patients were on monotherapy, and 51 patients - on combination therapy (irbesartan 150 mg + hydrochlorothiazide 12.5 mg). In the valsartan group, 96 patients received the drug at a dose of 80 mg once a day; Monotherapy was used in 8 patients, and a combination regimen (valsartan 80 mg + hydrochlorothiazide 12.5 mg) was used in 88 patients. When target blood pressure (BP) levels were achieved after 3 weeks of therapy (<140/90 mmHg; if well tolerated - <130/80 mmHg, but not <120/70 mmHg), patients continued taking the prescribed therapy regimen for three months. In case of insufficient BP control, therapy was intensified by doubling the dose of irbesartan or valsartan, both as part of monotherapy and as part of a combination regimen [12]. Venous blood sampling for determination of genetic polymorphisms of CYP2C9 rs1799853 (Arg144Cys, CYP2C9*2) and rs1057910 (Ile359Leu, CYP2C9*3) was performed 3 weeks after inclusion in the study. Office blood pressure measurement was performed at each visit: at the time of inclusion in the study (baseline), after 3 weeks and after 3 months of therapy. All patients were determined the genotype of polymorphisms Arg144Cys, Ile359Leu of the CYP2C9 gene. Among the examined patients, 141 patients (78.8%) had the wild homozygous genotype *1/*1 for the CYP2C9*2 locus, 34 patients (19.0%) had the heterozygous genotype *1/*2, and 4 patients (2.2%) had the mutant homozygous genotype *2/*2. The distribution of genotype frequencies for the CYP2C9*3 polymorphism was as follows: the wild homozygous genotype *1/*1 was found in 146 patients (81.6%), and the heterozygous *1/*3 was found in 33 patients (18.4%). The homozygous mutant genotype *3/*3 was not detected. In addition, 2 patients (1.1%) were found to have the compound heterozygous genotype *2/*3 for the CYP2C9 locus [14]. There were no statistically significant deviations of the observed genotype frequencies from the theoretical ones determined by the Hardy-Weinberg equilibrium: the Arg144Cys polymorphic locus of the CYP2C9 gene (χ²=0.62, p=0.43), the Ile359Leu polymorphic locus of the CYP2C9 gene (χ²=0.91, p=0.341) [15].
Tables 1 and 2 present the results of assessing the dynamics of office SBP, DBP, and HR in patients with different genotypes for the Arg144Cys and Ile359Leu polymorphic markers of the CYP2C9 gene in the irbesartan and valsartan patient groups.
Both the irbesartan and valsartan groups of patients showed a statistically significant decrease in office SBP during the observation period. Carriage of the *2 allele in patients receiving irbesartan was statistically significantly associated with a greater reduction in office SBP at the midterm assessment by an average of 8.3 [95% CI: -12.7–-3.8] mmHg, while in patients receiving valsartan, carriage of this allele was associated with a lesser effect of the drug (mean difference - 7.4 [95% CI: 1.6–13.2] mmHg). The CYP2C9 Arg144Cys genotype was not a statistically significant predictor of the effect of irbesartan at the end of the study (p=0.538), among patients receiving valsartan, a statistically significantly lesser reduction in office SBP was noted by an average of 5.2 [95% CI: 0.8–9.7] mmHg (Fig. 1).

Fig. 1. Comparative analysis of office SBP dynamics in patients with different genotypes of the CYP2C9 Arg144Cys polymorphic marker in the irbesartan and valsartan treatment groups

In both groups, a statistically significant decrease in office DBP was observed throughout the study (p < 0.001). The carriage of the *2 allele in patients receiving irbesartan was statistically significantly associated with a more pronounced decrease in office DBP by an average of 7.5 [95% CI: -11.7–-3.3] mmHg at the interim assessment of the effect; at the end of the study, no statistically significant association of the CYP2C9 Arg144Cys genotype with the effect of irbesartan was found (p = 0.65). In the group of patients receiving valsartan, no statistically significant association of the CYP2C9 Arg144Cys genotype was found either at the interim assessment (p = 0.384) or at the end of the study (p = 0.147) (Fig. 2).

Fig. 2. Comparative analysis of office DBP dynamics in patients with different genotypes of the CYP2C9 Arg144Cys polymorphic marker in the irbesartan and valsartan treatment groups

No statistically significant association of the CYP2C9 Arg144Cys genotype with the effect of irbesartan and valsartan on office HR was found at the interim assessment (p=0.641 and 0.774, respectively). No statistically significant association of the genotype at this locus with the change in office HR at the end of the study compared with baseline values ​​was found in patients receiving irbesartan (p=0.641); in patients receiving valsartan, carriage of the *2 allele was a statistically significant predictor of a more pronounced decrease in HR by an average of 4.4 [95% CI: -6.4–-2.4] beats per minute (Fig. 3).

Fig. 3. Comparative analysis of office HR dynamics in patients with different genotypes of the CYP2C9 Arg144Cys polymorphic marker in the irbesartan and valsartan treatment groups

Carriage of the *3 allele in patients receiving irbesartan was statistically significantly associated with a more pronounced reduction in office SBP at the interim assessment by an average of 11.8 [95% CI: -16.6–-7] mmHg, in patients receiving valsartan, carriage of this allele was associated with a more pronounced effect of the drug by an average of 10.5 [95% CI: -16.2–-4.9] mmHg. The CYP2C9 Ile359Leu genotype was not a statistically significant predictor of the effect after 3 months of pharmacotherapy, either with irbesartan (p=0.725) or with valsartan (p=0.146) (Fig. 4).

Fig. 4. Comparative analysis of office SBP dynamics in patients with different genotypes of the CYP2C9 Ile359Leu polymorphic marker in the irbesartan and valsartan treatment groups

The carriage of the *3 allele in patients receiving irbesartan was statistically significantly associated with a more pronounced reduction in office DBP at the interim assessment by an average of 7.3 [95% CI: -12.2–-2.4] mmHg, in patients receiving valsartan, the carriage of this allele was also associated with a more pronounced drug effect by an average of 9.7 [95% CI: -15.3–-4.1] mmHg. The CYP2C9 Ile359Leu genotype was not a statistically significant predictor of the effect on office DBP at the end of the study, either with irbesartan (p=0.604) or with valsartan (p=0.119) (Fig. 5).

Fig. 5. Comparative analysis of office DBP dynamics in patients with different genotypes of the CYP2C9 Ile359Leu polymorphic marker in the irbesartan and valsartan treatment groups

There was no statistically significant association between the CYP2C9 Ile359Leu genotype and the effect of irbesartan and valsartan on office HR at the interim assessment (p=0.188 and 0.946, respectively). In patients receiving valsartan, carriage of the *3 allele was a statistically significant predictor of a less pronounced decrease in HR by an average of 2.7 [95% CI: 0–5.3] bpm, while the effect was not mediated by steady-state drug concentrations (p=0.766), and there was no statistically significant association between genotype and the change in office HR at the end of the study compared with baseline values ​​in patients receiving irbesartan (p=0.313). It should also be noted that in carriers of the *3 allele who received irbesartan, the change in heart rate was not statistically significant either at the interim assessment or at the end of the study (Fig. 6).

Fig. 6. Comparative analysis of office HR dynamics in patients with different genotypes of the CYP2C9 Ile359Leu polymorphic marker in the irbesartan and valsartan treatment groups

Discussion
Genotypes *1/*2, *2/*2, *1/*3 and *3/*3 for CYP2C9 gene polymorphisms (Arg144Cys, Ile359Leu) are associated with reduced enzyme activity, with homozygous variants showing a marked decrease in its function. In the studied cohort of patients with newly diagnosed grade 1–2 hypertension, the frequency of CYP2C9*2 and CYP2C9*3 risk alleles was 11.7 and 9.2%, respectively, which is comparable to the frequency of these alleles in Europeans — 12 and 6%, respectively. [16]. A meta-analysis published in 2021 [17] pooled data from eight studies conducted up to March 2021 to evaluate the impact of CYP2C9 gene polymorphisms on the pharmacokinetic parameters of losartan and its active metabolite E-3174. In healthy volunteers carrying CYP2C9*2 or CYP2C9*3 alleles, there was a significant increase in the area under the pharmacokinetic curve (AUC) of losartan by 0.17 μg×h/mL (95% CI: 0.04–0.29) compared to carriers of the homozygous wild-type *1/*1. In this case, the AUC of the active metabolite E-3174 was reduced by 0.35 μg×h/mL (95% CI: -0.62 to -0.08), and the maximum concentration (Cmax) was reduced by 0.13 μg/mL (95% CI: -0.17 to -0.09). Also, in carriers of reduced-function alleles, an increase in the half-life was observed for both losartan (+0.47 h; 95% CI: 0.32 to 0.61) and the metabolite E-3174 (+0.68 h; 95% CI: 0.44 to 0.92). These data confirm the clinical significance of CYP2C9 genetic variants in the context of losartan metabolism and, probably, other angiotensin II receptor blockers metabolized with the participation of this enzyme.
According to Sinitsina II et al. [18], polymorphic variants CYP2C9*2 and CYP2C9*3 of the CYP2C9 gene affect the hypotensive effect of losartan in patients with hypertension. The study included 81 patients with newly diagnosed stage 1–2 hypertension, confirmed by 24-hour BP monitoring (ABPM), who were prescribed losartan therapy. Carriage of CYP2C9*2 and CYP2C9*3 alleles was associated with a decrease in the hypotensive effect of the drug (p <0.001; OR=8.13; 95% CI: 2.75–23.97), which was confirmed by significantly higher BP values ​​according to ABPM. At the same time, no significant differences in plasma uric acid levels and the E-3174/losartan ratio in urine were noted between the genotypes. The CYP2C9 isoenzyme plays a key role in the metabolism of not only losartan, but also irbesartan and azilsartan. Unlike losartan, the metabolism of irbesartan and azilsartan via CYP2C9 leads mainly to the formation of inactive metabolites, which causes a potential increase in the concentration of the parent drug in the blood plasma in carriers of alleles with reduced functional activity (CYP2C9*2, CYP2C9*3). This, in turn, may be associated with an increase in the pharmacodynamic effect and an increased risk of adverse drug reactions, which requires a cautious approach to dose selection. In the study by Hallberg P et al. [19], which included 49 patients receiving irbesartan for three months, a more pronounced decrease in diastolic blood pressure was recorded in carriers of the CYP2C9*2 allele: in the group with the CYP2C9*1/*1 genotype (n=33), the decrease was 7.5%, while in patients with the CYP2C9*1/*2 genotype it was 14.4% (p<0.05). This observation is likely associated with increased exposure of irbesartan in individuals with slow metabolism and emphasizes the clinical significance of taking into account CYP2C9 genetic variants when initiating therapy.
Additional data on the effect of CYP2C9 genetic variants on the pharmacokinetics of irbesartan were obtained in a study by Hong X et al. [20], which included 1087 patients of Chinese origin who received irbesartan at a dose of 150 mg once for 28 days. A total of 235 participants were selected for pharmacogenetic analysis. The CYP2C9*2 allele was not detected in the sample, and the frequency of the CYP2C9*3 allele carriage was 3.65%; all carriers had the heterozygous CYP2C9*1/*3 genotype. In this group of patients, the plasma concentration of irbesartan both after 24 hours (on the 27th day of therapy) and 6 hours after administration (on the 28th day) was statistically significantly higher compared to patients with the CYP2C9*1/*1 genotype. Despite the identified pharmacokinetic differences, no significant effect of the CYP2C9 genotype on the degree of blood pressure reduction was found in the study. These results highlight the complex nature of the interaction between pharmacokinetics and clinical response, as well as the need to take into account additional factors when interpreting pharmacogenetic data. In a subsequent analysis performed by Chen G et al. [21], 196 patients who demonstrated a pronounced maximal or minimal hypotensive response to irbesartan therapy were selected from the total number of participants in the Hong X et al. [20] study (n=1087). The frequency of the CYP2C9*3 allele in this subsample was 2.3%. According to the obtained data, in patients with heterozygous CYP2C9*1/*3 genotype, the concentration of irbesartan in blood plasma 6 hours after taking the drug, as well as the degree of reduction in diastolic blood pressure, were statistically significantly higher compared to carriers of the wild type (CYP2C9*1/*1). These results confirm the possible clinical significance of CYP2C9 genotyping in predicting the effectiveness of irbesartan therapy, especially at the initial stages of treatment.
In the study by Choi CI et al. [22], a comparative assessment of the pharmacokinetic parameters of irbesartan was performed in 28 healthy Korean volunteers with different CYP2C9 genotypes: CYP2C9*1/*1 (n=12), CYP2C9*1/*3 (n=10) and CYP2C9*1/*13 (n=6). In carriers of the CYP2C9*3 and CYP2C9*13 alleles, the maximum plasma concentration of irbesartan was significantly higher - by 1.56 and 1.5 times, respectively, the half-life of the drug was increased - by 1.64 and 1.79 times, and oral clearance was significantly lower (by 19.3 and 44.0%) compared to carriers of the "wild" type (CYP2C9*1/*1). The obtained data confirm that a decrease in the metabolic activity of the CYP2C9 enzyme in carriers of reduced-functional alleles can lead to an increase in the systemic exposure of irbesartan, which potentially affects both its efficacy and the safety of therapy. In the study by Dong H et al. [23], 598 patients with hypertension received irbesartan at a dose of 150 mg once a day for 4 weeks. In carriers of the CYP2C9*1/*3 and CYP2C9*3/*3 genotypes, a more pronounced decrease in SBP and DBP (by 34.9±15.5 versus 29.3±10.2 mmHg and by 22.8±9.0 versus 19.6±8.5 mmHg, respectively) was observed compared with patients with the CYP2C9*1/*1 genotype, which correlates with the results obtained among the studied sample of patients in the irbesartan group. Thus, CYP2C9 gene polymorphisms can have a significant impact on the antihypertensive effect of individual representatives of the angiotensin II receptor blockers (ARB) group. In carriers of reduced-functional CYP2C9 alleles, there is a decrease in the effectiveness of losartan therapy, up to the absence of a pronounced hypotensive response, while when using irbesartan and, probably, azilsartan, there is a tendency to increase the pharmacodynamic effect, accompanied by a potential increase in the risk of developing adverse drug reactions. ARB drugs that are not metabolized with the participation of cytochrome P450 isoenzymes (valsartan, candesartan, telmisartan, eprosartan and olmesartan) demonstrate stable antihypertensive activity independent of CYP2C9 genetic variants.
However, our study revealed an association between CYP2C9 genetic variants and the antihypertensive efficacy of valsartan, which contradicts the data on its predominantly extrahepatic elimination and metabolic independence from cytochrome P450 enzymes. This observation may be due to a number of factors, including individual features of liver enzyme expression and activity, involvement of other P450 isoenzymes in valsartan metabolism in individual patients, as well as the possible influence of CYP2C9 polymorphisms on pharmacodynamic parameters, such as angiotensin II receptor sensitivity or drug transport. In addition, it is possible that CYP2C9 genetic variants may indirectly affect the activity of other metabolic or regulatory pathways involved in the implementation of the hypotensive response. These data require further study using a larger sample and population pharmacokinetic analysis.

Conclusion
The obtained results demonstrate a statistically significant decrease in SBP and DBP in patients with newly diagnosed stage 1-2 hypertension during therapy with irbesartan and valsartan for three months. Carriage of polymorphic alleles *2 (Arg144Cys) and *3 (Ile359Leu) of the CYP2C9 gene is associated with a more pronounced decrease in SBP and DBP at the interim assessment of treatment efficacy (after 3 weeks), however, by the end of the observation period, CYP2C9 genotypes were not statistically significant predictors of the antihypertensive effect. The effect of genotype on HR was limited and depended on the combination of the allele and the drug used: carriage of the *2 allele was associated with a more pronounced decrease in HR in patients receiving valsartan, and the *3 allele - with a less pronounced decrease in HR in the same group. Thus, the results of the study confirm the potential role of CYP2C9 polymorphisms in modifying the early response to ARB therapy, which may be important in personalized selection of antihypertensive drugs.