Inhibitory Effects of Lansoprazole and Omeprazole on the CYP2C19-Mediated Metabolism of Arachidonic Acid to Epoxyeicosatrienoic Acids: Implications for Cardiovascular Risk

Abstract

Proton pump inhibitors (PPIs), such as lansoprazole and omeprazole, are associated with an increased risk of cardiovascular events; however, the underlying mechanisms remain unclear. We investigated the inhibitory effects of lansoprazole and omeprazole on the formation of cardioprotective epoxyeicosatrienoic acids (EETs) and their downstream metabolites, dihydroxyeicosatrienoic acids (DHETs), from arachidonic acid catalyzed by CYP enzymes. In vitro incubation studies were conducted using human liver microsomes (HLMs), recombinant CYP2C19 (rCYP2C19), and recombinant CYP2C9 (rCYP2C9). IC₅₀ values and R values (a clinical drug–drug interaction risk predictor) were estimated. In HLMs, lansoprazole inhibited the formation of 5,6-, 8,9-, and 11,12-EETs. Omeprazole showed limited inhibition in comparison. In experiments using recombinant enzymes, lansoprazole and omeprazole strongly inhibited EET formation via rCYP2C19, but not rCYP2C9. Lansoprazole exhibited more potent inhibition than omeprazole. Notably, R values for lansoprazole in rCYP2C19 exceeded 1.1 for total EET and DHET formation. Molecular docking analysis suggested that both PPIs may bind to CYP2C19 in a similar pose to a known inhibitor. Docking scores also supported the stronger inhibitory potential of lansoprazole compared to omeprazole. In conclusion, lansoprazole and omeprazole suppress EET formation primarily via CYP2C19 inhibition. The predicted high risk (R > 1.1) for lansoprazole observed in the rCYP2C19 assay suggests a potential mechanism for the increased cardiovascular risk. Further clinical studies are warranted to validate these findings.

INTRODUCTION

Cardiovascular disease (CVD) remains one of the leading causes of death worldwide. In recent years, progress in reducing CVD-related mortality and morbidity has stalled, underscoring the need for improved preventive and therapeutic strategies.^1–4) Several commonly used medications previously considered safe have been associated with an increased risk of cardiovascular events. For example, nonsteroidal anti-inflammatory drugs and denosumab have been implicated in cardiovascular adverse effects.^5,6) More recently, proton pump inhibitors (PPIs), widely prescribed as first-line treatments for gastroesophageal reflux disease and for the prevention of peptic ulcers, have also been linked to elevated cardiovascular risk, independent of concomitant antiplatelet therapy such as clopidogrel.^7–10) Although multiple mechanisms have been proposed, the precise molecular basis for this association remains unclear.

Epoxyeicosatrienoic acids (EETs) are endogenous lipid mediators with vasodilatory, anti-inflammatory, and cardioprotective properties, playing a vital role in maintaining cardiovascular homeostasis.^11,12) EETs are primarily synthesized from arachidonic acid by CYP enzymes, particularly those in the CYP2C and CYP2J subfamilies. They are subsequently metabolized by soluble epoxide hydrolase (sEH) into the less active dihydroxyeicosatrienoic acids (DHETs).¹³⁾ The sEH enzyme, therefore, plays a key role in regulating EET levels; its inhibition increases the bioavailability of EETs and enhances their biological activity. For example, small-molecule sEH-inhibitory metabolites derived from Stachybotrys microspora triprenyl phenols¹⁴⁾ have demonstrated therapeutic effects in various disease models, including cerebral embolism,¹⁵⁾ acute kidney injury,¹⁶⁾ diabetic neuropathy,¹⁷⁾ diabetic retinopathy,¹⁸⁾ and atherosclerosis.¹⁹⁾ These protective effects are believed to result from elevated EET concentrations due to sEH inhibition, supporting their role as cytoprotective mediators with anti-inflammatory and antioxidant properties.^20,21)

Conversely, suppression of CYP-mediated EET biosynthesis may impair these protective effects and contribute to disease progression. For instance, the cardiotoxicity of the anticancer drug sorafenib has been linked to an increased risk of arrhythmia and heart failure, attributed to reduced EET production via CYP2J inhibition.²²⁾ Similarly, several angiotensin II receptor blockers have been reported to inhibit CYP2C9, CYP2C8, and CYP2J2, resulting in decreased EET synthesis and potentially contributing to increased cardiovascular risk.^23,24) Given that EETs exert anti-inflammatory, anti-apoptotic, and vasodilatory effects within the cardiovascular system,²⁵⁾ inhibition of their biosynthesis via CYP enzymes is hypothesized to diminish these critical protective actions and ultimately elevate cardiovascular risk.

A recent study suggested that lansoprazole, among various PPIs, is associated with a higher risk of cardiovascular events, potentially through the inhibition of CYP-mediated EET biosynthesis.²⁶⁾ Among the CYP epoxygenases, the CYP2C subfamily enzymes account for approximately 18% of total hepatic CYP content, with CYP2C9 and CYP2C19 being the primary isoforms involved in EET formation.²⁷⁾ Lansoprazole and omeprazole have been reported to strongly inhibit CYP2C19 and to exhibit weak inhibitory effects on CYP2C9 as well.²⁸⁾ Furthermore, genetic polymorphisms that reduce the activity of these enzymes are associated with increased cardiovascular risk. For example, smokers carrying the heterozygous CYP2C9*2 allele have a 3.7-fold higher risk of coronary artery disease,²⁹⁾ while CYP2C19 poor metabolizers are more likely to develop microvascular complications due to decreased EET production.³⁰⁾ These findings collectively suggest that inhibition of CYP2C19 and CYP2C9 by PPIs may significantly impair EET biosynthesis, thereby diminishing their cardioprotective effects.

Therefore, this study focused on the PPIs lansoprazole and omeprazole to evaluate their inhibitory effects on EET formation using human liver microsomes (HLMs), as well as recombinant CYP2C19 (rCYP2C19) and CYP2C9 (rCYP2C9). This study aimed to investigate in vitro the potential of these PPIs to inhibit EET biosynthesis and to elucidate a possible molecular mechanism by which PPI use may contribute to increased cardiovascular risk.

MATERIALS AND METHODS

Reagents and Chemicals

Ultrapure water, acetonitrile, isopropanol, methanol, ethyl acetate (all LC/MS grade), formic acid (HPLC grade), potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium chloride hexahydrate (MgCl₂), 2-amino-2-hydroxymethyl-1,3-propanediol (Tris), hydrochloric acid, lansoprazole, and omeprazole were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Famotidine was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Reduced nicotinamide adenine dinucleotide phosphate (NADPH), glucose 6-phosphate (G-6-P), and glucose 6-phosphate dehydrogenase (G-6-PDH) were purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan). Arachidonic acid, 5,6-EET, 8,9-EET, 11,12-EET, 14,15-EET, 5,6-DHET, 8,9-DHET, 11,12-DHET, 14,15-DHET, and leukotriene B₄-d₄ (LTB₄-d₄) were obtained from Cayman Chemical (Ann Arbor, MI, U.S.A.). HLMs (BD Ultrapool^TM HLM150) were purchased from BD Biosciences (Woburn, MA, U.S.A.). rCYP2C19 and rCYP2C9 (Gentest^® Supersomes^®) were obtained from Discovery Life Sciences (Huntsville, AL, U.S.A.).

Inhibition Study in HLMs

The inhibition study was conducted based on previously published protocols,^22,23) with minor modifications. Famotidine, an H₂-receptor antagonist, was used as a negative control for PPIs. Lansoprazole, omeprazole, and famotidine were initially dissolved in 100% methanol. These stock solutions were then diluted to final concentrations of 0, 10, 30, 100, and 300 µM in the reaction mixture. Methanol was evaporated under a gentle stream of nitrogen, and the residue was reconstituted in 20 µL of water by vortexing. Control activity was measured using the same method, with methanol and water only. We added 90 mM potassium phosphate buffer (pH 7.4), 4 mM MgCl₂, 0.5 mg/mL HLM, and 20 µM arachidonic acid to the reconstituted solution. The mixture was preincubated at 37°C for 3 min. The reaction was initiated by adding 25 µL of an NADPH-generating system containing 1 mM NADPH, 8 mM G-6-P, and 0.5 U of G-6-PDH, bringing the final reaction volume to 200 µL. The NADPH-generating system was freshly prepared immediately before each experiment. After 10 min of incubation at 37°C, a 50-µL aliquot of the reaction mixture was transferred to 200 µL of ethyl acetate on ice to terminate the reaction. Each experiment was repeated 3 times. The final concentration of methanol in the reaction mixture was kept below 1% to minimize any effect on enzyme activity.

Inhibition Study in rCYPs

Inhibition studies were conducted using recombinant enzymes to identify isoform-specific effects. To establish appropriate experimental conditions for rCYP2C9 and rCYP2C19, the concentration of arachidonic acid was varied to determine the K_m and V_max values. Incubations involving rCYP2C9 were performed in Tris-HCl buffer, since potassium phosphate buffer has been reported to decrease rCYP2C9 enzymatic activity, whereas Tris-HCl does not.³¹⁾ For rCYP2C19, no substantial difference in enzymatic activity has been observed between these buffers within the concentration range used in the present study.³¹⁾ Therefore, Tris-HCl buffer was selected for the experiments using rCYP2C9 and rCYP2C19 in the present study. Each reaction mixture (final volume, 200 µL) contained 100 mM Tris-HCl buffer (pH 7.4), 3 mM MgCl₂, 50 pmol/mL rCYP2C19 or rCYP2C9, and arachidonic acid at final concentrations of 10, 20, 50, 80, or 120 µM. After preincubation at 37°C for 3 min, the reaction was initiated by adding 25 µL of an NADPH-generating system. The NADPH-generating system was freshly prepared immediately before each experiment. The reaction was allowed to proceed for 30 min, after which termination and extraction were performed as described for the HLM incubations. The final concentration of methanol in the reaction mixture was kept below 1% to minimize any effect on enzyme activity.

Subsequently, inhibition assays for EET and DHET formation were carried out to assess CYP inhibition by lansoprazole and omeprazole, using arachidonic acid at a final concentration of 20 µM for rCYP2C19 and 40 µM for rCYP2C9. These concentrations were selected based on the experimentally determined K_m values (26.54 µM for rCYP2C19 and 76.46 µM for rCYP2C9), while also considering previous reports indicating that high arachidonic acid concentrations may interfere with inhibition assays for rCYP2C9.³²⁾ Lansoprazole and omeprazole were prepared at each test concentration using the same method as that described in the “Inhibition Study in HLMs” section. The incubation procedures followed those used for the determination of K_m and V_max. The final concentration of methanol in the reaction mixture was kept below 1% to minimize any effect on enzyme activity.

Sample Preparation and Extraction

Extraction was performed by referring to previously published methods,^33,34) with minor modifications. To extract EETs and DHETs from the terminated reaction mixtures, 47.5 µL of methanol and 100 µL of 0.1% formic acid were added to the aliquots previously transferred to ethyl acetate. Then, 2.5 µL of LTB₄-d₄ (10 ng/mL) was added as an internal standard. The samples were vortexed thoroughly and centrifuged at 20400 × g for 5 min at 4°C. A 100-µL aliquot of the supernatant was collected and evaporated to dryness using a centrifugal evaporator. The residue was reconstituted in mobile phase A (ultrapure water/acetonitrile/formic acid = 63 : 37 : 0.02, v/v/v) and subjected to LC-electrospray ionization (ESI)-MS analysis. Both EETs and DHETs were quantified in this study. Although the conversion of EETs to DHETs is not catalyzed by CYP enzymes,¹³⁾ weak hydrolytic activity toward EETs by microsomal epoxide hydrolase present in HLM,³⁵⁾ as well as epoxide hydrolase–like activity reported in supersomes,³⁶⁾ can lead to secondary DHET formation under in vitro conditions. To account for these background reactions, DHETs were quantified together with EETs as downstream metabolites.

LC-ESI-MS Analysis

All MS analyses were conducted using a Prominence HPLC system (Shimadzu Corporation, Kyoto, Japan) coupled to a linear ion trap quadrupole mass spectrometer (QTRAP 5500; AB Sciex Pte. Ltd., Framingham, MA, U.S.A.), as previously described.^37–39) EETs and DHETs were analyzed using tandem MS in negative-ion mode by using multiple-reaction monitoring (MRM). The following m/z transitions were monitored: 319.084/191.100 for 5,6-EET, 319.080/155.000 for 8,9-EET, 319.054/167.000 for 11,12-EET, 319.062/219.000 for 14,15-EET, 337.149/144.950 for 5,6-DHET, 337.132/184.939 for 8,9-DHET, 337.103/157.200 for 11,12-DHET, 337.122/207.079 for 14,15-DHET, and 339.13/196.93 for LTB₄-d₄ (internal standard). The identities of these EET and DHET analytes were confirmed by comparing MRM transitions and LC retention times with those of authentic standards. Quantification was performed using calibration curves based on the ratio of each analyte’s peak area to that of the internal standard.

Data Analysis and R Value Calculation

The formation activity of each EET and DHET isomer was determined individually and expressed as a percentage of the control activity. For the determination of K_m and V_max, the data were presented as the total formation activity of all EET and DHET isomers. Inhibitory potency was evaluated by calculating IC₅₀ values. GraphPad Prism version 10.6.1 (GraphPad Software Inc., San Diego, CA, U.S.A.) was used to generate graphs and calculate K_m, V_max, and IC₅₀ values.

To predict the risk of in vivo drug–drug interactions from in vitro inhibition data, the R value was used as a threshold model.⁴⁰⁾ Because it was not feasible to independently assess the formation rates of individual EET and DHET isomers, or to perform experiments using substrate concentrations adjusted to their respective K_m values, the K_m derived from total EET and DHET production was used as a representative parameter, and the R values were calculated based on the combined formation of all EET and DHET isomers, calculated using the following equation:

  

$$R=1+\left[I\right]/K_{\text{i}}$$

where [I] represents the maximum plasma concentration (C_max) of the inhibitor.²³⁾ Because the C_max values of lansoprazole and omeprazole vary depending on genetic polymorphisms, this study used the reported values in extensive metabolizers.⁴¹⁾ The C_max values used in this study were 2.21 µM for lansoprazole and 1.07 µM for omeprazole.⁴¹⁾ Since lansoprazole and omeprazole act as competitive inhibitors of CYP2C19 and CYP2C9,²⁸⁾ the inhibition constant (K_i) was estimated as half of the experimentally determined IC₅₀ (K_i = IC₅₀/2).⁴²⁾ To avoid false-negative predictions, a cutoff value of R > 1.1 was applied.²³⁾

Protein–Ligand Docking Analysis

A previous study reported the X-ray crystal structure of the CYP2C19–0XV inhibitor complex (PDB ID: 4GQS).⁴³⁾ In chain A of the complex, the 0XV molecule binds to CYP2C19 via 2 water molecules, denoted as Wat1 and Wat2 (Supplementary Fig. S1). Wat1 binds to the Fe²⁺ ion of the heme group, while Wat2 interacts with the Asn107 residue of CYP2C19. Based on this structural insight, 4 CYP2C19 models were generated using chain A: (1) with both Wat1 and Wat2, (2) with Wat1 only, (3) with Wat2 only, and (4) without Wat1 or Wat2. Docking of lansoprazole and omeprazole was performed against all 4 CYP2C19 models. All calculations were conducted using the Schrödinger Suite program (ver. 2024-4; Schrödinger, LLC, New York, NY, U.S.A.). The 3D structures of small molecules were prepared using the LigPrep program (ver. 2024-4; Schrödinger, LLC), and docking was carried out in SP mode using the Glide program (ver. 2024-4; Schrödinger, LLC).

RESULTS

Inhibition Study in HLMs

As shown in Fig. 1A, the inhibitory effects of lansoprazole, omeprazole, and famotidine on EET formation in HLMs were evaluated. Similarly, Fig. 1B illustrates their effects on DHET formation. Lansoprazole inhibited the formation of 5,6-, 8,9-, and 11,12-EETs in a concentration-dependent manner, with over 50% inhibition observed at 300 µM compared to that of the control. In contrast, omeprazole and famotidine showed minimal or no inhibition (Fig. 1A). Lansoprazole inhibited the formation of 8,9-, 11,12-, and 14,15-DHETs in a concentration-dependent manner, with more than 50% inhibition at 300 µM compared to the control one. Omeprazole inhibited only 14,15-DHET formation to a similar extent, while famotidine had no effect on any DHET isomer (Fig. 1B).

Fig. 1. Inhibitory Effects of Lansoprazole, Omeprazole, and Famotidine on the Enzymatic Activity Responsible for the Formation of (A) EETs and (B) DHETs from Arachidonic Acid in HLMs, Expressed as the Percentage of Control Activity for Each EET and DHET Isomer

• and solid line, lansoprazole; ○ and dashed line, omeprazole; ▲ and dotted line, famotidine. Each experiment was repeated 3 times. Control values (pmol/mg protein/min) used for calculating the % of control (activity in the absence of inhibitor) were as follows: (A) EET isomers: 5,6-form: lansoprazole (3.08), omeprazole (6.88), famotidine (9.47); 8,9-form: lansoprazole (2.33), omeprazole (3.63), famotidine (4.86); 11,12-form: lansoprazole (2.55), omeprazole (5.32), famotidine (8.19); 14,15-form: lansoprazole (6.19), omeprazole (7.69), famotidine (11.59). (B) DHET isomers: 5,6-form: lansoprazole (1.16), omeprazole (1.17), famotidine (2.25); 8,9-form: lansoprazole (3.98), omeprazole (5.84), famotidine (8.37); 11,12-form: lansoprazole (16.13), omeprazole (20.94), famotidine (29.24); 14,15-form: lansoprazole (36.21), omeprazole (42.54), famotidine (50.81).

Table 1 summarizes the IC₅₀ values calculated from the data in Figs. 1 and 2A. The IC₅₀ values of lansoprazole for 5,6-, 8,9-, and 11,12-EET formation were 37.10, 101.90, and 137.30 µM, respectively; for 8,9-, 11,12-, and 14,15-DHET formation, the values were 86.99, 73.26, and 51.07 µM, respectively. For total EET and DHET formation, the IC₅₀ value was 79.07 µM. The IC₅₀ values for 14,15-EET and 5,6-DHET could not be determined, since 50% inhibition was not reached within the tested concentration range. For omeprazole, only the IC₅₀ for 14,15-DHET (40.19 µM) was determined, whereas IC₅₀ values for other EET and DHET isomers, as well as for total EET and DHET formation, could not be determined because 50% inhibition was not observed. Similarly, famotidine did not reach 50% inhibition for any EET or DHET isomer, and therefore, IC₅₀ values could not be calculated.

Table 1. Inhibitory Parameters (IC₅₀) of Lansoprazole, Omeprazole, and Famotidine in HLMs

	Lansoprazole	Omeprazole	Famotidine
	IC50 (µM)	IC50 (µM)	IC50 (µM)
Total EET + DHET	79.07	>300	>300
5,6-EET	37.10	>300	>300
8,9-EET	101.90	>300	>300
11,12-EET	137.30	>300	>300
14,15-EET	>300	>300	>300
5,6-DHET	>300	>300	>300
8,9-DHET	86.99	>300	>300
11,12-DHET	73.26	>300	>300
14,15-DHET	51.07	40.19	>300

Fig. 2. Inhibitory Effects of Lansoprazole, Omeprazole, and Famotidine on the Enzymatic Activity Responsible for the Formation of EETs and DHETs from Arachidonic Acid in HLMs (A), and Those of Lansoprazole and Omeprazole in rCYP2C19 (B)

Enzymatic activities are expressed as percentages of control activity based on the formation of total EET and DHET. • and solid line, lansoprazole; ○ and dashed line, omeprazole; ▲ and dotted line, famotidine. Each experiment was repeated 3 times. Control values used for calculating the % of control (activity in the absence of inhibitor) were as follows: (A) pmol/mg protein/min: lansoprazole (71.64), omeprazole (94.00), famotidine (124.79). (B) pmol/pmol protein/min: lansoprazole (1.74), omeprazole (2.15).

Inhibition Study in rCYPs

As shown in Fig. 3, the kinetic parameters for EET and DHET formation from arachidonic acid were determined. For rCYP2C19, the K_m and V_max values were 26.54 µM and 6.47 pmol/pmol P450/min, respectively. For rCYP2C9, the values were 76.46 µM and 10.19 pmol/pmol P450/min, respectively.

Fig. 3. Michaelis–Menten Plots of Total EET and DHET Formation from Arachidonic Acid by rCYP2C19 and rCYP2C9

The estimated K_m and V_max values were 26.54 µM and 6.47 pmol/pmol P450/min for rCYP2C19, and 76.46 µM and 10.19 pmol/pmol P450/min for rCYP2C9, respectively. Each experiment was repeated 3 times.

Incubation experiments using rCYP2C19 were conducted to evaluate the inhibitory effects of lansoprazole and omeprazole on total EET and DHET formation (Fig. 2B), as well as on the formation of individual EET (Fig. 4A) and DHET (Fig. 4B) isomers. Both compounds inhibited all EET and DHET isomers by more than 50% compared with the control.

Fig. 4. Inhibitory Effects of Lansoprazole and Omeprazole on the Enzymatic Activity Responsible for the Formation of (A) EETs and (B) DHETs from Arachidonic Acid in rCYP2C19, Expressed as the Percentage of Control Activity for Each EET and DHET Isomer

• and solid line, lansoprazole; ○ and dashed line, omeprazole. Each experiment was repeated 3 times. Control values (pmol/pmol protein/min) used for calculating the % of control (activity in the absence of inhibitor) for rCYP2C19 were as follows: (A) EET isomers: 5,6-form: lansoprazole (0.18), omeprazole (0.12); 8,9-form: lansoprazole (0.22), omeprazole (0.25); 11,12-form: lansoprazole (0.06), omeprazole (0.07); 14,15-form: lansoprazole (0.04), omeprazole (0.08). (B) DHET isomers: 5,6-form: lansoprazole (0.03), omeprazole (0.05); 8,9-form: lansoprazole (0.70), omeprazole (0.89); 11,12-form: lansoprazole (0.06), omeprazole (0.09); 14,15-form: lansoprazole (0.45), omeprazole (0.61).

Table 2 presents the IC₅₀ and R values calculated from Figs. 2B and 4. For lansoprazole, IC₅₀ values for 5,6-, 8,9-, 11,12-, and 14,15-EETs were 2.97, 8.19, 4.51, and 6.06 µM, respectively; for 5,6-, 8,9-, 11,12-, and 14,15-DHET formation, values were 4.81, 4.46, 17.68, and 5.96 µM, respectively. The IC₅₀ value for total EET and DHET formation was 2.87 µM, and the corresponding R value was 2.54, exceeding the cutoff value of 1.1. For omeprazole, IC₅₀ values for 5,6-, 8,9-, 11,12-, and 14,15-EETs were 10.46, 18.59, 21.29, and 6.07 µM, respectively; for 5,6-, 8,9-, 11,12-, and 14,15-DHET formation, values were 22.41, 16.35, 29.26, and 26.17 µM, respectively. The IC₅₀ value for total EET and DHET formation was 34.29 µM, and the corresponding R value was 1.06.

Table 2. Inhibitory Parameters (IC₅₀ and R Values) of Lansoprazole and Omeprazole in rCYP2C19

	Lansoprazole		Omeprazole
	IC50 (µM)	R value	IC50 (µM)	R value
Total EET + DHET	2.87	2.54	34.29	1.06
5,6-EET	2.97	—	10.46	—
8,9-EET	8.19	—	18.59	—
11,12-EET	4.51	—	21.29	—
14,15-EET	6.06	—	6.07	—
5,6-DHET	4.81	—	22.41	—
8,9-DHET	4.46	—	16.35	—
11,12-DHET	17.68	—	29.26	—
14,15-DHET	5.96	—	26.17	—

C_max values used for the calculations were as follows: lansoprazole, 2.21 µM; omeprazole, 1.07 µM.

In contrast, experiments using rCYP2C9 (Fig. 5) showed that neither lansoprazole nor omeprazole inhibited the formation of any EET or DHET isomer. Therefore, IC₅₀ and R values could not be determined.

Fig. 5. Inhibitory Effects of Lansoprazole and Omeprazole on the Enzymatic Activity Responsible for the Formation of (A) EETs and (B) DHETs from Arachidonic Acid in rCYP2C9, Expressed as the Percentage of the Control Activity for Each EET and DHET Isomer

• and solid line, lansoprazole; ○ and dashed line, omeprazole. Each experiment was repeated 3 times. Control values (pmol/pmol protein/min) used for calculating the % of control (activity in the absence of inhibitor) for rCYP2C9 were as follows: (A) EET isomers: 5,6-form: lansoprazole (0.02), omeprazole (0.03); 8,9-form: lansoprazole (0.38), omeprazole (0.35); 11,12-form: lansoprazole (0.67), omeprazole (0.64); 14,15-form: lansoprazole (2.18), omeprazole (2.16). (B) DHET isomers: 5,6-form: lansoprazole (0.008), omeprazole (0.004); 8,9-form: lansoprazole (0.22), omeprazole (0.18); 11,12-form: lansoprazole (0.17), omeprazole (0.13); 14,15-form: lansoprazole (0.37), omeprazole (0.22).

Molecular Docking Analysis of CYP2C19

Given the strong in vitro inhibitory effects of lansoprazole and omeprazole on CYP2C19, molecular docking analyses were performed to investigate their potential binding interactions. The top docking poses, ranked by docking score, are shown in Fig. 6. The docking scores for lansoprazole and omeprazole were −9.157 and −8.608, respectively. The analysis predicted that both compounds form hydrogen bonds with the Gly296 residue of CYP2C19.

Fig. 6. Top Docking Poses of Lansoprazole and Omeprazole Obtained from Docking Calculations against the 4 CYP2C19 Models

CYP2C19 is shown as a blue ribbon. The heme, lansoprazole and omeprazole, Gly296, and Wat1 are shown as stick models. Lansoprazole and omeprazole molecules are colored green, and the Fe²⁺ ion is shown as an orange sphere. Green dashed lines indicate hydrogen bonds.

DISCUSSION

In this study, we evaluated the inhibitory effects of lansoprazole and omeprazole on the formation of EETs from arachidonic acid, mediated by CYP enzymes in HLMs, rCYP2C9, and rCYP2C19. Since H₂ receptor antagonists have been associated with a lower risk of cardiovascular events compared with PPIs,⁴⁴⁾ famotidine was used as a negative control to determine whether CYP inhibition and the resulting reduction in EET formation would occur. As expected, famotidine showed no inhibitory effect (Figs. 1 and 2A), thereby validating the assay system.

In HLM incubations, lansoprazole inhibited the formation of 5,6-, 8,9-, and 11,12-EETs, as well as 8,9-, 11,12-, and 14,15-DHETs. Interestingly, the levels of 5,6-DHET remained unchanged despite increasing concentrations of lansoprazole. This may be attributed to the inherent instability of 5,6-EET, which readily undergoes spontaneous ring opening and oxidation. In addition, 5,6-EET is primarily metabolized by cyclooxygenase rather than sEH, and its low affinity for sEH likely contributes to the low levels of 5,6-DHET detected.⁴⁵⁾

In the rCYP2C19 experiments, both lansoprazole and omeprazole strongly inhibited EET and DHET formation (Figs. 2B and 4). Consistent with the HLM results, lansoprazole showed more potent inhibition at lower concentrations than omeprazole. This aligns with previous findings demonstrating that both compounds inhibit CYP2C19, with lansoprazole exhibiting greater potency.²⁸⁾

Molecular docking analysis supported these biochemical findings. Lansoprazole and omeprazole with CYP2C19 suggested that both PPI molecules bind to the active site of CYP2C19 with a binding pose similar to the known inhibitor (Supplementary Fig. S2). The docking score of lansoprazole was lower than that of omeprazole, which is consistent with lansoprazole’s stronger inhibitory potency against CYP2C19 compared to that of omeprazole.

Regarding the R values obtained in the rCYP2C19 assay, lansoprazole exceeded the cutoff value of 1.1 for total EET and DHET isomers, indicating a level of inhibition that warrants further clinical consideration, whereas the R values for total EET and DHET with omeprazole were below the cutoff value. In contrast, neither lansoprazole nor omeprazole inhibited EET or DHET formation in the rCYP2C9 incubation experiments (Fig. 5). This finding indicates that, under the present experimental conditions, CYP2C9 inhibition by these PPIs contributes minimally, if at all, to the suppression of EET biosynthesis. According to previous studies, PPIs generally exhibit strong inhibitory effects on CYP2C19, whereas their inhibition of CYP2C9 is relatively weak.²⁸⁾ In addition, CYP2C9 inhibition is known to be substrate-dependent, and the inhibitory potency of a given inhibitor can vary substantially depending on the probe substrate used.⁴⁶⁾ Therefore, the weak inhibition of CYP2C9 by PPIs observed in earlier studies using diclofenac as the substrate may become even less pronounced when arachidonic acid is used as the substrate for EET formation,²⁸⁾ as in the present study, and thus may not have manifested as a clear inhibitory effect under our experimental conditions. Moreover, CYP2C9 has been reported to produce 14,15-, 11,12-, and 8,9-EETs at a 2.3 : 1.0 : 0.5 ratio,⁴⁷⁾ indicating that 14,15-EET is the major product. Accordingly, the lack of PPI-mediated inhibition of CYP2C9 parallels the minimal inhibition of 14,15-EET formation observed in HLMs. Moreover, because the inhibitory effect on 14,15-EET formation was relatively weak even in rCYP2C19, the limited inhibition observed in HLMs (Figs. 1A, 4A, and 5A) is consistent with these enzymatic characteristics.

In this study, although lansoprazole and omeprazole exhibited potent inhibition of EET formation in rCYP2C19, their inhibitory effects were limited in HLM. In rCYP2C19, the enzyme is present as a single, highly expressed catalyst, allowing inhibitory effects to be directly and clearly reflected. In contrast, CYP2C19 accounts for approximately 4% of total CYP expression in HLM,⁴⁸⁾ and EET formation is maintained by multiple enzymes, including CYP2C9 and CYP2C8.¹³⁾ Thus, the impact of selective CYP2C19 inhibition on overall metabolite formation may be masked. In addition, substantial differences exist between recombinant systems and HLM in terms of membrane environment and protein binding.⁴⁸⁾ In particular, HLM exhibits high nonspecific protein binding, which can reduce the free concentration of inhibitors and consequently attenuate the apparent inhibitory activity.⁴⁹⁾ Moreover, the distribution of substrates and inhibitors among multiple CYP isoforms in HLM may also account for the discrepancy between the results obtained in the rCYP2C19 and HLM experiments. On the other hand, our findings suggest that although overall inhibition in HLM is limited due to the contribution of multiple CYPs, inhibition of CYP2C19 itself may still contribute to the modest reduction in EET formation. We acknowledge the importance of CYP2J2 in cardiac EET production. In contrast, since this study is primarily focused on the systemic circulatory effects of EETs, and CYP2J2 expression is highly tissue-specific (e.g., in the heart) rather than systemic (e.g., liver and vascular endothelium), particularly when compared with the CYP2C subfamily.⁵⁰⁾ However, the demonstrated CYP2C19-mediated inhibition represents a novel systemic pathway through which PPIs may reduce the overall circulating levels of cardioprotective EETs. These findings suggest that lansoprazole and omeprazole may alter the relative distribution of EET regioisomers, potentially affecting their integrated cardioprotective functions in vivo. For example, vasodilation is largely mediated by 14,15-EET and 11,12-EET, although 5,6-EET also contributes.⁵¹⁾ The anti-inflammatory effects of EETs, important in preventing CVD progression, are reported to be most selective for 11,12-EET.⁵²⁾ In addition, 11,12-EET exerts antithrombotic effects, and 8,9-, 11,12-, and 14,15-EETs inhibit platelet adhesion to endothelial cells, suggesting roles in reducing thrombotic event risk.^53,54) Furthermore, 11,12-EET suppresses epithelial cell sodium absorption, contributing to blood pressure regulation under high-salt conditions and supporting both renal and long-term cardiac protection. Therefore, although levels of the less functionally selective 14,15-EET may remain relatively unchanged, as observed in HLM experiments, reduction in other EET regioisomers could disrupt the broader balance of EET-mediated protective mechanisms. Such shifts may collectively contribute to an increased risk of cardiovascular events.

However, the IC₅₀ values obtained in HLM experiments exceeded the reported plasma C_max of these PPIs, indicating that the clinical relevance of the observed in vitro inhibition requires cautious interpretation. PPIs are activated in acidic environments and accumulate within parietal cells, but the activated sulfenamide species does not enter the systemic circulation⁵⁵⁾; thus, the PPI forms capable of interacting with CYP enzymes are the inactive (non-protonated) species present in plasma and the liver. Because EETs are primarily produced in vascular endothelial cells, inhibition of CYP enzymes by circulating inactive PPIs represents the mechanistically relevant pathway when considering the potential influence of PPIs on EET biosynthesis.

Although PPIs undergo extensive hepatic metabolism and their hepatic exposure may exceed plasma concentrations due to first-pass effects and tissue distribution, direct evidence on intrahepatic PPI concentrations in humans is scarce. Furthermore, increased plasma exposure has been reported in elderly individuals and in patients with impaired hepatic function, in whom reduced clearance and prolonged half-life of PPIs have been observed.⁵⁶⁾ These factors suggest that systemic exposure to PPIs may be higher in certain populations.

In conclusion, our in vitro findings strongly suggest that CYP2C19 inhibition by lansoprazole and omeprazole suppresses the formation of cardioprotective EETs from arachidonic acid. This inhibition was concentration-dependent and more pronounced with lansoprazole than with omeprazole. However, simple comparison of the IC₅₀ values obtained in this study with reported plasma C_max values is insufficient to determine whether CYP2C19 inhibition by PPIs has clinically meaningful in vivo consequences. As this study provides exploratory in vitro findings, additional in vivo investigations will be required to clarify the potential clinical risks associated with PPI-mediated suppression of EET production.

Acknowledgments

This research was supported by the Japan Society for the Promotion of Science (KAKENHI Grant Number: 22K08318) and by the Takeda Science Foundation (Grant Number: 2024065636 to K.S.).

DECLARATIONS

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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