Drug-Drug Interaction Between Eugenol and Repaglinide in Type 2 Diabetes Mellitus and Its Potential Mechanism

Summary

This study aimed to explore potential drug-drug interactions between eugenol (EUG) and repaglinide (RPG) in patients with type 2 diabetes mellitus (T2DM).

Abstract

This study investigated the potential drug-drug interaction (DDI) between eugenol (EUG) and repaglinide (RPG) in type 2 diabetes mellitus (T2DM). A T2DM rat model was established using a high-fat diet and streptozotocin (STZ). The single-dose group received 0.4 mg/kg RPG, and co-administration groups were pre-treated with EUG (50, 100, 150 mg/kg) for seven days before receiving a single dose of RPG (0.4 mg/kg). The blood glucose and pharmacokinetic parameters of RPG were assessed in the plasma samples of rats. The effect of EUG on RPG metabolic stability and cytochrome P3A (CYP3A) activity was evaluated in rat liver microsomes (RLM). In T2DM rats, RPG treatment showed a hypoglycemic effect, which was further promoted by EUG co-administration in a dose-dependent manner (p < 0.001). EUG co-administration affected the pharmacokinetics of RPG by increasing the area under the curve (AUC, increased 88.64%, 164.59%, and 244.72% vs single RPG, p < 0.001), plasma maximum concentration (C_max, increased 18.61%, 53.58%, and 77.63% vs single RPG, p < 0.01), maximum concentration (T_max, 1.0 h for EUG co-administration vs 0.5 h for single RPG), half-life (t_1/2, prolonged 33.86%, 49.61%, and 88.19% vs single RPG, p < 0.01), mean retention time (MRT, prolonged 17.62%, 26.42%, 44.04% vs single RPG, p < 0.001), and decreased the clearance rate (Clz/F, decreased 48.92%, 63.31%, and 74.10% vs single RPG, p < 0.001) in a dose-dependent manner. In vitro RLM study, EUG improved the metabolic stability of RPG by prolonging the half-life (44.09 ± 3.25 min) and reducing the intrinsic clearance (30.55 ± 3.69 µL/min/mg protein). EUG also inhibited the activity of CYP3A with an IC₅₀ of 7.90 ± 1.79 µM. Co-administration of EUG promoted the hypoglycemic effect of RPG through enhancing RPG systemic exposure in T2DM rats, potentially by improving RPG metabolic stability and inhibiting CYP3A activity.

Introduction

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder characterized by insulin resistance or reduced insulin efficiency, which affects millions of individuals worldwide¹. Complex interactions with genetic susceptibility and lifestyle factors are involved in T2DM, resulting in impaired glucose uptake, excessive hepatic gluconeogenesis, and chronic low-grade inflammation². T2DM can lead to serious complications if treated improperly, including cardiovascular diseases, kidney disease, retinopathy, and neuropathy, significantly affecting the patients’ life quality and increasing mortality². Current drug treatments of T2DM include metformin, sulfonylurea drugs, GLP-1 receptor agonists, and DPP-4 inhibitors, etc³. Repaglinide (RPG) is one kind of short-acting meglitinide that works by stimulating insulin secretion and has the characteristic of dose flexibility⁴. Compared with sulfonylureas, glinides offer superior control of postprandial hyperglycemia, reduce the risk of certain adverse effects such as hypoglycemia, and present a more favorable safety profile, particularly in patients with renal failure^5,6. RPG is rapidly absorbed after oral administration, reaching peak plasma concentrations typically within 30–60 min, and its short half‑life results in a brief stimulation of insulin release. Clinically, RPG is used either as monotherapy or in combination with other oral antihyperglycemic agents to optimize glycemic control⁷. However, its clinical application is limited by low oral bioavailability (about 56%), rapid liver metabolism, and potential risk of hypoglycemia^{8,9,10,11,12,13,14}. These drawbacks highlight the necessity of a comprehensive treatment strategy.

Eugenol (EUG), chemically known as 4-allyl-2-methoxyphenol, is an organic compound with the chemical formula C₁₀H₁₂O₂¹⁵. EUG is a colorless to pale yellow liquid and is an active phenolic compound mainly found in cloves (Eugenia caryophyllata Thunb.) and other aromatic plants¹⁶. Much attention has been attracted by EUG due to its various pharmacological properties, including antioxidant, anti-inflammatory, antibacterial, analgesic, and anti-tumor activities^17,18,19. Recent studies have demonstrated that EUG has shown the potential value as an adjunctive therapy for T2DM, as both in vitro and in vivo experimental evidence indicate that EUG improves insulin sensitivity, promotes glucose uptake in peripheral tissues, inhibits hepatic gluconeogenesis, and protects pancreatic β-cell function^20,21. These multifaceted measures, combined with relatively low toxicity²², position EUG as a highly promising natural drug candidate that can complement shortcomings of traditional clinical drugs and alleviate T2DM-related complications through a synergistic mechanism.

The cytochrome P450 enzymes (P450s) play a crucial role in the process of drug metabolism and mediate drug-drug interactions (DDIs) by either inducing or inhibiting specific subtypes, which is of vital importance for safe and effective drug treatment²³. Notably, RPG is mainly metabolized in the liver by CYP2C8 and CYP3A4^24,25. When co-administered with quercetin (QCT), QCT enhances the in vivo bioavailability of RPG by inhibiting hepatic first-pass and systemic metabolism via CYP450s²⁶. It has also been reported that EUG suppresses the activity of CYP450s, including 1A2, 2C9, 2D6, and 3A4 in a concentration-dependent manner, indicating potential interactions when EUG is co-administered with drugs metabolized by these enzymes²⁷. Despite the promising potential of EUG as an adjunctive therapy for T2DM and the known susceptibility of RPG to CYP‑mediated DDIs, it remains unclear whether EUG alters the systemic exposure, metabolic stability, or CYP3A‑mediated metabolism of RPG. Given that both EUG and RPG are likely to be co‑administered in clinical practice or dietary supplement settings, the lack of DDI data between them poses a potential risk of unintended alterations in RPG plasma concentrations. Such alterations could lead to either reduced therapeutic efficacy or an increased risk of hypoglycemia. Thus, this evidence gap represents a significant obstacle to the rational and safe combination of natural products with conventional antidiabetic drugs. Therefore, it is essential to systematically evaluate the effect of EUG on the pharmacokinetics and pharmacodynamics of RPG and to elucidate the underlying metabolic mechanism, with particular focus on CYP3A activity. Given the above evidence, it could be speculated that EUG may influence RPG metabolism by altering CYP450 activity. This is the first study to investigate the EUG–RPG interaction in a T2DM setting. The findings are expected to fill a critical knowledge gap and provide a mechanistic basis for optimizing the safe co‑administration of EUG and RPG in T2DM management. If co‑administration is considered, the potential for increased RPG exposure should be considered, and signs of hypoglycemia should be monitored, particularly when EUG is taken at high doses or as a concentrated supplement. In addition, although rats express CYP3A isoforms that are functionally analogous to human CYP3A4, direct extrapolation of quantitative findings to humans requires caution due to species differences in enzyme expression and catalytic activity.

Protocol

This study was approved by the Jilin Academy of Chinese Medical Sciences Ethics Committee (Date:2024.11.21/No.2024112) in compliance with the ARRIVE guidelines²⁸. The reagents and the equipment used are listed in the Table of Materials.

1. Animal housing conditions

Male Sprague-Dawley rats (200–250 g) were housed in a laboratory animal room under the following conditions: temperature: 22 °C ± 2 °C, relative humidity: 40–60%, and a 12-h light/dark cycle, with free access to food and water to allow one week of acclimatization^29,30.

2. Animal modeling, treatment, and sample processing

The sample size was determined based on a power analysis using the F-test (G*Power 3.1.9.7). A priori power analysis was performed with an effect size of 0.6, an α level of 0.05, and a desired power of 0.8, which yielded a total sample size of 55. This calculation indicated that at least 9 rats were required per group. Therefore, ten rats per group were used, achieving an actual power of 0.86.

Before the T2DM model was established, all rats were fasted for 12 h. Then they were randomly divided into two groups: the control group (n = 10) and the T2DM group (n = 50). The establishment of the T2DM rat model was carried out according to the method reported previously³¹. In brief, rats in the T2DM group were fed a high-fat diet (with a fat content of 60%) for 4 consecutive weeks and then received a single intraperitoneal injection of 30 mg/kg of STZ dissolved in sterile citrate buffer (pH 4.4). Three days after the STZ injection, the tail vein blood glucose of the rats was measured using a blood glucose meter, and rats with a blood glucose concentration exceeding 16.7 mmol/L were considered to have successfully established the T2DM. For the control group, the rats received a normal diet and received intraperitoneal injections of 0.9% normal saline at the same time period as the T2DM group.

T2DM rats were further divided into four groups with 10 rats in each group: the T2DM + RPG group (single RPG), the T2DM + RPG + EUG (50 mg/kg) group (co-RPG+EUG 50 mg/kg), the T2DM + RPG + EUG (100 mg/kg) group (co-RPG+EUG 100 mg/kg), and the T2DM + RPG + EUG (150 mg/kg) group (co-RPG+EUG 150 mg/kg). After fasting for 12 h, rats in the single RPG group were orally administered 0.4 mg/kg RPG (suspended in normal saline) via a gavage needle²⁶. Rats in the co-RPG+EUG groups orally received 50, 100, and 150 mg/kg EUG, respectively, once daily for seven consecutive days, and on the eighth day, after an additional 12‑h fasting period, the co-administration group was additionally given 0.4 mg/kg RPG via intragastric gavage. Subsequently, blood glucose parameters were assessed using blood samples collected from the tail vein.

For pharmacokinetic analysis, the blood sample (0.2 mL) was collected from the posterior orbital venous plexus of the rats into the heparinized microcentrifuge tubes at various time points (0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, and 8 h post-dose). After collection, the samples were centrifuged (1500 × g, 10 min, 4 °C) to separate the plasma (pale yellow, clear), which was stored at -80 °C until analysis. The plasma samples were subsequently analyzed for RPG concentration using the HPLC method described in step 4.

3. Metabolism study in the pooled rat liver microsomes

To investigate the mechanism underlying the in vivo pharmacokinetic changes, the effect of EUG on the RPG metabolic stability and CYP3A activity was evaluated in RLM. RPG (3 µM) was incubated with or without EUG in PBS buffer at 37 °C with a total volume of 200 µL, including RLM (0.5 mg/mL microsomal protein) and a NADPH regeneration system (1.3 mM NADP, 3.54 mM glucose 6-phosphate, 0.4 U/mL glucose 6-phosphate dehydrogenase, and 3.3 mM MgCl₂). After a 5‑min pre‑incubation at 37 °C without NADPH, the reaction was initiated by adding the NADPH regeneration system. The mixture was then incubated at 37 °C in a shaking water bath. At different incubation time points (0 min, 5 min, 15 min, 30 min, 45 min, 60 min), 30 µL samples were collected and transferred to a microcentrifuge tube with ice-cold acetonitrile to terminate the reaction. The samples were centrifuged (12000 × g for 10 min at 4 °C) to obtain the supernatant (clear). The resulting supernatants were then analyzed for RPG concentration using the same HPLC method detailed in step 4.

4. HPLC analysis

The RPG concentration in plasma (from step 2) and microsomal incubations (from step 3) was analyzed using the HPLC method reported by Kim et al.²⁶. The specific operation steps are as follows: samples (120 µL) were mixed with 300 µL of acetonitrile containing ketoconazole (internal standard, 100 ng/mL) to achieve protein precipitation. Then, the mixture was vortexed and centrifuged at 15000 × g for 10 min at 4 °C. The supernatant was transferred to a microcolumn centrifuge tube and evaporated at 25 °C under a nitrogen atmosphere. The residue after drying was re-dissolved with 50 µL mobile phase, then vortexed and centrifuged. Subsequently, 40 µL of the supernatant was injected into the HPLC column (250 mm × 4.6 mm, particle size 5 µm). The column was eluted at 25 °C in an isocratic elution mode with 10 mM phosphate buffer (pH 6.0) and acetonitrile (volume ratio 46.4: 53.6), with a flow rate of 1 mL/min. The eluent was monitored using a fluorescence detector with an excitation wavelength of 240 nm.

5. Statistical analysis

Pharmacokinetic analysis software was used to calculate pharmacokinetic parameters, including AUC, C_max, T_max, t_1/2, MRT, and clearance. Statistical analysis and graphing software were used for figure generation and data analysis. All data were presented as mean ± standard deviation. Differences among groups were analyzed using one-way ANOVA (for glucose level comparisons) or two-way ANOVA (for pharmacokinetic parameter comparisons), as appropriate, followed by Tukey’s post hoc test for multiple comparisons. The effect of EUG on CYP3A activity was evaluated using nonlinear regression analysis. A p-value < 0.05 was considered statistically significant.

Half-life of EUG on RPG metabolic stability in RLM was calculated by:
t_1/2 (min) = 0.693/ln(C_t/C₀ × 100).

Intrinsic clearance rate (µL/min/mg protein) = v × 0.693/t_1/2;
v (µL/mg) = incubation volume/protein concentration.

Results

EUG promoted the hypoglycemic effect of RPG in T2DM rats
The blood glucose level of the rats in the T2DM group was increased (p < 0.001, 95% CI: -13.90~-11.32) in comparison with that of the control group, indicating the successful conduction of the T2DM model (Figure 1A). Administration of a single RPG suppressed the blood glucose level in the T2DM rats (p < 0.001, 95% CI: 5.26~7.84). Co-administration of RPG and EUG further decreased the glucose...

Discussion

T2DM is a chronic metabolic disease that requires long-term treatment, and patients often need to take multiple medications due to underlying conditions such as cardiovascular disease and kidney disease³². The risk of DDIs significantly increases with drug co-administration, as it may affect the absorption, metabolism, or efficacy of hypoglycaemic drugs, which can lead to acute risks such as hypoglycaemic coma and uncontrolled blood sugar levels³³. It may also exacerbate or...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
acetonitrile	Merck, Germany	34851	≥99.9%
chromatographic column	Phenomenex, USA	00G-4041-E0	250 mm × 4.6 mm, particle size 5 μm
DAS	Beijing Bozhiyin, China	-	3.0
EUG	MUST Bio-technology, China	A0074	purity ≥99%
GraphPad Prism	GraphPad Software, USA	-	9.0.0
High Performance Liquid Chromatography	Shimadzu Corporation, Japan	228-45147-42	RF-20A
ketoconazole	Merck, Germany	K1003	Purity 99.0-101.0%
Male Sprague-Dawley rats	Shanghai Animal Center, China	-	200–250 g
NADPH	Merck, Germany	10107824001	97% (dry weight)
OneTouch Ultra2 blood glucose meter	Life Scan Inc., USA	https://shop.onetouch.com/onetouch-sup-reg-sup-ultra-sup-reg-sup-2-meter/product/OTSUS05_0019	-
PBS	Merck, Germany	P4474	ph 7.4
RLM	Merck, Germany	M9066	-
RPG	Merck, Germany	R9028	purity ≥98%
STZ	Merck, Germany	S0130	≥98% (HPLC)
EUG, eugenol; RPG, repaglinide; RLM, rat liver microsomes; NADPH, β-Nicotinamide adenine dinucleotide phosphate; STZ, streptozotocin; PBS, phosphate-buffered saline.