Pharmacokinetics and Tissue Distribution of Enavogliflozin in Mice Using a Validated Liquid Chromatography–Tandem Mass Spectrometry Method

Abstract

Enavogliflozin, a sodium–glucose cotransporter 2 inhibitor, was approved in 2022 by the Korean Ministry of Food and Drug Safety as a therapeutic agent for type 2 diabetes mellitus and has been investigated for expanded therapeutic efficacy in diabetic retinopathy and cardioprotection. In this study, we developed and validated an analytical method to precisely detect enavogliflozin in mouse plasma, employing liquid–liquid extraction combined with liquid chromatography–tandem mass spectrometry. Overall, the analytical method, covering a range of 5–3000 ng/mL, is reliable for investigating the time-concentration profiles of enavogliflozin, demonstrating acceptable accuracy, precision, extraction recovery, and minimal matrix effects without stability concerns as evidenced by assessments of post-treatment stability, freeze–thaw stability, and short-term stability of enavogliflozin. Pharmacokinetic profiles and all pharmacokinetic parameters of enavogliflozin in mice did not differ between fed and fasted states after oral administration of enavogliflozin (1 mg/kg). Additionally, no differences in the pharmacokinetic profiles of enavogliflozin were observed among single, 7-day repeated, and 14-day repeated oral administrations at 1 mg/kg. In the tissue distribution study, enavogliflozin showed the highest distribution in the kidneys, followed by the large intestine, stomach, small intestine, liver, heart, lungs, spleen, and testes after oral administration at both 1 and 3 mg/kg doses. Dose proportionality in tissue distribution was observed except for the kidneys. In conclusion, enavogliflozin can be administered without concern for pharmacokinetic changes, regardless of single or multiple dosing and whether in fed or fasted states. Furthermore, the tissue distribution profile may offer valuable insights into the therapeutic potential of this drug.

1. Introduction

Patients with diabetes who have increased renal glucose reabsorption capacity experience continuous rises in serum glucose levels [1]. SGLT2, a high-capacity and low-affinity glucose transporter, is responsible for nearly 90% of renal glucose reabsorption and is mainly expressed in the renal proximal tubule, where inhibitors of sodium–glucose cotransporter 2 (SGLT2) have therapeutic potential [1,2,3]. Polysaccharide-based drugs have shown high efficacy and specificity as novel therapeutic approaches [4,5]. Among these polysaccharide drugs, α-glucosidase inhibitors (e.g., voglibose, miglitol and acarbose) and SGLT2 inhibitors (e.g., dapagliflozin, empagliflozin, ertugliflozin, etc.) have a promising future as antidiabetic agents [5,6].

Enavogliflozin (DWP16001; IUPAC Name: 2S,3R,4R,5S,6R)-2-[7-chloro-6-[(4-cyclopropylphenyl)methyl]-2,3-dihydro-1-benzofuran-4-yl]-6-(hydroxymethyl)oxane-3,4,5-triol; molecular weight (MW): 446.9 g/mol; LogP value: 2.65; pKa value: 12.57), a selective SGLT2 inhibitor, received market approval in Korea in 2022 [7,8]. Enavogliflozin has a comparable IC₅₀ value (0.8 ± 0.3 nM) to other SGLT2 inhibitors such as dapagliflozin, empagliflozin, and ipragliflozin [7]. The solubility of enavogliflozin is 2.5 mg/mL and it is predicted to be rapidly absorbed with an absorption fraction of 0.866 using a physiologically based pharmacokinetic modeling technique [8]. Similarly, oral bioavailability was 84.5–97.2% for mice and 56.3–62.1% for rats [9]. Absorbed enavogliflozin is primarily metabolized by CYP3A4 and CYP2C19 to M1 (monohydroxylation in the dihydrobenzofuran group) and M2 (monohydroxylation in cyclopropyl group) [10]. After multiple administrations of enavogliflozin (0.1–2 mg) for 14 days, M1 and M2 levels in the human plasma were reported to be 20–25% and 1–6%, respectively, of parent enavogliflozin [11]. M3 and M4 were produced by the hydroxylation of M1 and M2 via CYP3A, and three glucuronide metabolites were identified in hepatocytes [10]. However, the pharmacological activities and plasma concentrations of these metabolites M1 and M2 have not yet been reported.

A multicenter phase III trial revealed that patients with type 2 diabetes mellitus (T2DM) taking 0.3 mg enavogliflozin for 24 weeks successfully achieved hemoglobin A1c levels below 7.0% and exhibited significant reductions in fasting plasma glucose levels (−40.1 mg/dL), body weight (−2.5 kg), blood pressure, and low-density lipoprotein cholesterol levels [12]. In multiple administrations, plasma exposure of enavogliflozin and urinary glucose excretion (UGE) over 24 h showed a clear pharmacokinetic and pharmacodynamic correlation in the dose range of 0.1–2.0 mg [11,13]. Additionally, as a result of high and sustained kidney distribution [7], enavogliflozin demonstrated long-lasting UGE up to 168 h and equivalent daily UGE compared with dapagliflozin 10 mg [11]. These results suggest the greater potency and long-lasting efficacy of enavogliflozin compared to dapagliflozin, which may reflect the importance of kidney distribution of in the pharmacodynamics of these SGLT2 inhibitors. Although about 2 years have passed since its market approval, a meta-analysis of enavogliflozin was performed using four clinical trials that included 684 T2DM patients for clinical outcomes over 12–24 weeks of clinical use. As a result, enavogliflozin is a well-tolerated and effective SGLT2 inhibitor for T2DM and could be superior to dapagliflozin with regard to hemoglobin A1c levels below 7.0% over 6 months of clinical use [11,14,15,16]. In addition to the antidiabetic effect of SGLT2 inhibitors, they have been reported to show beneficial effects on weight loss and cardio- and reno-protective effects in T2DM patients [17,18,19]. Based on these clinical outcomes of cardiorenal protection, the American Diabetes Association recommends that SGLT2 inhibitors and glucagon-like peptide-1 receptor agonists can be used—in combination with metformin—as first-line therapy for diabetic patients at high risk of heart failure, cardiovascular disease, and chronic kidney disease [20].

Moreover, recent studies have reported these cardioprotective and nephroprotective effects regardless of the presence of diabetes [20,21]. Canagliflozin has been shown to protect against cisplatin-induced acute kidney injury by activating AMPK (adenosine monophosphate–activated protein kinase) and inducing autophagy in renal proximal tubular cells [21]. Empagliflozin protected against methotrexate-induced renal toxicity via suppression of oxidative stress and inflammation in male rats [22]. Empagliflozin and dapagliflozin inhibited mTORC1 (mechanistic target of rapamycin complex 1) and HIF-α (hypoxia-inducible factor-1α), consequently showing protective effects in glomerular injury in lupus nephritis. Relevant clinical trials confirmed the acceptable safety profile of SGLT2 inhibitors for the treatment of lupus nephritis [23,24]. Considering the higher distribution of SGLT2 inhibitors to the kidneys and their mechanism of action in renal proximal tubular cells, the nephroprotective effects of SGLT2 inhibitors suggest their potential as promising therapeutics for acute kidney injury through reducing oxidative stress, increasing inflammatory mediators, and enhancing apoptotic reactions in the renal tissue [22].

Based on SGLT2 expression in retinal pericytes [25,26], SGLT2 inhibitors exert therapeutic effects by reducing glucose entry into pericytes, reducing oxidative stress, and restoring insulin signaling [25,26,27]. Jang et al. investigated the ocular and plasma pharmacokinetics of enavogliflozin in rabbits using enavogliflozin ophthalmic solution to evaluate its therapeutic potential for the treatment of diabetic retinopathy [25]. Additionally, enavogliflozin significantly decreased nanoplastic-induced premature endothelial senescence and dysfunction caused by upregulated cardiovascular SGLT2 [28]. It also improved vascular function by decreasing reactive oxygen species and upregulating endothelial nitric oxide synthase expression [28]. The study suggested that enavogliflozin improves vascular function through SGLT2 inhibition and inhibits the inflammatory response; enavogliflozin needs to be evaluated as a potential agent for the prevention of vascular aging-related disorders and cardiovascular disease caused by nanoplastic contaminants [29]. Rhee et al. evaluated the anti-obesity effect of enavogliflozin in naturally obese dogs. Body weight, body fat percentage, and fat thickness were significantly reduced in the enavogliflozin-treated group without affecting food consumption compared to the obese control group [30]. Taken together, the therapeutic potency of enavogliflozin could be expanded to include acute or chronic kidney injury, cardiovascular disease, retinopathy, and an anti-obesity effect in addition to T2DM treatment. In these cases, the expression of SGLT2 and tissue distribution kinetics of enavogliflozin could be important factors for the therapeutic expansion of this drug since its mechanism of action, which was reported as reduced oxidative stress, increased inflammatory mediators, and enhanced apoptotic reactions, is not tissue- or site-specific.

Therefore, we aimed to examine the pharmacokinetic characteristics of enavogliflozin, including differences between fasted and fed states and between single and repeated doses, which can be useful information to find a dose regimen for pharmacology study and the pharmacology–pharmacodynamic relationship regarding the new therapeutic efficacy of enavogliflozin. We also aimed to investigate dose dependency in the tissue distribution of enavogliflozin in mice. In addition, the analytical method of enavogliflozin using liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed and validated in this study. To validate the analytical method, specificity, linearity, intra- and inter-day precision and accuracy, matrix effect, extraction recovery, and stability of enavogliflozin were examined according to the US Food and Drug Administration (FDA) guidance on bioanalytical method validation (https://www.fda.gov/regulatory-information/search-fda-guidance-documents/bioanalytical-method-validation-guidance-industry, accessed on 3 December 2024).

2. Materials and Methods

2.1. Chemicals and Reagents

Enavogliflozin (Batch No. E2016-085-27-2, purity 100.01%) and d4-enavogliflozin (used as an internal standard, IS) were provided by Daewoong Pharmaceutical Co., Ltd. (Yongin, Republic of Korea). Water and methanol were purchased from Tedia (Fairfield, CT, USA). Methyl tertiary-butyl ether (MTBE), dimethyl sulfoxide, and formic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA). All solvents and chemicals used were of HPLC (high-performance liquid chromatography) or reagent grade.

2.2. Preparation of the Stock and Working Solutions

Stock solutions of enavogliflozin and d4-enavogliflozin (IS) were prepared at a concentration of 2 mg/mL in methanol. These stock solutions were diluted with methanol to prepare working solutions. The final concentrations of the enavogliflozin working solutions were 50, 150, 200, 500, 1500, 2500, 5000, 10,000, 20,000, and 30,000 ng/mL. The concentration of the d4-enavogliflozin working solution was 200 ng/mL.

2.3. Preparation of Calibration Standards and Quality Control Samples

To prepare calibration standards and quality control (QC) samples, 3 μL of the enavogliflozin working solution was spiked into 27 μL of blank plasma. The final concentrations of the enavogliflozin calibration standards were 5, 20, 50, 150, 500, 1000, and 3000 ng/mL, and the final concentrations of the QC samples were 5 ng/mL (lower limit of quantification, LLOQ), 15 ng/mL (Low QC), 250 ng/mL (Mid QC), and 2000 ng/mL (High QC). Calibration standards and QC samples were prepared following the sample preparation method described below.

An aliquot of 100 μL IS solution was added to 30 μL of plasma sample. Then, 500 μL of MTBE was added to the mixture, which was vigorously vortexed for 15 min. The solution was frozen at −80 °C for 1 h and then centrifuged at 16,000× g for 5 min. The supernatant was transferred to a clean tube and evaporated under a gentle stream of nitrogen gas. Finally, the residue was reconstituted with 150 μL of a methanol–water mixture (1:1, v/v). A 3 μL aliquot of the solution was directly injected into the LC–MS/MS system.

2.4. Instrument Conditions

Plasma concentrations of enavogliflozin and IS were analyzed using an Agilent Infinity 1260 HPLC system coupled with an Agilent 6430 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) equipped with an electrospray ionization source. The analytes were separated on a Synergi Polar RP column (4 μm particle size, 2.0 mm × 150 mm; Phenomenex, Torrance, CA, USA) with isocratic elution. The mobile phase consisted of water containing 0.1% formic acid and methanol containing 0.1% formic acid (15:85, v/v) at a flow rate of 0.25 mL/min. The column oven temperature was maintained at 30 °C. The gas flow rate was 10 L/min with a gas temperature of 310 °C. Nebulizer and capillary voltages were set at 35 psi and 4000 V, respectively. Multiple reaction monitoring (MRM) transitions were set to m/z 464.2 → 131.0 for enavogliflozin and m/z 468.1 → 135.2 for d4-enavogliflozin (IS) in a positive ion mode. The collision energy for both analytes was set to 25 eV.

2.5. Methodological Validation

Selectivity was assessed by comparing LLOQ samples (5 ng/mL of enavogliflozin) prepared from six different mice to the corresponding blank plasma samples. The ratios of analyte peak areas to IS peak areas was plotted against the concentration of enavogliflozin. The linearity of the calibration curve over the range of 5–3000 ng/mL was evaluated using least-squares linear regression with 1/concentration² as weighting factors. Acceptance criteria for calibration standard curve was as follows: non-zero calibration standards were ±15% of nominal concentrations, except at LLOQ (±20% of the nominal concentration) in each validation run. Moreover, 75% and a minimum of six non-zero calibration standard levels met the above criteria in each validation run. Six replicates of the four QC levels (5, 15, 250, and 2000 ng/mL) were analyzed on the same day to assess intra-day precision and accuracy. Inter-day precision and accuracy were determined by measuring the four QC levels (5, 15, 250, and 2000 ng/mL) over 5 consecutive days. Precision was represented as the coefficient of variance (CV, %), whereas accuracy was expressed as the percentage of the measured QC concentration to the nominal QC concentration.

Matrix effects for enavogliflozin were evaluated at three QC levels (15, 250, and 2000 ng/mL) using the six different mouse plasma by dividing the peak areas of post-extraction spiked samples by those from neat solutions of corresponding concentrations. Extraction recovery was determined at three QC levels (15, 250, and 2000 ng/mL) by comparing the mean peak areas of pre-extraction spiked samples with those of post-extraction spiked samples. The matrix effects and extraction recovery of IS was determined using the same procedure, with an IS concentration of 200 ng/mL. Post-treatment stability was assessed by placing processed QC samples at the three levels in the autosampler at 10 °C for 24 h. Freeze–thaw stability was evaluated by subjecting QC samples at the three levels to three freeze–thaw cycles, each consisting of freezing overnight at −80 °C and thawing completely at 25 °C. Bench-top stability was assessed by keeping QC samples at the three levels at 25 °C for 5 h.

2.6. Animals and Ethical Statement

Male ICR mice (7 weeks old, 225–250 g) were purchased from Samtako Co. (Osan, Gyeonggi-do, Republic of Korea). The animals underwent microbial monitoring including viral serology, parasite testing, and bacterial testing, and were maintained in a physiologically and pathologically healthy state, having passed major disease monitoring processes. The animals were acclimatized for 1 week in an animal facility at the College of Pharmacy, Kyungpook National University. Food and water were available ad libitum. All animal procedures were approved by the Animal Care and Use Committee of Kyungpook National University (IACUC No. KNU-2016-0036, KNU-2019-0036, and KNU-2022-0179) and carried out in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals. Ninety-eight mice were used for the validation of the analytical method and for the pharmacokinetic studies.

2.7. Pharmacokinetics Study

Twenty mice were randomly divided into two groups. One group was fasted for 16 h before oral administration of enavogliflozin, while the other group was not fasted. Enavogliflozin was dissolved in saline containing 10% dimethyl sulfoxide and administered orally at a dose of 1 mg/kg in a volume of 5 mL/kg via gavage to mice in both the fed and fasted states. Under 2% isoflurane anesthesia for 5 min, blood samples (approximately 80 μL) were collected from five fed mice at 0.5, 2, and 8 h via the right and left retro-orbital vein and abdominal artery, respectively. Another set of sparse blood samples (approximately 80 μL) was collected from five fed mice at 1, 4, and 24 h via the right and left retro-orbital vein and abdominal artery, respectively. The same protocol was followed for the 10 fasted mice. Blood samples were centrifuged at 12,000× g for 1 min to separate plasma, and aliquots (30 μL) of each plasma sample were stored at −80 °C until analysis of enavogliflozin.

Thirty nonfasted mice were randomly divided into three groups: single dose, 1-week repeated dose, and 2-week repeated dose. Enavogliflozin solution (1 mg/kg in 5 mL/kg of a mixture of 10% dimethyl sulfoxide and 90% saline) was orally administered via gavage to the single-dose group (10 mice). Under 2% isoflurane anesthesia for 5 min, blood samples (approximately 80 μL) were collected from five mice in the single-dose group at 0.5, 2, and 8 h via the right and left retro-orbital vein and abdominal artery, respectively. Another set of sparse blood samples (approximately 80 μL) was collected from five mice in the single-dose group at 1, 4, and 24 h via the right and left retro-orbital vein and abdominal artery, respectively. The same protocol was followed for the 1-week (10 mice) and 2-week (10 mice) repeated-dose groups. Blood samples were centrifuged at 12,000× g for 1 min to separate plasma, and aliquots (30 μL) of each plasma sample were stored at −80 °C until analysis of enavogliflozin.

2.8. Tissue Distribution Study

Forty-two mice were randomly divided into seven groups and fasted for 16 h before oral administration of enavogliflozin. Enavogliflozin dissolved in saline containing 10% dimethyl sulfoxide was orally administered at a dose of 1 mg/kg/5 mL. Blood and tissue samples were collected at 0.5, 1, 2, 4, 8, 24, and 48 h (six mice at each time point). Under 2% isoflurane anesthesia for 5 min, blood samples (approximately 80 μL) were collected from the abdominal artery at each time point. Blood samples were centrifuged at 12,000× g for 1 min to separate plasma. Subsequently, the stomach, small intestine, large intestine, liver, kidneys, brain, heart, lungs, spleen, and testis were isolated and washed with wet wiping towel and weighed by subtracting empty tube weight from total tube weight containing each tissue. All tissues were homogenized with four volumes of saline using an MM400 laboratory ball mixer mill (Retsch, Haan, Germany) by horizontal oscillation for 1 min at a maximum speed of 30 Hz to make 20% tissue homogenates. Aliquots (30 μL) of plasma samples and aliquots (50 μL) of 20% tissue homogenate samples were stored at −80 °C until analysis. An identical protocol was followed at 1, 2, 4, 8, 24, and 48 h.

To measure the concentration of enavogliflozin, the aliquots of plasma (30 μL) and tissue homogenates (50 μL) were mixed with 100 μL IS solution. Then, 500 μL of MTBE was added to the mixture, which was vigorously vortexed for 15 min. The solution was frozen at −80 °C for 1 h and then centrifuged at 16,000× g for 5 min. The supernatant was transferred to a clean tube and evaporated under a gentle stream of nitrogen gas. Finally, the residue was reconstituted with 150 μL of a methanol–water mixture (1:1, v/v). A 3 μL aliquot of the solution was directly injected into the LC–MS/MS system.

The calibration standard curves for enavogliflozin were separately prepared in the range of 2–3000 ng/mL in the plasma, stomach, small intestine, large intestine, lung, liver, kidney, lung, heart, brain, spleen, and testis homogenates. The inter-day and intra-day precision and accuracy were within 15% for all three levels of quality control samples.

2.9. Data Analysis and Statistics

Pharmacokinetic parameters such as terminal half-life (T_1/2), area under the plasma concentration–time curve to the last sampling time (AUC_last), AUC extrapolated to infinity (AUC_∞), and mean residence time (MRT) were calculated using noncompartmental analysis (WinNonlin version 5.2; Pharsight Co., Mountain View, CA, USA).

All data are expressed as the mean ± standard deviation (SD). Statistical analysis was performed using the Mann–Whitney U test or Kruskal–Wallis test to compare between two groups or among three groups, respectively, using IBM SPSS Statistics for Windows (version 27; IBM Corp., Armonk, NY, USA). Differences were considered significant at p < 0.05.

3. Results

3.1. LC–MS/MS Analysis of Enavogliflozin

To optimize the ESI conditions of enavogliflozin and IS, each analyte was directly injected into the MS ionization source. Both enavogliflozin and IS exhibited optimal ionization in the positive mode. The MRM transition of enavogliflozin was selected from the precursor ion ([M+NH₄]⁺, m/z 464.2) and the structurally predictable product ion (m/z 131.0). Since the IS is a structure in which enavogliflozin is substituted with four deuterium atoms (Figure 1), the MRM transition was selected from the precursor ion ([M+NH₄]⁺, m/z 468.1) and the most abundant product ion (m/z 135.2) (Figure 1). The selected precursor and product ions of enavogliflozin were consistent with previous results [7,9,11].

3.2. Selectivity and Linearity

Figure 2 shows the representative MRM chromatograms of double blank, zero blank, LLOQ sample (5 ng/mL), and plasma sample after oral administration of enavogliflozin. The retention times of enavogliflozin and IS were the same at 2.74 min. Compared to the blank samples, there was no significant matrix interference in the LLOQ samples at the retention times of enavogliflozin and IS. The signal-to-noise ratio of enavogliflozin was 13.3 in the LLOQ samples (Figure 2).

A calibration standard curve was linear within the enavogliflozin concentration range of 5–3000 ng/mL, with a coefficient of determination (r²) of 0.995 ± 0.002 determined from linear regression analysis using a weighting of 1/concentration². The calibration standard curve parameters are as follows: slope, 0.0077 ± 0.0021 (mean ± SD); intercept, 0.0061 ± 0.0159. Across all concentration ranges, the accuracy and coefficient of variation (CV) ranged from 95.50% to 107.8% and from 1.351% to 7.062%, respectively (

Table 1. Calculated concentrations of enavogliflozin in calibration standards.

Nominal Concentration (ng/mL)	Calculated Concentration (ng/mL)	Accuracy (%)	CV (%)
5	5.073 ± 0.069	101.5	1.351
20	19.13 ± 1.351	95.66	7.062
50	48.54 ± 3.127	97.08	6.442
150	143.2 ± 3.976	95.50	2.778
500	487.2 ± 24.43	97.44	5.015
1000	1046 ± 33.41	104.6	3.196
3000	3234 ± 101.0	107.8	3.124

Data represented as mean ± SD (n = 6).

).

3.3. Precision and Accuracy

The intra- and inter-day precision and accuracy for enavogliflozin from three levels of QC samples are summarized in

Table 2. Intra- and inter-day precision and accuracy of enavogliflozin.

	Nominal Concentration (ng/mL)	Measured Concentration (ng/mL)	Precision (CV, %)	Accuracy (%)
Intra-day (n = 6)	5	5.376 ± 0.386	4.601	110.4
	15	14.85 ± 0.844	5.680	99.00
	250	247.2 ± 16.19	6.551	98.88
	2000	2237 ± 61.09	2.731	111.8
Inter-day (n = 5)	5	5.520 ± 0.254	7.186	107.5
	15	14.15 ± 0.831	5.876	94.31
	250	248.2 ± 10.43	4.201	99.29
	2000	2117 ± 170.3	8.043	105.8

Data represented as mean ± SD (n = 5 or 6).

. The intra- and inter-day precision ranged from 2.731% to 8.043%, and the intra- and inter-day accuracy ranged from 94.31% to 111.8%, both of which fall within the acceptable criteria (Precision (%) ≤ 20% at the LLOQ and ≤ 15% at the other QC levels. Accuracy (%) of 80–120% at the LLOQ and 85–115% at the other QC levels).

3.4. Matrix Effect and Recovery

The matrix effects and extraction recovery can be affected by the organic solvent and endogenous compounds close to the analyte elution times [31,32]. The use of the protein precipitation method for preparation of plasma enavogliflozin samples represented the high extraction recovery but considerable matrix effects, due to the ion suppression. In order to quantify the concentration of enavogliflozin in plasma and various tissues, we tried a liquid–liquid extraction method with an isotopically labeled internal standard. For a liquid–liquid extraction, we tested an MTBE and ethyl acetate as an extraction solvent. The results showed that the liquid–liquid extraction with MTBE extracted enavogliflozin efficiently from mouse plasma and tissue samples with negligible matrix effects compared with the use of ethyl acetate. Therefore, the liquid–liquid extraction method with MTBE was chosen for the sample preparation method.

Table 3. Extraction recoveries and matrix effects for enavogliflozin and IS.

Analyte	Nominal Concentration (ng/mL)	Extraction Recovery (%) (n = 3)	CV (%)	Matrix Effects (%) (n = 6)	CV (%)
Enavogliflozin	15	88.79 ± 2.654	2.989	99.09 ± 5.104	5.151
	250	81.82 ± 2.861	3.497	103.6 ± 3.449	3.329
	2000	79.75 ± 3.190	4.000	103.2 ± 1.126	1.091
IS	200	85.76 ± 5.879	6.856	96.93 ± 6.236	6.433

Data represented as mean ± SD (n = 3 or 6).

summarizes the extraction recoveries and matrix effects of enavogliflozin and IS. The extraction recoveries for enavogliflozin were calculated at three levels of QC samples and were high and reproducible, ranging from 79.75% to 88.79%, with CVs from 2.989% to 4.000%. The extraction recovery of IS was 85.76 ± 5.879%. Therefore, the extraction process using MTBE, utilized in this study, was capable of efficiently extracting enavogliflozin and IS from mouse plasma. The matrix effects were 99.09% for Low QC, 103.6% for Mid QC, 103.2% for High QC, and 96.93% for IS, suggesting that coeluting substances did not interfere with the ionization of enavogliflozin. The low CVs (<15%) in the matrix effect indicated that there were no significant differences in the peak areas of enavogliflozin at the three concentrations across the six plasma samples. Therefore, it was possible to exclude any matrix effect on ion suppression or enhancement.

3.5. Stability

The results of the stability experiments are presented in

Table 4. Stability of enavogliflozin.

Nominal Concentration (ng/mL)	Measured Concentration (ng/mL)	Precision (%)	Accuracy (%)
Autosampler stability (10 °C, 24 h)
15	15.52 ± 0.907	5.846	103.5
250	255.8 ± 13.38	5.232	102.3
2000	1853 ± 54.45	2.939	92.63
Freeze–thaw stability (3 cycles)
15	15.55 ± 0.382	2.454	103.6
250	255.2 ± 10.07	3.945	102.1
2000	1716 ± 38.61	2.250	85.81
Bench-top stability (25 °C, 6 h)
15	15.03 ± 0.437	2.905	100.2
250	227.7 ± 11.56	5.074	91.09
2000	1711 ± 32.69	1.911	85.56

Data represented as mean ± SD (n = 3).

. The accuracy of enavogliflozin ranged from 85.56% to 103.6%, and the precision ranged from 1.911% to 5.846% under the three different stability test conditions. The acceptance criterion for all stability studies was an accuracy of 85–115% relative to the nominal concentration (100% by definition). As there were no significant differences observed between each measured concentration and the nominal concentration under various stability treatment conditions, these results provided evidence that enavogliflozin in mouse plasma was stable up to 6 h at 25 °C for bench-top stability, stable for 24 h in the autosampler after sample treatment, and remained stable over three freeze–thaw cycles.

3.6. Pharmacokinetics of Enavogliflozin in Fed and Fasted Mice

To investigate the pharmacokinetic differences of enavogliflozin between fed and fasted mice, enavogliflozin was administered orally (1 mg/kg) to both groups, and the results are shown in Figure 3 and

Table 5. Pharmacokinetic parameters of enavogliflozin in fasted and fed mice following oral administration of enavogliflozin (1 mg/kg).

Parameter	Group		p Value
	Fed	Fasted
Tmax (h)	1.800 ± 0.447	1.600 ± 0.548	0.690
Cmax (ng/mL)	261.7 ± 23.60	228.7 ± 93.06	0.222
T1/2 (h)	4.403 ± 0.228	4.449 ± 0.577	0.841
AUClast (h·ng/mL)	1772 ± 174.2	1633 ± 275.9	0.347
AUC∞ (h·ng/mL)	1809 ± 179.4	1671 ± 280.9	0.421
MRT (h)	5.991 ± 0.386	6.087 ± 0.651	0.841

Data are expressed as the mean ± SD (n = 5). p value was calculated by Mann–Whitney U test.

. The plasma profile of enavogliflozin in fed mice was similar to that in fasted mice. There were no statistically significant differences in T_max, C_max, T_1/2, AUC_24h, AUC_∞, and MRT (p > 0.05). Therefore, enavogliflozin appears to have similar pharmacokinetic characteristics regardless of fed or fasted states.

3.7. Pharmacokinetics of Enavogliflozin Following Single or Repeated Oral Administration

Enavogliflozin was administered once, repeatedly for 1 week, or repeatedly for 2 weeks to nonfasted mice. The overall plasma concentration–time profiles and pharmacokinetic parameters of enavogliflozin were quite similar, whether enavogliflozin was administered once or repeatedly for 1 or 2 weeks (Figure 4). As a result, there were no statistically significant differences in T_max, C_max, T_1/2, AUC_24h, AUC_∞, and MRT (p > 0.05) among the three groups, suggesting that the pharmacokinetic characteristics of enavogliflozin were not altered by repeated dosing within 2 weeks (

Table 6. Pharmacokinetic parameters of enavogliflozin in mice following single or repeated oral administration of enavogliflozin (1 mg/kg).

Parameter	Dose			p Value
	Single	Repeated for 7 Days	Repeated for 14 Days
Tmax (h)	2.400 ± 0.894	2.000 ± 0.000	1.600 ± 0.548	0.105
Cmax (ng/mL)	253.4 ± 28.69	258.5 ± 28.22	257.0 ± 74.37	0.914
T1/2 (h)	3.949 ± 0.674	4.070 ± 0.419	4.272 ± 0.554	0.357
AUClast (h·ng/mL)	2047 ± 389.1	1934 ± 253.7	2066 ± 516.8	0.533
AUC∞ (h·ng/mL)	2074 ± 380.4	1966 ± 263.9	2106 ± 506.3	0.613
MRT (h)	5.867 ± 0.242	5.763 ± 0.583	6.220 ± 0.368	0.174

Data are expressed as the mean ± SD (n = 5). p value was calculated among three groups by the Kruskal–Wallis test.

).

3.8. Enavogliflozin Tissue Distribution

To investigate the dose-dependent tissue distribution of enavogliflozin, concentrations in plasma and tissues—including the stomach, large intestine, small intestine, kidneys, liver, lungs, heart, testes, spleen, and brain—were analyzed after single oral administration of enavogliflozin at doses of 1 and 3 mg/kg in mice. Concentration–time profiles of each tissue and plasma are shown in Figure 5. Enavogliflozin was not detected in the brain; therefore, the brain was not included in this figure. The plasma concentration profiles were divided into three subsets according to the enavogliflozin concentrations in each tissue compared with its plasma concentration. Concentration profiles of enavogliflozin in the stomach, small intestine, large intestine, and liver showed similar patterns to that of plasma but with higher concentrations (Figure 5A,B at 1 mg/kg; Figure 5D,E at 3 mg/kg).

Kidney concentrations of enavogliflozin at 1 mg/kg and 3 mg/kg indicated significantly higher concentrations and longer elimination half-lives than plasma enavogliflozin, resulting in high AUC ratios for kidneys to plasma (71.56 ± 14.2 at 1 mg/kg and 28.64 ± 11.9 at 3 mg/kg) and T_1/2 values (

Table 7. Pharmacokinetic parameters of enavogliflozin in mice following single oral administration of enavogliflozin at doses of 1 and 3 mg/kg.

Dose	Tissue	Tmax (h)	T1/2 (h)	AUC∞/Dose (h·μg/g Tissue/Dose)	AUC Ratio
1 mg/kg	plamsa	1.917 ± 1.20	3.462 ± 0.24	1.340 ± 0.26	1.000 ± 0.00
	stomach	1.250 ± 1.37	4.557 ± 0.77	14.60 ± 6.60	11.94 ± 7.25
	small intestine	1.167 ± 1.40	3.055 ± 0.76	11.74 ± 3.77	8.961 ± 2.95
	large intestine	5.000 ± 2.45	5.559 ± 1.57	12.11 ± 3.41	9.461 ± 3.92
	kidney	3.000 ± 1.10	20.78 ± 0.65	92.88 ± 7.02	71.56 ± 14.2
	liver	0.833 ± 0.61	6.627 ± 1.30	8.077 ± 1.14	6.089 ± 0.45
	heart	2.167 ± 0.98	4.804 ± 1.61	2.213 ± 0.34	1.672 ± 0.34
	lung	1.667 ± 1.33	7.090 ± 1.49	2.580 ± 0.28	1.954 ± 0.19
	spleen	2.250 ± 1.47	3.608 ± 0.39	0.353 ± 0.11	0.259 ± 0.04
	testis	5.667 ± 2.66	11.26 ± 1.11	0.767 ± 0.17	0.575 ± 0.09
3 mg/kg	plasma	0.917 ± 0.20	4.186 ± 1.37	1.386±0.43	1.000 ± 0.00
	stomach	0.917 ± 0.58	5.366 ± 0.70	15.76 ± 4.97	12.63 ± 6.50
	small intestine	0.833 ± 0.26	2.974 ± 0.63	12.425 ± 2.92	9.970 ± 4.64
	large intestine	6.333 ± 2.66	4.866 ± 0.64	18.69 ± 8.61	14.79 ± 8.11
	kidney	1.167 ± 0.41 **	17.59 ± 0.48 ***	35.49 ± 1.22 ***	28.64 ± 11.9 ***
	liver	0.917 ± 0.20	5.771 ± 0.53	8.033 ± 1.30	6.214 ± 1.79
	heart	0.917 ± 0.20 *	5.370 ± 5.06	2.407 ± 0.95	1.730 ± 0.33
	lung	0.917 ± 0.20	6.266 ± 0.31	2.354 ± 0.20	1.849 ± 0.60
	spleen	0.917 ± 0.20	3.720 ± 0.85	0.583 ± 0.33	0.431 ± 0.19
	testis	8.000 ± 0.00	11.19 ± 2.00	0.862 ± 0.13	0.704 ± 0.35

Data are expressed as mean ± SD (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the 1 mg/kg dose group by Mann–Whitney U test.

). The AUC ratio, T_max, and T_1/2 of enavogliflozin in the kidneys were significantly different between the doses of 1 mg/kg and 3 mg/kg. However, the AUC ratio, T_max, and T_1/2 of enavogliflozin in the stomach, small intestine, large intestine, and liver were not significantly different between the two doses. The distribution of enavogliflozin in the lungs and heart was slightly higher than that in the plasma, yielding AUC ratios ranging from 1.672 to 1.954 (Figure 5B,E, Table 7). The distribution of enavogliflozin in the testes and spleen was lower than that in the plasma, yielding AUC ratios of about 0.5; however, the elimination profiles from the testes and spleen were different. Specifically, the T_1/2 of enavogliflozin in the testes was longer than that of plasma, but the T_1/2 in the spleen was similar to plasma.

4. Discussion

An analytical method for enavogliflozin was developed in this study using an LC–MS/MS system. Since a liquid–liquid extraction method using MTBE was applied for the extraction of enavogliflozin, we used a deuterium substitute of enavogliflozin as an IS. Consequently, the matrix effect ranged from 96.93% to 103.6%, which indicated no effect of the sample matrix on the ionization of enavogliflozin. Extraction recovery of enavogliflozin was high and reproducible, ranging from 79.75% to 88.79% with a CV of less than 4.000%. The method also demonstrated good linearity over a wide range of 5–3000 ng/mL for enavogliflozin, with a signal-to-noise ratio of >10.0 at the LLOQ. Collectively, the analytical method used in this study is reliable for concentration–time profile studies, showing acceptable accuracy, precision, recovery, matrix effect, and stability.

With a trend of expansion of the therapeutic potency of enavogliflozin to acute or chronic kidney injury, cardiovascular disease, retinopathy, and the anti-obesity effect [22,23,24,25,28,29,30], it is crucial to understand its distribution to target tissues [33]. We previously reported the linear pharmacokinetics of enavogliflozin in mice and rats following single intravenous and oral administration of enavogliflozin at doses of 0.3–3 mg/kg, with oral bioavailability ranging from 84.5% to 97.2% in mice and from 56.3% to 62.1% in rats. However, the pharmacokinetics were obtained from fasted animals [9]. Since therapeutic efficacy is usually investigated following repeated postprandial administration, we investigated the pharmacokinetic characteristics of enavogliflozin, including differences between fasted and fed states and between single and repeated administrations, providing useful information for the evaluation of the new therapeutic efficacy of enavogliflozin.

Plasma concentrations of enavogliflozin did not change regardless of whether it was administered to fed or fasted mice. Additionally, plasma concentrations were not altered by repeated oral administration of enavogliflozin for one or 2 weeks (Figure 3 and Figure 4). The pharmacokinetic differences between single and repeated doses of enavogliflozin within 2 weeks are acceptable, indicating that enavogliflozin does not accumulate in the body after repeated dosing within this time frame in mice. Taken together, these results suggest that plasma concentrations of enavogliflozin can be consistently maintained during repeated drug treatment, and thus the oral BA of enavogliflozin may not be changed under prandial and fasting conditions or under repeated dosing conditions, which provide the information for the dose regimen to evaluate its therapeutic potential in mice. We performed these experiments in male mice because we focused on the pharmacokinetic properties of enavogliflozin in fed and fasted conditions, as well as the pharmacokinetic differences between single and repeated doses. However, for future pharmacology and toxicity assessment, pharmacokinetic studies using male and female mice need to be performed.

We previously reported higher concentrations of enavogliflozin in the kidneys, which could contribute to long-lasting UGE up to 168 h after a single oral dose by inhibiting SGLT2-mediated glucose reabsorption in the renal proximal tubular cells [7,11]. In this study, enavogliflozin showed the highest distribution in the kidneys among the tested tissues and maintained its concentration for more than 48 h (Figure 5), consistent with our previous reports [7]. We also compared dose dependency in the tissue distribution of enavogliflozin using doses of 1 mg/kg and 3 mg/kg, since drug accumulation in tissues can alter therapeutic effects and increase the possibility of side effects. Although we were not able to evaluate it at a wider range of doses, tissue distribution of enavogliflozin exhibited dose proportionality except in the kidneys between 1 and 3 mg/kg PO dose (Table 7). Enavogliflozin levels in the kidneys were decreased at the higher dose (3 mg/kg) compared with 1 mg/kg, and T_max and T_1/2 at 3 mg/kg were significantly shorter than at 1 mg/kg. Considering that enavogliflozin is a substrate for OAT1 (organic anion transporter 1) and OAT3 [7], high enavogliflozin concentrations could cause saturation of these transporters, reducing drug influx into the kidneys at 3 mg/kg. Since these transporters are mainly expressed in the renal proximal tubular cells, enavogliflozin uptake was influenced only in the kidneys without affecting other tissues. The results suggest that enavogliflozin in the kidneys may not show a dose–tissue exposure correlation, whereas other tissues do, which provides important information for dose optimization. The rank order of enavogliflozin distribution was kidneys > stomach > small and large intestine > liver > heart and lungs > plasma > testes > spleen. However, enavogliflozin was not detected in brain samples. Compounds with MW > 400 and high polar surface area (PSA) > 80 Ų and a tendency to form more than six hydrogen bonds are limited in penetrating the blood–brain barrier [34]. Enavogliflozin has an MW of 446.9, a PSA value of 99.4 Ų, and a hydrogen bond number of 10, and contains chloride and several hydroxy substituents (Figure 1) [8]. This may explain its limited distribution to the brain.

SGLT2 is mainly expressed in the renal proximal tubules. It is also localized in pancreatic islet α-cells, prostatic and pancreatic cancer cells, glomerular mesangial cells, and retinal pericytes [25,26]. Ocular SGLT2 transports glucose and sodium to pericytes, and excessive glucose and sodium in pericytes lead to pericyte swelling and loss. Therefore, SGLT2 inhibitors exert therapeutic effects by reducing glucose entry into pericytes, reducing oxidative stress, and restoring insulin signaling [25,26,27]. Based on SGLT2 expression in retinal pericytes and ocular SGLT2 function, enavogliflozin is being developed as a therapeutic for diabetic retinopathy. However, orally administered enavogliflozin did not reach the retina. Therefore, the ocular and plasma pharmacokinetics of enavogliflozin in rabbits following ocular administration of enavogliflozin ophthalmic solution were evaluated. In this study, enavogliflozin administered via the ocular route distributed to the conjunctiva, cornea, and retina (the target site for retinopathy), and retinal enavogliflozin concentrations remained above its IC₅₀ value for SGLT2 for longer than 24 h [25].

Tissue distribution results also suggest that the therapeutic effects of enavogliflozin may be conferred in highly distributed tissues such as the kidneys, intestinal tract, liver, heart, and lungs. Previous research regarding the nephroprotective effects of SGLT2 inhibitors focused on the inhibition of induced AMPK/mTOR pathways as well as the HIF-1α pathway and the inhibition of immune inflammation. This subsequently delayed fibrosis in diabetic kidney disease and glomerular injury in lupus nephritis [20], and the clinical relevance for the treatment of lupus nephritis has been under investigation using dapagliflozin [24]. Regarding the use of SGLT2 inhibitors to treat acute nephropathy, dapagliflozin, canagliflozin, and empagliflozin have been reported to ameliorate renal fibrosis in hyperuricemic nephropathy and cisplatin-and methotrexate-induced acute nephropathy [21,22,35]. The nephroprotective effects of enavogliflozin have not yet been unveiled, but they need to be further investigated based on enavogliflozin’s effects of reducing reactive oxygen species and upregulating endothelial nitric oxide synthase expression, along with improving vascular function [28]. The highest but nonlinear kidney distribution of enavogliflozin in this study should be considered to support pharmacokinetic–response relationships of enavogliflozin in relation to nephroprotective effect [36].

Recent studies have reported these cardioprotective effects regardless of the presence of diabetes [20,28]. The use of SGLT2 inhibitors in T2DM patients with cardiovascular disease has been reported to decrease major cardiovascular events [37]. The FDA expanded the indication of dapagliflozin to include heart failure with reduced ejection fraction (HF-rEF) or heart failure with preserved ejection fraction (HF-pEF) [38]. Patients who started using empagliflozin experienced reduced cardiovascular mortality or hospitalization [39]. Enavogliflozin is also undergoing a clinical trial to investigate the composite of cardiovascular death or hospitalization for HF-pEF (NCT06027307). Enavogliflozin has been reported to improve cardiovascular function of pig coronary artery endothelial cells (PCAECs) by reducing the production of reactive oxygen species and upregulating the expression of endothelial nitric oxide synthase [28]. Establishing a pharmacokinetic–pharmacodynamic correlation between enavogliflozin pharmacokinetics and biomarkers of cardiovascular function and allometry combined in silico physiologically based approach awaits further investigation to extend the in vivo therapeutic efficacy of enavogliflozin regarding the improvement of cardiovascular function [36]. In this context, the study of the pharmacokinetics of enavogliflozin in the fed state and under repeated doses, tissue distribution information, and mode of action may be important.

5. Conclusions

An analytical method for enavogliflozin using an LC-MS/MS analytical method was developed and validated in accord with the FDA guidelines. The method was applied to study the pharmacokinetics of enavogliflozin in mice. Pharmacokinetic characteristics of enavogliflozin were not changed regardless of whether it was administered to fed or fasted mice or whether it was administered once or by repeated oral administration of enavogliflozin for one or 2 weeks. Orally administered enavogliflozin distributed to kidneys, stomach, small and large intestine, liver, heart, and lungs with higher concentrations than plasma enavogliflozin. And tissue distribution of enavogliflozin exhibited dose proportionality except in the kidneys between 1 and 3 mg/kg PO dose. This study provides useful information for dosing regimen decisions to evaluate novel therapeutic efficacies of enavogliflozin, including therapeutics for acute kidney injury and the improvement of cardiovascular function.