In vivo assessment of pharmacokinetic interactions of empagliflozin and henagliflozin with sorafenib: an animal-based study

Animals

Healthy adult male Sprague-Dawley (SD) rats, aged 10–12 weeks and weighing 230 ± 30 g, were supplied by Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China; license number SCXK (Jing) 2019-0008). All experimental procedures were reviewed and approved by the Animal Ethics Committee of Hebei General Hospital (Shijiazhuang, China) (No. 202328).

The rats were housed under controlled experimental conditions (23 ± 2 °C, 12-h dark-light cycle, relative humidity of 50 ± 10%) and were acclimatized for a week to affirm their health condition and minimize any influence. All rats were given free access to water, and the diet was prohibited for 12 h before administration.

Materials

Sorafenib (purity ≥ 98%, Lot C15090388) and donafenib (purity 99.9%, ZZS-20-X261-A1) were purchased from Shanghai Zhen Zhun Biotechnology Co., Ltd. (Shanghai, China). Empagliflozin (J19HS174525, ≥98%) was purchased from Shanghai Yuan ye Bio-Technology Co. Ltd. (Shanghai, China). Henagliflozin (purity ≥ 99%) was kindly provided by the Jiangsu Hengrui Medicine Co., Ltd. (Jiangsu, China). The internal standard (IS), dapagliflozin (purity ≥ 99%, Lot K1704045), was acquired from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).

Dimethyl sulfoxide (DMSO) was obtained from Beijing Solarbio Science Technology Co., Ltd. (Beijing, China). HPLC-grade acetonitrile, methyl tert-butyl ether, formic acid, and ammonium acetate were provided by Fisher Scientific (Pittsburgh, PA, USA). All experiments were conducted using ultrapure water, which was supplied by Wahaha Group Co., Ltd. (Hangzhou, China).

Pharmacokinetic study in rats

The experimental animals were randomly divided into seven drug treatment groups (n = 6). The doses were chosen by converting the clinically recommended doses for patients to animal doses. The animal equivalent dose was determined by dose-by-factor method: Human equivalent dose (mg/kg) = Animal does (mg/kg) × Km ratio (Km = 6.2 in rats) (Nair & Jacob, 2016). Sorafenib was suspended in 0.5% sodium carboxymethyl cellulose (CMC-Na) with 5% DMSO, whereas empagliflozin and henagliflozin were suspended in 0.5% CMC-Na. Groups I, Groups IV and Groups VI were administered sorafenib 100 mg/kg, empagliflozin 2.5 mg/kg, or henagliflozin 1 mg/kg by gavage, respectively. Group II received empagliflozin 2.5 mg/kg daily by gavage for 7 consecutive days, followed by sorafenib at 100 mg/kg; whereas Group III received henagliflozin 1 mg/kg daily by gavage for 7 consecutive days, followed by sorafenib at 100 mg/kg by gavage. Groups V and VII received sorafenib (100 mg/kg) daily by gavage for 7 consecutive days, followed by empagliflozin 2.5 mg/kg (Group VI) or henagliflozin 1 mg/kg (Group VII) by gavage (Table 1).

A total of 100 μL of the blood was collected into heparinized test tubes at the following time points: 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 24, 48, 72, 96, 120, and 144 h for sorafenib; 0, 0.083, 0.167, 0.25, 0.33, 0.5, 0.75, 1, 2, 4, 6, 8, 10, 12 h for empagliflozin; and 0, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 24, and 48 h for henagliflozin analysis. Blood samples were centrifuged at 3,500 rpm for 10 min, and the supernatant was collected and stored at −80 °C until processing for UPLC-MS/MS.

Following blood collection, rat liver and intestinal tissues were collected for molecular analysis on the seventh day post-treatment with the corresponding drugs for each group. The rats were anesthetized with chloral hydrate and were euthanized after that. The tissues were stored immediately at −80 °C until use.

Instruments and analytical conditions

The blood concentration of sorafenib was determined using our previously published article regarding the LC-MS/MS method (He et al., 2022). Empagliflozin and henagliflozin were quantified using UPLC-MS/MS with an LC-30A ultra-performance liquid chromatograph (Shimadzu, Kyoto, Japan) connected to a Sciex Triple Quad 5500 tandem triple quadrupole mass spectrometer (Sciex, Framingham, MA, USA). Chromatographic separation was performed on a Welch Boltimate column (2.1 mm × 100 mm, 2.7 μm), with the temperature maintained at 45 °C by gradient elution. The mobile phase composed of water containing 0.1% formic acid, 5 mM ammonium acetate (phase A), and acetonitrile (phase B). The flow rate was 0.25 mL/min and the gradient elution procedure was as follows: 0–1 min, 55% B; 1–2 min, 55–95% B; 2–3.5 min, 95% B; 3.5–3.6 min, 95–55% B; 3.6–4.1 min, 55% B. The injection volume was 6 μL.

The analytes were ionized using positive electrospray ionization and detected using multiple reaction monitoring (MRM). The m/z transitions ions for quantification were from 451.2→70.9 for empagliflozin, 455.3→290.9 for henagliflozin and 426.1→167.2 for dapagliflozin, respectively (Fig. 1). The conditions of the mass spectrometer were given as follows: ion source gas 1, 60.0 psi; ion source gas 2, 50.0 psi; curtain gas, 25.0 psi; source temperature, 500 °C; ion spray voltage, 5,500 V.

Preparation of calibration standards and quality control (QC) samples

Empagliflozin (1 mg/mL), henagliflozin (1 mg/mL), and IS (1 mg/mL) were separately prepared by dissolving them in DMSO. Working solutions for calibration were obtained by diluting the stock solutions with acetonitrile-water. Calibration standards were prepared by spiking 5 μL of the working solution with 45 μL of blank rat plasma. The final concentrations of the calibration curves were 5, 50, 200, 500, 1,000, and 2,000 ng/mL for empagliflozin and 5, 50, 200, 500, 1,000, and 2,000 ng/mL for henagliflozin. QC samples with concentrations of 15, 800, and 1,500 ng/mL for empagliflozin and 10, 800, and 1,500 ng/mL for henagliflozin were prepared independently in the same way. Stock solutions, working solutions, calibration standards, and QCs were stored at −20 °C until analysis.

Plasma sample preparation

The IS working solution (5 µL) was added to 50 µL of plasma sample, followed by the addition of 250 μL methyl tert-butyl ether (MTBE). After vortexing for 1 min, the tubes were centrifugated for 10 min at 12,000 rpm. The supernatant was then transferred to a clean centrifuge tube and evaporated to dryness under a stream of nitrogen at room temperature. The residue was re-dissolved in 100 μL of 50% acetonitrile-water and briefly vortexed for 1 min, after 6 μL was injected into the UPLC-MS/MS for analysis.

Method validation

The method was comprehensively validated according to the guidelines of the M10 Bioanalytical Method Validation and Study Sample Analysis for the US Food and Drug Administration (2022). The following elements were evaluated during the method validation process: selectivity, calibration curve, lower limit of quantification (LLOQ), accuracy, precision, matrix effects, recovery, and stability.

Quantitative real-time PCR (qRT-PCR) analysis

Frozen livers and intestines of rats were isolated using the TRNzol extraction method to extract total RNA, according to the manufacturer’s guidelines. Total RNA solution (2 μL) was taken and analyzed by Bio Tek Epoch (Bio Tek Instruments, Inc, Winooski, VT, USA) to determine the RNA concentration and purity using a 260/280 nm absorbance ratio. RNA samples (1 μg of RNA samples were converted to complementary DNA (cDNA) using the FastKing RT Kit. NADPH was served as an internal reference for normalization. PCR assays were performed using the SLAN-96S Real-time PCR system (Shanghai Hongshi Medical Technology Co., Ltd., Shanghai, China) under the following conditions: initial denaturation at 95 °C for 15 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 32 s. Data analysis was conducted by 2^−∆∆CT method. The PCR primer sequences used for amplification are listed in Table 2.

Statistical analysis

The number of animals (n = 6) was estimated using the resource equation method (Arifin & Zahiruddin, 2017). For or one-way analysis of variance (ANOVA), the minimun n = 10/k + 1 (k = number of groups, and n = number of subjects per group). In this study to compare the pharmacokinetic parameters between two treatment groups (experiment and control), the sample sizes per group are: the minimun n = 10/2 + 1 = 6. Non-compartmental analysis of plasma concentration–time data was performed using DAS 2.1.1 Software (Mathematical Pharmacology Professional Committee of China, Shanghai, China). The pharmacokinetic analysis included the area under the concentration-time curve (AUC), maximum plasma concentration (C_max), time to reach C_max (T_max), elimination half-life (t_1/2), plasma clearance volume per time unit (CL_z/F), apparent volume of distribution (V_z/F), and mean residence time (MRT). Pharmacokinetic variables were expressed as geometric mean values and standard deviation. SPSS software (SPSS Inc., Chicago, IL, USA) was used to statistically analyze the pharmacokinetic parameters. Levene’s test was used to assess the homogeneity of variances between groups, with a p-value threshold of 0.05. When variances were found to be equal (P > 0.05), t-tests were applied. In cases where variances were unequal (P < 0.05), the Satterthwaite correction was applied to adjust for unequal variances. Differences between the parameters which showed significant deviation from normality were tested with the Mann-Whitney U test.

Method development

Liquid–liquid extraction with ethyl acetate was used to process the samples because it provided better extraction than methyl tert-butyl ether. To obtain good peak symmetry, high detection sensitivity, and shortened retention times, we optimized many liquid chromatographic conditions, such as column type and mobile phase. Separation of analytes and the internal standard (IS) was achieved using a Welch Boltimate column (2.1 mm × 100 mm, 2.7 μm) maintained at 45 °C. The aqueous phase (A), containing 5 mM ammonium acetate and 0.1% formic acid, significantly improved chromatographic signals and peak profiles for empagliflozin and henagliflozin. In the organic phase (B), acetonitrile exhibited stronger elution capacity compared to methanol. A gradient elution method was implemented, starting with 55% acetonitrile at a flow rate of 0.25 mL/min, which enhanced detection sensitivity and reduced retention time. Mass spectrometry analysis was conducted in positive and multiple reaction monitoring (MRM) modes, with ion monitoring transitions at m/z 451.2→70.9, 455.3→290.9, and 426.1→167.2, respectively (Fig. 1). Further details on the optimization of mass parameters are provided in Table 3.

Method validation

Selectivity

To assess selectivity, six blank plasma samples from different rats were compared with plasma samples spiked with analytes and IS, as well as plasma samples from rats treated with empagliflozin or henagliflozin. Chromatograms were obtained from an unmodified blank sample, blank plasma spiked with analytes at the LLOQ and IS, and plasma samples from drug-treated rats (Fig. 2). The retention times of analytes in spiked samples were identical to those in drug-treated rat samples, demonstrating no interference from endogenous substances in rat plasma.

Pharmacokinetic interactions

The influence of empagliflozin and henagliflozin on the pharmacokinetics of sorafenib

The mean concentration–time profiles for sorafenib administered alone and in combination with multiple doses of empagliflozin or henagliflozin are shown in Fig. 3. The main pharmacokinetic parameters of sorafenib obtained using non-compartmental methods are summarized in Table 7. After the administration of empagliflozin to rats, the C_max, AUC_0–t and AUC_0–∞ of sorafenib increased by 92% (P = 0.019), 84% (P = 0.037), and 93% (P = 0.043) of that of the sorafenib alone group, respectively. The CL_z/F of sorafenib were significantly decreased by 45% (P = 0.037) compared in control group.

Table 7:

Pharmacokinetic parameters of sorafenib in rats when administered alone and following multiple doses of empagliflozin or henagliflozin (n = 6).

Parameters (Unit)	Sorafenib (100 mg/kg)
	Alone	After empagliflozin	After henagliflozin
AUC0–t (μg/L*h)	20,034.83 ± 9,089.00	36,992.26 ± 14,722.99*	33,531.19 ± 7,464.47*
AUC0–∞ (μg/L*h)	20,648.20 ± 9,598.13	39,936.00 ± 17,942.31*	37,215.92 ± 8,570.39**
Cmax (μg/L)	967.17 ± 175.53	1,859.17 ± 760.53*	1,231.17 ± 285.26
Tmax (h)	4.33 ± 2.07	5.50 ± 0.84	5.67 ± 2.58
t1/2 (h)	18.74 ± 4.85	47.56 ± 32.23	28.58 ± 21.71
CLz (L/h/kg)	5.82 ± 2.59	3.20 ± 2.01*	2.81 ± 0.65*
Vz (L/kg)	93.83 ± 63.20	201.55 ± 161.11	110.19 ± 73.12
MRT0–t (h)	37.22 ± 11.42	28.72 ± 11.50	51.01 ± 11.10
MRT0–∞ (h)	40.46 ± 13.37	39.80 ±2 2.93	65.97 ± 24.79

DOI: 10.7717/peerj.19662/table-7

Notes:

* P < 0.05.

** P < 0.01, compared to sorafenib alone.

Pharmacokinetic parameters are expressed as the mean ± standard deviation.

When coadministered with henagliflozin, the AUC_0–t and AUC_0–∞ of sorafenib significantly increased by 67% (P = 0.035) and 80% (P = 0.042), respectively. Furthermore, the CL_z/F of sorafenib was significantly decreased by 55% (P = 0.037) compared with that in the sorafenib alone group. However, no significant difference was observed among the groups in the following pharmacokinetic parameters of sorafenib: C_max, T_max, t_1/2z, V_z/F, MRT_0–t, and MRT_0–∞. In addition, 96 h after SOR gavage, the pharmacokinetic curves showed a second absorption peak as the concentration of SOR in rats increased again.

The influence of sorafenib on the pharmacokinetics of empagliflozin

The mean plasma concentration–time curves of empagliflozin when administered alone and when coadministered with sorafenib are presented in Fig. 4, and the main pharmacokinetic parameters of empagliflozin are displayed in Table 8. When coadministered with sorafenib, the AUC_0–t and AUC_0–∞ of empagliflozin increased by 44.65% (P = 0.009) and 30.59% (P = 0.044), respectively. The V_z/F of empagliflozin were significantly decreased by 50.68% (P = 0.024) compared to control group. In the group of rats receiving both drugs, C_max was higher than that in the control group, but the difference was not statistically significant.

Table 8:

Pharmacokinetic parameters of empagliflozin in rats after oral administration alone and following multiple doses of sorafenib (n = 6).

Parameters (Unit)	Empagliflozin (2.5 mg/kg)
	Alone	After sorafenib
AUC0–t (μg/L*h)	389.31 ± 76.86	563.12 ± 109.87**
AUC0–∞ (μg/L*h)	448.43 ± 101.99	585.59 ± 104.18*
Cmax (μg/L)	172.50 ± 16.26	231.17 ± 78.51
Tmax (h)	0.28 ± 0.12	0.40 ± 0.17
t1/2 (h)	5.03 ± 2.29	3.14 ± 1.09
CLz (L/h/kg)	5.84 ± 1.45	4.38 ± 0.75
Vz (L/kg)	40.92 ± 16.03	20.18 ± 8.16*
MRT0–t (h)	3.20 ± 0.29	2.86 ± 0.21*
MRT0–∞ (h)	5.42 ± 2.07	3.44 ± 0.52

DOI: 10.7717/peerj.19662/table-8

Notes:

* P < 0.05.

** P < 0.01, compared to empagliflozin alone.

Pharmacokinetic parameters are expressed as the mean ± standard deviation.

The influence of sorafenib on the pharmacokinetics of henagliflozin

The arithmetic means of the plasma concentrations of henagliflozin after oral administration alone or following multiple doses of sorafenib are presented in Fig. 5. The main pharmacokinetic parameters are listed in Table 9. When henagliflozin and sorafenib were administered, the AUC_0–t and AUC_0–∞ of henagliflozin increased by 79.1% (P = 0.002) and 77.7 % (P = 0.002), respectively. The CL_z/F of henagliflozin was significantly decreased by 55% (P = 0.002) compared to control group. There were no significant differences between T_max, t_1/2, V_z/F, MRT_0–t and MRT_0–∞.

Table 9:

Pharmacokinetic parameters of henagliflozin in rats after oral administration alone and following multiple doses of sorafenib (n = 6).

Parameters (Unit)	Henagliflozin (1 mg/kg)
	Alone	After sorafenib
AUC0–t (μg/L*h)	1,924.34 ± 376.44	3,452.28 ± 804.60**
AUC0–∞ (μg/L*h)	1,947.08 ± 367.13	3,460.48 ± 801.20**
Cmax (μg/L)	197.17 ± 36.94	357.67 ± 54.49**
Tmax (h)	3.25 ± 1.67	1.33 ± 1.36
t1/2 (h)	5.11 ± 3.62	5.10 ± 1.31
CLz (L/h/kg)	0.53 ± 0.1	0.30 ± 0.05**
Vz (L/kg)	3.88 ± 2.8	2.25 ± 0.77
MRT0–t (h)	7.84 ± 1.68	9.36 ± 3.63
MRT0–∞ (h)	8.49 ± 2.86	9.49 ± 3.57

DOI: 10.7717/peerj.19662/table-9

Notes:

** P < 0.01, compared to henagliflozin alone.

Pharmacokinetic parameters are expressed as the mean ± standard deviation.

Messenger RNA (mRNA) expression in rat liver and intestines

The relative mRNA expression levels in the liver and intestine of the rats are shown in Fig. 6. After the administration of sorafenib for seven consecutive days, the mRNA expression of Ugt2b7 in the intestine downregulated by 68.3% (P = 0.017); however, it did not significantly affect the expression of Ugt2b7 in the liver (Fig. 6A). The effect of empagliflozin after multiple doses on the mRNA expression of Ugt1a7 in liver decreased by 40.07% (P = 0.023) and demonstrated no significant inhibition in intestine. The mRNA expression of P-gp decreased by 61.19% (P = 0.036) and 22.77% (P = 0.042) in liver and intestine, respectively, and with a depression of 37.43% (P = 0.028) about that of Oatp1b2 (Fig. 6B). When treated with multiple doses of henagliflozin, the results revealed that the mRNA expression levels of P-gp and Oatp1b2 in rat liver were obviously decreased by 27.1% (P = 0.022) and 38.4% (P = 0.019), respectively. In addition, in intestine, the mRNA expression levels of P-gp shown a decreased by 43.2% (P = 0.018, Fig. 6C). However, there was no significant change in the expression of Ugt1a7 in either the liver or the intestine.

Effects of empagliflozin and henagliflozin on sorafenib pharmacokinetics

When coadministration with empagliflozin or henagliflozin, the increased exposure to sorafenib may be due to increased absorption in the intestine and inhibition of hepatic uptake. Sorafenib undergoes hepatic metabolism largely through two pathways: phase I oxidation, which is mediated by CYP3A4, and phase II conjugation, which is mediated by UGT1A9 (Gong et al., 2017). In addition, UGT1A9 is a pseudogene and is not expressed in rats, with its functions being compensated by Ugt1a7 in rats. Therefore, we assess the mRNA expression of Ugt1a7in rat to evaluate its role in the pharmacokinetic interactions (Dong et al., 2018). Similar with dapagliflozin, the PCR results indicated that empagliflozin suppressed the expression of Ugt1a7 in the liver of rats, which may affect the glucuronidation of sorafenib by UGT1A9 and enhance C_max (He et al., 2022). The PCR results showed that the mRNA expression of Ugt1a7 in the liver and intestine was not inhibited by henagliflozin in vivo, which may suggest that the metabolism mediated by UGT1A9 of sorafenib was not suppressed by henagliflozin. Therefore, transporters may have a significant impact on the overall elimination of sorafenib (Fig. 7).

The absorption of sorafenib, a P-gp substrate, may be influenced by this efflux transporter; thus, coadministration with another substrate or inhibitor of P-gp might result in a clinically significant DDI during the absorption process in intestine (Azam et al., 2020). For example, a previous study showed that the coadministration of paracetamol or morphine resulted in higher exposure to sorafenib, mostly due to P-gp (Karbownik et al., 2020a, 2021). In this study, empagliflozin and henagliflozin were substrates of P-gp, which indicates that increased exposure to sorafenib may be due to competition for the P-gp transport pathway. Furthermore, according to the PCR results, the decreased mRNA expression of P-gp in the liver and intestine after the administration of empagliflozin or henagliflozin for seven consecutive days might also be responsible for these changes. Sorafenib is absorbed into liver cells through OATP1B1 transporters. It is then metabolized by CYP3A4 to make sorafenib-N-oxide and conjugated by UGT1A9 to produce sorafenib-glucuronide (Zimmerman et al., 2012). Inhibition of OATP1B1 inhibits the hepatic uptake mechanism and leads to increased levels of sorafenib in the plasma (Vasilyeva et al., 2015). Thus, we speculate that the increased AUC of sorafenib when incubated with empagliflozin or henagliflozin is due to a decrease in hepatic OATP1B1 uptake, which is also consistent with the mRNA expression in vivo. Additionally, empagliflozin, as a substrate of OATP, may compete with sorafenib, which may also be responsible for the increased exposure to sorafenib.

According to FDA guidelines, an inducer or inhibitor of these key enzymes and transporters is typically associated with the toxicity or altered efficacy of a drug that is a substrate of a transporter and is used to assess or predict the risk of toxicity changes in DDI studies (Center for Drug Evaluation and Research (CDER), 2020). Understanding whether a drug is a substrate or inhibitor of these key enzymes and transporters can explain some clinical consequences. In conclusion, the increased systemic exposure of sorafenib may enhance the effective of anti-tumor treatment and fight with drug resistance in cancer cells (Tang et al., 2020). However, on the other hand, the higher C_max could be associated with the severity of such adverse events, such as hand-foot syndrome, alopecia, diarrhoea, hypertension, and hypocalcaemia, sacrificing quality of life for patients (Jian et al., 2020). Therefore, when these drugs are coadministered in clinical practice, doctors and pharmacists should pay more attention to this potential DDI.

Effects of sorafenib on empagliflozin pharmacokinetics

An increase in AUC and decrease in CL_z/F and V_z/F of empagliflozin were observed in rats for coadministration with sorafenib. First, empagliflozin and sorafenib are substrates of both P-gp and OATP, which means that comparison of the same transport pathway may contribute to increased exposure to empagliflozin (Fig. 7). In addition, coadministration of sorafenib with multiple doses increased plasma exposure to empagliflozin, which is consistent with the changes in pharmacokinetic parameters for tapentadol or dapagliflozin due to UGT1A9 when coadministration with sorafenib. Sorafenib is a highly effective inhibitor of UGT1A9 in vitro and in vivo analysis (Ge et al., 2019; He et al., 2022; Miners et al., 2017). Therefore, it is conceivable that sorafenib inhibits empagliflozin metabolism by UGT1A9. Furthermore, multiple doses of sorafenib inhibited the mRNA expression of UGT2B7, which was also associated with increased exposure to empagliflozin. In summary, the observed changes in empagliflozin pharmacokinetics may be due to inhibition of the metabolic pathways of empagliflozin. However, after oral administration of empagliflozin, approximately 60% of the parent drugs is eliminated unchanged in the urine, while each metabolite has a systemic exposure of less than 10% of the total drug-related material, which means that metabolic enzymes may play a relatively weak role in the process. Thus, inhibition of metabolic enzymes contributed to a slight increase in the duration of exposure. This fact may be verified by a continuation of the clinical study. This design is clinically meaningful, which may provide a valuable insight for clinical combination of these drugs and enhancing the safety of combination therapies. The clinical significance of this interaction cannot be overlooked, as the increased bioavailability of empagliflozin may help glycemic control but may also lead to more toxic effects.

Effect of sorafenib on henagliflozin pharmacokinetics

This study showed that exposure to henagliflozin increased significantly after administration of sorafenib for consecutive 7 days, however, no significant change was observed in the t_1/2 of henagliflozin. The AUC and C_max of henagliflozin increased significantly, demonstrating that the bioavailability of orally administered drugs significantly increased in rats. Interestingly, it is obvious that sorafenib showed a larger increase in the bioavailability of henagliflozin than empagliflozin, which may be because metabolic enzymes contribute to a different percentage of drug metabolism. Henagliflozin undergoes glucuronidation by UGT2B4/7, UGT1A9, UGT1A3, and UGT1A6, and the proportion of metabolites is wide; thus, metabolic enzymes play a vital role in metabolism. Thus, the inhibition of henagliflozin metabolism may noticeably affect systemic exposure. A previous study demonstrated that sorafenib increases the AUC and C_max of dapagliflozin, presumably due to UGT1A9-mediated DDIs (He et al., 2022). In contrast to the well-known inhibition of UGT1A, sorafenib decreased UGT2B expression in the intestine, which may decrease drug metabolism, increase drug absorption, and enhance bioavailability. Sorafenib has no effect on UGT2B in the liver; thus, a diminished glucuronidation pathway of sorafenib mediated by UGT1A may also compensate for the enhanced metabolic pathway mediated by UGT2B, causing no significant change in the t_1/2 of henagliflozin (Osborne et al., 2021).

In addition to the effects of metabolic enzymes, protein transporters, including P-gp /ABCB1 and BCRP /ABCG2, may play crucial roles in the absorption of this process. Sorafenib and henagliflozin, substrates of both P-gp and BCRP, may competitively decrease the functional expression of transporters when coadministered, which may also affect the absorption of henagliflozin. Coadministration of these drugs alters the pharmacokinetic profile of each drug due to DDIs, potentially leading to complex consequences, including heightened risk of severe adverse effects, reduced therapeutic efficacy, or challenges related to tolerability. Given the alterations in the AUC, C_max, or CL_z/F of henagliflozin when taken with sorafenib, doctors should be mindful of the possibility of this DDI and monitor complete blood counts closely when sorafenib and henagliflozin are coadministered.

However, it is crucial to address these limitations of this study. First, no experimental animal models for HCC and T2DM have been established to evaluate DDI. It is our understanding that the effect of cancer on hepatic and renal function might downregulate CYPs and UGTs, which may change drug pharmacokinetics, impaire the clearance of drugs (Cvan Trobec et al., 2015; Vasilogianni et al., 2022). Diabetic animals showed that T2DM increases exposure to sorafenib (Karbownik et al., 2020b). Thus, coadministration of these drugs may result in a strong pharmacokinetic-based DDI (Center for Drug Evaluation and Research (CDER), 2020). Futhermore, we acknowledge that further validation of protein expression was not conducted in this study. Additionally, the levels of sorafenib glucuronide were not quantified, and the pharmacokinetic profiles of sorafenib, empagliflozin, and henagliflozin may differ between rats and humans. In summary, the results provide essential information about the DDI potential of investigational drugs and can inform future clinical DDI studies, and by the way, all of these DDI studies in rats should be further evaluated in clinical settings to provide useful information about adjusting dose with concomitant drugs.