Inhibitory effect of resveratrol on famitinib metabolism in vitro and in vivo

2.1. Chemicals and reagents

Famitinib and N-desethylfaminitib (SHR116637) were sourced from Jiangsu Hengrui Pharmaceuticals Co., Ltd. (Jiangsu, China). Almonertinib (internal standard, IS, Figure 1c), 52 traditional Chinese medicines, and 75 additional pharmaceutical agents were procured from Shanghai Canspec Scientific Instruments Co., Ltd. (Shanghai, China). Detailed information on drugs as potential inhibitors can be found in the Table S1. Purity was ≥ 98% for each drug in this study. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Rat liver microsomes (RLM) were prepared in our laboratory. Human liver microsomes (HLM) were purchased from iPhase Pharmaceutical Services Co., Ltd. (Jiangsu, China). Ultrapure water was produced utilizing the Milli-Q water purification system (Millipore, Bedford, USA). Methanol and acetonitrile were provided by Merck Company (Darmstadt, Germany). Analytical grade or higher chemical solvents were used in the experiment.

2.2. Instrumentations and analytical conditions

The concentrations of famitinib and SHR116637 in the samples were accurately quantified by UPLC-MS/MS analysis. A Waters Acquity UPLC BEH C18 column (2.1 mm × 50 mm, particle size 1.7 μm) was used in the chromatographic system for separation, with the column temperature maintained at 40°C. The mobile phase, composed of 0.1% formic acid (mobile phase A) and acetonitrile (mobile phase B), was eluted at a gradient flow rate of 0.4 mL/min for a duration of 2.0 min. Quantification was conducted utilizing a Waters Xevo TQS triple quadrupole mass spectrometer (Milford, MA, USA) in positive ion mode with multiple reaction monitoring (MRM). The quantitative ion pairs and associated parameters for famitinib, SHR116637, and IS have been listed in Table 1. The mass transitions were m/z 411.14 → 338.38 for famitinib, m/z 383.11 → 338.06 for SHR116637, and m/z 526.01 → 72.04 for Almonertinib (IS).

2.3. RLM preparation

The RLM preparation was executed following the protocol by previous study with appropriate modifications [22]. The preparation procedure was outlined below. Livers from six Sprague-Dawley rats were weighed and subsequently added to 0.1 M ice-cold potassium phosphate buffer (PBS), which also contained 0.25 mM sucrose. The livers were then crushed into homogenates. Subsequently, the homogenate was centrifuged at 11,000 rpm for 15 mins, and then the supernatant was transferred sequentially into freshly prepared centrifuge tubes. The centrifugation operation was repeated, and the newly obtained supernatant was transferred to a centrifuge tube. Finally, the supernatant was centrifuged at 75,600 × g for 2 h at 4°C. At the end of centrifugation, the supernatant was discarded, and the precipitate was resuspended by adding PBS, while on ice, to form a homogeneous solution. Protein concentration was ascertained by employing the Pierce BCA Protein Assay Kits (Thermo Scientific, Waltham, MA, USA).

2.4. The construction of the enzyme culture system and determination of Michaelis-Menten constant (K_m)

The enzyme culture system has been constructed in previous studies [23]. K_m was determined by utilizing the generation rate of SHR116637 as an indicator. The culture system (200 μL) culture system contained famitinib, PBS (0.1 M), NADPH (1 mM), and RLM/HLM (0.3 mg/mL). The concentrations of famitinib were 1, 2, 5, 10, 20, 50, 100, and 200 μM in RLM and 1, 10, 100, 150, 200, 250, 300, 350, and 400 μM in HLM. Following an initial pre-culture for 5 mins at 37°C and the addition of NADPH, the enzyme reaction was initiated. After 30 mins, the reaction was stopped by placing the culture system at -80°C. At the end of the reaction, the culture system was added with 400 μL of protein precipitant (acetonitrile) and 20 μL of IS (200 ng/mL) working solution. Subsequently, the mixture was vortexed for 2 mins and then subjected to centrifugation at 13,000 rpm for 10 mins at 4°C. Ultimately, 100 μL of the supernatant was analysed by UPLC-MS/MS. In the in vitro experiments, the initiation and termination of reactions, as well as sample handling and analysis procedures, were consistent with this section.

2.5. Assessing the likelihood of potential DDI in RLM

The generation of the metabolite SHR116637 was employed as a means of investigating the effects of the inhibitors on the metabolic process of famitinib. Relative activity is the ratio of SHR116637 production in the presence versus absence of the inhibitor. The 200 μL culture system comprised NADPH (1 mM), famitinib, inhibitor (100 μM), RLM (0.3 mg/mL), and PBS (0.1 M). The concentration of famitinib was determined based on its K_m in RLM. A total of 52 traditional Chinese medicines and 75 additional pharmaceutical agents deemed to possess potential DDI capabilities were subjected to the screening experiments, the details of which have been provided in Table S1.

2.6. Study on the inhibitory mechanism of resveratrol on famitinib in vitro

The half-maximal inhibitory concentration (IC₅₀) of resveratrol on the metabolism of famitinib was determined from a 200 μL culture system that included NADPH (1 mM), famitinib, resveratrol, PBS (0.1 M), and RLM/HLM (0.3 mg/mL). The gradient concentrations of resveratrol were 0, 0.01, 0.1, 1, 10, 25, 50, and 100 μM. The concentration of famitinib was determined by the K_m in RLM and HLM, respectively.

The inhibition mechanism of famitinib by resveratrol was determined by a 200 μL culture system consisting of NADPH (1 mM), famitinib, resveratrol, PBS (0.1 M), and RLM or HLM (0.3 mg/mL). The concentration of famitinib was based on K_m, and the concentration of resveratrol was based on IC₅₀. In RLM, the concentrations of resveratrol were 0, 2.96, 5.92, and 11.83 μM, and the concentrations of famitinib were 2.93, 5.86, 11.71, and 23.42 μM. In HLM, the concentrations of resveratrol were 0, 4.03, 8.05 and 10.06 μM, while the concentrations of famitinib were 66.00, 99.00, 132.00, and 165.00 μM.

2.7. Effect of resveratrol on famitinib metabolism in vivo

The Animal Experimental Center of the First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China) (Approval No. WYYY-IACUC-AEC-2024-044, dated 2024-05-07) provided 12 male Sprague-Dawley rats weighing 200 ± 20 g. The rats were randomly assigned to two groups: the control group (famitinib alone) and the experimental group (famitinib and resveratrol). Prior to the initiation of the experiment, the rats were deprived of food for a period of 12 h, with unrestricted access to water. Famitinib and resveratrol were suspended in corn oil for oral administration. The experimental and control groups were given equal volumes of 50 mg/kg resveratrol and corn oil, respectively. Half an hour later, all rats received 1.5 mg/kg famitinib via gavage. After administering famitinib, 0.3 mL of blood from the tail vein was collected at 1, 2, 4, 6, 8, 12, 24, 48, and 72 hrs. The collected blood samples were promptly processed by centrifugation at 8,000 rpm for 10 mins at 4°C. This process allowed for the separation of the supernatant. The supernatant (100 µL) was subsequently transferred to a new centrifuge tube and stored at -80°C. Following the complete thawing of the sample at room temperature, 10 μL of IS (200 ng/mL) working solution and 300 μL of acetonitrile were added and thoroughly vortexed to ensure complete protein precipitation. Subsequently, the sample was subjected to centrifugation at 13,000 rpm for 10 mins at 4°C. Finally, 100 μL supernatant was obtained for UPLC-MS/MS analysis.

2.8. Statistics

The GraphPad 9.0 software generated various pharmacological curves, including those of K_m curve, the IC₅₀ curve, the Lineweaver-Burk plot, and the mean plasma concentration-time curve. Non-compartmental mode of DAS 3.0 (BontzInc., Beijing, China) was employed to obtain pharmacokinetic parameters of famitinib and SHR116637. A student’s t-test was employed in SPSS (version 27.0; SPSS Inc., Chicago, IL, USA) to conduct statistical comparisons within groups. A p-value of less than 0.05 was considered statistically significant.

3.1. Determination of famitinib and SHR116637 by UPLC-MS/MS

Xie et al. reported the presence of two peaks in the ion chromatograms of famitinib and SHR116637 by LC-MS/MS and identified the latter peak as either famitinib or SHR116637 [2]. In the UPLC-MS/MS analysis, we utilized the latter peak for the quantification of both famitinib and SHR116637. The separation of all analytes in the assay was achieved without the interference of endogenous substances. A standard calibration curve was established with a correlation coefficient greater than 0.99, and a range of 0.1 to 20 ng/mL for both famitinib and SHR116637 was constructed.

3.2. DDI study of famitinib in RLM

Figure 2 demonstrates the K_m of 11.71 and 132.00 μM for famitinib in RLM and HLM, respectively. Relative activity was used to compare the effects of these drugs with potential DDI on the metabolism of famitinib. Drugs with relative activity < 20% are considered to have strong metabolic inhibition and have potential for further study. Figure 3(a) and Figure 3(b), respectively, illustrate the distribution of inhibitory effects exerted by 52 traditional Chinese medicines and 75 additional pharmaceutical agents on famitinib metabolism. Figure 3(c) and Figure 3(d) showe the 5 traditional Chinese medicines and 14 additional pharmaceutical agents with relative activities < 20%, respectively. A comprehensive presentation of the relative activity of all drugs has been presented in Table S1.

Close

Figure 2.

Michaelis-Menten curves of famitinib in (a) RLM and (b) HLM. (n = 3, data are presented as mean ± SD).

Export to PPT

Close

Figure 3.

Comparison of the inhibitory effects of different drugs (100 μM) on the metabolism of famitinib in RLM. (a) Scatter plots of relative activities of 52 traditional Chinese medicines and (b) 75 additional pharmaceutical agents. (c) Histogram of relative activity < 20% in traditional Chinese medicines and (d) additional pharmaceutical agents. The X-axis indicates different drugs. The red stars and bars are markers for resveratrol and were used for further research Relative activity is the ratio of metabolite production in the presence versus absence of the inhibitor. (n = 2, data are presented as mean ± SD).

Export to PPT

3.3. Mechanistic study of resveratrol inhibition on famitinib metabolism in vitro

Table 2 provides a listing of the IC₅₀ and inhibitory mechanism of resveratrol against famitinib. The IC₅₀ curves and Lineweaver-Burk plot of resveratrol against famitinib have been displayed in Figures 4 and 5, respectively. The IC₅₀ of resveratrol on famitinib was found to be 11.83 μM in RLM and 8.05 μM in HLM, respectively. The mechanism of inhibition in RLM was un-competitive inhibition with αK_i of 1.13 μM. However, the mechanism of inhibition in HLM was mixed-type, consisting of un-competitive inhibition and non-competitive inhibition, having a K_i of 21.63 μM and an αK_i of 5.07 μM.

Close

Figure 4.

IC₅₀ curves of resveratrol on famitinib metabolism in (a) RLM and (b) HLM. n = 3, data are presented as mean ± SD.

Export to PPT

Close

Figure 5.

(i) Lineweaver-burk plot and (ii) secondary diagram of αK_i inhibiting famitinib metabolism at different concentrations of resveratrol in (a) RLM. (i) Lineweaver-burk plot, (ii) secondary diagram of K_i and (iii) secondary diagram of αK_i inhibiting famitinib metabolism at different concentrations of resveratrol in (b) HLM. (n = 3, data are presented as mean ± SD).

Export to PPT

3.4. Effect of resveratrol on the pharmacokinetic parameters of famitinib in vivo

The pharmacokinetic parameters of famitinib and SHR116637 have been presented in Tables 3 and 4, respectively. Figure 6 illustrates the mean plasma concentration-time curves of famitinib and SHR116637 in Sprague-Dawley rats. The experimental results demonstrated that resveratrol increased the AUC_(0-t), AUC_(0-∞), MRT_(0-t), MRT_(0-∞), t_1/2, T_max, and C_max of famitinib by 1.03-, 1.21-, 0.48-, 0.91-, 0.96-, 2.15-, and 0.62-fold, respectively, while CL_z/F was decreased by 56.4%. In addition, resveratrol prolonged the MRT_(0-t), MRT_(0-∞) and T_max of SHR116637 by 0.35-, 0.81-, and 1.86-fold, respectively, while there was no significant effect observed on other pharmacokinetic parameters. The findings indicated that resveratrol elevated plasma exposure of famitinib in Sprague-Dawley rats, with the potential to increase the risk of adverse effects.

Close

Figure 6.

Mean plasma concentration–time curves of (a) famitinib and (b) its metabolite SHR116637 in Sprague-Dawley rats.

Export to PPT

3.5. Discussion

CYP450 plays a central role in drug metabolism, by which the majority of clinically utilized drugs undergo metabolic processing [24]. Concomitant administration of multiple medications may result in inhibition of CYP450, leading to elevated levels of drug exposure and an increased risk of clinical adverse events [25]. Cancer patients typically require a plethora of medications to treat the myriad of symptoms that frequently accompany their illness [26]. A substantial number of pharmaceutical agents utilized in cancer treatment have been shown to induce elevated plasma concentrations of the drug due to CYP450 inhibition when employed in conjunction with other pharmaceutical agents [27]. This phenomenon has the capacity to precipitate deleterious consequences, an augmented financial burden, and a diminution in the overall health-related quality of the patient’s life.

Famitinib has emerged as a promising TKI for treating GIST, kidney cancer, and lung cancer. Previous studies have reported dose-limiting toxicity associated with the ingestion of high doses of famitinib [28]. The toxicity of the drug is associated with excessive drug exposure. The metabolism of famitinib is impacted by CYP450 inhibition. One study reported that itraconazole, a CYP3A inhibitor, significantly increased the AUC_(0-t) and C_max for famitinib by approximately 77.7% and 40.6%, respectively, and prolonged t_1/2 to 48.24 h [5]. There are fewer studies on DDI of famitinib. Therefore, further studies are necessary to reduce the risk of adverse effects due to DDI.

The initial screening process involved the evaluation of 52 traditional Chinese medicines and 75 additional pharmaceutical agents for their inhibitory effects. These drugs may be used in combination therapy for cancer patients to enhance anticancer effects or improve patient quality of life. For example, resveratrol has cardiovascular protective effects, anti-inflammatory, antioxidant, and anticancer capabilities, with great potential for combination use [15]. However, the ability of these drugs to inhibit famitinib metabolism is unclear. Thus, it is necessary to study the possibility of a potential DDI. Our results showed that 5 traditional Chinese medicines and 14 additional pharmaceutical agents exhibited relative activities below 20%, suggesting that these drugs had a greater ability to inhibit famitinib metabolism and had a greater propensity for inducing potential DDI. To our knowledge, heterocyclic nitrogen atoms in the structures of the antifungal drugs ketoconazole, isavuconazole, fluconazole, and posaconazole inhibit the enzyme activity by directly binding to iron atoms in CYP3A4 hemoglobin and are important factors influencing the metabolism of famitinib. This structural feature may be an important factor influencing CYP3A4. Among 52 traditional Chinese medicines, the relative activities of honokiol, myricetin, and resveratrol were 8.34%, 12.09%, and 18.18%, respectively. Given the strong inhibitory capacity of resveratrol in the screening experiments and its broad antitumor effects with potential for clinical combination use, further studies were conducted to elucidate its effect on famitinib metabolism.

The biological activities of resveratrol have been demonstrated to include anti-cancer, anti-inflammatory, cardiovascular protective, anti-diabetic, and neuroprotective effects [29]. A substantial body of current literature indicates that the inhibition of resveratrol on CYP450 results in increased plasma drug concentrations of co-administered other drugs. Resveratrol has been reported to significantly increase AUC, C_max, and t_1/2, while also markedly decreasing CL_z/F of carbamazepine [30]. Another study in healthy human volunteers showed that resveratrol caused significant increases in AUC, C_max, and t_1/2 was accompanied by a pronounced reduction in CL_z/F for diclofenac [31]. It was proposed that the inhibition of CYP3A4 and CYP2C9 in healthy subjects by resveratrol was the underlying cause of the observed metabolic inhibition of carbamazepine and diclofenac. Current evidence suggested that resveratrol may affect drug metabolism by inhibiting CYP450, which may result in an increased risk of adverse effects. Additionally, there is currently a lack of relevant studies that examine the impact of resveratrol on famitinib metabolism. Thus, it is imperative that further studies be conducted to elucidate the DDI.

In vitro findings demonstrated that resveratrol impeded the metabolic conversion of famitinib. The IC₅₀ for resveratrol was 11.83 μM in RLM and 8.05 μM in HLM, indicating its inhibitory effect on famitinib metabolism. Furthermore, the inhibitory effect of resveratrol on famitinib in RLM and HLM were found to be un-competitive inhibition and mixed-type inhibition (un-competitive and non-competitive inhibition), respectively. Differences in CYP450 caused by inter-species differences are the main reason for the different inhibitory mechanisms of RLM and HLM. CYP3A is predominantly CYP3A4/5 in human liver, whereas in rat liver, it is CYP3A1/2 [32]. The different enzyme catalytic activities of RLM and HLM may also be responsible for the different IC₅₀ and inhibition mechanisms [32]. In addition, resveratrol seems to inhibit different drugs by different mechanisms. Previous studies reported that resveratrol inhibited the metabolism of tofacitinib in RLM and HLM; however, the mechanism of inhibition was non-competitive inhibition [21]. The reasons for this discrepancy are unclear and require further research. Notably, resveratrol also has an inhibitory metabolic effect on erlotinib, another TKI. Therefore, resveratrol may also have inhibitory effects on other TKIs, and in-depth studies need to be conducted in the future [33]. The results of the in vivo study indicated a notable escalation in AUC, MRT, C_max, T_max, and t_1/2, and a significant reduction in CL_z/F for famitinib when combined with resveratrol. This may be attributed to the inhibitory effect of resveratrol on CYP3A4, which affects the metabolism of famitinib [19, 20]. The inhibition of famitinib metabolism by resveratrol may be responsible for significant alterations in the pharmacokinetics parameters of famitinib. Tofacitinib metabolism also undergoes CYP3A4, and the results are consistent with famitinib, which is inhibited by resveratrol [21]. Moreover, an obvious increase in T_max of the metabolite SHR116637 occurred in combination with resveratrol. Famitinib is metabolized into SHR116637 by CYP3A4, and the significant change in T_max may be due to the inhibition of SHR116637 metabolism by resveratrol. These significant alterations in pharmacokinetic parameters suggested that increased plasma exposure of famitinib occurs in vivo and that resveratrol may cause famitinib to lead to more serious adverse reactions. The rat’s CYP450 is similar to that of humans and is a plausible pharmacokinetic model. However, given the interspecies differences, further studies must be conducted in humans [34].

This study has identified the potential DDI when famitinib is combined with traditional Chinese medicines and additional pharmaceutical agents. The results of the study suggest that concomitant administration of famitinib and resveratrol carries a greater risk of DDI and increases the likelihood of adverse reactions. If concomitant medication cannot be avoided, clinicians should closely monitor drug concentrations and adverse effects and adjust drug dosages if necessary.