Materials and Methods
O-desmethyl tramadol (≥99%, Shanghai Canspec Scientific Instruments Co., Ltd, Shanghai, China); crizotinib, regorafenib and sorafenib (≥98%, Shanghai Canspec Scientific Instruments Co., Ltd, Shanghai, China); 20 other types of tyrosine kinase inhibitors (≥98%, Shanghai Macklin Biochemical Technology Co., Ltd, Shanghai, China); reduced nicotinamide adenine dinucleotide phosphate (NADPH) (≥99%, Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China); phosphate buffered saline (PBS) buffer (Shanghai Beyotime Biology Technology Co., Ltd, Shanghai, China); acetonitrile (ACN), formic acid, and methanol (Sigma-Aldrich, St. Louis, MO, USA); alanine aminotransferase (ALT) and aspartate transaminase (AST) activity assay kits (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China); human liver microsomes (HLM) (Corning Life Sciences Co., Ltd., Jiangsu, China); rat liver microsomes (RLM) were extracted by our team based on the previously reported references ( Simpson, 2010 ).

Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was utilized to measure the concentration of both tramadol and O-desmethyl tramadol. Separation was achieved using a Waters Acquity UPLC BEH C18 column, with dimensions of 2.1 mm × 50 mm and 1.7-µm particle size (Waters Corp., Milford, MA, USA), and quantitation was completed using a Waters XEVO TQD triple quadruple mass spectrometer. The mobile phase was composed of 0.1% formic acid (A) and ACN (B), and the gradient elution was carried out with a flow rate of 0.4 mL/min, following the procedure: 90–10% A (0–1.4 min), 10–90% A (1.4–1.5 min), and 90% A (1.5–2.0 min). Positive mode was used to detect the analytes with the following transitions: m/z 264.2 →58.0, m/z 250.2 →58.2, and m/z 285.0 →154.0 for tramadol, O-desmethyl tramadol, and diazepam (used as an internal standard, IS), respectively. The collision energy was 20 V for tramadol and O-desmethyl tramadol, and 25 V for diazepam. The standard for tramadol and O-desmethyl tramadol were dissolved in a small amount of DMSO (the concentration was 1 mg/mL) then diluted with acetonitrile (the final concentration was 1 ng/mL–1,000 ng/mL). Diazepam was diluted with methanol gradually to 500 ng/mL). All the substance meet the requirements for analysis.

To obtain the Michaelis kinetic parameters of tramadol in RLM and HLM, a 200 µL incubation system was established containing 2 µL RLM or HLM (0.2 mg/mL), 186 µL 1xPBS buffer (pH = 7.4), 2 µL tramadol (10–500 µM), and 10 µL NADPH (1 mM). The mixture was pre-incubated for 5 min without NADPH in a water bath shaking at 37 °C. Then, NADPH was added to initiate the reaction, and the mixture was incubated for another 30 min. The reaction was terminated by adding 400 µL of cold ACN and 20 µL of IS (500 ng/mL). After vortexing for 2 min and centrifuging at 16200 × g for 10 min at 4 °C, the supernatant was removed and subjected to UPLC-MS/MS analysis.

For drug-interaction screening, 100 µM of each drug (1.6 µL) was added to the system, and added PBS to the final volume of 200 uL. Tramadol concentration was set at 60 µM according to the K m .

To determine the half-maximal inhibitory concentration (IC 50 ), the concentration of crizotinib was set at 0.01, 0.1, 1, 10, 25, 50, and 100 µM, while the concentration of tramadol was constant at 60 µM in RLM or 100 µM in HLM (according to K m ). To determine the underlying mechanism of inhibition, the concentration of crizotinib was set at 0, 4, 8, and 16 µM (RLM) and 0, 5, 10, and 20 µM (HLM) according to the IC 50 value, while the concentration of tramadol was set at 15, 30, 60, and 120 µM (RLM) and 25, 50, 100, and 200 µM (HLM) according to the corresponding K m value.

SD male rats (280 ± 15 g) were purchased from the Shanghai Animal Experimental Center. Before the experiment, the rats were kept in the animal room for two weeks with adequate water and food in order to adapt to the new environment. The room temperature was kept at 20–25 °C and the humidity was kept at 50%–65%. The change period of light and dark conditions was 12 h, which simulates the change of day and night. All animal experiments were approved by the Ethics Committee of Wenzhou Medical University (Approval number: xmsq2022-0621).

Generally speaking, only when at least five or more animals are involved in the experiment can the results be convincing. Thus, twenty SD rats were randomly divided into four groups with five animals per group: group A received a single dose of 20 mg/kg of tramadol; group B received a single dose of 45 mg/kg of crizotinib and 20 mg/kg of tramadol; group C received multiple doses of 0.5% carboxymethylcellulose sodium salt (CMC-Na) for 7 days and a single dose of 20 mg/kg of tramadol; and group D received multiple doses of 45 mg/kg of crizotinib for 7 days and a single dose of 20 mg/kg of tramadol. Prior to the experiment, all rats were fasted for 12 h but allowed free access to water. Once the experiment began, 0.5% CMC-Na and crizotinib were administered to groups C and D for 7 days, respectively, then to groups A and B on the last day of the experiment. After 30 min, all rats were orally administered 20 mg/kg of tramadol. Blood samples were collected from the tail vein at 5 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h following tramadol administration. During the period, we paid attention to whether the state of rats were normal and no adverse reactions occurred once every hour (if not, we made records and further judged whether it was necessary to be exclude). The samples were centrifuged at 2400 × g for 10 min to obtain serum. Each 50 µL serum was mixed with 150 µL ACN and 20 µL IS (500 ng/mL). After vortexing for 2 min and centrifugation at 16200 × g for 10 min at 4 °C, the supernatant was obtained, then we used UPLC-MS/MS analysis to detect the concentration of tramadol and O-desmethyl tramadol. After the experiment, all the animals were euthanized by 5% isoflurane inhalation anesthesia.

Nine rats were randomly divided into three groups ( n = 3): group E received 0.5% CMC-Na for 7 days; group F received 45 mg/kg crizotinib for 6 days; and group G received 45 mg/kg of crizotinib for 7 days. On the 7th day, all rats were orally administered the substances and then euthanized after 6.5 h (T max of crizotinib). The blood, liver, kidney, and small intestine were harvested within 10 min and flash-frozen in liquid nitrogen before being transferred to −80 °C for later use.

Additionally, the tissues were fixed in 10% formalin solution and embedded in paraffin. Hematoxylin-eosin (H&E) staining was performed on paraffin sections, which had been dewaxed, dehydrated, and sealed with neutral gum. Pannoramic MIDI was then used to observe the tissues.

Serum was collected after centrifugation of blood sample at 2400 × g for 5 min at 4 °C. The levels of ALT and AST were detected according to the protocol of ALT and AST activity assay kits.

All liver tissue samples were extracted into liver microsomes by homogenization and centrifugation, following the same operational steps as previously reported ( Simpson, 2010 ). A new incubation system was established, which included 2 µL dextromethorphan or midazolam (the probe substrate of CYP2D1 or CYP3A2), 2 uL RLM (0.2 mg/mL) from different groups, 186 µL 1xPBS buffer, and 10 µL NADPH (1 mM). The concentrations of dextromethorphan and midazolam were set at 25 µM and 10 µM, respectively, according to the K m value obtained from group E. The subsequent steps were identical to those in the ‘Microsomal incubation assay’ section. UPLC-MS/MS was used to detect the concentration of dextrorphan and 1-hydroxymidazolam to determine whether the activities of CYP2D1 and CYP3A2 in the liver were affected by crizotinib.

The CO quantitative method was utilized to measure the total quantity of CYP in rat liver microsomes. The microsomes were transferred to a 1.5 mL centrifuge tube and CO gas was introduced for 60 s. Next, sodium dithionite powder was added and mixed thoroughly. After two minutes, the absorbance of the liquid was measured at 450 nm and 490 nm ultraviolet wavelengths using an ultraviolet and visible spectrophotometer to calculate the total amount of CYP.

The Michaelis–Menten, IC 50 , and Lineweaver-Burk plots were generated using GraphPad Prism 6.0 software (GraphPad Software, Boston, MA, USA) with corresponding values for K m , IC 50 , and K i (inhibition constant). The mean plasma concentration–time curve for tramadol and O-desmethyl tramadol was drawn using Origin 8.0, and the corresponding pharmacokinetic parameters were obtained using Drug and Statistics (DAS) software (version 3.0). All data were presented as mean ± SD (standard deviation). Statistical differences among the data were calculated using SPSS 24.0 (SPSS, Chicago, IL, USA), with p < 0.05 considered statistically significant. The excessive deviation of data value was considered for exclusion.