Results
Tramadol, O-desmethyl tramadol, and IS were detected using UPLC-MS/MS, and the chromatogram is shown in Fig. S1 . The three substances can be well-separated without mutual interference. The ranges of the standard calibration curves for tramadol and O-desmethyl tramadol were 1–1000 ng/mL and 0.1–500 ng/mL, respectively, with correlation coefficients greater than 0.99. To further verify the reliability of the method, we prepared six replicates at low, medium, and high concentrations to assess the accuracy, precision, stability, extraction recovery, and matrix effect of tramadol and O-desmethyl tramadol. The results are shown in Tables S1 – S3 .

The Michaelis–Menten curve for tramadol in RLM and HLM, along with the corresponding K m value, is shown in Fig. S2 . Figure 1A displays the inhibitory effect of 23 tyrosine kinase inhibitors on tramadol metabolism, with crizotinib, sorafenib, and regorafenib exhibiting the highest inhibitory rates of 97.22%, 96.66%, and 83.65%, respectively.

The IC 50 curves and Lineweaver-Burk plots of crizotinib on tramadol metabolism are presented in Figs. 1B and 2 . The IC 50 values were 8.74 ± 0.18 µM and 11.87 ± 0.25 µM in RLM and HLM, respectively. In addition, the Lineweaver-Burk plots indicated that crizotinib may inhibit tramadol metabolism in a non-competitive manner with K i values of 4.17 µM and 14.40 µM in RLM and HLM, respectively.

The mean concentration–time curves of tramadol and O-desmethyl tramadol are shown in Figure 3 , and the corresponding pharmacokinetic parameters are presented in Tables 1 and 2 . After a single dose of crizotinib was administered, the values of AUC (0−t) and AUC (0−∞) increased by 45.64% and 55.72%, respectively, compared to the control group for tramadol. However, there were no significant differences observed in the parameters of O-desmethyl tramadol. Upon administration of crizotinib for 7 days, the values of AUC (0−t) and AUC (0−∞) for tramadol increased by 112.01% and 109.01%, respectively, compared to the control group, while CL z/f decreased by 53.47%. Furthermore, the C max value for O-desmethyl tramadol decreased by 37.77%. All of the data indicate that crizotinib significantly inhibits the metabolism of tramadol, resulting in changes to its pharmacokinetic parameters.

The results of H&E staining were shown in the Figure 4A . Compared with the control group (group E), no obvious changes were found in the cell morphology, size, arrangement, nuclear morphology of liver, kidney and small intestine whether crizotinib was administered in a single dose or for 7 consecutive days, which indicated that crizotinib may not cause pathological changes in these tissues.

As shown in Figure 4B , when crizotinib was administered for a single dose, the level of serum ALT and AST have no significant change. However, when crizotinib was administered for consecutive 7 days, the value of both ALT and AST increased significantly.

Notes:

Group A: Tramadol single-use group; Group B: Tramadol with crizotinib (single dose).

AUC area under the blood concentration–time curve t 1/2z elimination half time T max peak time V z/F apparent volume of distribution CL z/F blood clearance C max maximum blood concentration

area under the blood concentration–time curve

elimination half time

peak time

apparent volume of distribution

blood clearance

maximum blood concentration

Notes:

Group C: Tramadol single-use group; Group D: Tramadol with crizotinib (multiple dose for 7 days).

* P < 0.05, ** P < 0.01, *** P < 0.001 in comparison with the control group.

AUC area under the blood concentration–time curve t 1/2z elimination half time T max peak time V z/F apparent volume of distribution CL z/F blood clearance C max maximum blood concentration

area under the blood concentration–time curve

elimination half time

peak time

apparent volume of distribution

blood clearance

maximum blood concentration

The incubation results of dextromethorphan and midazolam are shown in Figure 5A . When crizotinib was administered, the metabolic rate of dextromethorphan and midazolam decreased to different extents. These results suggest that the activities of CYP2D1 and CYP3A2 in SD rats may be inhibited by crizotinib. Additionally, as seen in Figs. 5B and 5C , the total amount of CYP also decreased upon crizotinib administration.