Discussion
As a commonly used analgesic in clinics, tramadol is often combined with other drugs, which can easily lead to drug-drug interactions (DDIs). Currently, there are reports showing that tramadol tends to interact with some drugs, such as terbinafine, venlafaxine, cimetidine, ketoconazole, and so on ( KuKanich, KuKanich & Black, 2017 ; Saarikoski et al., 2015 ; Szkutnik-Fiedler et al., 2017 ). However, there are no reports on the interaction between tyrosine kinase inhibitors and tramadol. Considering that both types of drugs are extensively used in clinical settings, it is meaningful to study the interaction between them and the underlying mechanism.

In this study, we evaluated the effect of 23 types of tyrosine kinase inhibitors on the metabolism of tramadol. We found that crizotinib, sorafenib, and regorafenib had a strong inhibitory effect on tramadol, with an inhibition rate of over 80%. Furthermore, we mainly focused on evaluating the inhibitory effect of crizotinib on tramadol metabolism. The IC 50 and K i values showed that crizotinib can strongly inhibit tramadol metabolism in both RLM and HLM. Besides, the Lineweaver-Burk shows that the straight lines intersect in the negative axis of X -axis (based on the α value), indicated that the underlying mechanism may be non-competitive inhibition.

In order to further study the interaction between crizotinib and tramadol, we carried out animal experiments. We found that whether crizotinib was administered as a single dose or multiple doses for 7 days, there was an increase in both the AUC and C max of tramadol, compared to the control group, while the CL decreased to varying degrees. The AUC of O-desmethyl tramadol did not show any significant change, but the C max decreased. This effect was more pronounced when crizotinib was administered for 7 days rather than a single dose. It is believed that the inhibition of tramadol metabolism caused by crizotinib resulted in the improvement of bioavailability and the accumulation of tramadol in the body, leading to an increase in its concentration, while the concentration of its metabolite O-desmethyl tramadol still have a similar level. The results were consistent with those obtained in vitro. However, as O-desmethyl tramadol is an active metabolite with a higher affinity to opioid receptors than tramadol, special attention should be paid to the dosage of tramadol when these two drugs are used in combination in order to avoid severe adverse reactions.

At present, crizotinib has been proven to cause liver damage, especially when continuously administered ( Duarte et al., 2021 ; Tsukita et al., 2015 ; Van Geel et al., 2016 ). Therefore, we conducted an evaluation of morphological and functional changes in certain tissues after crizotinib administration to further explore why crizotinib inhibits the metabolism of tramadol. We performed H&E staining experiments on liver, kidney, and small intestine to assess morphology. The results indicated that crizotinib did not cause any significant changes in morphology. However, it may have an impact on liver function. After oral administration of crizotinib for 7 days, there was an increase in the levels of ALT and AST, indicating that the liver may have suffered some degree of damage. This is consistent with previous reports ( Harada et al., 2021 ; Tsukita et al., 2015 ).

Considering that tramadol and crizotinib are primarily metabolized by CYP enzymes ( Grond & Sablotzki, 2004 ; Han et al., 2014 ; Ng et al., 2022 ; Shaw et al., 2020 ). We investigated whether the activity of CYP2D1 and CYP3A2 would be affected by crizotinib. Various studies have shown that crizotinib inhibits CYP3A enzymes ( Mao et al., 2013 ; O’Bryant et al., 2013 ; Timm & Kolesar, 2013 ; Xu et al., 2015 ), which is consistent with our findings. Furthermore, we observed a significant decrease in the metabolic rate of dextromethorphan, a probe substrate of CYP2D1, when crizotinib was administered for seven days. Therefore, we believe that crizotinib has the potential to inhibit the activity of not only CYP3A2 but also part of the activity of CYP2D1. In addition, we conducted CO quantification experiments to evaluate the expression of CYP enzymes in rat liver microsomes. The results indicated that crizotinib could reduce the overall abundance of CYP. This suggests that crizotinib may inhibit CYP activity by reducing the availability of CYP enzymes. As there are no relevant reports yet, we proposed this idea for the first time. Of course, further studies are required to confirm this.

In summary, this study demonstrates a high potential for drug-drug interactions (DDIs) between tramadol and crizotinib. Concurrent use of these drugs can alter the blood exposure of tramadol and impair liver function, leading to serious adverse reactions. Thus, it is advisable to avoid their simultaneous intake. However, this study also has some limitations: We did not evaluate the impact of tramadol on crizotinib metabolism; Additionally, our study was mainly conducted in male SD rats without further discussing the influence of gender differences, which may also can cause drug metabolism difference. Besides, the species differences between rats and humans is also a privacy worthy of attention. Therefore, further clinical studies are warranted to determine the effects of tramadol and crizotinib interaction.