Effect of tubeimoside I on the activity of cytochrome P450 enzymes in human liver microsomes

Abstract This study assessed the effect of tubeimoside I on CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 to reveal the potential of tubeimoside I to induce drug-drug interaction. The evaluation of cytochromes P450 enzyme (CYP) activity was performed in pooled human liver microsomes with probing substrates of CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. Typical inhibitors were employed as positive controls and the effect of 0, 2.5, 5, 10, 25, 50, and 100 μM tubeimoside I was investigated. The activity of CYP2D6, 2E1, and 3A4 was significantly inhibited by tubeimoside I with the IC50 values of 10.34, 11.58, and 9.74 μM, respectively. The inhibition of CYP2D6 and 2E1 was competitive with the Ki value of 5.66 and 5.29 μM, respectively. While the inhibition of CYP3A4 was non-competitive with the Ki value of 4.87 μM. Moreover, the inhibition of CYP3A4 was time-dependent with the KI and Kinact values of 0.635 μM−1 and 0.0373 min−1, respectively. Tubeimoside I served as a competitive inhibitor of CYP2D6 and 2E1 exerting weak inhibition and a non-competitive inhibitor of CYP3A4 exerting moderate inhibition.


Introduction
Rhizoma bolbostemmatis is the dried bulb of Bolbostemma paniculatum (Maxim.)Franquet, which is widely distributed in the North China Plain, loess plateau, and southwest of China (Zhou et al. 2022).According to the Compendium of Materia Medica, R. bolbostemmatis could dissolve knots and has been widely applied in the treatment of mastitis, scrofula, tuberculous cervical lymphadenitis, chronic lymphadenitis, and other diseases (Zhang and Zheng 2019;Wang and Mei 2021).The therapeutic efficiency of traditional Chinese medicine is the combination of co-administrated active ingredients and their processing.With the rapid development of extraction and separation technologies, multiple active ingredients of R. bolbostemmatis have been identified, including flavonoids, coumarins, steroids, triterpenes, and other heterocyclic compounds, where tubeimoside I was identified as a major active ingredient and has been suggested to play critical roles (Cheng et al. 2006).Recently, several studies have demonstrated the significance of tubeimoside I in alleviating myocardial injury, cardiac dysfunction, antitumor, and reversing drug resistance (Ruan et al. 2020;Wang et al. 2020;Cheng et al. 2021;Lv et al. 2021;Tang et al. 2022;Wang CL et al. 2022).
The combination of traditional Chinese medicine and Western medicines is cost-effective and easy to obtain, therefore it has been widely accepted in clinical prevention and therapies, which also improves the risk of drug-drug interaction.Metabolic interaction between co-administrated drugs or compounds is the most common drug-drug interaction.Cytochrome P450 enzymes (CYPs) have been considered the critical meditators for drug-drug interaction responsible for the phase I metabolism of various drugs, especially for the liver-metabolised drugs (Tornio and Backman 2018).For example, Shaoyao-Gancao-Fuzi decoction combined with tofacitinib is suitable for rheumatoid arthritis, but their coadministration could increase the systemic exposure of tofacitinib due to inhibited expression of CYP3A4 (Lin et al. 2022).The combination of phenytoin with thymoquinone could improve the protective effect on status epilepticus and benefit the nerve.However, thymoquinone significantly suppressed the activity of CYP2C9, which might induce adverse interaction with phenytoin (Wang Z et al. 2022).Different CYP enzymes are involved in positive or adverse drug-drug interactions (Gougis et al. 2021).For example, the inhibitory effect of quercetin on CYPs could enhance the anti-breast cancer effect of mycophenolate (Patel et al. 2020).Hesperetin and naringenin could inhibit CYP1A2 and therefore induce the prolonged systemic exposure of rasagiline mesylate during their combination in rats (Pingili et al. 2016).However, whether tubeimoside I affects CYPs activity remains unknown in previous studies, which would limit its co-prescription with other drugs and herbs.
Liver microsomes are optimised incubators that could lower the interference of external factors and could more intuitively indicate the selection of substrates by enzymes (Zhao 2008;Sun et al. 2021).In this study, pooled human liver microsomes were incubated with the probing substrates of CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4.The effects of tubeimoside I on the activity of these enzymes were investigated in this study with the help of HPLC method, aiming to guide the prescription of tubeimoside I sourced herbs.

Human liver microsomes incubation
The study design has been summarised in Figure S1.The incubation system was prepared at 4 � C on the ice and was composed of pooled human liver microsomes (50 donors, HLMs, BD Bioscience, USA), 4 mM MgCl 2 , 100 mM potassium phosphate buffer, 1 mM NADP þ , 10 mM glucose-6-phosphate, 1 U/mL glucose-6-phosphate dehydrogenase, and probing substrates.The effect of tubeimoside I (Figure S2, C 63 H 98 O 29 , putity > 98%, Chengdu Must Biotechnology Co., China) on CYPs was primarily evaluated with a concentration of 100 lM, and typical inhibitors of different CYP enzymes were employed as the positive controls.The concentrations of substrates, HLM proteins, and typical inhibitors are summarised in Table 1 according to previous studies (Lang et al. 2017;Zhang et al. 2017).All substrates and inhibitors for dextromethorphan and quinidine were dissolved in methanol, while dextromethorphan and quinidine were dissolved in water.
The incubation system was pre-incubated at 37 � C for 5 min, and then the NADPH-generating system (1 mM NADP þ , 10 mM glucose-6-phosphate, 1 U/mL of glucose-6phosphate dehydrogenase, and 4 mM MgCl 2 ) was immediately added to initiate the reaction.Acetonitrile was used to terminate the reactions, while the reaction system of CYP2A6 was stopped by trichloroacetic acid.The mixture was centrifugated at 12 000 rpm for 10 min and the supernatant was analysed with HPLC (Agilent 1260 series instrument with DAD and FLD detector, Agilent Technologies, USA) to detect the production of metabolites.The specific detection conditions for each CYP enzymes are summarised in Table S1.

Evaluation of IC50 values
Probing substrates of CYP2D6, 2E1, and 3A4 were further incubated with 0, 2.5, 5, 10, 25, 50, and 100 lM tubeimoside I according to the above incubating conditions.The inhibition results were fitted with non-linear regression analysis using GraphPad Prism 9.0 to obtain the values of IC 50 .

Kinetic fitting
Probing substrates of CYP2D6, 2E1, and 3A4 were incubated with 0, 2.5, 5, 10, 25, 50, and 100 lM tubeimoside I according to the above incubating conditions.The concentrations of substrates were: 10, 20, 40, and 50 lM dextromethorphan for CYP2D6, 25, 50, 200, and 250 lM chlorzoxazone for CYP2E1, and 20, 40, 60, and 100 lM testosterone for CYP3A4.The kinetic models were fitted with the following equations and analysed by the Lineweaver-Burk plots and Dixon plots to obtain the K i : S represents the substrate concentration, I represents tubeimoside I concentration, and K I is the inhibition constant obtained from the non-linear regression analysis.V max (the maximum velocity of the reaction) and K m (the concentration of substrate at half of V max ) were calculated with the Lineweaver-Burk plots with mean values.

Time-dependent assay
The incubation system was prepared as the above description with 20 lM tubeimoside I and 1 mg/mL (protein concentration) HLMs.A 30-min preincubation was performed at 37 � C and then 20 lL aliquot was transferred to another tube mixed with an NADPH-generating system and probe substrates (concentrations were approximate to estimated K m ).The incubation was conducted at 37 � C for 0, 5, 10, 15, and 30 min and terminated by acetonitrile.The mixture was placed on ice and analysed by HPLC after centrifugation.
The time-dependent parameters, K I and K inact , were evaluated with a higher concentration of probe substrates �4fold to estimated Km.The concentration of tubeimoside I was set as 0, 2, 5, 10, 20, and 50 lM incubating for 0, 5, 10, 15, 20, and 30 min according to the above description.

Statistical analysis
All data were presented as mean ± SD and analyzed by SPSS 26.0 and GraphPad Prism 9.0 for difference comparison (p < 0.05).

Tubeimoside I inhibited CYP2D6, 2E1, and 3A4 in a concentration-dependent manner
Compared with negative controls, positive controls significantly inhibited all CYP enzymes, while only CYP2D6, 2E1, and 3A4 were inhibited by 100 lM tubeimoside I (Figure 1(a)).The inhibitory effect of tubeimoside I was weaker than positive controls and was enhanced with increasing concentrations.The IC 50 values of CYP2D6 (Figure 1

Tubeimoside I served as a competitive inhibitor of CYP2D6 and 2E1
To fit the inhibition model of CYP2D6 and 2E1, the incubation systems were performed with several concentrations of tubeimoside I, where the highest concentration was �2-fold of IC 50 values.It was found that the inhibition of CYP2D6 (Figure 2

Tubeimoside I served as a non-competitive and time-dependent inhibitor of CYP3A4
A similar method to the fitting of CYP2D6 and 2E1 inhibition, the inhibition of CYP3A4 was best fitted with the non-competitive inhibition model with a constant value of K m (Figure 3(a)).The K i value of CYP3A4 was 4.87 lM (Figure 3(b)).
CYP3A4 in the presence of 20 lM tubeimoside I was reduced with time, but CYP2D6 and 2E1 showed no significant changes (Figure 4(a)).Furthermore, the time-dependent manner of CYP3A4 inhibition was evaluated with 2, 5, 10, 20, and 50 lM tubeimoside I (Figure 4(b)), and the time-dependent parameters, K I and K inact of CYP3A4 were obtained as 0.635 lM −1 and 0.0373 min −1 , respectively (Figure 4(c)).

Discussion
Although in vivo experiments are the indispensable way to finally evaluate the interaction between different co-administrated drugs, in vitro assessment still plays a critical role in the early research of new drugs.In vitro methods could eliminate the influence and interference of in vivo factors, providing reliable references for overall chemical experiments or in vivo tests.Liver microsomes are a commonly used experimental medium in in vitro studies, especially for evaluating CYP activities.To provide a reference for the clinical prescription of tubeimoside I and its sourced herbs, pooled human liver microsomes were employed in the present study.The activity of CYPs was evaluated through the production of corresponding metabolites using typical substrates with a single metabolic pathway.
In human liver microsomes, tubeimoside I showed a significant inhibitory effect on the activity of CYP2D6, 2E1, and 3A4, and the inhibitory effects were enhanced with increasing concentration.The inhibition behaviour of tubeimoside I on these enzymes was slightly different.The values of IC 50 could indicate the degree of inhibition.According to previous reports, IC 50 < 1 lM indicated a strong inhibition, 1 lM < IC 50 < 10 lM indicated a moderation inhibition, and IC 50 > 10 lM indicated a weak inhibition (Berry et al. 2013;Haupt et al. 2015;Ring et al. 2021).This study observed that the effect of tubeimoside I on CYP2D6 and 2E1 was a weak inhibition, but the inhibition of CYP3A4 was moderate.
Meanwhile, the inhibition of CYP2D6 and 2E1 was best fitted with the competitive model, and the inhibition of CYP3A4 was non-competitive.Additionally, IC 50 values also provide a reference for the prescription dosage of tubeimoside I to avoid adverse interactions with other herbs or drugs.
Although CYP2D6 accounts for a small percentage of liver CYPs in humans, it was involved in the metabolism of various clinical drugs (Waring 2020;Machalz et al. 2021).Differentially, CYP2E1 does not metabolise a large number of clinical drugs, but it is the secondly abundant CYP enzymes in HLMs after CYP3A4 (Achour et al. 2014).For instance, CYP2D6 was involved in the biotransformation of antidepressants, antiarrhythmic drugs, antipsychotics, b-blockers, and analgesics (van der Lee et al. 2021).The genetic polymorphism of CYP2D6 was demonstrated to regulate the activity of CYP2D6 and induced the increasing systemic exposure of dacomitinib and trazodone during their co-administration (Han et al. 2022).As a typical substrate of CYP2D6, the pharmacokinetics of metoprolol were significantly influenced by paroxetine and fluoxetine, which inhibits CYP2D6 activity (Bahar et al. 2018).The competitive inhibition of CYP2D6 and 2E1 by tubeimoside I might result from its similar structure and functional groups with the substrates of these two enzymes, such as aromatic nucleus.Although the inhibition of CYP2D6 and 2E1 by tubeimoside I is a weak inhibition, it still implies the potential of tubeimoside I affecting the pharmacokinetics of CYP2D6-or 2E1-metabolised drugs, especially during their co-prescription.
CYP3A is one of the most critical families of CYPs, which participates in the metabolism of over 70% of clinical drugs.CYP3A4 accounts for the largest percentage of the CYP3A family, which has been identified in the metabolism of over 30% of clinical drugs.Therefore, the activity of CYP3A4 in the presence of different compounds has become a research hot point (Zhou 2008).CYP3A4 has also been suggested to lead to interaction between co-administrated drugs in previous studies (Gougis et al. 2021).For example, the relative clearance rate of the CYP3A4 enzyme was revealed to be affected by the genetic polymorphism and nimodipine administration via a competitive and non-competitive mixed mechanism, which causes the reduced systemic exposure of istradefylline in rats (Hu et al. 2022).The moderate inhibition of CYP3A4 by tubeimoside I observed in the present study implies its potential inducing interaction with CYP3A4metabolised drugs.Also, the changes in CYP3A4 activity have not always induced drug-drug interactions.For instance, the inhibited CYP3A4 did not predict the interaction between tacrolimus and itraconazole (Vanhove et al.Taken together, tubeimoside I exerted a weak and competitively inhibitory effect on CYP2D6 and 2E1 and showed a moderate, non-competitive, and time-dependent inhibition on CYP3A4.The dosage of drugs metabolised by these CYP enzymes should be adjusted when co-prescribed with tubeimoside I or its sourced herbs.However, this study investigated the effect of tubeimoside I as an inhibitor.Whether tubeimoside I has the potential to induce CYPs activity has not been disclosed in the present study, which needs further exploration.Supersomes of CYP enzymes could provide more microcosmic and detailed insights into the changes of CYP enzymes in the presence of tubeimoside I, which could help explain the mechanism underlying the effect of tubeimoside I. Therefore, future studies could employ supersomes of CYP3A4, 2D6, and 2E1 to deeply understand the risk of tubeimoside I inducing adverse interactions. (b)), 2E1 (Figure 1(c)), and 3A4 (Figure 1(d)) were 10.34, 11.58, and 9.74 lM, respectively.
(a)) and 2E1(Figure 2(b)) was best fitted with the competitive inhibition model with a constant V max .The K i values of CYP2D6 and 2E1 were 5.66 and 5.29 lM, respectively.

Figure 2 .
Figure 2. The inhibition of CYP2D6 (a) and CYP2E1 (b) by tubeimoside I was fitted with the competitive model by the Lineweaver Burk plots.The Ki values were 5.66 and 5.29 lM, respectively.

Figure 3 .
Figure 3.The inhibition of CYP3A4 by tubeimoside I was fitted with the non-competitive model by the Lineweaver Burk plots (a).The Ki value was 4.87 lM (b).

Table 1 .
Reaction conditions of CYP isoforms tested, including maker reactions, incubation conditions, K m , and inhibitors used in the cocktail assay.