Physcion inhibition of CYP2C9, 2D6 and 3A4 in human liver microsomes

Abstract

Context

The effect of the active ingredients in traditional Chinese medicines on the activity of cytochrome P450 enzymes (CYP450s) is a critical factor that should be considered in TCM prescriptions. Physcion, the major active ingredient of Rheum spp. (Polygonaceae), possesses wide pharmacological activities.

Objectives

The effect of physcion on CYP450 activity was investigated to provide a theoretical basis for use.

Materials and methods

The experiments were conducted in pooled human liver microsomes (HLMs). The activity of CYP450 isoforms was evaluated with corresponding substrates and probe reactions. Blank HLMs were set as negative controls, and typical inhibitors were employed as positive controls. The inhibition model was fitted with Lineweaver Burk plots. The concentration (0, 2.5, 5, 10, 25, 50 and 100 μM physcion) and time-dependent (0, 5, 10, 15 and 30 min) effects of physcion were also assessed.

Results

Physcion suppressed CYP2C9, 2D6 and 3A4 in a concentration-dependent manner with IC₅₀ values of 7.44, 17.84 and 13.50 μM, respectively. The inhibition of CYP2C9 and 2D6 was competitive with the K_i values of 3.69 and 8.66 μM, respectively. The inhibition of CYP3A4 was non-competitive with a K_i value of 6.70 μM. Additionally, only the inhibition of CYP3A4 was time-dependent with the K_I and K_inact parameters of 3.10 μM⁻¹ and 0.049 min⁻¹, respectively.

Conclusions

The inhibition of CYP450s by physcion should be considered in its clinical prescription, and the study design can be employed to evaluate the interaction of CYP450s with other herbs.

Keywords:

• Rhubarb
• CYP450 isoforms
• competitive inhibition
• non-competitive inhibition
• time-dependent

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Correction

Introduction

Cytochrome P450 enzymes (CYP450s) are critical phase I enzymes in biotransformation and play vital roles in the metabolism of drugs, pollutions, carcinogens and endogenous substrates (Manikandan and Nagini 2018). CYP450s mainly exist in the human liver mediating the introduction of polar functional groups into drugs and promoting phase II reactions during drug metabolism (Kampe et al. 2014; Zimmerman et al. 2018). Due to their genetic diversity, wide substrate specificity, and catalytic versatility, CYP450s are responsible for about 3/4 of phase I-dependent drug metabolism. The CYP450 family includes a large number of members, and the CYP1, CYP2 and CYP3 families are most common in human liver microsomes (HLMs). The activity of CYP450s is one of the most susceptible factors during drug co-administration, which would induce drug-drug interaction and further affect drug efficacy (Lynch and Price 2007; Feng and He 2013). Drugs can be metabolized by a single CYP450 enzyme, or by several enzymes; inducers or inhibitors of CYP450s could also influence the activity of one or more CYP450 isoforms. Recently, the rapid development of traditional Chinese medicine, the use of Chinese patent medicine, and the widely accepted combination of traditional Chinese and western medicine, increases the occurrence of drug-drug interactions (Lu et al. 2015). Therefore, the effect of traditional Chinese medicine, Western medicine and their major active ingredients on the activity of CYP450s has attracted special attention.

In the treatment of endocrine diseases, traditional Chinese medicine has been widely used. Rheum officinale Baill. (Polygonaceae) has various pharmacological activities, such as anticancer, regulating lipids metabolism and alleviating liver injury (Huang Q et al. 2007; Zhuang et al. 2020; Huang H et al. 2021; Gao and Nan 2022). Rhubarb possesses a number of active ingredients, such as anthraquinone, anthrone, organic acid, tannin and polysaccharides. Physcion (Figure 1) is an anthraquinone compound that widely exists in the Polygonaceae family. Previous studies demonstrated that physcion could alleviate brain and nerve injury induced by ischemia and hypoxia and possessed the activities of antibacteria, anti-inflammation and antioxidants (Adnan et al. 2021; Dong et al. 2021; Ji et al. 2021; Qi et al. 2022). In endocrine diseases, physcion could prevent ethanol-induced liver injury, reduce lipid accumulation and prevent obesity, implying its potential in the therapy of fatty liver (Lee et al. 2019; Yao et al. 2020). Decocting is the major form of administration for traditional Chinese medicine, therefore investigating the influence of herb extracts on CYP450 activity is of great significance. The effect of physcion on CYP450s activity is an important factor that should be considered during the prescription of physcion-containing or sourced herbs.

Figure 1. Chemical structure of physcion.

The influence of physcion on the activity of major CYP450 isoforms was investigated in this study in HLMs. The kinetics and models of CYP450 inhibitions were also disclosed, aiming to reveal more environmental risk factors and provide theoretical reference for the clinical application of physcion and its sourced herbs, especially for their co-administration.

Materials and methods

Study design

Pooled HLMs were obtained from BD Gentest Co. (Franklin Lakes, NJ) from 50 donors with the protein concentration of 5 mg/mL. The activity of CYP450 isoforms was evaluated with corresponding probe reactions and specific substrates summarized in Table 1 (Mori et al. 2009; Dinger et al. 2014; Carrão et al. 2018). The HLMs were divided into negative control, positive control and physcion groups. The negative control group was the blank treatment incubated without inhibitors, while the positive control and physcion groups were treated with typical inhibitors of CYP450 isoforms and physcion, respectively. The concentration of inhibitors of different isoforms is listed in Table 1.

Table 1. Reaction conditions of CYP450 isoforms tested, including maker reactions, incubation conditions and Km used in the inhibition study.

CYPs	Marker reactions	Substrate concentration (μM)	Protein concentration (mg/mL)	Incubation time (min)	Estimated Km (μM)	Inhibitors	Concentration of inhibitors (μM)
1A2	Phenacetin O-deethylation	40	0.2	30	48	Furafylline	10
2A6	Coumarin 7-hydroxylation	1.0	0.1	10	1.5	Tranylcypromine	10
2C8	Paclitaxel 6α-hydroxylation	10	0.5	30	16	Montelukast	5
2C9	Diclofenac 4’-hydroxylation	10	0.3	10	13	Sulphaphenazole	10
2C19	S-Mephenytoin 4-hydroxylation	100	0.2	40	105	Tranylcypromine	50
2D6	Dextromethorphan O-demethylation	25	0.25	20	4.8	Quinidine	10
2E1	Chlorzoxazone 6-hydroxylation	120	0.4	30	126	Clomethiazole	50
3A4	Testosterone 6β-hydroxylation	50	0.5	10	53	Ketoconazole	1

Effect of physcion on the activity of CYP450 isoforms

The incubation system was composed of 100 mM potassium phosphate buffer (pH 7.4), NADPH generating system (1 mM NADP⁺, 10 mM glucose-6-phosphate, 1 U/mL of glucose-6-phosphate dehydrogenase and 4 mM MgCl₂), typical inhibitors or physcion and HLM at appropriate protein concentration (Table 1). The incubation volume was 200 μL and was initiated by the NADPH-generating system after a pre-incubation of 3 min. The reaction was terminated by acetonitrile or trichloroacetic acid (for CYP2A6). After centrifugation, the supernatant was analysed with a Agilent 1260 HPLC system with DAD and FLD detectors.

Inhibition model fitting

The isoforms inhibited by physcion were further studied to fit the inhibition models. First, the half-inhibition concentrations of the inhibited isoforms were obtained by incubating with different physcion concentrations (0–100 μM). While the K_i values were obtained by incubation with different substrate concentrations by Lineweaver–Burk plots. The competitive inhibition was fitted with the equation: v = (V_maxS)/[K_m(1+I/K_i)+S], while the non-competitive inhibition fitting was carried out with the equation: v = (V_maxS)/[K_m+S(1+I/K_i)], where v indicates reaction rate, I indicates the concentration of physcion, K_i is the inhibition constant, S indicates the concentration of substrates and K_m indicates the substrate concentration reaching the half of the maximum reaction rate (V_max).

Time-dependent inhibition

HLMs were pre-incubated with physcion for 30 min and then 20 μL of aliquot was transferred to another incubation tube and initiated by the NADPH-generated system as described above. The concentrations of substrates were approximately to K_m and the incubation was sustained for 5, 10, 15 and 30 min terminated by acetonitrile. The mixture was analysed by HPLC for residual activity after centrifugation.

Furthermore, the time-dependent inhibition was quantified by the values of K_I and K_inact parameters. The incubation system was performed with different concentrations of physcion (0–50 μM) higher concentration of substrate approximately 4-fold of K_m for 0–30 min. The two-step incubation scheme was conducted as described above.

Statistical analyses

Experiments were performed in triplicate, and data were analysed with GraphPad Prism version 9.0 (GraphPad Software, Inc, La Jolla, CA) and difference comparison was conducted with one-way ANOVA (p < 0.05). The values of IC₅₀ and K_i were calculated by the Lineweaver–Burk plot.

Results

Physcion inhibited CYP2C9, 2D6 and 3A4 in a concentration-dependent manner

Compared with negative controls, the activity of all studied isoforms was significantly suppressed by their typical inhibitors (positive controls). Only the activity of CYP2C9, 2D6 and 3A4 was inhibited by physcion (Figure 2(A)). The inhibitory effect of physcion was weaker than the positive inhibitors of CYP2C9, 2D6 and 3A4. Additionally, the inhibitory effect of physcion was enhanced with the increasing concentrations, where the IC₅₀ values of CYP2C9 (Figure 2(B)), 2D6 (Figure 2(C)) and 3A4 (Figure 2(D)) were 7.44, 17.84 and 13.50 μM obtained under 0, 2.5, 5, 10, 25, 50 and 100 μM physcion.

Figure 2. Effect of physcion on the major CYP450 isoforms (A) and its concentration-dependent manner in the inhibition of CYP2C9 (B), 2D6 (C) and 3A4 (D). ^****p < 0.0001 relative to the negative control. Negative control: blank treatment; physcion: treated with 100 μM physcion; positive control: treated with typical inhibitors (inhibitor types and concentrations are listed in Table 1).

Physcion served as a competitive inhibitor of CYP2C9 and 2D6

In the presence of 0, 2, 5, 10 and 15 μM physcion, the Lineweaver–Burk results fitted the inhibition of CYP2C9 (Figure 3(A)) and 2D6 (Figure 3(B)) with a competitive inhibition model with a constant value of V_max. Meanwhile, the K_i values of CYP2C9 and 2D6 were obtained as 3.69 and 8.66 μM, respectively.

Figure 3. The inhibition of CYP2C9 (A) and 2D6 (B) by physcion was best fitted with the competitive inhibition model by Lineweaver–Burk plot in the presence of 0, 2, 5, 10 and 15 μM physcion and the K_i values were 3.69 and 8.66 μM, respectively.

The inhibition of CYP3A4 by physcion was non-competitively and time-dependent

The inhibition of CYP3A4 by 0, 5, 10, 20 and 30 μM physcion was fitted with the non-competitive inhibition model with a constant value of K_m (Figure 4(A)). Moreover, the K_i value of CYP3A4 by physcion was obtained as 6.07 μM (Figure 4(B)).

Figure 4. The inhibition of CYP3A4 by physcion was best fitted the non-competitive inhibition model by Lineweaver Burk plot in the presence of 0, 5, 10, 20 and 30 μM physcion (A) and the K_i value was 6.70 μM (B).

CYP3A4 activity was decreased with the increasing incubation time, but the inhibition of CYP2C9 and 2D6 was not influenced, indicating the time-dependent characteristic of CYP3A4 inhibition by physcion (Figure 5(A)). Additionally, the time-dependent manner was observed in the presence of different concentrations of physcion (Figure 5(B)), and the K_I and K_inact parameters were obtained as 3.10 μM⁻¹ and 0.049 min⁻¹ (Figure 5(C)).

Figure 5. Only the inhibition of CYP3A4 showed a significant time-dependent manner (A), which was observed under various concentrations of physcion (B). The time-dependent K_I and K_inact parameters of CYP3A4 were obtained as 3.10 μM⁻¹ and 0.049 min⁻¹, respectively (C).

Discussion

Due to the complex composition of active ingredients, the therapeutic efficiency of TCM is always a result of the combined action of various components (Tang et al. 2008; Zhou P. 2010; Li et al. 2020). The activity of CYP450s might be suppressed or induced by co-administrated herbs and influence drug pharmacokinetics, therefore affecting the curative effects of TCM prescriptions (Almazroo et al. 2017). As a critical herb of liver injury treatment and anticancer therapy, the effect of rhubarb active ingredients on CYP450s activity is an important factor for its co-prescription with other herbs. Herein, the effect of physcion, a major extract of rhubarb, on CYP450s was investigated in HLMs. The activity of CYP2C9, 2D6 and 3A4 was dramatically suppressed by physcion, but the inhibitory effects of physcion were weaker than their positive inhibitors. The observed inhibitory effects of positive inhibitors are consistent with previous studies (Sun et al. 2021; Zhou J et al. 2022).

Both CYP2C9 and 2D6 are major members of the CYP2 subfamily. CYP2C9 is a critical mediator in the metabolism of non-steroidal anti-inflammatory drugs, oral anticoagulants, oral hypoglycemics and other therapeutic agents (Ma et al. 2008; Yildirim et al. 2008; Theken et al. 2020; Velizarova et al. 2022). CYP2D6 is responsible for the biotransformation of about a quarter of prescription drugs (Maréchal et al. 2008; Taylor et al. 2020). The activities of CYP2C9 and 2D6 were revealed to induce adverse drug-drug interaction during the co-administration of CYP2C9- or 2D6-metabolized drugs with CYP2C9 or 2D6 inhibitors or inducers (Bahar et al. 2017; Wang K et al. 2020). For example, the inhibitory effect of thymoquinone and its sourced herb, Nigella sativa L. (Ranunculaceae) on CYP2C9 resulted in their adverse interactions with phenytoin, and this kind of interaction might also occur during their combination with other CYP2C9 substrates (Wang Z et al. 2022). The genetic polymorphism of CYP2D6 significantly affected the metabolism of dacomitinib, and CYP2D6 was demonstrated to mediate the interaction between dacomitinib and trazodone (Han et al. 2022). Through fitting by Lineweaver Burk plots, the inhibition of CYP2C9 and 2D6 by physcion was revealed to be performed in a competitive manner, competing for binding sites with substrates and therefore suppressed enzyme activity.

The CYP3A subfamily is also a critical CYP450 enzyme in the liver and extrahepatic tissues, such as the intestinal and kidney, which plays vital roles in the oxidant of xenobiotics and contributes to the biotransformation of over 50% of clinical therapeutic drugs (Zhou SF 2008; Hohmann et al. 2016; Zhang N et al. 2018). CYP3A4 is the most important member of the CYP3A subfamily and could mediate various drug–drug interactions in recent studies (Kato 2008; Tian and Hu 2014; Kondža et al. 2021). For instance, the inhibition of CYP3A4 by tilmicosin was demonstrated to mediate its interaction with enrofloxacin (Zhang L et al. 2022). A clinical study in healthy individuals revealed that the inhibitory effect of itraconazole on CYP3A4 resulted in the increased systemic exposure of tropifexor (Chen et al. 2022). The inhibition of CYP3A4 by physcion was also observed in this study, which was non-competitive. Moreover, the inhibition of CYP3A4 by physcion showed a time-dependent manner. Therefore, the administration time and the half-life of physcion should be considered in its prescription. According to previous reports on the pharmacokinetics of physcion, the plasma concentration of physcion was 0.018 μg/mL after 1 h of rhubarb extract administration, and the maximum concentration was only 0.032 μg/mL, reached at 2 h of administration (Ding et al. 2003; Xun et al. 2019). Another study reported that physcion in rats reached the maximum plasma concentration of 461.85 ± 266.77 ng/mL at 0.167 ± 0.002 h after administration of Semen Cassiae extract (Yang et al. 2015). Although the pharmacokinetic parameters of physcion are much lower than the observed IC₅₀ and K_i in this study, its inhibitory effect on CYP2C9, CD6 and 3A4 should also be considered in its co-prescription with other drugs or herbs, especially the substrates metabolized by these CYP450s.

There were still several points that need to be confirmed in future studies. First, the activity of CYP450s is regulated by a series of genes, of which the expression influenced by physcion might be the potential molecular mechanism underlying the effect of physcion. The assessment of CYP450s activity is diverse, and some direct methods have been developed based on high throughput sequencing, fluorescence methods and molecular biological methods (Buters et al. 1993; Kozakai et al. 2014; Mattinen et al. 2014). Currently, molecular docking has attracted special attention, which could reveal the molecular mechanism underlying the inhibitory effect of physcion. Therefore, future studies could further explore the molecular mechanism and provide more information on the pharmacological characteristics of physcion and its source herbs and explore more direct evaluations of CYP450s.

CYP450s mainly exist in the human liver. The effect of physcion on the activity of CYP450s might also indicate changes in liver function. Hence, the effect of physcion on liver function should also attract special attention in future studies to further complete the attention in the prescription of physcion and its sourced or containing herbs.

Taken together, physcion was shown to be a competitive inhibitor of CYP2C9 and 2D6 and a non-competitive inhibitor of CYP3A4. The inhibitory effect of physcion was concentration-dependent and its inhibitory effect on CYP3A4 was time-dependent. The combined prescription of physcion or its sourced herbs should be careful, especially for their co-administration with CYP2C9-, 2D6- and 3A4-metabolized herbs or drugs. Further studies are needed to confirm the potential herb-herb or herb–drug interactions. Moreover, this study also provides a reference experimental design in the research direction of CYP450 isoforms.

Disclosure statement

The authors have no relevant financial or non-financial interests to disclose.

Data availability statement

The datasets used and/or analysed during this study are available from the corresponding author on reasonable request.

Additional information

Funding

This study was supported by Shanghai Pudong New Area Leading Talent Training Project [PWR12021-06], Outstanding Young Medical Talents Training Program of Shanghai Pudong New Area Health System [PWRq2021-11], Pudong New Area Health Commission Clinical Characteristic Discipline Construction Project [PWYts2021-13], Shanghai Pudong New Area Science and Technology Development Fund Special Project for People’s Livelihood Scientific Research [PKJ2021-Y01] and ‘QiMingXing’ Talent Training Program of Shanghai Seventh People’s Hospital [QMX2023-01].