Inhibitory effects of dioscin on cytochrome P450 enzymes

Abstract

Dioscin, a natural product, shows various pharmacological activities. However, the drug–drug interaction (DDI) potential of dioscin via the inhibition of cytochrome P450 (CYP) enzymes is still unknown. In this paper, the inhibitory effects of dioscin on six major CYP isoforms (1A2, 2A6, 2C9, 2D6, 2E1 and 3A4) were investigated. Using human liver microsomes (HLM), we found that dioscin inhibited the activities of CYP2C9, CYP2E1 and CYP3A4, with IC₅₀ values of 22.60, 17.40 and 12.59 μM, respectively. Further research indicated that dioscin was a typical competitive inhibitor for CYP2C9, CYP2E1 and CYP3A4 (K_i = 5.0, 10.4 and 15.5 μM, respectively), as demonstrated by Lineweaver–Burk plots. In addition, dioscin exhibited time- and NADPH-dependent inhibitions of CYP3A4, and weak inhibitions of CYP1A2, CYP2A6 and CYP2D6 were also observed. In cell and animal experiments, dioscin markedly down-regulated the protein expressions of CYP2C9, CYP2E1 and CYP3A4 in primary rat hepatocytes and livers in a dose-dependent manner. This is the first paper on the inhibitory effects of dioscin on CYP in HLMs to predict the potential for dioscin–drug interaction and toxicity. Nevertheless, the clinical significance of the data presented herein should be further evaluated with in vivo research.

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Introduction

Clinically, the use of combinations of medicines can increase the potential for drug–drug interactions (DDIs), which may alter drug metabolism in both phase I and phase II.^1,2 DDI is also a major aspect of pharmacokinetics in addition to adsorption, distribution and elimination.³ Furthermore, cytochrome P450 (CYP), a superfamily of metabolising enzymes located primarily in hepatocytes, is involved in DDI.⁴ In detail, in the phase I metabolism of endogenous and exogenous compounds, including dietary chemicals, food additives, and drugs, biotransformation processes can lead to unique pharmacological and toxic reactions in organisms.⁵ In addition, as CYP-mediated drug metabolism is a chief aspect of phase I metabolism, the discovery of potential inhibitors and inducers of CYP will be helpful to guide drug candidate selection and characterisation.⁶

In the field of drug discovery and development, a considerable number of traditional Chinese medicines (TCMs) and natural products have been successfully exploited as first-line drugs to treat human diseases.⁷ Dioscin (Fig. 1), a steroidal saponin that is extracted from some medicinal plants such as Dioscorea zingiberensis C. H. Wright and Discorea nipponica Makino,^8,9 has various pharmacological activities including anti-viral,¹⁰ anti-fungal,¹¹ hepatoprotective¹² and anti-cancer¹³ actions. Thus, this compound should be developed as a promising drug to treat multiple diseases in the future. Although the beneficial activities of dioscin have been investigated, dioscin–drug interactions have only recently become the focus of research.

Fig. 1 Chemical structure of dioscin.

Previous studies have shown that dioscin can decrease CYP2E1-mediated ethanol and CCl₄ metabolism in rats.^14,15 However, to the best of our knowledge, no papers have reported the modulatory effect of dioscin on CYP enzymes, which could increase the risk involved in the clinical application and medical preparation of dioscin.

Thus, in the present study, we investigated the inhibitory effects of dioscin on the activities of several CYP isozymes, with a focus on CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1 and CYP3A4. In addition, the parameters for in vitro and in vivo interactions were obtained from pooled human liver microsomes (HLM), primary rat hepatocytes and rat livers.

Results and discussion

Inhibition effects of dioscin on CYP activities in HLM

Drug metabolism is a main factor in pharmacokinetics, in addition to absorption, distribution and elimination.³ In the drug metabolism process, drug metabolising enzymes, including phase I enzymes comprised primarily of CYP and phase II enzymes, play important roles.^4,16 Furthermore, CYP enzymes are the major oxidative enzymes of endogenous substances such as fatty acids and ketones and exogenous compounds such as procarcinogens, drugs and environmental chemicals.^17,18 Changes in CYP expression can occur in various diseases, including obesity, diabetes, depression, infections and inflammation.^19,20 In addition, it is well known that about 20% of all harmful drug reactions can lead to the intracellular accumulation of free radicals, and other metabolic intoxicants are due to CYP-mediated DDIs.⁴ Therefore, it is important to estimate the effects of active compounds on CYP to predict DDIs and provide explanations for the lack of efficacy, or even toxicity.⁶

Dioscin, a natural product, has been shown to be an inhibitor of rat CYP2E1 in some previous reports.^14,15 However, information about dioscin as a CYP inhibitor is still relatively insufficient.

In the present study, the inhibitory effects of dioscin on the activities of six CYP isoforms were investigated. We found dioscin at a concentration of 200 μM inhibited the activities of CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 by 75.4%, 66.5%, 26.1%, 93.3%, 23.4% and 13.1%, respectively (Fig. 2). Moreover, dioscin strongly inhibited the activities of CYP2C9, CYP2E1 and CYP3A4 in pooled HLMs, with IC₅₀ values of 22.60, 17.40 and 12.59 μM, respectively (Fig. 3).

Fig. 2 Inhibitory effects of dioscin on CYP enzymes in pooled HLM. All data are expressed as the mean of triplicate incubations.

Fig. 3 Dioscin inhibition of CYP2C9-catalysed tolbutamide 4-hydroxylation, CYP2E1 catalysed-chlorzoxazone 6-hydroxylation, and CYP3A4-catalysed testosterone 6β-hydroxylation. All data are expressed as the means of triplicate incubations.

In general, CYP2C9 is an important enzyme in the metabolism of clinical drugs. The substrates of CYP2CP are numerous and include phenytoin, pioglitazone, warfarin, losartan and non-steroid anti-inflammatory drugs.²¹ In addition, several inhibitors of the CYP2C9 isozyme, including sulphaphenazole, amiodarone, omeprazole and fluconazole, may result in increased drug–substrate interactions in plasma, as well as reduced metabolism, leading to potentially detrimental and even life-threatening consequences for patients.²² Furthermore, the Lineweaver–Burk plots of inhibitory kinetic data indicated that dioscin is a potent and reversible competitive inhibitor of CYP2C9, with K_i = 5.0 μM (Fig. 4A).

Fig. 4 Lineweaver–Burk plots for dioscin inhibition of CYP-catalysed reactions in pooled HLM. (A) Data obtained from a 30 min incubation with tolbutamide (10–80 μM) in the absence or presence of dioscin (15–45 μM). (B) Data obtained from a 30 min incubation with chlorzoxazone (12.5–100 μM) in the absence or presence of dioscin (10–30 μM). (C) Data obtained from a 30 min incubation with testosterone (25–200 μM) in the absence or presence of dioscin (8–24 μM). The dextral figures show the re-plotted slopes obtained by linear regression of the data from the Lineweaver–Burk plots. All data are expressed as the means of triplicate incubations.

CYP2E1 comprises about six percent of the total P450 in human liver; it is also expressed in the lung, nasal epithelium and oropharynx.²³ Ethanol is an inducer and a substrate of CYP2E1.²⁴ Caffeine, acetaminophen and a large number of carcinogens, including acrylonitrile, benzene and styrene, are also metabolised by CYP2E1.²⁵ Their metabolism often results in the production of reactive oxygen species, causing liver injury and other detrimental effects.²⁶ The Lineweaver–Burk plots of inhibitory kinetics indicated that dioscin is a potent and reversible competitive inhibitor of CYP2E1, with K_i = 10.4 μM (Fig. 4B).

CYP3A4 catalyses biosynthetic reactions of cholesterol, lipids and steroid hormones, and its up-regulation may lead to failed pharmacotherapy or the elevated production of toxic intermediates.²⁷ Furthermore, many drugs, including carbamazepine, phenobarbital, rifampicin, phenytoin and dexamethasone, can induce CYP3A4, and some of them may also inhibit CYP3A4. Whether induction or inhibition is more prevalent is decided by the regulatory factors and the affinities of the drugs for the enzyme.²⁸ The grapefruit is a textbook example of a CYP3A4 inhibitor, and the grapefruit juice-mediated CYP3A4 reaction plays an important role in organic anion-transporting polypeptide (OATP) metabolism.²⁹ The Lineweaver–Burk plots of inhibitory kinetic data in this paper showed that dioscin is a potent and reversible competitive inhibitor of CYP3A4, with K_i = 15.5 μM (Fig. 4C).

In addition, we know that time-dependent inhibitors may generate more adverse effects than reversible inhibitors, as loss of enzyme activity occurs even after the elimination of the inhibitors.⁶ After pre-incubation of dioscin with HLM for 30 min in this paper, the inhibition effect of dioscin on CYP3A4 increased 21.3% in the presence of NADPH compared to in the absence of NADPH (Fig. 5). However, the inhibition of other CYP isoforms by dioscin was not time- or NADPH-dependent (Fig. 5).

Fig. 5 Time- and NADPH-dependent studies of dioscin. The concentrations of dioscin (10-times the IC₂₅) used for CYP2C9, CYP2E1, and CYP3A4 were 50, 40 and 22 μM, respectively. For other CYP isoforms, 100 μM of dioscin was adopted. All data represent the mean ± SD of triplicate incubations.

Inhibition effects of dioscin on CYP activities in primary rat hepatocytes and rat livers

Further studies were performed to evaluate the effects of dioscin on CYP2C9, 2E1 and 3A4. By using cell (Fig. 6A) and rat (Fig. 6B) experiments, we found that dioscin significantly reduced the protein expressions of CYP2C9, 2 × 10¹ and 3A4 in primary rat hepatocytes and rat livers in a dose-dependent manner compared to the control (p < 0.05 or p < 0.01), although dioscin was not directly toxic. Nevertheless, the down-regulation of these enzyme activities may significantly add to the risk of toxicity due to secondary exposure to other substances, but it may also decrease the risk for toxicity resulting from exposure to xenobiotics.³⁰

Fig. 6 Effects of dioscin on the protein expressions of CYP2C9, CYP2E1, CYP3A4 in primary rat hepatocytes (A) and rat livers (B). Values are expressed as mean ± SD (n ≥ 6). **p < 0.01 compared with control group.

Therefore, it is essential to pay attention to dioscin–drug interactions when using this compound clinically. Dioscin–drug interactions may alter therapeutic efficacy and produce potential toxicity since its clearance has been disturbed. These interactions should be evaluated, taking the following factors into account: the use of combinations of many medicines; the characteristics of substances with inhibitory effect; and the locations, expressions and functions of the influenced CYP enzymes.³¹

Further studies about the interactions of dioscin and P450 will be very valuable. A basic theory is that the existence of a reversible competitive intermolecular kinetic hydrogen isotope effect makes the C–H bond-breaking step at least partially rate-limiting in P450 reactions.³² We will attempt to identify the rate-limiting steps for the interactions of dioscin and CYP2C9, CYP2E1, and CYP3A4 in a future study. By using molecular dynamics (MD) simulation along with molecular mechanics Poisson–Boltzmann surface area and molecular mechanics generalised Born surface area (MM-PB/GBSA) analysis, we can search for appropriate pockets of P450 to dock with dioscin. Subsequently, we may also use Quantum Mechanics/Molecular Mechanics (QM/MM) simulation to research the mechanism of P450 chemical steps into electronic details by Gaussian software if the reactions of P450 and diosicn involves the key generation and fault.³³

Experimental section

Compounds and reagents

Dioscin (>98%) was obtained from the National Institute of Food and Drug Control of China (Beijing, China). Pooled HLM, the nicotinamide adenine dinucleotide phosphate (NADPH) generating system (1.3 mM NADP⁺, 3.3 mM glucose-6-phosphate, 0.4 U mL⁻¹ glucose-6-phosphate dehydrogenase and 3.3 mM MgCl₂), phenacetin, acetaminophen, 7-hydroxycoumarin, coumarin, 7-hydroxycoumarin, tolbutamide, 4-hydroxytolbutamide, dextromethorphan, dextrorphan, chlorzoxazone, 6-hydroxychlorzoxazone, testosterone and 6β-hydroxytestosterone were obtained from iPhase Pharmaceutical Services (Beijing, China). All other reagents were obtained from Sigma-Aldrich (St. Louis, Miss., USA). A tissue protein extraction kit was purchased from KEYGEN Biotech. Co., Ltd. (Nanjing, China). A bicinchoninic acid (BCA) protein assay kit and an enhanced chemiluminescence (ECL) kit were obtained from the Beyotime Institute of Biotechnology (Jiangsu, China). Rabbit anti-homo sapiens cytochrome P450, family 2, subfamily C, polypeptide 9 (CYP2C9), rabbit anti-homo sapiens cytochrome P450, family 2, subfamily E, polypeptide 1 (CYP2E1), rabbit anti-homo sapiens cytochrome P450, family 3, subfamily A, polypeptide 4 (CYP3A4), rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies were provided by Proteintech Group, Inc. (Chicago, USA).

Animals

Male Sprague Dawley (SD) wistar rats (180–220 g) eight weeks old were purchased from the Experimental Animal Center of Dalian Medical University, Dalian, China (SCXK: 2008-0002). The animals were housed under standard animal care conditions (temperature, 25 ± 3 °C; relative humidity, 55–70%; 12 h light and dark cycles) with free access to food and water. All experimental procedures were approved by the Animal Care and Use Committee of Dalian Medical University and performed in strict accordance with the PR China Legislation Regarding the Use and Care of Laboratory Animals.

Assay with HLM

The typical incubation system contained 0.5 mg mL⁻¹ HLM, the NADPH generating system, the corresponding probe substrate, dioscin and 0.1 mol L⁻¹ phosphate buffer (pH = 7.4) in a final volume of 200 μL. Probe substrates and dioscin were dissolved in 2 μL methanol, and 2 μL methanol was added to a control group without dioscin.

The incubation system was pre-incubated at 37 °C for 5 min followed by the addition of the NADPH-generating system and the initiation of the reaction. After 30 min, 100 μL of acetonitrile was added to terminate the reaction. Finally, the mixture was centrifuged at 20 000×g for 10 min, and 50 μL of supernatant was transferred for HPLC analysis on an Agilent Series 1200 HPLC system consisting of a quaternary delivery system, an auto-sampler, a ZORBAX SB-C18 column (250 mm × 4.6 mm i.d., 5 μm) and a diode array detector (DAD).^6,34 The chromatographic conditions for the corresponding metabolites are given in Table 1. Measurements were performed in triplicate, and the mean values are reported.

Table 1 The chromatographic conditions for the corresponding metabolites

CYP450	Probe substrates	Metabolites	Chromatographic conditions
CYP1A2	Phenacetin	4-Acetamidophenol	Methanol: 2% phosphoric acid = 10 : 90; 245 nm; 1 mL min^−1
CYP2A6	Coumarin	7-Hydroxycoumarin	Methanol: water = 30 : 70; 320 nm; 1 mL min^−1
CYP2C9	Tolbutamide	4-Hydroxytolbutamide	Methanol: 2% phosphoric acid = 40 : 60; 230 nm; 1 mL min^−1
CYP2D6	Dextromethorphan	Dextrorphan	Methanol: 2% phosphoric acid = 35 : 65; 220 nm; 1 mL min^−1
CYP2E1	Chlorzoxazone	6-Hydroxychlorzoxazone	Methanol: 2% phosphoric acid = 20 : 80; 296 nm; 1 mL min^−1
CYP3A4	Testosterone	6β-Hydroxytestosterone	Methanol: 2% phosphoric acid = 40 : 60; 240 nm; 1 mL min^−1

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Enzyme inhibition studies of dioscin

Dioscin (200 μM) was added into the incubation system to observe its effects on different human CYP isoforms. The half-inhibition concentrations (IC₅₀) of CYP isoforms whose activities were significantly inhibited by dioscin were determined. Kinetic imaging (K_i) values were obtained by incubating various concentrations of different probe substrates (10–80 μM tolbutamide, 12.5–100 μM chlorzoxazone and 25–200 μM testosterone) in the presence of 0–45 μM dioscin.

Time- and NADPH-dependent studies of dioscin

To determine whether dioscin is a time-dependent inhibitor of human CYP isoforms, dioscin was incubated with HLM (0.5 mg mL⁻¹) in the absence or presence of the NADPH-generating system for 30 min at 37 °C. For CYP2C9, CYP2E1 and CYP3A4, the concentrations of dioscin were 50 μM, 40 μM and 22 μM, respectively (10-times the IC₂₅). For other CYP isoforms, 100 μM dioscin was used.

Primary rat hepatocyte cultures

Male SD rats were anesthetised with chloral hydrate (300 mg kg⁻¹; i.p.), and livers were perfused in a two-step procedure according to the Seglen method. Isolated hepatocytes were seeded on rat tail collagen-coated multiwell plates at 37 °C and 5% CO₂ in Dulbecco's Minimum Essential Medium (DMEM) supplemented with 10% fetal bovine serum along with 100 U mL⁻¹ penicillin and 100 U mL⁻¹ streptomycin. In the experiments, cells were allowed to adhere and grow for 24 h in the culture medium prior to treatment with different concentrations of dioscin (0.3, 0.6 and 1.2 μg mL⁻¹) for 24 h.

Experimental design in vivo

After acclimation for one week, the animals were randomly divided into four groups (n = 6), and dioscin was administered intragastrically (i.g.) to the animals at doses of 0, 20, 40 and 60 mg kg⁻¹ once daily for seven consecutive days. On the eighth day, the animals were anesthetised, and the livers were perfused with 0.9% (w/v) sodium chloride before they were collected and stored at −80 °C.

Western blotting assay

Total protein was isolated from primary rat hepatocytes and rat livers of different groups using the tissue protein extraction kit, based on the manufacturer's instructions. The samples (containing 50 μg protein) were loaded onto the SDS-PAGE gel (12%), separated electrophoretically and transferred onto PVDF membranes (Millipore, USA). After blocking non-specific binding sites for 1 h with 5% dried skim milk in TTBS at 37 °C, the membranes were individually incubated overnight at 4 °C with primary antibodies including CYP2C9, CYP2E1 and CYP3A4 at dilutions of 1 : 1000. The membranes were then incubated at 37 °C for 1 h with horseradish peroxidase-conjugated antibodies (1 : 2000). Protein expression was detected by an ECL method and imaged using a Bio-Spectrum Gel Imaging System (UVP, USA). To evaluate the variations in protein expression, the data were adjusted to GAPDH expression (integral optical density (IOD) value of target protein versus IOD of GAPDH).

Statistical analysis

The data were compared among groups using one-way analysis of variance (ANOVA) coupled with LSD in post-hoc multiple comparisons with SPSS Statistics 18.0 (IBM, New York, USA). The results were considered significant at p < 0.05 or 0.01.

Conclusions

In conclusion, this in vitro study suggested that dioscin could strongly inhibit CYP2C9, CYP2E1 and CYP3A4 activities and had negligible influences on CYP1A2, CYP2A6 and CYP2D6. The in vivo results demonstrated that dioscin might cause dioscin–drug interactions when co-administrated with CYP2C9, CYP2E1 and CYP3A4 substrates. However, the deeper in vivo effects of dioscin on CYPs in humans require further study.

Competing interests

None declared.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (no. 81274195), the Doctorate in Higher Education Institutions of Ministry of Education (no. 20122105110004), the Foundation of innovation team of Education Ministry (IRT13049), the Program for Liaoning Innovative Research Team in University (LT2013019) and the Program for New Century Excellent Talents in University (NCET-11-1007).

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Footnote

† These authors contributed the same amount of work to this paper and are co-first authors.

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