Molecular docking and enzyme kinetic studies of dihydrotanshinone on metabolism of a model CYP2D6 probe substrate in human liver microsomes.ARTICLE INFO
Danshen (Salvia miltiorrhiza)
Human liver microsomes
The effects of Danshen and its active components (tanshinone I, tanshinone IIA, dihydrotanshinone and cryptotanshinone) on CYP2D6 activity was investigated by measuring the metabolism of a model CYP2D6 probe substrate, dextromethorphan to dextrorphan in human pooled liver microsomes. The ethanolic extract of crude Danshen (6.25-100 [micro]g/ml) decreased dextromethorphan O-demethylation in vitro ([IC.sub.50] =23.3 [micro]g/ml) and the water extract of crude Danshen (0.0625-1 mg/ml) showed no inhibition. A commercially available Danshen pill (31.25-500 [micro]g/ml) also decreased CYP2D6 activity ([IC.sub.50] = 265.8 [micro]g/ml). Among the tanshinones, only dihydrotanshinone significantly inhibited CYP2D6 activity ([IC.sub.50]= 35.4 [micro]M), compared to quinidine, a specific CYP2D6 inhibitor ([IC.sub.50] = 0.9 [micro]M). Crytotanshinone, tanshinone I and tanshinone IIA produced weak inhibition, with [IC.sub.20] of 40.8 [micro]M, 16.5 [micro]M and 61.4 [micro]M, respectively. Water soluble components such as salvianolic acid B and danshensu did not affect CYP2D6-mediated metabolism. Enzyme kinetics studies showed that inhibition of CYP2D6 activity by the ethanolic extract of crude Danshen and dihydrotanshinone was concentration-dependent, with K, values of 4.23 [micro]g/ml and 2.53 [micro]M, respectively, compared to quinidine, [K.sub.i] = 0.41 [micro]M. Molecular docking study confirmed that dihydrotanshinone and tanshinone I interacted with the Phel 20 amino acid residue in the active cavity of CYP2D6 through Pi-Pi interaction, but did not interact with Glu216 and Asp301, the key residues for substrate binding. The logarithm of free binding energy of dihydrotanshinone (-7.6 kcal/mol) to Phe120 was comparable to quinidine (-7.0 kcal/mol) but greater than tanshinone I (-5.4 kcal/mol), indicating dihydrotanshinone has similar affinity to quinidine in binding to the catalytic site on CYP2D6.
[c] 2012 Elsevier GmbH. All rights reserved.
Danshen (Salvia miltiorrhiza) has been widely used in China and other countries for the treatment of cardiovascular and cerebrovascular diseases (Wang et al., 2007; Zhou et al., 2005). Both the water-soluble and lipophilic compounds. of Salvia miltiorrhiza root extract appear to improve the infarction/reperfusion-induced vascular damage synergistically (Han et al., 2008). However, the use of Danshen has been associated with a number of clinically important herb-drug interactions leading to adverse outcome (Holbrook et al., 2005; Izzo et al., 2005; Yu et al., 1997). Interaction of Danshen with warfarin may be mediated via both pharmacodynamic and pharmacokinetic mechanisms (Lo et al., 1992). Danshen exaggerated the pharmacological effects of warfarin by prolonging the prothrombin time and increased the bioavailability and decreased the elimination of warfarin in the rat (Chan et al., 1995). The major tanshinones of Danshen inhibited warfarin hydroxylation and increased the steady-state plasma warfarin concentration (Wu and Yeung, 2010). The recent finding was in line with those from previous studies in which Danshen altered the metabolism of R- and S-warfarin (Chan et al., 1995), reactions widely accepted as being mediated through CYP isoforms such as 1A2, 2C9 and 3A4.
Advances in separation techniques have enabled isolation and characterization of the active components of Danshen (Liu et al., 2006; Ma et al., 2006; Shi et al., 2005; Zhang et al., 2005), followed by extensive research to investigate the pharmacology and therapeutic potential of the individual components of the herb. Tanshinone IIA, one of the major lipid soluble components of Danshen, has been reported to inhibit CYP1A2 activity in mouse, human and rat in vitro (He et al., 2007; Ueng et al., 2003; Wang et al., 2009b; Wang and Yeung, 2011c). Other major tanshinones such as tanshinone I, cryptotanshinone and dihydrotanshinone also exhibited different modes of inhibition on human CYPs 1A2, 2C9, 2E1 and 3A4 in vitro (Wang et al., 2010a), rat CYP2C11 (human CYP2C9 equivalent) and CYP3A2 in vitro and in vivo (Wang et al., 2010b, 2010c; Wang and Yeung, 2011a, 2011b). The aqueous extract from Danshen has also been shown to affect both human and rat CYP1A2 activity in vitro and in the rat after chronic treatment (Wang and Yeung, 2010, 2011c).
CYP2D6 is involved in the metabolism of approximately 25-50% of clinically used drugs, including antidepressants, neuroleptics, tamoxifen, HMG-CoA reductase inhibitors, anti-emetics, antiarrthymic drugs and beta-antagonists (De Gregori et al., 2010; Hsu, 2010; Prisant, 2008; Sideras et al., 2010; Vermes and Vermes, 2004; Wang et al., 2009a; Zhou, 2009). CYP2D6 is one of the highly polymorphic CYPs and the polymorphism can be translated into risk differences for drugs which are metabolised by these enzymes (Johansson and Ingelman-Sundberg, 2011). Approximately 5-14% of Caucasians, 0-5% Africans, and 0-1% of Asians lack CYP2D6 activity, and are known as poor metabolisers (Zhou et al., 2009). Given the widespread use of Danshen and Danshen-containing formulations, alone or in combination with many cardiovascular drugs which are CYP2D6 substrates, it would be of interest to study the effect of Danshen or its active components on CYP2D6 activity, especially when a fraction of the population being poor CYP2D6 metabolisers. The aim of this study was to investigate the effects of tanshinones (tanshinone I, tanshinone IIA, cryptotanshinone and dihydrotanshinone), danshensu, and salvianolic acid B (Fig. 1) on rat CYP2D6 activity, using dextromethorphan as the model probe substrate in vitro. The study design was similar to interaction studies with other CYP isoforms in the rat (Wang et al., 2009b, 2010b, 2010c; Wang and Yeung, 2010). A molecular docking study was used to determine the free binding energy and binding simulations of the tanshinones on the binding to the catalytic site on CYP2D6, which could be helpful for the understanding of the interaction between the tanshinones and human CYP2D6.
[FIGURE 1 OMITTED]
Materials and methods
Pooled human liver microsomes (HLMs) were obtained from GenTest Corporation (Woburn, MA, USA) and stored at -80 [degrees]C until use. Danshen was supplied by Winsor Health Products Ltd. (Hong Kong). Dried Danshen root was purchased from Eu Yan Sang Limited (Hong Kong) where an initial screening test has been carried out that the batch of Danshen was free from other contaminants. Cryptotanshinone, dihydrotanshinone, tanshinone I, tanshinone IIA, danshensu and salvianolic acid B were purchased from Chengdu Congon Bio-tech Co., Ltd. (China). Dextromethorphan, dextrorphan, quinidine, chlorpheniramine, [beta]-nicotinamide adenine dinucleotide phosphate (NADP), D-glucose 6-phosphate, glucose 6-phosphate dehydrogenase, heparin sodium, urethane, and phenacetin were from Sigma Chemical Co. (St. Louis, MO, USA). Acetonitrile (HPLC Grade) was purchased from Labscan Analytical Sciences (Bangkok, Thailand). Methanol (HPLC Grade) was from BDH Laboratory Supplies (Poole, UK), ethyl acetate (HPLC grade) was from Fisher Chemicals (Leicester, U.K.). Acetic acid, glacial, (HPLC grade) was from Scharlau Chemie (Barcelona, Spain). Phenobarbitone sodium was obtained from Universal Pharmaceutical Lab. (Hong Kong). Carbon monoxide was supplied by Hong Kong Special Gas Co.
Preparation of aqueous and ethanolic extracts of Danshen root
Aqueous and ethanolic extract of crude Danshen were prepared as previously described (Lee et al., 2012). For aqueous extract, Danshen root (200g) was cut into small pieces and boiled in 250 ml distilled water in reflux. After 1 h, the residue was mixed with distilled water (250 ml) and boiled for another hour. The filtrate was combined with the previous filtrate and cooled at room temperature. Water in the filtrate was removed by freeze-drying and about 35g (17.5% of yield) of the aqueous extract powder was obtained. For ethanolic fraction, Danshen root (200 g) was minced and boiled in 95% ethanol (250 ml twice under reflex condition. The filtrate was collected and dried using a rotary evaporator with warming below 50 [degrees]C. The brown residue was re-dissolved in ethyl acetate. The ethyl acetate layer was collected and dried by a rotary evaporator. The reddish brown crystals finally obtained represented the ethanolic fraction in which the percentage yield was about 1%. The content of individual tanshinones and phenolic acids in Danshen pill, ethanolic fraction and aqueous fraction was analyzed by HPLC (Wang et al., 2010b, 2010c). The major active constituents present in extracts of Danshen pill, aqueous extract and ethanolic extract of crude Danshen are shown in Table 1.
Table 1 Major active constituents present in extracts of Danshen pill, aqueous extract and ethanolic extract of crude Danshen determined by HPLC-DAD. Date is expressed as mean [+ or -] SD (n = 3). Danshen pill Aqueous extract Ethanolic extract (per g powder) (per g extract) (per g extract) Cryptotanshino 45.6 [+ or -] 34.6 [+ or -] 36.8 [+ or -] ne 2.37 [micro]g 0.86 [micro]g 0.56 mg Dihydrotanshin 99.4 [+ or -] 12.78 [+ or -] 9.3 [+ or -] 0.12 one 2.03 [micro]g 0.46 [micro]g mg Tanshinone I 26.6 [+ or -] 10.4 [+ or -] 17.9 [+ or -] 1.55 [micro]g 0.40 [micro]g 0.32 mg Tanshinone IIA 17.5 [+ or -] 22.6 [+ or -] 118.4 [+ or -] 1.15 [micro]g 0.51 [micro]g 2.91 mg Danshensu 28.7 [+ or -] 3.10 [+ or -] 2.70 [+ or -] 0.32 mg 0.03 mg 0.08 mg Salvianolic 38.5 [+ or -] 37.3 [+ or -] 209.3 [+ or -] acid B 0.73 mg 0.50 mg 5.48 mg Data from Lee et al. (2012).
Analysis of CYP2D6 (dextromethorphan O-demethylase) activity
Human liver microsome (1 mg/ml) was incubated in 0.05 M Tris/KC1 buffer, pH 7.4 with NADPH-regenerating system (10 mM NADP, 5 mM glucose-6-phosphate, 2 units/ml glucose-6-phosphate dehydrogenase, and 5 mM Magnesium chloride). For inhibition study, 100 [micro]M dextromethorphan was used. For kinetic study, dextromethorphan concentrations ranged from 50 [micro]M to 400 [micro]M. The concentrations of Danshen pill and water extract of crude Danshen used were from 0.0625 to 1.0 mg/ml. The concentration range of cryptotanshinone, dihydrotanshinone and tanshinone I and IIA were 6.25-100 [micro]M. The final concentration of DMSO was 1%. The concentration range for ethanolic extract of crude Danshen was 6.25-/DO The concentration ranges of danshensu and salvianolic acid B (SAB) were 6.25-100 [micro]M. Quinidine (6.25-100 [micro]M), CYP2D6 inhibitor, was used as positive control. The tubes were incubated in Eppendorf Thermomixer at 800 rpm, 37 [degrees]C. The reaction was initiated by adding ice-cold liver microsomes to the incubation mixture and the incubation time was 30 min. Incubations were terminated by adding ice cold acetonitrile (250 [micro]l). The tubes were then centrifuged at 13,000 rpm for 12 min to precipitate protein. The supernatant was then extracted with 250 pul ethyl acetate at 1400 rpm in a Thermomixer for 30 min at 25 [degrees]C. Chlorpheniramine (100 [micro]g/ml, 10 [micro]l) was added as internal standard. The tubes were then centrifuged at 8000 rpm for 8 min. The organic layer was transferred to a glass conical tube on a heat block at 40 [degrees]C and evaporated under a gentle stream of nitrogen gas. The residue was dissolved in 60 [micro]l phosphoric acid in water, pH 2.7 and then 50 [micro]l was used for HPLC analysis.
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
Inhibition kinetics studies
Ethanolic extract of crude Danshen (3.13-25 [micro]g/ml), Danshen pill (31.25-250 [micro]g/ml) or dihydrotanshinone (1.56-12.5 [micro]M) and dextromethorphan (50-400 [micro]M) were used for inhibition kinetics studies. Quinidine (0.156-1.25 [micro]M), a selective human CYP2D6 inhibitor, was used as positive control.
HPLC analysis of dextromethorphan and dextrorphan
Analysis of dextromethorphan and dextrorphan was a modified method. The HPLC equipment (Agilent 1050 Series) consisted of a quaternary pump and a multi-wavelength detector. Dextrorphan, dextromethorphan and chlorpheniramine (internal standard) were separated on an Agilent ZORBAX XDB-C8, 5 [micro]M (150 mm x 4.6 mm) column with Eclipse XDB-C8 guard column. The mobile phase was consisted of 30% acetonitrile in 0.1% triethylamine in water at pH 3.0, using a flow rate of 0.5 ml/min. Detection was by UV absorbance at 220 nm. Standard curve for dextrorphan was linear between 0.1 and 5.0 [micro]g/ml. Standard curve for dextromethorphan was linear between 1.0 and 40.0 [micro]g. Four different levels of quality control samples (0.1, 0.5, 1.0 and 2.0 [micro]g for dextrorphan and 4.63, 9.26, 18.52 and 37.04 [micro]g for dextromethorphan) were used to estimate the intra- and inter-day precision and accuracy. Precision tests gave good reproducibility of the HPLC analysis, the relative standard deviation was 5.7% at 50 [micro]g/ml and 2.8% at 100 [micro]g/ml. The accuracy of the standard curve is 100.8%, 100.2% and 101.1% at 0.1, 0.5 and 1.0 [micro]g/ml, respectively.
[FIGURE 4 OMITTED]
Molecular docking study
Molecular docking of cryptotanshinone, dihydrotanshinone, tanshinone I, tanshinone IIA dextromethorphan, quinidine to CYP2D6 was performed using the three dimensional (3-D) crystal structure of substrate free CYP2D6 (PDB code 2F9Q) obtained from the Protein Data Bank (Rowland et al., 2006). The software AutoDock Vina v.1.0.2 was used for all dockings in this study (Seeliger and de Groot, 2010). The docking parameters for AutoDock Vina were kept to their default values. The grid box was 20A x 14A x 12A, encompassing the active site cavity of CYP2D6. The binding modes were clustered through the root-mean square deviation (RMSD) among the Cartesian coordinates of the ligand atoms. The docking results were ranked by the binding free energy. The binding modes with lowest binding free energy and the most cluster members were chosen for the optimum docking conformation. The binding results were illustrated as three dimensional (3-D) and two dimensional (2-D) diagrams by PyMOL Molecular Graphics System Version 1.3 (Schrodinger, LLC) and Discovery Studio Visualizer (Accelrys, Inc., CA), respectively.
Statistical analysis of the data was carried out using ANOVA by a computer program (Statview 9.0, Abacus Concepts, USA). Enzyme kinetics data were fitted by non-linear regression analysis using GraphPad Prism 4 (GraphPad Software, CA, USA). [IC.sub.50] values (concentration of inhibitor to cause 50% inhibition of original enzyme activity) were determined by GraFit where appropriate using the following equation:
V = [V.sub.0] / 1 + [(1 / [IC.sub.50]).sup.5]
where [V.sub.0] is uninhibited velocity, V is observed velocity, S is slope factor and I is inhibitor concentration. A Lineweaver-Burk Plot is a double reciprocal plot in which varying substrate concentrations are plotted against reaction velocities to obtain linear transformation. The enzyme parameter Michaelis constant ([K.sub.m]) and [V.sub.max] values were obtained from Lineweaver-Burk Plot. The inhibition constant ([K.sub.i]), the inhibitor concentration at which the reaction is half of the maximal rate, was obtained by a secondary plot using the slope of the primary Lineweaver-Burk Plot and fitted by GraphPad Prism 4.
Effect of tanshinones and Danshen extracts on CYP2D6 activity in human pooled liver microsomes
The ethanolic extract of crude Danshen inhibited dextromethorphan metabolism concentration-dependently (Fig. 2a), while the water extract of crude Danshen (Fig. 2b) had no inhibitory effect on dextromethorphan metabolism. The commercially available Danshen pill also inhibited dextromethorphan metabolism concentration-dependently (Fig. 2c). The contents of the ethanolic extract of crude Danshen and the water extract of crude Danshen and the Danshen pill have been determined (Table 1) and contain the active lipid soluble and water soluble components of Salvia miltiorrhiza at relatively different proportions.
[FIGURE 5 OMITTED]
The effects of tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone on CYP2D6-mediated dextromethorphan metabolism in pooled human liver microsomes showed different degrees of concentration-dependent inhibition (Fig. 3), with dihydrotanshinone being the most potent, ([IC.sub.50] = 35.4 [micro]M), compared to quinidine, a specific CYP2D6 inhibitor ([IC.sub.50] = 0.91 [micro]M). The other tanshinones only showed weak inhibition on dextromethorphan metabolism. The results were confirmed with calculation of the metabolite/probe substrate ratio (dextrorphan/dextromethorphan) for dihydrotanshinone (Fig. 4).
The Lineweaver-Burke linear transformation of the enzyme velocities versus substrate concentration showed that the ethanolic extract of crude Danshen, Danshen pill and quinidine all show competitive inhibition (Fig. 5). Secondary plots of slopes of Primary Lineweaver-Burk Plots versus inhibitor concentrations (Fig. 6) and Dixon plots (Fig. 7) confirmed that the [K.sub.i] values were in the order: dihydrotanshinone (2.53 [micro]M), ethanolic extract (4.23 [micro]g/ml) and Danshen pill (29.7 [micro]g/ml). The [K.sub.i] of the specific CYP2D6 inhibitor, quinidine, was 0.41 [micro]M.
[FIGURE 6 OMITTED]
The inhibition parameters and [K.sub.i] values for all compounds tested are summarized in Table 2.
Table 2 [IC.sub.20], [IC.sub.50] and [K.sub.i] values for the inhibition of model CYP2D6 probe metabolism by tanshinones. [IC.sub.20] [IC.sub.50] [K.sub.i] Ethanolic extract 8.1 [micro]g/ml 23.3 [micro]g/ml 4.23 [micro]g/ml Danshen pill 116.5 [micro]g/ml 265.8 [micro]g/ml 29.7 [micro]g/ml Cryptotanshinone 40.8 [micro]M -- -- Dihydrotanshinone 11.0 [micro]M 35.4 [micro]M 2.53 [micro]M Tanshinone I 16.5 [micro]M -- -- Tanshinone 11A 61.4 [micro]M -- -- Quinidine 0.18 [micro]M 0.90 [micro]M 0.41 [micro]M
Molecular docking study
Results of molecular docking study (Table 3) showed that the logarithm of free binding energy of dihydrotanshinone (-9.1 kcal/mol) to the active cavity was comparable to quinidine (-9.0 kcal/mol) and greater than dextromethorphan (-7.4 kcal/mol) and tanshinone I (-8.0 kcal/mol). Quinidine binds to the same biding site on CYP2D6 as the model probe substrate dextromethorphan to cause competitive inhibition for the catalytic site (McLaughlin et al., 2005). Dihydrotanshinone showed similar affinity to quinidine in binding to the catalytic site on CYP2D6. Tanshinone IIA (-5.9 kcal/mol) and cryptotanshinone (-5.3 kcal/mol) showed much lower affinity than dextromethorphan and therefore unlikely to compete for the binding site on CYP2D6.
Table 3 Logarithm of free binding energies (kcal/mol) to the active cavity and the Phe120 amino acid residue of human CYP2D6 (PDB code 2F9Q). Active cavity Phe120 residue Cryptotanshinone -5.3 -- Dihydrotanshinone -9.1 -7.6 Tanshinone I -8.0 -5.4 Tanshinone IIA -5.9 -- Quinidine -9.0 -7.0 Dextromethorphan -7.4 -7.6
For better understanding of the protein-ligand interaction, molecular docking models of quinidine and tanshinones were performed. Fig. 8 illustrated the possible binding simulations of protein-ligand interaction. It has been shown that Phe120, G1u216 and Asp301 were key amino acid residues for substrate binding in the active site of CYP2D6 (McLaughlin et al., 2005; Unwalla et al., 2010). Alanine substitution of 216 and 301 played significant roles in binding activity; while alanine substitution of Phe120 had only a minor effect on the inhibition and binding activity. In this study, the interaction of these key amino acid residues with tanshinones was investigated and the results showed that Phe120 was critical for the binding of the tanshinones to the active cavity, but not G1u216 and Asp301. The benzene rings of dihydrotanshinone and tanshinone I provided a Pi-Pi interaction with Phe120, thus contributing to the lower binding energies of -7.6 and -5.4 kcal/mol, respectively. Interestingly, when quinidine interacted with Phel 20, it also showed a high affinity towards CYP2D6 (-7.0 kcal/mol). Tanshinone IIA and cryptotanshinone, with high binding energies in this docking study, did not show any interaction with Phe120 or any other amino acids residues such as G1u216 andAsp301. In summary, Phe120 may offer an optional conformation and lower binding energy due to the Pi-Pi interaction with the benzene rings of the tanshinones.
[FIGURE 7 OMITTED]
In this study, dihydrotanshinone inhibited dextromethorphan O-demethylation in pooled human liver microsomes. The [K.sub.i] value of dihydrotanshinone (2.53 [micro]M) was in the same range to that of quinidine, a specific CYP2D6 inhibitor (0.41 [micro]M). Cryptotanshinone, tanshinone HA and water soluble components such as salvianolic acid B and danshensu did not affect CYP2D6-mediated metabolism of dextromethorphan. Inhibition of dextromethorphan O-demethylase activity by the ethanolic extract of crude Danshen and dihydrotanshinone was concentration-dependent and mode of inhibition was competitive, with [K.sub.i] values of 4.23 [micro]g/ml and 2.53 [micro]M, respectively. A previous study in human liver microsomes showed that cryptotanshinone weakly inhibited human CYP2D6 activity, with a mixed inhibition mode in vitro (Qiu et al., 2008). Previous studies by Li et al. (2006) and Sun et al. (2007) have shown that tanshinones isolated from Danshen were metabolised mainly to hydroxy metabolites although the exact CYPs involved have not been elucidated.
The roles of CYPs in the metabolism of Danshen and its active ingredients have been confirmed in studies in which major tanshinones and different Danshen extracts affected metabolism of various human model CYP1A2, CYP2C9 and CYP3A4 probe substrates in vitro (Wang et al., 2010a) and their rat equivalent CYPs in vitro and in vivo (Wang et al., 2009b, 2010b, 2010c; Wang and Yeung, 2010). The potential of water soluble active ingredients such as danshensu and salvianolic acid B in causing potential herb-drug interaction, with underlying metabolic mechanism(s), is relatively lower than the tanshinones, as shown by this and previous studies. The four major tanshinones showed different modes of inhibition, possibly due to their differences in chemical structure. The structural difference between dihydrotanshinone and tanshinone I, also cryptotanshinone and tanshinone IIA, is only the presence of double bond at C-15 position of the furan ring (Fig. 1). Dihydrotanshinone is a competitive inhibitor of human CYP1A2 and CYP2C9, noncompetitive inhibitor of CYP3A4 and uncompetitive inhibitor of CYP2E1 (Wang et al., 2010a). Noncompetitive inhibition is a result of binding of the inhibitors containing electrophilic groups (imidazole or hydrazine group) to the haem protein of CYP (Lewis et al., 2002). In this study, the inhibition mode of dihydrotanshinone to human CYP2D6 was competitive. Competitive inhibition is a result of competition between probe substrates and substrates with higher affinity or inhibitors which bind to the catalytic site but not metabolized, like quinidine. Therefore, from the enzyme kinetic studies, it is suggested that dihydrotanshinone may bind to the catalytic site and prevent human CYP2D6 from metabolising the probe substrate.
[FIGURE 8 OMITTED]
Molecular docking was performed in this study and showed that dihydrotanshinone can bind to the active cavity of human CYP2D6 with similar high affinity to quinidine, a model CYP2D6 inhibitor, but it did not interact with Asp301 or G1u216 which generally interacted with typical CYP2D6 substrates (Kotsuma et al., 2008; Unwalla et al., 2010). As one of the key amino acid residues, Phe120 was very significant in the selectivity and region-specificity of substrates binding and catalysis in catalytic site of CYP2D6 (Keizers et al., 2004), and it directly prevented quinidine from a productive binding mode to CYP2D6 (McLaughlin et al., 2005). The benzene rings of dihydrotanshinone and tanshinone I provided a Pi-Pi interaction with Phe120, thus contributing to the lower binding energies of -7.6 and -5.4 kcal/mol, respectively. Moreover, since dihydrotanshinone and tanshinone I did not interact with the key residues for substrate binding, including G1u216 and Asp301, they may not be substrates of CYP2D6, but act as the competitive inhibitors in binding to the catalytic cavity with nonproductive biding mode. Accordingly, double bond at C-15 position in the furan ring of tanshinone I might decrease the Pi-Pi interaction with Phe120. Interestingly, when quinidine interacted with Phe120, it also showed a high affinity towards CYP2D6 (-7.0 kcal/mol). It is of interest that cryptotanshinone and tanshinone IIA also showed similar structural difference in the C-15 position but in this study, but cryptotanshinone only showed weak inhibition of CYP2D6. The enzyme kinetic studies confirmed the molecular docking study that dihydrotanshinone can inhibit the metabolism of model CYP2D6 probe substrate. Other tanshinones may contribute to the inhibitory effect of the ethanolic extract of Danshen on CYP2D6-mediated metabolism.
Given that both the water-soluble (salvianolic acids and danshensu) and lipophilic compounds (tanshinones) possess diverse pharmacological effects, an important issue would be the ways in which Salvia miltiorrhiza extracts are prepared. The composition of the water-soluble and lipophilic compounds present in different extracts of Salvia miltiorrhiza would be expected to differ in water-, alcohol- or alcohol/water-extracts. Salvianolic acid B and danshensu are the major ingredients in water extracts of Salvia miltiorrhiza while the tanshinones are most abundant in alcohol extract. Taken together the results from previous studies and the current study with dextromethorphan, it is clear that tanshinone-rich Danshen preparations may be more important in the herb-drug interaction potential. However, the significance of such metabolic interaction should be confirmed with in vivo studies, preferably combined PD-PK studies.
The authors gratefully acknowledge the supply of Danshen extract from Winsor Health Products Ltd. (Hong Kong). The work described in this paper was partly supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region, China (Project no. CUHK4517/06M).
Chan, K., Lo, A.C., Yeung, J.H., Woo, K.S., 1995. The effects of Danshen (Salvia miltiorrhiza) on warfarin pharmacodynamics and pharmacokinetics of warfarin enantiomers in rats. J. Pharm. Pharmacol. 47, 402-406.
De Gregori, M., Allegri, M., De Gregori, S., Garbin, G., Tinelli, C., Regazzi, M., Govoni, S., Ranzani, G.N., 2010. How and why to screen for CYP2D6 interindividual variability in patients under pharmacological treatments. Curr. Drug Metab. 11, 276-282.
Han, J.Y., Fan, J.Y., Horie, Y., Miura, S., Cui, D.H., Ishii, H., Hibi, T., Tsuneki, H., Kimura, I., 2008. Ameliorating effects of compounds derived from Salvia miltiorrhiza root extract on microcirculatory disturbance and target organ injury by ischemia and reperfusion. Pharmacol. Ther. 117, 280-295.
He, F., Bi, H.C., Xie, Z.Y., Zuo, Z., Li, J.K., Li, X., Zhao, L.Z., Chen, X., Huang, M., 2007. Rapid determination of six metabolites from multiple cytochrome P450 probe substrates in human liver microsome by liquid chromatography/mass spectrometry: application to high-throughput inhibition screening of terpenoids. Rapid Commun. Mass Spectrom. 21, 635-643.
Holbrook, A.M., Pereira, J.A., Labiris, R., McDonald, H., Douketis, J.D., Crowther, M., Wells, P.S., 2005. Systematic overview of warfarin and its drug and food interactions. Arch. Intern. Med. 165, 1095-1106.
Hsu, ES., 2010. A review of granisetron, 5-hydroxytryptamine3 receptor antagonists, and other antiemetics. Am. J. Ther. 17, 476-486.
Izzo, A.A., Di Carlo, G., Borrelli, F., Ernst, E., 2005. Cardiovascular pharmacotherapy and herbal medicines: the risk of drug interaction. Int. J. Cardiol. 98, 1-14.
Johansson, I., Ingelman-Sundberg, M., 2011. Genetic polymorphism and toxicology--with emphasis on cytochrome p450. Toxicol. Sci. 120,1-13.
Keizers, P.H., Lussenburg, B.M., de Graaf, C., Mentink, LM., Vermeulen, N.P., Commandeur, J.N., 2004. Influence of phenylalanine 120 on cytochrome P450 2D6 catalytic selectivity and regiospecificity: crucial role in 7-methoxy-4-(aminomethyl)-coumarin metabolism. Biochem. Pharmacol. 68, 2263-2271.
Kotsuma, M., Hanzawa, H., Iwata, Y., Takahashi, K., Tokui, 'F., 2008. Novel binding mode of the acidic CYP2D6 substrates pactimibe and its metabolite R-125528. Drug Metab. Dispos. 36, 1938-1943.
Lee, W.Y., Zhou, X., Or, P.M., Kwan, Y.W., Yeung, J.H., 2012. Tanshinone I increases CYP1A2 protein expression and enzyme activity in primary rat hepatocytes. Phytomedicine 19, 169-176.
Lewis, D.F., Modi, S., Dickins, M., 2002. Structure-activity relationship for human cytochrome P450 substrates and inhibitors. Drug Metab. Rev. 34, 69-82.
Li, P., Wang, G.J., Li, J., Hao, H.P., Zheng, C.N., 2006. Simultaneous determination of tanshinone HA and its three hydroxylated metabolites by liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 20, 815-822.
Liu, A.H., Lin, Y.H., Yang, M., Sun, J.H., Guo, H., Guo, D.A., 2006. High-performance liquid chromatographic determination of tanshinones in the roots of Salvia miltiorrhiza and related traditional chinese medicinal preparations. J. Pharm. Pharm. Sci. 9, 1-9.
Lo, A.C., Chan, K., Yeung, J.H., Woo, KS., 1992. The effects of Danshen (Salvia miltiorrhiza) on pharmacokinetics and pharmacodynamics of warfarin in rats. Eur. J. Drug Metab. Pharmacokinet. 17, 257-262.
Ma, L., Zhang, X., Guo, H., Gan, Y., 2006. Determination of four water-soluble compounds in Salvia miltiorrhiza Bunge by high-performance liquid chromatography with a coulometric electrode array system. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 833, 260-263.
McLaughlin, LA., Paine, M.J., Kemp, C.A., Marechal, J.D., Flanagan, J.U., Ward, C.J., Sutcliffe, M.J., Roberts, G.C., Wolf, C.R., 2005. Why is quinidine an inhibitor of cytochrome P450 206? The role of key active-site residues in quinidine binding. J. Biol. Chem. 280, 38617-38624.
Prisant, L.M., 2008. Nebivolol: pharmacologic profile of an ultraselective, vasodilatory betal-blocker. J. Clin. Pharmacol. 48, 225-239.
Qiu, F., Zhang, R., Sun, J., Jiye, A., Hao, H., Peng, Y., Ai, H., Wang, G., 2008. Inhibitory effects of seven components of danshen extract on catalytic activity of cytochrome P450 enzyme in human liver microsomes. Drug Metab. Dispos. 36, 1308-1314.
Rowland, P., Blaney, F.E., Smyth, M.G., Jones, J.J., Leydon, V.R., Oxbrow, A.K., Lewis, C.J., Tennant, M.G., Modi, S., Eggleston, D.S., Chenery, R.J., Bridges, A.M., 2006. Crystal structure of human cytochrome P450 2D6. J. Biol. Chem. 281, 7614-7622.
Seeliger, D., de Groot, B.L., 2010. Ligand docking and binding site analysis with PyMOL and Autodock/Vina. J. Comput. Aided Mol. Des. 24, 417-422.
Shi, Z., He, J., Yao, T., Chang, W., Zhao, M., 2005. Simultaneous determination of cryptotanshinone, tanshinone I and tanshinone 11A in traditional Chinese medicinal preparations containing Radix Salvia miltiorrhiza by HPLC. J. Pharm. Biomed. Anal. 37, 481-486.
Sideras, K., Ingle, J.N., Ames, M.M., Loprinzi, C.L., Mrazek, D.P., Black, J.L., Weinshilbourn, R.M., Hawse, J.R., Spelsberg, T.C., Goetz, M.P., 2010. Coprescription of tamoxifen and medications that inhibit CYP2D6. J. Clin. Oncol. 28, 2768-2776.
Sun, J., Yang, M., Han, J., Wang, B., Ma, X., Xu, M., Liu, P., Guo, D., 2007. Profiling the metabolic difference of seven tanshinones using high-performance liquid chromatography/multi-stage mass spectrometry with data-dependent acquisition. Rapid Commun. Mass Spectrom. 21, 2211-2226.
Ueng, Y.F., Kuo, Y.H., Peng, H.C., Chen, T.L., Jan, W.C., Peter Guengerich, F., Lin, Y.L., 2003. Diterpene quinone tanshinone IlA selectively inhibits mouse and human cytochrome p4501A2. Xenobiotica 33, 603-613.
Unwalla, R.J., Cross, J.B., Salaniwal, S., Shilling, A.D., Leung, L, Kao, J., Humblet, C., 2010. Using a homology model of cytochrome P450 2D6 to predict substrate site of metabolism. J. Comput. Aided Mol. Des. 24, 237-256.
Vermes, A., Vermes, I., 2004. Genetic polymorphisms in cytochrome P450 enzymes: effect on efficacy and tolerability of HMG-CoA reductase inhibitors. Am. J. Cardiovasc. Drugs 4, 247-255.
Wang, B., Yang, LP., Zhang, X.Z., Huang, S.Q., Bartlam, M., Zhou, S.F., 2009a. New insights into the structural characteristics and functional relevance of the human cytochrome P450 2D6 enzyme. Drug Metab. Rev. 41, 573-643.
Wang, X., Cheung, CM., Lee, W.Y., Or, P.M., Yeung, J.H., 2010a. Major tanshinones of Danshen (Salvia miltiorrhiza) exhibit different modes of inhibition on human CYP1A2, CYP2C9, CYP2E1 and CYP3A4 activities in vitro. Phytomedicine 17, 868-875.
Wang, X., Lee, W.Y., Or, P.M., Yeung, J.H., 2009b. Effects of major tanshinones isolated from Danshen (Salvia miltiorrhiza) on rat CYP1A2 expression and metabolism of model CYP1A2 probe substrates. Phytomedicine 16, 712-725.
Wang, X., Lee, W.Y., Or, P.M., Yeung, J.H., 2010b. Pharmacokinetic interaction studies of tanshinones with tolbutamide, a model CYP2C11 probe substrate, using liver microsomes, primary hepatocytes and in vivo in the rat. Phytomedicine 17, 203-211.
Wang, X., Lee, WY., Zhou, X., Or, P.M., Yeung, J.H., 2010c. A pharmacodynamicpharmacokinetic (PD-PK) study on the effects of Danshen (Salvia miltiorrhiza) on midazolam, a model CYP3A probe substrate, in the rat. Phytomedicine 17, 876-883.
Wang, X., Morris-Natschke, S.L., Lee, K.H., 2007. New developments in the chemistry and biology of the bioactive constituents ofTanshen. Med. Res. Rev. 27, 133-148.
Wang, X., Yeung, J.H., 2010. Effects of the aqueous extract from Salvia miltiorrhiza Bunge on caffeine pharmacokinetics and liver microsomal CYP1A2 activity in humans and rats. J. Pharm. Pharmacol. 62, 1077-1083.
Wang, X., Yeung, J.H., 2011a. Inhibitory effect of tanshinones on rat CYP3A2 and CYP2C11 activity and its structure-activity relationship. Fitoterapia 82, 539-545.
Wang, X., Yeung, J.H., 2011b. Effects of Salvia milriorrhiza extract on the liver CYP3A activity in humans and rats. Phytother. Res. 25, 1653-1659.
Wang, X., Yeung, J.H., 2011c. Investigation of cytochrome P450 1A2 and 3A inhibitory properties of Danshen tincture. Phytomedicine, doi:10.1016/j.phymed.2011.09.075.
Wu, W.W., Yeung, J.H., 2010. Inhibition of warfarin hydroxylation by major tanshinones of Danshen (Salvia miltiorrhiza) in the rat in vitro and in vivo. Phytomedicine 17, 219-226.
Yu, C.M., Chan, J.C., Sanderson, J.E., 1997. Chinese herbs and warfarin potentiation by idanshere. J. Intern. Med. 241, 337-339.
Zhang, J.L., Cui, M., He, Y., Yu, Hi, Guo, D.A., 2005. Chemical fingerprint and metabolic fingerprint analysis of Danshen injection by HPLC-UV and HPLC-MS methods. J. Pharm. Biomed. Anal. 36,1029-1035.
Zhou, L., Zuo, Z., Chow, MS., 2005. Danshen: an overview of its chemistry, pharmacology, pharmacokinetics, and clinical use. J. Clin. Pharmacol. 45,1345-1359.
Zhou, S.F., 2009. Polymorphism of human cytochrome P450 2D6 and its clinical significance. Part I. Clin. Pharmacokinet. 48, 689-723.
Zhou, S.F., Liu, J.P., Chowbay, B., 2009. Polymorphism of human cytochrome P450 enzymes and its clinical impact. Drug Metab. Rev. 41, 89-295.
Xuelin Zhou, Yan Wang, Penelope M.Y. Or, David C.C. Wan, Yiu Wa Kwan, John H.K. Yeungs *
School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAP, China
* Corresponding author. Tel.: +852 3943 6864; fax: +852 2603 5139. E-mail address: firstname.lastname@example.org (J.H.K. Yeung).
0944-7113/$--see front matter [c] 2012 Elsevier GmbH. All rights reserved.