Selective inhibitory effects of machilin A isolated from Machilus thunbergii on human cytochrome P450 1A and 2B6.
Background: The bark of Machilus thunbergii (Lauraceae) has been used as a folk medicine to treat abdominal pain and distension, and leg edema in Korea. Machilin A (MA), a lignan isolated from Machilus thunbergii, exhibits several biological activities including anti-oxidant and stimulatory effects on cell differentiation and proliferation.
Purpose: Potential drug-interactions with MA via inhibition of cytochrome P450 (CYP) activity in human liver microsomes (HLMs), have not been investigated.
Study design: The inhibitory effects of MA on the activities of CYPs were investigated using cocktail probe substrates in pooled HLMs and on human recombinant cDNA-expressed CYP isoforms.
Methods: The nine CYP-specific substrates were incubated in HLM or recombinant cDNA-expressed CYP 1A1, 1A2 and 2B6 with MA. After incubation, the samples were injected onto a C18 column for liquid chromatography-tandem mass spectrometry analysis. To investigate the binding poses between MA and CYP, we carried out structure-based docking simulations by using software and scripts written in-house (ALIS-DOCK; Automatic pLatform for Iterative Structure-based DOCKing).
Results: MA strongly inhibited CYPlA2-mediated phenacetin O-deethylation and CYP2B6-mediated bupropion hydroxylation with IC50 values of 3.0 and 3.9 [micro]M, respectively, while it did not significantly inhibit other CYPs. A Dixon plot indicated that MA competitively inhibits CYP1A2 and CYP2B6 with Ki values of 0.71 and 4.1 [micro]M, respectively.
Conclusion: Overall, this was the first investigation of the inhibitory effects of MA on CYP1A2 and CYP2B6 in HLMs, and it has identified that MA acts via competitive inhibition.
Human liver microsomes
Machilus thunbergii belongs to the Lauraceae family and is one of the most commonly cultivated broad-leaf evergreen trees in Asia. The bark of M. thunbergii has been used as a traditional medicine to treat abdominal pain and distension, and leg edema in Korea (Yu et al. 2000). Lignans, alkaloids, flavonoids, butanolides, and essential oils have been derived from M. thunbergii; some of these compounds are antioxidants with hepatoprotective and anti-bacterial activities (Karikome et al. 1991; Yu et al. 2000), while a few other show inhibitory effects on nitric oxide synthesis in activated macrophages (Kim and Ryu 2003) and neuroprotective activity against glutamate-induced neurotoxicity (Ma et al. 2004). Machilin A (MA), one of the lignans isolated from M. thunbergii, showed biological activities, including stimulation of osteoblast differentiation via activation of p38 mitogen-activated protein (MAP) kinases in an in vitro osteoblast differentiation model (Lee et al. 2007; Lee et al. 2009) and antioncogenic properties (Lee et al. 2004; Park et al. 2004).
Cytochrome P450 (CYP) is the largest groups of metabolic systems in the liver responsible for the metabolism of therapeutic drugs (Rendic 2002). The regulation of drug-metabolizing enzymes is a major cause of numerous drug-drug and herb-drug interactions (Guengerich et al. 1997). Moreover, the modulation of drugmetabolizing enzymes is an important factor in determining adverse drug effects during cancer chemotherapy (Chen et al. 2010; Rochat 2005). As such, CYP modulation is of considerable clinical importance and is known to occur through enzyme induction and direct inhibition.
In previous studies, lignans containing the methylenedioxyphenyl moiety showed inhibition of CYP activity (Lee et al. 2010). For example, piperonyl butoxide, isosafrole, kavalactones, podophyllotoxin, and epipodophyllotoxin are involved in the inhibition of CYP activity (Mathews et al. 2002; Pastrakuljic et al. 1997; Song et al. 2011; Usia et al. 2005). The presence of two methylenedioxyphenyl groups in MA may exert the desired effects on the enzyme activity of CYPs (Fig. 1). Until now, no metabolism studies have confirmed the effect of MA on CYP activity. Hence, the present study was conducted to investigate the potential inhibitory effects of MA on CYP activity in pooled human liver microsomes (HLMs) and on a human recombinant cDNA-expressed CYP isoform.
Materials and methods
Chemical reagents and enzymes
MA (chemical purity, 99.8%) was isolated from M. thunbergii. Pooled HLMs (BD UltraPool[TM] HLM 150[R], mixed gender) and human recombinant cDNA-expressed CYP 1A1,1A2, and 2B6 were obtained from Corning[R] Gentest[TM] (Woburn, MA). Glucose 6-phosphate (G6P), glucose 6-phosphate dehydrogenase (G6PDH) and [beta]-nicotinamide adenine dinucleotide phosphate ([beta]-NADPH) were obtained from Sigma (St. Louis, MO). All other chemicals were of analytical grade and were used in the form they were received.
CYP enzyme assays
The inhibitory effects of MA on the metabolism of nine CYP-specific substrates are listed in Table 1 (FDA Draft guidance, 2006). All incubations were performed in duplicate, and the data is presented as means. Briefly, each reaction was performed with 0.5 mg/ml pooled HLMs. The incubation medium consisted of 0.1 M potassium phosphate buffer (pH 7.4) containing MA, cocktail probe substrates, and an NADPH-regenerating system (NGS, including 0.1 M G6P, 1.0 U/ml G6PDH, and 10 mg/ml [beta]-NADPH) in a final incubation volume of 0.1 ml. After a mixture with MA at final concentrations of 0-100 [micro]M was preincubated for 15 min with cocktail substrates, the reaction was initiated by adding NGS. After 60 min incubation at 37[degrees]C the reaction was terminated by adding 100 pd of acetonitrile containing 0.1% formic acid and 5 [micro]l 10 [micro]M reserpine in methanol as an internal standard solution. After mixing and centrifuging at 13,000g for 10 min, a 10 [micro]l aliquot was injected onto a C18 column for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.
Determination of the inhibitory mode of MA
To investigate the inhibitory modes of CYP1A2 and 2B6 inhibition in response to MA, HLMs (0.5 mg/ml) were preincubated for 0-20 min in the absence of NGS in 0.1 M potassium phosphate buffer (pH 7.4). After preincubation, phenacetin (80 [micro]M) or bupropion (50 [micro]M) was added and incubated for 60 min at 37 [degrees]C. Furthermore, to characterize the mode of inhibition of CYP1A2 by MA, 0.5 mg/ml HLMs were incubated with MA at 0, 1.5, 3, or 6 [micro]M in 0.1 M potassium phosphate buffer (pH 7.4) for 60 min at 37 [degrees]C. Phenacetin was used as a probe substrate at 20,40, or 80 [micro]M. MA at O, 1.6,3.9, and 7.8 [micro]M was incubated to characterize the mode of inhibition of CYP2B6 by MA; bupropion was used as a probe substrate at 25,50, or 100 [micro]M.
Inactivation of human recombinant cDNA-expressed CYP1A1,1A2, and CYP2B6 by MA
To confirm the selective inhibition of CYP1A and 2B6 isoforms by MA, 10 pmol of human recombinant cDNA-expressed CYP1A1,1A2, or 2B6 was incubated with 0.1 -100 [micro]M MA and NGS for 60 min at 37[degrees]C after the addition of 80 pM phenacetin as a selective CYP1A substrate and 50 [micro]M bupropion as a selective CYP2B6 substrate. CYP1A1 and 1A2 activities were determined by phenacetin O-deethylation and CYP2B6 activity was determined by bupropion hydroxylation.
All measurement were performed using an Accela[TM] LC system coupled with a TSQ. Vantage triple quadrupole mass spectrometer (Thermo Fisher Scientific Inc., USA) equipped with a HESI-II spray source. In the multiple reactions-monitoring mode, electrospray ionization was performed in positive mode at a spray voltage of 3500 V, and hydroxyl chlorzoxazone was performed in negative mode. Nitrogen was used as a sheath and auxiliary gas at optimum values of 45 and 20 (arbitrary units), respectively. Vaporizer and capillary temperatures were 150 and 300[degrees]C, respectively. For LC analysis an inertsil[R] ODS-2,3-[micro]m column was used (2.1 x 150 mm, GL science). A gradient program was prepared using the linear gradient method from 5:95 to 95:5 for 7 min, and continued at 95:5 for 5 min with a flow rate of 220 [micro]l/min (100% LC grade acetonitrile containing 0.1% formic acid: 100% LC grade water containing 0.1% formic acid).
To determine the binding poses between MA and CYP, we carried out structure-based docking simulations. As template structures for CYP1A1 and CYP1A2, we employed X-ray crystal structures with the Protein Data Bank codes 4I8V (Walsh et al, 2013) and 2H14 (Sansen et al. 2007), respectively. Both structures contain [alpha]-naphthoflavone as an inhibitor. The coordinates of 2HI4 were first aligned with 4I8V for comparison. Following determination of the protonation states of the ionizable amino acids using the PDB2PQR server (Dolinsky et al. 2004), we parameterized the coordinates for docking simulations using AMBER ff99SB-ILDN (Lindorff-Larsen et al. 2010) and Amber compatible heme force-fields (Shahrokh et al. 2012). The antechamber program of the Amber package was used to prepare a force field for MA by semi-quantum calculation (Case et al. 2005). Thirty different conformers of the ligand were generated using the Sander program (Case et al. 2005) with the force-field, these were used for ensemble-based docking with DOCK version 6.6 (Brozell et al. 2012). After obtaining 30 ligand structures per protein by docking, using the anchor-and-grow algorithm, we clustered the resulting conformers based on structural similarities that were quantified by pairwise root mean square deviation values. We chose the centroid conformers in the most populated clusters as a docked pose. The lowest energies in each cluster were selected as the binding energy. Software and scripts (ALIS-DOCK; Automatic pLatform for Iterative Structure-based DOCKing) written in-house were used to automate all of the procedures.
All data are presented as means after duplicated incubations, and [IC.sub.50] values were obtained using percent activity versus log [I] concentration plots. Kinetic parameters were estimated by curve fitting using SigmaPlot (version 12.0, Systat Software, Inc.).
Inhibitory effects of MA on human liver microsomes
To investigate the inhibitory effects of MA on the activities of nine CYP isoforms, we performed CYP inhibition assays using a cocktail of probe substrates and an LC-MS/MS system. As shown in Table 1, MA strongly inhibited CYP1 A2-mediated phenacetin O-deethylation with an [IC.sub.50] value of 3.0 [micro]M and CYP2B6-catalyzed bupropion hydroxylation with an [IC.sub.50] value of 3.9 [micro]M in pooled HLMs. Therefore, MA selectively inhibited CYP1 A2-catalyzed phenacetin O-deethylation and 2B6-catalyzed bupropion hydroxylation; however, other CYPs were not significantly inhibited by MA.
Mode of inhibition of MA on CYP1A2 and CYP2B6
The [IC.sub.50] values of MA for CYP1A2 and 2B6 activities were determined with or without microsomal preincubation for 15 min at 37[degrees]C to investigate the inhibitory mechanism of MA. When there was no preincubation, the [IC.sub.50] values of MA for CYP1A2 and 2B6 were 3.0 and 3.9 [micro]M, respectively, whereas the [IC.sub.50] values were 3.2 and 6.0 [micro]M, respectively, after preincubation with MA (Table 1). Although the [IC.sub.50] values changed, the shifts were not significant. In addition, when HLMs were preincubated with MA for 0-20 min, MA showed strong dose-dependent inhibition of CYP 1A2-catalyzed phenacetin O-deethylation (Fig. 2A) and CYP2B6-catalyzed bupropion hydroxylation (Fig. 2B) in HLMs, instead of time-dependent inhibition. It is suggested that the mechanism of inhibition might be reversible. To further investigate the mechanism of CYP1A2 and 2B6 inhibition by MA in HLMs, Dixon plots were obtained by using a kinetic study of CYPlA2-catalyzed phenacetin O-deethylation (Fig. 3A) and CYP2B6-catalyzed bupropion hydroxylation (Fig. 3B) in the presence of MA at 0,1, 5, or 10 [micro]M. MA was a competitive inhibitor and the [K.sub.i] values calculated from a secondary plot were 0.71 [micro]M for CYP1A2 and 4.1 [micro]M for CYP2B6.
Selective inhibition of MA on CYPIA and CYP2B6
To confirm the selective inhibition of CYP1A1, 1A2, and 2B6 by MA, MA was incubated with human recombinant cDNA-expressed CYP1A1,1A2, and 2B6, respectively. MA decreased CYP1A1--and 1A2-catalyzed phenacetin O-deethylase activity with 1C50 values of 1.5 and 1.7 [micro]M, respectively, showing a non-selective inhibitory effect between CYP1 Al and 1A2 (Fig. 4A). In addition, MA decreased CYP2B6-catalyzed bupropion hydroxylation activity with an [IC.sub.50] value of 2.6[micro]M in human recombinant cDNA-expressed CYP2B6 (Fig. 4B).
Crystal structure of CYPIA in complex with MA
We revealed the binding poses between MA, CYP1A1, and 1A2 as shown in Fig. 5. For comparison, [alpha]-naphthoflavone, a known CYPIA inhibitor, was also determined (Fig. 5A). There was a difference in the docking scores of MA against CYP1A1 and CYP1A2, with values of -62.29 and -41.38 (kcal/mol), respectively, but the binding poses were quite similar (Fig. 5B). In particular, the positions and orientation toward the heme moieties were almost identical, while some diversity existed in the region away from the heme. These data are consistent with the experimental results. The crystal structure of MA with CYP2B6 was not calculated because the structure of CYP2B6 was not yet set.
Herbal extracts are used as alternative treatments for a number of ailments, but drug-interactions with herbs such as kava, St. John's wort, garlic, ginseng, and Ginkgo biloba are frequently reported (Cupp 1999). Recently, multi-drug therapies including herb-drug therapy have become common in patients with various complications; however, these therapies can induce drug interactions owing to abnormal alterations in CYP activity, which can lead to serious adverse drug reactions (ADR) (Lazarou et al. 1998). Pharmacological activity is achieved when the active components reach and sustain appropriate levels at their sites of action. We were concerned about the involvement of CYP enzymes in metabolism-based herb-drug interactions and the importance of mechanism-based understanding to avoid potential ADR. In the present study, we investigated the potential inhibitory effect of MA on CYP activity in pooled HLMs. MA selectively and competitively inhibited CYP1A-catalyzed phenacetin O-deethylation with a [K.sub.i] value of 0.71 [micro]M and CYP2B6-catalyzed bupropion hydroxylation with a [K.sub.i] value of 4.1 [micro]M in HLMs.
CYP1A1 and 1A2 are involved in the bioactivation and detoxification of carcinogens (Perera et al. 2012). Moreover, 20% of long-term pharmacotherapy drugs are metabolized by CYP1A2, including those used for the treatment of schizophrenia and depression (Bertilsson et al. 1994; Brosen et al. 1993; Wang and Zhou 2009).
In the present study, we compared the inhibitory effect of MA to anaphthoflavone, a known competitive inhibitor of CYP1A2 with an [IC.sub.50] value of 0.01-0.02 [micro]M, on phenacetin O-deethylase activity in HLMs, which also exhibited an [IC.sub.50] value of 0.01 [micro]M in our study (Table 1) (Tassaneeyakul et al. 1993; Zhou et al. 2009). Although it is known that a-naphthoflavone has approximately 10 fold more inhibition potential on CYP1A2 than CYP1A1, MA showed a non-specific inhibitory effect on both CYP1A1 and 1A2 in human recombinant cDNA-expressed CYP1A1 and 1A2 (Tassaneeyakul et al. 1993). The volume of the active site cavity in CYP1A2 is larger than CYP2A6 and smaller than CYP3A4 and 2C8 (Sansen et al. 2007). Substrates or competitive inhibitors of CYP1A2 contain a planar ring to fit the narrow and planar active site of the enzyme (Zhou et al. 2009). Therefore, the inhibitory effect of MA on CYP1A1 and 1A2 could be demonstrated using the 3D structure to show the interaction of MA with CYP1A1 and 1A2 (Fig. 5).
In addition, MA also inhibited CYP2B6 in HLMs and human recombinant cDNA-expressed CYP2B6 (Fig. 4). CYP2B6 is expressed in the liver and metabolizes approximately 8% of clinical drugs (n > 60) and endogenous materials (Mo et al. 2009). Recently the total hepatic CYP content of CYP2B6 was demonstrated to be 2-10% using specific antibodies; the interest in CYP2B6 research has increased from an ever-increasing substrate list for this enzyme (Wang and Tompkins, 2008). Therefore, CYP2B6 inhibition by MA might be important for blocking CYP-related carcinogenesis and/or producing potential herb-drug interactions with substrates that undergo CYP2Bmediated metabolism.
Taken together, the results of this study indicate that MA could be used as a novel inhibitor of CYP1A and CYP2B6 enzymes in HLMs for studies of CYP-dependent xenobiotic metabolism. In conclusion, we conducted the first investigation of potential drug-drug interactions associated with MA by evaluating the inhibitory effects of MA on CYP in HLMs. The results of this study showed that MA selectively inhibits CYP1A2 and CYP2B6 in HLMs by competitive inhibition.
Received 19 August 2014
Revised 5 March 2015
Accepted 26 March 2015
Conflict of interest
We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (grant no. All 2026)
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Sunju Kim, Jihye You, Hyun Gyu Choi, Jeong Ah Kim, Jun-Goo Jee, Sangkyu Lee *
College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daehak-ro 80, Daegu 702-701, South Korea
* Corresponding author. Tel.: +82 53 950 8571; fax; +82 53 950 8557. E-mail address: firstname.lastname@example.org, email@example.com (S. Lee).
Table 1 Inhibition of nine cytochrome P450 (CYP)-catalyzed activities by machilin A (MA) in pooled human liver microsomes (HLMs). Substrate reaction probes CYP450 Substrate isoforms conc. ([micro]M) Phenacetin O-deethylation CYP1A2 80 Coumarin 7-hydroxylation CYP2A6 2.0 Bupropion hydroxylation CYP2B6 50 Amodiaquine N-deethylation CYP2C8 5 Diclofenac 4'-hydroxylation CYP2C9 10 Omeprazole 5-hydroxyladon CYP2C19 20 Dextromethorphan O-deethylation CYP2D6 5 Chlorzoxazone 6-hydroxylation CYP2E1 50 Midazolam 1-hydroxylation CYP3A4 2.5 [alpha]-Naphthoflavone (a) CYP1A2 -- Substrate reaction probes [IC.sub.50] ([micro]-M) Without With preincubation preincubation Phenacetin O-deethylation 3.0 3.2 Coumarin 7-hydroxylation >100 >100 Bupropion hydroxylation 3.9 6.0 Amodiaquine N-deethylation >100 >100 Diclofenac 4'-hydroxylation >100 >100 Omeprazole 5-hydroxyladon 31.6 41.1 Dextromethorphan O-deethylation 26.0 36.2 Chlorzoxazone 6-hydroxylation >100 >100 Midazolam 1-hydroxylation >100 >100 [alpha]-Naphthoflavone (a) 0.01 -- To determine the inhibitory effects of MA on the activities of nine CYPs, a cocktail probe was incubated in HLMs after preincubation for 15 min with 0-100 [micro]M MA. The data shown represent the means of duplicate experiments. (a) Positive control as known competitive inhibitor of CYP1A2.