Selective Inhibition of Bakuchicin Isolated from Psoralea corylifolia on CYP1A in Human Liver Microsomes

Bakuchicin is a furanocoumarin isolated from Psoralea corylifolia and shows several biological activities. Although there have been studies on the biological effects of bakuchicin, its modulation potency of CYP activities has not been previously investigated. Here, we investigated the inhibitory effects of bakuchicin on the activities of CYP isoforms by using a cocktail of probe substrates in pooled human liver microsomes (HLMs) and human recombinant cDNA-expressed CYP. Bakuchicin strongly inhibited CYP1A-mediated phenacetin O-deethylation with an IC50 value of 0.43 μM in HLMs. It was confirmed by human recombinant cDNA-expressed CYP1A1 and CYP1A2 with a K i value of 0.11 μM and 0.32 μM, respectively. A Lineweaver-Burk plot indicated that the inhibition mechanism of bakuchicin was competitive inhibition. Overall, this is the first study to investigate the potential CYP1A1 and CYP1A2 inhibition associated with bakuchicin and to report its competitive inhibitory effects on HLMs.


Introduction
Cytochrome P450 (CYP) is a group of heme-containing enzymes embedded essentially in the lipid bilayer of the endoplasmic reticulum, and it acts in the metabolism of many drugs, steroids, and carcinogens [1]. They mainly contribute to the biotransformation of xenobiotics from a hydrophobic state to a more readily extractable hydrophilic form [2]. The regulations of CYP activities are the major cause of drug-drug or herb-drug interactions [3]. Moreover, the control of drugmetabolizing enzymes frequently causes adverse drug effects during cancer therapy [4].
The CYP1A subfamily consisted of two members CYP1A1 and CYP1A2. CYP1A is one of the important CYP subfamilies in humans and plays a role in the metabolism of some clinically used drugs including theophylline, clozapine, tacrine, and foodborne procarcinogens [5]. The CYP1A subfamily consisted of two members CYP1A1 and CYP1A2. CYP1A1 is found mainly in extra hepatic tissues such as lung, gastrointestinal tract, placenta, and skin [6]. CYP1A1 is not significantly expressed in the liver, whereas CYP1A2 is expressed mainly in the liver [7]. However, polycyclic aromatic hydrocarbons (PAHs) significantly induce the expression of CYP1A1 in the liver, lung, kidney, and gut of rats [8]. CYP1A1, a well-known aryl hydrocarbon hydroxylase, is involved in the metabolic activation of environmental procarcinogens. CYP1A2, one of the major CYPs in the human liver (∼13%), responds to the metabolism of a variety of arylamines and heterocyclic arylamines in therapeutic drugs [9,10]. In particular, the CYP1A subfamily has historically been thought to play important roles in the bioactivation of many procarcinogens including PAHs, heterocyclic aromatic amines, and mycotoxins [9,11]. Specific inhibition of CYP1A has been focused on the development of potential cancer preventative and therapeutic agents [6,9,12].
Bakuchicin is a furanocoumarin isolated from the seeds of Psoralea corylifolia which have been used in oriental medicine [13] (Figure 1). It has several biological effects including antivasorelaxant effect mediated by nitric oxidecyclic GMP (NO-cGMP) signaling in rat aortic rings [14], antibacterial activity against a number of bacteria [15], and antitumor effect induced by strong DNA polymerase inhibition [16]. Although pharmacological effects of bakuchicin have previously been studied and selective inhibitory effects of its structurally similar analogs, psoralen and isopsoralen, have been investigated, the effects of its CYP activities have not been studied until now ( Figure 1) [17]. Here, we investigated the selective inhibitory effect of bakuchicin on CYP1A in human liver microsomes (HLMs) and demonstrated its competitive inhibition.

Inhibitory Effects of Bakuchicin HLMs.
To investigate the inhibitory effects of bakuchicin on the activity of 9 CYP isoforms, we conducted CYP inhibition assays using a cocktail of probe substrates coupled with an LC-MS/MS system. The IC 50 values of bakuchicin on the 8 CYP activities were determined with or without microsomal preincubation for 15 min at 37 ∘ C to predict the inhibitory mechanism of bakuchicin. CYP2B and CYP2D6 were inhibited, but CYP1A was most strongly inhibited in HLMs compared to other CYPs at the IC 50 value of 0.43 M (Table 1). Therefore, bakuchicin selectively inhibited CYP1A-catalyzed phenacetin O-deethylation in HLMs.

Competitive Inhibition of CYP1A by Bakuchicin in HLMs.
In Table 1, the IC 50 of bakuchicin on CYP1A-catalyzed phenacetin O-deethylation activity was determined with or without preincubation for 15 min at 37 ∘ C to investigate the inhibitory mechanism. When there was no preincubation, the IC 50 value of bakuchicin on CYP1A was 0.43 M, whereas the IC 50 value was 0.22 M after preincubation either with or without -NADPH in pooled HLMs (Table 1). No significant change of IC 50 after preincubation with or without -NADPH indicates that bakuchicin is a reversible inhibitor of CYP1A-catalyzed phenacetin O-deethylation.
In addition, to investigate the mechanism responsible for inhibition of CYP1A-catalyzed phenacetin O-deethylation by bakuchicin, CYP1A inhibitory activity was determined depending on preincubation time for 0-20 min at 37 ∘ C in HLMs. Bakuchicin strongly and dose-dependently inhibited CYP1A-catalyzed phenacetin O-deethylation, but not timedependent inhibition in HLMs (Figure 2). The mechanism of CYP1A inhibition of bakuchicin was determined using a Lineweaver-Burk plot (Figure 3). The kinetics were obtained by measuring CYP1A-catalyzed phenacetin O-deethylation in the presence of bakuchicin at 0, 0.2, 0.4, or 0.8 M (Figure 3(a)). Bakuchicin was found to be a reversible competitive inhibitor and the value (0.09 M) was calculated from a secondary plot for CYP1A (Figure 3(b)). phenacetin O-deethylase activity which was 7-fold higher than the value of CYP1A1 (Figure 4).

Selective Inhibition of
The inhibition modes of bakuchicin on CYP1A1 and CYP1A2 were confirmed by kinetic studies in human recombinant cDNA-expressed CYP1A1 and CYP1A2 ( Figure 5). Lineweaver-Burk plots were obtained by a kinetic study of human recombinant cDNA-expressed CYP1A1 and CYP1A2catalyzed phenacetin O-deethylation in the concentration of bakuchicin at 0, 0.2, 0.4, or 0.8 M, respectively ( Figure 5(a)). The pattern showed that bakuchicin is a competitive inhibitor, and its value on CYP1A1 calculated from a secondary plot was found to be 0.11 M, whereas its value on CYP1A2 was 3-fold of that at 0.32 M (Figure 5(b)). This showed that bakuchicin inhibited CYP1A1 more selectively than CYP1A2 in HLMs.

Discussion
The CYP1A subfamily is well known to be involved in the bioactivation and detoxification of carcinogens [19]. CYP1A1 also plays an important role in the biotransformation of melatonin, arachidonic acid, eicosapentaenoic acid, dietary substrates, and environmental carcinogens [20][21][22]. CYP1A2 related to arylamine oxidation is important for demethylation of methylxanthines such as caffeine and theophylline, O-demethylation of naproxen, hydroxylation of tacrine, ropivacaine, R-warfarin and caffeine, dealkylation of phenacetin, and demethylation of tricyclic antidepressants [10,23,24]. Moreover, CYP1A2 is involved in the metabolism of medicines about 20% of clinically metabolized drugs such as those used in long-term treatment of schizophrenia and depression [25][26][27]. Because CYP1A is widely involved in drug metabolism, selective inhibition of CYP1A1 and CYP1A2 is important to prevent CYP-related drug-drug interactions with substrates that mostly undergo CYP1Amediated metabolism.
In addition, CYP1A1 catalyzes the metabolic activation of carcinogenic PAHs to reactive electrophiles in rodents [28]. For examples, benzo[a]pyrene, dibenzanthracene, and benzo[h]fluoranthene are activated by CYP1A1 to form epoxide intermediates, which are further activated to form diol epoxides [29]. The metabolic activation of heterocyclic amines is also metabolized by CYP1A1 [10]. The hydroxylation at position N2 of 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) by CYP1A1 initiates PhIP-DNA adduct formation, which induces carcinogenesis [11]. Therefore, the inhibition of CYP1A1 activity in order to inhibit the formation of CYP1A1-mediated carcinogenic reactive intermediates has been the focus on novel strategies in chemoprevention.
The inhibitory effect of bakuchicin on CYP1A was predicted from inhibitory effect of structural analogs, psoralen, and isopsoralen [17]. The psoralen and isopsoralen were identified as reversible and time-dependent inhibitor in human and rat liver microsomes. In the present study, we investigated selective inhibitory effects of bakuchicin on CYP1A in pooled HLMs. This was confirmed by selective inhibition of CYP1A-catalyzed phenacetin O-deethylation in HLMs, and human recombinant cDNA-expressed CYP1A1 and CYP1A2. In HLMs, bakuchicin showed the pattern of reversible competitive inhibitor with the value of 0.09 M for CYP1A. In addition, the Lineweaver-Burk plot indicated the inhibition mechanism of bakuchicin to be competitive inhibition with values of 0.11 M and 0.32 M in human recombinant cDNA-expressed CYP1A1 and CYP1A2, respectively. Understanding the basic mechanism of herb-drug interactions is useful not only in preventing drug toxicity, but also in developing novel cancer prevention therapies. This study showed that the metabolism of drugs that are CYP1A substrates, especially at high doses or when there is accumulation of the herb, may be important in metabolism-based herb-drug interactions [30,31].
P. corylifolia is an important medicinal plant widely used in traditional medicine in China, India, and the United States as a cardiotonic, vasodilator, pigmentor, antitumor, antibacterial, cytotoxin, or anthelmintic [28]. Bakuchicin (1.51 g) was extracted from P. corylifolia fruit powder (300 g) by ethanol extraction [32]. The concentration of bakuchicin in vivo can depend on variability in the environment, the preparation method, or the patient. Therefore, it is important to identify the modulation effect of bakuchicin on CYP activities in HLMs. In conclusion, we were the first to investigate potential herb-drug interactions associated with bakuchicin by evaluating its inhibitory effects on CYP1A in HLMs. Our study showed that bakuchicin was a strong and selective competitive inhibitor of CYP1A1 and CYP1A2 in HLMs.