Investigation on the metabolic characteristics of isobavachin in Psoralea corylifolia L. (Bu-gu-zhi) and its potential inhibition against human cytochrome P450s and UDP-glucuronosyltransferases

Abstract

Objectives

Isobavachin is a phenolic with anti-osteoporosis activity. This study aimed to explore its metabolic fates in vivo and in vitro, and to investigate the potential drug–drug interactions involving CYPs and UGTs.

Methods

Metabolites of isobavachin in mice were first identified and characterized. Oxidation and glucuronidation study were performed using liver and intestine microsomes. Reaction phenotyping, activity correlation analysis and relative activity factor approaches were employed to identify the main CYPs and UGTs involved in isobavachin metabolism. Through kinetic modelling, inhibition mechanisms towards CYPs and UGTs were also explored.

Key findings

Two glucuronides (G1 - G2) and three oxidated metabolites (M1 - M3) were identified in mice. Additionally, isobavachin underwent efficient oxidation and glucuronidation by human liver microsomes and HIM with CL_int values from 5.53 to 148.79 μl/min per mg. CYP1A2, 2C19 contributed 11.3% and 17.1% to hepatic metabolism of isobavachin, respectively, with CL_int values from 8.75 to 77.33 μl/min per mg. UGT1As displayed CL_int values from 10.73 to 202.62 μl/min per mg for glucuronidation. Besides, significant correlation analysis also proved that CYP1A2, 2C19 and UGT1A1, 1A9 were main contributors for the metabolism of isobavachin. Furthermore, mice may be the appropriate animal model for predicting its metabolism in human. Moreover, isobavachin exhibited broad inhibition against CYP2B6, 2C9, 2C19, UGT1A1, 1A9, 2B7 with K_i values from 0.05 to 3.05 μM.

Conclusions

CYP1A2, 2C19 and UGT1As play an important role in isobavachin metabolism. Isobavachin demonstrated broad-spectrum inhibition of CYPs and UGTs.

Introduction

The seeds of Psoralea corylifolia L. are widely used herbal medicines in clinical practice mainly for the treatment of bone diseases and dietary supplements.^[1] Prenylated flavonoids are responsible for the pharmacological activity and clinical efficacy.^[1] Among them, isobavachin (CAS No. 31524–62-6), one of the most abundant bioactive compounds in seeds of P. corylifolia, attracts tremendous interests because of its significant pharmacological effects. For instance, isobavachin can significantly stimulate osteoblasts proliferation and differentiation.^[2] In addition, isobavachin shows ability to facilitate mouse ES cells differentiating into neuronal cells through protein prenylation, and subsequently phos-ERK activation and the phosp38 off pathway.^[3] Besides, isobavachin could be a promising candidate for further evaluation for PCa prevention or management.^[4] Furthermore, isobavachin possesses cytotoxic effects on H4IIE hepatoma and metabolically poorly active C6 glioma cells.^[5] Owing to these remarkable activities, the in-vivo and in-vitro metabolism of isobavachin needs to be studied and has received increasing attention.

Poor bioavailability of prenylated flavonoids was mainly attributed to their poor intrinsic permeation across the gastrointestinal tract.^[6] Previous studies have proved that glucuronidation and sulfonation were the major clearance routes of these compounds in rats.^[7,8] In addition, after intragastric administration of Psoraleae Fructus extracts (1.2 g/kg), isobavachin exhibited weak exposure (about 13.2 nM), and readily penetrated the blood–brain barrier and distributed almost evenly to the cerebral nuclei.^[9] Nevertheless, the metabolic fates of isobavachin involving in the cytochromes P450 (CYP) and UDP-glucuronosyltransferases (UGT) enzymes still remain unknown. It is necessary to investigate the metabolic characteristics of isobavachin in vivo and in vitro.

On the other hand, the effects of isobavachin on drug metabolism or endogenous metabolism in human have not been well-investigated. Notably, the concomitant use of isobavachin or isobavachin-containing herbal products may bring clinically relevant herb–drug interactions, adverse reactions or even metabolic disorders, especially when isobavachin was co-administrated with those clinical drugs with narrow therapeutic window.^[10–12] Considering the widespread use of Psoraleae Fructus in patients with bone diseases, it is crucial to investigate the potential risks of drug–drug interactions.

The aim of this study was to investigate the metabolite characteristics of isobavachin in vivo and in vitro. Metabolites identification was of great importance for understanding metabolite pathways of isobavachin.^[11] In addition, co-incubation with human liver microsomes (HLM) and HIM was performed to better evaluate the metabolic contribution of CYP and UGT enzyme to the metabolism. Identification of the isoforms involved in isobavachin metabolism is of prime importance for predicting potential drug–drug interactions,^[13] and the results of isozymes identification can be used to guide clinical treatment and avoid side effects. Furthermore, considering that many clinical drugs are metabolized or eliminated by CYP1A2 (9%), 2B6 (2%), 2C8, 2C9 (16%), 2C19 (12%), 2E1 (2%), 3A4 (46%), and UGT1A1 (15%), 1A9, 2B7 (35%);^[14,15] we investigated the inhibitory effects of isobavachin against these enzymes. Hopefully, the preclinical metabolic fates of isobavachin in vivo and in vitro presented in this study will be helpful to reduce the potential risk of drug–drug interactions about CYP or UGT isozymes inhibition.

Materials and Methods

Chemicals and reagents

Isobavachin (purity above 98%) was purchased from Shanghai Winherb Medical Technology Co., Ltd (Shanghai, China). Alamethicin, D-saccharic-1, 4-lactone monohydrate, magnesium chloride (MgCl₂), nicotinamide adenine dinucleotide phosphate (NADPH) and uridine 5′-diphospho-glucuronosyltransferase (UDPGA) were all obtained from Sigma-Aldrich (St. Louis, MO, USA). Specific substrates and their corresponding metabolites including phenacetin, paracetamol, bupropion, hydroxybupropion, paclitaxel, 6α-hydroxy-paclitaxel, tolbutamide, 4-hydroxytolbutamide, mephenytoin, 4-hydroxymephenytoin, chlorzoxazone, 6-hydroxychlorzoxazone, nifedipine, oxidized nifedipine, β-estradiol, propofol and zidovudine were acquired from Aladdin Chemicals (Shanghai, China). β-estradiol-3-O-glucuronide, propofol-O-glucuronide and AZT-N-glucuronide used in this study were from Toronto Research Chemicals (North York, ON, Canada).

Pooled HLM, individual HLM (iHLM), recombinant human CYP isozymes (rCYP1A2, 2B6, 2C8, 2C9, 2C19, 2E1, 3A4) and UGT isoforms (rUGT1A1, 1A9, 2B7) were all provided by Corning Biosciences (Corning, NY, USA). Mice liver microsomes (MLM) and mice intestine microsomes (MIM) were prepared in our laboratory using the differential centrifugation method as described previously.^[16] Other chemicals and reagents were of analytical grade or better.

In-vivo treatment and samples preparation

Male KM mice (SPF grade, 6 – 8-week-old) were obtained from the Experimental Animal Center of Jinan University (Guangzhou, China). Before experiment, the mice were kept in an animal room with temperature (23 ± 2) °C and humidity (60 ± 5) % for a week. And all mice had free access to water and food ad libitum. The animal protocols were approved and conducted in accordance with the guidelines of Laboratory Animal Ethics Committee of Jinan University.

The mice in treated group received an oral dose of isobavachin dissolved in corn oil at 40 mg/kg just like the in-vivo metabolism of corylin.^[17] Blood samples (n = 3) were collected from orbital vein at 0.25, 1 and 6 h post-dosing, and placed at room temperature for 2 h to obtain serum samples after centrifugation at 900g for 10 min. Bile samples (n = 3) were also collected at 4, 8 and 12 h post-dosing under anesthesia operation. Urine and faeces samples (n = 3) were obtained at 0–12 h after oral administration. Fresh liver samples (n = 3) were collected and rinsed with normal saline. The bio-samples were all stored at −80 °C until analysis.

Serum sample (50 μl) was transferred to a 1.5-ml polypropylene tube, and 200 μl of methanol was added with one-step protein precipitation. After vortexing and mixing, samples were centrifuged at 13 800g for 10 min at 4 °C. And then, 4 μl aliquots of the supernatant were withdrawn for ultra-high-performance liquid chromatography (UHPLC)/Q-TOF-MS analysis. Similarly, mice urine (50 μl) and bile (50 μl) were precipitated with methanol at a volumetric sample-to-methanol ratio of 1 : 4. After centrifugation, the resulting supernatants were withdrawn for analysis. Faeces samples were soaked in methanol (1 : 10), while liver samples were homogenized in normal saline (1 : 10). Liver homogenate (50 μl) was pretreated with methanol to precipitin protein. Likewise, the supernatants were obtained for analysis.

Incubation systems

In brief, typical incubation system (100 μl) for phase I metabolism contained Tris-HCl buffer (50 mM, pH = 7.4), MgCl₂ (5 mM), appropriate concentration of HLM (or HIM, MLM, MIM, iHLM, expressed CYP isozymes) and substrates (isobavachin, or corresponding specific substrates).^[17] The reaction was initiated by addition of NADPH (1 mM) after pre-incubation at 37°C for 5 min. After incubation at 37 °C for 60 min, the reaction was terminated by the addition of 100 μl of ice-cold acetonitrile. The supernatant was obtained by centrifugation at 13 800g for 10 min and 8 μl aliquots of that was withdrawn for UHPLC system (Waters, Manchester, UK) analysis. The incubations without NADPH served were considered as control group to confirm that the transformed metabolites were NADPH-dependent.

For glucuronidation assays, a series of isobavachin solutions were incubated in 100 μl Tris-HCl buffer (50 mM, pH = 7.4) containing alamethicin (22 μg/ml), D-saccharic1, 4-lactone (4.4 mM), UDPGA (3.5 mM) and the appropriate concentration of microsomes (HLM, HIM, MLM, MIM, iHLM) or UGTs as described recently.^[18] Similarly, the reaction was terminated by ice-cold acetonitrile (100 μl) after incubation at 37 °C for 60 min. Supernatant (8 μl) was obtained as same as the above for determination by UHPLC system. Control incubations without UDPGA were performed to confirm that the metabolite produced were UDPGA-dependent.

Enzyme kinetics evaluation

Michaelis–Menten equation (1), substrate inhibition equation (2) and Hill equation (3) were fitted to the data of metabolic activities vs isobavachin concentrations.^[17–19] Appropriate models were selected by visual inspection of the Eadie–Hofstee plot.^[17–19] Model fitting and parameter estimation were performed using GraphPad Prism V5 software (SanDiego, CA, USA).

The detailed parameters were as follows. V is the formation rate of produced metabolites, and [S] is the concentration of substrate. V_max is the maximal velocity. K_m is the Michaelis constant, while K_si is the substrate inhibition constant. S₅₀ is the substrate concentration resulting in 50% of V_max, whereas n is the Hill coefficient. The intrinsic clearance (CL_int) values were calculated by V_max/K_m for Michaelis–Menten and substrate inhibition models, while the maximal clearance (CL_max) was obtained using equation (4).^[19]

$$V=\frac{V_{\max }\times [S]}{K_{\text{m}}+[S]}$$ (1)

$$V=\frac{V_{\max }\times [S]}{K_{\text{m}}+[S]\left(1+\frac{[S]}{K_{\text{si}}}\right)}$$ (2)

$$V=\frac{V_{\max }\times {[S]}^n}{S_{50}^n\times {[S]}^n}$$ (3)

$${\text{CL}}_{\max }=\frac{V_{\max }}{S_{50}\frac{n-1}{n{(n-1)}^{1/n}}}$$ (4)

Activity correlation analysis

The identification of expressed CYP or UGT isozymes was also performed by activity correlation analysis as described previously.^[17–19] The metabolic activities of isobavachin and several probe substrates were determined by a bank of individual HLM (n = 9) according to the assay protocol. For the oxidation activities, isobavachin (10 μM), phenacetin (200 μM, probe substrate of CYP1A2) and mephenytoin (50 μM, probe substrate of CYP2C19) were incubated with NADPH-supplemented individual HLM (n = 9), respectively.^[20] Similarly, β-estradiol (50 μM, probe substrate of UGT1A1) and propofol (500 μM, probe substrate of UGT1A9) were incubated with UDPGA-supplemented individual HLM (n = 9), respectively.^[17]

Furthermore, correlation activity analysis was performed between isobavachin mono-oxidation (M1), phenacetin-N-deacetylation and mephenytoin-4-hydroxylation, respectively. Likewise, correlation analysis was also investigated between isobavachin glucuronidation, β-estradiol-3-O-glucuronidation and propofol-O-glucuronidation. These correlation (Pearson) analyses were performed using GraphPad Prism V5 software.

Contribution of CYP isoforms

The contribution of individual CYP enzyme to the hepatic metabolism of substrates in HLM was evaluated by the relative activity factor (RAF) approach as described previously.^[18,21,22] The RAF value was calculated using equation (5). The relative amount of metabolic activities of isobavachin in HLM attributed to an expressed CYP enzyme (supersome) was estimated by multiplying the metabolic activities (i.e. the intrinsic clearance) derived with this enzyme by the corresponding RAF. The RAFs were derived for expressed CYP enzyme using the well-recognized probe substrates phenacetin and mephenytoin for CYP1A2 and 2C19, respectively. The contribution of individual CYP enzyme was calculated according to equation (6).

$$\text{RAF}=\frac{{\text{CL}}_{\text{int}}\left\{\text{probe}\text{substrate,}\text{HLM}\right\}}{{\text{CL}}_{\text{int}}\left\{\text{probe}\text{substrate,}\text{Supersome}\right\}}$$ (5)

$$\begin{gathered} \text{Con?tri?bu?tion}\text{of}\text{super?some} \\ =\frac{{\text{CL}}_{\text{int}}(\text{probesub?strates,}\text{Super?some})}{{\text{CL}}_{\text{int}}(\text{probesub?strate,}\text{pHLM})}\times \text{RAF} \end{gathered}$$ (6)

Inhibitory effects of isobavachin against human CYP and UGT isozymes

As described previously, phenacetin-N-deacetylation (100 μM for phenacetin), bupropion-hydroxylation (100 μM for bupropion), paclitaxel-6α-hydroxylation (60 μM for paclitaxel), tolbutamide-4-hydroxylation (200 μM for tolbutamide), mephenytoin-4-hydroxylation (100 μM for mephenytoin), chlorzoxazone-6-hydroxylation (200 μM for chlorzoxazone) and nifedipine-oxidation (40 μM for nifedipine) have been well-accepted as the specific reactions for CYP1A2, 2B6, 2C8, 2C9, 2C19, 2E1 and 3A4, respectively.^[20,23] The substrates were incubated with CYP isozyme (0.05 mg/ml for CYP1A2 and 3A4; 0.1 mg/mL for CYP2B6, 2C8, 2C9, 2C19 and 2E1) in the absence (control) and presence of isobavachin. Incubation procedures were same as above. Aliquots (4 μl) of the supernatant were further analyzed by UHPLC-TQD-MS.

Similarly, β-estradiol (60 μM), propofol (40 μM) and zidovudine (500 μM) were typically used as the specific probe substrates for UGT1A1, 1A9 and 2B7, respectively.^[24] The protein concentrations in incubations were 0.125, 0.05 and 0.1 mg/ml for UGT1A1, 1A9 and 2B7, respectively. Incubation procedures were same as above, and the supernatant (4 μl) was withdraw for UHPLC-TQD-MS analysis.

The inhibition effects towards human CYP and UGT enzymes were evaluated in the absence (0 μM, control group) or presence (1, 10, 100 μM) of isobavachin. The half-inhibition concentration (IC₅₀) values were determined by non-linear regression analysis.^[24] Based on the IC₅₀ values, the inhibitory effects against CYPs or UGTs were divided into four categories, potent (IC₅₀ values < 1 μM), moderate (IC₅₀ values between 1 and 10 μM), weak (IC₅₀ values over 10 μM) or no inhibition (IC₅₀ values over 100 μM).^[25] Only when the IC₅₀ values were <10 μM, the types of inhibition of isobavachin against corresponding CYP and UGT isozymes were further investigated.

Inhibition kinetic analysis of isobavachin on human CYP and UGT isozymes

The inhibition constant (K_i) values were determined by four concentrations of probe substrates in the absence or presence of inhibitor as described previously.^[23,24] Three kinetic models including competitive inhibition, non-competitive inhibition and mixed-type inhibition were used to calculate the K_i values by non-linear regression using the equation (7), equation (8) and equation (9), respectively.^[26,27] The inhibition kinetic types were assessed by the Akaike information criterion (AIC) and Schwartz information criterion (SC) values.^[26,27] The model with the smallest AIC and SC values was regarded as the best model.

The detailed parameters were as follow. V is the velocity of the reaction. [S] and [I] are the concentrations of probe substrate and inhibitor, respectively. K_i is the constant describing the affinity between the inhibitor and the enzyme. K_m is the substrate concentration at half of the maximum velocity (V_max) of the reaction. The αKi describes the affinity of the inhibitor to the complex of enzyme and substrate; when α is very large (α >> 1), the binding of inhibitor would prevent the binding of substrate, and the mixed inhibition model becomes identical to competitive inhibition.^[26,27]

$$V=\frac{V_{\max }\times [S]}{K_{\text{m}}\times \left(1+\frac{[I]}{K_i}\right)+[S]}$$ (7)

$$V=\frac{V_{\max }\times [S]}{\left(K_{\text{m}}+[S]\right)\times (1+\frac{[I]}{K_i}}$$ (8)

$$V=\frac{V_{\max }\times [S]}{K_{\text{m}}\times \left(1+\frac{[I]}{K_i}\right)+[S]\times \left(1+\frac{[I]}{\alpha K_i}\right)}$$ (9)

Analytical conditions

Chromatographic separation of isobavachin and its metabolites were achieved on a BEH C18 column (2.1 mm × 50 mm, 1.7 mm, Waters, Ireland) using Acquity UHPLC I-Class system (Waters Corporation, Manchester, UK). The mobile phase consisted of water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). The analytes were eluted with the following gradient elution programme: 0 – 2 min, 10 – 50% B; 2 – 3 min, 50 100% B; 3 – 3.2 min, 100% B; 3.2 – 3.5 min, 100 – 10% B; 3.5 – 4.0 min, 10% B. The flow rate was set as 0.5 ml/min, and column temperature was set to 35°C. Isobavachin and its metabolites were detected at 254, 270, 315 and 335 nm, and the sample injection volume was 4 μl.

LC-MS/MS analysis was performed on a UHPLC/Q-TOF-MS system (Waters Corporation, Manchester, UK), equipped with a hybrid quadrupole orthogonal time-of-flight (Q-TOF) tandem mass spectrometer (SYNAPT G2 HDMS, Waters Corporation, Manchester, UK) and an electrospray ionization (ESI) interface. Additionally, positive ionization mode was chosen for sample analysis. The operating parameters were optimized as follows: capillary voltage, −2500 V; sample cone voltage, 40 V; extraction cone voltage, 4 V; source temperature, 100 °C; desolvation temperature, 300 °C; cone gas flow, 50 l/h; and desolvation gas flow, 800 l/h. The full scan mass range was 50–1500 Da. The method employed lock spray with leucine enkephalin (m/z 556.2771 in positive ion mode) to ensure mass accuracy. All data were collected in centroid mode and processed using Masslynx 4.1 software (Waters Corporation, Manchester, UK).

Statistical analysis

Data are presented as the mean ± SD performed (n = 3). Model fitting and parameter estimation were performed by GraphPad Prism V5 software. The differences among treatment and control groups were analyzed by Kruskal–Wallis tests with P < 0.05 (∗) and P < 0.01 (∗∗).

Results

Identification of isobavachin-related metabolites in mice

After intragastric administration of isobavachin (40 mg/kg), a total of one parent compound and five metabolites were detected in mice (Table 1). P0, the parent compound, was unambiguously identified as isobavachin based on the reference standard. It gave the molecular ion at m/z 325.145, and product ions at m/z 269.081, 149.025 and 131.026 (Figure S1A), which kept in line with our previous study.^[7,8] The fragment ion of m/z 269.081 was generated by natural loss of a C4H8. Cleavage of Retro Diels Alder (RDA) is its main characteristic fragmentation of aglycones.^[7,8] The ^1,3A+ ion at m/z 149.025 was identified as the diagnostic ion (Figure S1A). According to the absence or presence of m/z 149.025, we could characterize the oxidation position of isobavachin.

Table 1.

Detailed information for isobavachin and its related metabolites in mice

No.	Time (min)	[M + H]+ ion	Formula	(+) ESI-MS/MS	Identification	Source
P0	2.70	325.145	C20H20O4	325.145, 269.081, 149.025, 131.026	isobavachin	S U B F L
G1	1.77	501.177	C26H28O10	501.177, 325.146, 269.082, 205.086, 149.024	isobavachin-glucuronide	S U B F L
G2	1.89	501.176	C26H28O10	501.176, 325.146, 269.082, 205.086, 149.024	isobavachin-glucuronide	S U B F L
M1	1.88	341.141	C20H20O5	341.141, 323.127, 305.118, 203.071	mono-oxidated isobavachin	S U F
M2	2.16	341.139	C20H20O5	341.139, 285.078, 267.065, 149.024, 131.024	mono-oxidated isobavachin	S U F
M3	2.31	341.138	C20H20O5	341.138, 285.075, 221.084, 165.018	mono-oxidated isobavachin	S U F

B, bile; F, faeces; L, liver; S, serum; U, urine.

Due to different ClogP values,^[17,22,28] two glucuronides, G1 (ClogP = 2.275, Figure S1B) and G2 (ClogP = 2.510, Figure S1C), were identified as isobavachin-7-O-glucuronide and isobavachin-4’-O-glucuronide, respectively. M1 (Figure S1D) was characterized as mono-oxidated isobavachin with the oxidation unit at the isopentenyl group. M2 (Figure S1E) was considered as mono-oxidated product with the oxidation position at the B ring due to the presence of fragment ions at m/z 149.024, 131.024, while M3 (Figure S1F) was also characterized as mono-oxidated isobavachin with the oxidation position at the A ring. The exposure of P0 and its metabolites in mice serum (Figure S2A), urine (Figure S2B), bile (Figure S2C), faeces (Figure S2D) and liver (Figure S2E) indicated that glucuronides were abundant than mono-oxidated products.

Quantification of isobavachin and its related metabolites

Because the reference standards of isobavachin-related metabolites were not commercially available, the quantification of produced metabolites was based on the standard curve of isobavachin according to the assumption that parent compound and its glucuronide have closely similar UV absorbance maxima.^[17–19] In this study, the detection wavelength of isobavachin and its metabolites were 254, 270, 315 and 335 nm. The linear range of isobavachin was 0.04 – 40 μM, with LOD (S/N = 3) and LOQ (S/N = 10) of 0.01 and 0.04 μM, respectively. And acceptable linear correlation (Y = 4672.5X) was confirmed by correlation coefficients (r²) of 0.9992. The accuracy and precision of the intra-day and inter-day error were both <3.6%. There were no matrix effects observed, and no other sample preparation performed except those mentioned in the manuscript.

Mono-oxidation of isobavachin by HLM, HIM, MLM, MIM and CYP enzymes

After incubation of isobavachin with separate pooled HLM, HIM, MLM, and MIM, M1, M2 and M3 all could be detected in mixture. Kinetic profiling revealed the formation of M1 in HLM (Figure S3A) and in MIM (Figure S3D) was fitted to the substrate inhibition equation, while the formation of M1 in HIM (Figure S3B) and MLM (Figure S3C) abided by the classical Michaelis–Menten equation (Figure S3A). Overall, M1 was the major metabolite with the intrinsic clearance (CL_int) values of 59.81, 93.97, 35.46 and 56.88 μl/min per mg in HLM, HIM, MLM and MIM, respectively, and with respective K_m values of 8.50, 2.74, 2.90 and 4.17 μM (Table 2). For the formation of M2 and M3, HIM contributed 19.71 and 43.79 μl/min per mg, respectively, and the CL_int values by MLM were 64.32 (M2) and 47.43 (M3) μl/min per mg (Table 2).

Table 2.

Kinetic parameters derived for the oxidation and glucuronidation of isobavachin by HLM, HIM, MLM, MIM, expressed CYP isozymes and recombinant UGT enzymes, respectively (Mean ± SD). All experiments were performed in triplicate

Enzyme	Meta.	Vmax (pmol/min per mg)	Km (S50) (μM)	Ki (μM)	n	CLint (CLmax) (μl/min per mg)	Model
HLM	M1	508.10 ± 116.90	8.50 ± 2.29	1.67 ± 0.45	N.A.	59.81 ± 21.18	SI
	M2	12.92 ± 0.46	2.34 ± 0.38	N.A.	N.A.	5.53 ± 0.93	MM
	M3	+	+	+	+	+
HIM	M1	257.10 ± 5.62	2.74 ± 0.21	N.A.	N.A.	93.97 ± 7.65	MM
	M2	63.34 ± 6.25	3.21 ± 0.65	77.80 ± 28.51	N.A.	19.71 ± 4.43	SI
	M3	154.00 ± 3.69	3.52 ± 0.29	N.A.	N.A.	43.79 ± 3.72	MM
MLM	M1	102.60 ± 4.56	2.90 ± 0.47	N.A.	N.A.	35.46 ± 5.98	MM
	M2	216.30 ± 25.66	3.36 ± 0.81	65.03 ± 25.52	N.A.	64.32 ± 17.18	SI
	M3	98.22 ± 6.54	2.07 ± 0.32	101.3 ± 32.28	N.A.	47.43 ± 7.99	SI
MIM	M1	237.3 ± 20.1	4.17 ± 0.72	134.8 ± 58.05	N.A.	56.88 ± 10.89	SI
	M2	++	++	++	++	++
	M3	79.5 ± 11.51	6.93 ± 1.68	46.66 ± 16.41	N.A.	11.48 ± 3.25	SI
CYP1A2	M1	311.60 ± 16.01	5.08 ± 0.54	96.53 ± 14.82	N.A.	61.37 ± 7.22	SI
	M2	++	++	++	++	++
	M3	173.30 ± 61.67	19.81 ± 9.47	17.76 ± 8.78	N.A.	8.75 ± 5.21	SI
CYP2C19	M1	2517.0 ± 627.9	56.54 ± 16.3	10.94 ± 3.27	N.A.	44.52 ± 16.97	SI
	M2	++	++	++	++	++
	M3	1300.0 ± 325.0	16.81 ± 5.02	4.48 ± 1.35	N.A.	77.33 ± 30.10	SI
HLM	G1	6948.0 ± 345.6	23.79 ± 1.36	N.A.	1.68 ± 0.15	148.79 ± 9.36	Hill
	G2	1600.0 ± 205.2	73.32 ± 11.19	30.98 ± 5.48	N.A.	21.82 ± 4.35	SI
HIM	G1	1188.00 ± 73.87	9.77 ± 1.11	134.80 ± 26.42	N.A.	121.62 ± 15.80	SI
	G2	394.90 ± 21.03	11.79 ± 1.10	123.60 ± 19.12	N.A.	33.49 ± 3.59	SI
MLM	G1	4022.0 ± 122.6	27.75 ± 1.97	N.A.	N.A.	144.94 ± 11.20	MM
	G2	988.4 ± 179.5	33.39 ± 8.14	48.7 ± 14.79	N.A.	29.60 ± 9.00	SI
MIM	G1	2432.0 ± 381.2	18.71 ± 4.52	84.26 ± 29.37	N.A.	129.98 ± 37.45	SI
	G2	632.3 ± 40.49	10.13 ± 1.16	110.10 ± 19.77	N.A.	62.42 ± 8.21	SI
UGT1A1	G1	7735.0 ± 1186.0	41.84 ± 8.25	40.82 ± 9.70	N.A.	184.87 ± 46.18	SI
	G2	++	++	++	++	++
UGT1A6	G1	3822.00 ± 782.40	67.66 ± 16.32	21.97 ± 5.84	N.A.	56.49 ± 17.87	SI
	G2	341.90 ± 55.04	31.85 ± 6.28	12.71 ± 2.60	N.A.	10.73 ± 2.73	SI
UGT1A7	G1	3593.00 ± 487.20	32.91 ± 5.76	27.54 ± 5.39	N.A.	109.18 ± 24.17	SI
	G2	+	+	+	+	+
UGT1A8	G1	1607.00 ± 241.00	34.71 ± 6.45	17.04 ± 3.36	N.A.	46.30 ± 11.05	SI
	G2	+	+	+	+	+
UGT1A9	G1	3167.00 ± 426.4	15.63 ± 3.12	38.39 ± 8.88	N.A.	202.62 ± 48.80	SI
	G2	++	++	++	++	++
UGT2B4	G1	81.29 ± 2.05	2.66 ± 0.28	N.A.	N.A.	30.59 ± 3.28	MM
	G2	+	+	+	+	+

+, under the limit of quantification; ++, unable to determine the kinetic parameters in the absence of a full kinetic profile; Hill, Hill equation; HIM, human intestine microsomes; HLM, human liver microsomes; Meta., metabolites; MIM, mouse intestine microsomes; MLM, mouse liver microsomes; MM, Michaelis–Menten model; N.A., not available; SI, substrate inhibition model.

Reaction phenotyping results demonstrated that CYP1A1, 1A2, 1B1, 2C8, 2C9, 2C19 and 2D6 all participated in the oxidation of isobavachin (Figure S4A). Because several concentrations of produced metabolites were under the limit of quantification, we were unable to determine the kinetic parameters in the absence of a full kinetic profile. In addition, the formation of M1 and M3 by CYP1A2 (Figure S5A) and CYP2C19 (Figure S5B) all followed the substrate inhibition equation. The CL_int values ranged from 8.75 to 77.33 μl/min per mg as shown in Table 2.

Glucuronidation of isobavachin by HLM, HIM, MLM, MIM and UGT enzymes

Two glucuronides were obviously detected after incubation of isobavachin with HLM, HIM, MLM and MIM. Except the formation of G1 in HLM (Hill equation, Figure S3E) and in MLM (substrate inhibition equation, Figure S3G), the glucuronidation of isobavachin in HLM, HIM, MLM and MIM all followed classical Michaelis–Menten equation (Figure S3E - S3H). G1 was the main glucuronide metabolite with CL_int values ranging from 121.62 to 148.79 μl/min per mg, while the CL_int values for G2 were from 21.82 to 62.42 μl/min per mg, respectively (Table 2).

Furthermore, reaction phenotyping results showed that UGT1A1, 1A6, 1A7, 1A8, 1A9 and 2B4 have an important role in glucuronidation reaction of isobavachin (Figure S4B). Except the formation of G1 by UGT2B4 followed Michaelis–Menten equation (Figure S6F), the glucuronidation of isobavachin by UGT1A1 (Figure S6A), 1A6 (Figure S6B), 1A7 (Figure S6C), 1A8 (Figure S6D) and 1A9 (Figure S6E) all followed substrate inhibition equation. For G1, the CL_int values ranged from 30.59 to 202.62 μl/min per mg (Table 2). Because the glucuronidation for G2 was significantly weak, we cannot get a full kinetic profile of G2 by these expressed UGT enzymes. Moreover, the CL_int values demonstrated that glucuronidation (Figure 1b) was more efficient than oxidation for isobavachin (Figure 1a).

Figure 1.

Intrinsic clearance (CL_int) values for metabolic activities of isobavachin by expressed CYP isoforms (a) and recombinant UGT enzymes (b). N.A., under the limit of quantification or unable to determine the kinetic parameters in the absence of a full kinetic profile. The data were presented as mean ± SD. All experiments were performed in triplicate. (* compared with those of HLM or HIM, ns P > 0.05, *P < 0.05, **P < 0.01). [Colour figure can be viewed at wileyonlinelibrary.com]

Activity correlation analysis

Results demonstrated that the production of M1 was significantly correlated with phenacetin-N-deacetylation with correlation factors (r = 0.721, P = 0.028) in a bank of iHLM (n = 9) (Figure S7A). Similarly, as shown in Figure S7B, the metabolic activity for M1 was also strongly correlation with mephenytoin-4-hydroxylation (r = 0.729, P = 0.026).

In a similar manner, there was significant correlation between the formation of G1 and β-estradiol-3-O-glucuronidation (r = 0.774, P = 0.014) (Figure S7C), and propofol-O-glucuronidation (r = 0.767, P = 0.016) (Figure S7D), respectively. These findings indicated that the contribution of CYP1A2, 2C19 and UGT1A1, 1A9 to oxidation and glucuronidation in liver was appreciable.

Contribution of individual CYP enzyme

The kinetic profiles of phenacetin and mephenytoin were shown in Figure S8A and S8B, respectively. And corresponding parameters were summarized in Table S1. The derived RAF values for CYP1A2 and CYP2C19 were 0.11 and 0.23, respectively (Table S1). The scaled CL_int value was 6.75 (=61.37 × 0.11) μl/min per mg for CYP1A2 that represented 11.3% of the CL_int value (59.81 μl/min per mg) in HLM. The scaled CL_int value was 10.24 (=44.52 × 0.23) μl/min per mg for CYP2C19 that represented 17.1% of the CL_int value (59.81 μl/min per mg) in HLM. Therefore, CYP1A2 and 2C19 contributed 11.3% and 17.1% to the hepatic metabolism of isobavachin, respectively.

Species difference

Mono-oxidation and glucuronidation of isobavachin were determined using HLM, HIM, MLM and MIM (Figure S3 and Table 2). For the formation of M1 (Figure 1a) and G1 (Figure 1b), there was no significant difference between HLM and MLM, or between HIM and MIM. In addition, the formation of M2, M3 and G2 between human and mice microsomes showed marked species difference (Figure 1). Mice could be the appropriate model animals for predicting the formation of M1 and G1 in human.

Effects of isobavachin towards human CYP and UGT isozymes

Notably, isobavachin exhibited negligible or weak inhibitory effects on CYP1A2, 2C8, 2E1 and 3A4, while it displayed significant inhibitory effects on CYP2B6, 2C9, 2C19 and UGT1A1, 1A9, 2B7 (Figure 2). Upon addition of 10 μM isobavachin, the residual activities of these three CYP and these UGT isozymes (CYP2B6, 2C9, 2C19 and UGT1A1, 1A9, 2B7) 30.86%, 1.04%, 0%, 4.58%, 16.51% and 19.32% of the negative control, respectively. In addition, the concentration-dependent inhibition curves of isobavachin against CYP2B6 (Figure S9A), 2C9 (Figure S9B), 2C19 (Figure S9C) and UGT1A1 (Figure S9D), 1A9 (Figure S9E), 2B7 (Figure S9F) were depicted. Furthermore, the inhibition data (Figure 3) were fitted to log (I) and normalized response equations to obtain the IC₅₀ values. The inhibition of these CYP and UGT isoforms by isobavachin was in a dose-dependent manner, and their IC₅₀ values were 5.00, 0.04, 0.27, 1.53, 1.21 and 0.09 μM for CYP2B6, 2C9, 2C19 and UGT1A1, 1A9, 2B7, respectively (Table 3). The inhibition kinetic types and the corresponding inhibition parameters of isobavachin were further evaluated for those CYP and UGT enzymes whose IC₅₀ values were <10 μM.

Figure 2.

The effects of isobavachin against seven expressed CYP isozymes (a) and three common UGT enzymes (b). The specific substrates were incubated at 37 °C in the absence (control) and presence of isobavachin (1, 10 and 100 μM). The data were presented as mean ± SD. All experiments were performed in triplicate determinations. (* compared with those of control, *P < 0.05, **P < 0.01, ***P < 0.001). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3.

The inhibition curves and IC₅₀ values of isobavachin against CYP2B6 (a), CYP2C9 (b), CYP2C19 (c), UGT1A1 (d), UGT1A9 (e), UGT2B7 (f). The data were fit to log (I) and normalized response equations. Each data point represented the mean value ± the SD of triplicate determinations. [Colour figure can be viewed at wileyonlinelibrary.com]

Table 3.

Inhibition parameters of isobavachin against several CYP and UGT isozymes (Mean ± SD). All experiments were performed in triplicate

Isozymes	Substrate	IC50 (μM)	Inhibition type	Ki (μM)	α	R 2	AIC	SC	Selection
CYP2B6	Bupropion	5.00 ± 0.48	Competitive	0.35 ± 0.11	–	0.9111	14.65	17.64
			Non-competitive	1.93 ± 0.18	–	0.9671	−5.19	−2.21	√
			Mixed-type	1.46 ± 0.62	1.49 ± 0.92	0.9678	−3.64	0.34
CYP2C9	Tolbutamide	0.04 ± 0.005	Competitive	0.11 ± 0.02	–	0.9623	−136.47	−133.48
			Non-competitive	0.22 ± 0.02	–	0.9793	−148.48	−145.49	√
			Mixed-type	0.27 ± 0.10	0.70 ± 0.49	0.9797	−146.85	−142.87
CYP2C19	Mephenytoin	0.27 ± 0.07	Competitive	1.55 ± 0.25	–	0.9724	−113.19	−110.20	√
			Non-competitive	3.34 ± 0.49	–	0.9625	−107.08	−104.10
			Mixed-type	1.55 ± 0.53	>>1	0.9724	−111.19	−107.21
UGT1A1	Estradiol	1.53 ± 0.39	Competitive	3.05 ± 0.41	–	0.9857	225.43	228.42	√
			Non-competitive	3.32 ± 0.38	–	0.9854	225.80	228.79
			Mixed-type	3.10 ± 1.23	8.01 ± 2.56	0.9856	227.49	231.47
UGT1A9	propofol	1.21 ± 0.30	Competitive	0.20 ± 0.03	–	0.9676	121.43	124.42
			Non-competitive	0.44 ± 0.04	–	0.9807	111.03	114.02	√
			Mixed-type	0.43 ± 0.14	1.05 ± 0.70	0.9807	113.02	117.01
UGT2B7	zidovudine	0.09 ± 0.01	Competitive	0.02 ± 0.00	–	0.9511	232.70	235.68
			Non-competitive	0.05 ± 0.01	–	0.9766	217.93	220.91	√
			Mixed-type	0.04 ± 0.01	1.80 ± 0.93	0.9781	218.55	222.54

Inhibition types of isobavachin against human CYP and UGT isozymes

Three conventional inhibition models (i.e. competitive, non-competitive and mixed-type) were applied to obtain the respective AIC and SC values. Based on the smallest AIC and SC values (Table 3), isobavachin demonstrated non-competitive inhibition mode against CYP2B6, 2C9 and UGT1A9, 2B7, and competitive inhibition against CYP2C19 and UGT1A1. Furthermore, the Dixon plots for CYP2B6 (Figure 4a), 2C9 (Figure 4b), 2C19 (Figure 4c) and UGT1A1 (Figure 4d), 1A9 (Figure 4e), 2B7 (Figure 4f) also provided strong evidences to support this judgement. And their corresponding K_i values were 1.93, 0.22, 1.55, 3.05, 0.44 and 0.05 μM, respectively (Table 3). These findings suggested that isobavachin was a potent non-selective inhibitor with low K_i values ranged from 0.22 – 1.93 μM for several CYP isozymes and 0.05 – 3.05 μM for three common UGT enzymes.

Figure 4.

The Dixon plots for the inhibition effects of isobavachin towards expressed CYP and UGT isozymes. (a) bupropion-hydroxylation for CYP2B6; (b) tolbutamide-4-hydroxylation for CYP2C9; (c) mephenytoin-4-hydroxylation for CYP2C19; (d) β-estradiol-3-O-glucuronidation for UGT1A1; (e) propofol-O-glucuronidation for UGT1A9; (f) zidovudine-N-glucuronidation for UGT2B7; All data represent the means ± SD of triplicate determinations. [Colour figure can be viewed at wileyonlinelibrary.com]

Discussion

Isobavachin a natural bioactive compound derived from Psoralea corylifolia got increasing attentions on pharmacological activities including anti-osteoporosis, anti-tumour and antioxidant activities.^[1–5] However, only three reports investigated its metabolism and pharmacokinetics in rats after intragastric administration of Psoralea corylifolia extracts or isobavachin-containing herbal preparation so far.^[7–9] The pharmacokinetic behaviours of isobavachin were characterized as poor bioavailability,^[9] and extensive conjugated glucuronides and sulfates in rats.^[7,8] These results inspired us to further explore the metabolic pathways of isobavachin in vivo and in vitro.

Our study demonstrated that isobavachin underwent extensive glucuronidation and oxidation in vivo, producing two glucuronide conjugates (G1 and G2) and three mono-oxidated isobavachin (M1–M3) in mice (Figure 5). As with other preliminary studies on metabolites of other active ingredients in natural medicines, UHPLC/Q-TOF-MS was selected and employed for the identification of metabolites.^[29–31] Nuclear magnetic resonance (NMR) can provide more accurate structure of metabolites that requires a long time to explore the preparation methods and purity.^[19,32] Therefore, considering the analysis efficiency comprehensively, these achievements of metabolites identification could support the preclinical pharmacokinetics studies of isobavachin. Furthermore, in vitro assays (Table 2) by pooled HLM and HIM also provided strong evidences to support the results in vivo. Obviously, the glucuronidation activity was more efficient than the oxidation activity, which kept in line with the results of the analogues of isobavachin, bavachin, corylin, neobavaisoflavone and corylifol A.^{[17,28,31,33]} This may be attributed to the fact that glucuronic acid group was more likely to conjugate in the phenolic hydroxyl group.^[6]

Figure 5.

Metabolic pathways of isobavachin involving in metabolic activities and inhibition effects against expressed CYP and UGT isozymes. [Colour figure can be viewed at wileyonlinelibrary.com]

In addition, reaction phenotyping results demonstrated that CYP1A1, 1A2, 1B1, 2C8, 2C9, 2C19 and 2D6 were all participated in the oxidation of isobavachin (Figure S4A), and UGT1A1, 1A6, 1A7, 1A8, 1A9 and 2B4 were responsible for its glucuronidation reaction (Figure S4B). Activity correlation analysis further confirmed the dominant roles of CYP1A2, 2C19 and UGT1A1, 1A9 in metabolism of isobavachin (Figure S7). RAF approaches also supported these results above (Table S1). The overall findings also indicated that extrahepatic tissue (i.e. intestine, kidney) played important roles in metabolism of isobavachin. In fact, CYP1A1 and 1B1 are absent in HLM, while mainly detected in extrahepatic tissues (i.e. intestine, lung), whereas CYP2C9, 2C19 and 2D6 were expressed both in human liver and intestine.^[34] Furthermore, UGT1A1, 1A6 and 1A9 were detected in liver and extrahepatic tissues,^[35] while UGT1A7 and 1A8 were absolutely expressed in extrahepatic tissues.^[36] These findings provided strong evidences about the roles of extrahepatic tissues (especially intestine) in metabolism of isobavachin.

Characterization of metabolic activities of parent compound is of great significance in the understanding of its pharmacokinetics behaviours and bioavailability.^[22] Oral bioavailability is a key factor in determining the pharmacological actions of phenolic compounds in vivo following oral administration.^[6] It is also worth mentioning that isobavachin underwent massive metabolism in HIM and MIM. The CL_int values of oxidation in HIM were 19.71–93.97 μl/min per mg, while those of glucuronidation in HIM were 33.49–144.94 μl/min per mg (Table 2). These results indicated that human intestine also played a vital role in the metabolism of isobavachin. Similar results were observed in MLM and MIM (Table 2). This suggested that isobavachin may undergo a first-pass metabolism during the absorption process.^[6] Thus, the roles of liver and intestine metabolism in influencing the oral bioavailability of isobavachin should not be underestimated.

In addition, the use of CL_int (= V_max/K_m) as an indicator for the activities of CYP and UGT isozymes is advantageous.^[37] Values of CL_int represent the catalytic efficiency or activity of the CYP or UGT enzyme and are independent with the substrate concentration. Furthermore, CL_int is more relevant in predicting the metabolism or clearance activity in vivo compared with other parameters (i.e. K_m and V_max).

On the other hand, drug–drug interactions due to the inhibition or activation of drug (or herbal compounds) against CYP or UGT enzymes bring significant clinical safety issues.^[10–12] Our results demonstrated that isobavachin displayed potent inhibitory effects against CYP2B6, 2C9 and 2C19 with K_i values of 1.93, 0.22 and 1.55 μM, respectively (Table 3). In clinical, these three CYP enzymes account for the metabolism of 30% drugs whose metabolism is catalyzed by CYP isozymes.^[14] These substrates contain the drugs with narrow therapeutic indices, such as warfarin. Traditionally, the magnitudes of inhibitory interactions mediated by isobavachin were predicted as the ratio of the areas under the plasma concentration-time curve (AUC) in the presence and absence of the isobavachin.^[23,38] However, there are no researches on the human plasma concentration of isobavachin so far. This limits the application of AUC increased (%) values for in-vitro–in-vivo extrapolation of isobavachin.^[23,38] Therefore, it is necessary to perform the pharmacokinetics of isobavachin in rats and mice, and develop a PB-PK model to predict the human pharmacokinetic behaviours of isobavachin in subsequent experiments.^[39,40]

Similarly, our results also showed that isobavachin was a strong inhibitor against UGT1A1, 1A9 and 2B7 with K_i values of 3.05, 0.44 and 0.05 μM, respectively (Table 3). In clinical, propofol is the well-accepted substrate of UGT1A9 and is a commonly used drug for the induction and maintenance of general anesthesia.^[41] In addition, UGT2B7 appeared to be of particular importance in the elimination of several therapeutic drugs (i.e. morphine, zidovudine, lorazepam).^[42] Meanwhile, UGT2B7 is the predominant contributor responsible for glucuronidation of morphine to M6G (morphine-6-glucuronide) and M3G (morphine-3-glucuronide).^[43] M6G exhibits 100 times more affinity with opioid receptor than morphine, while M3G is an opioid receptor antagonist.^[43] The therapeutic drug monitoring of UGT1A9 and 2B7 substrate drugs should get more attention to avoid clinical adverse issues due to drug–drug interactions.

Conclusion

In summary, the oxidation and glucuronidation pathway of isobavachin were well characterized in vivo and in vitro for the first time. Two glucuronides and three mono-oxidated metabolites were identified in mice. In addition, CYP1A2 and 2C19 were identified as main contributors for isobavachin oxidation, and UGT1A1, 1A6, 1A7 and 1A9 mainly contribute to the isobavachin glucuronidation. Activity correlation analysis and RAF approaches also supported these results above. Furthermore, mice were considered as the appropriate animal model to investigate the formation of M1 and G1 in human. Moreover, the inhibition assays demonstrated that isobavachin was a potent broad-spectrum inhibitor towards CYP2B6, 2C9, 2C19, and UGT1A1, 1A9, 2B7, which suggested that the inhibition of systemic clearance for these enzymes substrates should be paid more attention when isobavachin or its herbal preparations are coadministered with clinical drugs primarily cleared by these CYP and UGT enzymes.

Supplementary Material

Supple figs S1-S9 table

Figure S1. Mass spectra of isobavachin and its related metabolites after intragastric administration of isobavachin (40 mg/kg).

Figure S2. Exposure of isobavachin and its related metabolites in bio-samples after oral administration of isobavachin (40 mg/kg).

Figure S3. Kinetic profiles for oxidation and glucuronidation of isobavachin by pooled HLM, HIM, MLM and MIM.

Figure S4. Metabolic rates of isobavachin at 10 μM by expressed CYP (A) and UGT (B) enzymes.

Figure S5. Kinetic profiles for oxidation of isobavachin by CYP1A2 and 2C19.

Figure S6. Kinetic profiles for glucuronidation of isobavachin by expressed UGT enzymes.

Figure S7. Correlation analysis between the formation of isobavachin-related metabolites and the metabolic activity of substrates in a bank of individual HLM (n = 9).

Figure S8. Kinetic profiles for phenacetin-N-deacetylation (A) and mephenytoin-4-hydroxylation (B) by pooled human liver microsomes (HLM) and individual CYPs enzymes.

Figure S9. Concentration-dependent inhibition of isobavachin towards expressed CYP and UGT enzymes.

Table S1. Kinetic parameters derived for the phenacetin-N-deacetylation and mephenytoin-4-hydroxylation by pooled HLM, expressed CYP1A2 and 2C19, respectively (Mean ± SD).