Piperine enhances the bioavailability of silybin via inhibition of efflux transporters BCRP and MRP2

Accepted Manuscript

Piperine enhances the bioavailability of silybin via inhibition of efflux transporters BCRP and MRP2 Xiaoli Bi , Zhongwen Yuan , Biao Qu , Hua Zhou , Zhongqiu Liu , Ying Xie PII: DOI: Reference:

S0944-7113(18)30499-9 https://doi.org/10.1016/j.phymed.2018.09.217 PHYMED 52682

To appear in:

Phytomedicine

Received date: Revised date: Accepted date:

2 August 2018 6 September 2018 25 September 2018

Please cite this article as: Xiaoli Bi , Zhongwen Yuan , Biao Qu , Hua Zhou , Zhongqiu Liu , Ying Xie , Piperine enhances the bioavailability of silybin via inhibition of efflux transporters BCRP and MRP2, Phytomedicine (2018), doi: https://doi.org/10.1016/j.phymed.2018.09.217

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ACCEPTED MANUSCRIPT

Piperine enhances the bioavailability of silybin via inhibition of efflux transporters BCRP and MRP2

Xiaoli Bi

a, b, 1

, Zhongwen Yuan c, 1, Biao Qu a, Hua Zhou a, Zhongqiu Liu d and Ying Xie

a

State Key Laboratory for Quality Research of Chinese Medicines, Macau University of

Science and Technology, Taipa, Macau (SAR); b

Guangdong Province Engineering Technology Research Institute of Traditional Chinese

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Medicine, Guangzhou, Guangdong Province, China; c

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a,*

Department of Pharmacy, The Third Affiliated Hospital of Guangzhou Medical University,

Guangzhou, Guangdong Province, 510150, People’s Republic of China; d

Joint Laboratory for Translational Cancer Research of Chinese Medicine of the Ministry of

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Education of the People’s Republic of China, School of Chinese Medicine, Guangzhou

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University of Chinese Medicine, Guangzhou, Guangdong Province, China

These authors contributed equally to this work.

*

Corresponding authors:

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1

Ying Xie, Ph.D., Macau University of Science and Technology, Avenida Wai Long, Taipa, Macau (SAR), Tel: +853-88972426, E-mail: [email protected]

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ACCEPTED MANUSCRIPT ABSTRACT Background: Although silybin serves as a well-known hepatoprotective agent with prominent anti-inflammatory, anti-oxidant and anti-fibrotic activities, its low bioavailability limits its application in the treatment of chronic liver diseases. However, novel formulation products with increased solubility were not sufficient to achieve pharmacologically meaningful

Hypothesis/Purpose:

We

hypothesized

that

inhibiting

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concentrations of silybin in the clinical studies even used at high dosage. efflux

transporter(s)

and/or

glucuronidation by piperine might enhance the bioavailability and efficacy of silybin.

Methods: Pharmacokinetics of silybin given alone or in-combination with piperine was

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determined by a validated LC-MS method. A CCl4 induced rat model of liver injury was prepared and verified for comparing the effects of silybin and combination treatment. To investigate the underlying mechanism, the inhibition effects of piperine on transportation of

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silybin were performed in Caco-2 and transfected MDCKII cell lines as well as sandwich– cultured rat hepatocytes (SCH). Human liver microsomes incubation was used for exploring

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the modulation effects of piperine on the phase-2 metabolism of silybin. Results: In the present study, we demonstrated for the first time that piperine as a bioenhancer

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increased the bioavailability of silybin (146%-181%), contributing to a boosted therapeutic

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effect in CCl4-induced acute liver-injury rat model. The underlying mechanisms involved that piperine enhanced the absorption of silybin by inhibiting the efflux transporters including

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MRP2 and BCRP but not MDR1 in Caco-2 and transfected MDCKII cell lines. Moreover, piperine could inhibit the biliary excretion of silybin and conjugated metabolites in sandwich– cultured rat hepatocytes. Notably, we found that piperine did not affect the phase-2 metabolism of silybin. Conclusion: Efflux transporters play an important role in the pharmacokinetic behavior of flavolignans, and modulating these transporters by bioenhancer such as piperine could

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ACCEPTED MANUSCRIPT enhance the in vivo absorption of silybin, leading to more effective treatments. Keywords Silybin; piperine; bioenhancer; MRP2; BCRP; phase-2 metabolism;

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Abbreviations used in this paper: AUC, area under the plasma concentration-time curve; BCRP, breast cancer resistance protein; BEI, biliary excretion index; Clbiliary, biliary clearance; CCl4, carbon tetrachloride; HCV, hepatitis C virus; HE, hematoxylin and eosin; LC-MS/MS, Liquid chromatography

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tandem-mass spectrometry; LLOQ, lower limit of quantification; MRPs, multi-drug resistance proteins; SBa, Silybin A; SBb, silybin B; SCH, sandwich-cultured rat hepatocyte; SOD,

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superoxide dismutase; UGT1A1-competent; WT, wild-type phenotype.

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ACCEPTED MANUSCRIPT Introduction Silymarin, a complex extract from milk thistle (Silybum marianum), is the most frequently applied natural products for various liver disorders world widely (Flora K, 1998 ). It has been classified as liver therapy for toxic and inflammatory liver diseases by WHO anatomical therapeutic chemical (ATC) classification system (A05BA03) (Hellerbrand et al.,

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2016). Silybin (silibinin) is the primary and most active flavonolignan of silymarin and is composed of a mixture of two diastereoisomers, silybin A (SA) and silybin B (SB) at approximately 1:1 ratio (Fig. 1) (Federico et al., 2017; Kren V, 2000). The anti-inflammatory, antioxidant, antifibrogenic and anticancer properties of silybin have been demonstrated in

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various animal and in vitro experiments (Federico et al., 2017; Loguercio and Festi, 2011b). Although there is no conclusive data for the clinical efficacy of silybin in chronic liver diseases which is due to the poor-designed clinical trials, the different products, processing

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methods and manufacturing methods (Tamayo and Diamond, 2007), silybin seems a promising drug for chronic liver disease on the basis of literature data (Loguercio and Festi,

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2011a).

As low-bioavailability is quite common for flavolignans in clinical practice, several

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techniques have been utilized previously to improve the solubility and bioavaibility of silybin

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or silymarin (Ali et al., 2016; Mendez-Sanchez et al., 2017), leading to the development of commercial products such as Livergol, Silipide and Legalon (Karimi et al., 2011b). But, in a

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recent clinical study, high doses of Legalon (700 mg daily) did not significantly improve the serum ALT levels in patients with chronic HCV infection. Notably, author found that the active plasma concentrations at this high dosage of Legalon were lower than the in vitro effective concentrations for anti-inflammatory, anti-oxidant and anti-viral activities (Fried et al., 2012). Both human and animal studies suggested that following oral administration of silybin, the absorbed silybin undergoes rapid and extensive first-pass phase II conjugative

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ACCEPTED MANUSCRIPT metabolism with primarily biliary excretion, resulting in low bioavailability (0.73% in rat) which reduced its efficacy, resulted in inconsistent clinical trial results and limited its wide application in clinical practice (Hawke RL, 2010; Lo et al., 2014; Zhang et al., 2010). Therefore, using phase-2 metabolism and/or efflux transporters inhibitor(s) might increase plasma and tissue concentrations of silybin leading to enhanced therapeutic effects.

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In our primary study, silybin has been demonstrated to be a substrate of both breast cancer resistance protein (BCRP, ABCG2) and multi-drug resistance protein 2 (MRP2, ABCC2) (Miranda et al., 2008; Yuan et al., 2018). It is well known that membrane drug efflux transporters including multidrug resistance protein 1 (MDR1, also named P-glycoprotein,

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ABCB1), BCRP and MRP2 are mainly expressed in barrier tissues such as intestine, liver, kidney, blood-brain barrier and placenta, which could affect drug exposure and clearance in vivo. Inhibition of the efflux transporters may increase intestinal absorption of silybin as well

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as reduce biliary excretion, thus contributing to enhanced absorption and bioavailability (Amin, 2013; Hoosain et al., 2015; Yuan et al., 2018).

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Piperine (Fig. 1), a major component of black and long pepper, has gained increasing attention in recent years with the benefit of enhancing the bioavailability of a number of

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therapeutic drugs via modulation of metabolic enzymes and efflux transporter P-gp (Lee et al.,

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2018; Srinivasan, 2007). For example, the bioavailability of curcumin increased 2000% in human by co-administration with piperine (Shoba et al., 1998). It was reported that piperine

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inhibited efflux transporter P-gp (Bhardwaj et al., 2002) which altered the pharmacokinetic behavior of P-gp substrates. Additionally, Bcrp and Mrp2 in cancer cells were also inhibited by piperine (Basu et al., 2013; Berginc et al., 2012; Cheong et al., 2017). However, the specific role of piperine in the absorption and excretion of silybin and its conjugates via efflux transporters has not been investigated. Moreover, silybin was primarily catalyzed by UGT and SULT enzymes (Rickling et al., 1995) and the glucuronide and sulfate conjugates account for

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ACCEPTED MANUSCRIPT approximately 90% of the total amount metabolites in blood following oral administration (Gatti and Perucca, 1994; Weyhenmeyer et al., 1992). As UGT activity was significantly inhibited in the presence of piperine (Lee et al., 2018), we hypothesized that piperine might enhance the bioavailability of silybin by inhibiting

efflux transporter(s) and/or

glucuronidation. Therefore, in this study, we investigated the impact of piperine on the

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pharmacokinetic behavior and liver protective effects of silybin, and explored the underlying mechanisms involving the effects of efflux transporters and glucuronidation enzymes on the pharmacokinetics of silybin. Materials and Methods

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Chemicals and reagents

Silybin (48% SA and 52% SB), β-glucuronidase (type B-10 from bovine liver), β-glucuronidase (from Escherichia coli), sulfatase (type H-1 from Helix pomatia), Ko143,

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MK-571 and naringenin (NG) were purchased from Sigma-Aldrich (St. Louis, MO). The identity and purity of these chemicals were further verified by LC-MS in our laboratory. All

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other chemicals and reagents were of analytical grade or higher and were purchased through commercial sources. In in vitro study, silybin, piperine, Ko143, MK-571 were dissolved in

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DMSO and then diluted with Dulbecco's modified Eagle's medium to generate various

1‰.

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working concentrations. The percentage of DMSO in the final reaction solution was less than

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Determination of silybin and piperine by LC–MS/MS The concentration of silybin A, B and piperine as well as the conjugated silybin were

measured in plasma, medium or cell lysate sample using LC-MS/MS method as described previously with modification (Wen Z, 2008 ; Yuan et al., 2018). NG was used as internal standard because it has similar retention time with silybin and good peak shape, stability and recovery in this LC-MS detection method for silybin. In brief, 10 μl sample was mixed with 5

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ACCEPTED MANUSCRIPT times of ice-cold acetonitrile containing 1% glacial acetic acid and NG (25 ng/ml). After vortex, the mixture was centrifuged at 13,500 rpm for 15 min at 4°C. The supernatant was transferred into a new tube and evaporated to dryness under a nitrogen stream. The residue was reconstituted with 60 μl of 30% methanol solution. After centrifuged again at 13,500 rpm for 15 min at 4°C, 10 μl of the supernatant was used for analysis by LC-MS/MS. To estimate

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the concentration of total glucuronide and sulfate conjugates of silybin in plasma and cells, enzymatic hydrolysis with glucuronidase from bovine liver (8000 U/ml) and sulfatase (80 U/ml) was performed with 10μl aliquots of samples firstly. And the concentrations of glucuronides and sulfates in samples were calculated from the difference between the amount

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of total silybin released by incubation with both glucuronidase and sulfatase and the amount of free silybin (Wen et al., 2008).

The analysis of silybin and piperine were performed on an Agilent HP 1290 LC-MS/MS

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system (Agilent, CA) with a C18 analytical column (Waters ACQUITY UPLC® BEH C18 Colum ,1.7 μm, 2.1×50 mm, Waters Corp., Ireland). Silybin (silybin A and B) and piperine

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were well separated using optimized gradient elution with methanol/0.5% acetic acid (pH 4)

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as mobile phase at a flow rate of 0.35 ml/min with a running time of 10 min. The gradient employed to obtain the conjugation profiles was as follows (mobile phase B: methanol): 0

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min: 25%, 6 min: 40%, 9 min: 90%, 10 min: 25%. MS parameters: capillary voltage, ± 4000V; nebulizer pressure, 40 psi; drying gas, 9 L/min; drying gas temperature, 325°C;

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fragment voltage, 35 V; dwell time, 200 ms; scan mode, Multiple reaction monitoring (MRM) transition for silymarin flavonolignans (at m/z 481.00 → 301.20), mono-glucuronide (m/z 657.00 →481.00), piperine (m/z 286.0 →201.40) and NG (m/z 271.00→150.80), respectively. LC-MS/MS data were obtained by Agilent ChemStation Software. Pharmacokinetic studies in rats Male Sprague-Dawley (SD) rat weighing 200~220 g was purchased from the

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ACCEPTED MANUSCRIPT Guangdong Medical Laboratory Animal Center. Rats were housed in cage with food and water provided ad libitum and acclimated in the laboratory for one week prior to each experiment. All procedures involving animals and caring were approved by the Committee on Use of Human & Animal Subjects in Teaching and Research of Guangzhou University of Chinese Medicine (No # ZYYL20150808).

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Sixteen male SD rats (220-250g) were randomly divided into two groups (8 rats for each group): A group (single dose of 50 mg/kg silybin), and B group (oral 10 mg/kg piperine with single dose of 50 mg/kg silybin). Animals were fasted 24 h before oral drug administration to minimize variation in absorption. Multiple blood samples (100 μl) were collected from the tail

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vein at 0.25, 0.5 , 0.75, 1, 2, 4, 6, 8 and 12 h with a temporary cannula to dried heparinized tubes (Parasuraman et al., 2010). Immediately, the blood samples were centrifuged at 4,000 rpm (15 min, 4 C) and stored at -80 C until analysis.

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Pharmacokinetic parameters were calculated using the software (DAS) 2.1.1 pharmacokinetic program authorized by Chinese Pharmacology Society (Beijing, China) with

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non-compartmental analysis. The following non-compartmental pharmacokinetic parameters

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were calculated: half-life (T1/2), mean residence time (MRT), volume of distribution (Vd), oral clearance (CL/F), and area under the concentration-time curve (AUC).

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Acute liver damage induced by CCl4 in rats Male SD rats were randomly divided into seven groups (n=6 per group): 1) Group I

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orally received blank vehicle for 7 days, and intraperitoneally (i.p.) injected with olive oil (2 ml/kg body weight) on day 7; 2) Group II orally received blank solvent for 7 days; 3) Group III was treated with silybin (100 mg/kg bodyweight, p.o.) for 7 days; 4) Group IV was treated with silybin (100 mg/kg bodyweight, p.o.) and piperine (100 mg/kg bodyweight, p.o.) mixture for 7 days; 5) Group V was treated with silybin (100 mg/kg bodyweight, p.o.) and piperine (50 mg/kg bodyweight, p.o.) mixture for 7 days; 6) Group VI was treated with silybin (100

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ACCEPTED MANUSCRIPT mg/kg bodyweight, p.o.) and piperine (25 mg/kg bodyweight, p.o.) mixture for 7 days; 7) Group VII was treated with piperine (100 mg/kg bodyweight, p.o.) for 7 days. Rats in Group II-VII were injected with CCl4-olive oil mixture (40% CCl4, i.p., 2 ml/kg body weight) at 2 hours after administration of silybin or/and piperine in the 7th day. Model and control group were daily treated orally with vehicle (35% PEG400: 15 % Cremophor EL: 5 % ethanol: 45 %

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saline). 24 hours after the CCl4-induced toxic liver injury was initiated, rats were sacrificed under ether anesthesia. Blood was collected from the abdominal aorta for biochemical estimations. The liver was immediately removed and weighed. A large portion of liver was snap-frozen in

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the liquid nitrogen with remaining tissues fixed in 10% buffer formalin, processed and embedded in paraffin for histological examination after H&E staining. Steatohepatitis was evaluated by level of ALT, AST, triglycerides (TG), total cholesterol (TC) and superoxide

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dismutase (SOD) using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to manufacturer’s instructions and histological assessment and scoring

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according to standardized criteria by a pathologist blinded to the study. Cell culture

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Caco-2 cells were obtained from ATCC (American Type Culture Collection, Rockville,

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MD, USA) Parental MDCKII, and transfected MDR1-MDCKII, BCRP- MDCKII, MRP2MDCK II cells were kindly provided by the Netherlands Cancer Institute (Amsterdam, The

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Netherlands). The expression of human BCRP, MDR1 and MRP2 in transfected MDCKII cells has been confirmed in our laboratory by Western blotting (data not shown). The MDCKII cells and Caco-2 cells were cultured at 37°C/5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 100 U/ml penicillin and 100 μg/ml streptomycin. The Caco-2 cell monolayer was prepared by seeding 1.0×105 cells/cm2 onto polyester filter membranes in a 6-well Transwell® plate

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ACCEPTED MANUSCRIPT (Corning Costar Co., NY, USA.) for culture 21 to 23 days. The MDCK II cells were seeded on transwell plates at a density of 2.0×105 cells/ cm2 and grown for 4-5 days to form confluent monolayers (Yuan et al., 2018). Prior to transport study, cell monolayers were certified based on our internal criteria on transepithelial electrical resistance (TERR) values, and only monolayers that met the

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acceptance criteria (TERR >500 Ω · cm2 for Caco-2 cells, and >200 Ω · cm2 for MDCK cells) were used for transport studies.

Bidirectional transport studies with Caco-2 cell and MDCKII cells

The drug transport and efflux assay for silybin were carried out in the Caco-2 cells and

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transfected MDCKII cells in the absence or presence of piperine or inhibitor (such as Ko143, MK571 and QND) as described previously (Yuan et al., 2018). After the desired incubation time (30, 60, 90, 120,180 min) at 37°C, the AP/BL side solution (100 μl) was collected and

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methanol solution containing 1% formic acid added, and then stored at −80 °C until analysis by LC-MS/MS. For inhibition studies, Caco-2 or MDCK II cell monolayers were

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preincubated with the inhibitor in both apical and basolateral chambers for 30 min, and bidirectional transport of 10 μM silybin was conducted in the absence (control) or presence of

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the inhibitor in both chambers. The permeability coefficients (Papp) were calculated based on

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the appearance rate of silybin in the target side, while efflux ratio was the ratio of Papp in the B-A direction to Papp in the A-B direction.

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Transport studies using rat sandwich-cultured hepatocytes Sandwich-cultured rat hepatocytes (SCRHs) were established and used to determine

hepatobiliary accumulation according to previously described methods (Seglen, 1976) with minor modifications (Yuan et al., 2018). In brief, after isolation, hepatocytes were seeded onto six-well plates and cultured with William’s E medium containing fetal bovine serum (5%). Approximately 24 h later, hepatocytes were overlaid with Matrigel. The culture medium was

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ACCEPTED MANUSCRIPT changed daily, and the experiment was performed on day 5 of culture. 5-(and-6)-carboxy-2', 7’- dichlorofluorescein diacetate (CDCFDA) was used as canalicular marker for SCRHs. For transport study, SCRHs were pre-incubated in HBSS with or without Ca2+ at 37°C for 15 min. All wells were then incubated with 10 μM silybin in the absence and presence of inhibitor piperine or MK571 (50 μM) for 20 min at 37oC. After incubation, hepatocytes were

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rinsed three times with ice-cold buffer. Silybin were extracted from the cells + bill (for hepatocytes incubated with Ca2+ buffer) and cells (for hepatocytes incubated without Ca2+ buffer), and analyzed as described above. The biliary excretion index (BEI; %) and in vitro biliary clearance (Clbiliary; ml/min/kg) were calculated by using B-CLEAR technology

1999):

(Accumulation 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 − Accumulation𝐶𝑎𝑙𝑐𝑖𝑢𝑚 𝑓𝑟𝑒𝑒 )

Clbiliary =

Accumulation 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑

× 100%

Accumulation 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 − Acculumation 𝐶𝑎𝑙𝑐𝑖𝑢𝑚 𝑓𝑟𝑒𝑒 AUC

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BEI% =

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(Qualyst, Inc., Research Triangle Park, NC) based on the following equations(Liu et al.,

(1)

(2)

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where AUC represents the area under the substrate concentration-time curve, which was determined by multiplying the initial substrate concentration in the incubation medium by the

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incubation time (10 min). Clbiliary values were converted to milliliter per minute per kilogram

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based on previously reported values for protein content in liver tissue (200 mg/g liver weight for rats) and liver weight (40 g/kg body weight for rats)(Davies and Morris, 1993; Wilson et

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al., 2003).

Effects of piperine on the conjugates of silybin Glucuronide and sulfate conjugates of silybin were biosynthesized using pooled human

liver microsomes (HLMs) under our reported conditions (Xie et al., 2017). Reactions were performed in a shaking water bath and terminated with addition of acetonitrile containing 1% acetic acid and NG after 30 min incubation. MK571 (50 μM), a confirmed inhibitor of phase-2 conjugation of flavonols, was used as positive control (Barrington et al., 2015). 11

ACCEPTED MANUSCRIPT Mixtures were analyzed as description above. Statistical analysis Statistical analyses were performed with SPSS 11.5 (LEAD Technologies Inc.). One way ANOVA was used when multiple groups were compared and the two-sided unpaired Student’s t-test was used when treatments or differences between two groups were compared. During all

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statistical analyses, differences in group sizes were considered in the calculations. Differences were considered statistically significant when P<0.05. All data were presented as geometric mean ± SD. Results

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LC-MS/MS bioassay method development and validation

We developed and validated the LC-MS/MS method for analyzing silybin and its conjugated metabolites in plasma, media and cell lysate based on the reported method with

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modification (Xie et al., 2017). As shown in Fig. 1, silybin A, silybin B, piperine and NG were separated with the retention times of 5.1, 5.4, 8.3, and 5.7 min, respectively.

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The limit of quantitation for silybin A, B, and piperine was 1.0 ng/ml. The calibration curve in the range of 1–3000 ng/ml for silybin and range of 1–1500 ng/ml for piperine both

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had r2 value > 0.999 (Supplemental Table 1). The intra-or inter-day precision (RSD) of silybin

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A and B ranged were below 9.2 and 10.6%, respectively, with accuracy ranged between 89.0 and 108.0%. The recovery ratios of silybin A and B were above 92.5%. The accuracy and

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precision for piperine assay were ranged between 95.2% to110.3% and below 10.4%, respectively, with recovery more than 90.9%. The matrix effects were ranged around 81.3 to 88.62% for silybin, and 84.9 to 87.6% for piperine with testing QC samples (Supplemental Table 2). Both silybin A, B and piperine were stable in the rat blank plasma with recoveries > 85% under following storage conditions: 25 °C for 6 h (short-time), 4 °C for 12 h (postpreparation), -80 °C for 15 days (long-term), and three freeze- thaw cycles (Supplemental

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ACCEPTED MANUSCRIPT Table 3). These results demonstrate that this developed LC-MS method was suitable for evaluation of silybin and piperine in bio-samples. Piperine enhanced bioavailability of silybin An in vivo pharmacokinetic study was undertaken in rats to study whether piperine could increase the bioavailability of silybin using the established analytical LC-MS method. The (50 mg/kg) after oral

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resultant plasma concentration versus time profiles of silybin

administration alone or in combination with piperine (50 mg/kg) were shown in Fig. 2, and the corresponding pharmacokinetic parameters calculated with non-compartmental analysis were presented in Table 1.

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After oral administration, the peak plasma concentration Tmax of free silybin and total silybin were around 1 h, indicating a fast absorption and metabolism. And silybin was quickly metabolized to glucuronide and sulfate conjugates, while the plasma concentration of free

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silybin was very low, which was consistent with previous reports (Hawke RL, 2010). As expected, co-administration of silybin and piperine orally resulted in significantly increased

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plasma concentrations of silybin. The Cmax values of total silybin A and B were 1.49-fold and 1.39-fold higher than those of silybin given alone (p<0.001). The area under the plasma

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concentration-time curve AUC0-t were increased 146% for total silybin A (p<0.05) and 181%

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for total silybin B (p<0.01) in co-administration group compared with silybin administration group. Moreover, piperine also raised significant changes in free silybin pharmacokinetics.

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The Cmax and AUC0-t values of free silybin were increased with 1.76-fold and 1.37-fold for free silybin A and 1.60-fold and 1.26-fold for free B (p<0.05) respectively upon treatment with piperine. However, the Tmax, t1/2, and MRT of silybin were not different between the treatment groups. These results indicated that piperine had the potential to increase the bioavaibility of silybin, which may lead to enhanced therapeutical efficacy.

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ACCEPTED MANUSCRIPT Moreover, we evaluated the effect of silybin on the pharmacokinetic behaviour of piperine as shown in Fig. 2E and Supplemental Table 4. However, co-administration orally of silybin and piperine slightly reduced the plasma concentrations of piperine but without significance in statistical analysis compared to the group given piperine alone. Boosted hepatoprotective effects of silybin co-treated with piperine

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The therapeutic effects of silybin on acute liver injury induced by carbon tetrachloride (CCl4) in rats were further studied in the presence of piperine to demonstrate the enhanced therapeutic activities associated with increased absorption of silybin in vivo.

As shown in Fig. 3A, the liver weight and liver to body weight ratio were significantly

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elevated after CCl4-treatment, while those increases were abrogated in silybin-treated and silybin & piperine-treated groups (p < 0.05). The livers of CCl4- treated rats appeared larger with a pale and irregular surface, indicating severe hepatocellular damage as shown in Fig. 3B.

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Moreover, apparent liver injuries characterized by large areas of necrotic tissue, sever loss of hepatic architecture and a significant number of ballooning hepatocytes were observed in

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CCl4-treated rats with histopathological analysis of liver sections stained with hematoxylin and eosin (H & E), which was less prominent in silybin-treated animals. Notably,

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CCl4-induced macroscopic and histopathological changes were more significantly attenuated

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in silybin and piperine co-administrated animals compared with those of silybin given alone. Comparing with normal group, liver injury in the CCl4-treated group was also reflected

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by a marked increase of serum transaminases which were released into the blood once the structural integrity of the hepatocyte was damaged (Ding et al., 2012). After acute CCl4 challenge, the serum levels of ALT and AST in CCl4-treated group increased 5 to 6 times, respectively, over those in normal control group (Fig. 3). However, pre-administration of silybin-treated at 100 mg/kg for seven consecutive days significantly prevented the CCl4-induced increase of serum activity of ALT and AST and attenuated increases in the

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ACCEPTED MANUSCRIPT serum TG, and TC levels (p < 0.01). Similarly, we noticed that combination treatment of silybin-piperine showed a better hepatoprotective effect than single silybin treatment at the same dosage (Fig. 3). The antioxidant properties of silybin are considered to be responsible for its protective actions (Karimi et al., 2011a; Surai, 2015). The activity of the antioxidant enzymes

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superoxide dismutase (SOD) was assessed as indicators of oxidative stress in CCl4-induced liver injury rats. As shown in Fig. 3, CCl4 exposure induced a remarkable decrease in hepatic activities of SOD level (12.10 ± 1.13 versus 5.30 ± 1.32, p < 0.001), of those of control group. This depletion of endogenous antioxidants was markedly ameliorated in silybin-piperine

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co-treated group. But piperine given alone did not have any protection effects which were reflected on the similar histopathology, ALT, AST, SOD and cytokines levels as vehicle treated CCl4-induced liver injury rats.

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Piperine inhibited the efflux transport of SB

To investigate the underlying mechanism of increased oral bioavailability and

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bio-efficacy of silybin, cell permeability studies were carried out on Caco-2 cells and transporter-overexpressed MDCKII transfected cells. Prior to the transport studies, the MTT

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assay was performed to investigate the concentration-dependent cytotoxic effects of silybin

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and piperine in all cell lines. None of these compounds affect the growth of Caco-2, and MDCKII cells at the examined concentrations (data not shown).

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Firstly, the basic transport characteristics of silybin across the Caco-2 cell monolayer

were illustrated in Fig. 4. The transport rate of silybin in the basolateral to apical (BL-AP) direction was much greater than that of apical to basolateral (AP -BL) direction, and the efflux ratios were 5.04 ±0.48 for SBa and 4.61±0.39 for SBb indicating active efflux of silybin. Then, inhibition of efflux transporters was investigated by assessing bidirectional transport of silybin in the absence or presence of piperine. A dose-dependent increase in the AP-BL

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ACCEPTED MANUSCRIPT transport and decrease in efflux ratio of silybin were observed with piperine (10, 20, and 40 μM) added in Caco-2 cells (Fig. 4), suggesting that piperine inhibited the BCRP and/or MRPs mediated efflux transport of silybin in the Caco-2 cells. Piperine inhibited the efflux transporters The efflux transporter transfected cell line MDCKII-MRP2 and MDCKII-WT were used

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to identify the transporter(s) related to the bio-enhancing effect of piperine on silybin and confirm the results observed in Caco-2 cells. The mean efflux ratio for the reference MRP2 substrate CDCFDA (Siissalo et al., 2009) was 5.44±0.42 in the MDCKII-MRP2 cells, which was decreased to 1.05±0.06 after treatment with MK571 (MRP2 inhibitor) (Fig. 5A).

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Significant dose-dependent reductions in the efflux ratio of MRP2 substrate CDCFDA were found in the MDCKII-MRP2 cells in the presence of piperine from 3.58 ±0.52 to 1.40±0.02, suggesting that piperine is a MRP2 inhibitor. Consistent with the results observed

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in the Caco-2 cell monolayer, piperine markedly increased AP-BL transport and decreased efflux ratio of silybin in the MDCKII-MRP2 cells as shown in Fig. 5B.

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The MDCKII-BCRP cell monolayer model was validated by examining the bidirectional transport of pheophorbide A (Ph A, BCRP-specific probe) (Robey et al., 2004) as shown in

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Fig. 5A. The BL-AP direction transport of Ph A was significantly higher than that in the

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AP-BL direction with MDCKII-BCRP cells, while its efflux ratio significantly reduced from 3.22±0.11 to 1.06±0.03 by 0.5 μM Ko143, verifying the suitability of MDCKII-BCRP cell

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monolayer for investigating the function of BCRP in vitro. Piperine dose-dependently reduced the efflux ratio of BCRP substrate Ph A in the MDCKII-BCRP cells. The efflux ratio of silybin across the MDCKII-BCRP cell monolayer was markedly decreased from 2.34±0.17 to 1.55±0.38 for SBa and from 2.05±0.22 to 1.32±0.32 for SBb in the presence of piperine in a concentration dependent manner (Fig. 5 B). Therefore, piperine is a BCRP inhibitor and inhibits the efflux transport of silybin via BCRP transporter, which may partly contribute to its

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ACCEPTED MANUSCRIPT bio-enhancing effect. The MDCKII-MDR1 cell monolayer model was validated by examining the bidirectional transport of Rho123. A predominantly BL-AP transport of Rho123 was observed with efflux ratio at 2.52±0.18, which was significantly reduced to 1.41±0.09 by 10 μM verapamil verifying the credibility of using MDCKII-MDR1 cell monolayer in investigating the effect of

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P-gp in vitro. As expected, piperine reduced the efflux ratio of Rho123 in the MDCKII-MDR1 cells as shown in Fig. 5A, establishing piperine as a MDR1 inhibitor.

However, the bio-direction transport of silybin was not affected in the presence of P-gp inhibitor verapamil, quinidine and piperine in MDCKII-MDR1 cell and MDCKII-WT cell.

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Therefore, piperine could reduce the efflux of silybin and enhance its bioavaibility by inhibition of BCRP and MRP2.

Piperine inhibited biliary excretion of silybin and conjugates in SCRH model

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MRP2 and BCRP are the major canalicular efflux transporters for biliary excretion of drug and their metabolites. Therefore, we also investigated the effects of piperine on the biliary

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excretion of silybin by using sandwich-cultured rat hepatocytes whose function was confirmed by the hepatobiliary disposition of positive control CDCFDA. In the primary study,

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we evaluated the effect of substrate concentration on the hepatobiliary disposition of silybin in

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SCRH, and noticed that a concentration-dependent increase in silybin accumulation in standard buffer representing the mass of silybin accumulated in hepatocytes plus bile pockets.

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Then, a concentration of 10 μM silybin was chosen because it represented a concentration within a linear range of accumulation. As shown in Fig. 6, the average BEI for free silybin A and B from three different studies

were 30.74±1.38% and 32.97±0.44%, and the average Clbiliary for free silybin A and B were 0.55, and 0.76 ml/min/kg, respectively, while averages BEI for total silybin A and B were 27.37±1.39% and 27.00±1.08%, indicating about 30% silybin and its conjugates were

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ACCEPTED MANUSCRIPT excreted to biliary. The effects of piperine (10, 20, 40 μM) as well as the positive control MK571 (50 μM) on the accumulation, BEI, and Clbiliary of silybin were determined in SCRH, and the results are presented in Fig. 6. Compared with silybin given alone in SCHs, BEI of silybin was decreased by 79.04% for silybin A and 78.11% for silybin B in the presence of 40 μM piperine, and decreased by 99.91% for silybin A, 99.87% for silybin B in the presence of

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50 μM MK571, respectively. In the absence of test compound, the accumulation of free silybin in incubations with standard buffer (cells and bile pockets) and calcium-free buffer (cells only) was 86.16 ± 4.77 (silybin A) and 110.5 ± 3.98 (silybin B) ng/mg proteins and 59.69 ± 3.98 (silybin A) and

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74.08 ± 2.95 (silybin B) ng/mg proteins, respectively. The accumulation of total silybin A and B in incubations with standard buffer (cells and bile pockets) and calcium-free buffer (cells only) was 243.76 ± 13.86 and 333.37 ± 23.18 ng/mg proteins and 176.90 ± 6.61 and 240.90 ±

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19.57 ng/mg proteins, respectively, which means about 65% silybin were metabolized to silybin glucuronide and sulfates after 20 mins incubation with SRCHs. Moreover, MK571 and

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piperine significantly increased the accumulation of silybin in hepatocytes (cells) and decreased the excretion of silybin from hepatocytes to bile (Figs. 6 D and 6 E; S. tables 4 and

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5). Notably, the presence of piperine didn’t affect the phase-2 conjugates production by the

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hepatocytes, while MK571 significantly decreased the amounts of silybin conjugates (p<0.001) as shown in Fig. 6F. Thus, piperine inhibits the efflux transporters during the

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silybin absorption and excretion but not affect the UGT and SULT enzymes for the phase-2 metabolism of silybin. Piperine failed to regulate the phase II metabolism of silybin To confirm the impact of piperine on the metabolism of silybin in SCHs, human liver microsomes (HLMs) were incubated with silybin in the presence and absence of piperine which is a known inhibitor of hepatic and intestinal glucuronidation. As shown in

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ACCEPTED MANUSCRIPT Supplemental Fig. 1, 66.50±3.78% of silybin A and 66.58±3.86% of silybin B were metabolized to silybin conjugates after 30 mins incubation with HLMs. MK571 (50 μM) significantly inhibited the phase II metabolism of silybin that the percentages of conjugated silybin A and silybin B were reduced to 39.69±6.50% of 39.50±6.54%. However, piperine (40 μM) didn’t affect metabolism of silybin as there were 67.10±7.64% silybin A and 67.31±7.71%

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silybin B conjugated. Discussion

Enhancing the bioavaibility of the therapeutically potent compounds is critical for drug development as it reduces the drug dosage and frequency and thus reducing drug toxicity and

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cost for the patients(Kesarwani et al., 2013). Therefore, there has been a growing interest in improving the bioavaibility of chemicals with promising therapeutic potential by combination with natural bioenhancers (Ajazuddin et al., 2014). Co-administration with natural

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bioenhancers could increase the bioavailability of target drugs via inhibiting metabolic enzymes or transporters, representing a new strategy to improve therapeutic effects of drugs

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in co-administration (Basu et al., 2013). However, selection of an appropriate combination is crucial which requires elaborate understanding of the potential interactions between the

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bioenhancer and the target drug.

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Piperine, a major component of black and long pepper, has gained increasing attention in recent years with the benefit of enhancing the bioavailability of a number of therapeutic drugs.

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We noticed that the ratios for piperine used as bioenhancer with target drug were mostly ranged from 1/2 to 2/1 in the literature (Srinivasan, 2007). Moreover, the safety of piperine has been established in several animal studies with the LD50 around 514 mg/ kg in rats (Srinivasan, 2007). In the sub-acute toxicity studies, none toxicity was observed for piperine at the dose rate of 100 mg/kg for 7 days. Therefore, the dosage of piperine was selected at 1:1 with silybin, while no liver protection effect was observed for piperine in liver injury rats.

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ACCEPTED MANUSCRIPT However, piperine may cause gastrointestinal side-effects and bleeding at high doses (330-400 mg/kg) in toxicity study (Piyachaturawat et al., 1983). Therefore, to increase the bioavaibility and decrease the side-effects at the same time, new formulation containing both piperine and silybin should be developed for the clinical treatment. Our results showed that piperine enhanced the absorption and bioavaibility of silybin, and

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the combination treatment of silybin-piperine had a better hepatoprotective effect than single silybin treatment at the same dosage in rats with CCl4-induced acute liver injury via inhibition of MRP2 and BCRP. To the best of our knowledge, this is the first study to illustrate the bio-enhancing actions of piperine on silybin through inhibiting efflux transporter MRP2,

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BCRP instead of regulating phase-2 metabolism or MDR1 (P-gp). However, Wu etc. suggested that the active silybin efflux might be partially inhibited by P-glycoprotein as coadministration of cyclosporin A (CsA) with silybin significantly decreased the area under the

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concentration versus time curve (AUC) in bile. As CsA could inhibit both P-gp and BCRP (Li et al., 2014), therefor we believe that the biliary excretion inhibited by CsA should be partially

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inhibited by BCRP instead of P-gp.

Piperine could significantly inhibit the activity of UGT enzymes which are the main

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metabolic enzymes of silybin (Atal et al., 1985; Singh et al., 1986). However, experiments

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carried out in SCRHs and HLMs demonstrated that the presence of piperine didn’t affect the phase-2 conjugates production by the hepatocytes. Moreover, our previous research showed

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that alerted UGTs enzyme activity with gene polymorphism failed to regulate the metabolism or PK behavior of silybin in clinic (Xie et al. 2017). Therefore efflux transporters including MRP2 and BCRP play a key role in the pharmacokinetic behavior of silybin. This result is consistent with the observation reported by Dr. Hawke (Schrieber et al., 2011) as they found that patients with cirrhosis and nonalcoholic fatty liver disease achieved the higher exposures of silybin than health control which were due to the decreased protein expression of BCRP,

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ACCEPTED MANUSCRIPT MRP2 in alcoholic cirrhosis and hepatitis C cirrhosis (Wang et al., 2016) . In sum, both pharmacokinetic and pharmacological studies indicate that piperine serves as a promising bioenhancer for silybin and its mechanisms involved inhibition of efflux transporters including MRP2 and BCRP. New formulation containing both piperine and silybin with increased bioavaibility and safety will be expected to bring novel therapeutics

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into clinic. Acknowledgments This work was financially supported by the Macao Science and Technology Development Fund, Macau Special Administrative Region [Grant 003/2017/A1]. The authors declare no conflicts of interest.

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Conflict of interest

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ACCEPTED MANUSCRIPT Table 1. Mean pharmacokinetic parameters of free silybin after oral administration at a dose of 50mg/kg with or without simultaneous oral administration of 50mg/kg piperine to rats. Parameters a

Without Piperine Free SBa

Free SBa

Free SBb

176±34

179±57

310±127*

286±86*

Tmax (h)

0.33±0.13

0.38±0.14

0.25±0.00

0.25±0.00

t1/2 (h)

9.00±1.99

11.11±6.41

7.15±4.43

9.18±4.56

AUC(0-t) (ng ⋅ h /mL)

326.9±69.0

380.1±71.9

447.9±103.3*

478.8±149.0

AUC(0-∞)( ng ⋅ h /mL)

445.2±83.7

616.1±188.1

678.0±350.2

793.9±480.0

MRT (h)

3.67±0.38

4.04±0.44

3.68±0.35

4.28±0.64

CL/F( mL/h/kg)

106.5±17.9

87.1±27.1

89.3±37.7

80.3±38.3

Total SBa Cmax(ng/mL)

3130±693

Tmax (h)

0.79±0.459

t1/2 (h)

2.03±9.89

With Piperine

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Without Piperine

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Cmax(ng/mL)

Free SBb

With Piperine

Total SBb

Total SBa

Total SBb

5658±631

4677±263***

7852±471***

0.83±0.26

0.67±0.26

1.42±0.59

2.86±1.29

2.13±0.54

1.59±0.21

19482±5271

16266±4528*

35196±7130**

11146±3112

AUC(0-∞) (ng ⋅ h /mL)

15998±12670

20627±6112

16789±5134

35601±7436**

3.35±0.66

3.02±0.27

2.88±0.572

3.09±0.45

MRT (h)

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AUC(0-t) (ng ⋅ h /mL)

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CL/F( L/h/kg) 4.2±1.8 2.6±0.7 3.2±0.9 1.5±0.3 a : Cmax: Maximum plasma concentration; Tmax: Time to reach maximum plasma concentration;

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T1/2 E phase: Half-life of elimination phase; T1/2 D/A phase: Half-life of distribution phase; T1/2 A phase:

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Half-life of absorption phase; MRT: Mean residence time; Vd: Volume of distribution; CL/F:

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Oral clearance.

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Legends

Fig. 1. The typical chromatograms for detection of silybin A, silybin B, piperine and

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naringenin (internal standard) in a blank plasma sample (A); a plasma sample spiked with

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25ng/ml of silybin, and 50 ng/ml of piperine (B); and a plasma sample obtained 12h after

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co-administration of silybin and piperine in SD rats (C).

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Fig. 2. The mean plasma concentration versus time profiles of free silybin A (A), free silybin B (B), and total silybin A, total silybin B (C, D) as well as piperine (E) after oral

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administration of 50mg/kg silybin with or without oral administration of 10mg/kg piperine.

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Fig. 3. Silybin co-administrated with piperine prevented against CCl4-induced liver injury. A)

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Effects of silybin co-administrated with piperine on the histopathological changes of CCl4-induced acute liver injury. Liver sections were stained with hematoxylin-eosin (×200). Effects of silybin co-administrated with or without piperine on biochemical parameters

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B)

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in CCl4-treated rats (means± S.D., n=6). *P < 0.05 **P < 0.01; compared to the normal control group. #P <0.05，##P <0.01，

P <0.001 compared to the Model group (Student's t

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test).

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Fig. 4. Effects of piperine on the permeability of silybin in Caco-2 cells. Permeability Papp B-A (A) and Papp B-A (B) as well as efflux ratios (C) of silybin A and B were measured in

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the presence of piperine (10, 20, 40μM) in Caco-2 cells. Each data point represents the mean

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± S.D. of three independent experiments. * P<0.05, ** P<0.01, *** P<0.001, statistically

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significant compared with the silybin group.

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Fig. 5. Bidirectional permeability of substrate and the inhibitive effects of piperine in MDCKII cells overexpressing human transporters. (A) Efflux ratios of Rho 123, CDCFDA and Ph A were measured in the presence of piperine (10, 20, 40μM) in the transfected MDCK

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II cells. (B) Efflux ratios of silybin A and B were measured in the presence of piperine (10, 20, 40μM) in the transfected MDCK II cells. Data were expressed as the mean ± S.D. of three

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independent experiments. * P<0.05, ** P<0.01, *** P<0.001, statistically significant

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differences as compared to the control group.

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Fig. 6. The effects of piperine on the BEI and Clbiliary of silybin (10 μM) and hepatobiliary disposition of silybin in sandwich cultured hepatocytes. Data are expressed as mean ± S.E. (n ≥3). Piperine inhibits the BEI of free silybin (A) and conjugated silybin (B) as well as the

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biliary clearance of silybin (C) via significantly reducing the amount of free silybin (D) and conjugated silybin (E) excreted into biliary. However, piperine failed to inhibit the phase II * P<0.05, ** P<0.01, *** P<0.001 statistically significant

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metabolism (F) in hepatocytes.

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differences as compared to the control.

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Graphic Abstract

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