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. Author manuscript; available in PMC: 2022 Jun 1.
Published in final edited form as: Phytother Res. 2021 Feb 15;35(6):3286–3297. doi: 10.1002/ptr.7049

Hepatic organic anion transporting polypeptides mediate disposition of milk thistle flavonolignans and pharmacokinetic silymarin-drug interactions

Katherine D Lynch 1,#, Michelle L Montonye 1,#, Dan-Dan Tian 1, Tarana Arman 1, Victoria O Oyanna 1, Baron J Bechtold 1, Tyler N Graf 2, Nicholas H Oberlies 2, Mary F Paine 1, John D Clarke 1
PMCID: PMC8217340  NIHMSID: NIHMS1698858  PMID: 33587330

Abstract

Silybum marianum (L.) Gaertn. (Asteraceae), commonly known as milk thistle, is a botanical natural product used to self-treat multiple diseases such as type 2 diabetes mellitus and nonalcoholic steatohepatitis (NASH). An extract from milk thistle seeds (achenes), termed silymarin, is comprised primarily of several flavonolignans. Systemic concentrations of these flavonolignans can influence the potential biologic effects of silymarin and the risk for pharmacokinetic silymarin-drug interactions. The aims of this research were to determine the roles of organic anion transporting polypeptides (OATPs/Oatps) in silymarin flavonolignan disposition and in pharmacokinetic silymarin-drug interactions. The seven major flavonolignans from silymarin were determined to be substrates for OATP1B1, OATP1B3, and OATP2B1. Sprague Dawley rats were fed either a control diet or a NASH-inducing diet and administered pitavastatin (OATP/Oatp probe substrate), followed by silymarin via oral gavage. Decreased protein expression of Oatp1b2 and Oatp1a4 in NASH animals increased flavonolignan area under the plasma concentration-time curve (AUC) and maximum plasma concentration. The combination of silymarin inhibition of OATPs/Oatps and NASH-associated decrease in Oatp expression caused an additive increase in plasma pitavastatin AUC in the animals. These data indicate that OATPs/Oatps contribute to flavonolignan cellular uptake and mediate the interaction between silymarin and NASH on pitavastatin systemic exposure.

Keywords: flavonolignans, nonalcoholic steatohepatitis, organic anion transporting polypeptide, pharmacokinetics, silymarin, pitavastatin

1. Introduction

Silybum marianum (L.) Gaertn. (Asteraceae), commonly known as milk thistle, was ranked 10th and 23rd in U.S. natural channel and U.S. mainstream multi-outlet sales, respectively, amassing more than $26 million in sales in 2019 (Smith et al., 2020). An extract of milk thistle seeds, termed silymarin, consists of ~50-70% flavonolignans (e.g., silybin A [SA], silybin B [SB], isosilybin A [IA], isosilybin B [IB], silydianin [SD], silychristin [SC]) and flavonoids (e.g., taxifolin, quercetin), with the remaining ~30-50% consisting of chemically undefined polyphenolic compounds (Graf et al., 2016; Javed et al., 2011). Various forms of milk thistle have been used for centuries to treat multiple disorders, including liver diseases and mushroom poisoning (Abenavoli et al., 2018; Smith et al., 2020). More recently, silymarin has been investigated as a therapeutic agent for multiple diseases, including viral hepatitis, type 2 diabetes mellitus, alcoholic liver disease, cholestasis, drug- or toxin-induced liver disease, primary liver malignancies, nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH) (Abenavoli et al., 2018; Voroneanu et al., 2016). Silymarin is also marketed to the “healthy” population as a liver detoxification product, as well as to offset the effects of alcohol consumption (Feher & Lengyel, 2012; Lieber et al., 2003; Müzes et al., 1990).

Efficacy of silymarin as a treatment for various liver diseases is equivocal, but the doses and formulations that produce micromolar flavonolignan systemic plasma concentrations are well tolerated in healthy and liver diseased populations (Fried et al., 2012; Wah Kheong et al., 2017). For example, up to 2.1 grams per day of silymarin administered to NASH patients caused no adverse events (Wah Kheong et al., 2017). Similarly, 13 grams per day of a phytosome formulation consisting of silybin A, silybin B, and phosphatidylcholine produced flavonolignan plasma concentrations up to 75 μM and caused no adverse events in prostate cancer patients (Flaig et al., 2007). Flavonolignan systemic concentrations are influenced by uptake transporters, metabolizing enzymes, and efflux transporters and can influence the biologic effects and pharmacokinetic drug interaction liability of silymarin. Enteric absorption and hepatic clearance of flavonolignans are mediated by the efflux transporters multi-drug resistance-associated protein (MRP)2, MRP3, MRP4, and breast cancer resistance protein (BCRP) (Bi et al., 2019; Miranda et al., 2008; Schrieber et al., 2011; Wlcek et al., 2013; Xu et al., 2018). Organic anion transporting polypeptides (OATPs/Oatps) have been speculated as flavonolignan uptake transporters, although no direct mechanistic data have been collected.

OATPs/Oatps belong to a superfamily of transporters that mediate the sodium-independent transport of a wide range of amphiphilic organic compounds, including many hydroxymethylglutaryl-CoA reductase inhibitors (statins), angiotensin II receptor blockers, angiotensin converting enzyme inhibitors, chemotherapeutic agents, and anti-diabetic agents (Giacomini et al., 2010; Maeda, 2015). Common notation to distinguish human versus rodent OATP/Oatp isoforms is uppercase versus lowercase, respectively (Hagenbuch & Meier, 2004). The rate-limiting step in the hepatic clearance of OATP/Oatp substrates can be uptake from portal blood into the liver (Maeda, 2015), and silymarin has been investigated as a potential precipitant of natural product-drug interactions involving OATPs/Oatps (Deng et al., 2008). Intravenous administration of silymarin to Sprague Dawley rats was recently shown to inhibit Oatp-mediated pitavastatin uptake and increase the area under the plasma concentration-time curve (AUC) of intravenously administered pitavastatin, a probe for OATPs/Oatps (Montonye et al., 2019). Although these data provided fundamental pharmacokinetic information, both silymarin and pitavastatin are taken orally by patients with healthy livers and NASH patients, and risk for a pharmacokinetic interaction after oral consumption is unknown.

The purpose of the present study was to evaluate a probable natural product-disease-drug interaction in a preclinical model. The were two aims in this study. The first was to determine the role of OATPs/Oatps in flavonolignan disposition. The information gained can be used to predict populations with altered flavonolignan disposition or to exploit these mechanisms to alter flavonolignan disposition and augment potential beneficial effects. The second was to determine whether oral silymarin administration can precipitate pharmacokinetic drug interactions involving Oatps in healthy versus NASH animals. By using a NASH rodent model that recapitulates important clinical features related to drug disposition (Canet et al., 2014), information from this research can justify and inform investigation of this natural product-disease-drug interaction in humans.

2. Materials and methods

2.1. Chemicals and reagents

Pitavastatin calcium (cat.# ab141958, >98% purity) was purchased from Abcam (Cambridge, MA). Pitavastatin-d5 (cat.# P531022) was purchased from Toronto Research Chemicals (Ontario, Canada). Naringin (cat.# 71162-25G, >95% purity), dichlorofluorescein (cat.# 35848-5G, >90% purity), dibromofluorescein (cat.# 216720-5g, >95% purity), and silymarin (cat.# S0292, lot no. BCBT9170) were purchased from Sigma Aldrich (St. Louis, MO). The silymarin lot was characterized previously (Montonye et al., 2019). Fluorescein (cat.# 97062-186) and sodium butyrate (cat.# AAA11079-36, >98% purity) were purchased from VWR (Radnor, PA). Geneticin (cat.# 10-131-027) was purchased from Thermo Fisher Scientific (Waltham, MA). Flavonolignans were isolated from silymarin using a hybrid chromatographic/precipitative technique as detailed previously (Graf et al., 2007). Reference standards for glucuronides of the silymarin flavonolignans were generated using bovine liver microsomes as detailed previously (Gufford et al., 2015). All flavonolignan reference standards were validated to be greater than 95% pure, as measured by UPLC (Graf et al., 2016). Control diet (cat.# TD180070) and standard diet (cat.# 2016) were purchased from Envigo (Madison, WI). The methionine-and choline-deficient (MCD) diet (cat.# D518810) was purchased from Dyets Inc. (Bethlehem, PA). Wild-type Chinese hamster ovary (CHO) cells and CHO cells stably expressing human OATP1B1, OATP1B3, or OATP2B1 were provided by Dr. Bruno Stieger (University of Zurich, Zurich, Switzerland). Antibodies used in western blots were as follows: Oatp1b2 (cat.# 376904, Santa Cruz Biotechnology, Santa Cruz, CA) visualized with an anti-mouse HRP-labeled secondary antibody; and Oatp1a4 (cat.# OATP21-A, Alpha diagnostics, San Antonio, TX) visualized with an anti-rabbit HRP-labeled secondary antibody. SuperSignal West Pico and Femto (ThermoFisher Scientific) were used as chemiluminescent substrates to visualize proteins of interest. Guidelines relevant to best practices in natural products research were considered throughout the study (Heinrich et al., 2020; Izzo et al., 2020; Kellogg et al., 2019).

2.2. Animals

Handling, care, and maintenance of the animals took place in the Program of the Laboratory Animal Resources facility of Washington State University Health Sciences Spokane, which is accredited by the Association for the Assessment of Laboratory Animal Care International. The experimental protocol was approved by the Institutional Animal Care and Use Committee at Washington State University. Eight-week-old male Sprague Dawley rats (n = 26) were purchased from Envigo (Huntingdon, Cambridgeshire, UK). Animals were randomly placed in cages (two per cage) with diamond soft-paper bedding and fed a control diet for 16 weeks (n = 14) or standard diet for 8 weeks followed by an MCD diet for 8 weeks (n = 12). Silymarin and pitavastatin were prepared as previously described (Montonye et al., 2019). Silymarin vehicle consisted of 20% propylene glycol, 45% PEG400, and 35% molecular grade water. After the diet period, animals were fasted for 12 hours, then pitavastatin (2.5 mg/kg, 5 mL/kg), dissolved in DMSO, and diluted in sterile saline (2% DMSO final), was administered by oral gavage. Forty minutes after administration of pitavastatin, vehicle (10 mL/kg) or silymarin (500 mg/kg, 10 mL/kg) was administered by oral gavage. The dose of silymarin was selected to produce flavonolignan plasma concentrations observed in humans. Published clinical pharmacokinetic studies involving NAFLD and HCV patients reported average Cmax values for silybin A between 0.8-4.2 μM (Fried, 2012; Wah Kheong et al., 2017). This dose of silymarin was also shown to be safe in rodents (Gao et al., 2018). Blood (~0.2 mL) was collected through the tail vein into heparinized tubes at 0.083, 0.25, 0.5, 0.75, 0.83, 1, 1.5, 2, 4, 6, 9, and 12 hours after the first gavage. Animals were euthanized 27 hours after the first gavage. Plasma was isolated by centrifugation, and interconversion between the acid and lactone forms of pitavastatin was prevented by acidifying plasma samples as previously described (Montonye et al., 2019; Qi et al., 2013). After the terminal blood was collected, animals were euthanized by carbon dioxide asphyxiation and exsanguination, and livers were harvested as previously described (Montonye et al., 2019). Liver samples were formalin fixed and paraffin embedded or snap frozen in liquid nitrogen and stored at −80°C until further analyses.

2.3. Cell culture

Wild-type CHO cells and CHO cells stably expressing human OATP1B1 (passages 28-37), OATP1B3 (passages 27-36), or OATP2B1 (passages 20-29) were maintained at 37°C in a humidified 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium (low-glucose) containing 25 mM HEPES, supplemented with 10% fetal bovine serum, and 50 mg/mL L-proline culture without geneticin for wild-type cells and with 0.5 mg/mL geneticin for OATP-expressing cells. Cells were seeded onto 12-well plates at a density of 0.3 x 106 cells per well. Transporter expression was induced by incubating cells with 10 mM sodium butyrate for 24 hours prior to transport experiments. Waymouth transport buffer (135 mM sodium chloride, 1.3 mM HEPES, 2.8 mM D-glucose, 0.5 mM potassium chloride, 0.25 mM calcium dichloride, 0.12 mM magnesium dichloride, 80 mM magnesium sulfate, pH 7.4) was used in place of culture medium for all transport experiments. Prior to initiating transport, cells were washed three times with prewarmed transport buffer and allowed to equilibrate for 5 minutes at 37°C. Transport was normalized to total cellular protein as quantified by the Pierce BCA assay (Thermo Fisher Scientific) using a Synergy H1 (BioTek, Winooski, VT) plate reader.

2.3.1. IC50 determination

Uptake was initiated by addition of prewarmed transport buffer containing 5 μM dichlorofluorescein (OATP1B1 probe), 5 μM fluorescein (OATP1B3 probe), or 2 μM dibromofluorescein (OATP2B1 probe), with increasing silymarin concentrations (0 - 48 μg/ml). No change in cell viability was observed at these concentrations for this short incubation period (data not shown). For pre-incubation experiments, each cell line was incubated with transport buffer containing silymarin for 35 minutes at 37°C before addition of transport buffer containing fluorescent probe substrate. Transport was terminated at 5 minutes for OATP1B1 and OATP1B3, or 2 minutes for OATP2B1, with ice-cold transport buffer. Cells were washed quickly three times with ice-cold transport buffer before being lysed with 110 μL of milliQ water. Plates were subjected to a freeze-thaw cycle at −80°C before the wells were scraped and samples transferred to microcentrifuge tubes. Supernatant (80 μL) was analyzed using a fluorescence microplate reader (Synergy H1) at the following excitation/emission wavelengths: dichlorofluorescein, 460 nm/515 nm; dibromofluorescein, 485 nm/528 nm; and fluorescein, 460 nm/515 nm. Autofluorescence of cells without substrate was subtracted from the fluorescence measured in cells incubated with substrate before comparing to the calibration curve. Quantification of unknowns was accomplished using a calibration curve that was linear from 1.0 to 500 nM for each fluorescent compound. Dichlorofluorescein, fluorescein, and dibromofluorescein substrate choices were based on a previously published method (Izumi et al., 2016). Substrate concentrations and incubation times were validated prior to experimentation (data not shown). Best-fit IC50 values were determined by nonlinear least-squares regression using the standard Hill equation as previously described (Gufford et al., 2014) for all OATPs using GraphPad Prism (version 7.0; San Diego, CA).

2.3.2. Flavonolignans as substrates

Uptake was initiated by addition of prewarmed transport buffer containing 5 μM of flavonolignan; transport was terminated at 5 minutes with ice-cold transport buffer. Cells were washed quickly three times with ice-cold transport buffer before being lysed with 110 μL of 0.1% formic acid in HPLC water. Plates were subjected to a freeze-thaw cycle at −80°C before the wells were scraped, after which cell lysates were transferred to microcentrifuge tubes. Cell lysate (80 μL) was mixed with 320 μL of 0.028 μM naringin (internal standard) in acetonitrile; proteins were precipitated by centrifugation at 13,000 x g for 5 minutes at room temperature. The supernatant (380 μL) was dried under vacuum at 37°C, then reconstituted in 50 μL of 20% methanol with 0.1% formic acid and analyzed for flavonolignans as described below via liquid chromatography/mass spectrometry. OATP-mediated transport was calculated by subtracting intracellular substrate concentrations in wild-type CHO cells from intracellular substrate concentrations in OATP-expressing CHO cells.

2.4. Liquid chromatography-mass spectrometry for pitavastatin

Quantification of pitavastatin was performed as previously described (Montonye et al., 2019). Briefly, 10 μL of plasma was mixed with 0.1% formic acid water containing internal standards followed by protein precipitation. Chromatographic separation and quantification were performed using a QTRAP 6500 UHPLC-MS/MS (AB Sciex, Framingham, MA) system. A gradient of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) was passed through an HSS T3 column (1.8 mm, 2.1 x 50 mm, Waters Corporation, Waltham, MA) at 50°C. The turbo electrospray source was operated in positive ionization mode. The following multiple reaction monitoring transitions were used for compound quantification: pitavastatin, 422.1 → 290.1 m/z; pitavastatin-d5, 426.9 → 293.8 m/z. A second precursor-fragment ion combination was used to validate the presence of each compound. Plasma and liver samples were spiked with the internal standard, pitavastatin-d5 (0.24 μM). Powdered 50 mg liver tissue was homogenized in 100 μL of 0.1% formic acid water containing 1.2 μM pitavastatin-d5 and 6.2 μM pitavastatin lactone-d5 internal standard. Liver pitavastatin content was calculated based on total liver weight. Liver to plasma ratio was calculated by dividing total liver pitavastatin content by the final concentration of pitavastatin in the plasma at 27 hours. Quantification of pitavastatin was accomplished using a calibration curve that was linear from 0.018 to 4.75 μM, and quality control samples (0.5 μM) were run periodically to ensure consistent instrument performance.

2.5. Liquid chromatography-mass spectrometry for flavonolignans

Quantification of flavonolignans from silymarin was performed as previously described (Montonye et al., 2019). Briefly, 20 μL of plasma were mixed with internal standard and 0.1% formic acid in methanol. See section 2.3.2 for sample preparation methods after transport experiments. Chromatographic separation and quantification was performed on a QTRAP 6500. A gradient of 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B) was passed through an HSS T3 column at 50°C. The turbo electrospray source was operated in the negative ionization mode. The following multiple reaction monitoring transitions were used for compound quantification: silybin A, 480.9 → 301.9 m/z; silybin B, 480.9 → 125.0 m/z; silychristin, 480.9 → 325.1 m/z; silydianin, 480.9 → 178.9 m/z; isosilybin A, 480.9 → 125.0 m/z; isosilybin B, 480.9 → 125.0 m/z; isosilychristin, 480.9 → 463.0; taxifolin, 302.8 → 284.9 m/z; silybin A and silybin B glucuronides, 657.1 → 481.1; naringin, 578.9 → 270.9. A second precursor-product ion pair was used to validate the presence of each compound. Plasma samples were spiked with naringin as internal standard (0.17 μM). Flavonolignans in cell culture experiments were quantified as described above. Quantification of flavonolignans was accomplished using a calibration curve that was linear from 0.016 to 4.16 μM, and quality control samples (0.06 μM and 0.13 μM) were run periodically to ensure consistent instrument performance.

2.6. Western blot analysis

Liver protein samples were extracted as previously described (Montonye et al., 2019). Briefly, liver tissue was homogenized in NP-40 lysis buffer using a TissueLyzer II (Qiagen, Hilden, Germany). Total protein (20 μg) was prepared in Laemmli sample buffer containing 2.5% 2-mercaptoethanol and heated at 37°C for 30 minutes. Samples were loaded onto 7.5% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes using a Bio-Rad (Hercules, CA) Trans-Blot Turbo system at 25 V/1.0 A for 30 minutes. Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline/Tween 20 and incubated with the following antibodies: Oatp1b2 1:1000; mouse secondary 1:10,000; Oatp1a4 1:1,000; rabbit secondary 1:10,000. Signal was developed with SuperSignal West Pico for Oatp1b2 and Femto for Oatp1a4. Densitometry was performed using Image Lab (Bio-Rad, Standard Edition, Version 6.0.0 build 25). Proteins of interest were normalized to total protein as determined by Amido black staining (Aldridge et al., 2008).

2.7. Pharmacokinetic analysis

The pharmacokinetics of flavonolignans, taxifolin, and pitavastatin were determined via non-compartmental analysis methods using Phoenix (version 7.0; Certara, Princeton, NJ). Cmax and time to reach Cmax (tmax) were obtained directly from the concentration-time profile. Partial AUC was determined using the linear up/log down trapezoidal method (0 to 27 hours for flavonolignan glucuronides and pitavastatin and 0 to 2 hours for SC and TF). When appropriate (i.e., sufficient data points were available), terminal slope (λz) was recovered via linear regression of at least the last four data points, and terminal half-life (t1/2) was calculated as the ratio of 0.693 to λz. Total AUC (AUCinf) for SA, SB, IA, and IB was calculated as the sum of AUC0-27h and the ratio of the last measured concentration to λz. Oral clearance (Cl/F) was calculated as the ratio of dose to AUCinf.

2.8. Statistical analysis

Two-way analysis of variance (ANOVA) p-values are shown in tables accompanying the respective graph and in Table 2. Bonferroni post-hoc test was used when comparing pitavastatin pharmacokinetic and protein expression data among the control and NASH groups, with or without silymarin. Flavonolignan comparisons between control and NASH groups were made using the unpaired Student’s t test. AUC, Cmax, and Cl/F values were log-transformed prior to statistical analysis. Tmax values were analyzed with the Mann-Whitney U test. No randomization or blinding was performed for these analyses.

Table 2.

Pitavastatin plasma pharmacokinetics

AUC h*μmol/L Cmax μmol/L Tmax h
Control Vehicle 3.1 (2.5, 3.9) 0.8 (0.5, 1.3) 0.83 (0.5, 1.0)
Silymarin 5.7 (5.0, 6.4) 1.5 (1.2, 1.9) 1.0 (1.0, 1.0)
NASH Vehicle 9.1 (6.4, 13.1)b,c 3.1 (1.8, 5.1)b 0.50 (0.25, 0.75)c
Silymarin 15.9 (14.1, 18.1)a,b,c 4.2 (3.1, 5.6)b,c 1.25 (1.0, 2.0)a,b
Two-way ANOVA
NASH <0.05 <0.05 0.62
Silymarin <0.05 0.03 <0.05
Interaction 0.94 0.37 <0.05
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Data represent geometric means and upper and lower limits of 90% confidence intervals in parentheses for AUC and Cmax. Data for tmax represent median with minimum and maximum in parentheses. Oral clearance and terminal half-life could not be recovered due to insufficient data points in the terminal phase of the plasma concentration-time profile. All data shown are based on seven animals for the control group and six animals for the NASH group. Bonferroni post-hoc tests:

a

p-values ≤ 0.05 vs respective vehicle groups,

b

p-values ≤ 0.05 vs control vehicle group, and

c

p-values ≤ 0.05 vs control silymarin group.

3. Results and discussion

3.1. Role of OATPs/Oatps in silymarin disposition

Silymarin exposure time and concentration are important considerations for understanding potential beneficial or detrimental biologic effects. In the current study, average unconjugated flavonolignan plasma AUC and Cmax values after oral administration of silymarin (Figure 1 and Table 1) were congruent with relative proportions observed in healthy human volunteers (SA=SB=IA=IB>>SC=SD) (Wen et al., 2008). AUC and Cmax for individual flavonolignans and taxifolin were 2.2- to 2.5-fold and 2.4- to 3.5-fold higher, respectively, whereas corresponding Cl/F was 53% to 72% lower, in the NASH group compared to the control group (Table 1). These data are consistent with a previous clinical study in hepatitis C virus (HCV) and NAFLD patients in which total plasma flavonolignan AUC was 2.4- and 3.3-fold higher, respectively, than in healthy volunteers (Schrieber et al., 2011). AUC0–27h and Cmax for SA and SB glucuronides in the current study were 3.6-fold and 4.2- to 6.3-fold higher, respectively, in the NASH group compared to the control group (Table 1). These data show increased plasma concentrations of both the unconjugated flavonolignans and glucuronides in NASH, suggesting a mechanism involving enteric and/or hepatic transporters rather than phase II metabolism (Schrieber et al., 2008, 2011).

Fig. 1. Silymarin flavonolignan plasma concentration vs. time profiles and structures.

Fig. 1

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Plasma exposure to silymarin flavonolignans and glucuronides is higher in NASH compared to control animals. Average plasma concentration versus time profiles and chemical structures for silybin A (A), silybin B (B), isosilybin A (C), isosilybin B (D), silybin A-4”-O-β-D-glucuronide (E), and silybin A-7-O-β-D-glucuronide (F) following oral gavage of silymarin (500 mg/kg) to control and NASH animals. Symbols and error bars represent means and SDs, respectively, of control (n=7) and NASH (n=6) animals.

Table 1.

Silymarin flavonolignan and taxifolin plasma pharmacokinetics

AUC h*μmol/L Cl/F L/h/kg Cmax μmol/L t1/2 h tmax h
SA Control 2.3 (1.8, 2.9) 37.8 (29.9, 47.6) 1.6 (1.3, 2.0) 3.3 (2.7, 3.9) 0.8 (0.83, 1.5)
NASH 4.9 (3.8, 6.5) * 17.6 (13.4, 23.2) * 4.5 (3.4, 6.0) * 2.9 (2.0, 4.2) 1.0 (0.83, 1.0)
SB Control 3.1 (2.3, 4.1) 50.8 (37.7, 68.4) 2.4 (1.9, 3.0) 4.1 (2.7, 6.3) 0.83 (0.83, 1.5)
NASH 10.9 (6.3, 18.9) * 14.3 (8.2, 24.7) * 7.9 (6.2, 10.2) * 16.9 (2.9, 98.7) 0.83 (0.83, 1.0)
IA Control 2.4 (1.9, 3.1) 20.9 (16.5, 27.1) 1.7 (1.4, 2.0) 4.5 (2.9, 7.2) 0.83 (0.83, 1.5)
NASH 7.5 (5.3, 10.6) * 6.8 (4.8, 9.8) * 5.2 (4.1, 6.7) * 10.8 (0.6, 36.3) 0.83 (0.83, 1.0)
IB Control 3.0 (2.2, 4.0) 8.4 (6.2, 11.4) 1.9 (1.6, 2.4) 4.9 (3.0, 8.0) 0.83 (0.83, 1.50)
NASH 7.9 (4.0, 15.6) * 3.1 (1.6, 6.2) * 5.9 (4.6, 7.5) * 14.2 (3.9, 50.6) 1.0 (0.83, 1.0)
SC Control 0.02 (0.01, 0.02) NA 0.02 (0.02, 0.03) NA 1.0 (1.0, 1.5)
NASH 0.05 (0.04, 0.06) * NA 0.07 (0.05, 0.08) * NA 1.0 (0.83, 1.0)
TF Control 0.18 (0.14, 0.24) NA 0.3 (0.2, 0.3) NA 0.83 (0.75, 1.5)
NASH 0.44 (0.30, 0.63) * NA 0.7 (0.5, 0.9) * NA 0.83 (0.83, 1.0)
S4G Control 44.5 (40.9, 48.4) NA 2.0 (2.5, 3.6) NA 2.0 (1.0, 2.0)
NASH 160.3 (141.2, 182.1) * NA 12.6 (10.6, 14.9) * NA 2.0 (2.0, 2.0)
S7G Control 43.0 (38.2, 48.5) NA 3.0 (2.5, 3.6) NA 2.0 (1.0, 2.0)
NASH 154.3 (129.1,184.5) * NA 12.6 (10.6,14.9) * NA 2.0 (2.0, 2.0)
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Data represented as geometric means and upper and lower limits of 90% confidence intervals in parentheses for area under the plasma concentration time curve (AUC), oral clearance (Cl/F), maximum concentration (Cmax), and half-life (t1/2). Data for time to achieve Cmax (tmax) represent median with minimum and maximum in parentheses. NA = not applicable due to insufficient data points in the terminal phase of the plasma concentration-time profile. SB and IB are based on three animals in the NASH group, and IA is based on four animals in the NASH group. All other data shown are based on seven animals for control groups and six animals for NASH groups. Unpaired Student’s t test

*

p-values ≤ 0.05 vs control silymarin group. Abbreviations: silybin A (SA), silybin B (SB), isosilybin A (IA), isosilybin B (IB), silychristin (SC), taxifolin (TF), silybin A-4”-O-β-D-glucuronide (S4G), and silybin A-7-O-β-D-glucuronide (S7G).

The contribution of OATPs/Oatps to enteric absorption and hepatic clearance of flavonolignans has not been established. The International Transporter Consortium, composed of the world’s experts in drug transporters, highlighted OATP1B1, OATP1B3, and OATP2B1 as clinically important transporters for consideration in drug development, drug-drug interactions, and disease-related changes in drug disposition (Evers et al., 2018; Hillgren et al., 2013; Liang et al., 2011). All tested flavonolignans (SA, SB, IA, IB, SC, and SD) were identified as substrates for OATP1B1, OATP1B3, and OATP2B1 (Figure 2). The amount of SA in the silymarin powdered product used in this study was greater than the amount of SC (>30% versus <20%) (Montonye et al., 2019), but plasma AUC of SA was 110 times greater than that of SC (Table 1). OATP2B1 is the primary OATP expressed in enterocytes that contributes to the absorption of many drugs and nutrients (Chen et al., 2019). The apparent lower relative oral bioavailability of SC compared to SA may be partially explained by the lower transport activity of OATP2B1 for SC compared to SA (Figure 2). Species differences in OATP/Oatp isoforms between rodents and humans require analysis of species specific OATP/Oatp isoforms. We confirmed that MCD diet-induced NASH decreased the expression of Oatp1b2 and Oatp1a4 (Figure 3). Rodent Oatp1b2 is the single ortholog to human OATP1B1 and OATP1B3, and a previous report indicated decreased OATP1B3 expression in NASH patients (Clarke et al., 2017). Rodent Oatp1a4 is expressed on the sinusoidal membrane of hepatocytes and is involved in the disposition of many xenobiotics, including pitavastatin (Sano et al., 2018). The closest human ortholog to rodent Oatp1a4 is OATP1A2, but OATP1A2 is not expressed in the intestine nor on the sinusoidal membrane of hepatocytes and therefore was not tested (Lee et al., 2005). Decreased OATP/Oatp expression in NASH potentially contributes to higher flavonolignan systemic exposure. Patients with other liver diseases known to decrease OATP expression, such as hepatocellular carcinoma and alcoholic liver disease, may also experience increased flavonolignan systemic exposure, potentially increasing the risk for OATP-mediated silymarin-drug interactions (Obaidat et al., 2012; Wang et al., 2016). These data also suggest that the Sprague Dawley MCD NASH model recapitulates the clinical disposition of flavonolignans and taxifolin and can be used to investigate individual and combined effects of silymarin and NASH on the pharmacokinetics of co-consumed drugs.

Fig. 2. Silymarin flavonolignans are OATP substrates.

Fig. 2

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Average OATP-mediated uptake of SA, SB, IA, IB, SC, and SD in OATP1B1, OATP1B3 and OATP2B1 expressing cells. Horizontal lines represent means of duplicates.

Fig. 3. Liver Oatp expression.

Fig. 3

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Liver Oatp expression is lower in NASH compared to control animals. Western blot analysis of Oatp1b2 (A) and Oatp1a4 (B) in control and NASH livers 27 hours after pitavastatin oral gavage. Symbols and error bars (A, B) represent means and SDs, respectively, of control (n=7) and NASH (n=6) animals. One representative (n=2 for each group) western blot and amido black total protein stain is shown for each protein of interest. Two-way ANOVA p-values are shown in the table (C). Bonferroni post-hoc test bp-values ≤ 0.05 vs control vehicle group.

3.2. Additive pharmacokinetic interaction between silymarin and NASH on pitavastatin exposure

The FDA has made available several guidance documents describing recommendations related to drug development and investigation (FDA, 2020a, 2020b). The current study utilized two guidance documents to produce the most translatable and reliable data needed to evaluate potential hepatic OATP-mediated silymarin-drug interactions (FDA, 2020a, 2020b). First, the guidance for drug interaction studies states that the maximum dose of the precipitant should be used to maximize the possibility of observing a drug-drug interaction. The dose of silymarin in this study was selected to produce plasma flavonolignan concentrations comparable to those observed in humans. Published clinical pharmacokinetic studies in NAFLD and HCV patients showed that 560-700 mg doses of silymarin produced average Cmax values for SA between 0.8-4.2 μM (Fried et al., 2012; Schrieber et al., 2011), and we achieved similar average SA Cmax values of 1.6 μM for control and 4.5 μM for NASH animals. In addition, pitavastatin was administered first, followed by silymarin, to produce an overlap in tmax between pitavastatin and flavonolignans from silymarin (Table 1 and Table 2), maximizing the possibility of a hepatic OATP-mediated interaction. Second, the FDA guidance for in vitro drug interaction studies recommends preincubating cells with the inhibitor when investigating OATP-mediated drug interactions (FDA, 2020a). In the current study, compared to no preincubation, preincubation of OATP-expressing CHO cells with silymarin produced a 2.8-fold leftward shift in IC50 for OATP1B1 (2.8 ± 0.5 to 1.0 ± 0.1 μg/ml; no preincubation and preincubation, respectively; mean ± SD) and a 1.7-fold leftward shift in IC50 for both OATP1B3 (4.5 ± 1.2 to 2.6 ± 0.4 μg/ml) and OATP2B1 (1.2 ± 0.2 to 0.7 ± 0.1 μg/ml) (Figure 4). The FDA recommended R-value (ratio of unbound inhibitor concentration at the liver inlet to IC50) cut-off for hepatic OATP-mediated interactions is 1.1. Thus, the lower IC50 values with preincubation increase the risk of exceeding 1.1 for potential clinically relevant natural product-drug interactions (FDA, 2020a). These data highlight the importance of recapitulating clinical natural product constituent plasma concentrations and using preincubation for in vitro transporter inhibition experiments when investigating transporter-mediated natural product-drug interactions.

Fig. 4. Silymarin OATP IC50 with and without preincubation.

Fig. 4

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The IC50 of silymarin against OATPs shifts to the left with preincubation. IC50 curves for OATP1B1 (A), OATP1B3 (B), and OATP2B1 (C) using fluorescent probe substrates (dichlorofluorescein, fluorescein, and dibromofluorescein, respectively). Symbols and error bars represent means and SDs, respectively, of three replicates. Curves represent model-generated best fit to the data using the standard Hill equation.

Pharmacokinetic drug interactions can be precipitated by factors such as genetic polymorphisms, competition of one drug with another drug or a natural product for metabolizing enzymes or transporters, or disease states that alter metabolizing enzyme or transporter expression or function. Multifactorial pharmacokinetic interactions can additively or synergistically affect drug exposure (Clarke et al., 2014; Poller et al., 2019). The current study investigated the effects of orally administered silymarin and diet-induced NASH, both alone and in combination, on oral pitavastatin disposition. Silymarin increased pitavastatin AUC0-27h 1.8-fold in both the control silymarin and NASH silymarin groups, and NASH alone increased pitavastatin AUC0-27h 2.9-fold (NASH vehicle compared to control vehicle) (Figure 5 and Table 2). The combination of silymarin and NASH increased pitavastatin AUC0-27h 5.1-fold compared to the control vehicle group (Table 2), demonstrating an additive pharmacokinetic effect of silymarin and NASH on pitavastatin systemic exposure. Pitavastatin is metabolized to pitavastatin lactone by UDP glucuronosyl transferase (UGT) 1A3 and UGT2B7 (Schirris et al., 2015), but previous data indicate that inhibition of OATP1B-mediated pitavastatin uptake, rather than UGT-mediated metabolism, increased pitavastatin AUC and did not change pitavastatin lactone AUC (Prueksaritanont et al., 2014). In addition, silybin-mediated inhibition of UGT2B7 only occurs at millimolar concentrations (~0.60-1.5 mM) and was unlikely to contribute to increased pitavastatin AUC in the present study (Uchaipichat, 2018). The 40-minute offset between pitavastatin and silymarin administration produced a secondary increase in pitavastatin plasma concentrations from ~40 to 90 minutes after pitavastatin administration (Figure 5), resulting in no significant silymarin effect on pitavastatin Cmax (Table 2). Further research is needed to determine whether administration of silymarin simultaneously with pitavastatin produces a single pitavastatin peak and increases pitavastatin Cmax. Compared to control, NASH increased pitavastatin Cmax by 3.9-fold in the absence of silymarin and 2.8-fold in the presence of silymarin (Table 2). These NASH Cmax data are consistent with increased plasma concentration of the OATP substrate 99mTc-mebrofenin in NASH patients, suggesting that caution may be needed when prescribing narrow therapeutic index drugs dependent on OATPs for systemic clearance to NASH patients (Ali et al., 2018). Liver pitavastatin content was not different between the NASH and control groups; however, the liver to plasma ratio decreased in the NASH group due to higher plasma concentrations at the final time point (Figure 5). The current data did not identify a silymarin effect on pitavastatin liver content in control animals because liver samples were collected after silymarin constituents were largely cleared from the body. The retention of pitavastatin in the plasma in NASH groups at 27 hours suggests that daily administration of pitavastatin in NASH patients may result in higher steady state plasma concentrations and potentially increase the risk of myopathy. These data further support NASH as a determinant of OATP substrate disposition, and that silymarin at 700 mg doses could precipitate OATP-mediated silymarin-drug interactions. In addition, these data provide further evidence that the combination of two precipitants (silymarin and NASH) can cause additive pharmacokinetic effects on the object drug.

Fig. 5. Pitavastatin concentration vs. time profiles and liver content.

Fig. 5

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Plasma exposure to pitavastatin is higher in NASH compared to control animals in both the absence and presence of silymarin. Average pitavastatin plasma concentration-time profile in control and NASH animals over 27 hours (A) and the first four hours (B) after pitavastatin administration by oral gavage (2.5 mg/kg), followed 40 min later by silymarin administration by oral gavage (500 mg/kg). Liver pitavastatin content 27 hours after pitavastatin gavage (C). Ratio of liver pitavastatin content to plasma pitavastatin concentration at 27 hours (D) and corresponding two-way ANOVA p-values (E). Bonferroni post-hoc test p-values ≤ 0.05 vs control vehicle group. Symbols and error bars represent means and SDs, respectively, of control (n=7) and NASH (n=6) animals.

4. Conclusions

OATPs/Oatps transport flavonolignans and are important mediators of pharmacokinetic silymarin-drug interactions. Consuming silymarin doses at or above 700 mg may precipitate pharmacokinetic interactions with OATP drug substrates and place patients at risk for adverse drug reactions. In addition, decreased OATP function in liver diseases may be responsible for altered flavonolignan disposition, which may increase flavonolignan systemic exposure and the risk for pharmacokinetic interactions. Specifically, patients with NASH (which decreases OATP expression and function) taking silymarin may be susceptible to an additive pharmacokinetic interaction. Clinical investigation of this silymarin-OATP interaction is warranted to confirm these preclinical observations.

Acknowledgements

We kindly thank Dr. Bruno Steiger for providing cell lines.

Funding:

This work was supported in part by the National Institutes of Health National Center for Complementary and Integrative Health [Grant U54 AT008909] and the Washington State University College of Pharmacy and Pharmaceutical Sciences.

Footnotes

Conflicts of Interest

None

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