Differences in the Disposition of Silymarin between Patients with
Nonalcoholic Fatty Liver Disease and Chronic Hepatitis C
Sarah J. Schrieber,1Roy L. Hawke, Zhiming Wen, Philip C. Smith, K. Rajender Reddy,
Abdus S. Wahed, Steven H. Belle, Nezam H. Afdhal, Victor J. Navarro, Catherine M. Meyers,
Edward Doo, and Michael. W. Fried2
Division of Pharmacotherapy and Experimental Therapeutics (S.J.S., R.L.H.), and Division of Molecular Pharmaceutics (Z.W., P.C.S.),
University of North Carolina Eshelman School of Pharmacy, and Division of Gastroenterology and Hepatology, School of Medicine
(M.W.F.), University of North Carolina, Chapel Hill, North Carolina; Division of Gastroenterology, University of Pennsylvania, Philadelphia,
Pennsylvania (K.R.R.); Departments of Biostatistics (A.S.W.) and Epidemiology (S.H.B.), University of Pittsburgh, Pittsburgh, Pennsylvania;
Liver Center, Beth Israel Deaconess Medical Center, Boston, Massachusetts (N.H.A.); Division of Gastroenterology and Hepatology,
Thomas Jefferson University, Philadelphia, Pennsylvania (V.J.N.); National Center for Complementary and Alternative Medicine, National
Institutes of Health, Bethesda, Maryland (C.M.M.); and Liver Diseases Research Branch, Division of Digestive Diseases and Nutrition,
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland (E.D.)
Received April 22, 2011; accepted August 9, 2011
Silymarin, derived from the milk thistle plant Silybum marianum
and widely used for self-treatment of liver diseases, is composed
of six major flavonolignans including silybin A and silybin B, which
are the predominant flavonolignans quantified in human plasma.
The single- and multiple-dose pharmacokinetics of silymarin fla-
vonolignans were examined in patients with nonalcoholic fatty liver
disease (NAFLD) or hepatitis C virus (HCV) to determine whether
the disposition of silymarin and therefore its potential efficacy vary
among liver disease populations. Cohorts of eight subjects with
noncirrhotic liver disease were randomized 3:1 to oral silymarin or
placebo (280 or 560 mg) every 8 h for 7 days. Forty-eight-hour
blood sampling was conducted after the first and final doses. In
general, plasma concentrations of silybin A and silybin B were
higher, whereas concentrations of conjugates were lower in
NAFLD compared with HCV. After adjustment of the area under
plasma concentration-time curve from 0 to 8 h (AUC0–8 h) for
weight and dose, only silybin B and silybin B conjugates differed
significantly between disease types. For NAFLD, the adjusted
mean AUC0–8 hwas higher for silybin B (p < 0.05) but lower for
silybin B conjugates (p < 0.05) compared with that for HCV. At the
280-mg dose, steady-state plasma concentrations of silybin B con-
jugates for NAFLD subjects were characterized by 46% lower
AUC0–8 h(p < 0.05) and 42% lower Cmax(p < 0.05) compared with
HCV subjects. Evidence of enterohepatic cycling of flavonolignans
was only observed in NAFLD subjects. In summary, the efficacy of
silymarin may be more readily observed in NAFLD patients because of
their higher flavonolignan plasma concentrations and more extensive
enterohepatic cycling compared with those in HCV patients.
of the liver (Flora et al., 1998), and approximately one-third of patients seen
in U.S. liver clinics report the use of some complementary and alternative
medicine to self-treat their liver disease (Strader et al., 2002). Derived from
the milk thistle plant Silybum marianum, silymarin is a complex mixture of
and silydianin), as well as other minor polyphenolic compounds (Kim et al.,
2003). Silymarin has been shown to have antioxidant, anti-inflammatory/
models (Abenavoli et al., 2010). However, the antioxidant activity of sily-
marin is most likely to attenuate the pathologic effects initiated by oxidative
stress in the liver, which influence pathways of inflammation, necrosis, and
fibrosis in chronic liver disease (Galli et al., 2005; Medina and Moreno-
Silymarin may be the most potent antioxidant in nature by virtue of
its free radical scavenger reactivity and favorable membrane-lipid/
This work was supported by Cooperative Agreements from the National Insti-
tutes of Health National Center for Complementary and Alternative Medicine
[Grants U01-AT003571, U01-AT003560, U01-AT003573, U01-AT003566, U01-
AT003574], with cofunding from the National Institutes of Health National Institute
of Diabetes and Digestive and Kidney Diseases; and the National Institutes of
Health National Center for Research Resources [Grant RR00046] (General Clinical
Research Centers program). In addition, Rottapharm?Madaus, Italy, provided
silymarin and placebo and partly funded the trial.
This was an investigator-initiated trial, and Rottapharm?Madaus had no direct or
indirect involvement in the design of the trial, data collection, preparation, or submission
of the manuscript for this registered (http://clinicaltrials.gov/ct2/show/NCT00389376) in-
vestigator-initiated trial. None of the authors have a personal conflict of interest with the
manufacturer of any of the marketed silymarin formulations. No official endorsement by
the U.S. Food and Drug Administration is intended or should be inferred.
1Current affiliation: U.S. Food and Drug Administration, Silver Spring, Maryland.
2M.W.F. represents the SyNCH Trial Group.
Article, publicationdate,and citation
ABBREVIATIONS: NASH, nonalcoholic steatohepatitis; NAFLD, nonalcoholic fatty liver disease; HCV, hepatitis C virus; Mrp/MRP, multidrug resistance
protein; AUC, area under the plasma concentration-time curve; UGT, UDP-glucuronosyltransferase; OATP, organic anion-transporting polypeptide.
DRUG METABOLISM AND DISPOSITION
U.S. Government work not protected by U.S. copyright
DMD 39:2182–2190, 2011
Vol. 39, No. 12
Printed in U.S.A.
water partitioning (Gyo ¨rgy et al., 1992). Oxidative stress is thought to
play a central role in the etiology of nonalcoholic steatohepatitis
(NASH), a specific subset of nonalcoholic fatty liver disease
(NAFLD), and is hypothesized to represent a “second hit” triggering
the necroinflammatory response characteristic of NASH (Day and
James, 1998). Therefore, the antioxidant properties of silymarin may
be particularly beneficial as a treatment for NASH because patients
have significantly increased levels of serum lipid peroxidation prod-
ucts (Chalasani et al., 2004) as well as other oxidative stress markers
and decreased levels of antioxidant enzymes (Koruk et al., 2004). In
addition, oxidative stress is a key feature of disease activity in HCV
infection. Elevated levels of oxidative stress markers have been as-
sociated with the grade and stage of liver disease in HCV patients
(Jain et al., 2002), which suggests that antioxidant therapy may be
effective in slowing disease progression in the absence of antiviral
effects. These observations provide the rationale for current phase 2
trials on the effects of silymarin in HCV and NASH populations.
The type and stage of liver disease have been recently shown to
influence the single-dose pharmacokinetics of the major silymarin
flavonolignans (Schrieber et al., 2008). An unexpected finding was
that total silymarin flavonolignan exposures were 3- to 5-fold higher
for patient cohorts compared with healthy controls (Schrieber et al.,
2008). Whereas this study demonstrated that the pharmacokinetics of
silymarin depend upon the type and grade/stage of liver disease,
pharmacokinetic differences between patients with chronic HCV in-
fection and NAFLD were not fully elucidated because of the low
Silymarin flavonolignans are metabolized via phase 2 conjugation
pathways (Sridar et al., 2004; Janc ˇová et al., 2011), and the majority
of glucuronide and sulfate conjugates undergo hepatobiliary excretion
via multidrug resistance protein (Mrp) 2 (Miranda et al., 2008). In
obesity and NAFLD animal models, Mrp2 has been shown to have
altered hepatic expression and function (Geier et al., 2005; Cheng et
al., 2008). In addition, functional genetic polymorphisms in MRP2
have been associated with susceptibility to NAFLD and disease se-
verity (Sookoian et al., 2009). Therefore, disease-specific modulation
of silymarin-metabolizing enzymes or hepatic transporters may account
for alterations in silymarin pharmacokinetics that have been previously
observed in different types of liver diseases and therefore may have a
profound effect on the efficacy in different patient populations.
We have previously reported on the ascending multiple dose phar-
macokinetics of silymarin in noncirrhotic patients with chronic HCV
infection (Hawke et al., 2010) obtained from a double-blind, placebo-
controlled phase 1 trial that was conducted in patients with either
HCV or NAFLD. An unexpected finding was that dose proportional-
ity in the pharmacokinetics of parent silymarin flavonolignans was not
observed in HCV patients with well compensated liver disease at
silymarin doses greater than 560 mg when administered orally every
8 h (Hawke et al., 2010). Because the steady-state pharmacokinetics
of silymarin in patients with NAFLD have not been described previ-
ously and because the pharmacokinetics of silymarin may be different
in different types of liver diseases (Schrieber et al., 2008), we now
report on the pharmacokinetics of silymarin in NAFLD subjects
enrolled in the phase 1 trial. To determine whether the disposition of
silymarin differs between patients with NAFLD or HCV liver disease,
we also compare the single- and multiple-dose pharmacokinetics of
silybin A and silybin B and their conjugates between patients with
NAFLD or HCV. Finally, because the pharmacokinetics of silymarin
appear to be nonlinear in patients with HCV, the pharmacokinetics of
silymarin were evaluated at silymarin doses of 280 and 560 mg to
assess the interaction between dose and disease type. These trials were
conducted to optimize oral silymarin dosing for phase 2 efficacy trials
in patients with either HCV or NASH (Lang, 2006). In these phase 2
trials, which are now ongoing, oral doses higher than the customary
dose of 140 mg every 8 h are used in an attempt to overcome the high
first-pass metabolism of silymarin and achieve therapeutic, steady-
state plasma concentrations.
Materials and Methods
Subjects. Forty male and female subjects ?18 years of age with chronic
noncirrhotic NAFLD and HCV were enrolled in the study within 28 days of
screening (n ? 8/cohort). Subjects were required to have elevated alanine
aminotransferase levels ?65 IU/l within 1 year before screening and a creat-
inine clearance (calculated according to the Cockcroft-Gault equation) ?60
ml/min at screening as well as a negative urine pregnancy screen for women
of child-bearing potential who were also required to use barrier methods of
contraception during the study.
Subjects were excluded if they had either a history of or, in the clinical
opinion of the investigators, evidence of decompensated liver disease defined
by serum albumin ?3.2 g/dl, total bilirubin ?1.5 mg/dl, or prothrombin
time/international normalized ratio ?1.3 times normal or a history of or the
presence of ascites, encephalopathy, portal hypertension, or bleeding from
esophageal varices. Subjects were also excluded if they had evidence of other
chronic liver disease or serologic evidence of infection with human immuno-
deficiency virus. Other exclusion criteria included an allergy to milk thistle or
its preparations, use of silymarin or other milk thistle preparations, or use of
high doses of other antioxidants such as vitamin E, vitamin C, glutathione, or
?-tocopherol within 30 days of randomization through study completion.
However, use of standard doses of over-the-counter multivitamins or cough/
cold preparations was allowed. Also excluded was the chronic use of ?2 g/day
acetaminophen; use of oral contraceptives, warfarin, or metronidazole; or
concurrent use of the following cytochrome CYP3A4 inducers: aminoglute-
thimide, aprepitant, carbamazepine, dexamethasone, efavirenz, ethosuximide,
garlic supplements, glucocorticoids, glutethimide, griseofulvin, modafinil, naf-
cillin, nevirapine, oxcarbazepine, phenobarbital, phenytoin, primidone, rifabu-
tin, rifampin, rifapentine, and St. John’s wort; historical liver biopsy demon-
strating the presence of cirrhosis (Ishak stage 5 or 6) or ?15% steatosis or
evidence of steatohepatitis; positive urine screen for drugs of abuse; alcohol
consumption of ?12 g/day for ?6 months before screening; or other evi-
dence of alcohol or drug abuse within 6 months of screening. Women who
were pregnant or breast-feeding were also excluded. All subjects agreed not
to consume alcohol for 48 h before study randomization through study
Trial Design. Specific details on the design of this phase 1 study have been
described previously (Hawke et al., 2010). In brief, dose cohorts of eight
subjects each were randomized 3:1, via a web-based randomization system
used by each site’s pharmacist, to receive oral silymarin or placebo every 8 h
for 7 days. Forty-eight-hour pharmacokinetic samples were collected after an
initial single-dose administration before the 7-day treatment and a final dose
after the 7-day treatment for a total of 23 doses. Only pharmacists were
unblinded to treatment assignments until trial completion. The sample size was
selected to provide information on safety, tolerability, and pharmacokinetics of
silymarin and was based on historical experience for phase 1 trials and not on
statistical considerations. Cohorts were enrolled sequentially at doses of 280 or
560 mg of silymarin. The Legalon (Rottapharm?Madaus, Monza, Italy) brand
of silymarin was selected as the clinical trial material for the Silymarin Product
Development Program for use in National Institutes of Health-sponsored
clinical trials for liver diseases from competing bids in response to a Notice of
Opportunity by the National Center for Complementary and Alternative Med-
icine and the National Institute of Diabetes and Digestive and Kidney Diseases
of the National Institutes of Health.
The first and last doses for the pharmacokinetic studies were administered
on days 1 and 10, respectively. To control for potential variability induced by
fed versus fasted states, doses were administered with 240 ml of water 30 min
after breakfast to subjects who were fasted overnight. Subjects were allowed to
choose from a fixed list of items on the clinical research breakfast menu.
Grapefruit juice was not allowed. Subjects remained in the research unit for
48 h for collection of blood. Fourteen serial blood samples were collected at
0 h (predose) and 0.5, 1, 1.5, 2, 4, 6, 8, 12, 15, 18, 24, 32, and 48 h postdose.
DISPOSITION OF SILYMARIN IN NAFLD AND CHRONIC HCV
Twenty-one doses were dispensed to subjects upon discharge after collection
of the 48-h postdose sample on day 3. The first of these 21 doses was
self-administered under direct supervision in the clinical research center.
Eight-hour postdose trough plasma samples were collected during safety visits
on days 6 and 8. Patient adherence was assessed by patient drug diary, by pill
counts, and by maintaining records of drugs dispensed and returned.
Subjects were enrolled from December 2006 to July 2008 at four clinical
centers, which included University of North Carolina at Chapel Hill, Beth
Israel Deaconess Medical Center, University of Pennsylvania, and Thomas
Jefferson University. Institutional review boards of participating centers ap-
proved the protocol; all subjects provided written informed consent. The study
was conducted in accordance with the Declaration of Helsinki and guidelines
on Good Clinical Practice.
Safety Assessment. Safety, which consisted of clinical laboratory tests and
reports of clinical adverse events using a symptom assessment questionnaire,
was assessed before dosing on study days 1 (baseline), 6, 8, and 10. In addition,
on days 1 and 10, the questionnaire was also completed at approximately 24
and 48 h postdose. Common Terminology Criteria for Adverse Events (version
3.0) was used to grade the severity of adverse events. Physical examinations
and electrocardiograms were completed at baseline and at the end of the study.
Decisions to dose escalate were made after a safety evaluation by a designated
safety committee masked to treatment. The safety committee consisted of the
principal investigators from the four clinical centers and an external safety
Study Drug. Silymarin (Legalon) and matching placebo were manufactured
in hard capsules by Madaus Rottapharm Group (Cologne, Germany); all study
doses were administered from lot 0418901. Each dose consisted of five
silymarin and/or placebo capsules packaged in single-use medicine dose cups.
The flavonolignan content of each capsule was determined according to
previously published liquid chromatography-mass spectrometry methods as
follows: 23.2 mg, silybin A; 32.0 mg, silybin B; 11.8 mg, isosilybin A; 6.6 mg,
isosilybin B; 24.9 mg, silychristin; and 29.0 mg, silydianin (Wen et al., 2008).
These six flavonolignans account for 70.8% (127.5 mg of silymarin equivalent
to 140 mg of silymarin as determined by the manufacturer’s 2,4-dinitrophe-
nylhydrazine method) of the 180-mg milk thistle extract contained in each
capsule. Based on interim stability testing results performed by the manufac-
turer, Legalon capsules are stable under normal conditions (25°C, 60% relative
humidity) for at least 9 months. For the purpose of the pharmacokinetic
analyses described in this report, one Legalon capsule was considered equal to
140 mg of silymarin in accordance with the manufacturer’s specifications.
Analysis of Silymarin Flavonolignans. Whole-blood samples were col-
lected in two 3-ml EDTA-lined tubes (K2-EDTA tubes; BD, Franklin Lakes,
NJ) and centrifuged at 1200g for 10 min at 4°C. Plasma was aspirated and
transferred to polypropylene tubes. Plasma samples were temporarily stored at
?70°C by each clinical site for ?30 days before shipment to the University of
North Carolina where they were acidified by addition of glacial acetic acid
(final concentration 1% acetic acid) and stored at ?70°C until analysis.
For the determination of parent (i.e., nonconjugated) flavonolignan concen-
trations in plasma, a 125-?l aliquot of each patient sample was buffered using
sodium acetate (pH 5.0, 0.125 M) and incubated for 6 h at 37°C without
hydrolytic enzymes. A second 125-?l aliquot was also buffered using sodium
acetate (pH 5.0, 0.125 M) and incubated with a mixture of sulfatase (80 U/ml,
type H-1) and ?-glucuronidase (8000 U/ml, type B-10) (Sigma-Aldrich, St.
Louis, MO) for the determination of total (i.e., parent ? conjugates) flavono-
lignan concentrations, which were expressed as “parent flavonolignan equiv-
alents.” After incubation, 50 ng of naringenin (internal standard) in 25 ?l of
50% methanol was added to the samples, which were then deproteinized and
processed using a high-throughput protein filtration procedure as described
previously (Hawke et al., 2010). After filtration, 75 ?l of the plasma sample
supernatants were transferred to glass high-performance liquid chromatogra-
phy vials and concentrations of silymarin flavonolignans were quantified by
liquid chromatography-electrospray ionization-mass spectrometry as described
previously using a Luna C18 analytical column (50 ? 2.0 mm i.d., 3 ?m;
Phenomenex, Torrance, CA); an isocratic mobile phase consisting of 43%
methanol, 56% water, and 1% glacial acetic acid (pH 2.8); a flow rate of 0.3
ml/min; a 25-?l injection volume; and a 13-min run time (Wen et al., 2008).
For each silymarin flavonolignan, the limit of detection was 20 ng/ml, and the
quantitative ranges for parent and for total flavonolignan were 50 to 2500 and
100 to 20,000 ng/ml, respectively. The accuracy for each flavonolignan was
within 95.4 to 107.4% and intra- and interday precisions were 1.7 to 11 and 4.5
to 14%, respectively.
Data Analysis. Pharmacokinetic parameters including area under the
plasma concentration-time curve (AUC), maximum plasma concentration
(Cmax), time to Cmax(Tmax), and terminal half-life (t1/2) were calculated using
noncompartmental methods (WinNonlin Professional version 5.2; Pharsight,
Mountain View, CA). A constant dosing interval (?) of 8 h was assumed for the
calculation of steady-state AUC0–8 husing the linear up/log down trapezoidal
method. To obtain pharmacokinetic parameters for the conjugate flavonolignan
concentrations, the parent flavonolignan concentrations were subtracted from
the total flavonolignan concentrations at each time point over the entire
sampling period before pharmacokinetic analysis was performed. Pharmaco-
kinetic parameters are reported as geometric means with 95% confidence
intervals, except for Tmax, which is reported as median with minimum and
maximum values. For our primary analysis, differences in steady-state expo-
sures (i.e., AUC0–8 h) between disease cohorts were compared, after log
transformation, using a parametric two-sample t test. p ? 0.05 was used for
statistical significance. In addition, to eliminate weight as a potential con-
founder in the assessment of differences in flavonolignan exposures between
cohorts, a linear regression model with log AUC0–8 has outcome was used.
The model included dose, disease, and weight as independent variables to
adjust for variable weights across dose groups (280 mg versus 560 mg) or
disease type (HCV versus NAFLD) while comparing AUC0–8 h. Least-squares
means (adjusted means) were reported with 95% confidence intervals and
tested using t tests. All statistical analyses were performed by using SAS 9.2
or SAS JMP 9 (SAS Institute, Cary, NC).
Subjects. Baseline demographics are presented in Table 1. Study
subjects in the HCV cohorts were predominantly men with ages
ranging from 43 to 59 years, whereas men and women were more
equally represented in the NAFLD cohorts with ages ranging from 28
to 58 years. Subjects were characterized by well compensated, non-
cirrhotic liver disease as evidenced by total bilirubin (range 0. 3–2.6
mg/dl) and platelet counts (range 150–327 cells/mm3).
Efficacy and Safety Endpoints. Compared with their screening
baseline values, no reductions in serum transaminases for either HCV
or NAFLD subjects or reductions in HCV RNA titer for HCV subjects
were observed at the end of the 7-day treatment period (data not
There were no abnormal deviations from baseline laboratory values
reported with silymarin administration for any cohort. For the HCV
cohorts, three subjects who received a single 280-mg dose of sily-
marin reported a total of four adverse events. Three of the adverse
events were classified as neurologic (e.g., headache), whereas the
other was classified as gastrointestinal. Only one adverse event (diz-
ziness) was considered possibly related to silymarin administration
and resolved in less than 1 day.
For the NAFLD cohorts, 2 of 12 subjects (16.7%) receiving sily-
marin reported at least one adverse event compared with one of four
subjects (25%) receiving placebo. Adverse events reported with sily-
marin included upper respiratory infection and abdominal pain, both
of which occurred in the 560-mg dose cohort. All adverse events
reported with silymarin were determined to be mild to moderate and
self-limiting and were considered unrelated to treatment.
Single-Dose Pharmacokinetics of Silybin A and Silybin B. A
comparison of the pharmacokinetics of silybin A and silybin B be-
tween HCV and NAFLD cohorts after single oral doses of either 280
or 560 mg of silymarin are presented in Table 2. Silybin A was the
predominant flavonolignan in plasma for both HCV and NAFLD
cohorts and was characterized by a 2.7- to 3.3-fold greater Cmaxand
a 2- to 4.5-fold greater AUC0–48 hcompared with those for silybin B.
SCHRIEBER ET AL.
At the 280-mg dose, no differences were observed in the pharma-
cokinetics of silybin A or silybin B between HCV and NAFLD
subjects. Short elimination half-lives were observed for both silybin A
and silybin B (range 0.9–1.8 h).
However, at the 560-mg dose, pharmacokinetic differences were
observed between subjects with HCV and NAFLD. Compared with
HCV subjects, for NAFLD subjects, AUC0–48 hfor silybin A and
silybin B were 1.5-fold (p ? 0.05) and 2.1-fold (p ? 0.05) greater,
respectively. A similar trend was observed in the Cmaxfor silybin A
and silybin B, although the 1.4- to 1.6-fold differences between HCV
and NAFLD subjects did not achieve statistical significance. Elimi-
nation half-lives were similar between the disease groups (range
1.1–1.5 h), whereas Tmaxwas delayed by 1 h in NAFLD subjects.
Steady-State Pharmacokinetics of Silybin A and Silybin B. The
steady-state pharmacokinetics of silybin A and silybin B for the HCV
and NAFLD cohorts after chronic oral administration of either 280 or
560 mg of silymarin every 8 h for 7 days are presented in Table 3.
Similar to the data obtained after single doses, silybin A was the
predominant flavonolignan in plasma for both HCV and NAFLD
cohorts and was characterized by a 2.1- to 3.6-fold greater Cmaxand
a 2.6- to 4.9-fold greater AUC0–8 hcompared with those for silybin B.
In addition, there was no evidence of accumulation for either flavono-
lignan after repeated dosing with elimination half-lives ranging be-
tween 0.7 and 1.3 h. Also similar to the single-dose data, pharmaco-
kinetic differences between the HCV and NAFLD cohorts were only
observed at the 560-mg dose. The AUC0–8 hfor silybin A and silybin
B were 1.6- and 2.5-fold greater, respectively, in NAFLD subjects
than in HCV subjects at the 560-mg dose, whereas differences in the
Cmaxbetween cohorts ranged between 1.5- and 2.2-fold. After adjust-
ment for weight and disease type, silybin A and silybin B AUC0–8 h
differed significantly between the 280- and 560-mg dose groups (p ?
0.004), such that for either HCV or NAFLD or at any weight level, the
560-mg dose was associated with higher AUC0–8 h. With adjustment
for weight and dose, only silybin B differed significantly across
disease types such that the adjusted mean AUC0–8 hfor silybin B was
higher for NAFLD than for HCV (p ? 0.004). The higher silybin B
exposures in NAFLD subjects suggest the metabolism or hepatic
uptake of silybin B may be reduced in NAFLD compared with HCV.
Single-Dose and Steady-State Pharmacokinetics of Silybin A
and Silybin B Conjugates. To further explore the effect of NAFLD
on metabolism of silymarin, differences in the plasma concentrations
of silybin A and silybin B conjugates between HCV and NAFLD
subjects were examined. As defined under Materials and Methods,
plasma concentrations of conjugates were estimated from subtraction
of parent flavonolignan concentrations from total (parent ? conju-
gate) flavonolignan concentrations.
The single-dose and steady-state pharmacokinetic data for total
conjugates of silybin A and silybin B for both disease cohorts are
presented in Tables 4 and 5, respectively. Whereas plasma concen-
trations were observed to be greater for silybin A than for silybin B,
the converse was true for their conjugates. The Cmaxand AUC0–8 h
for silybin B conjugates were 3- to 4-fold greater than those for silybin
A conjugates across both dose levels and disease cohorts.
Differences between HCV and NAFLD subjects were observed in
the pharmacokinetics for plasma conjugates of silybin A and silybin B
at either dose level after single or chronic dosing. However, these
Single-dose pharmacokinetics of parent silybin A and silybin B
Results are shown as geometric mean (95% confidence interval), except for Tmax, which is shown as median (minimum, maximum). Data are for n ? 6 subjects.
HCVNAFLD HCV NAFLD
280 mgAUC0–8 h(ng ? h/ml)
AUC0–8 h(ng ? h/ml)
2.0 (1.0, 4.0)b
1.5 (1.0, 4.0)
2.0 (1.0, 6.0)a
2.7 (1.5, 4.0)
3.0 (1.0, 4.0)b
1.5 (0.5, 4.0)a
2.0 (2.0, 6.0)a
2.7 (1.0, 4.0)
SA, silymarin A; SB, silymarin B.
* p ? 0.05.
an ? 5.
bn ? 4.
Subject baseline demographics
Data are presented as medians (minimum, maximum).
Group and Cohort
560 mg280 mg560 mg
Total bilirubin (mg/dl)
50 (44, 59)
92 (67, 99)
28 (25, 42)
0.8 (0.3, 1.0)
95 (58, 288)
64 (43, 271)
208 (177, 327)
51 (43, 54)
104 (86, 123)
33 (26, 41)
0.6 (0.3, 1.4)
113 (81, 214)
89 (46, 110)
192 (150, 225)
51 (28, 58)
104 (79, 150)
38 (27, 42)
0.6 (0.3, 1.1)
99 (52, 322)
60 (38, 325)
275 (157, 341)
48 (28, 52)
101 (78, 143)
38 (27, 43)
0.5 (0.3, 2.6)
104 (77, 115)
66 (54, 104)
282 (162, 319)
BMI, body mass index; ALT, alanine aminotransferase; AST, aspartate aminotransferase.
aTwo cohorts of six subjects were used to study single and multiple dose pharmacokinetics.
DISPOSITION OF SILYMARIN IN NAFLD AND CHRONIC HCV
differences only achieved significance between HCV and NAFLD
cohorts dosed at 280 mg every 8 h, whereas conjugates of silybin B
in plasma of NAFLD subjects were characterized by 46% lower
AUC0–8 h(p ? 0.05) and 42% lower Cmax(p ? 0.05) compared with
HCV subjects. Figure 1 depicts the mean steady-state plasma concen-
tration versus time profiles for silybin B (inset) and silybin B conju-
gates for HCV and NAFLD subjects at the 280-mg dose. Plasma
concentrations of silybin B conjugates were lower in NAFLD subjects
compared with HCV subjects over the entire 8-h dosing interval (Fig.
1). In contrast, plasma concentrations of silybin B were higher in
NAFLD subjects until peak concentrations were achieved and then
declined similarly (Fig. 1, inset). These data suggest that reduced
silymarin metabolism may result in differences in silymarin exposures
between NAFLD and HCV subjects, rather than differences in
After adjustment for weight and disease type, the AUC0–8 hvalues
for silybin A conjugates and for silybin B conjugates differed signif-
icantly between the 280- and 560-mg dose groups (p ? 0.004), such
that for either HCV or NAFLD or at any weight level, the 560-mg
dose was associated with higher AUC0–8 h. With adjustment for
weight and dose, only silybin B conjugates differed significantly
across disease types such that the adjusted mean AUC0–8 hfor silybin
B conjugates was significantly lower for NAFLD compared with
HCV (p ? 0.03).
To further quantify differences in the extent of flavonolignan
conjugation between HCV and NAFLD subjects, steady-state met-
abolic ratios were calculated as the ratio of AUC0–8 hfor silybin B
divided by AUC0–8 hfor silybin B conjugates at the 560-mg dose.
Metabolic ratios differed 4-fold (p ? 0.05) between HCV and
NAFLD with means ? S.D. of 0.016 ? 0.011 and 0.060 ? 0.041,
respectively. These data suggest that there is less conjugation of
silybin B in NAFLD subjects then in HCV subjects at a silymarin
dose of 560 mg. In summary, plasma concentrations of silybin A
and silybin B were generally greater and the concentrations of their
conjugates were lower in NAFLD subjects than in HCV subjects
irrespective of the dose and frequency of oral silymarin adminis-
Flavonolignan Accumulation. The ratio of parent silybin A
steady-state AUC0–8 hdivided by single-dose AUC0–8 hwas calcu-
lated as an indication of the extent of accumulation after chronic three
times daily dosing. Silybin A ratios of 1.3 and 1.4 were calculated for
HCV and NAFLD, respectively, at the 560-mg dose, which indicates
no significant accumulation in either cohort with repeated dosing.
Similar ratios were calculated for silybin B. This finding is consistent
with the short half-life of the silymarin flavonolignans.
Although no evidence for parent silybin A and silybin B accumu-
lation was observed, the overall amount of parent flavonolignans in
plasma was significantly higher in NAFLD subjects than in HCV
subjects at the 560-mg dose because of the appearance of additional
parent flavonolignans. Figure 2 compares mean steady-state peak
plasma concentrations of the six parent silymarin flavonolignans for
HCV and NAFLD subjects at the 560-mg dose, as well as their sum
concentration. As seen in Fig. 2, plasma concentrations of isosilybin
A, isosilybin B, silychristin, and silydianin were significantly greater
in NAFLD subjects than in HCV subjects. Of interest, silychristin and
silydianin were not detected in the plasma of HCV subjects. To gain
insight into the mechanism(s) behind these observed differences, we
evaluated the plasma concentration versus time profile for each fla-
vonolignan over the 48-h sampling period after administration of the
last 560-mg dose (Fig. 3). Significant enterohepatic cycling of the six
flavonolignans was observed in NAFLD subjects as indicated by a
prominent second peak at 4 h after the absorption peak at 1 h. Most
Single-dose pharmacokinetics of silybin A conjugates and silybin B conjugates
Results are shown as geometric means (95% confidence interval), except for Tmax, which is shown as median (minimum, maximum). Data are for n ? 6 subjects.
Cohort PK Parameter
280 mg AUC0–48 h(ng ? h/ml)
AUC0–48 h(ng ? h/ml)
2 (1.5, 4.0)
3.0 (2.0, 4.0)
4.0 (2.0, 12.0)
4.0 (4.0, 6.2)
2.0 (1.5, 4.0)
4.0 (1.0, 4.0)
3.0 (2.0, 8.0)
3.0 (1.5, 6.0)
SA, silymarin A; SB, silymarin B.
Steady-state pharmacokinetics of silybin A and silybin B
Results are shown as geometric mean (95% confidence interval), except for Tmax, which is shown as median (minimum, maximum). Data are for n ? 6 subjects, except for the HCV 560-mg
steady-state cohort where n ? 5; one subject was dropped from the pharmacokinetic analysis because of incorrect dosing for pharmacokinetic sampling at steady-state on day 8.
HCV NAFLDHCV NAFLD
280 mg AUC0–8 h(ng ? h/ml)
AUC0–8 h(ng ? h/ml)
1.8 (1.0, 4.0)
1.5 (1.5, 2.0)
1.3 (0.5, 4.4)
3.0 (0.5, 4.0)
1.5 (0.5, 4.0)
1.5 (1.5, 2.0)
1.2 (0.5, 2.0)a
3 (0.5, 4.0)
SA, silymarin A; SB, silymarin B.
an ? 5.
SCHRIEBER ET AL.
flavonolignans also showed evidence of a third peak at 8-h postdose.
In contrast, there was less evidence of enterohepatic cycling in HCV
subjects in whom no secondary peaks were observed for either silybin
A or silybin B after the early absorption peak. Silychristin represented
a major flavonolignan in the plasma of NAFLD subjects at the
560-mg dose. The steady-state pharmacokinetics of silychristin (geo-
metric mean and 95% confident intervals) were characterized by a
Cmaxof 67 ng/ml (?2.5 to 174), an AUC0–8 hof 325 ng ? h/ml (?145
to 1100), and a t1/2of 3.1 h (1.2–6.3). The steady-state pharmacoki-
netics of the conjugates of silychristin in NAFLD subjects were
characterized by a Cmaxof 663 ng/ml (367–1394), an AUC0–8 hof
3800 ng ? h/ml (1628–8462), and a t1/2of 4.5 h (2.2–8.6).
The expression of drug disposition genes and their protein products
has been shown to be altered in liver disease (Congiu et al., 2002,
2009; Fisher et al., 2009), and effects of liver disease on the dispo-
sition of drugs have been demonstrated and tend to be more severe in
patients with more advanced cirrhotic disease (Chalon et al., 2003). In
contrast, significant differences in the disposition of drugs between
different types of liver disease have not been demonstrated. We have
shown that the disposition of silymarin, an herbal medicine widely
used by patients with liver disease, is significantly altered in patients
with liver disease (Schrieber et al., 2008). Concentrations of total
silymarin species found in plasma, which consist primarily of fla-
vonolignan conjugates, were found to be approximately 5-fold higher
in patients with chronic HCV infection or NAFLD compared with
those in healthy controls. Pharmacokinetic differences were also ob-
served between healthy subjects and patients with NAFLD or patients
with HCV cirrhosis. In contrast, differences were not observed be-
tween healthy subjects and patients with noncirrhotic HCV disease
possibly due to wide disease heterogeneity in patient cohorts or
reduced sensitivity as a result of low plasma concentrations of fla-
vonolignans associated with the low oral dose of a generic brand of
silymarin that was used in this study (Schrieber et al., 2008). These
results raised the possibility that the disposition of silymarin and its
potential beneficial effects may be different in various liver disease
populations with early-stage disease. To determine whether the dis-
position of silymarin is different among patients with different types
of the liver disease, this study examined the pharmacokinetics of
higher than customary oral doses of silymarin in noncirrhotic patients
with either chronic HCV infection or NAFLD. The results of our
study show that NAFLD patients are characterized by higher plasma
concentrations of certain silymarin flavonolignans and lower concen-
trations of flavonolignan conjugates compared with HCV patients
administered the same dose. Although silymarin flavonolignans ap-
pear to share common pathways of metabolism and transport, differ-
ences in their affinity for these processes have been noted (Sridar et
al., 2004; Miranda et al., 2008), which probably account for the
FIG. 1. Steady-state plasma concentration versus time profiles for silybin B conju-
gates and parent silybin B (inset) at 280 mg of silymarin in HCV (F) and NAFLD
(?) subjects. Forty-eight-hour plasma samples were obtained after a final single-
dose administration after an every 8 h for 7-day dose regimen. AUC0–8 hand Cmax
for silybin B conjugates were 46 and 42% lower, respectively, in NAFLD subjects
compared with HCV; p ? 0.05.
FIG. 2. Maximum steady-state plasma concentrations for silymarin flavonolignans
at 560 mg of silymarin in HCV (f) and NAFLD (?) subjects. Plasma concentra-
tions of isosilybin A, isosilybin B, silychristin, and silydianin were significantly
greater in NAFLD subjects compared with HCV subjects. Silychristin and silydia-
nin were not detected in the plasma of HCV subjects.
Steady-state pharmacokinetics of silybin A conjugates and silybin B conjugates
Results are shown as geometric means (95% confidence interval), except for Tmax, which is shown as median (minimum, maximum). Data are for n ? 6 subjects, except for the HCV 560-
mg steady-state cohort where n ? 5; one subject was dropped from the pharmacokinetic analysis because of incorrect dosing for pharmacokinetic sampling at steady state on day 8.
280 mgAUC0–8 h(ng ? h/ml)
AUC0–8 h(ng ? h/ml)
4 (0, 6)
2 (2, 4)
1.8 (0, 2)
3 (0, 4)
SA, silymarin A; SB, silymarin B.
* p ? 0.05.
DISPOSITION OF SILYMARIN IN NAFLD AND CHRONIC HCV
different relationships between AUC exposure and dose for silybin A
and silybin B observed in our study.
In vitro and in vivo studies suggest that silymarin flavonolignans
are primarily metabolized through glucuronidation and sulfation path-
ways with various UDP-glucuronosyltransferases (UGTs) sharing
overlapping specificity (Sridar et al., 2004; Janc ˇová et al., 2011). In
addition, the extent to which various flavonolignans undergo glucu-
ronidation or sulfation appears to vary (Wen et al., 2008). There are
several possibilities that could explain why the ratio of parent fla-
vonolignan (e.g., silybin B) to flavonolignan conjugates was higher in
patients with NAFLD than in those with HCV in our study. The
simplest explanation is that the expression or activity of UGTs is
decreased in NAFLD subjects. Nonalcoholic steatohepatitis, a specific
subset of NAFLD, is characterized by hepatic steatosis and varying
degrees of inflammation, which can lead to decreased UGT expres-
sion as has been observed in rodents (Richardson et al., 2006) and in
human liver tissue (Congiu et al., 2002). Therefore, it is plausible that
the major UGT isoforms involved in metabolism of silymarin may be
lower in NAFLD subjects, resulting in higher plasma levels of parent
flavonolignans and lower concentrations of conjugates. Because sily-
bin B conjugates represent 99% of the total (parent ? conjugates)
silybin B species in plasma of patients with HCV, metabolism stoi-
chiometry predicts that the 40% reduction in silymarin conjugates
observed in our NAFLD cohort should result in an ?30-fold increase
in silybin B plasma concentrations. However, plasma concentrations
of silybin B were comparable between HCV and NAFLD patients.
Therefore, reduced UGT activity does not appear to be a viable
explanation for the differences in silymarin pharmacokinetics between
HCV and NAFLD in our study. In addition, the lower plasma con-
centration of flavonolignan conjugates in NAFLD compared with
HCV does not appear to be related to reduced intestinal absorption
because parent flavonolignans would also be expected to be lower in
Alterations in the expression and function of hepatobiliary trans-
porters may be a more plausible explanation for the decrease in
flavonolignan conjugates and the higher plasma concentrations of
parent flavonolignans observed in the NAFLD cohorts. Evidence for
extensive enterohepatic cycling of silymarin and their conjugates has
been observed at high doses of silymarin (Schrieber et al., 2008;
Hawke et al., 2010). Enterohepatic cycling is regulated by hepatobi-
liary transporters involved in the active uptake of anionic and cationic
compounds from the blood such as the organic anion-transporting
polypeptides, OATP1B1 and OATP2B1, located on the basolateral
membrane of the hepatocyte (Chandra and Brouwer, 2004). In many
instances, these compounds undergo metabolism to more polar con-
jugates followed by transport and biliary excretion by ATP-binding
cassette transporters such as P-glycoprotein, MRP2, and breast cancer
resistance protein, located at the canalicular membrane of the hepa-
tocyte (Schinkel and Jonker, 2003; Leslie et al., 2005). Once delivered
to the small intestine, parent compounds can be reformed by bacterial
deconjugation and returned to portal blood for delivery to the liver for
reuptake. In competition with biliary efflux is the efflux of substrates
from the hepatocyte to blood by other members of the MRP family,
such as MRP3 and MRP4 (MRPs 3/4), which are located on the
basolateral (sinusoidal) membrane. It is generally thought that MRP2
and MRP3 work in concert in liver disease to promote hepatic efflux
and protect the hepatocyte from the effects of cholestasis (Wagner et
al., 2005; van de Steeg et al., 2010).
The most intriguing observation in the current study was the
suggestion of significant enterohepatic recycling of silymarin fla-
vonolignans in NAFLD subjects in contrast with HCV subjects in
whom there was no evidence of enterohepatic cycling (Fig. 3).
Silymarin flavonolignans demonstrate high affinity for MRP4 (Wu
et al., 2005), whereas silymarin conjugates but not parent flavono-
lignans appear to be better substrates for MRP2 (Miranda et al.,
2008). Glucuronides that are substrates for MRP2, such as conju-
gated bilirubin, can also be substrates for MRPs 3/4 (Borst et al.,
2006; Zelcer et al., 2006). Therefore, differences in the disposition
and enterohepatic cycling of silymarin flavonolignans may reflect
alterations in the function of hepatobiliary transporters as a result
of liver disease.
In obesity and NAFLD animal models, Mrp2 has been shown to
have altered hepatic expression and function (Geier et al., 2005;
Cheng et al., 2008). In addition, Mrp2, Mrp3, and Mrp4 protein
expression were significantly increased in a rodent model of NAFLD
(Lickteig et al., 2007). The biliary excretion of glucuronide and sulfate
conjugates of silymarin flavonolignans was shown to be dependent on
Mrp2 using isolated perfused livers, and some flavonolignans such as
silychristin and silydianin were almost quantitatively secreted into
bile (Miranda et al., 2008). Therefore, enterohepatic cycling of sily-
marin flavonolignans may be increased in NAFLD due to increased
MRP2-dependent biliary efflux and diversion of silymarin conjugates
away from sinusoidal efflux to blood. An increase in MRP4 would
also contribute to greater sinusoidal efflux of parent flavonolignans.
These changes would result in lower plasma concentrations of sily-
marin conjugates with higher concentrations of recycling silymarin
flavonolignans in patients with NAFLD compared with those with
As an alternative, the differences observed in the disposition of
silymarin between NAFLD and HCV patients may reflect HCV-
specific alterations in hepatobiliary function. HCV infection was
shown to be associated with increased hepatic expression of
MRP4, decreased expression of MRP2, and decreased expression
of OATP1B1 in cirrhotic and noncirrhotic liver, whereas the ex-
pression of MRP3 and OATP2B1 was similar to that in normal
human liver (Ogasawara et al., 2010). Therefore, the differences in
the disposition of silymarin between HCV and NAFLD subjects
observed in our study may reflect a diversion of silymarin conju-
gates to sinusoidal efflux in HCV patients due to reduced biliary
efflux by MRP2 or reduced uptake by OATP1B1, which would
FIG. 3. Steady-state plasma concentration versus time profiles for silymarin fla-
vonolignans at 560 mg of silymarin in HCV and NAFLD subjects. Forty-eight-hour
plasma samples were obtained after a final single dose administration after an every
8 h for 7-day dose regimen. Evidence of enterohepatic recycling of flavonolignans
by the appearance of secondary peaks was observed in NAFLD subjects (——),
whereas no evidence of enterohepatic recycling for silybin A or silybin B was
observed in HCV subjects (– – –). In addition to silybin A and silybin B, silychristin
(Œ) represented a major flavonolignan in NAFLD subjects. For presentation clarity,
error bars were not included.
SCHRIEBER ET AL.
also result in higher plasma concentrations of silymarin conjugates
and decreased enterohepatic cycling of silymarin flavonolignans
compared with those in patients with NAFLD. Although the results
of our study cannot delineate between these various potential
mechanisms, it is possible that the disposition of silymarin is
altered by different, disease-specific mechanisms in NAFLD and
HCV populations. This conclusion is supported by our previous
observation that plasma concentrations of silymarin conjugates are
significantly higher in both NAFLD and HCV patients compared
with concentrations found in healthy volunteers (Schrieber et al.,
In summary, differences in the disposition of silymarin between
NAFLD and HCV patients may reflect different disease-specific al-
terations in the function of hepatobiliary transport proteins. These
observations are significant because differences in the disposition of
drugs between different types of liver disease have not been demon-
strated, perhaps because of their more restrictive use indications. Of
importance, the antioxidant activity and potential anti-inflammatory
and antifibrotic effects of silymarin on disease progression will be
dependent on its hepatic disposition. Oxidative stress has been asso-
ciated with all stages of chronic HCV liver disease (Jain et al., 2002),
and recent data from the Hepatitis C Antiviral Long-Term Treatment
against Cirrhosis trial suggest that silymarin use among patients with
advanced HCV liver disease may be associated with reduced progres-
sion to cirrhosis (Freedman et al., 2011). Silymarin may demonstrate
greater benefits in patients with NAFLD compared with those with
HCV infection because oxidative stress is thought to play a central
role in the etiology of NASH (Day and James, 1998) and there are no
approved therapies. In addition, the results of this study suggest that
the effects of silymarin on liver disease progression may also be
greater in NAFLD patients that in HCV patients because of higher
flavonolignan plasma concentrations and more extensive enterohe-
patic cycling. These observations were critical in the design of a phase
2 silymarin trial in NASH, which is currently ongoing (Lang, 2006).
We are indebted to Drs. Josh Berman and Qi-Ying Liu for their important
early efforts in study design and to Dr. Ulrich Mengs for championing this
work. In addition, we thank the patients who volunteered for this trial, Dr. Tedi
Soule, Joseph Colagreco, Mary Hammond, and Deborah Moretti, who served
as the study coordinators, and Sharon Lawlor, who was the Data Coordinating
Center coordinator, for their invaluable assistance in the conduct of this trial.
We also thank Dr. Craig W. Hendrix, who graciously agreed to serve as the
independent safety monitor.
Participated in research design: Hawke, Reddy, Belle, Afdhal, Navarro,
Meyers, Doo, and Fried.
Conducted experiments: Schrieber and Wen.
Contributed new reagents or analytic tools: Hawke and Smith.
Performed data analysis: Schrieber and Wahed.
Wrote or contributed to the writing of the manuscript: Schrieber and
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Address correspondence to: Dr. Roy L. Hawke, Division of Pharmacotherapy
and Experimental Therapeutics, UNC Eshelman School of Pharmacy, CB #7569,
Kerr Hall Room 3310, Chapel Hill, NC 27599-7360. E-mail: rhawke@email.
SCHRIEBER ET AL.