Silymarin for HCV infection - Review

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Antiviral Therapy April 2013
Stephen J Polyak1,2,3,*, Nicholas H Oberlies4, Eve-Isabelle Pecheur5, Harel Dahari6,7,8, Peter Ferenci9, Jean-Michel Pawlotsky10,11

Silibinin Is a Potent Antiviral Agent in Patients With Chronic HCV not Responding to Pegylated Interferon/Ribavirin Therapy
http://www.natap.org/2008/HCV/090808_01.htm.........After 7 days of silibinin monotherapy the 5-mg/kg dose was marginally effective (n = 3; log drop, 0.55 ± 0.5), whereas the 10 mg/kg (n = 19 [including the patients in protocol 1], log drop 1.41 ± 0.59), 15 mg/kg (n = 5; log drop, 2.11 ± 1.15), and 20 mg/day doses (n = 9; log drop, 3.02 ± 1.01) led to a highly significant decrease in viral load (P < .001

Silibinin monotherapy prevents graft infection after orthotopic liver transplantation in a patient with chronic hepatitis C
http://www.natap.org/2011/HCV/021611_06.htm

Silymarin for HCV infection

Antiviral Therapy April 2013

Stephen J Polyak1,2,3,*, Nicholas H Oberlies4, Eve-Isabelle Pecheur5, Harel Dahari6,7,8, Peter Ferenci9, Jean-Michel Pawlotsky10,11

1Department of Laboratory Medicine, University of Washington, Seattle, WA, USA 2Department of Microbiology, University of Washington, Seattle, WA, USA 3Department of Global Health, University of Washington, Seattle, WA, USA 4Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, NC, USA 5UMR INSERM 1052/CNRS 5286, Lyon, France 6Department of Medicine, University of Illinois at Chicago, Chicago, IL, USA 7Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos, NM, USA 8Present address: Department of Medicine, Division of Hepatology, Loyola University Chicago, Maywood, IL, USA 9Internal Medicine 3, Medical University of Vienna, Vienna, Austria 10National Reference Center for Viral Hepatitis B, C and Delta, Department of Virology, Henri Mondor Hospital, University of Paris-Est, Creteil, France 11INSERM U955, Creteil, France

Abstract

Silymarin, an extract of milk thistle seeds, and silymarin-derived compounds have been considered hepatoprotective since the plant was first described in ancient times. Hepatoprotection is defined as several non-mutually exclusive biological activities including antiviral, antioxidant, anti-inflammatory and immunomodulatory functions. Despite clear evidence for silymarin-induced hepatoprotection in cell culture and animal models, evidence for beneficial effects in humans has been equivocal. This review will summarize the current state of knowledge on silymarin in the context of HCV infection. The information was collated from a recent workshop on silibinin in Germany.

Introduction

Despite clear evidence for silymarin-induced hepatoprotection in cell culture and animal models [1], evidence for beneficial effects in humans has been equivocal. This may be attributable to a relative dearth of well-designed and controlled clinical trials and a generalized pervasiveness of inadequate attention to silymarin nomenclature and composition. The potential of silymarin-derived natural products for hepatitis C originated from cell-culture-based studies showing that silymarin blocked HCV infection [2,3].

However, recent reports exemplify a bipolarity of silymarin action. On one hand, there is the striking observation by Ferenci et al. [4] that Silibinin-C-2', 3-dihydrogen succinate, disodium salt (Legalon SIL), an intravenous, aqueous soluble formulation of silybin A and silybin B (synonymously called silibinin A and B; Figure 1) that collectively comprise the mixture known as silibinin, reduces viral load in HCV-infected patients. This has led to other studies showing that Legalon SIL prevents re-infection of the graft during liver transplantation through its pretransplant antiviral effect [5-7]. At the opposite end of the spectrum is the report from the randomized double-blind placebo-controlled SyNCH trial using the highest oral doses of silymarin to date (700 mg per day) that showed lack of any demonstrable effect of orally administered silymarin in reduction of HCV viral loads and alanine aminotransferase in infected patients [8]. In the last few years, new investigators have entered the field with the goals of understanding how silymarin, silibinin, Legalon SIL and other silymarin-derived flavonolignans inhibit HCV infection, as well as defining other potential clinical applications of Legalon SIL.

With these goals in mind, the first workshop on silibinin was held on 10 February 2012, in Cologne, Germany. Organized by Ralf-Torsten Pohl from Madaus Rottapharm in Cologne, Germany, the manufacturer of Legalon SIL, and an affiliate of the international Rottapharm-Madaus-Group headquartered in Monza, Italy, the workshop was chaired by Peter Ferenci and Jean-Michel Pawlotsky, and attracted an international group of researchers in this small but growing field.

History and composition of silymarin and silibinin

Ralf Torsten-Pohl (Madaus Rottapharm) gave a brief overview of the history of silymarin and silibinin, stressing the ancient origins of silymarin, where the herbal extract has been referenced in early medical texts. He discussed briefly the taxonomy of milk thistle (Silybum marianum [L] Gaertn [Asteraceae]). Dr Pohl's talk also highlighted how silymarin is extracted from the seeds (achenes), purified to form the mixture of the two isomeric silybin components, and then esterified with succinic acid to form the disuccinate disodium salt, Legalon SIL. Moreover he reviewed the large body of published studies on silymarin components that describe their pleiomorphic biological and pharmacodynamic behaviour in different preclinical and clinical settings covering diverse fields of medicinal use.

Nicholas Oberlies (University of North Carolina at Greensboro, Greensboro, NC, USA) presented an overview of the components of silymarin, with particular emphasis on the structures and nomenclature of the various mixtures of flavonolignans. Focusing on the most well-studied mixtures, silymarin and silibinin, he compared and contrasted these through a series of structural diagrams, chromatograms and lists.

The mixture silymarin constitutes major flavonolignans silybin A, silybin B, isosilybin A, isosilybin B, silychristin, isosilychristin and silydianin. These flavonolignans are likely derived from the parent flavonoid, taxifolin, which represents the left-hand side of all those compounds, condensing with coniferol alcohol. The various ways in which coniferyl alcohol combines with taxifolin imparts a fair degree of molecular diversity. By contrast, silibinin is a two-component mixture, made up of only silybin A and silybin B, and as noted by Pohl, Legalon SIL results from a synthetic addition of two succinic acid moieties to both silybin isomers. Dr Oberlies also showcased some of the chromatographic methods his group has developed to purify the individual components to >98% purity [9]. In the structural descriptions, Dr Oberlies stressed the importance of proper nomenclature of silymarin-derived compounds [10]. Indeed, the literature is rife with misnaming of compounds, and this lack of attention to detail likely adds to the confusion surrounding the biology of these compounds in the literature. He stressed that in the interest of furthering the medical potential of these compounds, scientists should strive to be clear, correct, and consistent in the nomenclature of silymarin-derived compounds and mixtures. Please refer to Table 1 for a guide to milk thistle nomenclature.

Human clinical experience

Over 400 subjects have received oral silymarin in multiple clinical trials [8,11-17]. In approximately half of these studies, some improvement in liver histology or reduction in liver enzymes was observed [11-13,16]. In the HALT-C trial, approximately one-quarter of the 1,145 patients were using silymarin at baseline [18]. In a follow-up study of 1,049 HALT-C subjects [19], 34% of subjects were consuming silymarin at baseline, which was associated with lower hepatic collagen content on study biopsies and less histological progression. However, no effect was seen for clinical outcomes in this study. In all studies cited above, no reduction in HCV viral loads was observed.

Peter Ferenci (University of Vienna, Vienna, Austria) reviewed his pioneering studies on Legalon SIL administration to HCV-infected patients [4]. The first study demonstrated that Legalon SIL induced dose-dependent, log-fold reductions in HCV RNA level in 20 subjects. However, the virological response to Legalon SIL was heterogeneous from one patient to another. Particularly intriguing were his data showing that 2-3 weeks of daily Legalon SIL intravenous infusion may 'rescue' pegylated interferon plus ribavirin non-responders, obtaining 40% sustained virological response rates [20]. Similar data were recently described by Biermer et al. [21] who also successfully rescued patients ailing on treatment with protease inhibitor containing triple regimes. Thus, Legalon SIL (like other inhibitors of HCV replication) may be interferon-sensitizing, enabling the treatment of interferon non-responders with triple therapy. Moreover, Legalon SIL appears to suppress viral load in patients infected with genotypes 1, 3 and 4, whereas the effect of Legalon SIL on genotype-2-infected patients is currently unknown. Dr Ferenci also reviewed the cases in which Legalon SIL prevented re-infection of the graft following liver transplantation [5-7]. These data suggest that Legalon SIL may prove clinically valuable in the transplant setting, where therapeutic options are still limited.

Mechanisms of silibinin action based on in vitro investigations

Despite the clear reductions in viral load in HCV-infected patients treated with Legalon SIL, the mechanisms by which HCV suppression by silymarin, silibinin, and Legalon SIL occurs have not been fully characterized. In vitro studies have suggested multiple mechanisms may be operative.

Abdelhakim Ahmed-Belkacem, who is in Jean-Michel Pawlotsky's lab (INSERM U955, Henri Mondor University Hospital, Creteil, France), reviewed data showing that silymarin-derived compounds, Legalon SIL, and structurally related flavonoids can inhibit HCV RNA-dependent RNA polymerase (RdRp) activity in in vitro assays with recombinant HCV non-structural 5B (NS5B) protein with inhibitory concentrations 50% (IC50) in the range of 75-100 μM [22]. A focused screen of 44 compounds belonging to different subgroups of flavonoids, including flavones, flavonols, isoflavones, flavanols and flavonoid glycosides revealed that the flavonoid, quercetagetin, had the strongest RdRp inhibitory activity with IC50 of 6.7 ±1.0 μM and 4.3 ±0.7 μM against genotype 1a H77 and genotype 1b J4 NS5B isolates, respectively. Interestingly, quercetagetin was fivefold less efficient against RdRp from genotype 2a strain JFH1, with an IC50 of 20.5 ±2.8 μM. These data suggest that Legalon SIL may differentially modulate RNA polymerase of different HCV genotypes. Kinetic analyses showed non-competitive inhibition with nucleoside triphosphates. However, although the comparisons were made on key structural components of highly related flavonolignans, the compared structures differ in other regions of the molecules. Thus, it is not clear how changes at distant molecular positions affect overall structure and function of flavonoids.

Stephen J Polyak (University of Washington, Seattle, WA, USA) described the characterization of the hepatoprotective activities of silymarin and the seven major flavonolignans in silymarin using cell culture-based assays that measure antiviral, antioxidant and anti-inflammatory actions in liver cells, and immunomodulatory actions on T-cells [2,23]. A comparison of the effects of Legalon SIL versus the parent natural product mixture silibinin was also presented, focusing on the relationships between NS5B polymerase inhibition and antiviral activities. It was emphasized that Legalon SIL inhibits NS5B-1b better than silibinin, and silibinin is a poor inhibitor of NS5B-2a [24]. Regarding inhibition of HCV replication in subgenomic replicon cell lines, Legalon SIL inhibits genotype 1b but not 2a, while silibinin does not inhibit replication in 1b or 2a replicon cell lines. Since JFH-1 infection is more effectively inhibited by silibinin than by Legalon SIL, the data suggest that if indeed polymerase activity is involved in suppression of HCV infection by Legalon SIL, there may be genotype-dependent differential susceptibilities of NS5B polymerases to Legalon SIL. This is in line with the NS5B polymerase inhibition study described by Dr Ahmed-Belkacem, although in their study, silibinin and Legalon SIL had anti-HCV activity in both the genotype 1b subgenomic replicon and the JFH1 model [14]. These differences could be explained by different experimental conditions and/or cell lines. Moreover, Legalon SIL is by design soluble in aqueous solution while silibinin, silymarin, and all silymarin-derived flavonolignans are insoluble in aqueous solution and require organic solutions such as DMSO or methanol for solubilization. In this respect, Legalon SIL is thought to partition well into the membrane lipid-water interface [25]. Thus, the question arises as to whether differences in solubility contribute to the clear and demonstrable differences in HCV infection, replication, and polymerase inhibition between Legalon SIL and silibinin.

Julie Blaising, who is in Eve-Isabelle Pecheur's lab (UMR INSERM 1052/CNRS 5286, Lyon, France), presented exciting new data that continue the story on the silibinin inhibition of HCV entry at the fusion stage previously described by her group [3,24]. Using a variety of sophisticated techniques including spinning disk confocal microscopy, Ms Blaising showed that during HCV entry, cell culture-derived HCV virions (HCVcc) colocalize in clathrin-containing vesicles, and as a consequence of the interaction with vesicles that have markers of early endosomes, the virus particles transiently adopt low velocity movement. As the endosomes mature into late endosomes, the interaction with clathrin ceases, and HCVcc resume high velocity movement. Ms Blaising demonstrated that in presence of silibinin, HCVcc dissociation from clathrin vesicles does not occur, the particles become trapped in early endosomes, retain low velocity movement, and never become associated with late endosomes. Thus, silibinin inhibits key steps in the clathrin-dependent entry of HCV into hepatocytes. The data suggest that silibinin may impact fundamental cellular processes that could have profound influence on pathogens beyond HCV. These studies typify how one can use natural products or natural product containing mixtures to probe cellular functions to elucidate key biological processes and their effects on host-pathogen interactions.

Application of HCV kinetics to understanding how Legalon SIL works

Harel Dahari (University of Illinois, Chicago, IL, USA; Los Alamos National Laboratory, Los Alamos, NM, USA) reviewed the principles of mathematical modelling of HCV dynamics during antiviral treatment and examined the kinetics of HCV RNA changes observed during directly-acting antiviral (DAA) drug administration that might be relevant to understanding the modes of action of Legalon SIL against HCV. The original model published in 1998 [26] posits that HCV RNA decline is biphasic, consisting of an early, rapid phase lasting approximately 2 days that reflects the direct antiviral effects of interferon-α (IFN) in blocking virus production/release. The second phase is protracted in time (days-weeks), has a slower viral RNA decline, and presumably reflects the loss/death of infected cells. Dr Dahari reviewed the current state of modelling HCV kinetics and the challenges to the original biphasic viral decline model that arise when fitting HCV kinetics during DAA treatment [27].

Jeremie Guedj (Los Alamos National Laboratory) [28] described a recent publication presenting a modelling analysis of HCV RNA kinetics from 25 patients infected with HCV genotype 1 or 4, who were treated for 7 days with monotherapy of 10, 15 or 20 mg/kg/day of Legalon SIL. Drs Guedj, Dahari and colleagues attempted to resolve the controversy in the field of whether Legalon SIL inhibits viral production, which is expected if the RdRp activity is inhibited [3,22,24,29], or whether Legalon SIL blocks other components of the HCV lifecycle such as virus entry or release [3,24]. Interestingly, the antiviral profile of Legalon SIL resembles that of IFN during the first phase, characterized as a rapid, dose-dependent decrease in viral RNA levels in serum.

The second-phase decline induced by Legalon SIL is not dose-dependent and resembles that of an RdRp inhibitor [30]. Moreover, half of Legalon-SIL-treated patients had biphasic RNA decay profiles, while the other half had monophasic decay patterns. Thus, Legalon SIL appears to induce two classes of viral RNA decay profiles, suggesting the possibility that blocking both virus production/release (probably by RdRp inhibition and later steps in virion release) and virus entry mechanisms contribute to the anti-HCV effects of Legalon SIL.

Pharmacokinetics and pharmacodynamics of silymarin preparations

A recent study by Schrieber et al. [31], indicated that patients with non-alcoholic fatty liver disease show biphasic and in some cases triphasic plasma concentrations of silymarin flavonolignans. This may be due to enterohepatic recycling of silymarin flavonolignans, which involves the circulation of parent and conjugated (by glucuronidation and/or sulfation) versions of silymarin flavonolignans. The metabolized (that is, conjugated) flavonolignans are transported out of hepatocytes by hepatobiliary transporters into the bile. Upon encountering the small intestine, the parent flavonolignans can be reformed by deconjugation enzymes in intestinal flora. The deconjugated flavonolignans are then returned to the liver via the portal blood supply, where they are again taken up by hepatocytes. Thus, enterohepatic recycling can elicit profound differences in plasma flavonolignan pharmacokinetics, thereby influencing the hepatoprotective actions of Legalon SIL. Moreover, it has also been shown that patients with cirrhosis display higher plasma levels of silymarin flavonolignans [32]. Future studies should formally characterize flavonolignan metabolism following Legalon SIL versus silymarin administration.

Key issues and areas for future research

First, a key issue involves differences between in vitro and in vivo dosing of silymarin-derived compounds. The in vitro antiviral activities of silymarin and silymarin-derived flavonolignans are observable at concentrations generally >20 μM. In healthy subjects, oral dosing of silymarin results in very low plasma concentrations of major flavonolignans in the range of 50-300 ng/ml due to their rapid metabolism to glucuronide and sulfate conjugates [8,32-37]. The key point is that silymarin as an oral formulation is not highly bioavailable, with a pharmacokinetic profile that achieves peak plasma concentrations 1-2 h post-dosing, with elimination in 4-6 h. However, in HCV patients with advanced liver disease, three- to fivefold higher plasma concentrations of flavonolignan conjugates are achieved compared to healthy subjects [38], while in patients with prostate cancer, plasma levels of silybin A and B up to 40 μg/ml (approximately 80 μM) are achieved with high oral doses of silipide, a formulation of silibinin with phospholipids [39]. Thus, it is possible that with the correct formulation for oral dosing, plasma levels of flavonolignans that approach in vitro concentrations can be achieved. However, results from the SyNCH trial, which administered the highest oral doses of silymarin to date, were presented at the AASLD meeting in 2011 and recently published [40]. The patients achieved 2-2,000 ng/ml of silymarin flavonolignans and there was no significant change in serum alanine aminotransferase activity or RNA levels in the silymarin treatment arms [8]. While it is logical to assume that intravenous dosing of Legalon SIL leads to higher serum levels of silybin A and B as compared with oral dosing, it is not clear how much higher. Thus, there is a need for additional clinical trials of Legalon SIL administration to patients.

The effects of Legalon SIL on different HCV genotypes should also be evaluated. These studies should have frequent sampling so that pharmacokinetic profiles can be accurately assessed. The investigators at the workshop also urged the company to publish existing Legalon SIL pharmacokinetic data. Although the SyNCH trial provided the highest oral silymarin dosing to date and was shown to be well-tolerated and safe, it will likely be impractical to increase pill burden to increase dose in future studies. Therefore, improved formulations of silymarin and silibinin for oral dosing that overcomes the bioavailability limitations are also urgently needed.

Second, hepatic levels of silymarin flavonolignans following oral and intravenous dosing in humans are unknown. Historically, liver biopsies have not been performed in patients receiving Legalon SIL and in the recently completed SyNCH trial [41]. However, it might be possible to justify studies in future trials, especially if Legalon SIL is used as a rescue therapy for previous non-responders to interferon-based therapies.

Third, despite the clear antiviral effects of silymarin and silymarin-derived compounds on HCV in vivo and in vitro, the mechanism(s) remain incompletely understood. While polymerase inhibition is demonstrable in vitro using purified NS5B proteins, the activity is modest. Cumulatively, the data support an important role for inhibition of virus entry in inhibition of HCV infection.

Silymarin, Legalon SIL, and silibinin also appear to inhibit release of progeny viruses [3,24], so it is possible that these compounds target other steps in the virus lifecycle that are dependent on host cell functions. Thus, additional studies should be performed to more clearly elucidate the mechanisms of antiviral action of silymarin. In doing so, these studies may reveal whether one or multiple mechanisms predominate.

Fourth, it is not known if Legalon SIL, which is the disuccinate versions of silybin A and silybin B, or silybin A and silybin B are the bioactive components. It is likely that the succinate moities on Legalon SIL could be cleaved by intracellular esterases, meaning that the parent silybin A and silybin B flavonolignans are the actual intracellular biological effectors of Legalon SIL. This issue is quite important because if it turns out that Legalon SIL is cleaved into silybin A and silybin B in cells, it likely means that the bioactivity of silibinin is more relevant than that of Legalon SIL, and as such, this information might help to settle the controversy over antiviral mechanisms of action of Legalon SIL versus silibinin. The situation becomes even more complex considering that silymarin flavonolignans are metabolized primarily through glucuronidation and also by sulfation [8,42], with multiple possible sites available for modification on each compound. Thus, metabolic studies on liver tissue are required to resolve these issues.

Fifth, Legalon SIL has shown promise in prevention of HCV infection during orthotopic liver transplantation [5-7]. Additional studies with recently FDA-approved protease inhibitors and other emerging (DAA) compounds are required to formally demonstrate the efficacy of Legalon SIL in conjunction with new antivirals.

Sixth, at present, Legalon SIL is administered daily for only 1-2 weeks before treatment is stopped. Despite having robust anti-HCV activity, this dosing regimen could conceivably create a scenario for development of resistance to Legalon SIL. Indeed, a recent study shows resistance to SIL can be selected both in vitro and in vivo [43]; thus, further studies on SIL resistance should be conducted.

With the recent approvals of new DAA compounds and second-generation drugs on the horizon, the renewed excitement and interest in compounds derived from the ancient botanical medicine known as silymarin may, at first glance, seem to be 'too little too late'. For patients who can afford and tolerate standard of care therapy, this statement may be true. However, many patients experience side effects that require cessation of therapy, and Legalon SIL may be useful as salvage therapy for prior interferon non-responders. Moreover, many developing countries may be unable to afford DAA combination therapy, let alone pegylated interferon plus ribavirin therapy. Thus, silymarin-derived compounds may provide clinical utility in these situations. Moreover, if hepatoprotective mechanisms of action and molecular targets for silymarin flavonolignans can be identified, this may lead to the structure-based development of potent new compounds that are orally bioavailable. The efficacy of Legalon SIL in orthotopic liver transplantation has been demonstrated, so continued research into this area may lead to identification of biomarkers of silymarin treatment and efficacy, novel targets for antiviral and anti-inflammatory drug design, and guide refinements in natural product-derived treatments for liver diseases in HCV- and HCV-HIV-coinfected patients. In this regard, we have recently shown that Legalon SIL inhibits in vitro HIV-1 infection of multiple cell types [44]. Furthermore, the antioxidant and anti-inflammatory effects of silymarin [45,46] may reduce pathogenesis of liver diseases of non-viral origin, as well as show efficacy in inflammatory diseases including cancer.

Source

Milk Thistle No Help in Tough Hep C Cases

By Kristina Fiore, Staff Writer, MedPage Today

Published: July 17, 2012

Reviewed by Dori F. Zaleznik, MD; Associate Clinical Professor of Medicine, Harvard Medical School, Boston and Dorothy Caputo, MA, BSN, RN, Nurse Planner

Silymarin, a milk thistle extract, probably will not be of much help to patients with chronic hepatitis C virus (HCV) who have already failed interferon therapy, researchers found.

In a randomized, controlled trial, there were no differences in improvements in alanine aminotransferase (ALT) levels between two doses of silymarin -- often used as an alternative therapy -- and placebo in these hard-to-treat patients, Michael Fried, MD, of the University of North Carolina at Chapel Hill, and colleagues reported in the July 18 issue of the Journal of the American Medical Association.

Currently, standard therapy for HCV involves a basic regimen of peginterferon and ribavirin, now buffered by two newer protease inhibitors. Still, a large proportion of patients don't respond to these therapies, and many others can't be treated with them because of medical comorbidities, the researchers said.

Patients often turn to alternative therapies such as silymarin in hopes of some added benefit. According to some estimates, about a third of HCV and cirrhosis patients report using the milk thistle extract for their disease -- but studies have yielded inconsistent results regarding its benefit.

So Fried and colleagues conducted a double-blind, placebo-controlled trial at four medical centers in the U.S., totaling 154 patients with chronic HCV who'd already failed interferon therapy. Just over half (56%) had never used milk thistle before.

Patients were enrolled between May 2008 and May 2010, with last follow-up in May 2011. The median age was 54, almost three-quarters (71%) of patients were male, and the median body mass index (BMI) was 29. Most patients (91%) had HCV genotype 1 infection.

They were randomly assigned to placebo or one of two doses of silymarin (420 mg or 700 mg) three times a day for 24 weeks. The primary outcome was a serum ALT of 45 U/L or less, or under 65 U/L provided that was at least a 50% drop from baseline values.

By study end, two participants from each treatment group met the primary outcome measure -- as did two patients in the placebo group.

The mean decline in serum ALT didn't differ significantly across the groups, either:

• -4.3 U/L for placebo
• -14.4 U/L for 420 mg silymarin
• -11.3 U/L for 700 mg silymarin

Nor were there any significant differences in HCV RNA levels or in quality-of-life measures, the researchers reported.

Adverse events were similar across all groups, with the most frequent being gastrointestinal symptoms, occurring in 12% of both silymarin groups compared with 5% of those on placebo.

The percentage of patients with serious adverse events was numerically higher in the silymarin groups, but Fried and colleagues noted that the power to detect differences was limited by the small number of patients in each group.

They concluded that oral silymarin "used at higher than customary doses did not significantly alter biochemical or virological markers of disease activity in patients with chronic HCV infection who had prior treatment with IFN-based regimens."

Recently, researchers reported that intravenous silibinin, another milk thistle extract, helped patients with liver toxicity resulting from mushroom poisoning. A trial of the compound in this indication is ongoing.

The study was supported by the National Center for Complementary and Alternative Medicine and the National Institute of Diabetes and Digestive and Kidney Diseases.

Rottapharm/Madaus donated the silymarin and placebo.

Abbott Molecular donated the HCV assays.

The researchers reported relationships with Genentech, Merck, Vertex, Gilead, Tibotec, Janssen, Bristol Myers Squibb, Abbott, Rottapharm/Madaus, Novartis, GlaxoSmithKline, Springbank, Medgenics, Boehringer Ingelheim, Ikaria, Anadys, and Gore.

Primary source: Journal of the American Medical Association
Source reference:
Fried MW, et al "Effect of silymarin (milk thistle) on liver disease in patients with chronic hepatitis C unsuccessfully treated with interferon therapy" JAMA 2012; 308(3): 274-282.

Source

Silymarin Is Ineffective for Chronic Hepatitis C Virus Infection

From Journal Watch > Journal Watch Gastroenterology

Atif Zaman, MD, MPH

Posted: 09/21/2012; Journal Watch © 2012 Massachusetts Medical Society

Abstract and Introduction

Abstract

In the most rigorous trial to date, oral silymarin was not superior to placebo in decreasing disease activity.

Introduction

Silymarin is a botanical extract of milk thistle commonly used by patients with liver disease. In vitro studies have demonstrated antiviral, anti-inflammatory properties of silymarin in hepatitis C virus (HCV) replicon systems. However, the few efficacy studies conducted in patients with chronic HCV infection have produced mixed results.

In a new multicenter, double-blind, placebo-controlled efficacy trial, investigators randomized 154 patients (median age, 54; 71% men) with chronic HCV infection who previously failed interferon-based therapy to receive 420 mg of silymarin, 700 mg of silymarin, or placebo three times daily for 24 weeks. The two oral doses of pure silymarin were determined by earlier dose finding studies and were three to five times higher than concentrations used in previous studies. The primary endpoint was a serum alanine aminotransferase (ALT) level of ≤45 U/L or a 50% reduction from baseline ALT to a level <65 U/L. Secondary endpoints included HCV RNA levels and quality-of-life indicators.

After 24 weeks of treatment, only two patients in each group achieved the primary endpoint. The mean decline in ALT levels at the end of treatment, the mean change in HCV RNA levels, and the quality-of-life indicators did not differ among the three groups.

Comment

This trial definitively demonstrates that silymarin, even at three to five times the typical dose, is ineffective in treating patients with chronic HCV infection. Unlike previous trials, this study used a pure, quantifiable formulation of silymarin and well-defined outcomes, its cohort was large and representative of the patient population, the treatment period was sufficiently long, and both medication and visit adherence rates were high. Clinicians should quote this study when addressing patients' questions regarding the use of milk thistle for treating HCV infection.

References

1. Fried MW et al. Effect of silymarin (milk thistle) on liver disease in patients with chronic hepatitis C unsuccessfully treated with interferon therapy: A randomized controlled trial. JAMA 2012 Jul 18; 308:274.

Source

Spirulina platensis versus silymarin in the treatment of chronic hepatitis C virus infection. A pilot randomized, comparative clinical trial

Published on: 2012-04-12

Spirulina platensis, a cynobacterium used frequently as a dietary supplement had been found to exhibit many immune-stimulating and antiviral activities. It had been found to activate macrophages, NK cells, T cells, B cells, and to stimulate the production of Interferon gamma (IFN-gamma) and other cytokines.

Natural substances isolated from Spirulina platensis had been found to be potent inhibitors against several enveloped viruses by blocking viral absorption/penetration and some replication stages of progeny viruses after penetration into cells. We aimed to study whether this dietary supplement possesses any therapeutically feasible activity worthy of further larger controlled clinical evaluation.

Methods: Sixty six patients with chronic hepatitis C virus infection and eligible for inclusion had been randomized to either Spirulina or Silymarin treated groups for a period of six months treatment.The two groups were followed up and blindly compared for early (after 3 months) and end of 6 months treatment virological response.

The effects of both treatments on each of alanine aminotransferase (ALT), Chronic Liver Disease Questionnaire scores (CLDQ), Arizona Sexual Experience Scale scores (ASEX) and the occurrence of any attributable adverse events were also compared.

Results: Among the 30 patients who had been treated with Spirulina and completed the 6 months protocol, 4 patients (13.3%) had a complete end of treatment virological response and 2 patients (6.7%) had a partial end of treatment response defined as significant decrease of virus load of at least 2-logs10. Though the proportion of responders in Spirulina group was greater than in the Silymarin group, the difference was not statistically significant at the end of both 6 months (p = 0.12) and 3 months treatment (p = 0.22) by Exact test.

Alanine aminotransferase as well as CLDQ and ASEX scores were found to be more significantly improved in Spirulina than in Silymarin treated group.

Conclusions: Our results could suggest a therapeutically feasible potential for Spirulina platensis in chronic HCV patients, worthy to conduct a larger sized and longer study to confirm these safety and efficacy encouraging results.WHO Clinical Trial Registration ID:ACTRN12610000958088 http://apps.who.int/trialsearch/trial.aspx?trialid=ACTRN12610000958088

Author: Mostafa YakootAmel Salem
Credits/Source: BMC Gastroenterology 2012, 12:32

Source

Silymarin Flops in Treatment of Chronic Hepatitis C

By: SUSAN LONDON, Family Practice News Digital Network

SAN FRANCISCO – Oral silymarin, an extract of milk thistle, is well tolerated but not efficacious for treating chronic hepatitis C virus infection, according to results from a randomized phase II trial reported at the annual meeting of the American Association for the Study of Liver Diseases.

After 24 weeks, patients taking high doses of the botanical agent – which is known to have anti-inflammatory, immunomodulatory, and antiviral activity in vitro – did not show improvement in serum levels of alanine aminotransferase (ALT) compared with their counterparts taking placebo.

A botanical agent – and milk thistle extract – has been proven ineffective for treating chronic hepatitis C.

There were also no significant differences between groups in a variety of measures of symptoms and quality of life.

"We must conclude that oral silymarin used at higher-than-customary doses did not significantly alter biochemical or virological markers of disease activity in hepatitis C patients previously treated with interferon-based regimens," said first author Dr. Michael W. Fried, director of hepatology at the University of North Carolina at Chapel Hill. But he added that "this was a fairly difficult group – these were nonresponders [to conventional therapy] – they were older."

The study is noteworthy for its many strengths, such as its use of a well characterized silymarin product, prolonged treatment, excellent adherence, and focus on a specific liver disease, he said.

Recent studies finding a benefit of silymarin in treating HCV infection used a different, intravenous formulation, according to Dr. Fried. That formulation contains a succinate moiety that may be important and likely achieves blood levels of silybin A, a major active component, higher than can be achieved with the oral formulation.

The oral doses used in the trial were probably not sufficient to inhibit the virus’ replicative enzymes, but giving higher doses was not practical. "We had to balance that with the pill burden: These were five capsules given three times a day, so we were not able to push the dose currently," he explained. "There may be attempts to try higher[-dose] formulations. There is also some way, interestingly, that’s being investigated to try to boost the silymarin levels with other botanical products ... because of their interactions on metabolism."

Dr. T. Jake Liang, president of the AASLD and chief of the liver diseases branch at the National Institute of Diabetes and Digestive and Kidney Diseases in Bethesda, Md., noted that the market for herbal and related products "is a big business in this country, about $30 billion, and it’s really not that well regulated, and there are a lot of unsubstantiated claims."

He pointed out that the trial focused on a more modest disease outcome. "The goal was not so much to see whether silymarin can eradicate the hepatitis C virus; the goal was really to see whether it could improve the damage to the liver," he explained in a press conference.

"I think this study clearly shows that the use of milk thistle is not effective in treating patients with hepatitis C. It’s a very important negative study – I think that’s key. We really want to make sure that we prevent any unwarranted use of herbal products despite the [health claims]," Dr. Liang concluded.

The trial, known as SyNCH and funded by the National Institutes of Health, enrolled 154 adult patients from four U.S. centers with chronic HCV who had not had a sustained virologic response to prior interferon-based therapy, had quantifiable HCV RNA, and had an ALT level of at least 65 IU/L.

Patients who had decompensated cirrhosis or moderate to severe steatosis or steatohepatitis were excluded, as were those who were positive for HIV or hepatitis B surface antigen, or who had used silymarin in the past month.

The patients were assigned to double-blind treatment in three groups: silymarin 420 mg three times daily, silymarin 700 mg three times daily, or matching placebo three times daily. The silymarin product used was a standardized preparation (Legalon, manufactured and supplied by Rottapharm Madaus in Monza, Italy, and Cologne, Germany) that is approved as a prescription drug in some European and Asian countries. Doses three- to fivefold higher than the customary one were chosen to achieve the highest likelihood of finding a benefit, according to Dr. Fried.

The patients studied were about 55 years old, on average, and three-fourths were white. Their mean ALT was 125 IU/L. Forty-four percent had used silymarin previously.

"Adherence throughout the treatment trial was quite excellent," he reported; across groups, 92%-98% of participants took more than 80% of planned doses. And random testing confirmed that patients in the silymarin group had silybin A in plasma while those in the placebo group did not.

However, final intent-to-treat analyses showed no significant difference between groups at 24 weeks in the primary end point, defined as achievement of a serum ALT level of 45 IU/L or lower (approximately the upper limit of normal) or a drop in serum ALT level of at least 50% to less than 65 IU/L (approximately 1.5 times the upper limit of normal).

Just 3.8% of patients in the placebo group met this end point, as did 4% in the silymarin 420-mg group and 3.8% in the silymarin 700-mg group.

"We really looked at many different subpopulations, and we could not find any signal at all," Dr. Fried said; additionally, the findings were much the same in per-protocol analyses.

The treatment groups were also statistically indistinguishable with respect to the changes from baseline in ALT levels and in HCV RNA levels, and with respect to changes from baseline in scores on questionnaires assessing depression, physical and mental health, and quality of life specific to chronic liver disease.

"Silymarin was well tolerated with an adverse event profile similar to placebo," Dr. Fried said. The three treatment groups did not differ significantly with respect to rates of adverse events (most of which were mild or moderate) or serious adverse events.

Dr. Fried reported that he is a consultant to Genentech, Tibotec, Vertex, Merck, Abbott, and Pharmasset; receives grant or research support from Genentech, Tibotec, Vertex, Merck, Anadys, Abbott, and Bristol-Myers Squibb; is an adviser or reviewer for GlaxoSmithKline; and has stock in Pharmasset. Dr. Liang reported that he had no relevant conflicts of interest.

Source

Milk Thistle Extract Comparable to Placebo in Hep C Liver Enzyme Study

November 15, 2011

Silymarin, an extract of milk thistle (Carduus marianus), had no benefit on levels of the liver enzyme alanine aminotransferase (ALT) in people living with hepatitis C, according to a new study reported Tuesday, November 8, by researchers at the 62nd annual meeting of the American Association for the Study of Liver Diseases in San Francisco.

Originally a native of Southern Europe and Asia, the milk thistle plant is now found throughout the world. The medicinal parts of the plant are the ripe seeds—slymarin is a complex of milk thistle seed extracts—which have been used for centuries to help manage a variety of liver diseases.

Whether sylimarin has been proved scientifically to treat chronic hepatitis is debatable. Though it has established activity in test tube studies, notably in calming immune system cells that contribute to liver inflammation, studies involving humans taking the drug have produced mixed results. One reason is that that supplement hasn't been systematically studied in a specific population of people living with liver disease, notably chronic HCV infection.

The study, reported at AASLD by Michael Fried, MD, of the University of North Carolina at Chapel Hill and his colleagues, randomized three groups of people who had chronic hepatitis C and had tried interferon-based treatment in the past but were unable to cure their infection and had elevated ALT levels. For the study, they received one of two doses of Legalon-brand silymarin or placebo for 24 weeks.

To participate in the study, volunteers had to have ALT levels exceeding 65 international units per liter, or IU/L (normal is 45 IU/L); the average ALT level, upon entering the study, was 106 IU/L.

The Legalon doses employed in the study were 420 milligrams (mg) or 700 mg, to be taken three times daily. These doses, which are 4.5 to 7.5 times higher than the customary amount, were selected for the study based on the findings of an earlier Phase I study of the supplement.

The study’s primary goal was to get participants’ ALT levels below 45 IU/L, or to decrease the ALT level to below 65 IU/L, provided this was at least a 50 percent decline from pre-treatment measurements.

Four U.S. clinical centers enrolled a total of 154 people, of whom 90 percent completed the requisite 24 weeks of follow-up. Roughly 71 percent of the study volunteers were male, and on average they were 54 years old.

Unfortunately, average declines in ALT levels after 24 weeks of treatment did not differ significantly between the three groups. Six study volunteers—two receiving 420 mg Legalon, two receiving 700 mg Legalon and two receiving placebo—met one of the primary goals of the study.

Knowing that adherence can be a problem, particularly with a treatment requiring three-times-daily dosing, Fried’s group examined whether failure to take the Legalon as prescribed contributed to the lack of efficacy. Yet, more than 90 percent of the study volunteers met or exceeded an 80 percent adherence threshold, determined by counting the number of dose cups returned to the clinic sites. And when the analysis was restricted only to those who maintained at least 80 percent adherence, there were still no statistically significant differences between the three groups.

Fried noted, however, that Legalon treatment was well tolerated—side effects were similar in all three study groups.

“Although well tolerated,” Fried’s team concluded, “oral silymarin administered at higher-than-customary doses did not significantly alter biochemical markers of disease activity in patients with chronic hepatitis C who had failed prior treatment with interferon-based regimens.”

Source

Inhibition of HCV 3a core gene through Silymarin and its Fractions

Published on: 2011-04-01

Hepatitis C is a major health problem affecting 270 million individuals in world including Pakistan. Current treatment regimen, interferon alpha and ribavirin only cure half of patients due to side effects and high cost.

Results: In the present study Silybum marianum (Milk thistle) seeds were collected, extracted and analyzed against HCV 3a core gene by transiently transfecting the liver cells with HCV core plasmid.

Our results demonstrated that Silymarin (SM) dose dependently inhibit the expression or function of HCV core gene at a non toxic concentration while the GAPDH remained constant. To identify the active ingredient, SM was fractioned by thin layer chromatography (TLC), column chromatography and HPLC.

Purified fractions were tested for HCV core gene and western blotting results showed that two factions of SM (S1 and S2) inhibit HCV 3a core expression or function in liver cells.

Conclusion: Our results suggest SM and its fractions (S1 and S2) inhibit HCV core gene of 3a genotype and combination of SM and its fractions with interferon will be a better option to treat HCV infection.

Author: Usman AshfaqTariq JavedSidra RehmanZafar NawazSheikh Riazuddin

Credits/Source: Virology Journal 2011, 8:153

Source

Effects of silybum marianum on patients with chronic hepatitis C

Journal of Research in Medical Sciences, Vol 16, No 3 (2011)

Hamid Kalantari, Zahra Shahshahan, Mehdi Hejazi, Taghi Ghafghazi, Vahid Sebghatolahi

Abstract

• BACKGROUND: Silymarin derived from silybum marianum (milk thistle), a flowering member of the daisy family, may benefit liver function in people infected with the hepatitis C virus. The aims of this pilot study were to assess the efficacy and safety of silymarin on serum hepatitis C virus (HCV) RNA, serum aminotransferases (ALT, AST) levels, liver fibrosis and well-being in patients with chronic hepatitis C (CHC).

• METHODS: This prospective self-controlled trial study was conducted from March to September 2006 at Department of Gastroenterology, Isfahan University of Medical Sciences, Isfahan, Iran. 55 patients with HCV (10 female and 45 male) with a mean age of 31.8 ± 6.4 years (10-67 years) were participated in the study. Patients received 24 weeks of silymarin (630 mg/day). Baseline virological biochemical, liver fibrosis (by a serum fibrosis markers, including YKL–40 and Hyaluronic acid), and SF-36 questionnaire were performed with biochemical tests repeated at the end of the treatment period.

• RESULTS: There was statistically difference in mean of ALT (108.7 ± 86.6 vs 70.3 ± 57.7) before and after the treatment (p < 0.001). The means of AST were 99.4 ± 139.7 and 59.7 ± 64.32 before and after the treatment with statistically differences (p = 0.004). After the treatment, nine patients were found with negative HCV-RNA (p = 0.004) and statistically significant improvement in results of liver fibrosis markers were found only in fibrosis group (p = 0.015). Quality of life was improved significantly (p < 0.001).

• CONCLUSIONS: This study indicated that in patients with CHC performing silymarin (650 mg/day) for 6 months, improved serum HCV-RNA titer, serum aminotransferases (ALT, AST), hepatic fibrosis and patient’s quality of life. More future studies are warranted.

• KEYWORDS: Hepatitis C Virus (HCV), Quality of life, Serum Aminotransferases.

Source

Silymarin use and liver disease progression in the Hepatitis C Antiviral Long-Term Treatment against Cirrhosis trial

Alimentary Pharmacology & Therapeutics
Volume 33, Issue 1, pages 127–137, January 2011

N. D. Freedman 1, T. M. Curto 2, C. Morishima 3, L. B. Seeff 4, Z. D. Goodman 5, E. C. Wright 6, R. Sinha 1, J. E. Everhart 7, the HALT-C Trial Group

Article first published online: 2 NOV 2010
DOI: 10.1111/j.1365-2036.2010.04503.x
Published 2010. This article is a US Government work and is in the public domain in the USA

Author Information

1 Nutritional Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Department of Health and Human Services, Rockville, MD, USA.
2 New England Research Institutes, Watertown, MA, USA.
3 Division of Virology, Department of Laboratory Medicine, University of Washington, Seattle, WA, USA.
4 Division of Digestive Diseases and Nutrition, and Liver Diseases Branch, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, MD, USA.
5 Division of Hepatic Pathology, Armed Forces Institute of Pathology, Washington, DC, USA.
6 Office of the Director, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, MD, USA.
7 Division of Digestive Diseases and Nutrition, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, MD, USA.

* Correspondence: Dr N. D. Freedman, Nutritional Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, 6120 Executive Blvd, EPS/320, MSC 7232, Rockville, MD 20852, USA. E-mail: freedmanne@mail.nih.gov

Abstract

Aliment Pharmacol Ther 2011; 33: 127–137

Summary

Background  Silymarin is the most commonly used herbal product for chronic liver disease; yet, whether silymarin protects against liver disease progression remains unclear.

Aim  To assess the effects of silymarin use on subsequent liver disease progression in 1049 patients of the Hepatitis C Antiviral Long-Term Treatment against Cirrhosis (HALT-C) trial who had advanced fibrosis or cirrhosis and had failed prior peginterferon plus ribavirin treatment.

Methods  Patients recorded their use of silymarin at baseline and were followed up for liver disease progression (two point increase in Ishak fibrosis score across baseline, year 1.5, and year 3.5 biopsies) and over 8.65 years for clinical outcomes.

Results  At baseline, 34% of patients had used silymarin, half of whom were current users. Use of silymarin was associated (P < 0.05) with male gender; oesophageal varices; higher ALT and albumin; and lower AST/ALT ratio, among other features. Baseline users had less hepatic collagen content on study biopsies and had less histological progression (HR: 0.57, 95% CI: 0.33–1.00; P-trend for longer duration of use=0.026). No effect was seen for clinical outcomes.

Conclusions  Silymarin use among patients with advanced hepatitis C-related liver disease is associated with reduced progression from fibrosis to cirrhosis, but has no impact on clinical outcomes (Clinicaltrials.gov #NCT00006164).

Introduction

An estimated 130–170 million individuals are chronically infected with hepatitis C virus worldwide.1 Pegylated interferon and ribavirin treatment results in an approximate sustained virological response rate of 55%.2, 3 However, individuals who fail to respond or who are unable to tolerate treatment have few additional options. As such, many patients have turned to complementary and alternative medications (CAM) instead of, or in addition to, standard therapy.4, 5

An extract of the milk thistle plant, silymarin (Silybum marianum), has been used to treat chronic liver disease since the time of the ancient Greeks.6 Silymarin is the most commonly used herbal product by individuals with chronic liver disease, and a recent publication from the Hepatitis C Antiviral Long-Term Treatment against Cirrhosis (HALT-C) trial indicated that nearly one-third of patients in the trial were former or current users.7 Although the exact chemical composition of preparations varies, silymarin consists of a mixture of flavonoids termed flavonolignans.8 Results from laboratory, animal and clinical studies suggest that silymarin may have anti-inflammatory,9–11 anti-viral,11–14 antioxidant,10, 15 and antifibrotic effects in the liver.10, 16, 17 However, clinical efficacy, particularly in the context of chronic hepatitis C, remains unproven, and results from most previous studies, including randomised trials, are inconsistent.5, 6

As an a priori hypothesis, information on baseline silymarin use was collected as part of the HALT-C trial. In the current report, we examined the association of baseline silymarin use with subsequent liver disease progression in 1049 patients with advanced chronic hepatitis C.

Patients and methods

The HALT-C trial was designed to evaluate the efficacy of long-term treatment with low-dose peginterferon alpha-2a for patients with hepatitis C-related bridging fibrosis and cirrhosis who had failed standard of care peginterferon plus ribavirin therapy.18 Patients were recruited from ten US medical centres and met the following criteria: detectable HCV RNA; nonresponse to prior peginterferon/ribavirin therapy; hepatic bridging fibrosis or cirrhosis on liver biopsy (Ishak fibrosis stage ≥2); and the absence of defined exclusion criteria (such as liver disease other than hepatitis C or history of hepatic decompensation or HCC).

Study design

A detailed description of the design of the HALT-C Trial is published.18 Patients whose previous failed treatment had not included peginterferon plus ribavirin were treated with this therapeutic combination as part of the ‘lead-in’ phase of the trial. If patients had detectable HCV RNA at 20 weeks of treatment, they were considered nonresponders and included in the randomised phase of the trial. Responders, who had undetectable HCV RNA at 20 weeks of treatment, received peginterferon plus ribavirin treatment for 48 weeks. Patients experiencing breakthrough, defined by detectable HCV RNA between 20 and 48 weeks of treatment, and patients who relapsed after completion of 48 weeks of therapy, could also enrol in the randomised phase of the trial. Finally, patients who upon recruitment had already failed peginterferon plus ribavirin therapy were immediately entered into the randomised phase of the trial (express patients).

During the randomised phase of the trial, patients were randomised to peginterferon alpha-2a 90 mcg weekly or no treatment. Liver biopsies were repeated 1.5 and 3.5 years after randomisation. A panel of twelve hepatic pathologists reviewed all biopsies and scored inflammation (0–18) and fibrosis (0–6) using the Ishak scoring system.19 As peginterferon therapy did not affect clinical outcome or histological progression,18 treated and untreated participant data from the randomised phase of the trial were combined.

Assessment of silymarin use

At baseline, trained study coordinators obtained patient medication use by way of an in-person interview. In addition to assessing prescription and nonprescription drugs, interviewers assessed CAM (herbal medications, dietary supplements and botanical products). Patients who used CAM at least once a week for 1 month or longer in their lives were defined as users. Duration of use was also recorded. To facilitate recall, patients were shown a card indicating 37 examples of herbal products in alphabetical order. Silymarin was one of these products. Patients also had the opportunity to indicate use of herbal products not listed on the card. Every 3 months after baseline, and as often as every 2 weeks during the lead-in phase, patients were asked whether they had stopped using any herbal products since their last visit or whether they were taking any new herbal products. Of 1050 randomised participants, we excluded one patient who lacked a medication record. We considered current users to be those using silymarin on the day of study randomisation. Former users could have used silymarin at any time prior to randomisation, including the lead-in phase. Duration of silymarin use included all months of use up to the day of randomisation. To analyse duration, we created a three level variable which included never users as the reference category. Months of use were then split at the median (16.6 months).

Morphometric image analysis of hepatic collagen content

Collagen in liver biopsy sections was stained with Sirius red and the degree of staining, which is proportional to the collagen content, was assessed using Image pro plus 6.0 imaging software (Media Cybernetics, Silver Spring, MD, USA) as previously described.20, 21 Sirius red values are expressed in arbitrary units, but reflect collagen content on a continuous scale. Analyses of collagen content were restricted to unfragmented biopsies with more than 10 mm2 of liver tissue in the section to avoid artefact and to reduce sampling variability. In total, 558 patients had morphometric image analysis performed on baseline biopsies, 550 patients had year 1.5 biopsies assessed by morphometry and 409 patients had year 3.5 biopsies analysed in this way. Of these patients, 183 had all three biopsies read.

Assessment of fibrosis and clinical outcomes

Patients were seen every 3 months in the randomised phase, at which point, complete blood counts, a liver chemistry panel and alpha-fetoprotein were tested at each clinical site. Patients also had at least one abdominal ultrasound examination every 12 months. Clinical outcomes included ascites, Child-Turcotte-Pugh score of ≥722 on two consecutive study visits, liver disease-related death, hepatic encephalopathy, hepatocellular carcinoma (HCC), spontaneous bacterial peritonitis, or variceal haemorrhage. Outcome reports were reviewed by an Outcomes Review Panel consisting of three investigators from the participating clinical centres. Participants with bridging fibrosis at baseline and a ≥2 point increase in Ishak fibrosis score (TPI) on either of the follow-up biopsies were considered to have fibrosis progression. Results are presented separately for clinical outcomes and for a two point increase in Ishak fibrosis score as well as for the two endpoints combined. HCC was diagnosed via ultrasound, AFP and histological confirmation as previously described.23

All details of this study were approved by the local Institutional Review Board at each participating institution, and all participants gave written informed consent.

Statistical analyses

We performed analyses using sas release 9.1 (SAS Institute, Cary, NC, USA). All tests were two-sided and an alpha level of <0.05 was considered statistically significant.

We tabulated baseline demographic, behavioural and clinical factors by categories of silymarin use (never, former, and current). Statistically significant variation across categories of silymarin use was assessed for categorical variables by the Mantel–Haenszel test for trend and for continuous variables with the Jonckheere–Terpstra test of trend.

Relative risks and 95% confidence intervals for the association of silymarin use with disease progression were calculated by use of Cox proportional hazards regression.24 Person-time was calculated from baseline to first outcome, end of study, or date of patient withdrawal. For clinical outcomes and the combined endpoint, follow-up time was up to 8.65 years. For TPI, follow-up was until first (1.5 years) or second biopsy (3.5 years). For clinical outcomes, Kaplan–Meier curves were generated for never, former and current silymarin users and were compared with the log-rank test.

Linear trend tests across increasing categories of silymarin use were performed by creating an ordinal variable for each category and entering the term as a continuous variable into the regression model. We tested the proportional hazards assumption by modelling interaction terms of time with trend variables for silymarin use. No significant deviations were found for silymarin use with either TPI or clinical outcomes; however, a significant deviation was found for HCC (P = 0.011).

Relative risks for liver disease progression were estimated from crude models and two different multivariate adjusted models. The first multivariate model included continuous age, lifetime alcohol use and coffee intake along with categorical variables for education (high school or less, some post high school, completed college), race/ethnicity (Caucasian, African American, Hispanic and other), gender, diabetes and ever use of other herbal products besides silymarin, such as green tea, garlic, ginseng, or echinacea. A second multivariate model was additionally adjusted for continuous mental and physical short-form (SF)-36 summary quality of life scores,25 and serological predictors of liver disease progression26– AST/ALT ratio, albumin, platelets, bilirubin, as well as categorical variables for cirrhosis status at baseline and presence or absence of oesophageal varices. For analyses of silymarin use during the trial, we updated silymarin use at the time of each follow-up biopsy. As for the main analyses, patients with a TPI at biopsy one (year 1.5) were censored at this time.

We assessed possible effect modification (interaction) by randomisation group, cirrhosis at baseline, median mental, and physical SF-36 quality of life scores and gender using stratification. Risk estimates did not vary by stratification group (P > 0.23 for all). Results stratified by cirrhosis at baseline are presented in the results section.

Finally, we analysed changes in morphometric collagen content across study biopsies using repeated analysis of variance, assuming an autoregressive covariance structure with the PROC MIXED function of sas 9.1. Again, analyses were restricted to patients who had not had an outcome prior to a particular scheduled study biopsy. Time (baseline, biopsy one, and biopsy two) and silymarin use (former and current) were included in the model as fixed effects. Adjustment for age and gender did not alter risk estimates and so were not included in the final models. Possible differences between the collagen content of biopsies taken from former or current silymarin users were compared with the collagen content of biopsies taken from never silymarin users by the Mann–Whitney test.

Results

At baseline, 17% (178/1049) of patients were former users of silymarin and 16% (170/1049) of patients were current users compared to 67% (701/1049) who reported never using silymarin (Table 1). The median duration of use for current users up to study entry was 35 months, whereas the median duration of use for former users was 6 months. Baseline silymarin use was associated with Caucasian race, completing college, male gender, lower prevalence of diabetes mellitus, higher lifetime alcohol and coffee consumption, and higher physical quality of life score. Silymarin was also modestly associated with a lower AST/ALT ratio and alkaline phosphatase levels and higher ALT, albumin levels, and prevalence of oesophageal varices (P < 0.05 for all). No association was observed for age, treatment group, patient cohort, body mass index, mental summary SF-36 score, serum AST, bilirubin, platelets, prothrombin time, HCV genotype or log RNA level, hepatic cirrhosis, collagen content, steatosis grade, or Ishak inflammation score.

At baseline, 621 patients had fibrosis and 428 patients had cirrhosis. During 4758 person-years of follow-up (median: 5.5 years per patient, interquartile range: 3.0–6.6 years), 384 patients had a two point increase in fibrosis score (TPI) from baseline or had a clinical outcome for liver disease. Combining these endpoints, we observed an inverse association between baseline silymarin use and liver disease progression (Table 2). In crude models, the relative risk (RR) associated with former use of silymarin was 0.87 (95% CI: 0.66–1.15), whereas the RR for current use was 0.73 (95% CI: 0.54–0.98; P-trend across categories = 0.029). Upon stratification by outcome, current silymarin use was associated with less TPI (RR for current vs. never use of silymarin, 0.54, 95% CI: 0.32–0.93; P-trend = 0.015), but had no association with clinical outcomes (RR for current vs. never, 0.86, 0.61–1.20; P-trend = 0.42). Multivariate adjustment for age, education, race/ethnicity, gender, lifetime alcohol use, diabetes, coffee intake, ever use of other herbal products besides silymarin, mental and physical quality of life scores, baseline cirrhosis, AST/ALT ratio, albumin, platelets, bilirubin and oesophageal varices only modestly affected risk estimates. After multivariate adjustment, the RR for current vs. never use of silymarin was 0.57 (95% CI: 0.33–1.00; P-trend = 0.042) for TPI and 1.09 (95% CI: 0.77–1.56; P-trend = 0.89) for clinical outcomes.

Duration of silymarin use, prior to baseline, was also assessed. Compared to never users, patients who used silymarin for up to the median duration (16.6 months) had an RR for TPI of 0.93 (0.58–1.51), whereas patients who used silymarin for greater than the median duration had an RR of 0.51 (95% CI: 0.30–0.90; P-trend = 0.026). The RRs for clinical outcomes for the same two categories of silymarin use were 0.86 (95% CI: 0.61–1.21) and 0.94 (95% CI: 0.66–1.35; P-trend = 0.57) respectively (data not shown in table).

In addition to silymarin use at baseline, we examined silymarin use over the course of the study. At the time of the second biopsy, three and a half years after randomisation, 69% of baseline users continued to use silymarin (88/128), whereas only 3% of baseline non-users (15/477) had started use. The risk of TPI among patients with fibrosis who continued to use silymarin throughout the study was 0.55 (95% CI: 0.29–1.03; 68 patients, 11 events), whereas the risk of TPI among patients who stopped using silymarin during follow-up was 0.66 (95% CI: 0.23–1.87; 20 patients, 4 events) (data not shown in table).

Silymarin was the most commonly used herbal product in the HALT-C trial. Fourteen percent of patients used an herbal product other than silymarin (n = 142). Use of a nonsilymarin herbal product had no association with either TPI (0.92, 95% CI: 0.57–1.48) or clinical outcomes (0.87, 95% CI: 0.60–1.25) (data not shown in table).

Among those with fibrosis, comparing current users of silymarin with never users, the RR for TPI was 0.19 (95% CI: 0.02–2.05; 16 events) for patients with an Ishak score of 2 at baseline, 0.48 (95% CI: 0.22–1.04; 87 events) for patients with an Ishak score of 3 at baseline, and 1.04 (95% CI: 0.37–2.90; 49 events) for patients with an Ishak score of 4 at baseline (data not shown in table). For those with fibrosis at baseline, we also examined the distribution of Ishak scores at year 1.5 and year 3.5 protocol biopsies. The distribution of Ishak scores was similar between former and never silymarin users for both biopsies (P > 0.30). For current silymarin users vs. never silymarin users, P-values for differences in the distribution of Ishak scores were 0.097 and 0.0059, for year 1.5 and year 3.5 biopsies respectively (Figure 1).

No association for clinical outcomes was found for those with either fibrosis or cirrhosis at baseline (RR for current use vs. never use, 1.36, 95% CI: 0.74–2.50, P-trend = 0.32, 98 events and 0.97, 95% CI: 0.61–1.53, P-trend = 0.41, 176 events, for fibrosis and cirrhosis respectively). The association between silymarin and clinical outcomes was also similar for outcomes occurring during years zero through four (RR for current vs. never use, 1.20, 95% CI: 0.78–1.86; P-trend = 0.85, 173 events) and five through eight of follow-up (0.88, 95% CI: 0.47–1.63; P-trend = 0.88, 101 events) (data not shown in table). Kaplan–Meier curves for clinical outcomes among current, former and never users of silymarin were similar (Figure 2; P = 0.657). In a secondary analysis of 88 incident cases of HCC, compared to never use, the RR for former and current users was 1.15 (95% CI: 0.62–2.13) and 1.60 (95% CI: 0.93–2.76) respectively. This possible effect was restricted to events occurring during the first 4 years (HR: 1.96, 95% CI: 0.95–4.05; 47 events), but not years five-eight of follow-up (HR: 1.26, 95% CI: 0.54–2.94; 41 events).

Finally, we examined the association between silymarin use and biopsy collagen content as measured by morphometric image analysis (Table 3). The collagen content of each study biopsy appeared generally similar in former and never users of silymarin. But, the study biopsies of baseline silymarin users tended to have a lower collagen content than study biopsies of never users. For example, the mean collagen content on the year 3.5 biopsy was 0.071 (standard deviation = 0.069) among current silymarin users and 0.090 (standard deviation = 0.085) among never users, P-value = 0.061. The overall P-value comparing the change in collagen content across repeated biopsies in baseline silymarin users relative to never users was 0.037. After stratification by baseline cirrhosis status, the association between silymarin use with change in collagen content across repeated study biopsies persisted in both patients with fibrosis (overall P-value = 0.034) and those with cirrhosis (overall P-value = 0.011) at baseline.

Discussion

In a large prospective cohort of individuals with advanced hepatitis C- related chronic liver disease, no clinical benefit was found for current silymarin use at baseline. In addition, we observed a nonsignificant increase in HCC risk among current silymarin users, which was present only in the first 4 years of follow-up. Baseline silymarin use was associated with less liver disease progression as measured by a two-point increase in Ishak fibrosis score as well as in the distribution of fibrosis scores in follow-up biopsies. A dose–response with duration of use was observed. Current use of silymarin at baseline, but not former use prior to baseline, was associated inversely with biopsy collagen content, regardless of whether patients had fibrosis or cirrhosis.

Silymarin has been used to treat liver disease for thousands of years.6, 27 Furthermore, results from animal, in vitro and clinical studies suggest that silymarin has possible anti-inflammatory,9–11 anti-viral,11–14 antioxidant10, 15 and antifibrotic effects.10, 16, 17 Yet, few clinical and observational studies have evaluated the effect of silymarin on liver disease progression and clinical outcomes in humans. Previous studies had small size, limited power to detect associations, and yielded mixed results.6, 27 For example, one trial of 170 patients with alcohol-related liver disease showed an effect of silymarin on survival,28 whereas a second trial of 200 patients showed similar survival rates in the randomised and control arms.29 Even fewer data are available for hepatitis C-related liver disease. Data from an Egyptian randomised trial of 141 patients showed no effect for silymarin on outcomes.30, 31

It is not clear why silymarin was associated with a reduction in rate of fibrosis progression, but not with clinical outcomes in our study. One possibility is that in order to exert an effect, silymarin must be used early in the disease progression process. In support of this hypothesis, silymarin seemed to have an effect on histological progression if patients had an Ishak score of 2 or 3 at baseline, but no effect on individuals with a score of 4 at baseline. On the other hand, silymarin had no effect on clinical outcomes for individuals with either cirrhosis or fibrosis at baseline, or for outcomes occurring during the first 4 years, or years five-eight of follow-up. It remains possible, however, that follow-up was too short to see an effect on clinical outcomes.

Fibrosis progression is not the sole determinant of subsequent decompensation or complications of portal hypertension. As such, it is also possible that silymarin does not have a beneficial effect on other determinants of clinical outcomes.32 Alternatively, differences between histological progression and clinical outcomes could simply reflect chance.

Strengths of our study include assessment of silymarin use before disease progression, the large number of patients with advanced hepatitis C-related liver disease, comprehensive assessment of clinical, demographic and lifestyle information, and careful assessment of clinical and histological outcomes. The major limitation was a complete lack of information on the amount of silymarin patients used per day. We also lacked information on how silymarin was prepared. Patients in the HALT-C trial probably used many different dosages and formulations of silymarin and even for individual patients, preparations probably varied day by day and week by week. Furthermore, it is unlikely that patients would have ingested pharmacological doses of silymarin as have shown effect in vitro, clinical, and animal studies. For example, a recent study of 36 patients observed an effect of intravenously (i.v.) administered silymarin (as silibinin) on hepatitis C viral level,14 although a study with similar dosing of orally administered silymarin showed no effect.33 Most likely, patients in HALT-C used less silymarin than those in the i.v. study. Further complicating interpretation is that the pharmacokinetics of silymarin may be altered by fibrosis. A recent study administered a standard silymarin dose to cirrhotics and healthy volunteers. In response to silymarin treatment, serum flavonolignans were higher in the cirrhotic volunteers.34 Finally, not all study biopsies were large enough to have morphometric analysis performed, a potential source of selection bias. Indeed, patients with cirrhosis were less likely to receive all biopsies.21 Yet, as we observed an apparent inverse association between silymarin use and collagen content, such a bias, if anything, would probably attenuate the observed association between silymarin use and collagen content.

In the HALT-C trial, use of silymarin was associated with Caucasian race, completing college and a higher SF-36 physical quality of life score, suggesting that silymarin use might be a marker for a large number of other lifestyle factors. We adjusted our risk estimates for these and other possible confounders. After adjustment, risk estimates were only modestly altered. In addition, the observed effect of silymarin does not simply reflect a propensity to use herbal products. Using herbal products, other than silymarin, had no association with either histological progression or clinical outcomes in our study. Nevertheless, as an observational study, the inverse association observed between silymarin use and histological progression could reflect another exposure or chance. We did not have any information on brand or dosage of silymarin. However, this limitation is reflective of the difficulty in detailing patient behaviour outside controlled studies. Many, if not most, patients with currently incurable liver disease seek alternative, unapproved therapies that cannot be easily quantified, yet deserve evaluation.

In summary, among individuals with advanced hepatitis-C-associated liver disease, we observed an inverse association between silymarin use and the progression of liver disease from fibrosis to cirrhosis, but no evidence for an effect on clinical outcomes. As our results are from an observational study, it is possible that the observed beneficial effect on liver disease progression is due to chance. Future studies with a comprehensive assessment of silymarin dose are needed to replicate these findings. Nevertheless, our results provide support for conducting additional studies of silymarin, including intervention trials with defined dosage regimens and standard silymarin product. Such studies would be most appropriate for patients who have not responded to or are not candidates for anti-viral therapy and have limited other treatment options. Importantly, our results do not support the use of ad hoc dosing of silymarin by patients with chronic liver disease.

Acknowledgements

Declaration of personal interests: None. In addition to the authors of this manuscript, the following individuals were instrumental in the planning, conduct and/or care of patients enrolled in this study at each of the participating institutions as follows:

University of Massachusetts Medical Center, Worcester, MA: (Contract N01-DK-9-2326) Gyongyi Szabo, MD, Barbara F. Banner, MD, Maureen Cormier, RN, Donna Giansiracusa, RN.

University of Connecticut Health Center, Farmington, CT: (Grant M01RR-06192) Herbert L. Bonkovsky, MD, Gloria Borders, RN, Michelle Kelley, RN, ANP.

Saint Louis University School of Medicine, St Louis, MO: (Contract N01-DK-9-2324) Adrian M. Di Bisceglie, MD, Bruce Bacon, MD, Brent Neuschwander-Tetri, MD, Elizabeth M. Brunt, MD, Debra King, RN.

Massachusetts General Hospital, Boston, MA: (Contract N01-DK-9-2319, Grant M01RR-01066; Grant 1 UL1 RR025758-01, Harvard Clinical and Translational Science Center) Jules L. Dienstag, MD, Raymond T. Chung, MD, Andrea E. Reid, MD, Atul K. Bhan, MD, Wallis A. Molchen, David P. Lundmark.

University of Colorado Denver, School of Medicine, Aurora, CO: (Contract N01-DK-9-2327, Grant M01RR-00051, Grant 1 UL1 RR 025780-01), Gregory T. Everson, MD, Thomas Trouillot, MD, Marcelo Kugelmas, MD, S. Russell Nash, MD, Jennifer DeSanto, RN, Carol McKinley, RN.

University of California - Irvine, Irvine, CA: (Contract N01-DK-9-2320, Grant M01RR-00827) Timothy R. Morgan, MD, John C. Hoefs, MD, John R. Craig, MD, M. Mazen Jamal, MD, MPH, Muhammad Sheikh, MD, Choon Park, RN.

University of Texas Southwestern Medical Center, Dallas, TX: (Contract N01-DK-9-2321, Grant M01RR-00633, Grant 1 UL1 RR024982-01, North and Central Texas Clinical and Translational Science Initiative) William M. Lee, MD, Thomas E. Rogers, MD, Peter F. Malet, MD, Janel Shelton, Nicole Crowder, LVN, Rivka Elbein, RN, BSN, Nancy Liston, MPH.

University of Southern California, Los Angeles, CA: (Contract N01-DK-9-2325, Grant M01RR-00043) Karen L. Lindsay, MD, MMM, Sugantha Govindarajan, MD, Carol B. Jones, RN, Susan L. Milstein, RN.

University of Michigan Medical Center, Ann Arbor, MI: (Contract N01-DK-9-2323, Grant M01RR-00042, Grant 1 UL1 RR024986, Michigan Center for Clinical and Health Research) Anna S. Lok, MD, Robert J. Fontana, MD, Joel K. Greenson, MD, Pamela A. Richtmyer, LPN, CCRC, R. Tess Bonham, BS.

Virginia Commonwealth University Health System, Richmond, VA: (Contract N01-DK-9-2322, Grant M01RR-00065) Mitchell L. Shiffman, MD, Richard K. Sterling, MD, MSc, Melissa J. Contos, MD, A. Scott Mills, MD, Charlotte Hofmann, RN, Paula Smith, RN.

Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD: Marc G. Ghany, MD, T. Jake Liang, MD, David Kleiner, MD, PhD, Yoon Park, RN, Elenita Rivera, RN, Vanessa Haynes-Williams, RN.

National Institute of Diabetes and Digestive and Kidney Diseases, Division of Digestive Diseases and Nutrition, Bethesda, MD: Patricia R. Robuck, PhD, Jay H. Hoofnagle, MD.

University of Washington, Seattle, WA: (Contract N01-DK-9-2318) David R. Gretch, MD, PhD, Minjun Chung Apodaca, BS, ASCP, Rohit Shankar, BC, ASCP, Natalia Antonov, M. Ed.

New England Research Institutes, Watertown, MA: (Contract N01-DK-9-2328) Kristin K. Snow, MSc, ScD, Anne M. Stoddard, ScD, Margaret C. Bell, MS, MPH.

Armed Forces Institute of Pathology, Washington, DC: Fanny Monge, Michelle Parks.

Data and Safety Monitoring Board Members: (Chair) Gary L. Davis, MD, Guadalupe Garcia-Tsao, MD, Michael Kutner, PhD, Stanley M. Lemon, MD, Robert P. Perrillo, MD.

Declaration of funding interests: This study was funded in part by the National Institute of Diabetes & Digestive & Kidney Diseases (contract numbers are listed below). Additional support was provided by the National Institute of Allergy and Infectious Diseases (NIAID), the National Cancer Institute, the National Center for Minority Health and Health Disparities and by General Clinical Research Center and Clinical and Translational Science Center grants from the National Center for Research Resources, National Institutes of Health (grant numbers are listed in the Acknowledgement). This research was also supported in part by the Intramural Research Program of the National Cancer Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health. Additional funding to conduct this study was supplied by Hoffmann-La Roche, Inc., through a Cooperative Research and Development Agreement (CRADA) with the National Institutes of Health.

References

1 Lavanchy D. The global burden of hepatitis C. Liver Int 2009; 29(Suppl. 1): 74–81.

2 Fried MW, Shiffman ML, Reddy KR, et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 2002; 347: 975–82.

3 Manns MP, McHutchison JG, Gordon SC, et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet 2001; 358: 958–65.

4 Stickel F, Schuppan D. Herbal medicine in the treatment of liver diseases. Dig Liver Dis 2007; 39: 293–304.

5 Verma S, Thuluvath PJ. Complementary and alternative medicine in hepatology: review of the evidence of efficacy. Clin Gastroenterol Hepatol 2007; 5: 408–16.

6 Rambaldi A, Jacobs BP, Iaquinto G, Gluud C. Milk thistle for alcoholic and/or hepatitis B or C liver diseases – a systematic cochrane hepato-biliary group review with meta-analyses of randomized clinical trials. Am J Gastroenterol 2005; 100: 2583–91.

7 Seeff LB, Curto TM, Szabo G, et al. Herbal product use by persons enrolled in the Hepatitis C Antiviral Long-Term Treatment against Cirrhosis (HALT-C) trial. Hepatology 2008; 47: 605–12.

8 Shibano M, Lin AS, Itokawa H, Lee KH. Separation and characterization of active flavonolignans of Silybum marianum by liquid chromatography connected with hybrid ion-trap and time-of-flight mass spectrometry (LC-MS/IT-TOF). J Nat Prod 2007; 70: 1424–8.

9 Morishima C, Shuhart MC, Wang CC, et al. Silymarin inhibits in vitro T-cell proliferation and cytokine production in hepatitis C virus infection. Gastroenterology 2010; 138: 671–81.

10 Trappoliere M, Caligiuri A, Schmid M, et al. Silybin, a component of sylimarin, exerts anti-inflammatory and anti-fibrogenic effects on human hepatic stellate cells. J Hepatol 2009; 50: 1102–11.

11 Polyak SJ, Morishima C, Shuhart MC, et al. Inhibition of T-cell inflammatory cytokines, hepatocyte NF-kappaB signaling, and HCV infection by standardized Silymarin. Gastroenterology 2007; 132: 1925–36.

12 Ahmed-Belkacem A, Ahnou N, Barbotte L, et al. Silibinin and related compounds are direct inhibitors of hepatitis C virus RNA-dependent RNA polymerase. Gastroenterology 2010; 138: 1112–22.

13 Bonifaz V, Shan Y, Lambrecht RW, et al. Effects of silymarin on hepatitis C virus and haem oxygenase-1 gene expression in human hepatoma cells. Liver Int 2009; 29: 366–73.

14 Ferenci P, Scherzer TM, Kerschner H, et al. Silibinin is a potent antiviral agent in patients with chronic hepatitis C not responding to pegylated interferon/ribavirin therapy. Gastroenterology 2008; 135: 1561–7.

15 Pietrangelo A, Borella F, Casalgrandi G, et al. Antioxidant activity of silybin in vivo during long-term iron overload in rats. Gastroenterology 1995; 109: 1941–9.

16 Boigk G, Stroedter L, Herbst H, et al. Silymarin retards collagen accumulation in early and advanced biliary fibrosis secondary to complete bile duct obliteration in rats. Hepatology 1997; 26: 643–9.

17 Dehmlow C, Erhard J, De Groot H. Inhibition of Kupffer cell functions as an explanation for the hepatoprotective properties of silibinin. Hepatology 1996; 23: 749–54.

18 Di Bisceglie AM, Shiffman ML, Everson GT, et al. Prolonged therapy of advanced chronic hepatitis C with low-dose peginterferon. N Engl J Med 2008; 359: 2429–41.

19 Ishak K, Baptista A, Bianchi L, et al. Histological grading and staging of chronic hepatitis. J Hepatol 1995; 22: 696–9.

20 Goodman ZD, Becker RL Jr, Pockros PJ, Afdhal NH. Progression of fibrosis in advanced chronic hepatitis C: evaluation by morphometric image analysis. Hepatology 2007; 45: 886–94.

21 Goodman ZD, Stoddard AM, Bonkovsky HL, et al. Fibrosis progression in chronic hepatitis C: morphometric image analysis in the HALT-C trial. Hepatology 2009; 50: 1738–49.

22 Pugh RN, Murray-Lyon IM, Dawson JL, Pietroni MC, Williams R. Transection of the oesophagus for bleeding oesophageal varices. Br J Surg 1973; 60: 646–9.

23 Lok AS, Seeff LB, Morgan TR, et al. Incidence of hepatocellular carcinoma and associated risk factors in hepatitis C-related advanced liver disease. Gastroenterology 2009; 136: 138–48.

24 Cox DR. Regression models and life-tables. J R Stat Soc Series B Stat Methodol 1972; 34: 187.

25 Ware JE Jr, Sherbourne CD. The MOS 36-item short-form health survey (SF-36). I. Conceptual framework and item selection. Med Care 1992; 30: 473–83.

26 Ghany MG, Lok AS, Everhart JE, et al. Predicting clinical and histologic outcomes based on standard laboratory tests in advanced chronic hepatitis C. Gastroenterology 2010; 138: 136–46.

27 Mayer KE, Myers RP, Lee SS. Silymarin treatment of viral hepatitis: a systematic review. J Viral Hepat 2005; 12: 559–67.

28 Ferenci P, Dragosics B, Dittrich H, et al. Randomized controlled trial of silymarin treatment in patients with cirrhosis of the liver. J Hepatol 1989; 9: 105–13.

29 Pares A, Planas R, Torres M, et al. Effects of silymarin in alcoholic patients with cirrhosis of the liver: results of a controlled, double-blind, randomized and multicenter trial. J Hepatol 1998; 28: 615–21.

30 Strickland GT, Tanamly MD, Tadros F, et al. Two-year results of a randomised double-blinded trial evaluating silymarin for chronic hepatitis C. Dig Liver Dis 2005; 37: 542–3.

31 Tanamly MD, Tadros F, Labeeb S, et al. Randomised double-blinded trial evaluating silymarin for chronic hepatitis C in an Egyptian village: study description and 12-month results. Dig Liver Dis 2004; 36: 752–9.

32 Cho YK, Yun JW, Park JH, et al. Deleterious effects of silymarin on the expression of genes controlling endothelial nitric oxide synthase activity in carbon tetrachloride-treated rat livers. Life Sci 2009; 85: 281–90.

33 Gordon A, Hobbs DA, Bowden DS, et al. Effects of Silybum marianum on serum hepatitis C virus RNA, alanine aminotransferase levels and well-being in patients with chronic hepatitis C. J Gastroenterol Hepatol 2006; 21: 275–80.

34 Schrieber SJ, Wen Z, Vourvahis M, et al. The pharmacokinetics of silymarin is altered in patients with hepatitis C virus and nonalcoholic fatty liver disease and correlates with plasma caspase-3/7 activity. Drug Metab Dispos 2008; 36: 1909–16.

 

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