Showing posts with label silymarin. Show all posts
Showing posts with label silymarin. Show all posts

Thursday, January 26, 2017

Review - Silymarin/Silybin and Chronic Liver Disease: A Marriage of Many Years

Molecules 2017, 22(2), 191; doi:10.3390/molecules22020191
Silymarin/Silybin and Chronic Liver Disease: A Marriage of Many Years
Alessandro Federico *, Marcello Dallio and Carmelina Loguercio           
Department of Clinical and Experimental Medicine, Second University of Naples, 80131 Naples, Italy

Received: 6 December 2016 / Accepted: 18 January 2017 / Published: 24 January 2017 

View Full-Text Article

Silymarin is the extract of Silybum marianum, or milk thistle, and its major active compound is silybin, which has a remarkable biological effect. It is used in different liver disorders, particularly chronic liver diseases, cirrhosis and hepatocellular carcinoma, because of its antioxidant, anti-inflammatory and antifibrotic power. Indeed, the anti-oxidant and anti-inflammatory effect of silymarin is oriented towards the reduction of virus-related liver damages through inflammatory cascade softening and immune system modulation. It also has a direct antiviral effect associated with its intravenous administration in hepatitis C virus infection. With respect to alcohol abuse, silymarin is able to increase cellular vitality and to reduce both lipid peroxidation and cellular necrosis. Furthermore, silymarin/silybin use has important biological effects in non-alcoholic fatty liver disease. These substances antagonize the progression of non-alcoholic fatty liver disease, by intervening in various therapeutic targets: oxidative stress, insulin resistance, liver fat accumulation and mitochondrial dysfunction. Silymarin is also used in liver cirrhosis and hepatocellular carcinoma that represent common end stages of different hepatopathies by modulating different molecular patterns. Therefore, the aim of this review is to examine scientific studies concerning the effects derived from silymarin/silybin use in chronic liver diseases, cirrhosis and hepatocellular carcinoma.

Keywords: silymarin; silybin; antioxidants; alcoholic liver disease; viral hepatitis; non-alcoholic fatty liver disease; hepatocellular carcinoma

Continue to full text article @ Molecules

About This Journal
Molecules (ISSN 1420-3049, CODEN: MOLEFW) is an open access journal covering all aspects of organic chemistry. Originally conceived as a forum for papers on synthetic organic chemistry and natural product chemistry, like the field, Molecules has evolved over its 20 years, with increasing numbers of papers on more theoretical subjects, physical organic chemistry, nanomaterials and polymer chemistry and applied studies. All articles are peer-reviewed and published continuously upon acceptance. Molecules is published by MDPI, Basel, Switzerland.

Tuesday, May 14, 2013

Is There a Future for Milk Thistle In HCV?

This summary highlights current information on the use of oral silymarin (the active ingredient in milk thistle found in the seeds of the plant) for hepatitis C.

As once a HCV patient myself who successfully treated the virus with conventional therapy in 2000, I never used the supplement, nor do I endorse it, but I had a few close friends who did. They found it to be well tolerated when used according to the recommended dose, as for the benefits? I can only comment that it seemed to help my friends endure the virus, especially when standard therapy failed them.

Today I point you to the research, a few studies which offer a better understanding of the popular supplement, including the possible benefits. Personally I have no opinions of my own to share, except a grave concern for anyone using milk thistle under the pretense it will eradicate the virus. Still, a part of me can't help to wonder if HCV patients will continue to use milk thistle after interferon free therapies become available? Yes, no, maybe. 

When It All Began

In 1990 when blood screening for hepatitis C began there was little hope for people infected with the virus. Back then milk thistle was commonly used by HCV patients, although the use of milk thistle is not as common as it once was, in regards to liver disease it is still used today by people living with the virus.

Why Was Milk Thistle Considered An Alternative To HCV Treatment?

In 1990 HCV was still in its early infancy, we didn't have many treatment options, from 1991-1997 interferon was the only therapy available. Not until 1998 was ribavirin added to interferon with the approval of  Rebetron - Schering’s combo of Intron A and ribavirin. Later in 2001 the first pegylated interferon went to market, it was Schering’s Peg-Intron followed in 2002 by Genentech’s Pegasys.

A decade later standard therapy for genotype 1 HCV patients still involves peginterferon and ribavirin, used along with (Incivek) Telaprevir or (Vicrelis) Boceprevir both protease inhibitors were approved in 2011. However, not everyone will respond to triple therapy or a regimen of peginterferon and ribavirin alone, plus some people are unable to tolerate interferon. With few options left milk thistle is generally explored by HCV patients.

Use of Herbal Supplements for Hepatitis C

Research on silymarin suggests that it may protect the liver from inflammation. But it does not have a direct effect on viruses that cause hepatitis, such as the hepatitis C. The National Institutes of Health, National Center for Complementary and Alternative Medicine this month updated - Hepatitis C and Dietary Supplements:What the Science Says

Another great resource on the use of herbs and supplements including their interactions is available at HCV Advocate, download the PDF here, updated April 2013.

A few words about the supplements listed in this glossary:

The goal was to choose supplements that may be particularly pertinent to those with liver disease, especially viral hepatitis

The information applies to supplements, NOT food. For instance, under artichoke, it says,

“Avoid with bile duct obstruction or gallstones. Use cautiously in liver patients with clotting problems as artichoke may increase risk of bleeding.” This means to use caution when taking extracts and formulations that have high doses of the active ingredients

Heads Up

This information is not intended as medical advice or endorsement of the use of dietary supplements. Always talk to your medical provider before taking any herbs or supplements. All herbs, drugs, and other substances have potential side effects. Allergic reactions have been reported for nearly every herb, sometimes with life-threatening consequences. If you suspect you are having an allergic reaction or other serious side effect, stop taking the substance and seek immediate medical advice. If you have trouble breathing or feel faint, call 911.

If you are scheduled for a medical or surgical procedure, particularly if anesthesia will be used, or plan to undergo chemotherapy, report supplement use to your medical provider. You may need to stop supplement use for a week or more before the procedure since many supplements interfere with anesthesia and/or blood clotting.
Download PDF 

Commercial Milk Thistle

The U.S. Food and Drug Administration (FDA) does not regulate supplements in the same way it regulates medicines. A dietary supplement can be sold with limited or no research on how well it works. The form you buy in health food or grocery stores may not be the same as the form used in research. For instance in one article "Antioxidant and Anti-Hepatitis C Viral Activities of Commercial Milk Thistle Food Supplements" researchers analyzed 45 products from local stores for their silymarin content, antioxidant activities, and antiviral activity against HCV. Some of these samples were found to vary widely in their silymarin content.

The 2013 research paper Antioxidant and Anti-Hepatitis C Viral Activities of Commercial Milk Thistle Food Supplements - Anthony, K.; Subramanya, G.; Uprichard, S.; Hammouda, F.; Saleh, M. is available online @ MDPI AG - Antioxidants
*About the journal.

Excerpt from a 2010 article found in the HCV Advocate Newsletter, written by Lucinda K. Porter, RN

All milk thistle is not alike and what is in the bottle may not match what is promised on the label. In a startling report published by (CL), only one of 10 products passed the necessary tests in order to carry the CL seal of approval.

CL is an independent organization that provides information and testing of nutritional products. They have been around for about ten years. Some information is free, but product reports are available only to subscribers. A one year subscription is $30 and worth every penny.

The Research

In 2013 patients with hepatitis C still use alternative therapies in hopes of some added benefit. The most common as mentioned above is silymarin, an antioxidant that exhibits anti-inflammatory properties.  According to research close to a third of HCV and cirrhosis patients report using the milk thistle extract, even when studies have shown inconsistent results regarding its benefit.

The most rigorous trial to date to test the herbal extract milk thistle (silymarin), was published in the 2012 issue of  the Journal of the American Medical Association (JAMA),  researchers reported oral silymarin was not superior to placebo (A substance containing no medication) in decreasing disease activity or its symptoms.

The Study

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. 

How many patients were in the study?

154 patients (median age, 54; 71% men) with chronic HCV infection who previously failed interferon-based therapy.

What dose of silymarin did the patients receive?

The researchers randomly assigned the patients to one of three groups, two of which took high doses of a standardized form of silymarin at 420mg or 700mg three times daily for 24 weeks. The third group took a placebo.

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.

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.

What was the baseline ALT of patients who participated in the study?

The patients in the study had serum alanine aminotransferase (ALT) enzyme levels greater than 65 IU/L, with a median of 106 IU/L at baseline. A normal level is 45 IU/L.

Did patients respond, was ALT lowered?

Of the 138 patients who completed the 24-week study, 90% were able to adhere to at least 80% of the pill regimen. In spite of the compliance, however, the mean drop in serum ALT was not significantly different between the three groups.

After six months, one person in the placebo group had a serum ALT less then 45 IU, as did two patients in each of the silymarin arms. Additionally, two patients in the placebo and high-dose silymarin groups had at least a 50% decline in baseline ALT to less then 65 IU, as did one patient in the 420-mg silymarin arm.

Side effects

The most common types of adverse events were gastrointestinal, musculoskeletal, dermatologic, infection, and physical injury. Six patients in the 420-mg silymarin arm and five in the 700-mg arm had serious adverse events, compared with one in the placebo group.

This trial found that oral silymarin used at higher than customary doses (three to five times the typical dose) did  not significantly alter biochemical or virological markers of disease activity.

MedPage Today
 2012 issue of JAMA

Video summary of trial published in the Journal of the American Medical Association

Is There a Future for Milk Thistle In HCV?

In the June 2012 issue of Gastroenterology & Endoscopy News, Christina Frangou reported on the AASLD 2011 meeting and the above study, the author noted commentary from Joseph K. Lim, MD on the future role of milk thistle for patients with liver disease and hepatitis C. The director of the Yale Viral Hepatitis Program cited three studies of interest where the extract was given in higher doses or different formulations

With the latest study results, it is unlikely that oral silymarin will have a future role in the care of patients with hepatitis C infection, although it still will be considered for other liver conditions for which no therapeutic options exist, said Joseph K. Lim, MD, associate professor of medicine and director of the Yale Viral Hepatitis Program at the Yale University School of Medicine, New Haven, Conn.

“Although the study cannot entirely exclude the possibility of a benefit from much higher doses of oral silymarin or intravenous silymarin, in this era of rapid advances in high-potency direct-acting antiviral agents, which may soon eliminate interferon, clinical interest in adjunctive therapies with significant pill burden or intravenous formulations with unproven benefit will be very limited.”

Dr. Lim added, “Once simplified nontoxic, oral–oral regimens with high sustained viral response rates materialize in the next several years, supplements such as silymarin may lose relevance.”
Attendees at the meeting said that they hope patients pay attention to the results of the study.
“Thank you for saving our patients from financial stress and false hope, possibly leading to delays in definitive therapy,” one attendee tweeted during the session.

However, to others, the results were disappointing. In poorer areas of the world where the newer hepatitis drugs are simply too expensive, the silymarin compound could be extremely useful, said one hepatologist.

It may be that there is a future role for silymarin or silibinin in hepatitis treatment when the extract is given in higher doses or different formulations than used in the current randomized study. In one study published in January in the Journal of Hepatology, investigators studied changes in HCV RNA levels in 25 patients receiving 10, 15 or 20 mg/kg per day of Legalon SIL, a chemically hydrophilized version of silibinin (Guedj J et al. 2012;56:1019-1024). Patients showed a significant drop in viral load, particularly between days 2 and 7. The investigators concluded that Legalon SIL may affect hepatitis virus by blocking both viral infection and production/release. In another study, investigators found that IV-administered silibinin had a “substantial antiviral effect” against HCV in 20 IFN nonresponders (Ferenci P et al. Gastroenterology 2008;135:1561-1567). And, still another study showed that oral Silybum marianum significantly affected serum HCV RNA levels, ALT levels, quality of life and psychological well-being (Gordon A et al. J Gastroenterol Hepatol 2006;21:275-280).

Mushroom Poisoning and intravenous silibinin

In 2011 Georgetown University researchers reported that intravenous silibinin, another milk thistle extract, helped patients with liver toxicity resulting from mushroom poisoning.

Treating HCV without Interferon

HCV drugs currently in clinical trials without interferon have shown great promise, Gilead Sciences Inc and Abbvie Inc are two pharmaceutical companies that seem to be leading the race, with treatments that are predicted to reach the market some time next year. Pegylated interferons will hopefully one day be obsolete, however most likely, treatment may still include ribavirin, a drug with difficult side effects. The future is looking promising for the millions of people currently afflicted with HCV. But these therapies may come to late for HCV patients in need of a liver transplant or battling liver cancer. Dr. Scott Biggins with the University of Colorado School of Medicine recently reported there were 126,862 new candidates for first liver transplant registered with Organ Procurement and Transplantation Network (OPTN), with 41% of these having HCV, the article is available here.

This brings us full circle to a paper published in the March 2013 issue of Journal of Hepatology - Silibinin: An old drug in the high tech era of liver transplantation. 

The study concluded:
Intravenous silibinin (Iv-SIL) is safe but by the currently used applications cannot prevent graft reinfection or eradicate HCV from the transplanted liver in most patients. At present there is no alternative of an interferon-free approach in the peritransplant setting and silibinin may be the ideal drug until safe and effective HCV RNA polymerase inhibitors with a high genetic barrier become available. Nevertheless, there is a need for improvements in treatment schedules before such a treatment can be recommended. Flushing the liver graft with a solution containing silibinin as it has been done to prevent oxidative damage to the liver [15] should be tested in order to determine if it helps prevent recurrence.

In the future hepatitis C patients will be spared the debilitating side effects of interferon. The dream begins. Fast forward to today with online analysts predicating interferon-free regimens reaching patients by 2014. However, some people infected with HCV will still be left behind, for these people silymarin will most likely be used and yes - even relevant.

Additional Links

More on silibinin

Expert's Picks: Silymarin for NAFLD

From National Institutes of Health • National Center for Complementary and Alternative Medicine
Updated May 2013
Hepatitis C and Dietary Supplements

Wednesday, December 5, 2012

Hepatitis C virus and natural compounds: a new antiviral approach?

Viruses. 2012 Oct 17;4(10):2197-217. doi: 10.3390/v4102197.

Hepatitis C virus and natural compounds: a new antiviral approach?

Calland N, Dubuisson J, Rouillé Y, Séron K.

Inserm U1019, CNRS UMR8204, Center for Infection & Immunity of Lille (CIIL), Institut Pasteur de Lille, Université Lille Nord de France, Lille, France.

Download Full Text here 


Hepatitis C is a major global health burden with an estimated 160 million infected individuals worldwide. This long-term disease evolves slowly, often leading to chronicity and potentially to liver failure. There is no anti-HCV vaccine, and, until recently, the only treatment available, based on pegylated interferon and ribavirin, was partially effective, and had considerable side effects. With recent advances in the understanding of the HCV life cycle, the development of promising direct acting antivirals (DAAs) has been achieved. Their use in combination with the current treatment has led to encouraging results for HCV genotype 1 patients. However, this therapy is quite expensive and will probably not be accessible for all patients worldwide. For this reason, constant efforts are being made to identify new antiviral molecules. Recent reports about natural compounds highlight their antiviral activity against HCV. Here, we aim to review the natural molecules that interfere with the HCV life cycle and discuss their potential use in HCV therapy.

1. Introduction
Plants have been used for centuries for the treatment of human diseases. Historically, numerous important modern drugs have been developed from molecules originally isolated from natural
sources [1,2]. Among them, the most popular is aspirin, based on a natural product salicin isolated from Salix alba. Morphine and codeine were extracted from opium poppy Papaver somniferum, and quinine, traditionally used as an anti-malaria treatment, from cinchona tree. In the past decades, taxol, a molecule extracted from the bark of the Pacific yew tree, Taxus brevifolia, has become one of the most used anti-cancer agent worldwide [3].

The search for new bioactive molecules in plants in key therapeutic areas such as immunosuppression, infectious diseases, oncology and metabolic diseases is still an active part of pharmaceutical research [4]. About 40 new drugs launched on the market between 2000 and 2010, originate from terrestrial plants, terrestrial microorganisms, marine organisms, and terrestrial vertebrates and invertebrates [5]. The World Health Organization (WHO) estimates that approximately 80% of the world’s population rely mainly on traditional medicine, predominantly originated from plants, for their primary health care.

Traditional medicines, including Chinese herbal formulations, can serve as the source of potential new drugs. Active plant compounds used either for prophylactic or therapeutic treatments are orally administrated to patients as teas, powders, and other herbal formulations [6,7]. Phenolic compounds are often responsible for the bioactivities of the plant crude extracts. During the last decades, people have tried to identify more precisely the active molecules of these traditional medicines. Another approach was to systematically screen natural molecules present in plant extracts and test the activity of these phytochemicals using the appropriate assays (depending on the pathology studied).

The main advantage of using natural molecules from plant extracts is a reduced cost of production, with no need of chemical synthesis. This mode of production might lead to less expensive treatments, available for populations of low-income countries.

Some natural medicines have been shown to possess antiviral activities against herpes simplex virus [8,9], influenza virus, human immunodeficiency virus [10–12], hepatitis B and C viruses [13,14]. The screening of natural products has led to the discovery of potent inhibitors of in vitro viral growth [15]. Antiviral activities of several hundred natural compounds have been identified worldwide. In addition, dozens of herbs are known to have hepatoprotective activities. During the last decades, scientists have tried to analyze more precisely the active molecules present in this traditional medicine that is frequently used for the treatment of hepatitis in China [16].

The last few years have seen a flurry of reports on the identification of natural molecules of plant origin with anti-hepatitis C activities. The aim of this review is to give an overview of these different compounds with a special focus on the most promising molecules.

 2. Hepatitis C Virus
Hepatitis C is a major healthcare problem worldwide caused by a viral infection with a high tendency to become chronic. Chronic hepatitis C is linked to the development of cirrhosis and hepatocellular carcinoma. The virus responsible for this disease was discovered more than 20 years ago [17]. Its transmission is thought to be essentially parenteral, and has been linked to blood transfusions before its discovery. Since hepatitis C virus (HCV) discovery, blood screening diagnostics have greatly reduced the blood-borne transmission of the virus. However, the transmission still occurs through other modes of contamination and the slow development of the disease results in many
persons not knowing their infected status. It is estimated that about 160 million persons (2.35% of the world population) are infected with HCV [18].

Currently, there is no vaccine against HCV and the high diversity of viral isolates will probably make it very difficult to develop a vaccine. On the other hand, we know that, in contrast to hepatitis B and human immunodeficiency viruses, HCV can be eradicated from chronically infected patients with antiviral treatments. However, the standard therapy, which is based on a combination of pegylated interferon alpha (IFN-α) and ribavirin [19], results in highly variable outcomes [20], is very expensive and has severe side effects that are difficult to endure for the patients. Nevertheless, it is currently thought that efficient anti-HCV therapies will be achieved with direct acting antivirals (DAAs) [21]. The recent addition of protease inhibitors to the standard anti-HCV therapy has already improved sustained virological response rates in patients infected with genotype 1 HCV. New drugs targeting other viral proteins are in clinical trials and will probably also help improving response to HCV therapy [22,23]. A combination of DAAs will reduce the risk of selecting viral escape mutants. DAAs combinations in the absence of interferon will probably enable to greatly reduce side effects of the therapy, which are mainly associated with the use of interferon and contribute to the failure of the treatment. Ideally, such a combination should include DAAs targeting different steps of the HCV life cycle and should be efficient against all HCV genotypes. Moreover, to have a chance of eradicating HCV, the therapy should be cheap so as to be able to cure infected patients from low-income countries and stop the transmission of the virus.

Hepatitis C virus is a small, enveloped virus belonging to the Hepacivirus genus of the Flaviviridae family [24]. Its single-stranded genomic RNA contains a single open reading frame surrounded by two untranslated regions (UTR) that are necessary for the translation and the replication of the viral genome [25–27]. The translation of the open reading frame is under the control of an internal ribosome entry site (IRES), located in the 5’UTR. It gives rise to a polyprotein precursor, which is cleaved by host- and viral-encoded proteases into ten polypeptides. The N-terminal part of the polyprotein contains structural proteins: the core protein C, a component of the viral capsid, and the two envelope glycoproteins E1 and E2. The C-terminal part of the polyprotein contains non-structural proteins required for RNA replication: NS3, which has protease and helicase activities; NS4A, a co-factor of NS3 protease; NS4B, a polytopic membrane protein; NS5A, a phosphoprotein; and NS5B, the viral RNA-dependent RNA polymerase. Between the structural proteins and the non-structural proteins involved in RNA replication, the polyprotein also contains two additional polypeptides required for viral assembly, which are dispensable for RNA replication: the viroporin p7 and the NS2 protein, which has an autoprotease activity during the maturation of the polyprotein precursor.

The structure of the viral particle is still unknown. In patients, circulating HCV particles are associated with apolipoproteins (Apo) B and E, and have highly variable buoyant densities, the lighter ones being the most infectious [28]. It is currently thought that infectious HCV particles are initially secreted as very low-density lipoprotein (VLDL)-like particles by infected hepatocytes and then potentially undergo lipolysis in the bloodstream, which progressively converts them into intermediate density lipoprotein (IDL)- and LDL-like particles. However, it is not yet clear what in this process reduces the specific infectivity of HCV viral particles.

Our knowledge of the HCV life cycle has greatly improved in recent years, following the finding of a viral strain (JFH-1) able to replicate in cell culture [29–31]. The JFH-1-based cell culture model has
been named HCVcc. For reasons that are still unknown, other viral isolates do not efficiently replicate in cell culture. Before the HCVcc model was established, specific steps of the HCV life cycle had been studied with other experimental systems recapitulating RNA replication, with the subgenomic replicon model [32,33], or viral entry, with the HCV pseudoparticles (HCVpp) model [34–36]. The replicon model is based on a modified HCV genome, in which the coding region of the structural proteins is replaced by a selection marker. In vitro synthesized subgenomic replicon RNA is introduced in cells by electroporation and the cells replicating it express the selection marker and can thus be selected. There is no release of viral particles, and this model only allows studying cellular and molecular mechanisms involved in viral RNA replication. Hepatitis C virus pseudoparticles are retroviral particles pseudotyped with HCV envelope glycoproteins E1E2. In this system, only E1E2-dependent, early entry steps (virus binding, uptake and fusion) are HCV specific, whereas later steps depend on retroviral function.

The life cycle of HCV can be divided into three major steps: entry of the virus into its target cells by receptor-mediated endocytosis, cytoplasmic and membrane-associated replication of the RNA genome, and assembly and release of the progeny virions (Figure 1). Hepatitis C virus entry is a very complex process, which involves a series of host entry factors [37]. On the viral particle, envelope glycoproteins E1E2 play a major role during entry. The viral particle probably initially binds to glycosaminoglycans (GAG) on the surface of the target cell. It has been proposed that interactions between the LDL receptor (LDL-R) and apolipoproteins of the viral particle might also participate in the initial binding to the cell surface. Following these rather non-specific initial binding events, several host entry factors are specifically involved in the entry process [38]. The tetraspanin CD81 [39], the scavenger receptor class B type I (SR-BI) [40], and the tight junction proteins claudin-1 (CLDN1) [41] and occludin (OCLN) [42,43] are mandatory for HCV entry. Epidermal growth factor receptor, ephrin receptor A2 [44], and the cholesterol transporter Niemann-Pick C1-like 1 also participate to the entry process [45]. The particle is internalized by clathrin-mediated endocytosis [46] and the viral genome is released into the cytosol of the cell following the fusion of the viral envelope and the endosomal membrane.

Once in the cytosol, the viral genome is translated. Non-structural viral proteins NS3/4A, NS4B, NS5A, and NS5B assemble into replication complexes [47] that generate new viral genomic RNA molecules through the prior synthesis of negative RNA strands, complementary to the genomic RNA. Much like for many positive stranded RNA viruses, HCV replication occurs in host cell cytoplasm in association with rearranged membranes, named ‘membranous webs’ [48]. A large number of host factors probably participate to the formation and the functioning of HCV replication complexes, which are recruited through interactions with viral proteins. A major host cell factor regulating HCV replication recently identified is the class III phosphatidylinositol 4-kinase alpha [49,50]. The protease NS3/4A and the RNA polymerase NS5B are the two major druggable viral factors involved in HCV replication, which have been used in antiviral screens.

Figure 1.
Hepatitis C virus (HCV) life cycle and targets of the most potent natural inhibitors. First, HCV binds to non-specific factors glycosaminoglycans (GAG) and LDL receptor (LDL-R) present at the cell surface (attachment step). Then, the viral particle is directed to specific entry factors (entry step), the scavenger receptor class B type I (SR-BI), the tetraspanin CD81 and the tight junction proteins claudin-1 (CLDN1) and occludin (OCLN). The virus is internalized by endocytosis and the viral genome is released into the cytosol of the cell after fusion with endosomes (fusion step). Next, the translation and the polyprotein processing take place and RNA is replicated (replication step). In the late stages of the cycle, the virion is assembled (assembly step) in the vicinity of cytoplasmic lipid droplets (LD) and is released from the cell. Finally, the released virions can infect adjacent cells by cell-free transmission or cell-to-cell transmission. The affected steps of the viral cycle are in black. The natural compounds are in red. EGCG: epigallocatechin-3-gallate; ER: endoplamic reticulum.

The assembly step of the HCV life cycle occurs in the vicinity of cytoplasmic lipid droplets (LD). The core protein, which is localized on the surface of LD [51], recruits replication complexes through interaction with NS5A [52]. It was recently shown that p7 and NS2 are involved in the assembly step by interacting with E1E2 envelope glycoproteins and non-structural proteins, mainly NS3, and that these interactions are crucial for the formation of assembly sites [53–57]. Host factors critical for HCV assembly include diacylglycerol acyltransferase-1, a triglyceride-synthesizing enzyme required for core trafficking to LD [58], and VLDL secretion machinery [59,60]. For years, the production of HCV in cell culture has been impossible and the search for DAAs was essentially limited to host and viral targets involved in the replication step of the virus. Most of the early screens were performed based on the in vitro protease activity of NS3/4A. With the recent introduction of various assays based on the HCVcc system, the search for DAAs has been highly stimulated and can now be performed in the context of a complete HCV life cycle. During the last few years, this led to a substantial increase of reports on natural compounds displaying an anti-HCV activity. The identified molecules belonging to different chemical families are summarized in Table 1, and the different affected steps of the HCV life cycle depicted in Figure 1. In this review, we have chosen to classify these molecules according to the advances in their proof of concept and to their chemical family.

3. Flavonoids
Flavonoids or bioflavonoids are a class of plant secondary metabolites. They are naturally present in numerous plants. More than 4,500 flavonoids have been characterized so far. They have been classified according to their chemical structure and are usually subdivided into different subgroups. Some of them are described as potential anti-HCV molecules.

3.1. Silymarin/Silibinin
Silymarin is extracted from the seeds of milk thistle Silybum marianum. This plant native of Southern Europe and Asia is now found throughout the world. The seed extract of milk thistle is an ancient herbal remedy used as hepatoprotectant and to treat liver disease. It contains at least seven flavonolignans (silybin A, silybin B, isosilybin A, isosilybin B, silychristin, isosilychristin, silydianin) and one flavonoid (taxifolin). Flavonolignans are natural polyphenols composed of flavonoid and lignan moieties. The major component of silymarin, silibinin (a mixture of the two diastereoisomers silybin A and silybin B) has also been reported to have anti-HCV activity.

Silymarin has multiple effects on HCV. Silymarin appears to inhibit HCV infection at least at two different levels: it inhibits HCV replication in cell culture [61] and it also displays anti-inflammatory and immunomodulatory actions that may contribute to its hepatoprotective effects [84]. By screening the seven major flavonolignans, Polyak et al. showed that specific compounds present in silymarin are responsible for the different anti-HCV activities [61,64]. The inhibition of HCV replication was attributed to the inhibitory action of silibinin on the NS5B RNA-dependent RNA polymerase [63,65]. Half inhibitory concentrations (IC50) in the order of 75–100 μM and 40–85 μM were reported in these studies for a succinate-conjugated form of silibinin, which is more soluble in aqueous solutions than natural silibinin. An inhibition of entry was also reported with HCVpp and liposome fusion assays [62,63]. Another potential anti-HCV activity of silymarin has been described by Ashfaq et al. [66]. Using a heterologous expression system, they reported an NS5B-independent inhibition of HCV genotype 3a core expression by silymarin.

Low bioavailability of silymarin components has been reported [85]. This is probably the reason why clinical studies dealing with oral administration of silymarin have been unsuccessful in curing patients from HCV [85–87]. Because silibinin is rapidly metabolized after oral administration [88], clinical studies were also attempted with the water-soluble, succinate-conjugated silibinin formulated for intravenous injection. In this case, silibinin monotherapy showed a substantial antiviral effect in patients with chronic hepatitis C not responding to standard pegylated interferon/ribavirin therapy [67,68]. Two cases of successful prevention of liver graft infection with silibinin monotherapy in patients with chronic hepatitis C have also been reported [69,70], and a case of sustained virological response after treatment with intravenous silibinin was reported for a HCV/HIV co-infected patient not responding to the standard HCV therapy [71]. Therefore, although oral administration of silymarin is not effective for the treatment of HCV patients, intravenous silibinin formulation may represent a potential therapeutic option.   

3.2. (−)-Epigallocatechin-3-gallate (EGCG)
EGCG is the most abundant flavonoid from the subclass of catechin present in green tea extract. It has been shown that a single cup of tea contains up to 150 mg of this molecule and its administration is safe in healthy individuals [89]. Very recently, three different groups have independently identified EGCG as a new inhibitor of HCV entry [72–74]. These studies showed that EGCG present during infection of Huh-7 cells with HCVcc resulted in dose-dependent inhibition of infection. Different IC50 (between 2.5 μg/mL and 9.7 μg/mL, corresponding to 5 μM and 21 μM) were obtained in the three studies, probably at least in part reflecting differences in experimental setups. The half cytotoxic dose was comprised between 150 and 175 μM in Huh-7 cells, depending on the exposure time. Two groups found no additional effect of EGCG on HCV RNA replication and on release of HCV infectious particles [72,73], despite reported inhibitory activities of EGCG on NS3 and NS5B in in vitro assays [90,91], while the third group reported an additional activity of EGCG on the RNA
replication step [74]. Hepatitis C virus pseudoparticles were used to further confirm the impact of EGCG on HCV entry. EGCG inhibited HCVpp entry in a genotype-independent manner in hepatoma-derived cells [72–74], as well as in primary human hepatocytes [72]. 

The mechanism of action of EGCG on HCV entry is still being investigated. EGCG inhibits HCV entry only when it is present during the inoculation period, or when viral particles have been pre-incubated with it [72–74]. In contrast, the pre-incubation of target cells has no impact on HCV infection. Moreover, EGCG does not change the expression levels of cellular entry factors (CD81, CLDN1, OCLN, SR-BI) [72,74]. Therefore it is very likely that EGCG acts directly on the viral particle. EGCG inhibits the binding of the virus to the cell surface [72,73] and has no effect when added post-binding. It does not appear to alter physical properties of HCV virions, such as their density profile or lipoprotein association [72]. Although antiviral activities of EGCG have also been reported against other viruses, such as herpes simplex virus and influenza virus [92–94], the antiviral effect of EGCG on HCV cannot be generalized to the other members of the Flaviviridae family, because this molecule is inactive against bovine viral diarrhea virus (BVDV, a pestivirus) or yellow fever virus (YFV, a flavivirus) [73]. Based on its action on both HCVcc and HCVpp, it can be speculated that EGCG interacts with E1E2 glycoproteins and that this interaction inhibits virion binding to target cell surface. However a direct experimental evidence for this interaction is still missing. 

Interestingly, EGCG could inhibit cell-to-cell transmission [72–74] in addition to its action on cell-free particle binding. Cell-to-cell transmission is probably a major route of spreading of HCV in the liver of infected patients. It was also reported that EGCG could be used in combination with boceprevir or cyclosporin A (two known inhibitors of HCV replication), with an increased efficiency [72]. Finally, it was shown that the anti-HCV effect of EGCG can lead to undetectable levels of virions in the supernatant of Huh-7 infected-cells after a few passages [73,74]. Recently, Fukazawa
et al. [95] by developing a new anti-HCV molecule-screening assay, have confirmed the anti-HCV activity of EGCG. Moreover, consumption of up to 800 mg of EGCG is safe and increases the concentration of EGCG detected in the plasma [89], indicating its potential use in clinical trials. 

All these data indicate that EGCG is a new anti-HCV molecule with interesting properties. It directly inactivates HCV particle, is not genotype-specific (unlike currently used protease inhibitors), and also prevents cell-to-cell transmission. These properties make it an especially interesting molecule to prevent HCV recurrence and spread in chronically infected liver transplant patients. Future clinical trials should investigate whether it could actually prevent the re-infection of patients undergoing orthotopic liver transplantation, and whether it could be used in combination with other DAAs to treat infected patients.

3.3. Ladanein 

Recently, Haid et al. have isolated a molecule with anti-HCV activity in a screen of a library of natural phenolic compounds from plant extracts [75]. From the most active plant extract, they characterized and re-synthesized the component exhibiting the highest antiviral activity. Ladanein (and its synthetic equivalent BJ486K) was identified as the active anti-HCV component. Ladanein, extracted from Marrubium peregrinum L. (Lamiaceae), is a flavone, a molecule belonging to a subgroup of the flavonoid family. Ladanein inhibited HCV entry with an IC50 of 2.5 μM in a genotype-independent manner. In contrast to EGCG, ladanein did not appear to inhibit the binding of the viral particle (although results of a direct binding assay were not reported), but rather inhibited a later, yet uncharacterized step of viral entry. Interestingly, when used in combination with cyclosporin A, a known inhibitor of HCV replication, ladanein acted synergistically on HCV infection. Ladanein also exhibited an antiviral activity in primary human hepatocytes, but with an increased IC50 (10 μM). Very importantly, this molecule was shown to be orally bioavailable in mice with a peak of plasma level of 329 nM after a single oral dose of 0.25 mg/kg. These data are encouraging for a potential use of ladanein as an anti-HCV molecule in patients. 

3.4. Naringenin

Naringenin is a dietary supplement demonstrated to possess anti-oxidant, anti-inflammatory, and anti-carcinogenic properties both in vitro and in vivo. This molecule belongs to the flavonoid family. It is the predominant flavanone present in the grapefruit and is responsible for its bitter taste. Naringenin has been previously shown to reduce cholesterol levels both in vitro [96] and in vivo [97]. Furthermore, naringenin inhibits ApoB secretion by reducing the activity and the expression of the microsomal triglyceride transfer protein (MTP) and the acyl-coenzyme A cholesterol acyltransferase 2 (ACAT) [96,98]. Due to the close link between HCV assembly/secretion and lipoprotein metabolism, Nahmias et al. have studied the impact of naringenin on the secretion of HCV particles [76]. A concomitant dose-dependent decrease of core protein, HCV-positive strand RNA, infectious particles, and ApoB was observed in the supernatant of infected Huh-7 cells after naringenin treatment [76]. The inhibitory activity of naringenin was also observed in primary hepatocytes in culture. Naringenin blocked the assembly of intracellular infectious viral particles without affecting intracellular levels of the viral RNA or protein. The maximal inhibition (74% of inhibition) of secretion of both ApoB and HCV RNA is observed at 200 μM naringenin with an IC50 of 109 μM [77]. 

The mechanism of action of naringenin was proposed to be through the inhibition of ApoB secretion. In a first study, this inhibition was correlated to a reduction of the activity of the MTP and an inhibition of the transcription of 3-hydroxy-3-methyl-glutaryl-coenzyme reductase (HMGR) and ACAT, three enzymes involved in the production of VLDL [76]. The authors later observed that naringenin also induces peroxisome proliferator-activated receptor alpha (PPARα) and that naringenin inhibition of HCV secretion can be reversed by a PPAR inhibitor [77]. Naringenin caused an increased expression of PPARα and its target gene acyl-CoA oxydase and a concomitant decrease in sterol regulatory element-binding protein (SREBP) and its target HMGR. PPARα induction is known to inhibit cholesterol synthesis through SREBP and its target gene, HMGR. These data suggest that naringenin effect is at least partially mediated by PPARα activation.

3.5. Quercetin

Quercetin is a flavonol, a plant-derived flavonoid, present in fruits, vegetables, leaves and grains. This molecule has been described as an anti-HCV molecule by two different teams. In 2009, Gonzalez et al. were looking for novel cellular proteins that interact with the viral protein NS5A (from H77 strain (genotype 1a)) of HCV [78]. By co-immunoprecipitation and co-localisation assays, they detected an interaction between NS5A and the heat shock proteins (HSP) HSP40 and HSP70. In order to confirm the implication of these proteins in the HCV life cycle, they tested the impact of quercetin, a known inhibitor of HSP synthesis. Using a cell culture-based bicistronic reporter system, quercetin was found to decrease IRES activity either in absence or in presence of NS5A. Quercetin also had a strong inhibitory effect at 50 μM on HCV production in cell culture. However, its mechanism of action is not clear, because siRNA-mediated depletion of HSP proteins had no effect on HCV particle production, and because quercetin had only a modest effect on replication in the HCVcc system, and did not inhibit HCV replication in a subgenomic replicon system. Therefore, the anti-HCV action of quercetin could be related to an impairment of viral morphogenesis or secretion, rather than to a direct action on the replication step of the HCV life cycle.

3.6. Luteolin and Apigenin

Luteolin and apigenin, two other natural flavone molecules, were identified as anti-HCV agents via a pharmacophore search [80]. A pharmacophore corresponds to a theoretical description of molecular features, which can be used for probing specific interactions between a ligand and a biological molecule. In this study, the designed pharmacophore was established from eight NS5B inhibitors selected from the literature according to different criteria. The resulting pharmacophore was tested against 15,568 compounds from an in-house database. Only 31 compounds were potentially relevant and were evaluated for their anti-HCV activities in vitro. Finally, 20 compounds showed a significant activity against HCV (half maximal effective concentration, EC50 < 50 μM). Among them, the most potent molecules were luteolin and apigenin. Luteolin and apigenin displayed an anti-HCV activity with EC50 values of 4.3 μM and 7.9 μM respectively in a cell-based antiviral assay. Finally, the authors showed that luteolin exhibited a good inhibition of NS5B polymerase enzymatic function with an IC50 of 1.12 μM according to the method used [99]. 

4. Lignans

The lignans are a group of chemical compounds found in plants. Lignans are one of the major classes of phytoestrogen, which are estrogen-like chemicals and act as antioxidants.

4.1. Honokiol

Honokiol is a lignan present in the cones, the bark and the leaves of Magnolia officinalis. This plant has been used in the traditional Japanese medicine Saiboku-to. In 2011, Lan et al. showed that honokiol inhibits HCV infection [81]. The effect of honokiol on HCV infection, entry, translation and replication was assessed in Huh-7 cells using HCVcc, HCVpp and subgenomic replicons. Honokiol strongly inhibited HCVcc infection (EC50 = 1.2 μg/mL, corresponding to 4.5 μM, and EC90 = 6.5 μg/mL) at non-toxic concentrations (median lethal dose = 35 μg/mL). Combined with IFN-α, its inhibitory effect on HCVcc was more profound than that of ribavirin combine with interferon. Honokiol-mediated inhibition of HCV infection was shown to result from multiple effects on the HCV life cycle. Honokiol inhibited the entry of HCVpp from genotypes 1a, 1b and 2a. Honokiol dose-dependently inhibited the expression levels of NS3, NS5A and NS5B. It also inhibited the replication of genotypes 1a and 2a subgenomic replicons in a dose-dependent manner. The authors conclude that the inhibition of both entry and replication by honokiol provides the impetus to fully explore the clinical utility of honokiol as an adjunct to current standards of treatment for HCV infection. 

4.2. 3-Hydroxy Caruilignan C

In 2012, Wu et al. reported the anti-HCV effect of 3-hydroxy caruilignan C (3-HCL-C) isolated from Swietenia macrophylla stems [82]. Swietenia macrophylla belongs to the Meliaceae family and its fruits are used as a folk medicine in Malaysia. 3-HCL-C reduced both protein (NS3) and RNA levels of HCV with an EC50 value of 10.5 μg/mL (corresponding to 37.5 μM) in the subgenomic replicon system. Moreover, combinations of 3-HCL-C and IFN-α, 2'-C-methylcytidine (NM-107, an NS5B polymerase inhibitor) or telaprevir (VX-950, an NS3/4A protease inhibitor) increased the suppression of HCV RNA replication. 3-HCL-C interfered with HCV replication by inducing IFN-stimulated response element transcription and IFN-dependent anti-viral gene expression. Therefore, 3-HCL-C has the potential to be developed into a potent adjuvant for anti-HCV therapy. 

5. Other Polyphenols

Other polyphenols have been reported to have potential anti-HCV activity using in vitro assays. Suzuki et al. identified 3',4',5,6,7,8-hexamethoxyflavone, also known as nobiletin, as the active compound responsible for the anti-HCV activity of Citrus unshiu peel (Aurantii nobilis pericarpium) extract, an ingredient of traditional Japanese Kampo medicine [100]. Nobiletin displayed an anti-HCV activity at 10 μg/mL in MOLT-4 cells infection assay. Hegde et al. isolated and characterized two novel oligophenolic compounds, named SCH 644343, and SCH 644342 from the Peruvian plant Stylogne cauliflora [101]. These two compounds were identified as inhibitors of HCV NS3 protease activity in vitro with IC50 of 0.3 μM and 0.8 μM respectively. SCH 644343 was also active in an NS3 binding assay (IC50 = 2.8 μM). Zuo et al. identified the 1,2,3,4,6-penta-O-galloyl-β-D-glucoside as a potent inhibitor of NS3 protease activity from the plant Saxifraga melanocentra Franch [102]. The IC50 was 0.68 μM and the molecule was not toxic up to 6 mg/mL (corresponding to 6.4 mM) on COS cells. Duan et al. identified three polyphenol components from the ethyl acetate fraction of the traditional Chinese medicine Galla Chinese [103]. These polyphenols molecules, 1,2,6-tri-O-galloyl-β-D-glucose, 1,2,3,6-tetra-O-galloyl-β-D-glucose and 1,2,3,4,6-penta-O-galloyl-β-D-glucose, were shown to inhibit NS3 protease in vitro with IC50 values of 1.89, 0.75 and 1.60 μM, respectively.

All these compounds were identified before the HCVcc model was established and to our knowledge, they have not been evaluated since then. Therefore, they will need to be tested in cell-based assays, before considering them as new potential anti-HCV agents. This is illustrated by the study of Li et al. who identified four polyphenolic compounds inhibiting NS3 protease in vitro, from the Chinese mangrove plant Excoecaria agallocha L. (Euphorbiaceae) [83]. Among them, only two, excoecariphenol D and corilagin had a significant inhibitory action in the replicon assay with an IC50 of 12.6 and 13.5 μM respectively.

6. Crude Plant Extracts from Traditional Medicines
Traditional medicines represent a broad source of natural polyphenols molecules. In 2003, Liu et al. assessed beneficial and harmful effects of medicinal herbs against HCV infection. Thirteen randomized trials have evaluated fourteen medicinal herbs. Only four trials had an adequate evaluation method [104]. Even if traditional medicine represents an attractive source of new natural antivirals, studies with herbs need to be standardized in order to clearly evaluate the effects due to the plant extracts on HCV infection, and should provide all methodological details. Compounds isolated from these herbs could be used for designing and developing drugs for treatment of hepatitis C.

A study, which evaluated the benefice of a Far-Eastern traditional herbal formulation for patients with chronic hepatitis C [105], found some improvement of circulating aminotransferases, with no effect on HCV RNA levels. Several other studies evaluated the effect of plant extracts from traditional medicine using various assays, which found extracts with anti-NS3 protease activity [106,107], anti-NS5B activity [16,107] and with anti-replicative effect in the replicon model [107–109]. None of these studies tried to evaluate other steps of the HCV life cycle. In two studies, additive or synergistic actions in combination with interferon [107,108], telaprevir or 2'-C-methylcytidine [108,109] were reported. Future studies on these plant extracts may lead to the identification of new anti-HCV molecules.

7. Concluding Remarks
In this review, we discussed the diverse and broad actions of natural molecules issued from plants as potential anti-HCV antivirals. It is important to note that some other natural compounds, even if they do not target the virus directly, might also be used to improve HCV therapy. Glycyrrhizin, for instance, a component of licorice roots extract, has been shown to prevent the development of hepatocellular carcinoma in patients with chronic hepatitis [110].

Although a number of natural compounds with anti-HCV activities were identified in recent years, many aspects concerning their mechanisms of action remain unknown. Very often, the replication was the only step of the viral life cycle that was investigated and, for older reports, the conclusions are only based on in vitro models, mostly NS3 protease assays. Yet, ladanein and EGCG proved to be potent entry inhibitors, and reports on quercetin anti-HCV activity suggest that it may in fact be more active on the assembly, than on the replication step. The HCVcc model now allows to identify anti-HCV molecules in the context of a complete life cycle and then to pinpoint the inhibited step, with additional and more specific assays. Importantly, we should keep in mind that the in vitro inhibition of the enzymatic activity of a viral protein by a natural compound does not conclusively demonstrate that this viral protein is the bona fide target of the compound. Additional binding and crystallization studies are required to prove the point. For example, in the case of silymarin/silibinin, high concentrations are required to inhibit NS5B in vitro. It is possible instead that a cellular protein target could mechanistically be involved in the antiviral action of these compounds. More investigations are clearly needed to validate numbers of these molecules in vivo. As illustrated by silymarin, the bioavailability is an important point to consider, even for molecules extracted from ancient herbal medicines. In conclusion, even if we do not expect that natural molecules may replace the current anti-HCV therapy, treatments could be more likely supplemented, and perhaps lightened by adapted diet, limiting their cost.


We thank Sandrine Belouzard (Center for Infection & Immunity of Lille) for critical reading of the manuscript and Sophana Ung for preparing the illustration. HCV research conducted by the authors is supported by the French "Agence Nationale de Recherche sur le Sida et les hépatites virales" (ANRS).

Conflict of Interest

The authors declare no conflict of interest.

References and Notes

Friday, September 21, 2012

Silymarin Is Ineffective for Chronic Hepatitis C Virus Infection

From Journal Watch > Journal Watch Gastroenterology

Silymarin Is Ineffective for Chronic Hepatitis C Virus Infection

Atif Zaman, MD, MPH
Posted: 09/21/2012; Journal Watch © 2012 Massachusetts Medical Society

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

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.

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.

Friday, August 26, 2011

Differences in the Disposition of Silymarin Between Patients with Non-Alcoholic Fatty Liver Disease and Chronic Hepatitis C

DMD #40212 1 Title Page Title:

Differences in the Disposition of Silymarin Between Patients with Non-Alcoholic Fatty Liver Disease and Chronic Hepatitis C

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Authors: Sarah J. Schrieber1, Roy 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. Fried for The SyNCH Trial Group Affiliations: Division of Pharmacotherapy & Experimental Therapeutics (S.J.S., R.L.H.), and Division of Molecular Pharmaceutics (Z.W., P.C.S.), UNC 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 (K.R.R.); Department of Biostatistics (A.S.W.), and Department of Epidemiology (S.H.B.), University of Pittsburgh; Liver Center, Beth Israel Deaconess Medical Center (N.H.A.); Division of Gastroenterology and Hepatology, Thomas Jefferson University (V.J.N.); National Center for Complementary and Alternative Medicine (C.M.M.), and Liver Diseases Research Branch, Division of Digestive Diseases and Nutrition, National Institute of Diabetes and Digestive and Kidney Diseases (E.D.), National Institutes of Health, Bethesda, Maryland. DMD Fast Forward.

Published on August 24, 2011 as doi:10.1124/dmd.111.040212
Copyright 2011 by the American Society for Pharmacology and Experimental Therapeutics.


Silymarin, derived from the milk thistle plant Silybum marianum and widely used for self-treatment of liver diseases, is comprised 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 flavonolignans were examined in patients with nonalcoholic fatty liver disease (NAFLD) or hepatitis C virus (HCV) to determine if silymarin’s disposition, and therefore its potential efficacy, varies between liver disease populations.

Cohorts of eight subjects with non-cirrhotic liver disease were randomized 3:1 to oral silymarin or placebo (280 or 560 mg) every 8 hours for 7 days. 48-Hour blood sampling was conducted following the first and final doses.

In general, plasma concentrations of silybin A and silybin B were higher while concentrations of conjugates were lower in NAFLD compared to HCV. After adjusting AUC0-8h for weight and dose, only silybin B and silybin B conjugates differed significantly between disease types. For NAFLD, the adjusted mean AUC0-8h was higher for silybin B (p<0.05) but lower for silybin B conjugates (p<0.05) compared to HCV.

At the 280 mg dose, steady-state plasma concentrations of silybin B conjugates for NAFLD subjects were characterized by 46% lower AUC0-8h (p<0.05) and 42% lower Cmax (p<0.05) compared to HCV subjects.

Evidence of enterohepatic cycling of flavonolignans was only observed in NAFLD subjects.

In summary, silymarin’s efficacy may be more readily observed in NAFLD patients due to higher flavonolignan plasma concentrations and more extensive enterohepatic cycling compared to patients with HCV.

DMD #40212 4


Silymarin is an herbal product that has been used for centuries for diseases of the liver (Flora et al., 1998), and approximately one-third of patients seen in US liver clinics report the use of some CAM to self-treat their liver disease (Strader et al, 2002). Derived from the milk thistle plant, Silybum marianum, silymarin is a complex mixture of six major flavonolignans (silybins A and B, isosilybins A and B, silychristin, and silydianin), as well as other minor polyphenolic compounds (Kim et al., 2003).

Silymarin has been shown to have antioxidant, anti-inflammatory/immunomodulatory, and anti-fibrotic properties in various in vitro and animal models (Abenavoli et al., 2010). However, it is the antioxidant activity of silymarin that 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-Otero, 2005).

Silymarin may be the most potent antioxidant in nature by virtue of its free radical scavenger reactivity and favorable membrane-lipid/water partitioning (György 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 since patients have significantly increased levels of serum lipid peroxidation products (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 associated with the grade and stage of liver disease in patients with HCV (Jain et al., 2002) which suggests that antioxidant therapy may be effective in slowing disease DMD #40212 5 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 has 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 to healthy controls (Schrieber et al., 2008).

While 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 infection and NAFLD were not fully elucidated due to the low plasma concentrations. Silymarin flavonolignans are metabolized via phase 2 conjugation pathways (Jancova et al., 2011; Sridar et al., 2004) and the majority of glucuronide and sulfate conjugates undergo hepatobiliary excretion via multi-drug resistance protein-2 (Mrp2) (Miranda et al., 2008). In obesity and NAFLD animal models, Mrp2 has been shown to have altered hepatic expression and function (Cheng et al., 2008; Geier et al., 2005). In addition, functional genetic polymorphisms in MRP2 have been associated with susceptibility to NAFLD and disease severity (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 pharmacokinetics 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. Unexpectedly, dose proportionality in the pharmacokinetics of parent silymarin flavonolignans was not DMD #40212 6 observed in HCV patients with well-compensated liver disease at silymarin doses above 560 mg when administered orally every eight hours (Hawke et al., 2010). Since the steady-state pharmacokinetics of silymarin in patients with NALFD has not been previously described, and because silymarin’s pharmacokinetics 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 if 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, since silymarin’s pharmacokinetics appears to be nonlinear in patients with HCV, the pharmacokinetics of silymarin was evaluated at silymarin doses of 280 mg 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 hours are utilized in an attempt to overcome silymarin’s high firstpass metabolism and achieve therapeutic, steady-state plasma concentrations. DMD #40212 7


Subjects Forty male and female subjects ≥ 18 years of age with chronic noncirrhotic NAFLD and HCV were enrolled into the study within 28 days of screening (N=8 per cohort). Subjects were required to have elevated alanine aminotransferase levels ≥ 65 IU/L within 1 year prior to screening, and a creatinine clearance (calculated according to Cockcroft-Gault equation) > 60 ml/min at screening as well as a negative urine pregnancy screen for females 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 investigator’s, evidence of decompensated liver disease defined by: serum albumin < 3.2 g/dl, total bilirubin > 1.5 mg/dl, or PT/INR > 1.3 times normal, history or 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 immunodeficiency 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, alpha-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 acetaminophen > 2 grams/day; use of oral contraceptive, warfarin, metronidazole, or concurrent use of the following cytochrome CYP3A4 inducers: aminoglutethimide, aprepitant, carbamazepine, dexamethasone, efavirenz, ethosuximide, garlic supplements, glucocorticoids, glutethimide, griseofulvin, modafinil, nafcillin, nevirapine, oxcarbazepine, phenobarbital, phenytoin, primidone, rifabutin, rifampin, rifapentine, and St. John's Wort; historical liver DMD #40212 8 biopsy demonstrating 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 grams/day for ≥ 6 months prior to screening; or other evidence 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 48 hours prior to study randomization through study completion.

Trial Design Specific details on the design of this Phase 1 study have been previously described (Hawke et al., 2010). Briefly, 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 hours for 7 days. 48- hour pharmacokinetic samples were collected following an initial single dose administration before the 7 day treatment and a final dose following 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 based on historical experience for Phase 1 trials and not on statistical considerations.

Cohorts were enrolled sequentially at doses of 280 mg or 560 mg Legalon®. Legalon® (Madaus, Germany now RottapharmMadaus, Italy) brand of silymarin was selected as the clinical trial material for the Silymarin Product Development Program for use in NIH-sponsored clinical trials for liver diseases from competing bids in response to a Notice of Opportunity by the National Center for Complementary and Alternative Medicine 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 DMD #40212 9 administered with 240 ml of water 30 minutes 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 hours for collection of blood. Fourteen serial blood samples were collected at 0 (pre-dose), 0.5, 1, 1.5, 2, 4, 6, 8, 12, 15, 18, 24, 32, and 48 hours post-dose. Twenty-one doses were dispensed to subjects upon discharge after collection of the 48 hour post-dose sample on day 3. The first of these 21 doses was self-administered under direct supervision in the clinical research center. 8-Hour post-dose trough plasma samples were collected during safety visits on days 6 and 8.

Patient adherence was assessed by patient drug diary, pill counts, and by maintaining records of drugs dispensed and returned. Subjects were enrolled from December 2006 to July 2008 at 4 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 approved 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 was assessed before dosing on study days 1 (baseline), 6, 8, and 10, which consisted of clinical laboratory tests and reports of clinical adverse events using a symptom assessment questionnaire.

Additionally, on days 1 and 10, the questionnaire was also completed at approximately 24 and 48 hours postdose. Common Terminology Criteria for Adverse Events (CTCAE v3.0) was utilized to grade the severity of adverse events.

Physical examinations and electrocardiograms were completed at baseline and at end-of-study. Decisions to dose escalate were made after a safety DMD #40212 10 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 monitor. 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 No. 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 LCMS 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 silymarin equivalent to 140 mg of silymarin as determined by the manufacturer’s DNPH method) of the 180 mg milk thistle extract contained in each capsule. Based on interim stability testing results performed by the manufacturer, 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 Flavononlignans Whole blood samples were collected in two 3 ml EDTA-lined tubes (K2-EDTA tubes; BD, Franklin Lakes, NJ, USA) and centrifuged at 1200 x g for 10 minutes at 4oC. Plasma was aspirated and transferred to polypropylene tubes. Plasma samples were temporarily stored at -70°C by each clinical DMD #40212 11 site for < 30 days prior to 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 concentrations in plasma, a 125 μl aliquot of each patient sample was buffered using sodium acetate (pH 5.0, 0.125 M) and incubated for 6 hours at 37ºC without hydrolytic enzymes. A second 125 μl aliquot was also buffered using sodium acetate (pH 5.0, 0.125 M) and incubated with a mixture of sulfatase (80 U/ml, S9626 Type H-1) and β-glucuronidase (8000 U/ml, G0501 Type B-10) (Sigma-Aldrich, St. Louis, MO) for the determination of total (i.e. parent + conjugates) flavonolignan concentrations which were expressed as “Parent Flavonolignan Equivalents”.

After incubation, 50 ng of naringenin (internal standard) in 25 μl of 50% MeOH was added to the samples which were then deproteinized and processed using a highthroughput protein filtration procedure as previously described (Hawke et al., 2010). Following filtration, 75 μl of the plasma sample supernatants were transferred to glass HPLC vials and concentrations of silymarin flavonolignans were quantified by LC-ESI-MS as previously described using a Luna C18 analytical column (50 × 2.0 mm i.d., 3 μm; Phenomenex, Torrance, CA); an isocratic mobile phase consisting of 43% methanol, 56% water, and 1% glacial acetic acid (pH 2.8); a flow rate, 0.3 ml/min; a 25 μl injection volume; and a 13 minute 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 – 2,500 ng/ml and 100 – 20,000 ng/ml, respectively. The accuracy for each flavonolignan was within 95.4 – 107.4% and intra- and inter-day precisions were 1.7 – 11% and 4.5 – 14%, respectively. Data Analysis DMD #40212 12 Pharmacokinetic parameters including: area under plasma concentration-time curve (AUC); maximum plasma concentration (Cmax); time to Cmax (Tmax); and terminal half-life (T½) were calculated using noncompartmental methods, WinNonlin-Pro (v5.2; Pharsight Corp, Mountain View, CA).

A constant dosing interval (tau) of 8 hours was assumed for the calculation of steady-state AUC0-8h using 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 prior to performing a pharmacokinetic analysis. Pharmacokinetic 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 exposures (i.e., AUC0-8h) between disease cohorts were compared, following 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 confounder in the assessment of differences in flavonolignan exposures between cohorts, a linear regression model with log AUC0-8h as outcome was used.

The model included dose, disease, and weight as independent variables in order to adjust for variable weights across dose groups (280 mg vs. 560 mg) or disease type (HCV vs. NAFLD) while comparing AUC0-8h. Least-square 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 Inc., Cary, NC). DMD #40212 13


Subjects Baseline demographics are presented in Table I.

Study subjects in the HCV cohorts were predominantly males with ages ranging from 43 – 59 years, while males and females were more equally represented in the NAFLD cohorts with ages ranging from 28 – 58 years. Subjects were characterized by well-compensated, noncirrhotic 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

When compared to 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 shown).

There were no abnormal deviations from baseline laboratory values reported with silymarin administration for any cohort. For the HCV cohorts, 3 subjects who received a single 280 mg dose of silymarin reported a total of 4 adverse events.

Three of the adverse events were classified as neurological (e.g., headache) while the other was classified as gastrointestinal. Only one adverse event (dizziness) was considered possibly related Legalon® administration and resolved in less than 1 day. For NAFLD cohorts, two out of 12 subjects (16.7%) receiving silymarin reported at least one adverse event compared to 1 out of 4 subjects (25%) receiving placebo.

Adverse events reported with silymarin 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, self-limiting, and were considered unrelated to treatment.

DMD #40212 14

Single dose Pharmacokinetics of Silybin A and Silybin B A comparison of the pharmacokinetics of silybin A and silybin B between HCV and NAFLD cohorts following single oral doses of either 280 or 560 mg silymarin are presented in Table II. 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 Cmax and a 2- to 4.5-fold greater AUC0-48h compared to silybin B. At the 280 mg dose, no differences were observed in the pharmacokinetics 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 hours).

However, at the 560 mg dose, pharmacokinetic differences were observed between HCV and NAFLD subjects. Compared to HCV subjects, AUC0-48h for silybin A and silybin B were 1.5-fold (p > 0.05) and 2.1-fold (p < 0.05) greater, respectively, for NAFLD subjects. A similar trend was observed in the Cmax for silybin A and silybin B, although the 1.4- to 1.6-fold differences between HCV and NAFLD subjects did not achieve statistical significance.

Elimination half-lives were similar between the disease groups (range 1.1 – 1.5 hours), while Tmax was delayed by 1 hour 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 following chronic oral administration of either 280 or 560 mg silymarin every 8 hours for 7 days are presented in Table III.

Similar to the data obtained following 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 Cmax and a 2.6- to 4.9-fold greater AUC0-8h compared to silybin B. In addition, DMD #40212 15 there was no evidence of accumulation for either flavonolignan following repeated dosing with elimination half-lives ranging between 0.7 to 1.3 hours.

Also similar to the single dose data, pharmacokinetic differences between HCV and NAFLD cohorts were only observed at the 560 mg dose. The AUC0-8h for silybin A and silybin B were 1.6-fold and 2.5-fold greater, respectively, in NAFLD subjects compared to HCV subjects at the 560 mg while differences in the Cmax between cohorts ranged between 1.5- to 2.2-fold. After adjusting for weight and disease type, silybin A and silybin B AUC0-8h 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 AUC0-8h. When adjusted for weight and dose, only silybin B differed significantly across disease types such that adjusted mean AUC0-8h for silybin B was higher for NAFLD compared to 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 to HCV.

Single dose and Steady-State Pharmacokinetics of Silybin A and Silybin B Conjugates To further explore the effect of NAFLD on silymarin’s metabolism, differences in the plasma concentrations of silybin A and silybin B conjugates between HCV and NAFLD subjects were examined. As defined in Methods, plasma concentrations of conjugates were estimated from the subtraction of parent flavonolignan concentrations from total (parent + conjugate) 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 IV and V, respectively. Whereas plasma concentrations were observed to be greater for silybin A than for silybin B, the converse was true for DMD #40212 16 their conjugates.

The Cmax and AUC0-8h for silybin B conjugates were 3- to 4-fold greater than 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 following single or chronic dosing. However, these differences only achieved significance between HCV and NAFLD cohorts dosed at 280 mg every 8 hours whereas conjugates of silybin B in plasma of NAFLD subjects were characterized by 46% lower AUC0-8h (p < 0.05) and 42% lower Cmax (p < 0.05) compared to HCV subjects.

Figure 1 depicts the mean steady-state plasma concentration versus time profiles for silybin B (inset) and silybin B conjugates for HCV and NAFLD subjects at the 280 mg dose. Plasma concentrations of silybin B conjugates were lower in NAFLD subjects compared to HCV subjects over the entire 8 hour dosing interval (Figure 1). In contrast, plasma concentrations of silybin B were higher in NAFLD subjects until peak concentrations were achieved and then declined similarly (Figure 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 absorption. After adjusting for weight and disease type, the AUC0-8h for silybin A conjugates and for silybin B conjugates 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 AUC0-8h. When adjusted for weight and dose, only silybin B conjugates differed significantly across disease types such that adjusted mean AUC0-8h for silybin B conjugates was significantly lower for NAFLD subjects compared to HCV (p = 0.03).

To further quantify differences in the extent of flavonolignan conjugation between HCV and NAFLD subjects, steady-state metabolic ratios were calculated as the ratio of AUC0-8h for silybin B DMD #40212 17 divided by AUC0-8h for silybin B conjugates at the 560 mg dose. Metabolic ratios differed 4-fold (p < 0.05) between HCV and NAFLD with means (± SD) 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 compared to HCV 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 lower in NAFLD subjects compared to HCV subjects irrespective of the dose and frequency of oral silymarin administration.

Flavonolignan Accumulation

The ratio of parent silybin A steady-state AUC0-8h divided by single-dose AUC0-8h was calculated as an indication of the extent of accumulation following 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. While no evidence for parent silybin A and silybin B accumulation was observed, the overall amount of parent flavonolignans in plasma was significantly higher in NAFLD subjects compared to HCV subjects at the 560 mg dose due to 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 Figure 2, plasma concentrations of isosilybin A, isosilybin B, silychristin, and silydianin were significantly greater in NAFLD subjects compared to HCV subjects. Interestingly, silychristin and silydianin were not detected in the plasma of HCV subjects.

To gain insight into the mechanism(s) behind these observed DMD #40212 18 differences, we evaluated the plasma concentration versus time profile for each flavonolignan over the 48 hour sampling period following administration of the last 560 mg dose (Figure 3). Significant enterohepatic cycling of the six flavonolignans were observed in NAFLD subjects as indicated by a prominent second peak at 4 hours following the absorption peak at 1 hour.

Most flavonolignans also showed evidence of a third peak at 8 hours post dose. In contrast, there was less evidence of enterohepatic cycling in HCV subjects where no secondary peaks were observed for either silybin A or silybin B following the early absorption peak. Silychristin represented a major flavonolignan in the plasma of NAFLD subjects at the dose 560 mg dose. Silychristin’s steady-state pharmacokinetics (geometric mean and 95% confident intervals) were characterized by a Cmax of 67 ng/ml (-2.5, 174), an AUC0-8h of 325 ng
●hr/ml (-145, 1100), and a T½ of 3.1 hr (1.2, 6.3). The steady-state pharmacokinetics of the conjugates of silychristin in NAFLD subjects were characterized by a Cmax of 663 ng/ml (367, 1394), an AUC0-8h of 3800 ng
●hr/ml (1628, 8462), and a T½ of 4.5 hr (2.2, 8.6). DMD #40212 19


The expression of drug disposition genes and their protein products have been shown to be altered in liver disease (Congiu et al., 2009; Fisher et al., 2009; Congiu et al., 2002), and effects of liver disease on the disposition 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 flavonolignan conjugates, were found to be approximately 5-fold higher in patients with chronic HCV infection or NAFLD when compared to healthy controls. Pharmacokinetic differences were also observed between healthy subjects and patients with NAFLD or patients with HCV cirrhosis. In contrast, differences were not observed between 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 flavonolignans 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 if the disposition of silymarin is different between 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 concentrations of flavonolignan conjugates compared to HCV patients administered the same DMD #40212 20 dose. While silymarin flavonolignans appear to share common pathways of metabolism and transport, differences in their affinity for these processes have been noted (Miranda et al., 2008; Sridar et al., 2004) which likely account for the different relationships between AUC exposure and dose for silybin A and silybin B observed in our study. In vitro and in vivo studies suggest silymarin flavonolignans are primarily metabolized through glucuronidation and sulfation pathways with various UDP-glucuronosyltransferases (UGTs) sharing overlapping specificity (Sridar et al., 2004; Jancova et al., 2011). In addition, the extent in which various flavonolignans undergo glucuronidation or sulfation appears to vary (Wen et al., 2008).

There are several possibilities that could explain why the ratio of parent flavonolignan (e.g., silybin B) to flavonolignan conjugates was higher in patients with NAFLD compared to 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 expression which 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. Since silybin B conjugates represents 99% of the total (parent + conjugates) silybin B species in HCV patient plasma, metabolism stoichiometry 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 DMD #40212 21 lower plasma concentration of flavonolignan conjugates in NAFLD compared to HCV does not appear to be related to reduced intestinal absorption since parent flavonolignans would also be expected to be lower in plasma.

Alterations in the expression and function of hepatobilary transporters 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 (Hawke et al., 2010; Schrieber et al., 2008). Enterohepatic cycling is regulated by hepatobiliary 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 et al., 2004). In many instances, these compounds undergo metabolism to more polar conjugates followed by transport and biliary excretion by ATP-binding cassette transporters such as Pglycoprotein , multidrug resistance associated protein 2 (MRP2), and breast cancer resistance protein, located at the canalicular membrane of the hepatocyte (Leslie et al., 2005; Schinkel and Jonker, 2003).

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 (Van de Steeg et al., 2010; Wagner et al., 2005).

The most intriguing observation in the current study was the suggestion of significant enterohepatic recycling of silymarin flavonolignans in NAFLD subjects in contrast to HCV subjects DMD #40212 22 where there was no evidence of enterohepatic cycling (see Figure 3). Silymarin flavonolignans demonstrate high affinity for MRP4 (Wu et al., 2005) while silymarin conjugates, but not parent flavonolignans, appear to be better substrates for MRP2 (Miranda et al., 2008).
Glucuronides that are substrates for MRP2, such as conjugated 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 hepatobilary transporters as a result of liver disease.

In obesity and NAFLD animal models, Mrp2 has been shown to have altered hepatic expression and function (Cheng et al., 2008; Geier et al., 2005). 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 silymarin 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 silymarin conjugates with higher concentrations of recycling silymarin flavonolignans in NAFLD patients compared to HCV. Alternatively, 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 while the expression of MRP3 and DMD #40212 23 OATP2B1 were 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 conjugates to sinusoidal efflux in HCV patients due to reduced biliary efflux by MRP2 or reduced uptake by OATP1B1, which would also result in higher plasma concentrations of silymarin conjugates and decreased enterohepatic cycling of silymarin flavonolignans compared to patients with NAFLD.

While the results of our study cannot delineate between these various potential mechanisms, it is possible that silymarin’s disposition is altered by different, diseasespecific 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 to concentrations found in healthy volunteers (Schrieber et al., 2008).

In summary, differences in the disposition of silymarin between NAFLD and HCV patients may reflect different disease-specific alterations 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 demonstrated, perhaps because of their more restrictive use indications.

Importantly, the antioxidant activity and potential antiinflammatory and antifibrotic effects of silymarin on disease progression will be dependent on its hepatic disposition. Oxidative stress has been associated with all stages of chronic HCV liver disease (Jain et al., 2002) and recent data from the HALT-C trial suggest that silymarin use among patients with advanced HCV liver disease may be associated with reduced progression to cirrhosis (Freedman et al., 2011). Compared to HCV infection, silymarin may demonstrate greater benefits in patients with NAFLD since oxidative stress is thought to play a central role in the etiology of NASH (Day et al., 1998) and there are no approved therapies.

In addition, the results of this study suggest silymarin’s effects on liver disease progression may also be greater in DMD #40212 24 NAFLD patients due to higher flavonolignan plasma concentrations and more extensive enterohepatic cycling compared to patients with HCV.

These observations were critical in the design of a Phase 2 silymarin trial in NASH which is currently ongoing (Lang, 2006). DMD #40212 25

Acknowledgements The authors are indebted to Dr. Josh Berman and Dr. Qi-Ying Liu for their important early efforts in study design and to Dr. Ulrich Mengs for championing this work. In addition, the authors wish to thank the patients who volunteered for this trial, and Dr. Tedi Soule, Joseph Colagreco, Mary Hammond, and Deborah Moretti, who served as the study coordinators, and Sharon Lawlor, who was the DCC coordinator, for their invaluable assistance in the conduct of this trial. The authors would also like to thank Dr. Craig W. Hendrix, M.D. who graciously agreed to serve as the independent safety monitor. DMD #40212 26 Authorship Contributions Participated in research design: Hawke, Reddy, Belle, Afdhal, Navarro, Meyers, Doo, Fried Conducted experiments: Wen, Schrieber Contributed new reagents or analytic tools: Hawke, Smith Performed data analysis: Schrieber, Wahed Wrote or contributed to the writing of the manuscript: Schrieber, Hawke DMD #40212 27