Thirty-two male and female patients ≥18 years of age with chronic noncirrhotic HCV who previously failed interferon-based therapy were recruited into the study. Patients were required to have serologic evidence of HCV infection documented by quantifiable HCV ribonucleic acid (RNA) in serum by polymerase chain reaction (PCR) assay and elevated alanine aminotransferase (ALT) levels ≥65 IU/L within 1 year prior to screening and ALT ≥45 IU/L during screening. Patients were required to have a platelet count ≥130 cells/mm3, a creatinine clearance (calculated according to Cockcroft-Gault) >60 mL/min, serum albumin ≥3.2 g/dL, total bilirubin ≤1.5 mg/dL, prothrombin time (PT)/international normalized ratio (INR) ≤1.3 times normal at screening, and negative urine pregnancy screen for women of childbearing potential who were also required to use barrier methods of contraception during the study. Use of standard doses of over-the-counter multivitamins or cough/cold preparations was allowed.
Patients were excluded if they had evidence of portal hypertension or other chronic liver disease, had serologic evidence of infection with human immunodeficiency virus, or received antiviral therapy for HCV within 6 months of screening. Patients were excluded for the following reasons: allergy to milk thistle or its preparations; use of silymarin or other milk thistle preparations or use of antioxidants within 30 days of screening; chronic use of acetaminophen >2 g/d; use of oral contraceptive, warfarin, or metronidazole; concurrent use of the following cytochrome P450 3A4 inducers: aminoglutethimide, aprepitant, carbamazepine, dexamethasone, efavirenz, ethosuximide, garlicsupplements, glucocorticoids, glutethimide, griseofulvin, modafinil, nafcillin, nevirapine, oxcarbazepine, phenobarbital, phenytoin, primidone, rifabutin, rifampin, rifapentine, and St. John’s wort; historical liver biopsy demonstrating the presence of cirrhosis (Ishak stage 5 or 6), ≥15% steatosis, or evidence of steatohepatitis; positive urine drug screen for drugs of abuse; alcohol consumption of >12 g/d for ≥6 months prior to screening; or other evidence of alcohol or drug abuse within 6 months of screening. Women who were pregnant or breastfeeding were also excluded. All patients agreed not to consume alcohol 48 hours prior to study randomization through study completion.
Four dose cohorts of 8 patients 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. Patients and all study personnel, except for pharmacists, were blinded to treatment assignments until trial completion. No significant adverse events have been observed in prior studies with silymarin at oral doses up to 1200 mg per day. Therefore, to verify the safety of oral doses of silymarin ~2 times higher than previously studied, we chose a simple, 3:1 randomized active to placebo, sequential dose cohort escalation design with a sample size that would provide information on safety, tolerability, and pharmacokinetics, which is customary for phase I investigations. Cohorts were enrolled sequentially at doses of 140 mg, 280 mg, 560 mg, or 700 mg Legalon. Legalon (Madaus, Germany [now Rottapharm|Madaus, Italy]) brand of silymarin was selected as the clinical trial material for the Silymarin Product Development Program for use in National Institutes of Health (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 NIH. The 140-mg dose is the customary dose of Legalon and was included in the trial design as a reference to previous investigations.
Patients were recruited from December 2006 to July 2008, and no protocol deviations were granted. Twenty-one doses were dispensed to patients upon randomization. The first dose was self-administered under direct supervision in a clinical research. The last study dose was administered on day 8 with 240 mL of water to patients fasted overnight. Patients remained in the unit for 48 hours for collection of blood and urine samples. Three daily meals were served in the unit with breakfast served ≥2 hours postdose on day 8. Fourteen serial blood samples were collected at 0 (predose), 0.5, 1, 1.5, 2, 4, 6, 8, 12, 15, 18, 24, 32, and 48 hours postdose. On day 8, 3 timed urine collections were obtained at 0 to 12 hours, 12 to 24 hours, and 24 to 48 hours postdose determined in urine collections from patients in the 140-, 280-, and 560-mg dose groups. Eight-hour post-dose trough plasma samples were collected during safety visits on days 1, 4, and 6. The phase I trial design also included a single-dose pharmacokinetic study that was performed for each participant prior to the start of the 7-day dosing period. The single-dose pharmacokinetic data obtained from this phase I trial will be described in a separate report.
Serum HCV RNA level was determined on day 1 prior to the first dose and following the final dose on day 8 by quantitative PCR (Roche AMPLICOR assay, Nutley, New Jersey). Patient adherence was assessed by patient drug diary, pill counts, and by maintaining records of drugs dispensed and returned.
The study was conducted at 4 clinical centers: 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 patients provided written informed consent. The study was conducted in accordance with the Declaration of Helsinki and guidelines on good clinical practice.
Safety was assessed during safety visits on study days 1, 4, 6, and 8, which consisted of clinical laboratory tests and reports of clinical adverse events using a standardized symptom assessment questionnaire. In addition, on day 8, the questionnaire was completed at predose (0 hours) and at approximately 24 and 48 hours postdose. Common Terminology Criteria for Adverse Events (CTCAE v3.0) was used 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 evaluation by a safety committee masked to treatment, which consisted of the principal investigators from the 4 clinical centers and an external safety monitor.
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 5 silymarin and/or placebo capsules packaged in single-use medicine dose cups. The flavonolignan content of each capsule was determined according to previously published liquid chromatography/mass spectrometry (LC/MS) 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.17
These 6 flavonolignans account for 70.8% (127.5 mg silymarin equivalent to 140 mg of silymarin as determined by the manufacturer’s 2,4-dinitrophenylhydrazine [DNPH] method) of the 180-mg milk thistle extract contained in each capsule. Based on interim stability testing results performed by the manufacturer, Legalon 140 capsules are stable under normal conditions (25°C, 60% relative humidity) for at least 9 months. For the purpose of this report, we refer to the 1-, 2-, 4-, or 5-capsule doses of Legalon as equal to 140, 280, 560, and 700 mg of silymarin, respectively.
Analysis of Silymarin Flavonolignans
Whole-blood samples were collected in two 3-mL ethylenediaminetetraacetic acid–lined tubes (K2-EDTA tubes; BD, Franklin Lakes, New Jersey) and centrifuged at 2400 rpm for 10 minutes at 4°C. Plasma was aspirated and transferred to polypropylene tubes. Urine weight and volume for each of the 3 timed intervals were recorded, and four 10-mL aliquots for each interval were stored in polypropylene tubes. Plasma and urine samples were temporarily stored at −70°C by each clinical 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 (ie, nonconjugated) flavonolignan concentrations in plasma, a 125-μL aliquot of each patient sample was incubated for 6 hours at 37°C without hydrolytic enzymes. A second 125-μL aliquot was incubated with a mixture of sulfatase (80 U/mL) and β-glucuronidase (8000 U/mL) (Sigma-Aldrich, St. Louis, Missouri) for the determination of total (ie, 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 high-throughput protein filtration procedure. Briefly, 140-μL aliquots of the samples (equal to 100 μL plasma) were transferred to preequilibrated 96-well protein filtration plates (0.2 μm cutoff, Orochem Technologies, Lombard, Illinois) containing 700 μL ice-cold acetonitrile/1% HAc in each well. Filtration plates were placed on the top of a CaptiVac (Varian, Palo Alto, California) vacuum collar with a 96-well collection plate on the bottom. After ~5 minutes at room temperature, the filtration was performed under a slight vacuum over 5 to 8 minutes until all filtration plate wells were dried. Collection plates were removed, sealed, and kept at room temperature for 30 minutes before the filtrates were dried using a Zipvap 96-well evaporator (ChromTech, Apple Valley, Minnesota) at ~45°C under a stream of nitrogen. To reconstitute the dried sample residues, we added 100 μL of MeOH/1% HAc (40:60, v/v) to each well, and then collection plates were vortexed on a microplate shaker for ~2 minutes at 1600 rpm. Each sample was then transferred to 0.5-mL microfuge tubes and centrifuged at 15 000 g and 4°C for 10 minutes. Finally, 75 μL of the supernatants was transferred to glass high- performance liquid chromatography (HPLC) vials.
Plasma concentrations of silymarin flavonolignans were quantified by liquid chromatography electron spray ionization mass spectrometry (LC-ESI-MS), as previously described using a Luna C18
analytical column (50 × 2.0 mm i.d., 3 μm; Phenomenex, Torrance, California); an isocratic mobile phase consisting of 43% methanol, 56% water, and 1% glacial acetic acid (pH 2.8); a flow rate of 0.3 mL/min; a 25-μL injection volume; and a 13-minute run time.17
For the analysis of flavonolignans in urine, 0.5 mL of urine sample was transferred into a 1.5-mL vial and centrifuged at 3000 g for 5 minutes. Aliquots of 100 μL of urine were then transferred to 1.5-mL polypropylene tubes, buffered using sodium acetate (pH 5.0, 0.125 M in a final incubation volume of 120 μL), and incubated at 37°C with gentle shaking for 6 hours with or without a mixture of sulfatase (80 U/mL) and β-glucuronidase (8000 U/mL) for the determination of total (parent + conjugates) or parent (nonconjugated) flavonolignans, respectively. After incubation, 250 ng of naringenin (internal standard) was added to each sample, followed by addition of 380 μL of ice-cold methanol containing 0.1% acetic acid to precipitate protein. Tubes were then vortexed and centrifuged at 15 000 g and 4°C for 15 minutes, and then 100 μL of the supernatants was transferred to LC vials, diluted with 100 μL of MeOH/H2O (50/50, v/v), and vortexed before 25 μL was injected for LC-ESI-MS analysis (final dilution ratio of urine sample: 1/10). Calibration curves in blank urine were treated similarly. For authentic reference standards, the composition of silybin (silibinin, Sigma-Aldrich, St. Louis, Missouri) was confirmed to be a mixture of silybin A (SA) and silybin B (SB) by LC-ESI-MS, and the specific contents of SA and SB were analyzed to be 48% and 52%, respectively. Silychristin (SC) was obtained from ChromaDex (Santa Ana, California), and silydianin (SD) was purchased from U.S. Pharmacopoeia (USP, Rockville, Maryland). Isosilybin A (ISA) and isosilybin B (ISB) reference standards were obtained as a generous gift from Ulrich Mengs (Madaus GmbH).
Separation of silymarin flavonolignans in urine samples was performed using an Agilent HP 1050 LC system (Palo Alto, California) with a BrownLee RP C18 guard column (15 × 3.2 mm i.d., 7 μm; PerkinElmer, Shelton, Connecticut) and a AQUA C18 (2) analytical column (150 × 4.6 mm i.d., 5 μm; Phenomenex, Torrance, California) and a methanol/0.1% acetic acid mobile phase gradient from 55% methanol to 60% methanol over 20 minutes followed by a column wash with 80% methanol, a flow rate of 0.4 mL/min (70 bars pressure), an injection volume of 25 μL, and a run time of 25 minutes. Typical retention times for SC, SD, SBA, SBB, ISBA, ISBB, and naringenin were 8.3, 10.1, 15.0, 16.4, 19.4, 20.5, and 17.4 minutes, respectively. MS detection was conducted using a PE Sciex API 100 LC/MS system (PE Sciex, Toronto, Canada) with a TurboIonspray (Applied Biosystems, Foster City, California) interface in the negative ESI ionization mode. MS parameters used for qualitative analysis included the following: ionspray voltage, −3100 V; ion-spray temperature, 450°C; orifice voltage, −30 V; focusing ring voltage, −200 V; entrance potential (EP), −10 V; nebulizer gas, 10 L/min; curtain gas, 8 L/min; channel electron multiplier, 2400; dwell time, 300 ms; scan speed, 0.61 s; and scan mode, selective ion monitoring (SIM) with [M-H]− for silymarin flavonolignans (m/z 481), silymarin sulfates (m/z 561), silymarin glucuronides (m/z 657), and naringenin (m/z 271).
Concentrations of silymarin flavonolignans in the samples were estimated with 1/x2 weighted least squares regression equations derived from the peak area ratios of individual silymarin flavonolignans to that of the internal standard. For each silymarin flavonolignan, the limit of detection was 20 ng/mL, and the quantitative ranges for parent and for total flavonolignan were 50 to 2500 ng/mL and 100 to 20 000 ng/ml, respectively. Intra- and interday precisions were 1.7% to 11% and 4.5% to 14%, respectively.
Pharmacokinetic parameters, including area under plasma concentration-time curve from time 0 to 48 hours (AUC0–48 h), maximum plasma concentration (Cmax), time to Cmax (tmax), renal clearance (CLR), apparent clearance (total oral clearance divided by bioavailability, [CL/F]), and terminal half-life (t1/2), were calculated using noncompartmental methods, WinNonlin-Pro (Version 5.2; Pharsight Corp, Mountain View, California). A constant dosing interval (tau) of 8 hours was assumed for the calculation of steady-state AUC0–8 h using the linear up/log down trapezoidal method. To obtain pharmacokinetic parameters for the sum of all parent flavonolignan concentrations and for the sum of all total flavonolignan concentrations, we summed the parent or total flavonolignan concentration for each of the 6 flavonolignans at each time point over the entire sampling period prior to performing a pharmacokinetic analysis. For CL/F calculations, the dose of each silymarin flavonolignan was determined from their specific content in the milk thistle product as described above. Apparent CLR(0–8 h) was approximated from the amount of flavonolignans excreted in a 12-hour urine collection divided by the AUC0–8 h for each patient. All pharmacokinetic parameters are reported as medians with minimum and maximum values, except where indicated as a mean ± standard deviation. Pharmacokinetic parameter estimates across more than 2 dose groups were compared using a permutation test based on the Kruskal-Wallis statistic. For pairwise comparison, a permutation test based on 2-sample Wilcoxon’s statistic was used. For multiple comparisons, P values were adjusted using Holm’s step-down method. P < .05 was used for statistical significance. All of the analyses were done using SAS Version 9.1.3 (SAS Institute, Inc, Cary, North Carolina).