Roy L. Hawke

In vivo and in vitro induction of human cytochrome P4503A4 by dexamethasone

The aims of these experiments were to determine the effect of a therapeutic regimen of dexamethasone on cytochrome P4503A4 (CYP3A4) activity in healthy volunteers; and the concentration‐effect relationship between dexamethasone and CYP3A4 activity in primary human hepatocyte cultures.

Pharmacokinetics and metabolic profile of free, conjugated, and total silymarin flavonolignans in human plasma after oral administration of milk thistle extract.

Silymarin, a mixture of polyphenolic flavonoids extracted from milk thistle (Silybum marianum), is composed mainly of silychristin, silydianin, silybin A, silybin B (SBB), isosilybin A (ISBA), and isosilybin B. In this study, the plasma concentrations of free (unconjugated), conjugated (sulfated and glucuronidated), and total (free and conjugated) silymarin flavonolignans were measured using liquid chromatography-electrospray ionization-mass spectrometry, after a single oral dose of 600 mg of standardized milk thistle extracts to three healthy volunteers. Pharmacokinetic analysis indicated that silymarin flavonolignans were rapidly eliminated with short half-lives (1–3 and 3–8 h for free and conjugated, respectively). The AUC0→∞ values of the conjugated silymarin flavonolignans were 4- to 30-fold higher than those of their free fractions, with SBB (mean AUC0→∞ = 51 and 597 μg · h/l for free and conjugated, respectively) and ISBA (mean AUC0→∞ = 30 and 734 μg · h/l for free and conjugated, respectively) exhibiting higher AUC0→∞ values in comparison with other flavonolignans. Near the plasma peak times (1–3 h), the free, sulfated, and glucuronidated flavonolignans represented approximately 17, 28, and 55% of the total silymarin, respectively. In addition, the individual silymarin flavonolignans exhibited quite different plasma profiles for both the free and conjugated fractions. These data suggest that, after oral administration, silymarin flavonolignans are quickly metabolized to their conjugates, primarily forming glucuronides, and the conjugates are primary components present in human plasma.

Silymarin Ascending Multiple Oral Dosing Phase I Study in Noncirrhotic Patients With Chronic Hepatitis C

Silymarin, derived from the milk thistle plant Silybum marianum, is widely used for self‐treatment of liver diseases, including hepatitis C virus (HCV), and its antiviral activity has been demonstrated in vitro and in HCV patients administered an intravenous formulation of the major silymarin flavonolignans, silybin A and silybin B. The safety and dose‐exposure relationships of higher than customary oral doses of silymarin and its acute effects on serum HCV RNA were evaluated in noncirrhotic HCV patients. Four cohorts of 8 patients with well‐compensated, chronic noncirrhotic HCV who failed interferon‐based therapy were randomized 3:1 to silymarin or placebo. Oral doses of 140, 280, 560, or 700 mg silymarin were administered every 8 hours for 7 days. Steady‐state exposures for silybin A and silybin B increased 11‐fold and 38‐fold, respectively, with a 5‐fold increase in dose, suggesting nonlinear pharmacokinetics. No drug‐related adverse events were reported, and no clinically meaningful reductions from baseline serum transaminases or HCV RNA titer were observed. Oral doses of silymarin up to 2.1 g per day were safe and well tolerated. The nonlinear pharmacokinetics of silybin A and silybin B suggests low bioavailability associated with customary doses of silymarin may be overcome with doses above 700 mg.

Effect of Silymarin (Milk Thistle) on Liver Disease in Patients With Chronic Hepatitis C Unsuccessfully Treated With Interferon Therapy: A Randomized Controlled Trial

CONTEXT The botanical product silymarin, an extract of milk thistle, is commonly used by patients to treat chronic liver disease, despite scant and conflicting evidence of its efficacy. OBJECTIVE To determine the effect of silymarin on liver disease activity in patients with chronic hepatitis C virus (HCV) infection unsuccessfully treated with interferon-based therapy. DESIGN, SETTING, AND PARTICIPANTS Multicenter, double-blind, placebo-controlled trial conducted at 4 medical centers in the United States. Participants included 154 persons with chronic HCV infection and serum alanine aminotransferase (ALT) levels of 65 U/L or greater who were previously unsuccessfully treated with interferon-based therapy. Enrollment began in May 2008 and was completed in May 2010, with the last follow-up visit completed in March 2011. INTERVENTION Participants were randomly assigned to receive 420-mg silymarin, 700-mg silymarin, or matching placebo administered 3 times per day for 24 weeks. MAIN OUTCOME MEASURES The primary outcome measure was serum ALT level of 45 U/L or less (considered within the normal range) or less than 65 U/L, provided this was at least a 50% decline from baseline values. Secondary outcomes included changes in ALT levels, HCV RNA levels, and quality-of-life measures. RESULTS After 24 weeks of treatment, only 2 participants in each treatment group (P ≥ .99) met the primary outcome measure (3.8% [95% CI, 0.5% to 13.2%] for placebo, 4.0% [95% CI, 0.5% to 13.7%] for 420-mg silymarin, and 3.8% [95% CI, 0.5% to 13.2%] for 700-mg silymarin). The mean decline in serum ALT activity at the end of treatment did not differ significantly (P = .75) across the 3 treatment groups (mean decline, -4.3 [95% CI, -17.3 to 8.7] U/L for placebo, -14.4 [95% CI, -41.6 to 12.7] U/L for 420-mg silymarin, -11.3 [95% CI, -27.9 to 5.4] U/L for 700-mg silymarin); there likewise were no significant differences in HCV RNA levels (mean change, 0.07 [95% CI, -0.05 to 0.18] log10 IU/mL for placebo, -0.03 [95% CI, -0.18 to 0.12] log10 IU/mL for 420-mg silymarin, 0.04 [95% CI, -0.08 to 0.16] log10 IU/mL for 700-mg silymarin; P = .54) or quality-of-life measures. The adverse event profile of silymarin was comparable with that of placebo. CONCLUSION Higher than customary doses of silymarin did not significantly reduce serum ALT levels more than placebo in participants with chronic HCV infection unsuccessfully treated with interferon-based therapy. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00680342.

The Pharmacokinetics of Silymarin Is Altered in Patients with Hepatitis C Virus and Nonalcoholic Fatty Liver Disease and Correlates with Plasma Caspase-3/7 Activity

Silymarin, used by 30 to 40% of liver disease patients, is composed of six major flavonolignans, each of which may contribute to silymarins hepatoprotective properties. Previous studies have only described the pharmacokinetics for two flavonolignans, silybin A and silybin B, in healthy volunteers. The aim of this study was to determine the pharmacokinetics of the major silymarin flavonolignans in liver disease patients. Healthy volunteers and three patient cohorts were administered a single, 600-mg p.o. dose of milk thistle extract, and 14 blood samples were obtained over 24 h. Silybin A and B accounted for 43% of the exposure to the sum of total silymarin flavonolignans in healthy volunteers and only 31 to 38% in liver disease cohorts as a result of accumulation of silychristin (20–36%). Area under the curve (AUC0–24h) for the sum of total silymarin flavonolignans was 2.4-, 3.3-, and 4.7-fold higher for hepatitis C virus (HCV) noncirrhosis, nonalcoholic fatty liver disease (p ≤ 0.03), and HCV cirrhosis cohorts (p ≤ 0.03), respectively, compared with healthy volunteers (AUC0–24h = 2021 ng · h/ml). Caspase-3/7 activity correlated with the AUC0–24h for the sum of all silymarin conjugates among all subjects (R2 = 0.52) and was 5-fold higher in the HCV cirrhosis cohort (p ≤ 0.005 versus healthy). No correlation was observed with other measures of disease activity, including plasma alanine aminotransferase, interleukin 6, and 8-isoprostane F2α, a measure of oxidative stress. These findings suggest that the pharmacokinetics of silymarin is altered in patients with liver disease. Patients with cirrhosis had the highest plasma caspase-3/7 activity and also achieved the highest exposures for the major silymarin flavonolignans.

Differences in the Disposition of Silymarin between Patients with Nonalcoholic Fatty Liver Disease and Chronic Hepatitis C

Silymarin, derived from the milk thistle plant Silybum marianum and widely used for self-treatment of liver diseases, is composed of six major flavonolignans including silybin A and silybin B, which are the predominant flavonolignans quantified in human plasma. The single- and multiple-dose pharmacokinetics of silymarin flavonolignans were examined in patients with nonalcoholic fatty liver disease (NAFLD) or hepatitis C virus (HCV) to determine whether the disposition of silymarin and therefore its potential efficacy vary among liver disease populations. Cohorts of eight subjects with noncirrhotic liver disease were randomized 3:1 to oral silymarin or placebo (280 or 560 mg) every 8 h for 7 days. Forty-eight-hour blood sampling was conducted after the first and final doses. In general, plasma concentrations of silybin A and silybin B were higher, whereas concentrations of conjugates were lower in NAFLD compared with HCV. After adjustment of the area under plasma concentration-time curve from 0 to 8 h (AUC0–8 h) for weight and dose, only silybin B and silybin B conjugates differed significantly between disease types. For NAFLD, the adjusted mean AUC0–8 h was higher for silybin B (p < 0.05) but lower for silybin B conjugates (p < 0.05) compared with that for HCV. At the 280-mg dose, steady-state plasma concentrations of silybin B conjugates for NAFLD subjects were characterized by 46% lower AUC0–8 h (p < 0.05) and 42% lower Cmax (p < 0.05) compared with HCV subjects. Evidence of enterohepatic cycling of flavonolignans was only observed in NAFLD subjects. In summary, the efficacy of silymarin may be more readily observed in NAFLD patients because of their higher flavonolignan plasma concentrations and more extensive enterohepatic cycling compared with those in HCV patients.

Hepatic metabolism and biliary excretion of silymarin flavonolignans in isolated perfused rat livers: Role of multidrug resistance-associated protein 2 (Abcc2)

Silymarin, an extract from seeds of Silybum marianum, is used by 8 to 33% of patients to self-treat chronic viral hepatitis C in the United States. Studies in humans and rodents suggest that biliary excretion of glucuronide and sulfate conjugates is the major route for silymarins elimination. To determine the role of multidrug resistance-associated protein 2 (Mrp2) (Abcc2) in the biliary excretion of silymarin, the hepatobiliary disposition of the six major silymarin flavonolignans was studied using isolated perfused livers (IPRLs) from wild-type (WT) and Mrp2-deficient (TR-) Wistar rats. For all the flavonolignans, approximately 96% of the dose was cleared from perfusate within 30 min in both WT and TR- livers, and <5% of parent was recovered in bile or perfusate by the end of the perfusion. In WT livers, the percentage of dose excreted as conjugates into bile varied for each flavonolignan (silychristin, 51.6 ± 9.3%; silydianin, 101.5 ± 28.3%; silybin A, 21.0 ± 8.3%; silybin B, 31.7 ± 13.2%; isosilybin A, 50.5 ± 23.6%; isosilybin B, 42.8 ± 19.3%). Among the flavonolignans, only silydianin was primarily glucuronidated and almost completely recovered in bile. In TR- livers, biliary excretion of flavonolignan conjugates was reduced 80 to 92%, with 30 to 83% of each flavonolignan conjugate recovered in perfusate compared with only 5 to 30% at 90 min. Biliary excretion of glucuronide and sulfate conjugates of all the flavonolignans were reduced by 94 to 98% and 73 to 84%, respectively, in TR- IPRLs. These data indicate a primary role for Mrp2 in the biliary elimination of silymarin flavonolignan conjugates.

The influence of CYP3A5 expression on the extent of hepatic CYP3A inhibition is substrate-dependent: an in vitro-in vivo evaluation.

Despite several studies suggesting that CYP3A5 expression can influence the extent of hepatic CYP3A-mediated inhibition, a systematic in vitro-in vivo evaluation of this potential clinically important issue has not been reported. Using representative probes from two distinct CYP3A substrate subgroups (midazolam, erythromycin), the inhibitory potency of fluconazole was evaluated in pooled human liver microsomes (HLM) with a low or high specific CYP3A5 content, in recombinant CYP3A enzymes (rCYP3A), and in healthy volunteers lacking or carrying the CYP3A5*1 allele. Fluconazole was a slightly more potent inhibitor of CYP3A activity in CYP3A5-HLM than in CYP3A5+ HLM with midazolam (Ki of 15 and 25 μM, respectively) but not with erythromycin (IC50 of 70 and 54 μM, respectively). In comparison, fluconazole was a much more potent inhibitor of rCYP3A4 than rCYP3A5 with both midazolam (Ki of 7.7 and 54 μM, respectively) and erythromycin (IC50 of 100 and 350 μM, respectively). As predicted from HLM, with i.v. midazolam, the average (± S.D.) in vivo Ki (Ki,iv) was significantly higher in CYP3A5*1 carriers (24 ± 17 and 17 ± 8 μM for homozygous and heterozygous groups, respectively) than in noncarriers (13 ± 6 μM) (p = 0.02). With the erythromycin breath test, the average Ki,iv was not different between homozygous CYP3A5*1 carriers (30 ± 12 μM) and noncarriers (58 ± 53 μM). In conclusion, the effect of CYP3A5 on hepatic CYP3A-mediated inhibitory drug-drug interactions is substrate-dependent, and HLM, rather than rCYP3A, are the preferred in vitro system for predicting these interactions in vivo.

Interaction of Silymarin Flavonolignans with Organic Anion-Transporting Polypeptides

Organic anion-transporting polypeptides (OATPs) are multispecific transporters mediating the uptake of endogenous compounds and xenobiotics in tissues that are important for drug absorption and elimination, including the intestine and liver. Silymarin is a popular herbal supplement often used by patients with chronic liver disease; higher oral doses than those customarily used (140 mg three times/day) are being evaluated clinically. The present study examined the effect of silymarin flavonolignans on OATP1B1-, OATP1B3-, and OATP2B1-mediated transport in cell lines stably expressing these transporters and in human hepatocytes. In overexpressing cell lines, OATP1B1- and OATP1B3-mediated estradiol-17β-glucuronide uptake and OATP2B1-mediated estrone-3-sulfate uptake were inhibited by most of the silymarin flavonolignans investigated. OATP1B1-, OATP1B3-, and OATP2B1-mediated substrate transport was inhibited efficiently by silymarin (IC50 values of 1.3, 2.2 and 0.3 µM, respectively), silybin A (IC50 values of 9.7, 2.7 and 4.5 µM, respectively), silybin B (IC50 values of 8.5, 5.0 and 0.8 µM, respectively), and silychristin (IC50 values of 9.0, 36.4, and 3.6 µM, respectively). Furthermore, silymarin, silybin A, and silybin B (100 µM) significantly inhibited OATP-mediated estradiol-17β-glucuronide and rosuvastatin uptake into human hepatocytes. Calculation of the maximal unbound portal vein concentrations/IC50 values indicated a low risk for silymarin-drug interactions in hepatic uptake with a customary silymarin dose. The extent of silymarin-drug interactions depends on OATP isoform specificity and concentrations of flavonolignans at the site of drug transport. Higher than customary doses of silymarin, or formulations with improved bioavailability, may increase the risk of flavonolignan interactions with OATP substrates in patients.

The Influence of CYP3A5 Genotype on Dexamethasone Induction of CYP3A Activity in African Americans

The CYP3A5*1 allele has been associated with differences in the metabolism of some CYP3A substrates. CYP3A5 polymorphism may also influence susceptibility for certain drug interactions. We have previously noted a correlation between basal CYP3A activity and the inductive effects of dexamethasone using the erythromycin breath test (ERBT). To determine whether CYP3A5 polymorphism influences induction of CYP3A activity, we examined the effect of an antiemetic regimen of dexamethasone, and the prototypical inducer rifampin, on the ERBT in African American volunteers prospectively stratified by CYP3A5*1 allele carrier status. Mean basal ERBTs were significantly higher in CYP3A5*1 carriers (2.71 ± 0.53%) versus noncarriers (2.12 ± 0.37%, P = 0.006). Rifampin increased ERBTs in CYP3A5*1 carriers (4.68 versus 2.60%, P = 0.0008) and noncarriers (3.55 versus 2.11%, P = 0.0017), whereas dexamethasone increased ERBTs only in CYP3A5*1 noncarriers (3.03 versus 2.14%, P = 0.031). CYP3A5 polymorphism appears to influence susceptibility to induction-type drug interactions for some inducers, and CYP3A5*1 noncarriers may be more susceptible to the inductive effects of dexamethasone as a result of lower basal CYP3A activity.