Original Paper Received: April 17, 2012 Accepted: June 11, 2012 Published online: September 5, 2012
Digestion 2012;86:244–249 DOI: 10.1159/000341420
Combined Rifampicin and Ursodeoxycholic Acid Treatment Does Not Amplify Rifampicin Effects on Hepatic Detoxification and Transport Systems in Humans Hanns-Ulrich Marschall a Martin Wagner c Gernot Zollner c Peter Fickert c Dagmar Silbert c Ulf Gustafsson b Staffan Sahlin b Michael Trauner c, d a
Department of Internal Medicine, The Sahlgrenska Academy, Institute of Medicine, Gothenburg, and Department of Surgery, Danderyd Hospital, Karolinska Institutet, Stockholm, Sweden; c Division of Gastroenterology and Hepatology, Department of Medicine, Medical University of Graz, Graz, and dDivision of Gastroenterology and Hepatology, Department of Medicine III, Medical University of Vienna, Vienna, Austria b
Key Words Fibroblast growth factor ⴢ Hepatic detoxification ⴢ Laparoscopic cholecystectomy ⴢ Primary biliary cirrhosis ⴢ Rifampicin ⴢ Ursodeoxycholic acid
Abstract Background: Rifampicin (RIFA) and ursodeoxycholic acid (UDCA) were found to stimulate different but complementary hepatobiliary detoxification pathways in gallstone patients. Aim: To study whether single drug effects are sustained or even enhanced by combination of both drugs and whether possible effects are mediated by circulating fibroblast growth factor 19 (FGF19), which has recently been identified as a master regulator of bile acid biosynthesis. Methods: 20 patients scheduled for laparoscopic cholecystectomy were randomized to a combination of UDCA (1 g/day during 3 weeks before surgery) and RIFA (600 mg/day during 1 week before surgery), or no treatment. Routine biochemistry, lipids, bile acid synthesis (7␣-hydroxy-4-cholesten-3one, C-4) and FGF19 were measured in serum. Bile acids were analyzed in serum and bile. A wedge liver biopsy was taken for determination of expression of hepatobiliary ABC trans-
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porters on mRNA and protein levels and of enzymes and regulatory transcription factors involved in the metabolism of biliary compounds on mRNA levels. Results: Combination treatment with both RIFA and UDCA significantly stimulated bile acid and bilirubin detoxification (CYP3A4, p ! 0.001), conjugation (UGT1A1, p ! 0.001) and elimination (MRP2, p ! 0.05), as well as bile acid synthesis (p ! 0.05), as compared to untreated controls. Notably, serum FGF19 levels in RIFA- and UDCA-treated patients did not differ from controls. Conclusion: Combined treatment with RIFA and UDCA preserves the previously observed beneficial effects of single treatment with RIFA, including stimulation of bile acid synthesis. Most notably, the latter effect in humans is not mediated by FGF19. Copyright © 2012 S. Karger AG, Basel
Introduction
The hydrophilic bile acid ursodeoxycholic acid (UDCA) improves liver function tests in a number of cholestatic liver diseases. Evidence from randomizedcontrolled trials has established UDCA as the treatment Hanns-Ulrich Marschall, MD, PhD Department of Internal Medicine, Gastroenterology and Hepatology The Sahlgrenska Academy, Institute of Medicine SE–413 45 Gothenburg (Sweden) E-Mail hanns-ulrich.marschall @ gu.se
of choice for patients with primary biliary cirrhosis (PBC) and as first-line treatment for women with intrahepatic cholestasis of pregnancy [1]. The antibiotic rifampicin (RIFA) is recommended for treatment of cholestatic pruritus not responding to bile acid sequestrates [1]. In order to identify potential molecular mechanism(s) of action of UDCA and RIFA in humans, we had performed a comprehensive biochemical and molecular study of the effects of these agents on bile acids, biliary lipids, and genes involved in bile acid/bilirubin metabolism and transport in otherwise healthy patients with gallstone disease [2]. We found that RIFA enhanced bile acid detoxification as well as bilirubin conjugation and excretion as reflected by enhanced expression of CYP3A4, UGT1A1 and MRP2. UDCA on the other hand was found to stimulate the expression of canalicular transporters for bile acids and phospholipids, BSEP and MDR3, and the basolateral alternative conjugate transporter MRP4 [2]. These effects are considered to result from different stimulation of nuclear receptors CAR, FXR and PXR [3], which are major targets for more recent drug developments in liver disease [4, 5]. Since RIFA and UDCA have overlapping in vitro affinities to NR, in particular PXR, combined treatments with these drugs may have different effects than those observed for individual compounds. We therefore now studied the combined effects of RIFA and UDCA on hepatobiliary detoxification and elimination mechanisms in gallstone patients also addressing the effects on circulating fibroblast growth factor 19 (FGF19) that is tightly involved in the regulation of bile acid metabolism [6]. Material and Methods Patients Otherwise healthy patients scheduled for laparoscopic cholecystectomy for symptomatic gallstone disease were invited to participate in a clinical study of the metabolic and molecular effects of UDCA and RIFA. Potential candidates completed a clinical research file consisting of a detailed questionnaire about the patient’s history and lifestyle. Previous or ongoing liver, kidney, intestinal or metabolic diseases were exclusion criteria as well as the use of medications known to affect liver function and metabolism, e.g. lipid or glucose-lowering drugs, P450 enzyme inducers. After giving a written informed consent, 20 patients between 21 and 70 years of age were randomized by drawing lots to combined therapy with UDCA (Ursofalk쏐, 1 g/day for 3 weeks) and RIFA (Rimactan쏐, 600 mg/day during the third week) or no medication (controls) before surgery. Study drugs were administered open-label in one (RIFA) or two (UDCA) daily doses until the day before surgery. The study protocol was approved by the Ethics Committee at Karolinska Institutet and the Swedish Medical Products agency.
Combined RIFA and UDCA Treatment
Serum Clinical Chemistry, Bile Acids, C4 and FGF19 Routine parameters including serum lipids and lipoproteins as well as liver and kidney function tests were analyzed using standard clinical biochemical procedures. Serum 7␣-hydroxy-4cholesten-3-one (C4), a sensitive marker of endogenous bile acid synthesis [7], was measured by HPLC in comparison to the internal standard 7-hydroxy-4-cholesten-3-one. Serum bile acids were analyzed by isotope-dilution gas chromatography-mass spectrometry as previously described [2]. Serum FGF19 was measured in the fasting state at 08:00 h by ELISA [6]. In addition to the samples from patients now having been administered RIFA and UDCA in combination, we analyzed for comparison serum FGF19 in the samples from our previous study of single RIFA and UDCA treatment [2] since this assay was not available at that time. Biliary Lipid Analyses During surgery, gallbladder bile was sampled for the determination of bile acids as described previously [8]. mRNA and Protein Expression Analyses A wedge liver biopsy was taken during surgery and immediately frozen in liquid nitrogen. mRNA and protein were then prepared for the determination of mRNA expression levels of enzymes involved in bile acid synthesis and metabolism (CYP3A4, CYP7A1, UGT1A1), regulatory nuclear transcription factors (CAR/ NR1I3, FXR/NR1H4, PXR/NR1I2, RXR/NR1B1), and protein as well as mRNA levels of hepatobiliary ABC transporters (ABCG5/8, BSEP/ABCB11, MDR3/ABCB4, MRP2/ABCC2, MRP3/ABCC3, MRP4/ABCC4) using recently in detail described methodology [9–11]. Protein expression levels from RIFA- and UDCA-treated patients were estimated in comparison to proteins prepared from controls (‘Reference’ in fig. 3) that were run on each gel. We re-estimated mRNA expression of the indicated genes in samples from our previous study of RIFA vs. UDCA vs. controls [2] simultaneously with the new samples on combination therapy for direct comparison. Statistical Analysis Data are expressed as mean 8 SD values. Differences within patient groups were analyzed using Student’s paired t test and among patient groups unpaired Student’s t test using the Sigma Stat쏐 statistic program (Jandel Scientific, San Rafael, Calif., USA). A p value of !0.05 was considered significant.
Results
Patients’ Demographics and Routine Biochemistry The patients randomized to control or RIFA + UDCA groups did not differ significantly in mean age (range 21– 70 years of age) or body mass index (range 22.9–34.7). Females outnumbered males (in line with the target population in cholestatic liver diseases such as PBC) and there were more women in the RIFA + UDCA group (n = 8) than in the control (n = 6) groups (table 1). All patients had cholesterol gallstones. Digestion 2012;86:244–249
245
60
** Serum (μmol/l)
8 6 4 2
Gallbladder bile (%)
10
0
a
* p < 0.05 **p < 0.01 vs. control
**
40
*
*
30 20
**
10 0
LCA
DCA
CDCA
CA
UDCA
Fig. 1. Effects of RIFA and UDCA combined on bile acids in serum (a) and gallbladder (b) bile. Serum bile acid concentrations (a) and gallbladder bile acid composition (b) at the end of treat-
ment with RIFA and UDCA, compared to controls. For easier
No side effects from study medications were reported. Serum lipids and lipoproteins as well as liver and kidney function tests remained in the normal range in all study patients and did not change significantly from the initial value (table 1). However, serum bilirubin decreased statistically significant (p = 0.002) in the RIFA + UDCA group. Compliance Compliance of all patients randomized to RIFA + UDCA was confirmed by pill counts (less than 10% tablets and capsules, respectively, returned), reddish stained urine from RIFA and UDCA enrichment in serum and bile. Effects of RIFA + UDCA on Serum and Biliary Bile Acids Total serum levels of bile acids in controls (2.8 8 1.0 M) and the RIFA + UDCA treatment group at baseline did not differ (1.4 8 0.9 M). After 3 weeks of treatment, UDCA was significantly increased to 5.4 8 3.2 M (p ! 0.01), consisting 72.3 8 0.07% of total serum bile acids, which is not different from our previous study where we had found an enrichment of UDCA of 78.9 8 7.0% [2]. Serum levels of lithocholic (LCA), deoxycholic (DCA), chenodeoxycholic (CDCA) and cholic (CA) acids did not change significantly during treatment. However, there was a trend to higher serum concentrations of primary bile acids CDCA and CA that together increased by 83% from 0.72 to 1.32 M (p = 0.19). In gallbladder bile of controls, LCA, DCA, CDCA, CA and UDCA accounted for 1.0 8 0.4, 32.0 8 7.9, 37.1 8 5.2, 27.6 8 12.9 and 2.0 8 246
50
Digestion 2012;86:244–249
b
LCA
DCA
CDCA
CA
UDCA
comparison, baseline serum data that did not differ between groups are not shown. LCA, DCA, CDCA, CA, UDCA are respectively lithocholic, deoxycholic, chenodeoxycholic, cholic, and ursodeoxycholic acids.
Table 1. Demographics and routine serum biochemistry of pa-
tients studied Control
Gender (F/M) Age, years BMI Total cholesterol [1.4 mM] APO B [>0.8 mM] Lp(a) [
