Adjunct Fenofibrate Up-regulates Bile Acid Glucuronidation and Improves Treatment Response For Patients With Cholestasis

Abstract

Accumulation of cytotoxic bile acids (BAs) during cholestasis can result in liver failure. Glucuronidation, a phase II metabolism pathway responsible for BA detoxification, is regulated by peroxisome proliferator-activated receptor alpha (PPARα). This study investigates the efficacy of adjunct fenofibrate therapy to up-regulate BA-glucuronidation and reduce serum BA toxicity during cholestasis. Adult patients with primary biliary cholangitis (PBC, n = 32) and primary sclerosing cholangitis (PSC, n = 23), who experienced an incomplete response while receiving ursodiol monotherapy (13-15 mg/kg/day), defined as serum alkaline phosphatase (ALP) ≥ 1.5 times the upper limit of normal, received additional fenofibrate (145-160 mg/day) as standard of care. Serum BA and BA-glucuronide concentrations were measured by liquid chromatography-mass spectrometry. Combination therapy with fenofibrate significantly decreased elevated serum ALP (-76%, P < 0.001), aspartate transaminase, alanine aminotransferase, bilirubin, total serum BAs (-54%), and increased serum BA-glucuronides (+2.1-fold, P < 0.01) versus ursodiol monotherapy. The major serum BA-glucuronides that were favorably altered following adjunct fenofibrate include hyodeoxycholic acid-6G (+3.7-fold, P < 0.01), hyocholic acid-6G (+2.6-fold, P < 0.05), chenodeoxycholic acid (CDCA)-3G (-36%), and lithocholic acid (LCA)-3G (-42%) versus ursodiol monotherapy. Fenofibrate also up-regulated the expression of uridine 5'-diphospho-glucuronosyltransferases and multidrug resistance-associated protein 3 messenger RNA in primary human hepatocytes. Pearson's correlation coefficients identified strong associations between serum ALP and metabolic ratios of CDCA-3G (r² = 0.62, P < 0.0001), deoxycholic acid (DCA)-3G (r² = 0.48, P < 0.0001), and LCA-3G (r² = 0.40, P < 0.001), in ursodiol monotherapy versus control. Receiver operating characteristic analysis identified serum BA-glucuronides as measures of response to therapy. Conclusion: Fenofibrate favorably alters major serum BA-glucuronides, which correlate with reduced serum ALP levels and improved outcomes. A PPARα-mediated anti-cholestatic mechanism is involved in detoxifying serum BAs in patients with PBC and PSC who have an incomplete response on ursodiol monotherapy and receive adjunct fenofibrate. Serum BA-glucuronides may serve as a noninvasive measure of treatment response in PBC and PSC.

FIG. 1

(A) Quantitative real‐time PCR analysis shows that fenofibrate (50 µM) treatment for 24 hours up‐regulates PPARα, UGT1A1, UGT1A3, UGT1A4, UGT2B4, CYP3A4, MRP2, MRP3, and CYP4A11 mRNA expression in cryopreserved hepatocytes compared with DMSO (vehicle control). Relative mRNA expression was calculated using the ∆∆Ct method, normalized to PPIA and expressed as a fold‐change over DMSO. Data are expressed as mean ± SEM (n = 3‐4 individual cases of hepatocytes), analyzed by two‐tailed unpaired Student t test. *P < 0.05, **P < 0.01, and ****P < 0.0001 versus DMSO. Combination treatment with fenofibrate decreases total serum BAs (B) and increases total serum BA‐glucuronides (C). Log₂‐transformed data were analyzed by one‐way ANOVA. **P < 0.01 and ****P < 0.0001. Total serum BAs and BA‐glucuronides are defined in the Supporting Materials and Methods.

FIG. 2

Combination treatment with fenofibrate alters serum BA‐glucuronides in patients with PBC and PSC. Scatter plots show individual patient serum concentrations of HCA‐6G (A), HDCA‐6G (B) (B), CDCA‐3G (C), LCA‐3G (D), and DCA‐3G (E). Log₂‐transformed data were analyzed by one‐way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Abbreviations: Combo, combination; and Mono, monotherapy.

FIG. 3

Combination treatment with fenofibrate shifts the percentage and composition of total 3G and 6G‐glucuronides (CDCA‐3G, DCA‐3G, LCA‐3G, HCA‐6G, and HDCA‐6G) in patients with PBC and PSC, closer to values observed in control subjects: healthy controls (A), PBC monotherapy (B), PBC combined (C), PSC monotherapy (D), and PSC combined (E).

FIG. 4

Combination treatment with fenofibrate alters metabolic ratios (BA‐glucuronide/parent BA) in patients with PBC and PSC versus ursodiol monotherapy: CDCA‐3G/CDCA (A), DCA‐3G/DCA (B), LCA‐3G/LCA (C), HCA‐6G/HCA (D), and HDCA‐6G/HDCA (E). Data were analyzed by the Kruskal‐Wallis test with Dunn’s multiple comparisons. *P < 0.05, **P < 0.01, and ***P < 0.001.

FIG. 5

Pearson’s correlation coefficient of BA‐glucuronide metabolic ratios with ALP levels: CDCA‐3G/CDCA, DCA‐3G/DCA, and LCA‐3G/LCA (A). ROC analysis in ursodiol monotherapy versus control: CDCA‐3G and LCA‐3G (B), CDCA‐3G/CDCA and DCA‐3G/DCA ratios (C). ROC curve analyses in combined fenofibrate treatment versus control: HDCA‐6G and HCA‐6G (D) and HDCA‐6G/HDCA and HCA‐6G/HCA ratios (E). Data was log₂‐transformed before analysis.

FIG. 6

(A) Two PCA score plots of individual BA‐glucuronides from the serum of healthy controls, ursodiol monotherapy, and combination fenofibrate‐treated patients to account for approximately 68% of variability. Data was log₂‐transformed before PCA analysis and grouped as Control, PBC Mono, PBC Combo, PSC Mono, and PSC Combo. (B) Heatmap analysis of individual glucuronides from the serum of healthy subjects, ursodiol monotherapy, and combination fenofibrate‐treated patients. Data were log₂‐transformed before constructing the heatmap. Rows represent BA‐glucuronides. Columns represent individual samples and are clustered based on cohort (Control, PBC Mono, PBC Combo, PSC Mono, and PSC Combo).