Background

Longitudinal studies are needed to decipher mechanistic links between the gut microbiome and nonalcoholic steatohepatitis (NASH). We examined shifts in the gut microbiome in persons with NASH with improvement in liver stiffness measurement (LSM) by magnetic resonance (MR) elastography.

Methods

Gut microbial profiling was performed at baseline and study completion (24 weeks) using 16 S rRNA gene sequencing in 69 adults with biopsy-confirmed NASH and significant fibrosis (stages 2-3) enrolled in a multi-center randomized controlled trial evaluating selonsertib alone or in combination with simtuzumab. Differential abundance of bacterial taxa at baseline and end of study were examined in participants with and without longitudinal improvement in LSM. Gut microbial shifts that correlated with secondary outcomes, including reduction in MR imaging-derived proton density fat faction (MRI-PDFF) and histologic fibrosis regression were evaluated. Fecal samples from 32 healthy adults were profiled and genus-level multidimensional scaling was used to determine if microbial shifts in persons with NASH improvement represented a shift toward a healthy gut microbiome.

Results

Shifts in abundance of 36 bacterial taxa including Lactobacillus (log2_FC = -4.51, FDR < 0.001), Enterococcus (log2_FC = -6.72, FDR < 0.001), and Megasphaera (log2_FC = 7.74, FDR < 0.001) were associated with improvement in LSM. Improvement in LSM was associated with microbial shifts toward healthy reference (p = 0.05). Significant shifts in 10 and 12 bacterial taxa were associated with improvement in LSM in addition to MRI-PDFF and fibrosis regression, respectively, indicating consistent taxonomic changes across multiple clinical endpoints.

Conclusion

Longitudinal changes in the gut microbiota are observed in adults with NASH and clinical improvement and represent a shift toward a healthy microbiome.

Background

Eicosanoid and related docosanoid polyunsaturated fatty acids (PUFAs) and their oxygenated derivatives have been proposed as noninvasive lipidomic biomarkers of nonalcoholic steatohepatitis (NASH). Therefore, we investigated associations between plasma eicosanoids and liver fibrosis to evaluate their utility in diagnosing and monitoring NASH-related fibrosis.

Methods

Our analysis used baseline eicosanoid data from 427 patients with biopsy-confirmed nonalcoholic fatty liver disease (NAFLD), and longitudinal measurements along with liver fibrosis staging from 63 patients with NASH and stage 2/3 fibrosis followed for 24 weeks in a phase II trial.

Results

At baseline, four eicosanoids were significantly associated with liver fibrosis stage: 11,12-DIHETE, tetranor 12-HETE, adrenic acid, and 14, 15-DIHETE. Over 24 weeks of follow up, a combination of changes in seven eicosanoids [5-HETE, 7,17-DHDPA, adrenic acid, arachidonic acid (AA), eicosapentaenoic acid (EPA), 16-HDOHE, and 9-HODE) had good diagnostic accuracy for the prediction of ⩾1 stage improvement in fibrosis (AUROC: 0.74; 95% CI: 0.62-0.87), and a combination of four eicosanoids (7,17-DHDPA, 14,15-DIHETRE, 9-HOTRE, and free adrenic acid) accurately predicted improvement in hepatic collagen content (AUROC: 0.72; 95% CI: 0.50-0.77).

Conclusion

This study provides preliminary evidence that plasma eicosanoids may serve as noninvasive biomarkers of liver fibrosis and may predict liver fibrosis improvement in NASH.

Background & aims

Patients with advanced fibrosis due to nonalcoholic steatohepatitis (NASH) are at high risk of morbidity and mortality. We previously found that a combination of the farnesoid X receptor agonist cilofexor (CILO) and the acetyl-CoA carboxylase inhibitor firsocostat (FIR) improved liver histology and biomarkers in NASH with advanced fibrosis but was associated with hypertriglyceridemia. We evaluated the safety and efficacy of icosapent ethyl (Vascepa) and fenofibrate to mitigate triglyceride elevations in patients with NASH treated with CILO and FIR.

Methods

Patients with NASH with elevated triglycerides (≥150 and <500 mg/dL) were randomized to Vascepa 2 g twice daily (n = 33) or fenofibrate 145 mg daily (n = 33) for 2 weeks, followed by the addition of CILO 30 mg and FIR 20 mg daily for 6 weeks. Safety, lipids, and liver biochemistry were monitored.

Results

All treatments were well-tolerated; most treatment-emergent adverse events were Grade 1 to 2 severity, and there were no discontinuations due to adverse events. At baseline, median (interquartile range [IQR]) triglycerides were similar in the Vascepa and fenofibrate groups (median, 177 [IQR, 154-205] vs 190 [IQR, 144-258] mg/dL, respectively). Median changes from baseline in triglycerides for Vascepa vs fenofibrate after 2 weeks of pretreatment were -12 mg/dL (IQR, -33 to 7 mg/dL; P = .09) vs -32 mg/dL (IQR, -76 to 6 mg/dL; P = .012) and at 6 weeks were +41 mg/dL (IQR, 16-103 mg/dL; P < .001) vs -2 mg/dL (IQR, -42 to 54 mg/dL; P = .92). In patients with baseline triglycerides <250 mg/dL, fenofibrate was more effective vs Vascepa in mitigating triglyceride increases after 6 weeks of combination treatment (+6 vs +39 mg/dL); similar trends were observed in patients with baseline triglycerides ≥250 mg/d (-61 vs +99 mg/dL).

Conclusions

In patients with NASH with hypertriglyceridemia treated with CILO and FIR, fenofibrate was safe and effectively mitigated increases in triglycerides associated with acetyl-CoA carboxylase inhibition.

Clinicaltrials

gov, Number: NCT02781584.

Treatment with acetyl-CoA carboxylase inhibitors (ACCi) in nonalcoholic steatohepatitis (NASH) may increase plasma triglycerides (TGs), with variable changes in apoB concentrations. ACC is rate limiting in de novo lipogenesis and regulates fatty acid oxidation, making it an attractive therapeutic target in NASH. Our objectives were to determine the effects of the ACCi, firsocostat, on production rates of plasma LDL-apoB in NASH and the effects of combined therapy with fenofibrate. Metabolic labeling with heavy water and tandem mass spectrometric analysis of LDL-apoB enrichments was performed in 16 NASH patients treated with firsocostat for 12 weeks and in 29 NASH subjects treated with firsocostat and fenofibrate for 12 weeks. In NASH on firsocostat, plasma TG increased significantly by 17% from baseline to week 12 (P = 0.0056). Significant increases were also observed in LDL-apoB fractional replacement rate (baseline to week 12: 31 ± 20.2 to 46 ± 22.6%/day, P = 0.03) and absolute synthesis rate (ASR) (30.4-45.2 mg/dl/day, P = 0.016) but not plasma apoB concentrations. The effect of firsocostat on LDL-apoB ASR was restricted to patients with cirrhosis (21.0 ± 9.6 at baseline and 44.2 ± 17 mg/dl/day at week 12, P = 0.002, N = 8); noncirrhotic patients did not change (39.8 ± 20.8 and 46.3 ± 14.8 mg/dl/day, respectively, P = 0.51, N = 8). Combination treatment with fenofibrate and firsocostat prevented increases in plasma TG, LDL-apoB fractional replacement rate, and ASR. In summary, in NASH with cirrhosis, ACCi treatment increases LDL-apoB100 production rate and this effect can be prevented by concurrent fenofibrate therapy.

De novo lipogenesis (DNL) converts carbon substrates to lipids. Increased hepatic DNL could contribute to pathogenic liver triglyceride accumulation in nonalcoholic steatohepatitis (NASH) and therefore may be a potential target for pharmacological intervention. Here, we measured hepatic DNL using heavy water in 123 patients with NASH with fibrosis or cirrhosis, calculated the turnover of hepatic triglycerides to allow repeat labeling studies, and determined the associations of hepatic DNL with metabolic, fibrotic, and imaging markers. We found that hepatic DNL was higher in patients with fibrotic NASH [median (IQR), 40.7% contribution to palmitate (32.1, 47.5), n=103] than has been previously reported in healthy volunteers and remained elevated [median (IQR), 36.8% (31.0, 44.5), n=20] in patients with cirrhosis, despite lower liver fat content. We also showed that turnover of intrahepatic triglyceride pools was slow (t_½ >10 days). Furthermore, DNL contribution was determined to be independent of liver stiffness by magnetic resonance imaging but was positively associated with the number of large very low density lipoprotein (VLDL) particles, the size of VLDL, the lipoprotein insulin resistance score, and levels of ApoB100, and trended toward negative associations with the fibrosis markers FIB-4, FibroSure, and APRI. Finally, we found treatment with the acetyl-CoA carboxylase inhibitor firsocostat reduced hepatic DNL at 4 and 12 weeks, using a correction model for residual label that accounts for hepatic triglyceride turnover. Taken together, these data support an important pathophysiological role for elevated hepatic DNL in NASH and demonstrate that response to pharmacological agents targeting DNL can be correlated with pretreatment DNL.

Background and aims

Pruritus is associated with multiple liver diseases, particularly those with cholestasis, but the mechanism remains incompletely understood. Our aim was to evaluate serum IL-31 as a putative biomarker of pruritus in clinical trials of an farnesoid X receptor (FXR) agonist, cilofexor, in patients with NASH, primary sclerosing cholangitis (PSC), and primary biliary cholangitis (PBC).

Approach and results

Serum IL-31 was measured in clinical studies of cilofexor in NASH, PSC, and PBC. In patients with PSC or PBC, baseline IL-31 was elevated compared to patients with NASH and healthy volunteers (HVs). IL-31 correlated with serum bile acids among patients with NASH, PBC, and PSC. Baseline IL-31 levels in PSC and PBC were positively correlated with Visual Analog Scale for pruritus and 5-D itch scores. In patients with NASH, cilofexor dose-dependently increased IL-31 from Week (W)1 to W24. In patients with NASH receiving cilofexor 100 mg, IL-31 was higher in those with Grade 2-3 pruritus adverse events (AEs) than those with Grade 0-1 pruritus AEs. IL-31 weakly correlated with C4 at baseline in patients with NASH, and among those receiving cilofexor 100 mg, changes in IL-31 and C4 from baseline to W24 were negatively correlated. IL-31 messenger RNA (mRNA) was elevated in hepatocytes from patients with PSC and NASH compared to HVs. In a humanized liver murine model, obeticholic acid increased IL-31 mRNA expression in human hepatocytes and serum levels of human IL-31.

Conclusions

IL-31 levels correlate with pruritus in patients with cholestatic disease and NASH, with FXR agonist therapy resulting in higher serum levels in the latter group. IL-31 appears to derive in part from increased hepatocyte expression. These findings have therapeutic implications for patients with liver disease and pruritus.

Primary sclerosing cholangitis (PSC) represents a major unmet medical need. In a phase II double-blind, placebo-controlled study, we tested the safety and efficacy of cilofexor (formerly GS-9674), a nonsteroidal farnesoid X receptor agonist in patients without cirrhosis with large-duct PSC. Patients were randomized to receive cilofexor 100 mg (n = 22), 30 mg (n = 20), or placebo (n = 10) orally once daily for 12 weeks. All patients had serum alkaline phosphatase (ALP) > 1.67 × upper limit of normal and total bilirubin ≤ 2 mg/dL at baseline. Safety, tolerability, pharmacodynamic effects of cilofexor (serum C4 [7α-hydroxy-4-cholesten-3-one] and bile acids), and changes in liver biochemistry and serum fibrosis markers were evaluated. Overall, 52 patients were randomized (median age 43 years, 58% male, 60% with inflammatory bowel disease, 46% on ursodeoxycholic acid). Baseline median serum ALP and bilirubin were 348 U/L (interquartile range 288-439) and 0.7 mg/dL (0.5-1.0), respectively. Dose-dependent reductions in liver biochemistry were observed. At week 12, cilofexor 100 mg led to significant reductions in serum ALP (median reduction -21%; P = 0.029 versus placebo), gamma-glutamyl transferase (-30%; P < 0.001), alanine aminotransferase (ALT) (-49%; P = 0.009), and aspartate aminotransferase (-42%; P = 0.019). Cilofexor reduced serum C4 compared with placebo; reductions in bile acids were greatest with 100 mg. Relative reductions in ALP were similar between ursodeoxycholic acid-treated and untreated patients. At week 12, cilofexor-treated patients with a 25% or more relative reduction in ALP had greater reductions in serum alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, tissue inhibitor of metalloproteinase 1, C-reactive protein, and bile acids than nonresponders. Adverse events were similar between cilofexor and placebo-treated patients. Rates of grade 2 or 3 pruritus were 14% with 100 mg, 20% with 30 mg, and 40% with placebo. Conclusion: In this 12-week, randomized, placebo-controlled study, cilofexor was well tolerated and led to significant improvements in liver biochemistries and markers of cholestasis in patients with PSC.