ABSTRACT
-
Background/Aims
Berberine ursodeoxycholate (HTD1801) has been shown to significantly reduce liver fat content (LFC) in an 18-week, placebo-controlled Phase 2 study in patients with metabolic dysfunction-associated steatohepatitis (MASH) and type 2 diabetes mellitus. The purpose of this assessment was to establish proof of concept in liver histologic improvement with HTD1801 treatment based on preclinical and clinical evidence.
-
Methods
The efficacy of HTD1801 was evaluated in a preclinical MASH/dyslipidemia model (golden hamsters fed a high fat diet, eight/group) after six weeks of daily treatment. Additionally, in a secondary analysis of a Phase 2 clinical study, 100 patients with presumed MASH were evaluated by multiple noninvasive markers associated with MASH resolution and/or fibrosis improvement. These include magnetic resonance imaging proton density fat fraction (MRIPDFF; ≥30% LFC reduction), iron-corrected T1 (≥80 ms reduction), alanine aminotransferase (≥17 U/L reduction), weight loss (≥5% reduction), Fibrosis-4 index (shift to <1.3), and MASH resolution index (achieving ≥–0.67).
-
Results
Preclinical findings in the MASH/dyslipidemia hamster model showed that HTD1801 significantly improved histologic fibrosis and the Nonalcoholic Fatty Liver Disease Activity Score to such a degree that improvements approximated the appearance of the normal controls. In the clinical study, 52% of HTD1801-treated patients achieved MRI response criteria compared to 24% of placebo (P<0.05). Dose-dependent improvements were observed across biomarkers, with more HTD1801-treated patients achieving response criteria associated with improvements in the histologic features of MASH.
-
Conclusions
These findings suggest that HTD1801 has strong potential to produce histological improvements in patients with MASH.
-
Keywords: Noninvasive biomarkers; Liver histology; Metabolic dysfunction-associated steatohepatitis
Study Highlights
• In a secondary analysis of a Phase 2 clinical trial in patients with presumed MASH and T2DM HTD1801 demonstrated the potential to induce clinically relevant improvements in noninvasive markers closely correlated with the histologic features of MASH and fibrosis.
• In a preclinical model of MASH in hamsters, HTD1801 improved fibrosis, inflammation and ballooning such that histologic features resembled normal controls.
• In the clinical trial, 52% of HTD1801-treated patients achieved MRI response criteria compared to 24% of placebo (P<0.05).
• Dose-dependent improvements were observed across biomarkers, with more HTD1801-treated patients achieving thresholds correlated with histologic improvement and lower disease activity.
Graphical Abstract
INTRODUCTION
The complex and interrelated pathophysiology underlying metabolic liver diseases calls for treatments that address multiple targets. Metabolic dysfunction-associated steatohepatitis (MASH), a severe form of metabolic dysfunctionassociated steatotic liver disease (MASLD), is the hepatic manifestation of metabolic syndrome and is characterized by dysregulation of glucose, lipid and bile acid metabolism, and progressive fibrosis [
1]. The disease burden of MASLD is gradually increasing and parallels the obesity and type 2 diabetes (T2DM) epidemics [
1]. In patients with MASLD, concomitant cardiometabolic risk factors such as T2DM drastically increase the risk of both cardiovascular and liver-related mortality [
2].
Liver histology has been used as the surrogate endpoint to assess treatment response in late-stage clinical trials. However, there are well-reported limitations and associated risks with the invasive procedure [
3]. Additionally, many noninvasive tests have repeatedly been shown to predict major adverse liver outcomes as good as, if not better than, histologic fibrosis staging [
4,
5].
Promising imaging biomarkers include the use of magnetic resonance imaging proton density fat fraction (MRI PDFF) and iron-corrected T1 (cT1) mapping. MRI-PDFF is currently the gold standard for quantifying hepatic steatosis [
1]. In a meta-analysis of seven clinical studies (n=346), a ≥30% relative decline in liver fat content (LFC) by MRIPDFF was strongly associated with histologic response including a ≥2-point improvement in the Nonalcoholic Fatty Liver Disease Activity Score (NAS) with ≥1-point improvement in lobular inflammation or ballooning (odds ratio 6.98,
P<0.001) and MASH resolution (odds ratio 5.45,
P<0.01) [
6].
Liver cT1 has been shown to correlate with fibroinflammatory activity present in histology and has high diagnostic accuracy in identifying patients with MASH across the disease spectrum [
7]. In a cross-sectional investigation of 264 patients, it was estimated that an approximately 80 ms reduction in cT1 corresponded to a ≥2-point decrease in NAS with no worsening of fibrosis [
8]. In a follow-up analysis of data pooled from three clinical trials (n=150), a cT1 reduction of 80 ms was associated with a 5-fold increase in the odds of achieving MASH resolution [
9].
In addition to imaging, other noninvasive laboratorybased markers and weight loss have been associated with a histologic response in patients with MASH. A secondary analysis of the FLINT study found that a decrease in alanine aminotransferase (ALT) of ≥17 U/L was significantly associated with ≥2-point improvement in NAS with no worsening of fibrosis (odds ratio 11.0,
P<0.001) [
10]. Similarly, in a prospective evaluation on the impact of lifestyle changes on the histologic features of MASH, a reduction in body weight of ≥5% was associated with higher rates of resolution of steatohepatitis (58% vs. 10%,
P<0.001) and ≥2‑point improvement in NAS (82% vs. 32%,
P<0.001) [
11-
13]. Lastly, the fibrosis-4 index (FIB-4) was developed to predict the severity of liver fibrosis in lieu of performing a liver biopsy using routine laboratory assessments and, in an analysis of 541 adults with MASLD, was shown to be superior to other noninvasive markers of fibrosis [
12,
13]. A FIB-4 of ≤1.3 has been used to reliably exclude patients with advanced fibrosis with a negative predictive value of 88–95% [
13,
14].
Composite scores based on a combination of imaging and serum biomarkers have also been developed to better predict an improvement in histologic response. In a multivariable analysis of 108 patients, a ≥30% decrease in MRIPDFF and a ≥17 U/L decrease in ALT was a predictor of achieving a ≥2-point improvement in NAS with no worsening of fibrosis (adjusted odds ratio 11.3,
P<0.01) and MASH resolution (adjusted odds ratio 7.4,
P<0.01) [
15]. Loomba et al. [
16] used the strong association of ALT and MRI-PDFF with MASH resolution to develop a more robust noninvasive score, the MASH resolution index, which combines these with serum aspartate aminotransferase (AST). The MASH resolution index outperformed the individual ALT or MRIPDFF response criteria in predicting MASH resolution with an area under the receiver-operating characteristic curve of 0.83 compared to 0.58 and 0.76, respectively [
16].
Berberine ursodeoxycholate (HTD1801) is a gut-liver antiinflammatory metabolic modulator, a new molecular entity composed of an ionic salt of ursodeoxycholic acid and berberine that has the potential to offer a comprehensive benefit to diseases driven by insulin resistance and inflammation. With its unique dual mechanism of action (adenosine monophosphate kinase activation and NLRP3 inflammasome inhibition), HTD1801 was designed on the premise that it would have the benefits of both active moieties, but with improved and novel pharmacological effects resulting from its unique molecular structure. As a result, HTD1801 has the potential to become a novel treatment option for multiple chronic metabolic and liver diseases. The safety and efficacy of HTD1801 have been evaluated in Phase 1 and Phase 2 clinical studies globally and have demonstrated beneficial effects on glycemic parameters, lipid metabolism, markers of liver injury and cholestasis, body weight, and noninvasive markers of liver fibrosis [
17-
20].
In an 18-week study of HTD1801 in patients with presumed MASH and T2DM, patients treated with HTD1801 had significant reductions in LFC per MRI-PDFF, glycated hemoglobin (HbA1c), lipids, and body weight [
18]. While liver biopsies were not conducted in this study, several different non-invasive techniques have been evaluated to estimate the potential of HTD1801 on histologic improvements in patients with MASH. Noninvasive tests were selected based on a review of the most commonly used noninvasive biomarkers and treatment response criteria reported to be associated with histological improvements of MASH (steatosis, inflammation and fibrosis) and whose outcomes were also assessed in the present Phase 2 clinical study.
The purpose of this manuscript is to establish proof of concept of liver histologic improvement following treatment with HTD1801 based on histologic evaluation in a preclinical model of metabolic disease and assessment of noninvasive biomarkers associated with histologic improvements in patients with MASH.
MATERIALS AND METHODS
Preclinical study: hamster model of MASH and dyslipidemia
Male Syrian golden hamsters (n=32; Vital River Laboratories, Beijing, China) were utilized in a preliminary evaluation of histologic and biochemical efficacy of HTD1801. Hamsters received a high fat diet containing 1.25% cholesterol and 20% grease (2.79% soybean oil and 17.27% cocoa butter) for 2 weeks prior to treatment administration to induce MASH/dyslipidemia and maintained this diet through Week 6. After a 2-week high fat diet feeding period, prior to dosing and grouping, 4 hamsters were sacrificed for histological evaluation. The remaining hamsters were randomly stratified by total cholesterol level prior to treatment into the following groups: normal control (n=8), model control (n=8), and HTD1801 (high dose: 200 mg/kg, n=8 and low dose: 100 mg/kg, n=8). Hamsters designated to the normal control group received normal chow throughout the study period. Food intake was monitored for all groups throughout the study period. Hamsters received daily treatment for 6 weeks. Blood was drawn by orbital vein for assessment of biochemical parameters (AST, ALT, total bilirubin, low-density lipoprotein cholesterol [LDL-C], total cholesterol) at Day 0 and Week 2, 4 and 6. Livers were resected for analysis at Week 6 after the animals were sacrificed. Livers were sectioned (paraffin embedded blocks into 4 μm thickness sections) and stained with hematoxylin and eosin and Masson’s trichrome for evaluation of the NAS and the Nonalcoholic Steatohepatitis Clinical Research Network fibrosis score by a pathologist blinded to group assignment [
21]. The liver index was calculated as liver weight (g) divided by body weight (kg). All animal experiments were conducted with Laboratory Animal Management and Ethics Committee of Zhejiang University of Traditional Chinese Medicine approvals.
For statistical testing of biochemical parameters in the preclinical hamster model, an analysis of variance (ANOVA) was used to evaluate differences between groups. Only for biochemical parameters, the Grubbs test was used to determine the outliers for each group with a confidence interval of 97.5% [
22]. No more than one outlier was removed from each group. For histologic parameters in the preclinical hamster model, a non-parametric Kruskal–Wallis test was used. P-values less than 0.05 were considered to be significant. SPSS 22.0 software (IBM Co., Armonk, NY, USA) was used for statistical analyses and PRISM 9.5 (GraphPad Software, San Diego, CA, USA) was used for plotting data.
This secondary analysis used data from a Phase 2, randomized, double-blind, placebo-controlled study of the efficacy and safety of HTD1801 in patients with presumed MASH and T2DM. The primary results, including the study design and patient disposition were previously published by Harrison et al. [
18] (NCT03656744). In brief, 100 patients with presumed MASH based on an LFC ≥10%, cT1 ≥830 ms, AST ≥20 U/L, and a documented clinical diagnosis of T2DM (at least 6 months prior to randomization) were randomized 1:1:1 using permuted blocks without additional stratification factors and treated orally with placebo (n=33), HTD1801 500 mg BID (n=33), and HTD1801 1,000 mg BID (n=34) for 18 weeks. HTD1801 and placebo were provided as matching white tablets (supplied by HighTide Therapeutics Inc.). All patients, study personnel, and the sponsor were blinded to treatment assignment. MRIs were performed at baseline and at Week 18. Biochemistry and body weight were assessed at baseline and every visit throughout the study.
For this analysis, MRI response was defined as achieving either ≥30% reduction in LFC by MRI-PDFF or an improvement in fibroinflammation as determined by ≥80 ms reduction in cT1 at 18 weeks. MRI-PDFF data was collected prospectively for evaluation of the primary endpoint. A cT1 segmented analysis (Perspectum, Oxford, UK) was evaluated following completion of the study in patients who had been randomized to HTD1801 1,000 mg BID or placebo only.
FIB-4 was derived from a patient’s age, AST, ALT, and platelet count using the formula developed by Sterling et al. [
12] with a shift in FIB-4 from >1.3 at baseline to ≤1.3 at Week 18 representing an improvement in fibrosis stage [
13,
14].
The MASH resolution index, a composite score comprised of MRI-PDFF, ALT, and AST, was calculated using the equation published by Loomba et al. [
16] For this analysis, a histologic improvement was defined as achieving a MASH resolution index of ≥–0.67.
The clinical study was conducted in accordance with the Declaration of Helsinki 2013, approved by the institutional review boards and written informed consent was obtained from all participants.
Statistical analysis for the comparisons between active treatment groups and placebo were tested using an estimation based on a Cochran-Mantel-Haenszel model. Analyses were performed using SAS V9.4.
RESULTS
Preclinical study: hamster model of MASH and dyslipidemia
Macroscopic observations and histological evaluation prior to dosing for golden hamsters fed a high fat diet for 2 weeks are presented in
Supplementary Figure 1. Following dosing, the model control had marked elevation in aminotransferases, total bilirubin and lipids (
Fig. 1) compared with normal controls. Each of the markers of liver inflammation and hepatocellular damage (AST, ALT) were improved with HTD1801 treatment by Week 2, with significant mean improvements in the HTD1801 high dose group to a level resembling the normal controls at Week 6 (
Table 1). Furthermore, significant reductions with HTD1801 were also observed in key cardiometabolic parameters (LDL-C and total cholesterol) with mean LDL‑C approaching the same values as the normal controls. Additionally, a clear dose-dependent effect is observed with greater reductions across each biochemical parameter with the higher dose of HTD1801 approximating the level of the normal control (
Fig. 1).
Improvements in HTD1801 could be readily viewed in resected hamster livers without the aid of a microscope. As shown in Figure 2A, HTD1801-treated livers most closely resembled the normal control group macroscopically. The liver index in the model control increased significantly compared with the normal control (mean [standard error of means, SEM]: 51.9 g/kg [2.8] vs. 27.9 g/kg [1.1]; P<0.01). Significant reductions in the liver index were observed with HTD1801 100 mg/kg (33.5 g/kg [0.9]; P<0.01) and HTD1801 200 mg/kg (30.2 g/kg [0.6]; P<0.01) compared to the model control.
Following hematoxylin and eosin staining (
Fig. 2B), the structure of the liver in the normal control group was clear, the central vein of the liver was intact, the hepatic cords were arranged neatly, and the hepatic cells were normal in shape. In contrast, for the model control group, the boundary of hepatic lobules was unclear, hepatocytes were arranged mussily, cord structures disappeared, and a large number of hepatocytes showed swelling. Overall, the morphology in the HTD1801 groups improved compared to the model control group. In the HTD1801 200 mg/kg group, the hepatic lobular structure and cord structure were clear, and there was no obvious swelling of hepatocytes.
The model controls developed significant fibrosis, steatosis, inflammation, and ballooning compared to the normal controls (
Fig. 2C). Treatment with both doses of HTD1801 resulted in significant reductions in fibrosis (by approximately 1 stage) as well as improvements in each component of the NAS with almost no residual ballooning. A clear dose-dependent effect was observed with HTD1801 resulting in greater histologic improvements at the highest dose across each histologic parameter.
A total of 100 patients were randomized and received at least one dose of study drug (placebo n=33, HTD1801 500 mg BID n=33, and HTD1801 1,000 mg BID n=34) [
18]. Overall, 88 patients (87%) completed the study, 32 patients (97%) in the placebo group, 29 patients (85%) in the HTD1801 500 mg BID group and 27 patients (79%) in the HTD1801 1,000 mg BID group. The most common reason for study discontinuation was due to adverse events (6 patients), primarily gastrointestinal adverse events, followed by lost to follow-up [
18]. Demographic characteristics were generally well balanced across all three treatment groups (
Supplementary Table 1). The median age overall was 57 years (range: 26 to 75 years), and 72% were female. Most patients were White (91%) and not Hispanic or Latino (62%). Median baseline weight and body mass index were similar across treatment groups. Baseline values for HbA1c, ALT, γ-glutamyl transferase, LDL-C, triglycerides, and LFC were similar across treatment groups. As described in Harrison et al. [
18], the primary endpoint was achieved with a significant reduction in LFC (via MRIPDFF) with HTD1801 1,000 mg BID compared to placebo (–4.8% vs. –2.0%,
P=0.011). The beneficial effect of HTD1801 on liver fat was accompanied by improvements in liver biochemistry and weight (
Supplementary Table 2).
As shown in
Supplementary Table 3, treatment with HTD1801 was generally safe and well tolerated. More patients in the HTD1801 1,000 mg BID group experienced a treatment emergent adverse event during the study compared to placebo (26 [76%] vs. 20 [61%]). The most common adverse events were diarrhea and nausea which were generally mild to moderate in severity. During the study, 3 patients (3%) experienced serious adverse events of decreased oxygen saturation (1), myocardial infarction (1), and bladder transitional cell carcinoma (1) in the HTD1801 500 mg BID, 1,000 mg BID, and placebo groups, respectively. All serious adverse events were considered not related to study drug by the Investigator.
At baseline, patients receiving placebo and HTD1801 1,000 mg BID had elevated mean LFC by MRI-PDFF (20% and 19%, respectively) and cT1 (938 ms and 942 ms, respectively). As shown in
Figure 3 and
Table 2, after 18 weeks of treatment, the MRI response criteria (either MRIPDFF or cT1) was achieved by 52% in the HTD1801 group and 24% in the placebo group (
P=0.03). Of these patients, 22% in the HTD1801 group and 12% in the placebo group achieved both the MRI-PDFF and cT1 response criteria.
In an analysis of biochemical and anthropometric response criteria, treatment with HTD1801 resulted in a larger proportion of patients achieving thresholds associated with a ≥2-point improvement in NAS (
Fig. 4A) or fibrosis (
Fig. 4B) compared to placebo (
Table 2). A clear dose response was observed in each criterion evaluated with HTD1801 treatment. For HTD1801 1,000 mg BID compared to placebo, a ≥17 U/L reduction in ALT was achieved by 12 patients (44%) vs. 3 (9%); a ≥5% reduction in body weight was achieved by 6 patients (22%) vs. 3 (9%); and a shift in FIB-4 to <1.3 was achieved by 3 patients (27%) vs. 1 (8%).
More patients treated with HTD1801 compared to placebo achieved the composite response criteria of MRI-PDFF and ALT which is associated with achieving ≥2-point improvement in NAS and MASH resolution. A dose-dependent effect was observed, with 7% (n=2) and 33% (n=9) of patients in the HTD1801 500 mg BID and 1,000 mg BID group, respectively, achieving both a ≥30% reduction in LFC and a ≥17 U/L reduction in ALT compared to none in the placebo group (
Fig. 5A and
Table 2).
Additionally, more than 3 times as many patients treated with HTD1801 1,000 mg BID (41%) than placebo (12%) achieved MASH resolution index ≥–0.67 (
Fig. 5B and
Table 2).
DISCUSSION
Using a preclinical model and secondary analysis of a Phase 2 clinical study with consistent findings across multiple noninvasive surrogates, HTD1801 demonstrated the potential to induce clinically relevant histologic improvements in patients with MASH.
Preclinical findings in the MASH/dyslipidemia hamster model showed that HTD1801 significantly reduced ALT, AST, total bilirubin, LDL-C and total cholesterol after 6 weeks of treatment. Furthermore, HTD1801 improved histology including fibrosis and NAS to such a degree that the final histologic features were similar to control animals fed with normal chow. The improvements in liver size and steatosis were also obvious macroscopically.
This secondary analysis of a randomized, placebo-controlled Phase 2 study in patients with presumed MASH examined biomarkers and treatment response criteria that are associated with improvements across the spectrum of histologic features of MASH including steatosis (MRI-PDFF and weight loss), inflammation (cT1, MRI-PDFF, ALT, and weight loss) and fibrosis (cT1 and FIB-4). The MRI response criteria was achieved by twice as many patients treated with HTD1801 as placebo, with nearly half in the HTD1801 1,000 mg BID group achieving response criteria in both LFC and cT1. Dose-dependent improvements were observed across biomarkers, with more HTD1801-treated patients achieving thresholds correlated with histologic improvement and lower disease activity. Treatment with HTD1801 was generally safe and well tolerated. Mild to moderate diarrhea and nausea were the most frequently reported events and no serious adverse events were considered related to study drug.
HTD1801 is believed to work through multiple pathways that address the core aspects of MASH, including activation of adenosine monophosphate kinase, inhibition of the NLRP3 inflammasome, induction of the insulin receptor, and induction of the LDL receptor. The results on fibrosis and metabolic control are consistent with adenosine monophosphate kinase activation and suppression of NLRP3 inflammasomes— addressing MASH fibrosis in two ways— improving the metabolic drivers of disease and potentially a direct anti-fibrotic effect through suppressing NLRP3, which is an inflammatory pathway reported to be tied to fibrogenesis [
23].
In MASLD, biomarkers are generally developed for diagnostic, prognostic, monitoring, and response assessment purposes [
24]. While numerous studies have confirmed the diagnostic and prognostic capabilities of various noninvasive tests, few have been validated as response biomarkers. This limitation arises because developing response biomarkers necessitates not only serial noninvasive tests paired with liver biopsies but also effective treatment between assessments. Among the proposed response biomarkers, an MRI-PDFF response of ≥30% relative reduction has the most robust evidence base, validated in clinical trials such as the Phase 3 MAESTRO-NASH study for resmetirom [
25]. However, it is crucial to understand that MRIPDFF is a steatosis marker, with its association to improvements in inflammation and fibrosis being correlational. Conversely, while weight reduction has been linked to MASH resolution and fibrosis improvement in natural history or lifestyle intervention cohorts [
11,
26], the relationship between weight loss and histologic response may become dissociated when a patient is undergoing effective drug treatment for MASH. Additionally, some biomarkers, such as liver stiffness measurement by vibration-controlled transient elastography, are influenced by weight changes, further complicating their use as response biomarkers [
27]. Lastly, liver biopsies themselves are an imperfect reference standard for assessing treatment response in MASH, as imprecision in histologic scoring may result in dilution of treatment effect size and misevaluation of therapeutic effects [
28]. Thus, consistency in response across multiple biomarkers would enhance confidence in identifying genuine therapeutic effects, as demonstrated in the current study.
Currently, the only pharmacological treatment to receive approval by the United States Food and Drug Administration for the treatment of MASH is resmetirom, a small molecule thyroid hormone receptor beta agonist. In the pivotal Phase 3 study, treatment with resmetirom resulted in histologic improvements in MASH and fibrosis. While histologic data are not yet available from clinical studies of HTD1801, treatment with HTD1801 may provide a broader metabolic benefit, including significant improvements in glycemic control and weight loss, which have not been shown with resmetirom [
18,
20].
Limitations of this evaluation include the post hoc nature of the analysis of the clinical study, short treatment duration with HTD1801 (only 18 weeks), and limited number of biomarkers/imaging tools evaluated. Additionally, in this study the presumption of MASH was based on the combination of LFC ≥10%, cT1 ≥830 ms, and AST ≥20 U/L. Although these thresholds have been correlated with the presence of high-risk MASH and are higher than the clinical diagnostic criteria for MASLD, as liver biopsies were not performed, the presence of MASH was presumed [
1,
7]. The strengths of this evaluation include the fact that the data are from a randomized, placebo-controlled, multi-site clinical study.
While these data are preliminary, it suggests that ongoing treatment with HTD1801 would be expected to result in improvements in the key histologic targets in patients with MASH. A Phase 2b study is currently ongoing to evaluate the histologic effects of HTD1801 in patients with MASH and T2DM or prediabetes (NCT05623189).
FOOTNOTES
-
Authors’ contributions
Conception and design: Di Bisceglie, Bai, Cheng, Yu, Liberman, Liu. Acquisition, analysis or interpretation of the data: Wong, Neff, Di Bisceglie, Bai, Cheng, Yu, Liberman, Gunn. Drafting of the manuscript: Wong, Bai, Liberman. Critical revision of the manuscript: all authors. Final approval: all authors.
-
Acknowledgements
The authors would like to acknowledge Dr. Minli Chen’s lab from Zhejiang Chinese Medical University for their contributions to this study. Additionally, the authors would like to acknowledge the patients and investigators, including Dr. Stephen Harrison (Lead Principal Investigator), who participated in the Phase 2 study of HTD1801 in MASH and T2DM (NCT03656744). Statistical and programming support was provided by Pacific Northwest Statistical Consulting (Abigail Flyer) and Hangzhou TigerMed Consulting Co. (Xiaoxia Wu). Medical writing support was provided by Kjersti Swearingen of HighTide Therapeutics Inc. The preclinical and clinical studies were funded by HighTide Therapeutics, Inc.
-
Conflicts of Interest
Vincent Wong served as a consultant or advisory board member for AbbVie, AstraZeneca, Boehringer Ingelheim, Echosens, Eli Lilly, Gilead Sciences, Intercept, Inventiva, Merck, Novo Nordisk, Pfizer, Sagimet Biosciences, TARGET PharmaSolutions, and Visirna; and a speaker for Abbott, AbbVie, Echosens, Gilead Sciences, Novo Nordisk, and Unilab. He has received a research grant from Gilead Sciences, and is a co-founder of Illuminatio Medical Technology. Adrian M. Di Bisceglie has received consulting fees from HighTide Therapeutics, Inc. and Intercept Pharmaceuticals, Inc. Ru Bai, Junwei Cheng, Meng Yu, Alexander Liberman, and Liping Liu are employees and stock shareholders of HighTide Therapeutics, Inc.
SUPPLEMENTARY MATERIAL
Supplementary material is available at Clinical and Molecular Hepatology website (
http://www.e-cmh.org).
Supplementary Figure 1.
Macroscopic and histologic evaluation of hamster livers after 2 weeks of a high fat diet. Golden hamster liver tissues: (A) macroscopic observations (representative photos); (B) hematoxylin and eosin staining where the nucleus is blue, and the cytoplasm and intercellular substance were pink (representative photos); (C) fibrosis score and Nonalcoholic Fatty Liver Disease Activity Score. Fibrosis scores were based on the Nonalcoholic Steatohepatitis Clinical Research Network staging criteria. Analysis was performed with a non-parametric Kruskal–Wallis test.
Figure 1.Liver biochemistry and lipids after 6 weeks of treatment in hamsters fed with a high fat diet. Outliers were determined using the Grubbs test and excluded from the analysis. An analysis of variance was used to evaluate differences between groups. *P<0.05, **P<0.01 vs. the model control group. ##P<0.01 vs. the normal control group. ALT, alanine aminotransferase; AST, aspartate aminotransferase; HTD1801, berberine ursodeoxycholate; LDL-C, low-density lipoprotein-cholesterol; SEM, standard error of the mean.
Figure 2.Macroscopic and histologic evaluation of hamster livers after 6 weeks of treatment. Golden hamster liver tissues: (A) macroscopic observations (representative photos); (B) hematoxylin and eosin staining where the nucleus is blue, and the cytoplasm and intercellular substance were pink; (C) change in fibrosis score and Nonalcoholic Fatty Liver Disease Activity Score. Fibrosis scores were based on the Nonalcoholic Steatohepatitis Clinical Research Network staging criteria. Analysis was performed with a non-parametric Krusakal–Wallis test. *P<0.05, **P<0.01 vs. the model control group. ##P<0.01 vs. the normal control group. HTD1801, berberine ursodeoxycholate; NAFLD, nonalcoholic fatty liver disease; SEM, standard error of the mean.
Figure 3.Percentage of patients achieving MRI response criteria. Percentage of patients achieving an MRI response defined as achieving either ≥30% reduction in liver fat content by MRI-PDFF or an improvement in fibroinflammation as determined by ≥80 ms reduction in cT1 at 18 weeks. CMH model was used for statistical analysis. BID, twice daily; CMH, Cochran–Mantel–Haenszel; cT1, corrected T1; HTD1801, berberine ursodeoxycholate; MRI, magnetic resonance imaging; MRI-PDFF, MRI-proton density fat fraction.
Figure 4.Percentage of patients achieving the biochemical and anthropometric response criteria. Percentage of patients achieving biochemical and anthropometric response criteria associated with a (A) ≥2 point improvement in NAS and (B) fibrosis improvement, which was limited to patients with a FIB-4 ≥1.3 at baseline. CMH model was used for statistical analysis. ALT, alanine aminotransferase; BID, twice daily; CMH, Cochran–Mantel–Haenszel; FIB-4, fibrosis-4 index; HTD1801, berberine ursodeoxycholate; NAFLD, nonalcoholic fatty liver disease; NAS, NAFLD Activity Score.
Figure 5.Percentage of patients achieving composite response criteria. Percentage of patients achieving composite response criteria associated with a (A) ≥2 point improvement in NAS and (B) MASH resolution. CMH model was used for statistical analysis. ALT, alanine aminotransferase; BID, twice daily; CMH, Cochran–Mantel–Haenszel; HTD1801, berberine ursodeoxycholate; LFC, liver fat content; MASH, metabolic dysfunction-associated steatohepatitis; NAFLD, nonalcoholic fatty liver disease; NAS, NAFLD Activity Score.
Table 1.ALT and AST over time - preclinical hamster model of MASH/dyslipidemia
Table 1.
|
Mean±SEM |
Normal control |
Model control |
HTD1801 (100 mg/kg) |
HTD1801 (200 mg/kg) |
|
ALT (mmol/L) |
|
|
|
|
|
Baseline |
85.8±5.0 |
1,423.6±284.9‡
|
1,419.8±220.7 |
1,421.6±217.3 |
|
Week 2 |
93.3±8.1 |
1,160.0±242.1‡
|
165.0±30.5**
|
233.3±53.4**
|
|
Week 4 |
147.6±46.1 |
959.9±114.8‡
|
489.4±125.4*
|
165.3±35.6**
|
|
Week 6 |
86.1±15.4 |
1,334.7±133.0‡
|
754.9±134.8*
|
96.6±17.5**
|
|
AST (mmol/L) |
|
|
|
|
|
Baseline |
52.6±3.7 |
413.6±105.0‡
|
366.2±80.2 |
305.3±64.5 |
|
Week 2 |
48.1±2.9 |
312.4±57.6‡
|
61.9±13.4**
|
81.7±7.6**
|
|
Week 4 |
49.3±6.7 |
204.5±27.8‡
|
117.2±16.8*
|
73.1±9.6**
|
|
Week 6 |
66.0±2.9 |
270.60±25.3‡
|
147.7±21.0 |
66.4±4.6**
|
Table 2.Proportion of patients achieving noninvasive biomarker response at week 18 - Phase 2 study in MASH
Table 2.
|
Proportion of patients achieving |
Placebo (n=33) |
HTD1801
|
|
500 mg BID (n=33) |
1,000 mg BID (n=34) |
|
≥30% reduction in MRI-PDFF only |
33 |
NA*
|
27 |
|
Number (%) |
3 (9.1) |
NA*
|
4 (14.8) |
|
≥80 ms reduction in cT1 only |
33 |
NA*
|
27 |
|
Number (%) |
1 (3.0) |
NA*
|
4 (14.8) |
|
Both MRI and cT1 response |
33 |
NA*
|
27 |
|
Number (%) |
4 (12.1) |
NA*
|
6 (22.2) |
|
MRI or cT1 response |
33 |
NA*
|
27 |
|
Number (%) |
8 (24.2) |
NA*
|
14 (51.9) |
|
95% CI†
|
- |
NA*
|
1.12, 10.08 |
|
P-value†
|
- |
NA*
|
0.029 |
|
≥17 U/L reduction in ALT |
33 |
30 |
27 |
|
Number (%) |
3 (9.1) |
6 (20.0) |
12 (44.4) |
|
95% CI†
|
- |
0.57, 11.05 |
1.96, 32.73 |
|
P-value†
|
- |
0.220 |
0.002 |
|
≥5% reduction in body weight |
33 |
30 |
27 |
|
Number (%) |
3 (9.1) |
4 (13.3) |
6 (22.2) |
|
95% CI†
|
- |
0.31, 7.52 |
0.64, 12.73 |
|
P-value†
|
- |
0.596 |
0.160 |
|
Shift in FIB-4 to <1.3 |
13 |
14 |
11 |
|
Number (%) |
1 (7.7) |
3 (21.4) |
3 (27.3) |
|
95% CI†
|
- |
0.29, 36.31 |
0.39, 51.30 |
|
P-value†
|
- |
0.325 |
0.209 |
|
≥30% reduction in MRI-PDFF and a ≥17 U/L reduction in ALT |
33 |
30 |
27 |
|
Number (%) |
0 |
2 (6.7) |
9 (33.3) |
|
95% CI†
|
- |
NR |
NR |
|
P-value†
|
- |
0.135 |
0.0004 |
|
MASH resolution index ≥–0.67 |
33 |
30 |
27 |
|
Number (%) |
4 (12.1) |
7 (23.3) |
11 (40.7) |
|
95% CI†
|
- |
0.57, 8.47 |
1.36, 18.23 |
|
P-value†
|
- |
0.246 |
0.012 |
Abbreviations
aspartate aminotransferase
berberine ursodeoxycholate
low-density lipoprotein cholesterol
metabolic dysfunction-associated steatohepatitis
metabolic dysfunction-associated steatotic liver disease
magnetic resonance imaging
MRI-proton density fat fraction
Nonalcoholic Fatty Liver Disease Activity Score
REFERENCES
- 1. Rinella ME, Neuschwander-Tetri BA, Siddiqui MS, Abdelmalek MF, Caldwell S, Barb D, et al. AASLD Practice Guidance on the clinical assessment and management of nonalcoholic fatty liver disease. Hepatology 2023;77:1797-1835.
- 2. Golabi P, Otgonsuren M, de Avila L, Sayiner M, Rafiq N, Younossi ZM. Components of metabolic syndrome increase the risk of mortality in nonalcoholic fatty liver disease (NAFLD). Medicine (Baltimore) 2018;97:e0214.
- 3. Tong XF, Wang QY, Zhao XY, Sun YM, Wu XN, Yang LL, et al. Histological assessment based on liver biopsy: the value and challenges in NASH drug development. Acta Pharmacol Sin 2022;43:1200-1209.
- 4. Lin H, Lee HW, Yip TC, Tsochatzis E, Petta S, Bugianesi E, et al. Vibration-controlled transient elastography scores to predict liver-related events in steatotic liver disease. Jama 2024;331:1287-1297.
- 5. Mózes FE, Lee JA, Vali Y, Alzoubi O, Staufer K, Trauner M, et al. Performance of non-invasive tests and histology for the prediction of clinical outcomes in patients with non-alcoholic fatty liver disease: an individual participant data meta-analysis. Lancet Gastroenterol Hepatol 2023;8:704-713.
- 6. Stine JG, Munaganuru N, Barnard A, Wang JL, Kaulback K, Argo CK, et al. Change in MRI-PDFF and histologic response in patients with nonalcoholic steatohepatitis: a systematic review and meta-analysis. Clin Gastroenterol Hepatol 2021;19:2274-2283.e2275.
- 7. Andersson A, Kelly M, Imajo K, Nakajima A, Fallowfield JA, Hirschfield G, et al. Clinical utility of magnetic resonance imaging biomarkers for identifying nonalcoholic steatohepatitis patients at high risk of progression: a multicenter pooled data and meta-analysis. Clin Gastroenterol Hepatol 2022;20:2451-2461.e2453.
- 8. Dennis A, Kelly MD, Fernandes C, Mouchti S, Fallowfield JA, Hirschfield G, et al. Correlations between MRI biomarkers PDFF and cT1 with histopathological features of non-alcoholic steatohepatitis. Front Endocrinol (Lausanne) 2020;11:575843.
- 9. Alkhouri N, Beyer C, Shumbayawonda E, Andersson A, Yale K, Rolph T, et al. Decreases in cT1 and liver fat content reflect treatment-induced histological improvements in MASH. J Hepatol 2025;82:438-445.
- 10. Loomba R, Sanyal AJ, Kowdley KV, Terrault N, Chalasani NP, Abdelmalek MF, et al. Factors associated with histologic response in adult patients with nonalcoholic steatohepatitis. Gastroenterology 2019;156:88-95.e85.
- 11. Vilar-Gomez E, Martinez-Perez Y, Calzadilla-Bertot L, Torres-Gonzalez A, Gra-Oramas B, Gonzalez-Fabian L, et al. Weight loss through lifestyle modification significantly reduces features of nonalcoholic steatohepatitis. Gastroenterology 2015;149:367-378.e365 quiz e314-365.
- 12. Sterling RK, Lissen E, Clumeck N, Sola R, Correa MC, Montaner J, et al. Development of a simple noninvasive index to predict significant fibrosis in patients with HIV/HCV coinfection. Hepatology 2006;43:1317-1325.
- 13. Shah AG, Lydecker A, Murray K, Tetri BN, Contos MJ, Sanyal AJ. Comparison of noninvasive markers of fibrosis in patients with nonalcoholic fatty liver disease. Clin Gastroenterol Hepatol 2009;7:1104-1112.
- 14. McPherson S, Stewart SF, Henderson E, Burt AD, Day CP. Simple non-invasive fibrosis scoring systems can reliably exclude advanced fibrosis in patients with non-alcoholic fatty liver disease. Gut 2010;59:1265-1269.
- 15. Huang DQ, Sharpton SR, Amangurbanova M, Tamaki N, Sirlin CB, Loomba R. Clinical utility of combined MRI-PDFF and ALT response in predicting histologic response in nonalcoholic fatty liver disease. Clin Gastroenterol Hepatol 2023;21:2682-2685.e2684.
- 16. Loomba R, Amangurbanova M, Bettencourt R, Madamba E, Siddiqi H, Richards L, et al. MASH resolution index: development and validation of a non-invasive score to detect histological resolution of MASH. Gut 2024;73:1343-1349.
- 17. Di Bisceglie AM, Watts GF, Lavin P, Yu M, Bai R, Liu L. Pharmacokinetics and pharmacodynamics of HTD1801 (berberine ursodeoxycholate, BUDCA) in patients with hyperlipidemia. Lipids Health Dis 2020;19:239.
- 18. Harrison SA, Gunn N, Neff GW, Kohli A, Liu L, Flyer A, et al. A phase 2, proof of concept, randomised controlled trial of berberine ursodeoxycholate in patients with presumed nonalcoholic steatohepatitis and type 2 diabetes. Nat Commun 2021;12:5503.
- 19. Kowdley KV, Forman L, Eksteen B, Gunn N, Sundaram V, Landis C, et al. A randomized, dose-finding, proof-of-concept study of berberine ursodeoxycholate in patients with primary sclerosing cholangitis. Am J Gastroenterol 2022;117:1805-1815.
- 20. Ji L, Ma J, Ma Y, Cheng Z, Gan S, Yuan G, et al. Berberine ursodeoxycholate for the treatment of type 2 diabetes: a randomized clinical trial. JAMA Netw Open 2025;8:e2462185.
- 21. Brunt EM, Kleiner DE, Wilson LA, Belt P, Neuschwander-Tetri BA. Nonalcoholic fatty liver disease (NAFLD) activity score and the histopathologic diagnosis in NAFLD: distinct clinicopathologic meanings. Hepatology 2011;53:810-820.
- 22. Grubbs FE. Procedures for detecting outlying observations in samples. Technometrics 1969;11:1-21.
- 23. Zhang WJ, Chen SJ, Zhou SC, Wu SZ, Wang H. Inflammasomes and fibrosis. Front Immunol 2021;12:643149.
- 24. Sanyal AJ, Castera L, Wong VW. Noninvasive assessment of liver fibrosis in NAFLD. Clin Gastroenterol Hepatol 2023;21:2026-2039.
- 25. Harrison SA, Bedossa P, Guy CD, Schattenberg JM, Loomba R, Taub R, et al. A phase 3, randomized, controlled trial of resmetirom in NASH with liver fibrosis. N Engl J Med 2024;390:497-509.
- 26. Wong VW, Wong GL, Chan RS, Shu SS, Cheung BH, Li LS, et al. Beneficial effects of lifestyle intervention in non-obese patients with non-alcoholic fatty liver disease. J Hepatol 2018;69:1349-1356.
- 27. Wai-Sun Wong V, Anstee QM, Nitze LM, Geerts A, George J, Nolasco V, et al. FibroScan-aspartate aminotransferase (FAST) score for monitoring histological improvement in non-alcoholic steatohepatitis activity during semaglutide treatment: post-hoc analysis of a randomised, double-blind, placebo-controlled, phase 2b trial. EClinicalMedicine 2023;66:102310.
- 28. Shah A, MacConell L, Liberman A, Di Bisceglie AM, Shapiro D. Challenges in histological endpoints for MASH therapies: an exercise in statistical modelling. Aliment Pharmacol Ther 2025;61:1489-1499.
, Guy W. Neff2, Adrian M. Di Bisceglie3, Ru Bai4, Junwei Cheng4, Meng Yu4, Alexander Liberman3, Liping Liu4, Nadege Gunn5
