All animal experiments conformed to the internationally accepted principles for the care and use of laboratory animals (license no. 2013-15-2934-00784, The Animal Experiments Inspectorate, Denmark). C57Bl6/J and B6.V-Lep^ob/JRj (ob/ob) mice (5-6 wk-old) were from Janvier Labs (Le Genest Saint Isle, France) and housed in a controlled environment (12 h light/dark cycle, light on at 3 AM, 21 °C ± 2 °C, humidity 50% ± 10%). Each animal was identified by an implantable microchip (PetID Microchip, E-vet, Haderslev, Denmark). Mice had ad libitum access to tap water and either regular rodent chow (C57Bl6/J mice, Altromin 1324, Brogaarden, Hoersholm, Denmark), or a diet high in fat (ob/ob mice, 40%, containing 18% trans-fat), 40% carbohydrates (20% fructose) and 2% cholesterol (AMLN diet; D09100301, Research Diets, New Brunswick, NJ)[19]. ob/ob mice (n = 87) were fed the AMLN diet for 9 or 15 wk prior to treatment start and during drug treatment. All ob/ob animals underwent liver biopsy prior to treatment, see below.

The biopsy procedure was applied to all mice approximately three weeks before completion of the dieting period, as detailed previously[19]. In brief, mice were anesthetized with isoflurane, a small abdominal incision in the midline was made, and the left lateral lobe of the liver was exposed. A cone-shaped wedge of liver tissue (50-100 mg) was excised from the distal part of the lobe. The cut surface of the liver was closed by electrosurgical bipolar coagulation. The liver was returned to the abdominal cavity, the abdominal wall was sutured and skin stapled. Carprofen (Rimadyl^®, 5 mg/mL, 0.01 mL/10 g; Pfizer, NY) and enrofloxazin (5 mg/mL, 1 mL/kg, i.p.) were administered at the time of surgery and at post-operative day one and two. Animals were single-housed after the procedure and recovered for three weeks prior to treatment.

INT-767 and OCA (Intercept Pharmaceuticals, New York, NY, United States) were dissolved in 0.5% carboxymethyl cellulose, and orally administered in a dosing volume of 5 mL/kg. Animals were stratified (n = 11-12 per group) based on mean fibrosis as assessed by collagen 1a1 staining. In one study, after 15 wk on diet mice were treated with INT-767 (3.0 or 10 mg/kg) for 8 wk. In a second study, after 9 wk on diet mice were treated with either vehicle, INT-767 (3.0 or 10 mg/kg), or OCA (10 or 30 mg/kg) for 16 wk. A longer duration of administration was selected for this second study to (a) examine the durability of INT-767 NASH histological improvements, and (b) because in our experience OCA requires extended dosing (e.g., at least 12 wk at 10-30 mg/kg) to elicit histological anti-fibrotic responses (data on file) and shorter treatment periods with OCA are not always sufficient to promote antifibrotic effects in ob/ob-NASH mice[20]. For analysis of liver and intestinal compound levels, as well as hepatic gene expression, lean C57Bl6 mice received INT-767 (1-3-10-30 mg/kg, PO, QD) or OCA (1-3-10-30 mg/kg, PO, QD) for 14 d.

Body weight was monitored once daily during the intervention period. Whole-body fat mass was analyzed at baseline (week -1) and week 8 of the intervention period by non-invasive EchoMRI scanning using EchoMRI-900 (EchoMRI, Houston, TX).

Plasma and liver biochemistry was assessed according to methods described in detail previously[19]. Baseline and terminal blood samples from non-fasted mice were assayed for blood glucose levels as well as plasma concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), total cholesterol (TC), and insulin. Terminal liver samples (about 100 mg) were analyzed for TG and TC content.

An oral glucose tolerance test (OGTT) was performed in week 4 of the treatment period according to previously reported procedures[19]. In brief, animals were fasted for 4 h prior to the OGTT. At t = 0 an oral glucose load (2 g glucose/kg) was administered via oral gavage. Succesive blood samples for measuring blood glucose (BG) were collected from the tail vein at t = 0, 15, 30, 60 and 120 min. Glucose area-under-the-curve (AUC) was determined from the sampling period of 0 to 120 min.

Baseline liver biopsy and terminal samples (both from the left lateral lobe) were fixed overnight in 4% paraformaldehyde. Liver tissue was paraffin-embedded and sectioned (3 μm thickness). Sections were stained with hematoxylin-eosin (HE), PicroSirius Red (PSR, Sigma-Aldrich, Broendby, Denmark), anti-type I collagen (Col1a1; cat. 1310-01, Southern Biotech, Birmingham, AL), anti-galectin-3 (cat. 125402, Biolegend, San Diego, CA, United States), or anti-laminin (cat. Z0097, Agilent Technologies, Glostrup, Denmark) using standard procedures[19]. The NAFLD activity score (NAS) and fibrosis staging system was applied for scoring of steatosis (score 0-3), lobular inflammation (score 0-3), hepatocyte ballooning (score 0-2), and fibrosis (stage 0-4)[21]. Mice with fibrosis stage ≥ 1 and steatosis score ≥ 2 were included in the study. Liver sections stained for fat, Col1a1, galectin-3 and laminin were analyzed using digital imaging software (Visiomorph^®, Visiopharm, Hørsholm, Denmark). Immunoreactive fractional area was expressed relative (%) to total parenchymal area by subtracting corresponding fat area determined on adjacent HE-sections. In addition, deparaffinized and unstained tissue sections of similar thickness where imaged by second harmonic generation (SHG, label free, specific to collagen 1 and 3, in transmission at 780 nm) and concurrently by 2-photon fluorescence excitation (2-PE at 780 nm, collected at 550 nm ± 88 nm to delineate the tissue structure). Both optical methods were performed on a mid-throughput, fully automated, nonlinear optical imaging system at 20 X objective and 0.39 μm resolution (Genesis 200^®, HistoIndex, Singapore), as described previously[22]. Fat droplets appear as circular dark objects (no intrinsic fluorescence) and can thus be segregated from the parenchyma and quantified (droplet count, area, eccentricity, and size), Collagen fiber data (area, density, reticulation index) were expressed relative (%) to corresponding parenchymal area. The collagen fiber network complexity (reticulation index) was expressed, as the ratio of skeleton fibers nodes to the total length of the equivalent skeleton. Imaging parameters (laser intensity, photomultiplier tube gains, scanning) and image analysis parameters (signal intensity and morphometric thresholding and pruning) where kept constant for the entire study after optimization. SHG was optimized to use the full 8-bit dynamic range of the instrument and avoid saturation. All histological assessments were performed by pathologists and operators blinded to the experimental groups.

A mass dependent volume of homogenization solution (seven volumes of 80/20 ACN/HBSS) was added to each tube containing a pre-weighed, intact tissue specimen. Tissue samples were homogenized using an Omni Bead Ruptor 24 tissue homogenizer set to 4 m/s for two cycles of 30 s each. Sample tubes were centrifuged at 3500 rcf for 3 min and a 200 μL aliquot of supernatant was transferred to a 96 deep-well plate. Samples were dried down under nitrogen and reconstituted in 60/40 methanol/10 mmol/L ammonium acetate containing 25 nmol/L of d5-GCA internal standard. Reconstituted samples were transferred to a Millipore 0.45 μm filter plate (Millipore MSHVN45) and filtered into a Costar 3957 plate by centrifugation and sealed with a silicone capmat prior to LC-MS/MS analysis. This extract was injected onto an UHPLC system equipped with a ultra-high resolution, accurate mass, mass spectrometer (UHRAM) (Thermo Fisher Scientific Q Exactive with Ion Max M2 with HESI-II probe) detector operated in negative electrospray mode. Separation of test article from extracted matrix materials was accomplished using a Thermo Fisher Hypersil Gold C18 column (1 mm x 100 mm, 1.9 μm particle size) operating at 45 °C. The gradient mobile phase system consisted of 0.25 mmol/L ammonium acetate in 40% methanol (mobile phase A) and 0.25 mmol/L ammonium acetate in 95% methanol (mobile phase B) at a total flow rate of 0.100 mL/min.

Twenty analytical runs were performed during the study sample analysis. Calibration standards, prepared in matrix matched mouse control tissues purchased from BioreclamationIVT (Baltimore, MD), were used to construct standard curves for all analytes. Typically, quadratic weighted (1/x) regression analysis of peak area ratio vs theoretical concentration was used to produce calibration curves.

Hepatic and ileal transcriptome analysis was performed by RNA sequencing on RNA extracts from terminal liver samples, as described[19]. RNA sequence libraries were prepared with NeoPrep (Illumina, San Diego, CA, United States) using Illumina TruSeq stranded mRNA Library kit for NeoPrep (Illumina, San Diego, CA, United States) and sequenced on the NextSeq 500 (Illumina, San Diego, CA) with NSQ 500 hi-Output KT v2 (75 CYS, Illumina, San Diego, CA, United States). Reads were aligned to the GRCm38 v84 Ensembl Mus musculus genome using STAR v.2.5.2a with default parameters[23]. Differential gene expression analysis was performed with DEseq215[24].

All data were analyzed using GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, United States). Results are presented as mean ± SE. A chi-square (χ²) test was used to test for within-subject changes in qualitative histology scores before and after treatment, compared to vehicle controls. Responders were defined as having a ≥ 1 point change in the indicated score. An unpaired two-tailed t-test was applied to quantitative histological analyses after treatment. A two-way ANOVA with Bonferroni’s post-hoc test was used for analysis of changes in gene expression and quantitative histology analytes (fractional area of fat, galectin-3, and Col1a1) before and after treatment, compared to vehicle controls. A P-value less than 0.05 was considered statistically significant.

All ob/ob-NASH mice included in the experiment had liver biopsy-confirmed NASH (NAS 5-7) and fibrosis (stage 1-4) prior to initiation of treatment (Table 1).

INT-767 promoted dose-dependent improvements in liver histopathology after eight wk of treatment in ob/ob-NASH mice. INT-767 10 mg/kg significantly reduced steatosis, inflammation and hepatocyte ballooning degeneration scores compared to vehicle controls (Table 1). All INT-767 (10 mg/kg) treated mice exhibited reductions in NAS and fibrosis stage (Figure 1, Table 1). In contrast, ob/ob-NASH control mice maintained composite NAS and fibrosis scores compared to pre-treatment levels. INT-767 also reduced quantitative hepatic collagen and fat, as indicated by lowered Col1a1 immunoreactivity (Figure 2B) and liver fat area (Figure 3B). This reduction of collagen and fat by INT-767 was also shown in the IHC staining images (Figures 2A and 3A, respectively) and confirmed by label free SHG/2-PE images and processed images for fat content (Figures 2C and 3C, respectively).

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Figure 1 INT-767 treatment for 8 wk improves liver histopathology in ob/ob-NASH mice with biopsy-confirmed liver pathology. A: Composite NAS and fibrosis stage before and after treatment intervention; B: Representative HE and PSR stainings. ^aP < 0.05, ^bP < 0.001 (χ² test, vs vehicle controls).

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Figure 2 INT-767 treatment for 8 wk reduces hepatic collagen deposition in ob/ob-NASH mice with biopsy-confirmed liver pathology. A: Collagen 1a1 (immunohistochemistry); B: Fractional area of collagen (immunohistochemistry); C: Label free SHG/2-PE images for collagen fiber deposition (green) in hepatic parenchyma (red). Data are expressed as % of total parenchymal area (subtraction of fat area); D: Fractional area of collagen fiber, collagen fiber density, and collagen fiber reticulation index (SHG analysis). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 (unpaired t-test).

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Figure 3 INT-767 treatment for 8 wk reduces hepatic lipid deposition in ob/ob-NASH mice with biopsy-confirmed liver pathology. A: Liver lipid (HE staining); B: Fractional area of liver fat (HE staining); C: Processed (color coded) images to display hepatic lipid content. Lipid droplets (white), rejected structures (yellow), collagen (green), auto-fluorescence (blue); D: Fractional area of liver lipid area ratio (%), lipid droplet number (count in 15 mm² tissue area), and lipid droplet area (1 pixel = 0.39 μm, 2-PE analysis). ^bP < 0.001 (unpaired t-test).

SHG/2-PE morphometric assessments of collagen were positively correlated with Col1a1 assessment by immunohistochemistry (r² = 0.88, P <0.0001) and demonstrated that INT-767 predominantly impacted collagen fiber density, but not collagen fiber network complexity (reticulation index, Figure 2D). With respect to steatosis, INT-767 reduced mean fat droplet area, but not droplet count (Figure 3D). The size distribution of fat droplets in INT-767 treated ob/ob-NASH mice were left-shifted, suggesting a vesicular fat redistribution towards a less macro-steatotic phenotype (Figure 4).

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Figure 4 Histogram depicting lipid droplet size distribution in INT-767 vs vehicle treated ob/ob-NASH mice. The “equivalent diameter” is the diameter of a circle of equivalent area as the one measured on the image (often not a perfect circle). Unit is in pixels, with 1 pixel = 0.39 μm.

In line with these morphometric changes, and similar to PPAR agonists[25], INT-767 10 mg/kg significantly reduced mRNA levels of cell death-inducing DNA fragmentation factor alpha-like effector c (CIDEC; a lipid-droplet associated protein that promotes intracellular storage) relative to vehicle (in RPKM, 109.4 ± 10 (vehicle) vs 40.8 ± 10.8 (INT-767), P = 0.0018).

The liver histological changes with INT-767 (10 mg/kg) treatment were accompanied by significant improvements in liver enzymes (ALT and AST). INT-767 also reduced whole-body fat mass (without changing body weight), hepatomegaly and intrahepatic concentrations of TC and TG (Table 1). In addition, INT-767 reduced terminal liver concentrations of TC and TG, and plasma TC (Table 1).

Hepatic and ileal concentrations of INT-767 and OCA were determined after 14 d of dosing (1-30 mg/kg, PO) in chow-fed C57Bl6 mice only. Both compounds showed similar dose-dependent increases in hepatic and ileal exposure. For both compounds, ileal drug concentrations were approximately two-fold higher compared to the liver (Figure 5).

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Figure 5 INT-767 and obeticholic acid (OCA) distribute similarly in the liver and ileum after 2 wk of administration in lean C57Bl/6 mice. A: Liver concentration; B: Ileum concentration.

Based on RNAseq data, prototypic FXR target genes were selected for analysis upon acute dosing of INT-767 and OCA (1-30 mg/kg, PO) in C57Bl6 mice. Both INT-767 and OCA regulated hepatic FXR genes, however, INT-767 more strongly regulated these genes at the highest dose administered (Figure 6A-D). By contrast, within the ileum, OCA had more pronounced effects that INT-767 to stimulate SHP and FGF-15 (Figure 6E and F).

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Figure 6 Obeticholic acid (OCA) and INT-767 modulate ileal and hepatic FXR target gene expression to a similar degree after 2 wk of dosing in lean C57Bl/6 mice. A-D: Hepatic expression of BSEP, OST, CYP8b1, and SHP; E and F: Ileal mRNA expression of FGF-15 and SHP. ^aP < 0.05, ^bP < 0.01, ^eP < 0.001 vs no treatment (dose = 0); ^cP < 0.05, ^dP < 0.01, ^fP < 0.001 vs corresponding INT-767 dose (two-way ANOVA with Bonferroni’s post-hoc test).

Given that INT-767 showed similar efficacy on hepatic FXR target gene expression, albeit at a lower dose compared to OCA (see above), a comparative study on the pharmacodynamics of potency-adjusted doses of INT-767 (3.0, 10 mg/kg) and OCA (10, 30 mg/kg) was performed in ob/ob-NASH mice. To better capture effects on progression of fibrosis, treatment was initiated in ob/ob-NASH mice fed AMLN diet for 6 wk (vs 12 wk in the previous study) and these mice presented with milder baseline NASH (NAS ≥ 4, fibrosis ≥ stage 1). All tested doses of INT-767 and OCA significantly reduced composite NAS, as compared to baseline (Figure 7 and 8). Administration of the highest doses of each compound effectively reduced NAS in all treated ob/ob-NASH mice (Table 2), but also significantly reduced fibrosis scores in the animals (Figure 7 and Figure 8). The ratio of mice achieving > 1 points improvement in score from baseline were 82% (9/11 animals, INT-767 10 mg/kg) and 57% (4/7 animals, OCA 30 mg/kg), see Table 2.

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Figure 7 INT-767 and obeticholic acid (OCA) treatment for 16 wk improves liver histopathology in ob/ob-NASH mice with biopsy-confirmed liver pathology. Representative hematoxylin-eosin (HE), picro-Sirius red, collagen 1a1 and galectin-3 stainings; NASH: Non-alcoholic steatohepatitis.

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Figure 8 INT-767 and obeticholic acid (OCA) treatment for 16 wk improves liver histopathology in ob/ob-NASH mice with biopsy-confirmed liver pathology. Composite NAS and fibrosis stage before and after treatment intervention. ^aP < 0.05, ^bP < 0.001 (χ² test, vs vehicle controls), NASH: Non-alcoholic steatohepatitis.

INT-767 and OCA showed corresponding effects on quantitative measures (fractional area) of hepatic steatosis (lipid %), inflammation (galectin-3 %), and collagen deposition (Col1a1 %) in ob/ob-NASH mice. INT-767 showed consistently greater maximal effects on all three parameters, as compared to OCA (Figure 9, Table 3).

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Figure 9 INT-767 or obeticholic acid treatment for 16 wks improves hepatic fat deposition, collagen 1a1 and galectin-3 levels in ob/ob-NASH mice with biopsy-confirmed liver pathology. Data are indicated before (baseline) and after treatment intervention. A: Fractional area of fat (HE staining); B: Galectin-3; C: Collagen 1a1. Data are expressed as % of total parenchymal area (subtraction of fat area); NASH: Non-alcoholic steatohepatitis.

Studies within this manuscript detail the efficacy of the dual in vitro FXR/TGR5 agonist INT-767 upon multiple histological endpoints in a mouse model of diet-induced non-alcoholic steatohepatitis (NASH). Prior pharmacology studies using INT-767 had not been conducted in models of established and biopsy-confirmed NASH with sufficient fibrosis.

INT-767 is in early clinical development for NASH. These preclinical studies provide proof-of-concept for efficacy in NASH and preclinical superiority relative to obeticholic acid (OCA; an FXR agonist in late stage development for NASH). The present studies also aimed to shed light on the impact of INT-767 on morphometric features of steatosis (droplet size and number) and fibrosis (fiber density) using quantitative histological methods.

The primary objective was to characterize improvements in NASH histopathology using qualitative (e.g., NAS stage scoring for steatosis, inflammation, and ballooning and fibrosis) and quantitative (percent fractional area; %FA) endpoints. The secondary objective was to understand the relative efficacy of INT-767 to and mechanistic differences from OCA. This objective was achieved by comparing drug distribution and gene expression profiles in the liver and ileum.

Therapeutic effects in NASH were measured using blinded qualitative (HE stained sections) and quantitative (%FA of IHC-stained sections) methods. The inclusion of a biopsy at both baseline and endpoint is unique and enabled a within-subjects, repeated-measures study design. Most studies do not include a biopsy and rely on endpoint measurements only. Morphometric assessments were also performed using label-free second harmonic generation imaging (for fibrosis) and two-photon emission (for steatosis) which are novel in NASH. The rigor of measuring the same samples using multiple histological techniques also allows the reader to consider how well these methods correlate with one another. mRNA levels of FXR regulated candidate genes were measured using RNA sequencing. LC/MS/MS was used to determine compound levels in liver and ileum.

In an 8-wk monotherapy study, INT-767 significantly improved qualitative features of NASH as demonstrated by a blinded assessment of NAS and fibrosis stage scores. Quantitatively, INT-767 significantly improved %FA for steatosis, inflammation (assessed by galectin-3 immunohistochemistry; IHC), fibrosis (Col1a1 IHC) and key components of basement membrane formation (laminin IHC). In a 16-wk comparative treatment study, NASH mice treated with INT-767 (3 and 10 mg/kg) exerted greater therapeutic potency and efficacy than OCA (10 and 30 mg/kg). Mechanistically, both OCA and INT-767 accumulate to a similar extent within the liver and ileum. INT-767 drives hepatic, but not ileal, FXR gene expression profiles more strongly than OCA, implying that the liver is a key site of action for INT-767.

INT-767 improved key histological features of diet-induced and biopsy-confirmed NASH across studies using multiple methods and was shown to be more potent and efficacious than OCA. Novel insights from morphometric analyses include: (1) not only does INT-767 improve fibrosis %FA, but also the intensity of the collagen fiber signal consistent with reducing fiber density; and (2) in addition to reducing steatosis %FA, INT-767 induced a clear shift to reduce vesicular lipid droplet size believed to be more a healthful form of steatosis. Together, these preclinical findings confirm durable histological benefits with INT-767 dosing and suggest that INT-767 may exert greater efficacy than OCA.

The present studies confirm and extend upon the extant literature validating FXR as a pharmacological target for NASH. Future research should consider (1) the mechanistic contribution of TGR5 to the in vivo effects of INT-767, and (2) the necessity and sufficiency of activating hepatic vs ileal FXR when targeting NASH, as INT-767 was more efficacious than OCA yet elicited only minimal ileal FXR activation. Finally, other models of diet- or toxin-induced NASH should consider incorporating a baseline biopsy to exclude mice that did not develop the desired phenotype prior to treatment initiation and gain a deeper mechanistic understanding of any pharmacologic intervention. Ongoing research continues to elucidate the role of FXR agonism on features of the basement membrane in NASH.