The SCD inhibitor MTI-301 reduces steatohepatitis and ratio of C18:1/C18:0 levels in diet-induced murine models of MASH

Abstract

Metabolic dysfunction-associated steatohepatitis (MASH) and metabolic dysfunction-associated steatotic liver disease (MASLD) develop because of fat disposition and excess accumulation of lipid droplets in hepatocytes and hepatic stellate cells. The endoplasmic reticulum enzyme stearoyl-CoA desaturase (SCD) is the rate-limiting enzyme that converts saturated fatty acids (SFAs) to monounsaturated fatty acids (MUFAs). MUFAs are an essential building block for triglycerides found in lipid droplet deposits in fatty livers. Increased deposit of triglycerides in the liver contributes to the sequelae of events, including inflammation and fibrosis, that culminates in the development of MASH. Due to the key role of SCD in lipid droplet formation, SCD is an attractive target for management of MASLD/MASH. Two independent diet induced MASH models were used to test the efficacy of the novel SCD inhibitor MTI-301. MTI-301 treatment decreased lipid droplets in both models, a finding that correlated with reduced desaturation indices (16:1/16:00 and 18:1/18:0) measured in the liver. Collectively our data suggests that the MTI-301 is an attractive drug for further development for the treatment of patients with fatty liver disease.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-54346-x.

Introduction

Increases in the intake of high-fat diets and lifestyle changes are major contributors to the emergence of fatty liver disease as an emerging health care crisis. The estimated global prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) is approximately 30%, an increase of 39–50% from 1990 to 2006¹. As excessive lipids continue to accumulate in the liver, leading to cell injury and inflammation, MASLD progresses to metabolic dysfunction-associated steatohepatitis (MASH). MASH is associated with an increased risk of fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Currently, two drugs are FDA-approved for the treatment of patients with MASH. Resmetirom, a hepatic thyroid receptor beta (THR-β) agonist, has been approved by the FDA for the treatment of MASH². THR-β is a nuclear hormone receptor, the expression of which is predominantly localized in the liver. It is also a key regulator of lipid synthesis and metabolism. Treatment with THR-β agonists is associated with decreased triglyceride and cholesterol levels. Phase III clinical data indicated that patients receiving 80 and 100 mg of Resmetirom showed 25.9 and 29.9% resolution of MASH compared to 9.7% on placebo control². In addition, the fibrosis score was improved by at least one stage in 24.2 and 25.9% of patients receiving 80 and 100 mg of Resmetirom, respectively². Despite the success of Resmetirom leading to accelerated conditional approval by the FDA in March of 2024, the majority of patients did not observe significant benefits, and thus additional therapeutics are required to further improve patient outcomes.

The second therapeutic for the treatment of MASH is the GLP-1 agonist Semaglutide³. In the Phase III trial, 63.9% of patients had resolution of steatohepatitis without worsening of fibrosis. Based on the interim results, semaglutide was conditionally approved in August, 2025. Thus, confirmatory trials are required for both FDA-approved drugs for full FDA approval. The most common side effect of semaglutide is damage to the gastrointestinal tract. In addition, patients with a family history of thyroid cancer are recommended not to take semaglutide because of the increased risk of thyroid cancer associated with animal models, although this has yet to be confirmed in human studies⁴. Moreover, the discontinuation rate of GLP-1 agonist at one year has been reported to be high owing to side effects, costs, and comorbidities, ranging from 37 to 81% of patients discontinuing the use of semaglutide.

Another attractive target for MASH treatment is stearoyl-CoA desaturase-1 (SCD1). Endoplasmic reticulum SCD catalyzes the conversion of saturated fatty acids (SFAs) to monounsaturated fatty acids (MUFAs). MUFAs are major substrates in the synthesis of triglycerides, membrane phospholipids, wax, and cholesterol esters⁵. Abnormal SCD expression alters lipoprotein metabolism, adiposity, and membrane fluidity. SCD1 is the predominant isoenzyme expressed in the liver of mice. Its expression can be increased by feeding fasted mice a fat-free diet rich in carbohydrates⁶. SCD2 is expressed predominantly in the brain in mice⁷, while both SCD1 and SCD2 are expressed in adipose tissue and eyelids. SCD1, SCD2, and SCD3 are all expressed in the skin and Harderian and preputial glands⁸, whereas SCD4 is expressed in the heart in mice⁷. In humans, two isoenzymes have been identified: SCD1 is expressed predominately in the liver and SCD5 is expressed in the brain⁹. Mammalian SCDs catalyze the first double bond in the cis-delta9 position of SFA acyl-CoAs. The predominant substrates are SFAs palmitoyl CoA and stearoyl CoA, yielding palmitoleic and oleic acid, respectively. Several studies have reported that SCD1 plays a major role in regulating lipid metabolism in the liver¹⁰. Experimental evidence indicates that mice deficient in SCD1 demonstrate a reduction in body fat, increased insulin sensitivity, and resistance to diet-induced weight gain¹¹. The SCD1 inhibitor Aramchol, with a structure comprising of a SFA conjugated to cholinic acid, directly inhibits SCD1 activity in mouse microsomes. However, there are no reports on the IC₅₀ value of inhibition of SCD1 activity by Aramchol treatment. Aramchol inhibits SCD1 expression and activates cholesterol efflux^12,13. Aramchol showed promising Phase IIb results, with reductions in hepatic fat and fibrosis when using digital pathology as an endpoint, and is currently in a Phase III clinical trial (NCT04104321) for the treatment of MASH at doses of 300 mg twice daily^14,15. High doses of Aramchol are required, suggesting that Aramchol is not a potent inhibitor of SCD1, which may lead to additional off-target effects of treatment and an increase in side effects.

We are developing MTI-301, a potent and selective orally bioavailable small-molecule inhibitor that blocks the enzymatic activity of SCD1¹⁶. MTI-301 is FDA-approved for the initiation of a Phase I clinical trial for the treatment of refractory solid tumors (NCT06911008), and the trial is currently open for enrollment. In the present study, we tested MTI-301’s activity in fatty liver disease using two independent in vivo models of MASH and benchmarking the results with Aramchol. Our data indicates that MTI-301 potently reverses lipid accumulation, CRN score and collagen deposition in male mice fed a western diet (WD).

Materials and methods

Study drug, formulation and administration: Scale up synthesis of MTI-301 was performed by STA Pharmaceuticals (San Diego, Ca). The purity of MTI-301 was determined to be 99.2% by HPLC analysis. The clinical route of administration in the ongoing clinical trial for solid refractory cancer indication is a liquid oral formulation (NCT06911008). The stability of MTI-301 as a liquid formulation has been tested for 6 months and no degradation of MTI-301 was noted at 4 degrees using HPLC analysis. For this study the route of administration is IP to reduce concerns of stress on mice with administration of repeated oral gavage. The formulation for this study was 9:1 ratio of PEG400:DMSO. The highest concentrated stock solution was 0.8 mg/ml of MTI-301. The solution was clear and final volume of administration was 100 µl for each dose via IP injections. The final DMSO concentration for each dose was 1%. Control animals were treated with diluent only. For both diet MASH models dosing was performed 7 days a week for a total of 14 days starting on day 42.

Mouse models of MASH: This study was designed to test the effects of MTI-301 on lipid metabolism and deposition in the liver, using two mouse models of diet-induced MASH. The study protocol was approved by the West Virginia University IACUC Committee. The study was reported in accordance with ARRIVE guidelines. C57BL/6 mice, aged 2–4 months, were procured from the Jackson Laboratory and allowed to acclimate for one week prior to experimentation. Mice were housed in a temperature-regulated environment under a 12-hr light-dark cycle. Equal proportions of male and female mice were used. To induce fatty liver and fibrosis, mice were fed either a Western Diet (WD: Research Diets, Inc., cat. no. D12079B) or a High Fat Methionine and Choline Deficient Diet (HFMCDD: 0.1% Methione; Research Diets, Inc., cat. no. A06071302) for a total 8 weeks:, two well-established murine models for investigating the sequelae of events associated with MASH, with liver fibrosis increased in the HFMCDD model compared to the WD model^17,18. Choline- and methionine-deficient diets impair fatty acid β-oxidation, very low-density lipoprotein (VLDL) particle production, and hepatic VLDL secretion, leading to increased fibrosis, hepatic fat accumulation, hepatocyte death and oxidative stress¹⁹.

In the WD model, following 42 days of feeding, mice were randomized into 6 treatment groups of 10 mice (N = 5 F and 5 M mice per group). The treatment groups consisted of 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, or 4.0 mg/kg of MTI-301, 10 mg/kg Aramchol (Sigma-Aldrich: SML2352) for benchmarking, response and one group received diluent only treatment). In additional control group consisted of mice fed a regular diet (RD). Mice were fed the HFMCDD for 42 days prior to randomization into treatment groups. For HFMCDD model treatment consisted of 0.5 mg/kg or 2 mg/kg per day MTI-301; 10 mg/kg per day for benchmarking, and one group received diluent only (Supp. Figure 1). An additional control consisted of mice fed a regular diet (RD) (N = 5 F, 5 M mice per group). The frequency of drug treatment was once daily, and the duration of treatment was 14 days. The dosing of 10 mg/kg Aramchol was based on previous reports using mouse models²⁰. During dietary feeding of the western diet 1 male and 2 female mice were removed from study due to health concerns per IACUC guidelines and 1 male mice during Aramchol treatment was removed from the study. During feeding on HDMCDD 1 female and 1 male mouse were removed from the study prior to drug treatment due to health concerns.

Fig. 1.

MTI-301 attenuates hepatic lipid deposition in mice fed a Western diet (WD): Histological assessment and quantification of hepatic steatosis in male and female mice fed a WD 1A: Representative hematoxylin-eosin (H&E)-stained liver sections from male mice 1B: H&E-stained liver sections were quantified using ImageJ software and expressed as the percentage of lipid area per section. 1C: The increase in lipid droplets was reduced in female compared to male mice. 1D:. Data are presented as mean ± SEM, N = 4–5 mice in each group. p-values were determined by one-way ANOVA and post hoc Tukey test to correct for multiple testing.

The body weights of mice were recorded weekly throughout the study period (Supp. Figure 2A &B). At 8 weeks (24 h post last drug treatment, all mice were euthanized (deep anesthesia via inhalation 3–5% isoflurane followed by terminal cardiac blood collection) according to the West Virginia University IACUC guidelines. At the completion of the study, serum was separated and stored on dry ice. Liver specimens were excised, washed in PBS, sectioned, snap-frozen in liquid nitrogen, formalin-fixed, and stored in TRIzol reagent for further analysis.

Fig. 2.

MTI-301 reduces hepatic lipid deposition in mice fed a high-fat methionine and choline-deficient diet (HFMCDD): Histological assessment and quantification of hepatic steatosis in male and female mice fed a regular diet (RD) or HFMCDD. A-D: Representative H&E-stained liver sections and quantification show marked lipid accumulation in HFMCDD-fed mice compared with RD controls, which was substantially reduced by MTI-301 or Aramchol treatment. Quantification of lipid area (%) confirmed a significant decrease in hepatic fat deposition with MTI-301 or Aramchol compared to HFMCDD alone in both male and female mice. Data are presented as mean ± SEM (n = 4–5/group), p-values were determined by one-way ANOVA and post hoc Tukey test to correct for multiple testing.

Measurement of lipid deposition. Formalin-fixed liver tissues (10%) were embedded in paraffin. The sections were stained with hematoxylin and eosin (H&E) and scanned at 10x magnification using a digital slide scanner (MIF Olympus). Quantification of staining areas and the percentage of lipid deposition was performed using ImageJ software (https://imagej.net/ij/docs/guide/index.html) and expressed as a percentage of the area of the liver analyzed. For males fed Normal Diet (ND) or fed WD and treated with Aramchol (10 mg/kg) or MTI-301 (0.5–4 mg/kg); the threshold was set at 190–255, particle size as 100–6000 micron2, circularity was set at 0.1-1.0. For females fed a RD or WD and treated with Aramchol or MTI-301 as described above; the analysis was set at the same threshold of 190–255, but particle size was 75-6000 micron2 and circularity was set at 0.25 -1.0. Ten random fields of view were analyzed and averaged for each sample. For HFMCDD the threshold was set at 170–255 and particle size as 500–500,000 circularity was set at 0.1–1.5. Five random fields were captured and averaged (Supp. Figure 3).

Histologic assessment

Assessment of liver biopsies was performed using the NASH Clinical Research Network (CRN) scoring system by a board-certified pathologist. Each biopsy was evaluated for steatosis, lobular inflammation, and hepatocellular ballooning to calculate the NAFLD Activity Score (NAS), with scores ranging from 0 to 8. H&E slides were reviewed by a board-certified liver pathologists blinded to the data sets. The scoring system was applied in accordance with established NASH CRN criteria, ensuring standardized and reproducible evaluation of disease activity²¹.

Picro-Sirius Red (PSR) Staining & Analysis. All materials and reagents were equilibrated to room temperature prior to use and gently agitated during incubations. FFPE sections were deparaffinized in xylene (2 × 5 min) followed by rehydration through graded ethanol (100%, 2 × 3 min; 95%, 1 × 2 min; 70%, 1 × 1 min) and rinsed in distilled water. Slides were incubated in Picro-Sirius Red solution (Abcam, Cat# ab246832) for 60 min and subsequently washed in two changes of 0.5% glacial acetic acid in distilled water (2 min each). Sections were then dehydrated in 100% ethanol (2 × 1 min) and mounted using mounting medium (Epredia, Cat# 4112). Slides were imaged using brightfield microscopy using 10x magnification and a digital slide scanner (MIF Olympus). Image segmentation and PSR stain quantification were performed in MATLAB (MathWorks). The custom code for picrosirius red staining segmentation is deposited at https://github.com/maringa780/PSRsegmentation. Briefly, tissue boundaries were detected using Otsu’s thresholding method implemented with the multithresh function, as we previously described²². For PSR stain detection, we applied our previously established approach²³, in which RGB images were converted to Lab* color space and K-means clustering was performed on the chromaticity channels to group color clusters. Red intensity corresponding to PSR staining was then algorithmically extracted. Morphological operations (bwmorph) were applied to the PSR binary images to exclude perivascular staining. The remaining PSR-positive areas within the parenchyma were quantified and expressed as a percentage of total tissue area (Supp. Figure 4).

Fig. 4.

Diet type differentially regulates hepatic SCD isoforms, RT-PCR analysis of SCD1 and SCD2 expression in liver tissues from male and female mice fed a regular diet (RD), Western diet (WD), or high-fat methionine and choline-deficient diet (HFMCDD). A-B: Expression of SCD1 and SCD2 and SCD2 was elevated in WD-fed mice compared to RD. C-D: In contrast mice fed HFMCDD significantly suppressed SCD1 by 100-fold at both the RNA and protein level. This observation correlated with an 8-fold increase in SCD2 expression. Data are presented as mean ± SEM (n = 4–5/group). p-values were determined by one-way ANOVA and post hoc Tukey test to correct for multiple testing.

mRNA analysis. Total mRNA was isolated from the liver by homogenization on ice with the addition of 1 ml of TRIzol reagent (Invitrogen, cat. no. 15596026). The homogenate was centrifuged at 11,500 rpm at 4 °C in a Qiagen QIAshredder according to the manufacturer’s instructions. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using the Applied Biosystems QuantStudio 3 Real-Time PCR system and PowerUp SYBR Green Master Mix (cat. no. A25742) with 5000 ng of total RNA to quantify mRNA levels. The thermal cycling conditions were 2 min at 50 °C, 2 min at 95 °C, and 40 cycles of 15 s at 95 °C, 15 s at 58 °C, and 1 min at 72 °C. The expression of the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize the target genes. The following primers were used: SCD-1 and, SCD-2, CoL1a1, and α-SMA.

SCD-1.

Forward primer: 5’-TGACTATCATCATGCCGGCC-3’.

Reverse primer: 5’-CTTTGACAGCCGGGTGTTTG-3’.

CoL1a1.

Forward primer: 5’-GGAGAGAGCATGACCGATGG-3’.

Reverse primer: 5’-AAGTTCCGGTGTGACTCGTG-3’.

GAPDH.

Forward primer: 5’-AGGTCGGTGTGAACGGATTTG-3’.

Reverse primer: 5’-TGTAGACCATGTAGTTGAGGTCA-3’.

SCD2.

Forward primer: 5’-ATGGGAGCTGTGGGCGAG-3’.

Reverse primer: 5’-AATCCTGGCCAAGACAGCAG-3’.

SCD3.

Forward primer: 5’-GTTTGTGCCAACACCTAGCTT-3’.

Reverse primer: 5’-TGTACGAAGGCGTCATCTCTT-3’.

Quantitative Real-Time PCR (qRT-PCR) analysis

Relative gene expression levels were quantified using the comparative C_t (2^−∆∆C_t) method. Briefly, the cycle threshold (C_t) values for the target gene (e.g., SCD1) and the internal housekeeping reference gene (GAPDH) were averaged across replicates. The ∆C_t was calculated for both experimental and control groups as the difference between the C_t of the target gene and the C_t of the housekeeping gene (∆C_t = C_t, target gene - Ct, housekeeping gene). The double delta C_t (∆∆C_t) wa determined by subtracting the average ∆C_t of the control group from the ∆Ct of the experimental group (∆∆C_t = ∆C_t, experimental sample - Average ∆C_t, control group. The final relative expression levels were expressed as fold changes calculated using the formula 2^−∆∆Ct, where the control condition was normalized to a value of 1. Data are presented as the mean ± standard deviation (SD), and statistical significance was evaluated using two-way ANOVA and subsequently performing a Tukey test to correct for multiple comparisons.

Western blotting

For each condition 50 mg of liver tissue was homogenized in 300ul of cold RIPA buffer containing protease and phosphatase to investigate protein expression. The homogenates underwent a 10-minute, 10,000-g centrifugation at 4 °C, after which the supernatant was collected. Lysates were quantified Using the BCA protein assay kit (Thermo Fisher 23228; Rockford, IL, USA). The lysate was resolved on a 4–20% SDS–polyacrylamide gradient gel (Bio-Rad) and then transferred to a PVDF membrane for immunoblotting. After blocking non-specific binding sites with 5% non-fat dry milk, the membranes were incubated with primary antibodies overnight at 4 °C on a rocker. SCD-1 primary antibody, Cell Signaling 2794 S (1:1000), SCD-2 primary antibody Santa Cruz biotech 518,034 (1:1000), Goat anti-rabbit IgG, Cell Signaling 7074P2 (1:10000), Goat anti-mouse IgG, Invitrogen PA1-74421 (1:10000) were the secondary antibodies. Immobilon ECL ultra western HRP substrate (EMD Millipore, Burlington, MA, USA) was used to visualize the bands. Three individual mice samples per group were used to run western blot and the average percentage intensity was calculated and normalized with GAPDH.

Liver fatty acid analysis. Fatty acids were analyzed by gas chromatography–mass spectrometry (GC-MS) using the fatty acid methyl ester (FAMES) method. Liver samples were cut into pieces weighing approximately 5–7 mg, placed into 2-ml tubes and kept on dry ice. Cold methanol (50 µl/1 mg of sample) was added and the samples were homogenized by sequentially adding chloroform (50 µl/1 mg of sample) and water (20 µl/1 mg of sample). The samples were kept on dry ice for 30 min, vortexed, and centrifuged at 10,000 rpm at 4 °C for 5 min. The chloroform layer at the bottom was extracted into glass inserts and D27-Myristic Acid at final concentration of 100 µM was added into each sample as internal standard and subsequently dried under nitrogen. For derivatization using the FAMES method, 200 µL of 2% H₂SO₃ in methanol was added to the inserts, vortexed, and incubated at 50 °C for 80 min. The solution was then transferred to a new insert (Insert 1), 100 µL of saturated salt solution and 150 µL of hexane were added, the solution was vortexed, and the top hexane layer was transferred to another insert (Insert 2). Another 150 µL of hexane was added to insert 1, the solution was vortexed, and the top hexane layer was moved to insert 2. Samples were dried under nitrogen, resuspended in 75 µL of hexanes, and transferred to vials for analysis by the Agilent 7890B/5977B GC/MS system with a DB-5MS column (30-m × 0.25-mm × 0.25-µm film). Mass spectra were collected from 80 to 600 m/z in selective ion monitoring mode. Data were analyzed using MassHunter Workstation Quantitative Analysis, as reported for GCMS version B.07.01, and abundance values were obtained by measuring the area under the curve (AUC) presented as relative abundance after normalization with internal standard²⁴.

Statistical analysis

Statistical analyses were carried out using GraphPad Prism version 7.0.1 for Windows (GraphPad Software, Boston, Massachusetts USA). Numerical variables were summarized using means and standard errors. Significant differences for multi-group comparisons by one-way or two-way ANOVA followed by post hoc Tukey test to correct for multiple comparisons.

Results

Body weight

In the WD model, male mice showed a significant increase in body weight at 8 weeks compared with mice fed a regular diet (p < 0.05, Two Way ANOVA corrected with post-hoc Tukey’s test for multiple testing Supp. Figure 2A.). Treatment with either MTI-301 or Aramchol significantly decreased the body weight when male mice were fed a high fat diet (p < 0.05, two-way ANOVA, and post-hoc Tukey test to correct for multiple testing). These results align with the literature demonstrating that either pharmacological inhibition of SCD1 or SCD1 KO mice effectively reduce high fat diet induced obesity²⁵. Female mice demonstrated a trend toward increased body weight which was not found to be significant (See supp Fig. 2A). However, Mice treated with MTI-301 or Aramchol demonstrated a significant decrease in body weight. Performing a two-way ANOVA the interaction term based on Sex showed no significant interaction differences between the groups (F score 0.9449 and p = 0.762). In contrast in HFMCDD model, male mice showed a significant decrease in body compared to mice fed a regular diet (Supp. Figure 2B), and inhibition of SCD1 further decreased body weight after 2 weeks of treatment. Again, this is consistent with the literature that although a more aggressive model with respects to liver fibrosis and inflammation this model does not mimic the human disease of MASH with respect to obesity and weight gain but rather mice lose weight when fed a HFMCDD²⁶. Interestingly, females did not show a significant decrease in body weight when fed a HFMCDD diet but treatment with SCD1 inhibitors did significantly reduce the body weight of female and male mice (Supp. Figure 2B). Together these data indicate that inhibition of SCD1 with either MTI-301 or Aramchol treatment reduces body weight when fed a high fat diet. Again, there were no sex difference using a two way when mice were fed HFMCDD compared to a regular diet (ANOVA F score 1.932 and p = 0.126).

Quantification of fat deposits in the liver

Histological analysis revealed that male and female mice fed WD demonstrated increased deposit of lipids, compared to the control group fed RD (Fig. 1A-D) (Supp. Figure 5). H&E staining demonstrated a 6- and 4-fold increase in lipid accumulation in the livers of male and female mice fed a WD, respectively. MTI-301 reduced lipid accumulation in a dose-dependent manner in male mice, and the treatment effect was more pronounced in male mice compared to female mice. This may be due to the 2-fold increased accumulation of lipid droplets in male mice compared to female mice. There was a significant difference in males versus female in the measurement of lipid droplets when mice were fed a WD as measured by 2-way ANOVA (F-score 4.956, p = 0.004). The difference in staging based on sex in mice fed a western diet was also observed by a board-certified pathologist using the CRN scoring method with a difference in steatosis and ballooning but not inflammation (See Supp Table 1). Interestingly, Aramchol treatment was not effective in reducing the CRN score in female mice while MTI-301 did reduce the CRN score in female mice fed a WD. These studies suggest that female and male mice potentially due to hormonal differences may respond differently to lipid accumulation as well as to treatment and this warrants a larger study to further understand differences in treatment strategies based on sex.

Fig. 5.

Quantification of SCD1 and SCD2 protein levels by western blot analysis: In WD (A-D) and HFCDD (E-H). SCD2 protein levels remained similar across all treatment groups as detected by western blot analysis whereas SCD1 protein level expression was suppressed in HFMCDD. Shown are the mean and SE of 3 independent experiments. p-values were determined by one-way ANOVA and post hoc Tukey test to correct for multiple testing.

We observed a 30-fold increase in lipid accumulation in the liver of both male and female mice fed HFMCDD (Fig. 2A-D) (Supp. Figure 6). Even at the lowest dose (0.5 mg/kg), treatment with MTI-301 reduced lipid deposits by 2- and 3-fold in female and male mice, respectively. Overall, treatment with 0.5 mg/kg and 2 mg/kg of MTI-301 and 10 mg/kg of Aramchol had similar effects in reducing lipid droplets. There was not a significant difference when comparing males versus female mice fed a HFMCDD (F-score = 0.334 and p = 0.853 two-way ANOVA) with respect to accumulation of lipid droplets.

Fig. 6.

SCD1 inhibition mitigates liver damage induced by Western Diet in male mice. Serum was collected at termination (24 h post last drug treatment) and underwent comprehensive chemistry panel analysis (IDEXX BioAnalytics). A) Western Diet (WD) significantly elevated the levels of cholesterol and glucose in male mice. SCD1 inhibition with MTI-301 and Aramchol mitigated increases induced by a WD. The increase of liver enzymes, ALT, and LDH in male mice fed with WD compared to normal diet was significantly reduced with SCD1 inhibitors, MTI-301 and Aramchol, treatment indicating a reversal of liver damage induced by the WD. A similar increase in liver enzymes and LDH mediated by WD was not observed in female mice. Two-way ANOVA was performed to determine statistical significance and p values were corrected for multiple testing by Tukey test.

Collagen synthesis and deposition

Picrosirius red detects both deposition of collagen type 1 and Type III deposition in formalin embedded tissue. Picrosirius red Staining demonstrated that mice fed either the WD or the HFMCDD resulted in increased expression of COL1A1 and deposition of collagen (Fig. 3A-E) (Supp. Figs. 7 and 8). Interestingly, although female mice demonstrated protection from accumulation of lipid deposits in the WD female mice demonstrated similar deposits of collagen deposition compared to male mice (2 Way ANOVA, F-Score 0.1599 p=0.9223). Overall Inhibition of SCD attenuated collagen deposition in both male and female mice fed a WD (See Fig. 3A-B). In contrast in mice fed a HFMCDD diet demonstrated increased fibrosis but inhibition of SCD did not significantly decrease fibrosis in this model. Together, these data suggest that inhibition of SCD reverses mild fibrosis but does not reverse more severe fibrotic disease during two weeks of treatment. Treatment with MTI-301 and Aramchol significantly reduced the expression of COL1A1 in male mice fed a HFMCDD (Fig. 3F). It is feasible that a longer duration of treatment will reduce COLA1A1 levels more robustly at lower doses in both sexes. Together these data indicate that mice fed HFMCDD that pharmacological inhibition of SCD for two weeks is not sufficient to reverse fibrotic disease but may halt progression of fibrosis as indicated by inhibition of Col1a1 expression.

Fig. 3.

A: Expression of COL1A1 and quantification of collagen deposition. Mice fed a WD showed significant increase in percent positive Pico-Sirius Red (PSR) staining which was reduced with inhibition of SCD (A-B). COL1A1 expression was increased but was not found to be significant in mice fed a WD (C). In contrast mice fed a HFMCDD showed a robust increase in the percent of positive PSR staining but inhibition of SCD1 did not significantly reduce percent of PSR staining (D-E). HFMCDD caused an increase in COL1A1 expression. Data are presented as mean ± SEM (n = 4–5/group). p-values were determined by one-way ANOVA and post hoc Tukey test to correct for multiple testing.

Fig. 7.

Elevated ratio of C18:1/C18:0 were observed in GCMS analysis of mice liver fed with western diet (WD) and high fat methionine-choline deficient diet (HFMCDD): GC-MS analysis of hepatic monounsaturated fatty acids revealed significantly elevated levels of the ratio of C18:1/C18:0 which was inhibited by inhibition of SCD1. In contrast mice fed a HFMCDD diet showed decreased C16:1/C16:0 ratios which was further reduced by inhibition of SCD1. p-values were determined by one-way ANOVA and post hoc Tukey test to correct for multiple testing.

SCD1 and SCD2 Expression

WD was associated with approximately 3- and 2-fold increases in SCD1 mRNA expression in male and female mice, respectively. This finding corresponded to an increase in SCD1 protein levels albeit the finding was not significant perhaps due to the small sample size of 3 independent mice for each sex (See Fig. 4A and Fig. 5A-B). SCD2 expressions were also increased in the mice fed a WD, however, SCD2 protein levels remained similar across all samples (See Fig. 4B and Fig. 5C-D). In contrast, HFMCDD was associated with an approximately 100-fold decrease in SCD1 expression in both male and female mice. This remarkable decrease in SCD1 was consistent at the protein levels (See Fig. 4C and Fig. 5E-F). In mice fed a HFMCDD a 9- and 2-fold increase in SCD2 expression was observed in male and female mice, respectively (See Fig. 4D). However, changes in SCD2 at the protein levels were not noted. (See Fig. 5G-H). Together, these data point to the differential regulation of SCD1 versus SCD2 expression in the liver based on diet and potentially the amount of damage or fibrosis to the liver. Considering the dramatic decrease in SCD1 at the RNA and protein levels our data indicates that SCD2 is likely the main isoform responsible for desaturation in the liver in mice fed a HFMCDD. SCD3 expression was not detected in mice fed RD, WD or HFMCDD (data not shown).

Plasma markers of liver damage

Consistent with male mice accumulating more fatty acid deposits male, but not female, mice fed the WD showed increases in cholesterol, alkaline, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) activity in the plasma compared to mice fed the RD (Fig. 6A and B). Although AST levels were increased, they were not found to be significant. This trend was increased in male compared to female mice fed a WD. However, a two-way ANOVA test did not indicate a significant difference between sexes across all treatment groups for this study. Treatment with either MTI-301 or Aramchol decreased levels to near or below the levels in mice fed RD for markers of liver damage. Interestingly, circulating triglyceride levels were not increased when mice were fed a WD. Future studies will determine whether measuring MUFA/PUFA ratio is decreased in the plasma following drug treatment.

Saturated/monounsaturated fatty acids ratio in liver

Male mice fed a WD (Fig. 7A and B) demonstrated increases in the ratio of C18:1/C18:0 and C16:1/C16:0 fatty acids suggesting increased SCD activity based on diet. Treatment with MTI-301 or Aramchol significantly reduced the ratios of both C18:1/C18:0 and C16:1/C16:0monounsaturated (One Way ANOVA, Tukey test for multiple comparisons). In contrast, male mice fed a HFMCDD ratio of C18:1/C18:0 was increased but a reduction of 16:1/16:0 ratio was observed (Fig. 7C and D). The reduction in C16:1/C16:0 ratio in mice fed a HFMCDD is consistent with previous reports²⁰. In the HFMCDD inhibition of SCD significantly inhibited C18:1/C18:0 ratios and further reduced the levels of C16:1/C16:00 albeit the finding was not significant. Together these data indicate that dosing with either Aramchol or MTI-301 resulted in good target coverage based on the ratio of C18:1/C18:0 in the liver.

Discussion

Fatty liver disease is an emerging health care crisis that, if left untreated, can progress to MASH with an increased risk of developing hepatocellular cancer. Risk factors for progression from MASLD to MASH include insulin resistance, oxidative stress, hepatic stellate activation, inflammatory cytokines, and adipokine secretion. In the current study, we evaluated the effects of treatment with the novel SCD inhibitor, MTI-301, in two independent in vivo mouse models of diet-induced MASH. The results indicated that MTI-301 significantly reduced lipid deposits mice in male mice in both models at doses as low as 0.5 mg/kg. In the Western Diet model, we showed significant increase in lipid droplet formation in male versus female mice which was confirmed by the pathologist scoring of CRN. Interestingly, although we did not observe significant changes in the decrease in lipid area with inhibition of SCD in female mice fed a WD the CRN score was reduced with MTI-301 treatment as reviewed by the pathologist. The differences in sex with respects to hallmarks of MASH included a decrease in lipids but not inflammation or fibrosis in female mice compared to male mice. More studies are warranted to understand the mechanism underlying the difference in diet induced hallmarks of MASH in male and female mice. In addition, our data indicates that inhibition of SCD was more effective in the WD diet compared to the more aggressive fibrotic HFMCDD in reversing key hallmarks of MASH progression including the CRN score and deposition of collagen. Of note the HFMCDD diet induced weight loss questioning the relevance to human pathology, however as anticipated the fibrosis score indicative of advanced disease was increased when mice were fed the HFMCDD compared to WD.

In 2013, Kurikawa et al. examined the SCD-1 inhibitor compound (N-(2-hydroxy-2-phenylethyl)-6-[4-(2-methylbenzoyl) piperidin-1-yl]pyridazine-3-carboxamide) in a Col1a1-Luc Tg rat model, administering a methionine- and choline-deficient diet (HFMCDD) to induce steatosis, and noted lipid deposition and elevated triglyceride levels in the liver, which is consistent with our current study. Treatment with SCD1 inhibitor resolved the increase in ALT, AST and triglyceride levels in the liver albeit the doses administered where 30 and 100 mg/kg compared to our dosing of 0.5-2 mg/kg in mice again demonstrating the potency of MTI-301²⁷. Interestingly, we did not observe MTI-301 treatment changed total triglycerides in the plasma but observed decreased C18:1/C18:0 ratio indicating target coverage in the liver.

Mice fed with HFMCDD showed a dramatic decrease in SCD1 expression and an increase in SCD2 expression. These data indicate that regulation of SCD1 is distinct from that of SCD2. In mice, SCD2 is increased in liver tumor-initiating cells (LTICs) and is required for lipid synthesis during early skin and liver development^28,29. Thus, our findings suggested that HFMCDD may lead to increased stem-like properties in hepatocytes. These results also highlight the need to consider all SCD isoenzymes in MASH and potential cancer models. The effects of MTI-301 treatment, although variable, trended toward reversal of fibrosis markers. The treatment time was relatively short (14 days for MTI-301), and it is feasible that if the duration of MTI-301 treatment was extended, the results would be more dramatic. Future studies will increase the duration of treatment and determine whether C18:1/C18:0 levels in the blood can be used as a minimally invasive pharmacodynamic marker to assess target coverage.

Inhibition of SCD remains an attractive target for the treatment of MASH patients. A recent clinical study indicates that Aramchol is sufficient to improve fibrotic score with increased effectiveness correlating with increased duration of treatment¹⁵. The transition from Aramchol to Aramchol Megulmine which shows increased bioavailability and lower dosing of 200 mg is ongoing and likely to be promising. MTI-301 has recently been approved by the FDA to initiate a phase I clinical trial for the treatment of patients with refractory solid tumors (NCT06911008). The preclinical data reported here support that MTI-301 is an attractive agent to consider for the treatment of MASH. Considering the polypharmacy of MASH patients, it will be important for clinicians to have choices based on drug-drug interactions and side effects. We propose that understanding potential combination strategies of MTI-301 with agents in clinical trials such as GLP1 agonist will be important to evaluate pre-clinically for efficient design of clinical trials.

Electronic Supplementary Material

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Author contributions

Lori, Karen, Sridhar, Santhosh, John and Werner: Conceptualization, Data curation, Resource, Supervision and Experimental Design. Vincent, Mark, Jianhai, Sijin and Devanand: Methodology and data interpretation. Lori, John, Santhosh, Karen, and Sridhar: Manuscript writing, Data visualization, editing and rewriting.

Funding

The work was supported by the NIDDK 1R41DK131705-01A1.

Data availability

All data supporting the findings of this study are available within the paper and its Supplementary Information.

Declarations

Competing interests

Modulation Therapeutics has licensed MTI-301 and LAH is a cofounder and KH SK are employees at Modulation Therapeutics.

Conflict of interest

LAH is cofounder of Modulation Therapeutics and MTI-301 is licensed to Modulation Therapeutics. SK and KH are employees of Modulation Therapeutics.

Compliance with Ethics Requirements

All animal experiments were carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals.

Informed consent

N/A.