Introduction to MASH and MASLD
Metabolic disorders, including obesity and type 2 diabetes (T2D), have dramatically increased over the last few decades, affecting approximately 13% and 10% of the global population, respectively. While lifestyle and environmental factors play key roles, genetic and biological factors contribute significantly to these pathologies. Research aiming to uncover novel biological mechanisms sheds light on these complex and multifactorial disorders and provides new therapeutic avenues. Some notable successes include incretin mimetics (e.g., Dulaglutide, Semaglutide, etc.) for treating obesity and T2D.
Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) and Metabolic Dysfunction-Associated Steatohepatitis (MASH) are liver-associated metabolic disorders and are often linked to obesity and type 2 diabetes (T2D). MASLD is a hepatic manifestation of a metabolic syndrome characterized by lipid accumulation in hepatocytes, typically asymptomatic. This silent pathology is estimated to affect approximately 25% of the global adult population. A subset of individuals with MASLD can progress to MASH, characterized by inflammation and fibrosis, potentially leading to cirrhosis or hepatocellular carcinoma. The underlying mechanisms of these complex disorders are not fully understood.
Advancements in MASH Treatments
Unlike obesity and T2D, MASH had no FDA-approved drug therapies until 2024. This milestone was achieved by Madrigal Pharmaceuticals with their drug Resmetirom (Rezdiffra), a selective agonist of the liver thyroid hormone receptor-beta (THR-β) that works by increasing fatty acid oxidation. Resmetirom met the FDA criteria in the MAESTRO-NASH clinical trial, demonstrating a reduction of one stage in liver fibrosis and an improvement in lipid profiles. While the mechanisms underlying the reduction of fibrosis are not entirely understood, the reduction in liver lipids likely contributes to protecting against further progression to inflammation and fibrosis.
InSphero's Role and Expertise: 3D InSight™ In Vitro MASH Model
Despite the failure of multiple drug candidates along the way, the approval of Resmetirom represents a paradigmatic example and offers hope for future and improved treatments. Drug testing using translational preclinical models is key to driving the success of clinical candidates into regulatory approval. The selection of preclinical models is also a complex process, as they all have advantages and disadvantages. Among all preclinical models, the emergence of 3D in vitro human-based models is notable by representing a cost-effective benefit accompanied by a recapitulation of human-specific biology and pathology. These approaches are already reducing drug discovery spending, time, and drug attrition rates.
InSphero has more than 15 years of experience providing 3D in vitro models for different applications and is among the key leaders in the preclinical research field. Metabolic disorders are a main focus area where we provide research platforms for obesity, T1D, T2D, MASLD, and MASH. These multicellular and spheroid-based human models are cultured in our proprietary multiwell Akura™ plates. The key principle for the success of 3D modeling is “as simple as possible and as complex as needed”. Reproducibility, scalability, and translational ability are the essential foundations for drug discovery of novel candidates, and these criteria are fulfilled by all InSphero models.
MASH CALL Initiative: Comprehensive Drug Screening Strategy
One of the challenges in drug discovery is to reduce drug attrition rates by identifying the best candidates early on. There is a need for advanced in vitro 3D models that predict future clinical responses reliably. These models need to be scalable at a low cost, automation-compatible, and able to provide a wide variety of readouts for complex phenotypic analysis. Having this in mind, we wanted to tackle the next avenue of drug discovery for MASH by offering solutions from screening of candidates to advanced phenotypic characterization. We initiated a MASH CALL campaign offering drug testing screening at 3 announced fixed dates spread over the calendar year.
MASH CALL: Results and Insights from Recent Screenings
Our standard 3D InSight™ MASH model consists of primary human hepatocytes, Kupffer cells, endothelial cells, and stellate cells. These healthy liver spheroids are exposed to steatotic and pro-inflammatory media conditions, which over 10 days result in the main hallmarks of human MASH (steatosis, inflammation, and fibrosis). This model has been described in peer-reviewed publications and used successfully by the industry over the last 5 years. The robustness and reproducibility of this model are based on the InSphero know-how collected over the years and the use of well-curated and controlled primary human cells.
Figure 1: 3D InSight™ MASH model by InSphero
Peer-reviewed paper: Digital pathology with artificial intelligence analysis provides insight to the efficacy of anti-fibrotic compounds in human 3D MASH model
Peer-reviewed paper: A 3D primary human cell-based in vitro model of non-alcoholic steatohepatitis for efficacy testing of clinical drug candidates
The MASH CALL initiative consists of a first screening phase using a multiparametric endpoint approach to evaluate simultaneously potential hepatotoxic effects, steatosis and fibrosis by determining extracellular LDH release, intracellular triglycerides, and secretion of pro-collagen 1 (PC-1) respectively. Drugs are tested with a 6-dose concentration range in triplicates (Figure 2).
Figure 2: MASH CALL Initiative. Multiparametric approach for drug testing of customer drug candidates and comparison to clinical candidates.
InSphero successfully performed the screening of more than 60 MASH potential drug candidates for multiple companies in February 2024 including different modalities such as siRNA, antibodies or ASOs. The InSphero MASH CALL initiative is currently performing a retrospective approach by screening current clinical candidates. The project has started with the testing of 20 clinical candidates covering different mechanisms of action including anti-metabolic (PPAR agonists, de novo lipogenesis inhibitors, THR agonists, etc.) or anti-fibrotic agents.
Heather Hsu, CSO at Inipharm
"For our lead target at Inipharm, a human-centric system is extremely important for proof of concept. InSphero’s primary human liver spheroids under culture conditions that resulted in fibrotic and inflammatory processes was able to reproduce known human genetic phenotypes relating to our target of interest. Additionally, the MASH CALL testing platform was an efficient way for us to assess multiple leads for potential efficacy."
The results of the clinical candidate’s screen are shared with the participants of the MASH CALL initiative and provide an invaluable comparative tool for the efficacy of their candidates. An example of a public clinical candidate is the avb1 Integrin inhibitor PLN-1474 from Pliant Therapeutics, which showed a strong dose-dependent decrease of PC-1 suggesting an anti-fibrotic effect (Figure 3). PLN-1474 has completed a Phase 1 study with favorable results.
Another example is the Fatty Acid Synthase (FASN) inhibitor TVB-2640 (Denifanstat) from Sagimet Biosciences, which showed a decrease in total triglycerides (Figure 4). This recapitulates the clinical findings announced recently in the phase 2b FASCINATE-2 clinical trial demonstrating a reduction of liver fat.
Figure 3. MASH CALL Initiative Dose-response effect of PLN-1474 on reduction of PC-1 at day 7 and day 10 of a 10-day treatment.
Figure 4. MASH CALL Initiative - Treatment of increasing concentrations of TVB-2640 for the determination of total intracellular triglycerides.
We have analyzed all tested clinical candidates with a matrix of anti-steatotic and anti-fibrotic effects compared to MASH control. This approach is particularly helpful in identifying potential combinations of drugs with individual anti-steatotic or anti-fibrotic effects which could work to improve MASH hallmarks (Figure 5).
MASH CALL Follow-up: Enhanced Phenotypic Analysis and Validation
The MASH CALL initiative allows for follow-up on the selected candidates from the screening phase to building a comprehensive phenotypic analysis package. Imaging acquisition of spheroids followed by AI-based lipid droplet size quantification analysis is used to investigate effects of compounds on micro- versus macrovesicular steatosis. Inflammation is a key hallmark of MASH, triggered by an initial steatosis and involves a crosstalk between hepatocytes, Kupffer cells, and stellate cells. Inflammatory markers are determined by a panel of cytokines and chemokines secreted into the supernatant. Fibrosis is considered the most dangerous histological feature of MASH, and therefore resolution of fibrosis is one of the cornerstones of MASH treatment. Several secreted fibrotic markers help monitor the degree of fibrosis, including MMPs, TIMP1, or PC-3, the latter two being in clinical use as part of the ELF (Enhances Liver Fibrosis) biomarker blood test. Methods that go beyond surrogate markers of collagen synthesis or degradation are needed to capture the extent of collagen deposition at a histological level. InSphero spheroids are compatible with a high level of histological analysis and immunohistochemistry. We have partnered with Pharmanest to apply Fibronest, the first single-fiber, high-resolution, and fully translational quantitative AI digital pathology image analysis method for the phenotypic quantification of fibrosis and its associated features (link reference paper collaboration). Finally, spheroids are also compatible with transcriptomics methods. InSphero has optimized single-spheroid transcriptomic sequencing and analysis. Several transcriptomics options are available including TempOseq, low-input RNAseq or DRUG-seq.
Figure 5. MASH CALL Initiative - Anti-steatotic and anti-fibrotic effects of your compounds
InSphero MASH CALL Initiative - In a Nutshell
Altogether, the MASH CALL is an integrated solution from candidate selection to the extensive characterization of highly optimized endpoints. This approach helps to narrow down candidates and validate their effectiveness with an independent study and additional endpoints.
While the challenge of identifying better drugs for MASH, InSphero is working towards providing the best biological models and approaches to test the efficacy and safety of candidates. Providing clinically relevant endpoints using a translational model proven to predict clinical responses is the holy grail of drug discovery. Providing models validated by drug regulatory agencies is also crucial and we are working in that direction to facilitate the process of revision of IND applications.
We are continuously improving our models and readouts to adapt to novel biological mechanisms underlying metabolic disorders. Stay tuned and join the MASH CALL initiative for a fascinating journey of drug discovery, InSphero can be the key catalyzer for your next drug identification.
