A composition comprising a saponin and / or an anthelmintic and / or a mitochondrial uncoupler for the treatment of liver disease

Abstract

The present invention pertains to a composition for use in a method of treating a liver disease, preferably metabolic dysfunction-associated steatohepatitis (MASH), the composition comprising an effective amount of at least one saponin agent, preferably ginsenoside or diosgenin, and / or an effective amount of at least one anthelmintic agent, preferably niclosamide or nitazoxanide and / or an effective amount of at least one mitochondrial uncoupling agent.

Description

[0001] A COMPOSITION COMPRISING A SAPONIN AND / OR AN ANTHELMINTIC AND / OR A MITOCHONDRIAL UNCOUPLER FOR THE TREATMENT OF LIVER DISEASE

[0002] The present invention relates to the field of treating liver disease. In particular, the present disclosure relates to a combination or monotherapy treatment for a liver disease as well as compositions to be used in the treatment of liver disease.

[0003] In the following, the technical background of the present invention is described.

[0004] Metabolic dysfunction-associated steatotic liver disease (MASLD; and formerly Non-alcoholic fatty liver disease, NAFLD) has seen a significant rise in prevalence from 22% to 37% between 1991 and 2019 (1 ,2). It encompasses a spectrum of conditions, ranging from Metabolic dysfunction-associated simple steatosis (accumulation of fat in the liver) to the more severe Metabolic dysfunction-associated steatohepatitis (MASH; and formerly Non-alcoholic steatohepatitis, NASH).

[0005] MASH is characterized by the addition of hepatocyte injury (ballooning) and inflammation, with or without fibrosis, in the absence of significant alcohol consumption. With the escalating prevalence of obesity and metabolic syndrome, MASH has emerged as a leading cause of chronic liver disease and a major risk factor for cirrhosis and hepatocellular carcinoma (HCC). Due to this increase, MASH is now the leading indication for liver transplant listing for women and second-leading for men and is expected to overtake alcoholic liver disease as the leading liver transplant indication for all patients within the next few years (3). Lifetime direct medical costs for US patients with MASH in 2017 were estimated at $222 billion (4). These estimate does not include indirect medical or societal costs and will only increase as the prevalence of MASH rises. In 2016, it was estimated that the annual cost for patients diagnosed with MASLD in the Europe-4 countries (Germany, France, Italy, and United Kingdom; ~52 million patients) was ~ €35 billion (5).

[0006] Central to the development of MASLD / MASH is the aberrant accumulation of triglycerides within hepatocytes, predisposing to lipotoxicity and cellular injury. Insulin resistance further exacerbates hepatic steatosis by promoting de novo lipogenesis and impairing lipid oxidation. Concurrently, dysregulated adipokine secretion and chronic low-grade inflammation contribute to hepatocyte injury and fibrogenesis (6). Moreover, emerging evidence implicates gut dysbiosis and alterations in intestinal permeability in the pathogenesis of MASH (7). Additionally, genetic factors play a key role in the susceptibility and progression of MASLD. To date, the PNPLA3 and TM6SF2 gene variants are the major determinants of interindividual differences in liver steatosis and susceptibility to progressive MASH (8).

[0007] MASH and liver-specific disease outcomes are strongly associated with degree of hepatic fibrosis (9). The severity of fibrosis and not the diagnosis of MASH, is predictive of long-term outcomes including overall mortality in patients with MASLD (10, 11 ). Around 40% of MASH patients have progression of their fibrosis over time, at a rate of about 1 stage per decade (4) and ~25% of patients’ fibrosis is staged at F2 or greater at the time of MASLD diagnosis (Figure 1 and 2) (11 ). In 1 case series, at 15 years of follow-up, 11 % of patients with MASH developed cirrhosis (12).

[0008] Patients with MASH develop HCC at significantly higher rates than the general population and have an annual rate that is 12 times higher than patients with MASLD (4). Although HCC typically develops in the background of cirrhosis, patients with noncirrhotic MASH are still at increased risk (13). Long-term studies have shown that compared with the general population, patients with MASLD have higher overall and liver-specific mortality (14). MASH has an annual mortality of 1.7 times higher than MASLD (25.56 vs 15.44 events per 1000 person-years), and liver-specific mortality is 15 times higher than in MASLD (11.77 vs 0.77 events per 1000 person-years) (4). Additionally, approximately 80% of NASH patients suffer from at least one comorbidity. The five most prevalent comorbidities had rates above 50% in the NASH group: dyslipidaemia (82.6 %), hypertension (78.7 %), GERD (69.9%), Type 2 Diabetes (62.2%) and cardiovascular disease (56.0%) (15). Hence, despite increased liver-related mortality, cardiovascular disease, a well-documented co-morbidity, is the primary cause of death for patients with MASH, and increased risk of cardiovascular death appears to be the most significant factor related to the elevated risk of all-cause death for patients with MASH (16,17).

[0009] Current diagnosis of MASLD involves a comprehensive assessment combining clinical evaluation, laboratory tests, imaging studies, and sometimes liver biopsy (when MASH is suspected). Clinical assessment involves obtaining a comprehensive medical history focusing on risk factors like obesity, diabetes, and dyslipidemia, alongside assessing for nonspecific symptoms such as fatigue and abdominal discomfort. Laboratory tests include liver function tests (LFTs) measuring ALT, AST, ALP, and bilirubin levels, commonly elevated in liver injury, along with markers of insulin resistance and lipid profile. Ultrasonography serves as the primary imaging modality for hepatic steatosis, while CT or MRI may further characterize liver fat content and assess for complications. Exclusion of other liver diseases, including viral hepatitis and autoimmune conditions, is crucial. Advanced fibrosis or cirrhosis suspicion warrants non-invasive tests like Fibroscan, NAFLD Fibrosis Score (NFS), Fibrosis-4 Score (FIB-4), and Enhanced Liver Fibrosis (ELF) test, aiding in diagnosis and management. While liver biopsy is the only test that can prove a diagnosis of MASH and clearly present the severity of the disease. Diagnosis chart adapted from UK (22) and USA (23) publications is illustrated in Figure 4. However, it is important to note that with the new nomenclature (e.g. NAFLD to MASLD), there has also been a switching from a definition based on the exclusion of any other liver disease (i.e., NAFLD / NASH) to a definition based on inclusion criteria (i.e MASLD / MASH). This in turn has slightly modified the diagnostic criteria for the diseases. The new diagnostic criteria were selected to align with cardiometabolic risk factors believed to be associated with insulin resistance, and already well established and validated in the context of cardiovascular disease.

[0010] Up until March 2024, there was no FDA- or EMEA- approved therapy exists for MASH, and the AASLD (American Association) and EASL (European Association) guidelines recommend weight loss, achieved by hypocaloric diets in conjunction with increased physical activity, for treatment of MASLD / MASH (17,25). While lifestyle modification consisting of diet, exercise, and weight loss is advocated to treat those patients such modifications are difficult to sustain long-term (26). Moreover, weight loss alone may not suffice for the majority of patients, particularly those with advanced hepatic fibrosis (27). However, recently, the FDA approved Madrigal Pharmaceuticals’ resmetirom for the treatment of MASH, with moderate or severe liver scarring, or fibrosis, consistent with stage F2 and F3 disease.

[0011] The present invention addresses the objective technical problem of alleviating or abolishing the problems of the prior art. In particular, a novel way of treating liver disease has been sought. This objective technical problem is solved by the subject- matter of claim 1. Further embodiments of the invention are the subject-matter of the dependent claims.

[0012] Liver diseases can be categorised into: (i) viral hepatitis, (ii) genetic, (iii) drug or toxins-related, (iv) autoimmune disorders, (v) metabolic dysfunction-associated ste- atotic liver disease and (vi) cancer. Although originated by different aetiologies all will lead, if untreated, to the formation of chronic liver fibrosis / cirrhosis which will ultimately affect the microanatomy and function of the liver.

[0013] MASH represents a complex and dynamic progression from the initial phase of simple steatosis to a more aggressive phenotype marked by various distinctive pathological changes. An initial hallmark of MASH is hepatocellular injury, a consequence of cellular stress responses triggered by excessive lipid accumulation. This injury sets the stage for a series of events, including oxidative stress, inflammation, and disruptions in metabolic pathways (28,29).

[0014] The inflammatory aspect of MASH is characterized by the infiltration of immune cells into the liver, giving rise to localized inflammation within the hepatic lobules. This lobular inflammation contributes to the unique histological pattern observed in MASH biopsies, often featuring clusters of inflammatory cells surrounding ballooned hepatocytes. These inflamed regions can further exacerbate hepatocellular injury and progress the disease (30,31 ).

[0015] Hepatocellular ballooning, a prominent hallmark of MASH, entails the swelling and distortion of hepatocytes due to various insults, including oxidative stress and metabolic dysfunction. Ballooning signifies a state of cellular stress and impending damage, frequently serving as a precursor to apoptosis and heightened inflammation. This ballooning phenomenon is a pivotal point in the progression of MASH from MASLD. Patients with hepatocyte ballooning, with or without coexisting fibrosis, had an increased risk of adverse liver-related outcomes (32), however, it is not a significant predictor of mortality (32,33).

[0016] An important downstream consequence of hepatocellular injury and hepatic inflammation is the activation of HSCs, the principal fibrogenic cell type in the liver leading to the excessive accumulation of extracellular matrix (ECM) and as result stiffening of the liver. Fibrosis severity mirrors disease progression and is the only histologic feature that predicts liver-related mortality in MASH patients (11).

[0017] When all these events come together it creates a “closed” positive feedback loop perpetuating a vicious cycle of hepatocyte damage, inflammation, HSC activation and ECM accumulation, ultimately driving MASH progression (see Figure 5). Targeting only one aspect of this complex network, such as inflammation or hepatocyte lipotoxicity, may not be sufficient for effective treatment due to the interconnected nature of these processes. Thus, the present invention targets multiple aspects of this complex network. Therefore, the comprehensive therapeutic approach of the present invention addresses multiple aspects of MASH pathogenesis, including inflammation, fibrosis, and metabolic dysfunction (hepatocytes), which are necessary to achieve meaningful clinical outcomes and halt disease progression.

[0018] In the following, amode of action of compounds disclosed herein is described.

[0019] The inventors of the present invention sought to achieve a combination therapy for MASH using at least two drugs (conceptionally referred to as Drug A and Drug B) capable of alleviating the four key drivers of the disease, as highlighted in Figure 5.

[0020] The mechanisms underlying the therapeutic actions of for example Ginsenoside (that can be regarded as a “Drug A” as shown in Fig. 12, similar to diosgenin) encompass the modulation of several signalling pathways involved in lipid accumulation and lipotoxicity of hepatocytes.

[0021] Ginsenoside has been shown to trigger the oxidative decomposition of fatty acid, suppressed FA synthesis, increased ketone level, and decreased glucocorticoid secretion in NSCLC mice models with hepatic metabolism disorders and altered lipid metabolism (34). Ginsenoside significantly downregulated the expression of FASN. Further mechanistic exploration revealed that Ginsenoside suppressed the expression and SREBP-1 , and disturbed the SREBP-1-FASN interaction (34). Ginsenoside has also been shown to increase the expression of HCBP6, thereby ameliorating metabolic disorders in HFD-induced mouse model of MASLD. It was found that Ginsenoside to alleviates liver steatosis and improve glucose tolerance even if mice kept on a HFD (35). Furthermore, Ginsenoside has been shown to significantly reduce fatty acid accumulation in hepatocytes of MCD-induced MAFLD, Additionally, it inhibits NLRP3 inflammasome activation in Kupffer cells, and hepatocytes, hence attenuating liver inflammation and fibrosis and restored liver function in MASH mice (36). Finally, Ginsenoside is able to promote hepatic lipolytic genes (CPT-1a), inhibited lipogenic genes (SREBP-1 c, FAS, ACC-1 ) and improves leptin resistance (37). The mechanisms underlying the therapeutic actions of Niclosamide (that can be regarded as a “Drug B” as shown in Fig. 12, similar to Nitazoxanide) encompass the modulation of several signalling pathways involved in the activation of HSCs, including Wnt / [3-catenin, mTORCI , STAT3, NF-KB, and Notch. It has been demonstrated to significantly reduce liver enzymes, oxidative stress, inflammation, and phosphorylated STAT3 (p-STAT3) levels. It also significantly decreases components of the NOTCH pathway (Jaggedl , NOTCH2, NOTCH3, HES1 , SOX9), the Wnt pathway (Wnt5B and WntlOA), as well as fibrotic markers (TGF-|31 ), a-SMA, and collagen deposition (38). Additionally, it is able to decrease total bilirubin, alanine transaminase (ALT), aspartate transaminase (AST), [3-catenin, I- hydroxyproline, l-glutaminase activity, and gene expression of TGF-[31 and Dvl2, thereby demonstrating the ability of Niclosamide to protect against liver fibrosis by inhibiting the Wnt / [3-catenin and NOTCH pathway (39).

[0022] Additionally, Niclosamide exerts potent immunomodulatory effects by targeting multiple key pathways involved in inflammation and immune regulation. Firstly, it inhibits the activation of NF-KB, a crucial transcription factor responsible for orchestrating inflammatory responses, thereby suppressing the expression of pro-inflammatory cytokines such as TNF-a, IL-6, and IL-1 [3 (40). Additionally, Niclosamide blocks STAT3 signalling (41), further reducing the production of pro-inflammatory cytokines and fostering an anti-inflammatory environment. Furthermore, Niclosamide modulates the function of dendritic cells, impairing their maturation and activation (40), thereby dampening immune responses and mitigating inflammation. These multifaceted immunomodulatory properties underscore Niclosamide’s potential as a therapeutic agent for inflammatory and autoimmune conditions.

[0023] Both fibrosis and its latest stage, cirrhosis, affects and alters the quantity and quality of ECM, specifically collagen, leading to the formation of scar tissue (Williams). Myofibroblasts are the source of ECM overproduction during the development of fibrosis (Lim). Myofibroblasts are not found in a normal, healthy liver and their origin is hepatic stellate cells (Kisseleva). Additionally, inflammation and autophagy occur during hepatic fibrosis development. Chronic inflammation in liver is an immune response that persists for months leading to tissue remodelling, and repair pro- cesses. Regardless of aetiology, chronic liver inflammation induces hepatic fibrosis that eventually leads to cirrhosis and hepatocellular carcinoma which is a leading cause of death worldwide (Dhar et al.).

[0024] The pivotal event in preventing the progression of hepatic fibrosis is to reduce the activation and transition of hepatic stellate cells into myofibroblasts or to promote hepatic stellate cells’ senescence and apoptosis (Higashi et al.). Activated hepatic stellate cells cause strong cell proliferation and dramatic changes in their stellate morphology, and are accompanied by lipid deposition, degradation imbalance of extracellular matrix, and over-secretion of specific marker protein, such as alphasmooth muscle actin (a-SMA) (Schinagl et al.). A study has shown that the activated hepatic stellate cells migrate with the infiltrating leukocytes into damaged area of the liver (Yagai et al.), whereas inflammatory cells evoke hepatic inflammation (Wan et al.) and secret proinflam matory, as well as profibrotic factors, including transforming growth factor (31 (TGF-[31) (Zhan et al.).

[0025] Niclosamide and Ginsenoside or Niclosamide and Diosgenin or Nitazoxanide and Ginsenoside or Nitazoxanide and Diosgenin have been surprisingly found by the present inventors to have a positive effect in the treatment of liver disease, for example MASH. The likely mechanism of action of these coumpounds is further explained below:

[0026] Niclosamide demonstrates low acute oral toxicity, as documented in Andrews et al.'s review from 1982 (71 ). However, comprehensive data regarding its long-term usage remains scarce. Preliminary findings from a phase II clinical trial (NIKOLO, NCT02519582) investigating niclosamide for metastasized colorectal cancer revealed Cmax ranging from 429 to 1777 ng / mL following the administration of 2 g of niclosamide (Yomesan®) (1 ,2). However, since this study has not yet been published its results are not available for further analysis and discussion.

[0027] However, in a phase lb clinical trial conducted in 2021 (NCT02807805), significant pharmacokinetic findings involving the use of a niclosamide formulation (niclosa- mide / PDMX1001 ) at a dosage of 1200 mg administered orally twice daily (BID), in conjunction with 1000 mg of abiraterone orally daily and 5 mg of prednisone BID. Remarkably, all patients in the study tolerated this regimen so well that none discontinued treatment due to toxicities (72). Commonly reported Grade 1 and 2 side effects included diarrhea, fatigue, headache, and anorexia. Grade 3 toxicity side effects reported after receiving this drug combination included abdominal pain, fatigue, hypoalbuminemia, anemia, and hyperglycemia. Notably, some side effects were mitigated by reducing the dose of abiraterone. Additionally, two patients received 1600 mg orally three times daily (TID) for more than two years without experiencing relevant side effects (72).

[0028] There is only one recorded clinical trial that involved diosgenin. The clinical pilot study was conducted to investigate the efficacy and safety profile of Diallyl Thiosulfinate (20 mg), Nuciferine (137.5 mg) and Diosgenin (45 mg) in the cure of primary and secondary erectile dysfunction. A total of 143 candidates (age 18-39 Y) were selected and treated orally for up to three months (single dose, alternative days). In the study, the treatment with the combination was well tolerated by all patients, not showing any side effect reported during the period study (73).

[0029] In vivo studies of steroidal saponins of different doses from D. zingiberensis were given to mice that were experimental in another in vitro investigation. Diosgenin was the metabolite and the main component of these saponins. There were no harmful effects up to a level of 562.5 mg / kg, according to the findings. However, steroidal saponins, which include diosgenin, displayed deleterious consequences and even death in a dose-dependent manner at dosages of 1125 mg / kg and higher. Interestingly, the steroidal saponins dosage, which is traditional, is 510 mg / kg / day, implying that steroidal saponins, together with diosgenin, have no significant toxicity at this dosage (74). A recent study also discovered that diosgenin derivatives had antithrombotic properties. In vivo experiments revealed that they appeared to be protective and comparable to aspirin, with a decreased risk of bleeding and less stomach mucosal injury (75). Furthermore, investigations have shown that diosgenin has a modest inhibitory impact on cytochrome P450 enzymes (CYPs), suggesting diosgenin fused with any other medicine would be safe to consider that they would have no toxicity (76). These investigations demonstrated that diosgenin and its derivatives are nontoxic and have underlined their utility in the medicaments of chronic disorders including cancer.

[0030] Furthermore, a composition according to the present invention can further comprise mebendazole.

[0031] Mebendazole, which is an antimicrotubular agent that possesses a high affinity for tubulin, has been shown to exert an inhibitory effect on collagen biosynthesis and secretion in cell cultures (Soto et al.). These changes were reflected in an intracellular accumulation of total proteins and collagen in fibroblasts and result in a marked decrease of its deposit in the extracellular matrix (Soto et al.), therefore this can be directly related to hepatic stellate cells in liver disease. Another study in pancreatic cancer have shown a reduced connective tissue deposition and reduced a-SMA expression in samples treated with Mebendazole in comparison to untreated samples (Williamson et al.). Therefore, when translated to hepatic stellate cells, this proves again the benefit of mebendazole as an anti-fibrotic agent. Further it was shown that Mebendazole has the ability to reduce the levels of TGF-[31 , which is known to stimulate fibrogenesis (Guerini et al.).

[0032] However, mebendazole has also been proven to induce cytokine release from PBMC cultures. Stimulated PBMCs released several pro-inflammatory cytokines including TNFa, IL1 (3, IFNy, IL6 from PBMCs activated by IL2 and anti-CD3 stimulation (Rubin et al.). Previous studies by the present inventors have also demonstrated that mebendazole enhanced the secretion of IL6, TNFa and IFNy which is known to promote inflammation and in the long term can enhance fibrosis. Therefore, to maintain the anti-fibrotic properties of mebendazole while eliminating the negative pro-inflammatory aspects, mebendazole can also be combined with at least one ginsenoside.

[0033] Additionally, fibrotic patients can benefit from Ginsenoside as it has been demonstrated that it can attenuate hepatic fibrosis through regulating autophagy processes (Liu et al.). Another study has demonstrated ginsenosides ability to inhibit the TGF[31 / SMAD pathway, thereby improving liver fibrosis (Hafez et al.). Further, by regulating c-FLIP pathway-mediated NF-KB activation, ginsenoside can induce the apoptosis of activated hepatic stellate cells (Wu et al.).

[0034] Additionally, the compounds and compositions disclosed have been studied in the context of cancer and it has emerged that the compounds and compositions can be useful in treating liver diseases and cancer. It has thus emerged surprisingly that the same plausible mechanism of action may be employed for treating liver diseases and for treating cancer. Thus, insights regarding the mechanism of action of the compounds with respect to cancer cells can underpin the mechanism of actions of these compounds regarding the treatment of liver disease. The compounds and their mechanisms of action are further described as follows:

[0035] Many attempts to treat malignant neoplastic cells through conventional therapies have been met with limited success due to inter- and intra-tumor heterogeneity within the mosaic of tumor subclones.

[0036] Multi-clonal tumors are major setbacks to moleclarly-targeted therapies (which only inhibit subsets of certain tumor clones that host the druggable oncogenic targets), thus rendering other competing tumor subclones (with undruggable oncogenic targets) resistant to such treatments.

[0037] Moreover, some tumor cells exhibit stem like properties with limited proliferation particularly upon exposure to treatment yet retain self-renewal capacity (cancer stem cells or CSCs) which could result in tumor relapse. CSCs could also migrate to distant organs and form distant metastatic tumors with more diverse tumor subclones, particularly under increased selective pressures of survival, thus certain emerging subclones will develop increased resistance to chemotherapy and molecular targeted therapy which will further progress to aggressive tumor progression, worse prognosis and poor survival.

[0038] From a patient point of view, chemotherapy leads to poor quality of life due to well- documented side effects, including cancer cachexia (muscle wasting and loss of appetite), associated with high pro-inflammatory TNF-alpha levels, chemotherapy induced alopecia due to non-specific targeting of high proliferative cells, fatigue and physical (including bone cancer pain) and neurological pain through IL-6-TRPA1 and TNF-alpha-TRPA1 pathways (Liu et al., 2019). Blocking IL-6 and TNF-alpha was shown to be beneficial in inhibiting pain in in-vivo models including chemotherapy-induced pain.

[0039] Thus, of great interest, was the paradigm shift towards antigen-independent im- muno-oncology for cancer treatment to concurrently target different subclones of the heterogenous tumor mosaic simultaneously.

[0040] However, despite efforts of non-antigen specific immune-oncology (particularly immune checkpoint inhibitors ICIs) showing promise in achieving partial and even some complete responses in can-cer patients, a high risk of autoimmune-like immune related adverse events (iRAEs) commonly arises in patients. Persistence in T-cell responses, through in-hibiting T-cell exhaustion (via ICIs) could prolong the expression of inflammatory cytokines and could trigger self-antigen presentation alongside tumor antigen presentation; thus, targeting both tumor and surrounding inflamed target organ or tissue (Kdnig & Laubli, 2020). Post-mortem studies showed that melanoma cancer patients receiving ICIs led to immune infiltration in the myocardial tissue which developed into myocarditis (Gurdogan, 2020).

[0041] Among the most common inflammatory cytokines as biomarkers of iRAEs is IL-6 which is also known to be a surrogate of immune response, inflammation, tumor progression and pain. Serum IL-6 is associated with worse prognosis and poor survival in cancer patients.

[0042] Among the first treatments of iRAEs such severe arthritis, myocarditis, uveitis, great vasculitis, severe pneumonia, great vasculitis, and myasthenia gravis is Tocili- zumab (Anti IL-6 Receptor antibody) which inhibits IL-6 signalling. Targeting IL-6 pathway does not activate tumor progression.

[0043] Therefore, tumor cell killing without over-expression of IL-6 and / or activate IL-6 signalling pathways and other inflammatory cytokines pose as great benefit for pa- tients such as inhibit-ing cancer cachexia, physical and neuropathic pain and fatigue- thus contributing towards improving quality of life.

[0044] A combination of Ginsenoside and Mebendazole can be used to trigger immune- mediated tumor cell killing without the secretion of IL-6. Therefore, Ginsenoside and Mebendazole combination reduces cytokine release commonly associated with cancer cachexia, physical and neuropathic pain, fatigue and poor quality of life.

[0045] Ginsenoside, a bioactive saponin compound found in Panax ginseng in trace amounts, poses anti-proliferative and pro-apoptotic effects in tumors. Ginsenoside contributes to both extrinsic apoptosis (via Death receptors such as FAS and TRAIL) and intrinsic mitochondrial apoptosis, resulting in caspase 8 and caspase 3 / 7 activation downstream.

[0046] Additionally, activated caspase 8 could also indirectly activate caspase 3 / 7 by truncating BID, a pro-apoptotic protein, into its functional form tBID; tBID then contributes to translocating BAX to the mitochondria to re-lease mitochondrial cytochrome c thus triggering intrinsic apoptosis as well.

[0047] With respect to extrinsic apoptosis, tumors generally live in harsh pathophysiological conditions such as low glucose and hypoxia which trigger intracellular ER stress and impairment in protein folding, lipid metabolism and calcium level regulation. Ginsenoside increases ER stress towards which expressing death receptors such as DR4 and DR5 receptors in tumors via PERK-ATF4-CHOP pathway; this primes cancer cells for extrinsic apoptosis upon interaction with cells that strongly express TRAIL (ligand) particularly, activated T cells and monocytes (Lam et al., 2020; Martin-Perez et al., 2011 ).

[0048] However, there are several TRAIL-resistant cancer cells which are due to increased pro-survival anti-apoptotic proteins. Nevertheless, these resistant cells are sensitized to apoptosis upon inhibition of the pro-survival Akt- pathway (commonly expressed in many tumors) inhibition resulting in a de-crease of pro-survival proteins (such as Bcl-2 and BcL-xL) compared to pro-apototic counterparts (BAX). Akt also phosphorylates BAX at Serine 184 residue (S184) which causes BAX to inhibit apoptosis. Ginsenosides inhibit Akt-pathway and disrupt plasma membrane lipid rafts (sphingolipid and cholesterol enriched micro domains of transmembrane proteins found clustered together which serve as external components of cell signalling pathways). This inactivates the pro-surivival Akt pathway and helps to rapidly and strongly translocate BAX into the mitochondria, thus further triggering mitochondrial-based intrinsic apoptosis.

[0049] To further sensitise TRAIL apoptosis, three strategies could be approached: (i) By further decreasing pro-survival proteins in tumors through further inhibition of prosurvival proteins, (ii) By increasing TRAIL- ligand expression in cells surrounding tumors, by enhancing immune activation, (iii) Convert non-immunological 'cold' tumors to immune-infiltrative hot tumors.

[0050] Mebendazole, an anti-helminth drug, addresses the three strategies above by (i) phosphorylating and deactivating BCL-2 protein, (ii) enhance T-cell activation through increased clustering and interaction between CD14+monocytes / macrophages and T-cells and (iii) re-polarize immunosuppressive tumor-promoting M2 macrophages to M1 classically activated antitumor macrophages which can directly kill the tumor and provide chemokine signals to increase immune infiltration of activated immune cells expressing TRAIL could interact with TRAIL-sensitized tumor cells to induce cancer cell death.

[0051] Hence in summary, the tumor cell death is based on extrinsic death receptor-based and intrinsic mitochondrial-based apoptotic pathways, sug-gestively through direct interaction of death ligands in activated immune cells and death receptors in tumor cells; where ginsenosides up-regulate death receptors in stressed malnourished or hypoxic cells (such as tumor cells), while mebendazole enhances T-cell activation and limits intracellular anti-apoptotic proteins in cancer cells.

[0052] Rather than systemically targeting highly proliferative cells which results in off-target toxicities, such as chemotherapy-induced alopecia, selective tumor cell killing based upon cross-sectional ER stress-based apoptotic pathways among different tumors.

[0053] A first aspect of the present invention pertains to a composition for use in a method of treating a liver disease, preferably metabolic dysfunction-associated steatohepa- titis (MASH), the composition comprising an effective amount of at least one saponin agent and I or an effective amount of at least one anthelmintic agent and I or an effective amount of at least one mitochondrial uncoupling agent.

[0054] The mitochondrial uncoupling agent can in some cases exhibit anthelmintic activity, as some anthelmintics are effective through mitochondrial uncoupling.

[0055] Thus, in some cases the anthelmintic agent and the mitochondrial uncoupling agent can be the same compound. However, it can also be the case that the anthelmintic agent and the mitochondrial uncoupling agent are separate compounds and the composition according to the present invention comprises only one of these two separate compounds.

[0056] Similarly, the saponin agent, the anthelmintic agent, and the mitochondrial uncoupling agent can be the same compound or can each be a separate compound.

[0057] An anthelmintic agent preferably is preferably a compound exhibiting worm-killing actions when administered to a human being in a physiologically acceptable dosage. Exemplary anthelmintic agents can be found in class P02 of the ATC classification.

[0058] A saponin agent preferably is a compound composed of glycone and aglycone moieties. Saponins can be classified as steroidal, steroidal alkaloids, and triterpenoidal based on the carbon skeleton of the aglycones.

[0059] A saponin to be used in the context of the present invention can be for example (i) Hederagenin (a Oleanane triterpenoid saponin) (1), (ii) Ursolic acid (a Ursane triterpenoid saponin) (2), (iii) Betulinic Acid (a Lupine triterpenoid saponin) (3), (iv) Ginsinosides (a Dammarane triterpenoid saponin) (4), and (v) Daucosterol (a Cho- lestane steroid saponin) (5), (vi) Dioscin (a Spirostane steroid saponin) (6), (vii) Pro- todioscin (a Furostane steroid saponin) (7) and (viii) Digoxin (a Cardenolide steroid saponin) (8)

[0060] A mitochondrial uncoupling agent preferably is an agent causing mitochondrial uncoupling. Exemplary mitochondrial uncoupling agents that can be used in the con- text of the present invention are shown in the table below alongside their mechanisms of action.

[0061] Category Mechanism Specific examples:

[0062] Classical I Dissipate proton gradi2,4-DNP, CCCP, FCCP

[0063] Protonophoric ent by carrying H+ Uncouplers across membrane Fatty Acid Fatty acids shuttle H+ Oleic acid (with LICP1 or ANT) Cycling Un- with help of proteins couplers (e.g., UCPs, ANT) Protein- Uncoupling proteins UCP1 , UCP2, UCP3

[0064] Mediated Un- form regulated proton couplers channels lonophoric Facilitate transport of Valinomycin, Nigericin Uncouplers ions, disrupting membrane potential

[0065] Synthetic I Engineered molecules BAM15, SR4, Niclosamide,

[0066] Novel Unfor controlled / mild unNitazoxanide, Flubendazole couplers coupling

[0067] The specific examples disclosed in the table, however, are only examples and in the context of the present invention, other mitochondrial uncouplers, in particular acting by one of the exemplary mechanisms shown in the table above, may be used.

[0068] In the following, aspects of the present disclosure are described that may be realised individually or in combination in an embodiment or embodiments of the pre- sent invention. It is readily apparent to the person skilled in the art that the present invention is not limited to embodiments explicitly disclosed but features of the invention can also be claimed in isolation or in other combinations.

[0069] A second aspect relates to the composition of aspect 1 , wherein the saponin agent is a saponin, preferably a ginsenoside or a diosgenin. The ginsenoside may be an extract derived from red, black, or white ginseng, notoginseng or ginseng.

[0070] Generally, in the context of the present invention, steroidal and I or triterpene saponin agents may be used. In principle, also other saponin agents can be used.

[0071] A third aspect relates to the composition of aspect 1 or 2, wherein the anthelmintic agent is a niclosamide and / or a nitazoxanide.

[0072] As niclosamide and nitazoxanide also have a mitochondrial uncoupling effect, these compounds can also be regarded as mitochondrial uncoupling agents.

[0073] A comprision according to the present invention can contain a mitochondrial uncoupling agent. The mitochondrial uncoupling agent can exhibit an anthelmitic effect, however, this is optional.

[0074] The anthelmintic agent and the mitochondrial uncoupling agent are preferably configured to exhibit the anthelmitic or mitochondrial uncoupling effect at physiologically acceptable dosages in a human being.

[0075] The mitochondrial uncoupling agent comprised in a composition according to the present invention can be a protonophoric uncoupler, in particular 2,4-dinitrophenol (2,4-DNP), Carbonyl cyanide m-chlorophenylhydrazone (CCCP) or carbonylcya- nide-p-trifluoromethoxyphenylhydrazone (FCCP), a fatty acid cycling uncoupler, in particular oleic acid, a protein-mediated uncoupler, in particular mitochondrial uncoupling protein 1 (LICP1 ), mitochondrial uncoupling protein 2 (LICP2), mitochondrial uncoupling protein 3 (LICP3), a ionophoric uncoupler, in particular Valinomyin, Nigericin, and / or a synthetic uncoupler, in particular N5,N6-Bis(2- fluorophenyl)[2,1 ,3]oxadiazolo[4,5-b]pyrazine-5,6-diamine (BAM15), SR4, Niclosamide, Nitazoxanide, Flubendazole.

[0076] A composition according to the present invention can comprise a saponin agent and a mitochondrial uncoupling agent, wherein the saponin agent and the mitochondrial uncoupling agent can be the same or separate compounds.

[0077] A composition according to the present invention can comprise a saponin agent and an anthelmitic, wherein the saponin agent and the anthelmitic agent can be the same or separate compounds.

[0078] A composition according to the present invention can comprise a mitochondrial uncoupling agent and an anthelmitic agent, wherein the anthelmitic agent and the mitochondrial uncoupling agent can be the same or separate compounds.

[0079] A fourth aspect relates to the composition according to one of the preceding aspects, wherein the at least one saponin agent is linked or tethered to a targeting molecule selected to selectively deliver the saponin agent to human hepatocytes.

[0080] A fifth aspect relates to the composition of aspect 4, wherein the targeting molecule is a bile acid.

[0081] Alternatively or additionaly, the targeting molecule can be an antibody configured with the at least one saponin agent as an antibody-drug-conjugate, wherein the antibody preferably is selective to a target protein expressed exclusively or predominantly in human hepatocytes.

[0082] For example, the antibody can be a monoclonal antibody selective to a sodium I bile acid cotransporter family integral cell membrane glycoprotein ex-pressed in human hepatocytes, in particular to SLC10A1.

[0083] A sixth aspect relates to the composition according to one of the preceding aspects, wherein the at least one anthelmintic agent and / or the at least one mito- chondrial uncoupling agent is linked or tethered to a targeting molecule selected to selectively deliver the anthelmintic agent to human stellate cells and I or hepatocytes.

[0084] According to a seventh aspect, the targeting molecule can be an amino acid, preferably Ergothioneine.

[0085] In other words, another aspect of the present invention pertains to a composition for use in a method of treating a liver disease, preferably metabolic dysfunction- associated steatohepatitis (MASH), the composition comprising an effective amount of at least one saponin agent, wherein the at least one saponin agent is linked or tethered to a targeting molecule selected to selectively deliver the saponin agent to human hepatocytes, and I or an effective amount of at least one anthelmintic agent and I or at least one mitochondrial uncoupling agent, wherein the at least one anthelmintic agent and I or at least one mitochondrial uncoupling agent is linked or tethered to a targeting molecule selected to selectively deliver the saponin agent to human hepatocytes.

[0086] Generally, each compound or agent can be linked or tethered to one or more targeting molecules using fixed or flexible linkers that may be configured to be selectively cleaved by specific enzymes in situ.

[0087] According to an eigth aspect, a composition according to one of the preceding aspects can further comprise the anthelmintic agent methyl N-(6-benzoyl- IH- benzimidazoi-2-yl)carbamate (mebendazole).

[0088] According to an nineth aspect, a composition according to one of the preceding aspects can further comprise the anthelmintic agent methyl N-[6-(4-fluorobenzoyl)-1 H- benzimidazol-2-yl]carbamate (flubendazole) and / or methyl N-(6-propylsulfanyl-1 H- benzimidazol-2-yl)carbamate (albendazole). A tenth aspect relates to the composition of one of the preceding aspects, wherein the composition is a nanocarrier formulation, wherein the nanocarrier preferably is PEGylat-ed or non-PEGylated.

[0089] An eleventh aspect relates to the composition of aspect 11 , wherein the nanocarrier comprises SMEEDs, SNEDDS, SEDDS, solid lipid nanopartice, nanostructured lipid carrier, microemulsions, liposome, micelles, polymeric nanoparticle, polymeric micelle, dendrimer and I or mesoporous nanoparticles, amorphous solid dispersions, solid dispersions, micronised particles, hydrogels, dendrimers, cyclodextrins, polymer drug conjugates, iron oxide nanoparticle (magnetic carrier), gold nanoparticle

[0090] A twelvth aspect relates to the composition of one of the preceding aspects, wherein the saponin agent, preferably a saponin agent, such as a ginsenoside or dios- genin, is administered at an amount of from 0.001 mg / Kg body weight to 200 mg / Kg body weight, preferably from 0.01 mg / Kg body weight to 100 mg / Kg body weight, in particular from 0.1 mg / Kg body weight to 50 mg / Kg body weight (for mice and rats); and administered at an amount of from 0.001 mg / Kg body weight to 20 mg / kg body weight, preferably from 0.01 mg / Kg body weight to 15 mg / kg body weight, in particular from 0.01 mg / Kg body weight to 10 mg / kg body weight (for humans). The ginsenoside and diosgenin may be applied in the same amounts or different amounts.

[0091] The anthelmintic agent, preferably niclosamide or mebendazole, and / or the mitochondrial uncoupling agent may also be administered at an amount of from 0.001 mg / Kg body weight to 200 mg / Kg body weight (for mice and rats); and administered at an amount of from 0.001 mg / Kg body weight to 20 mg / kg body weight (for humans) with the preferred dosage as recited above. The anthelmintic agent and I or the mitochondrial uncoupling agent, e.g. niclosamide or mebendazole, may be applied in the same amounts or different amounts.

[0092] Similarly, the biguanide agent, preferably metformin, may also be administered at an amount of from 0.001 mg / Kg body weight to 200 mg / Kg body weight for (mice and rats); and administered at an amount of from 0.001 mg / Kg body weight to 20 mg / kg body weight (for humans) with the preferred dosage as recited above.

[0093] The saponin agent, the anthelmintic agent and I or the mitochondrial uncoupling agent may be present at the same or at different amounts in a composition according to the pre-sent disclosure.

[0094] The saponin agent, the anthelmintic agent and I or the mitochondrial uncoupling agent may be present in the same or at different compunds in a composition according to the present disclosure.

[0095] The saponin agent, the anthelmintic agent and I or the mitochondrial uncoupling agent and / or the biguanide agent may be administered at the same or at different amounts or dosages in a method of treatment according to the present disclosure.

[0096] Saponin maybe administered at an amount of from 0.001 mg / Kg body weight to 200 mg / Kg body weight (for mice and rats); and administered at an amount of from 0.001 mg / Kg body weight to 20 mg / kg body weight (for humans) in combination with antihelmentic agent and / or biguanide agent, each preferably administered at the same range of an amount of from 0.001 mg / Kg body weight to 200 mg / Kg body weight for mice and rats); and administered at an amount of from 0.001 mg / Kg body weight to 20 mg / kg body weight (for humans). Each component is preferably administered at an amount of from 0.001 mg / Kg body weight to 200 mg / Kg body weight, preferably from 0.01 mg / Kg body weight to 100 mg / Kg body weight, in particular from 0.1 mg / Kg body weight to 50 mg / Kg body weight (for mice and rats); and administered at an amount of from 0.001 mg / Kg body weight to 20 mg / kg body weight, preferably from 0.01 mg / Kg body weight to 15 mg / kg body weight, in particular from 0.01 mg / Kg body weight to 10 mg / kg body weight (for humans).

[0097] A fifthteenth aspect relates to the composition of one of the precedings aspects, wherein the effective amount of at least one saponin agent and the effective amount of at least one anthelmintic agent and I or the at least on emitochondrial uncoupling agent and / or the effective amount of at least one biguanide agent are present in a single formulation or are present in at least two separate formula-tions, wherein preferably the saponin agent, e.g. a ginsenoside, is administered at an amount of from 0.001 mg / Kg body weight to 200 mg / Kg body weight (for mice and rats); and administered at an amount of from 0.001 mg / Kg body weight to 20 mg / kg body weight (for humans). The saponin agent is preferably administered at an amount of from 0.001 mg / Kg body weight to 200 mg / Kg body weight, preferably from 0.01 mg / Kg body weight to 100 mg / Kg body weight, in particular from 0.1 mg / Kg body weight to 50 mg / Kg body weight (for mice and rats); and administered at an amount of from 0.001 mg / Kg body weight to 20 mg / kg body weight, preferably from 0.01 mg / Kg body weight to 15 mg / kg body weight, in particular from 0.01 mg / Kg body weight to 10 mg / kg body weight (for humans).

[0098] The anthelmintic agent, preferably niclosamide or nitazoxanide or mebendazole, and I or the mitochondrial uncoupling agent may also be administered at an amount of from 0.001 mg / Kg body weight to 200 mg / Kg body weight for mice and rats); and administered at an amount of from t0.001 mg / Kg body weight to 20 mg / kg body weight (for humans).

[0099] The effective amount of at least one saponin agent and the effective amount of at least one anthelmintic agent agent and I or the effective amount of the at least one mitochondrial uncoupling agent can be administered sequentially or concurrently.

[0100] The present disclosure also encompasses the aspect of a method of preparation of a composition according to one of the preceding aspects.

[0101] Also, the present disclosure encompasses intermediate stages, such as an anthelmintic agent, preferably niclosamide, and / or a mitochondrial uncoupling agent and / or a saponin agent linked or tethered to an amino acid, preferably ergothioneine, for use in the treatment of liver disease.

[0102] Similarly, the present disclosure encompasses intermediate stages, such as a saponin agent, preferably ginsensoside or diosgenin,and / or a mitochondrial uncoupling agent and / or an anthelmitic agent linked or tethered to a bile acid, for use in the treatment of liver disease. The saponin agent can be e.g. a steroidal or triterpene saponin agent.

[0103] Another aspect of the disclosure relates to a method of treating liver disease, the method comprising administering to a subject in need thereof an effective amount of at least one preferably steroidal or triterpene saponin agent, in particular ginsen- soside or diosgenin, and an effective amount of at least one anthelmintic agent and I or mitochondrial uncoupling agent, in particular niclosamide. Preferably, a composition according to the present invention is administered.

[0104] The subject is preferably human. The liver disease can be a disease involving a pathology of hepatocytes and I or stellate cells.

[0105] The composition can be or comprise a nanocarrier formulation, wherein the nanocamer preferably is PEGylated or non-PEGylated. The nanocarrier can comprise SMEEDs, SNEDDS, SEDDS, solid lipid nanopartice, nanostructured lipid carrier, microemulsions, liposome, micelles, polymeric nano-particle, polymeric micelle, dendrimer and I or mesoporous nanoparticles, amor-phous solid dispersions, solid dispersions, micronized particles, hydrogels, den-drimers, cyclodextrins, polymer drug conjugates, iron oxide nanoparticle (magnetic carrier), gold nanoparticle

[0106] Another aspect of the disclosure relates to a kit for treating liver disease, preferably MASH, in a human subject, the kit comprising a composition according to the present invention and instructions for use.

[0107] The compositions according to the present invention can be used in a method of treating liver disease, wherein the liver disease is preferably selected from the group consisting of alcohol-related liver disease, non-alcoholic fatty liver disease, hepatitis, primary biliary cirrhosis and / or liver fibrosis.

[0108] The applicability of the composition or compositions disclosed herein for the treatment of liver diseases is preferably based on the mechanism of action of the compounds used described in the the present application. There is thus a plausible mechanism of action proposed herein supporting the effect of the claimed compositions in the treatment of liver diseases. Further experiments relating thereto are being performed.

[0109] The present disclosure is not limited to the features or aspects of the invention as described above, but also encompasses any such feature or aspect in isolation as well as any combination of features or aspects described above.

[0110] In the context of the present invention, an anthelmintic preferably is a compound selectively exhibiting anti-helminth properties. Thus, preferably, an “anthelmintic agent” as used in the context of this disclosure does not equate to any compound exhibiting any anti-helminth properties at any concentration (such as most compounds do if the concentration or time of exposure is only chosen to be high enough, e.g. peppermint oil, sodium chloride or HCI) but is a compound exhibiting specific and I or selective anti-helminth effects. The anthelmintic can be a compound not merely suitable but configured to exhibit specific and I or selective antihelminth effects. The anthelmintic can be a coumpound classified as an anthelmitic according to the ATC classification.

[0111] Saponin agents as mentioned in the context of this disclosure can comprise steroid glycosides and triterpene glycosides or any other saponins.

[0112] Mitochondrial uncoupling agents can preferebaly be any agents exhibiting mitochondrial uncoupling effect in humans at a physiologically viable dosage.

[0113] The disclosure is further illustrated drawings and by exemplary experimental data that are dis-cussed in the following section. Further features, effects and advantages of the present invention become apparent from the following discussion referring to Figs. 1 to 12.

[0114] Figure 1 illustrates the regression from MASLD / NAFLD to MASH / NASH to HCC. F0-F4 signifies fibrosis stage: stratified on liver biopsy to stages 0 to 4 using the METAVIR scoring system: FO — no fibrosis, F1 — portal fibrosis, F2 — periportal fibrosis, F3 — bridging fibrosis, F4 — cirrhosis. (18).

[0115] Figure 2 shows the revalence of significant liver fibrosis (F2-3) and cirrhosis (F4) by MASLD and MASH status. * p < 0.05; ** p < 0.001 . p-values refer to chi-square test between patients with MASH (third column) and those with MASLD but without MASH (second column) or those with no MASLD (first column) and are considered significant when < 0.05 (19).

[0116] Figure 3 indicates the verall MASH prevalence in the general population. MASH non-alcoholic steatohepatitis, UK United Kingdom, USA United States, a Values rounded to 1 decimal point (21 ).

[0117] Figure 4 illustrates an algorithm for diagnosis of MASLD / NAFLD and non- invasive assessment of liver fibrosis [1], * NFS threshold <0.12 for patients > 65 years # FIB-4 threshold <2.0 for patients >65 years old. Abbreviations: ELF - enhanced liver fibrosis, FIB-4 - Fibrosis 4, NFS - NAFLD fibrosis core, NPV - negative predictive value, PPV - positive predictive value (24).

[0118] Figure 5 illustrates key mechanisms underlying MASH - a complex positive feedback loop involving four key components - hepatocyte damage, inflammation, HSC activation and ECM accumulation.

[0119] Figure 6 shows a Schematic image of microscopic features of quiescent vs activated HSCs.

[0120] Figure 7 shows an immunostaining in that primary HSCs that present wildtype PNPLA3 and wildtype SM6SF2 were stained for F-actin cytoskeleton (Phalloidin; red), Lipid droplets (BODIPY; green) and nuclei (DAPI; blue) in untreated control (A) and treated (combination) conditions (B). Lipid droplets (BODIPY; green) were not evident in the untreated cells (C) and were highly evident in the treated (combination) cells (D). All images are 10X magnification, scale bar: 100 pm. Figure 8 shows an immunostaining in that primary HSCs that present mutant PNPLA3 (rs738409) and mutant SM6SF2 (rs58542926) were stained for F- actin cytoskeleton (Phalloidin; red), Lipid droplets (BODIPY; green) and nuclei (DAPI; blue) in untreated control (A) and treated (combination) conditions (B). Lipid droplets (BODIPY; green) were not evident in the untreated cells (C) and were highly evident in the treated (combination) cells (D). All images are 10X magnification, scale bar: 100 pm.

[0121] Figure 9 shows the mRNA expression of Primary HSCs that present mutant PNPLA3 (rs738409) and mutant SM6SF2 (rs58542926) for (A) aSMA, (B) PPARG, (C) COL3A1 , (D) MMP9, (E) PDGFRB and (F) TGFBR1. n = 4 per condition, ** p < 0.01 , *** p < 0.001 , and **** p < 0.0001 .

[0122] Figure 10 illustrates a FACS analysis of PBMCs stimulated with CD3 and CD28 comparing treated (combination) and untreated cells for (A) cell viability of T-cells, (B) Ki67 expression of CD4+ T-cells, (C) Ki67 expression of CD8+ T-cells, (D) INFy expression of CD4+ T-cells, and (E) TNFa expression of CD8+ T-cells n = 4 per condition, * p < 0.05.

[0123] Figure 11 gives an overview of MASH therapeutic agents in advanced clinical trials. Comparison of therapeutic target (Hepatocyte lipotoxicity, HSC activation and fibrosis, and / or inflammation; yellow circles indicate secondary indirect effect), multiple molecular targets, route of administration (oral=green vs injectable=red), and targeted delivery of therapeutic. Category is based on mode of action, A=Glucose metabolism, B= Lipid metabolism and C=HSC activation / Fibrosis.

[0124] Figure 12 illustrates the concept of selectively targeting therapeutic agents according to the present invention to target cells, such as hepatocytes and stellate cells. Bile acid transporters are highly expressed in intestinal cells and hepatocytes and are ideal transporters for a “Trojan horse” strategy. Similarly, the amino acid Ergothioneine is mainly delivered to the liver after oral ingestion. However, interestingly, Ergothioneine is minimally taken up into hepatocytes but taken up into HSCs.

[0125] For the experiments leading to the experimental data shown in Fig. 7 to Fig. 10, primary human hepatic stellate cells (pHSCs) from different donors were genotyped for the PNPLA3-rs738409 and TM6SF2-rs58542926 SNP mutations and two donors were chosen for this study; Donor 1 has wildtype PNPLA3 and TM6SF2, and Donor 2 had the aforementioned mutations in both genes. Human liver tissues were acquired from consented patients undergoing resection at a Central London Hospital. Isolation of pHSCs from tissue sections was performed immediately, following the protocol detailed in Mederacke et al. (158).

[0126] As mentioned above, activation of HSCs is crucial to the development of fibrosis in MASH. During activation, HSCs transform into myofibroblast-like cells with concomitant loss of their lipid droplets and production of excessive ECM. The loss of ability to store lipid droplets is a defining feature of activated HSCs. Accumulating evidence supports the proposal that recovering the retention of lipids would inhibit the activation of HSCs and revert them into their “healthy” quiescent state (Figure 6).

[0127] Initially, the viability of activated pHSCs (both donors) were assessed when treated with the compounds Ginsenoside and Niclosamide as monotherapies and in combination, as well as microscopic changes in morphology under brightfield microscopy to determine the most appropriate drug concentrations that maintained the pHSCs around or above 50% viability while changing the morphology back towards a quiescent state.

[0128] Next, F-actin cytoskeleton (Phalloidin; red), lipid droplets (BODIPY; green) and nuclei (DAPI; blue) were stained in treated (combination of Ginsenoside and Niclosamide) and untreated pHSCs from both donors. It was evident in the PNPLA3 and TM6SF2 wildtypes pHSCs a change in cytoskeleton morphology (Figure 7 A, B) and more strikingly an increase in lipid droplets within the treated cells compared to the untreated cells (Figure 7 C, D). The effects of the PNPLA3-I148M variant on MASLD / MASH severity have been extensively studied (42,43). In previous studies, the rs738409 variant increased the risk for hepatic steatosis, liver fibrosis / cirrhosis, as well as HCC in a wide spectrum of liver disease (44). The PNPLA3 protein is highly expressed in the liver and has lipase activity towards triglycerides in hepatocytes and towards retinyl esters in HSCs (45). The I148M mutation leads to loss of function resulting in hepatic fat and retinol retention (42). Additionally, it has been demonstrated that a TM6SF2 variant negatively affects aSMA expression in HSCs, and the TM6SF2 isoform associated with the rs58542926 SNP affects HSC activation sensitivity and hence, plays a role in the process of HSC activation and liver fibrosis in MASH (46).

[0129] In the inventor's experiments, when pHSCs carrying both these mutations were treated (combination) and stained for F-actin cytoskeleton (Phalloidin; red), Lipid droplets (BODIPY; green) and nuclei (DAPI; blue), it was also evident that there were change in cytoskeleton morphology (Figure 8 A, B) and additionally it was able to revert these cells to significantly store lipid droplets in the treated cells compared to the untreated cells (Figure 8 C, D).

[0130] Next, the changes in expression levels of HSC markers on the treated (combination of Ginsenoside and Niclosamide) and untreated PNPLA3 / TM6SF2 mutant pHSCs were investigated. It is well documented that aSMA is up-regulated and maintained in HSCs as they transition to activated myofibroblast-like phenotype during liver injury and the wound healing response (47,48).

[0131] In our results, treated cells had a 20-fold significant reduction (p<0.001) in aSMA expression compared to untreated cells (Figure 9 A). PPAR-y (PPARG) is expressed in quiescent healthy HSCs, and its expression gradually decreases with increased HSC activation stimulated by inflammation or injury, indicating that PPAR-y inhibits HSC activation and maintains its quiescence (49). Indeed, PPAR-y has been a highly studied therapeutic target for liver disease (50).

[0132] In our results, treated cells had a 10-fold significant increase (p<0.001 ) in PPARG expression compared to untreated cells (Figure 9 B). Liver fibrosis is associated with major alterations in both the quantity and composition of ECM (34). In advanced stages, the liver contains approximately 6 times more ECM than normal, including collagens (I, III, and IV) (51 ).

[0133] In our results, treated cells had a 5-fold significant decrease (p<0.0001 ) in COL3A1 expression compared to untreated cells (Figure 9 C). Additionally, it is important to be able to reverse the fibrotic damage in the liver by eliminating and remodelling the ECM. Matrix metalloproteinases (MMPs) are involved in the degradation of various proteins in the ECM. MMP9 is one of the most widely studied MMP and plays a vital role in various fibrotic diseases, and it can affect the progression and regression of fibrotic diseases through different signalling pathways (52).

[0134] In our results, untreated cells had almost lost their ability to express MMP9, and indeed treated cells had a 170-fold significant increase (p<0.0001) in MMP9 expression compared to untreated cells (Figure 9 D). Among mitogenic pathways in HSCs, signalling by [3-PDGFR (PDGFRB) is the most potent (53). Expression of PDGF receptors is low in healthy liver but increases in HSCs during injury. In our results, treated cells had a 1.8-fold significant decrease (p<0.01 ) in PDGFRB expression compared to untreated cells (Figure 9 E). Similar to PDGF, the TGF-[3 signalling is considered the key fibrogenic pathway that drives HSC activation and induces ECM production (54). Hence downregulating its receptor on HSCs is an important factor in reducing activation.

[0135] In our results, treated cells had a 2-fold significant decrease (p<0.001) in TGFBR1 expression compared to untreated cells expression compared to untreated cells (Figure 9 E).

[0136] As mentioned above, the hepatic inflammatory response is an important driving force of MASH disease progression, as it promotes sustained hepatic fibrogenesis, which ultimately leads to cirrhosis. Indeed, there is sufficient evidence to support the concept that hepatic inflammation drives liver fibrosis. Therefore, it was important to investigate the effect of our treatment on immune cells, and specifically T- cells involved in MASH inflammation. We initially investigated the effect of our treatment on T-cell viability, which demonstrated no significant difference in the treated (combination) vs untreated cells (Figure 10 A). Further, increasing evidence indicates that T cell-mediated adaptive immunity also provokes inflammation and fibrosis in MASH via cytotoxicity, cytokines and other proinflammatory and profibrotic mediators.

[0137] It has recently been revealed through single-cell transcriptome profiling that the populations of CD4+ T cells and CD8+ T cells are expanded in the livers with MASH (55). Therefore, we investigated if our treatment would reduce proliferation of these cells, which indeed indicated a significant reduction in Ki67 (a proliferation marker; p>0.05) expression in both CD4+ T-cells (Figure 10 B) and CD8+ T-cells (Figure 10 C) in the treated (combination) cells compared to untreated cells.

[0138] Furthermore, in a study in humanized mice to identify human-specific immune response in MASH, CD4+ T cells were found to be crucial in promoting liver steatosis-fibrosis transition (56). Moreover, in vivo depletion of human CD4+ T cells can efficiently reduce proinflammatory cytokine production and fibrosis in the humanized MASH mice, further confirming the importance of CD4+ T cells in the pathogenesis of MASH (56). Other evidence also supports a potential role of CD4+ T cells in promoting MASH by releasing proinflammatory cytokines, because MCD- HFD-induced MASH can be significantly attenuated in mice deficient for IFNy (57). It is well known that CD4+ T cells have several functionally diverse subsets, such as Th1 , Th2, Th17, Th22, and Tregs, which are characterized by expression of different cytokines respectively. Th1 cells are solely known to be profibrotic and are characterized by the expression of IFNy (57,58). In our experiments, the percentage of stimulated CD4+ T-cells expressing IFNy was significantly reduced (p<0.05) in treated (combination) vs untreated cells (Figure 10 D).

[0139] Finally, growing evidence shows that CD8+ T-cells accumulate in the liver of patients with MASH (59-61) and mouse models of the disease (60,62). In patients with MASH, the increased number of CD8+ T cells in the liver correlates with elevated frequency of blood CD8+ T cells expressing perforin, IFNy, and TNFa, raising the possibility of systemic activation or crosstalk with other tissues (30). Experimental depletion of total CD8+ T-cells in animal models ameliorates MASH (63) and subsequent transition to HCC (60), suggesting that CD8+ T-cells directly promote disease progression. Indeed, improved MASH in CD8+ T-cell-deficient mice is accompanied by restored hepatic insulin sensitivity (64), decreased liver damage (60), and reduced fibrosis (62).

[0140] In our experiments, the percentage of stimulated CD8+ T-cells expressing TNFa was significantly reduced (p<0.05) in treated (combination) vs untreated cells (Figure 10 E).

[0141] It is now evident that MASH is a complex "closed" positive feedback loop involving four key components: hepatocyte damage, inflammation, HSC activation, and ECM accumulation. Consequently, once all four factors have been initiated, halting only one event would prove insufficient to resolve the disease. For instance, resolving solely hepatocyte injury would momentarily slow disease progression, yet activated immune cells (inflammation) would eventually reinitiate injury to the hepatocytes. Similarly, the stiffened and accumulated ECM would continue to exert pressure on hepatocytes, leading to re-injury. Likewise, another example would be treating solely the HSCs, however, without addressing hepatocyte injury and inflammation, it would likely reactivate HSCs post-treatment, and the stiff dense ECM would also significantly contribute to their re-activation.

[0142] Therefore, the comprehensive therapeutic approach of the present invention concurrently addresses multiple aspects of MASH pathogenesis, encompassing inflammation, fibrosis (HSC activation and ECM accumulation), and metabolic dysfunction (hepatocytes). We firmly believe that such a multi-pronged strategy is imperative to attain clinically significant outcomes and impede disease progression.

[0143] Further studies are being performed at the moment to corroborate the therapeutic effect of the compositions disclosed herein for the treatment of liver disease: In order to maximize success while minimizing risk, the inventors have meticulously crafted the next phase of development. This plan is structured into three distinct stages, each strategically designed to achieve our objectives efficiently and effectively.

[0144] Stage 1 : This study aims to assess the efficacy of unconjugated drugs as monotherapies and in combination within a MASH mouse model. Assessment criteria encompass bi-weekly monitoring of body weight, determination of liver weight, and measurement of plasma ALT / AST / TC / TG / HDL-C / LDL-C levels at the study's conclusion. Additionally, hepatic TG levels and liver inflammation, assessed via H&E staining and scoring, as well as fibrosis percentage, determined via Sirius red staining scoring, will be evaluated. Proposed mice models for consideration include cheaper and faster studies such as (i) diet supplemented with chemical induction (e.g., high-fat diet + CCL4-induced MASH model), or (ii) spontaneously genetically induced MASH (e.g., GemPharmatech's Chow Diet B6-Alms1-del model).

[0145] Output: The accepted outcome to move forward from the study will be (i) a reduction of overall cholesterol and LDL, (ii) an improvement of at least 2 stages in the NAFLD activity score (NAS), and (iii) an improvement of at least 1 stage of fibrosis.

[0146] Stage 2: As an approach according to the present invention will entail oral administration targeted to the designated site of action, "Drug A" will undergo conjugation with a bile acid, thereby augmenting liver bioavailability and facilitating direct targeting to hepatocytes. In a similar manner, "Drug B" will be conjugated to an amino acid selectively taken up by HSCs (this is conceptionally shown in Figure 12).

[0147] Bile acid transporters are highly expressed in intestinal cells and hepatocytes, rendering them optimal candidates for a "Trojan horse" approach. Likewise, the amino acid Ergothioneine is predominantly delivered to the liver post-oral ingestion. Intri- guingly, Ergothioneine demonstrates minimal hepatocyte uptake but is actively internalized by HSCs. Therefore, this subsequent phase of the inventors developmental strategy will involve the conjugation of our pharmaceutical compounds with their respective carriers. The inventors have pinpointed the requisite functional groups on the compounds, ensuring that such conjugation does not compromise their intrinsic activity post-release. Presently, the focus lies on exploring various linker chemistries to optimize the method of drug release from carriers after cellular uptake, while precluding extracellular drug release.

[0148] As a contingency plan, “Drug A” will be developed as an Antibody-Drug-Conjugate (ADC). A useful antibody identified, SLC10A1 , a sodium / bile acid cotransporter family integral cell membrane glycoprotein, is only expressed in hepatocytes and nowhere else in the human body. Advances in ADCs and ADC-linker chemistry make this option much easier and with a higher chance of success. However, for long-term MASH treatments, ADCs are less favourable, as they can only be administered through injectables and would also be significantly more expensive.

[0149] Furthermore, the inventors are presently investigating an alternative to “Drug A", namely Diosgenin that has the advantage of easy conjugation due to it having only 1 functional group.

[0150] Diosgenin also demonstrates attenuation of lipid metabolism as a result of inhibiting the hepatic fatty acid and triglyceride accumulation via activation of the AMPK / ACC / CPT-1A pathway and suppressing the SREBP-1c and FASN genes (37)(38).

[0151] Output: the inventors will test the conjugates’ (prodrugs) abilities to selectively target their desired cell types in vitro, i.e. , Prodrug A to hepatocytes and Prodrug B to HSCs. Additionally, the inventors will investigate their ability to retain their known drug functions through viability assays, immunofluorescence and gene expression changes, as presented in the results above. The inventors will additionally evaluate the stability of conjugates in blood plasma and. Accordingly, the conjugates with the best stability in blood and desired effect in the target cells will be chosen for Stage 3. Stage 3: This study aims to assess the efficacy of conjugated drugs as monotherapies and in combination within two MASH mouse model. Similar to Stage 1 , the assessment criteria encompass bi-weekly monitoring of body weight, determination of liver weight, and measurement of plasma ALT / AST / TC / TG / HDL-C / LDL-C levels at the study's conclusion. Additionally, hepatic TG levels and liver inflammation, assessed via H&E staining and scoring, as well as fibrosis percentage, determined via Sirius red staining scoring, will be evaluated. Initially, the first MASH model will be the same model chosen in Stage 1 . Accordingly, if the desired outcome is achieved the study will be repeated in Gubra's GAN DIO-MASH model.

[0152] Output: The accepted outcome to move forward will be (i) a reduction of overall cholesterol and LDL, (ii) an improvement of at least 2 stages in the NAFLD activity score (NAS), and (iii) an improvement of at least 1 stage of fibrosis for the initial mouse model. Further, the outcome from the Gubra GAN DIO-MASH model will be (i) a reduction of overall cholesterol and LDL, (ii) an improvement of at least 2 stages in the NAFLD activity score (NAS), and (iii) an improvement of at least 2 stages of fibrosis.

[0153] References

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Claims

Claims1 . A composition for use in a method of treating a liver disease, preferably metabolic dysfunction-associated steatohepatitis (MASH), the composition comprising an effective amount of at least one saponin agent and I or an effective amount of at least one anthelmintic agent and I or an effective amount of at least one mitochondrial uncoupling agent.

2. The composition for use of claim 1 , wherein the saponin agent is a steroidal saponin, preferably a ginsenoside or a diosgenin, and / or a triterpene saponin and I or a hederagenin and / or a oleanane triterpenoid saponin and I or ursolic acid and I or an ursane triterpenoid saponin and I or betulinic acid and / or a lupine triterpenoid saponin and / or a dammarane triterpenoid saponin and I or daucosterol and I or a cholestane steroid saponin and / or a dioscin and / or a spirostane steroid saponin and I or protodioscin and / or a Furostane steroid saponin and I or digoxin and / or a cardenolide steroid saponin.

3. The composition for use according to claim 1 or claim 2, wherein the anthelmintic agent is a mitochondrial uncouler, in particular niclosamide or nitazoxanide.

4. The composition for use according to one of the preceding claims, wherein the mitochondrial uncoupling agent is a protonophoric uncoupler, in particular 2,4- dinitrophenol (2,4-DNP), Carbonyl cyanide m-chlorophenylhydrazone (CCCP) or carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a fatty acid cycling uncoupler, in particular oleic acid, a protein-mediated uncoupler, in particular mitochondrial uncoupling protein 1 (LICP1 ), mitochondrial uncoupling protein 2 (LICP2), mitochondrial uncoupling protein 3 (LICP3), a ionophone uncoupler, in particular Valinomyin, Nigericin, and / or a synthetic uncoupler, in particular N5,N6-Bis(2-fluorophenyl)[2,1 ,3]oxadiazolo[4,5- b]pyrazine-5,6-diamine (BAM15), SR4, Niclosamide, Nitazoxanide, Flubendazole.

5. The composition for use according to one of the preceding claims, wherein the composition comprises a saponin agent and a mitochondrial uncoupling agent, wherein the saponin agent and the mitochondrial uncoupling agent are separate compounds.

6. The composition for use according to one of the preceding claims, wherein the at least one saponin agent is linked or tethered or conjugated to a targeting molecule selected to selectively deliver the saponin agent to human hepatocytes.

7. The composition for use according to claim 6, wherein the targeting molecule is a bile acid, preferably chenodeoxycholic acid, or the targeting molecule is an antibody configured with the at least one saponin agent as an antibody-drug- conjugate, wherein the antibody preferably is a monoclonal antibody selective to a sodium I bile acid cotransporter family integral cell membrane glycoprotein expressed in human hepatocytes, in particular to SLC10A1.

8. The composition for use according to one of the preceding claims, wherein the at least one anthelmintic agent and I or the at least one mitochondrial uncoupling agent is linked or tethered or conjugated to a targeting molecule selected to selec-tively deliver the anthelmintic agent and I or the at least one mitochondrial uncoupling agent to human stellate cells.

9. The composition for use according to claim 8, wherein the targeting molecule is an amino acid, preferably Ergothioneine, or the targeting molecule is an antibody configured with the at least one anthelmintic agent and I or the at least one mitochondrial uncoupling agent as an antibody-drug-conjugate, wherein the antibody preferably is a TH1 -antibody.

10. The composition for use according to one of the preceding claims, further comprising the anthelmintic agent methyl N-(6-benzoyl- IH-benzimidazoi-2- yljcarbamate (mebendazole).11 . The composition for use according to one of the preceding claims, further comprising the anthelmintic agent methyl N-[6-(4-fluorobenzoyl)-1 H-benzimidazol- 2-yl]carbamate (flubendazole) and / or methyl N-(6-propylsulfanyl-1 H-benzimidazol- 2-yl)carbamate (albendazole).

12. The composition for use according to one of the preceding claims, wherein the composition is a nanocamer formulation, wherein the nanocamer preferably is PEGylated or non-PEGylated.

13. The composition for use of claim 12, wherein the nanocamer comprises a liposome, micelles, polymeric nanoparticle, polymeric micelle, dendrimer and I or mesoporous nanoparticles.

14. The composition for use according to one of the preceding claims, wherein the saponin agent, preferably a steroidal saponin agent, in particular diosgenin or gin- senoside, is administered at an amount of from 0.001 mg / Kg body weight to 20 mg / Kg body weight, preferably from 0.01 mg / Kg body weight to 15 mg / kg body weight, in particular from 0.01 mg / Kg body weight to 10 mg / kg body weight.

15. The composition for use according to one of the preceding claims, wherein the anthelmintic agent and I or the mitochondrial uncoupling agent, preferably a niclosamide, is administered at an amount of from 0.001 mg / Kg body weight to 20 mg / Kg body weight, preferably from 0.01 mg / Kg body weight to 15 mg / kg body weight, in particular from 0.01 mg / Kg body weight to 10 mg / kg body weight.

16. The composition for use according to one of the preceding claims, wherein the effective amount of at least one saponin agent and the effective amount of at least one anthelmintic agent and I or the at least one mitochondrial uncoupling agent are present in a single formulation or are present in at least two separate formulations and I or are present in a single compound or are present in at least two compounds.

17. The composition for use according to one of the preceding claims, wherein the effective amount of at least one saponin agent and the effective amount of at least one anthelmintic agent and I or the effective amount of at least one mitochondrial uncoupling agent are administered sequentially or concurrently.

18. The composition for use according to one of the preceding claims, wherein the at least one saponin agent and I or the least one anthelmintic agent and the I or the at leats one mitochondrial uncoupling agent are administered in the same amount.

19. The composition for use according to one of the preceding claims, further comprising an effective amount of at least one antifungal agent, wherein the antifungal agent preferably is (R-(R*,S*))-alpha-(2,4-difluorophenyl)-5-fluoro-beta-methyl- alpha-(1 H-1 ,2,4-triazol-1-ylmethyl)-4-pyrimidineethanol(aR,[3S)-a-(2,4- difluorophenyl)-5-fluoro-[3-methyl-a(1 H-1 , 2,4-triazol-1 -ylmethyl)-4-pyrimidineethanol (Voriconazole), and / or, 4-[4-[4-[4-[[(3R,5R)-5-(2,4-difluorophenyl)-5-(1 ,2,4-triazol-1 - ylmethyl)oxolan-3-yl]methoxy]phenyl]piperazin-1 -yl]phenyl]-2-[(2S,3S)-2- hydroxypentan-3-yl]-1 ,2,4-triazol-3-one (Posaconazole).

20. The composition for use of according to one of the preceding claims, wherein the composition further comprises a bioavailability enhancer, wherein the bioavailability enhancer is a CYP inhibitor and / or a drug transport inhibitor.

21. The composition dor use according to one of the preceding claims, wherein the liver disease is selected from the group consisting of alcohol-related liver disease, non-alcoholic fatty liver disease, metabolic dysfunction-associated steatotic liver disease (MASLD) hepatitis, hepatocellular carcinoma, primary biliary cirrhosis and / or liver fibrosis.

22. A composition for use in a method of treating a liver disease, preferably metabolic dysfunction-associated steatohepatitis (MASH), preferably a composition for use according to one of the preceding claims, the composition comprising an effective amount of at least one saponin agent, wherein the at least one saponin agent is linked or tethered to a targeting molecule selected to selectively deliver the saponin agent to human hepatocytes, and I or an effective amount of at least one anthelmintic agent, wherein the at least one saponin agent is linked or tethered to a targeting molecule selected to selectively deliver the saponin agent to human hepatocytes and I or an effective amount of at least one mitochondrial uncoupling agent, wherein the at least one mitochondrial uncoupling agent is linked or tethered to a targeting molecule selected to selectively deliver the mitochondrial uncoupling agent to human hepatocytes.

Images