Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease worldwide. With no Food and Drug Administration approved drugs, current treatment options include dietary restrictions and lifestyle modification. NAFLD is closely associated with metabolic disorders such as obesity, type 2 diabetes, and dyslipidemia. Hence, clinically various pharmacological approaches using existing drugs such as antidiabetic, anti-obesity, antioxidants, and cytoprotective agents have been considered in the management of NAFLD and nonalcoholic steatohepatitis (NASH). However, several pharmacological therapies aiming to alleviate NAFLD-NASH are currently being examined at various phases of clinical trials. Emerging data from these studies with drugs targeting diverse molecular mechanisms show promising outcomes. This review summarizes the current understanding of the pathogenic mechanisms of NAFLD and provides an insight into the pharmacological targets and emerging therapeutics with specific interventional mechanisms. In addition, we also discuss the importance and utility of new approach methodologies and regulatory perspectives for NAFLD-NASH drug development.

Insights into the molecular targets and emerging pharmacotherapeutic interventions for nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease worldwide. With no Food and Drug Administration approved drugs, current treatment options include dietary restrictions and lifestyle modification. NAFLD is closely associated with metabolic disorders such as obesity, type 2 diabetes, and dyslipidemia. Hence, clinically various pharmacological approaches using existing drugs such as antidiabetic, anti-obesity, antioxidants, and cytoprotective agents have been considered in the management of NAFLD and nonalcoholic steatohepatitis (NASH). However, several pharmacological therapies aiming to alleviate NAFLD-NASH are currently being examined at various phases of clinical trials. Emerging data from these studies with drugs targeting diverse molecular mechanisms show promising outcomes. This review summarizes the current understanding of the pathogenic mechanisms of NAFLD and provides an insight into the pharmacological targets and emerging therapeutics with specific interventional mechanisms. In addition, we also discuss the importance and utility of new approach methodologies and regulatory perspectives for NAFLD-NASH drug development. 

Insights into the molecular targets and emerging pharmacotherapeutic interventions for nonalcoholic fatty liver disease

Liver cancer results in a high degree of mortality, especially among men. As fatty liver disease is a risk factor for development of hepatocellular carcinoma, we investigated the role of dietary fat type in tumor promotion by high-fat diets in mice after initiation with the chemical carcinogen diethyl nitrosamine. Tumor incidence and multiplicity were significantly greater in males than those in females. In males, fat type had complex effects on tumorigenesis. Preneoplastic foci were most prevalent in mice fed a polyunsaturated fat diet enriched in docosahexaenoic acid, whereas carcinomas and large visible liver tumors were significantly greater in mice fed a saturated fat diet made with cocoa butter relative to mice fed mono- or polyunsaturated fats. Different mechanisms thus seemed involved in early and late tumor promotion. The hepatic transcriptome and gut microbiome were assessed for traits associated with tumorigenesis. Hepatic expression of more than 20% of all genes was affected by sex, whereas fat type affected fewer genes. In males, the saturated fat diet induced expression of the proto-oncogene Agap2 and affected the expression of several cytochrome P450 genes, and genes involved in lipid, bile acid and fatty acid metabolism. The gut microbiome had a higher level of genus Akkermansia and a lower level of Firmicutes in females than in males. Males fed saturated fat had an altered microbiome, including an enrichment of the genus Coprococcus. In conclusion, sex and the dietary fat type affect the gut microbiome, the hepatic transcriptome and ultimately hepatic tumor growth.

Liver tumorigenesis is promoted by a high saturated fat diet specifically in male mice and is associated with hepatic expression of the proto-oncogene Agap2 and enrichment of the intestinal microbiome with Coprococcus

Advanced hepatic fibrosis, driven by the activation of hepatic stellate cells (HSCs), affects millions worldwide and is the strongest predictor of mortality in nonalcoholic steatohepatitis (NASH); however, there are no approved antifibrotic therapies. To identify antifibrotic drug targets, we integrated progressive transcriptomic and morphological responses that accompany HSC activation in advanced disease using single-nucleus RNA sequencing and tissue clearing in a robust murine NASH model. In advanced fibrosis, we found that an autocrine HSC signaling circuit emerged that was composed of 68 receptor-ligand interactions conserved between murine and human NASH. These predicted interactions were supported by the parallel appearance of markedly increased direct stellate cell-cell contacts in murine NASH. As proof of principle, pharmacological inhibition of one such autocrine interaction, neurotrophic receptor tyrosine kinase 3–neurotrophin 3, inhibited human HSC activation in culture and reversed advanced murine NASH fibrosis. In summary, we uncovered a repertoire of antifibrotic drug targets underlying advanced fibrosis in vivo. The findings suggest a therapeutic paradigm in which stage-specific therapies could yield enhanced antifibrotic efficacy in patients with advanced hepatic fibrosis. Description A repertoire of autocrine signaling modalities in advanced hepatic fibrosis constitutes potential therapeutic targets. Targeting advanced liver fibrosis Nonalcoholic steatohepatitis (NASH) has no approved therapy and advanced, fibrotic stages of the disease are in particular difficult to target. Wang et al. show that in both human and murine NASH, a profibrotic autocrine signaling loop comprising dozens of receptor-ligand pairs emerges in hepatic stellate cells specifically during late-stage disease. Pharmacological targeting of one of these receptor-ligand interactions reduced advanced fibrosis in a NASH mouse model, establishing this interaction as an antifibrotic target and suggesting the possibility of a disease stage-specific treatment. —CAC

An autocrine signaling circuit in hepatic stellate cells underlies advanced fibrosis in nonalcoholic steatohepatitis

Hepatocellular carcinoma (HCC) is the fastest growing cancer worldwide in part due to the obesity epidemic and fatty liver disease, particularly nonalcoholic steatohepatitis (NASH). Chronic inflammation with the release of cytokines and chemokines with activation of hepatic stellate cells results in changes of the liver extracellular matrix (ECM) that predisposes to the development of HCC. Blood levels of the gastrointestinal peptide cholecystokinin (CCK) are increased in humans and mice consuming a high-fat diet. We found that the CCK-B receptor (CCK-BR) expression increased in the livers of mice with NASH. Treatment of mice with a CCK-BR antagonist, proglumide, prevented NASH, lowered hepatic inflammatory cytokines and chemokines, reduced oxidative stress, decreased F4/80+ hepatic macrophages, and prevented HCC. CCK-AR and CCK-BR expression was increased in both murine and human HCC cell lines compared with that of normal liver, and CCK stimulated the growth of wild-type and CCK-A receptor knockout HCC cells in vitro, but not CCK-BR knockout cells suggesting that the CCK-BR mediates proliferation. Proglumide therapy significantly reduced growth by 70% and 73% in mice bearing Dt81Hepa1-6 or in RIL-75 HCC tumors, respectively. IHC of a human liver tissue array with a selective CCK-BR antibody revealed staining of human HCC and no staining in normal liver. PREVENTION RELEVANCE: This investigation demonstrates the role of the gastrointestinal peptide cholecystokinin (CCK) in hepatocellular carcinoma (HCC) and how CCK-BR blockade reverses the premalignant state of the hepatic extracellular matrix hence, rendering it less susceptible to the development of HCC. Thereby, CCK-BR blockade is a novel approach for the prevention/treatment of HCC.

https://cancerpreventionresearch.aacrjournals.org/content/canprevres/early/2020/10/28/1940-6207.CAPR-20-0220.full.pdf

Targeting the Cholecystokinin Receptor: A Novel Approach for Treatment and Prevention of Hepatocellular Cancer

Nonalcoholic steatohepatitis (NASH) is a common inflammatory liver condition that may lead to cirrhosis and hepatocellular carcinoma (HCC) Risk factors for NASH include a saturated fat diet, altered lipid metabolism, and genetic and epigenetic factors, including microRNAs Serum levels of cholecystokinin (CCK) are elevated in mice and humans that consume a high-saturated fat diet CCK receptors (CCK-Rs) have been reported on fibroblasts which when activated can induce fibrosis; however, their role in hepatic fibrosis remains unknown We hypothesized that elevated levels of CCK acting on the CCK-Rs play a role in the development of NASH and in NASH-associated HCC We performed a NASH Prevention study and Reversal study in mice fed a saturated fat 75% choline-deficient–ethionine-supplemented (CDE) diet for 12 or 18 weeks In each study, half of the mice received untreated drinking water, while the other half received water supplemented with the CCK-R antagonist proglumide CCK-R expression was evaluated in mouse liver and murine HCC cells CCK receptor antagonist treatment not only prevented NASH but also reversed hepatic inflammation, fibrosis, and steatosis and normalized hepatic transaminases after NASH was established Thirty-five percent of the mice on the CDE diet developed HCC compared with none in the proglumide-treated group We found that CCK-BR expression was markedly upregulated in mouse CDE liver and HCC cells compared with normal hepatic parenchymal cells, and this expression was epigenetically regulated by microRNA-148a These results support the novel role of CCK receptors in the pathogenesis of NASH and HCC

/pdf/a-cholecystokinin-receptor-antagonist-halts-nonalcoholic-21buqntdjf.pdf

A Cholecystokinin Receptor Antagonist Halts Nonalcoholic Steatohepatitis and Prevents Hepatocellular Carcinoma.

Pancreatic cancer is resistant to chemotherapy in part due to the dense desmoplastic fibrosis surrounding the tumor, the immunosuppressive cells in the tumor microenvironment (TME), and the early rate of metastases. In this study, we examined the effects of a CCK receptor antagonist, proglumide, alone and in combination with gemcitabine in murine models of pancreatic cancer. Tumor growth rate, metastases, and survival were assessed in mice bearing syngeneic murine or human pancreatic tumors treated with PBS (control), gemcitabine, proglumide, or the combination of gemcitabine and proglumide. Excised tumors were evaluated histologically for fibrosis, immune cells, molecular markers, and uptake of chemotherapy by mass spectroscopy. Peripheral blood was analyzed with a microRNAs biomarker panel associated with fibrosis and oncogenesis. Differentially expressed genes between tumors of mice treated with gemcitabine monotherapy and combination therapy were compared by RNAseq. When given in combination the two compounds exhibited inhibitory effects by decreasing tumor growth rate by 70%, metastases, and prolonging survival. Proglumide monotherapy altered the TME by decreasing fibrosis, increasing intratumoral CD8+ T-cells, and decreasing arginase-positive cells, thus rendering the tumor sensitive to chemotherapy. Proglumide altered the expression of genes involved in fibrosis, epithelial–mesenchymal transition, and invasion. CCK-receptor antagonism with proglumide renders pancreatic cancer susceptible to chemotherapy.

Cholecystokinin Receptor Antagonist Improves Efficacy of Chemotherapy in Murine Models of Pancreatic Cancer by Altering the Tumor Microenvironment.

https://doi.org/10.1016/j.jaccas.2022.09.014

Anita Safronenka

Papers

A Cholecystokinin Receptor Antagonist Halts Nonalcoholic Steatohepatitis and Prevents Hepatocellular Carcinoma.

Targeting the Cholecystokinin Receptor: A Novel Approach for Treatment and Prevention of Hepatocellular Cancer

Cholecystokinin Receptor Antagonist Improves Efficacy of Chemotherapy in Murine Models of Pancreatic Cancer by Altering the Tumor Microenvironment.

Autoimmune Reaction Associated With Long COVID Syndrome and Cardiovascular Disease

Growth Hormone-releasing Hormone Receptor Antagonist MIA-602 Reduces Chimeric SARS-CoV-2-induced Cardiopulmonary Inflammation in K18-hACE2tg Mice