Periodic Reporting for period 4 - HEPASPHER (Mimicking liver disease and regeneration in vitro for drug development and liver transplantation)
Okres sprawozdawczy: 2022-03-01 do 2023-06-30
Problem and importance. A serious and important cause of liver damage is adverse drug reactions (ADRs). It is estimated that adverse drug reactions cost as much as the drug treatment itself, cause 4% of new chemical agents to be withdrawn from the market and are the 4th to 5th leading cause of death. The prevalence of non-alcoholic fatty liver disease (NAFLD) is estimated at 24% worldwide, and the prevalence of non-alcoholic steatosis (NASH) among NAFLD patients is reported at 21%. This suggests an overall prevalence of NASH in the population of 4 %. The prevalence of comorbidities associated with NASH in these individuals is obesity (82 %), type 2 diabetes mellitus (T2DM) (48 %), metabolic syndrome (76 %) and hypertension (70 %). Many of these liver diseases require liver transplantation. Indeed, recent studies show that NASH/liver fibrosis is now the leading cause of liver transplantation. Therefore, new tools are needed to study drug-induced hepatotoxicity, insulin resistance, liver formation and liver disease.
Objectives. The overall objectives of the project were to identify novel mechanisms, novel mediators, novel biomarkers and novel drugs for the development and treatment of steatosis, NASH, fibrosis and drug-induced hepatotoxicity based on in vivo experiments such as 3D spheroid models with different types of liver cells.
Objectives. The overall objectives of the project were to identify novel mechanisms, novel mediators, novel biomarkers and novel drugs for the development and treatment of steatosis, NASH, fibrosis and drug-induced hepatotoxicity based on in vivo experiments such as 3D spheroid models with different types of liver cells.
The project focused on the use of a novel in vitro 3D liver spheroid system for the prediction and mechanistic assessment of chronic hepatotoxicity of drugs, liver diseases such as NAFLD, NASH and fibrosis, and for studies on liver regeneration and hepatocyte plasticity.
The development of new therapies for chronic liver diseases is currently hampered by the lack of understanding of liver regeneration in humans. By using this model to study the molecular mechanisms behind liver regeneration, we found that the β-catenin signalling pathway plays an essential role in the expansion of liver cells during liver regeneration.
In the development of drugs, the autoinduction of metabolism by the drug in question plays an important role. We indentified new indirect mechanisms for drug-dependent induction of the important enzyme CYP3A4, in which the drug interacts with intracellular signal transduction systems that can indirectly activate the nuclear receptor PXR and cause induction of these drug-metabolising enzymes. This type of induction was relevant to at least 4 other drugs and cannot be mimicked in 2D hepatocyte systems.
A major achievement of the project was the development of an HTS-compatible system to study the aetiology, mechanisms and treatment of liver fibrosis. This has been used for screening chemical libraries to find new candidates for antifibrotic drugs and for the treatment of NASH, as well as to study in detail the mechanisms behind FFA-induced fibrosis, including the identification of released biomarkers such as miRNAs and cytokines, which may be new biomarkers for tracking the development of NASH and fibrosis in vivo in humans.
We evaluated the 3D spheroid system for predictions of drug induced hepatotoxicity. We found that this system could predict the chronic hepatotoxicity at a sensitivity of 72 % and specificity of 100 %, based on screening of 120 different drugs on the market, known to be hepatotoxic or without hepatotoxic effects which constitutes the best in vitro system published for this purpose. We also found formation of spheroid thorns as a new endpoint for drug induced hepatotoxicity to a major extent caused by alteration of the keratin proteins in the spheroids.
Overview of the results and their exploitation and dissemination
In summary, we have developed a new in vitro model for studying human liver with respect to function, diseases and drug-induced injuries. In this 3D hepatic spheroid model we have been able i) to reconstitute and explain mechanisms of chronic drug hepatotoxicity not previously understood, ii) to use the system for high accuracy prediction of hepatotoxic drugs, iii) to develop protocols to study the influence of genetic, dietary and endocrine factors of importance for generating and for treatment of steatosis, iv) to find conditions using various liver donors to establish a HTS compatible model for generation and treatment of NASH and liver fibrosis, v) to identify novel mechanisms by which drugs can induce their own hepatic metabolism, a problem of importance for drug development, vi) to initiate liver regeneration in the spheroids by cytotoxic drugs, wnt ligands or kinase inhibitors making it a nice model for studies of mechanisms of liver regeneration, vii) to identify the formation of thorns as a novel endpoint for drug induced hepatotoxicity, viii) to find the role of CTGF in FFA induced liver fibrosis, ix) to find the specific mechanism and consequence of inflammatory mediators for expression of different drug transporters and drug metabolising enzymes and x) to identify the polymorphic nuclear protein NFIB as a regulator of cytochrome P450 expression with clinical implications for drug treatment.
We have furthermore developed a new triple-culture based spheroid model for identification of the role of LSECs in formation and degradation of liver fibrosis. Here we have found that the metalloproteinase TIMP-1 is present in a subset of LSECs in the human liver and in the spheroids secreted by activated LSECs and contribute to enhanced liver fibrosis. We have been able to see that soluble components are secreted from LSECs which activate the Stellate cells for collagen production but also affect the phenotype of the hepatocytes causing e.g. diminished expression of key drug metabolising enzymes such as CYP3A4. This regulation is partly mediated by IL-6 and implicate important phenotypic alterations in the hepatocytes of patients with steatosis and liver fibrosis leading to increased risk for drug related hepatic adverse reactions.
The development of new therapies for chronic liver diseases is currently hampered by the lack of understanding of liver regeneration in humans. By using this model to study the molecular mechanisms behind liver regeneration, we found that the β-catenin signalling pathway plays an essential role in the expansion of liver cells during liver regeneration.
In the development of drugs, the autoinduction of metabolism by the drug in question plays an important role. We indentified new indirect mechanisms for drug-dependent induction of the important enzyme CYP3A4, in which the drug interacts with intracellular signal transduction systems that can indirectly activate the nuclear receptor PXR and cause induction of these drug-metabolising enzymes. This type of induction was relevant to at least 4 other drugs and cannot be mimicked in 2D hepatocyte systems.
A major achievement of the project was the development of an HTS-compatible system to study the aetiology, mechanisms and treatment of liver fibrosis. This has been used for screening chemical libraries to find new candidates for antifibrotic drugs and for the treatment of NASH, as well as to study in detail the mechanisms behind FFA-induced fibrosis, including the identification of released biomarkers such as miRNAs and cytokines, which may be new biomarkers for tracking the development of NASH and fibrosis in vivo in humans.
We evaluated the 3D spheroid system for predictions of drug induced hepatotoxicity. We found that this system could predict the chronic hepatotoxicity at a sensitivity of 72 % and specificity of 100 %, based on screening of 120 different drugs on the market, known to be hepatotoxic or without hepatotoxic effects which constitutes the best in vitro system published for this purpose. We also found formation of spheroid thorns as a new endpoint for drug induced hepatotoxicity to a major extent caused by alteration of the keratin proteins in the spheroids.
Overview of the results and their exploitation and dissemination
In summary, we have developed a new in vitro model for studying human liver with respect to function, diseases and drug-induced injuries. In this 3D hepatic spheroid model we have been able i) to reconstitute and explain mechanisms of chronic drug hepatotoxicity not previously understood, ii) to use the system for high accuracy prediction of hepatotoxic drugs, iii) to develop protocols to study the influence of genetic, dietary and endocrine factors of importance for generating and for treatment of steatosis, iv) to find conditions using various liver donors to establish a HTS compatible model for generation and treatment of NASH and liver fibrosis, v) to identify novel mechanisms by which drugs can induce their own hepatic metabolism, a problem of importance for drug development, vi) to initiate liver regeneration in the spheroids by cytotoxic drugs, wnt ligands or kinase inhibitors making it a nice model for studies of mechanisms of liver regeneration, vii) to identify the formation of thorns as a novel endpoint for drug induced hepatotoxicity, viii) to find the role of CTGF in FFA induced liver fibrosis, ix) to find the specific mechanism and consequence of inflammatory mediators for expression of different drug transporters and drug metabolising enzymes and x) to identify the polymorphic nuclear protein NFIB as a regulator of cytochrome P450 expression with clinical implications for drug treatment.
We have furthermore developed a new triple-culture based spheroid model for identification of the role of LSECs in formation and degradation of liver fibrosis. Here we have found that the metalloproteinase TIMP-1 is present in a subset of LSECs in the human liver and in the spheroids secreted by activated LSECs and contribute to enhanced liver fibrosis. We have been able to see that soluble components are secreted from LSECs which activate the Stellate cells for collagen production but also affect the phenotype of the hepatocytes causing e.g. diminished expression of key drug metabolising enzymes such as CYP3A4. This regulation is partly mediated by IL-6 and implicate important phenotypic alterations in the hepatocytes of patients with steatosis and liver fibrosis leading to increased risk for drug related hepatic adverse reactions.
We have developed a very useful human in vitro liver model to study the regulation of hepatic gene expression by proinflammatory cytokines that make predictions about drug development affected by this regulation. We have succeeded in showing that the liver spheroid model can be used for studies of liver regeneration. We have shown that the model developed is currently the most predictive in vitro liver model for determining drug-induced hepatotoxicity and the most versatile model for predicting drug-induced expression of liver enzymes involved in drug metabolism and toxicity. This model is also well suited to elucidate the mechanism of drug-induced hepatotoxicity, e.g. based on the ability to specifically silence different gene products to identify the responsible genes. The novel LSEC-based liver spheroid model is most useful for studying the effect of putative anti-NASH compounds and formed the basis for the successful ERC-PoC grant 101123215, which was launched on 1 September. 2023, in which we will use the model to identify new targets and drug components that could be of clinical value for the treatment of NASH in vivo in humans.
The majority of papers generated are shown in https://pubmed.ncbi.nlm.nih.gov/?term=ingelman-sundberg+m+AND+spheroids&sort=date&size=100
The majority of papers generated are shown in https://pubmed.ncbi.nlm.nih.gov/?term=ingelman-sundberg+m+AND+spheroids&sort=date&size=100