Periodic Reporting for period 3 - HEPASPHER (Mimicking liver disease and regeneration in vitro for drug development and liver transplantation)
Reporting period: 2020-09-01 to 2022-02-28
We are developing 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 develop protocols to study the influence of genetic, dietary and endocrine factors of importance for generating and for treatment of steatosis, iii) found conditions using various liver donors to establish a HTS compatible model for generation and treatment of NASH and liver fibrosis, iv) identified a novel mechanisms by which drugs can induce their own hepatic metabolism, a problem of importance for drug development and v) to initiate liver regeneration in the spheroids by cytotoxic drugs or kinase inhibitors making it a nice model for studies of mechanisms of liver regeneration.
The overall objectives in the project are to identify novel mechanisms, novel mediators, novel biomarkers and novel drugs for the development and treatment of steatosis, NASH, fibrosis and drug-induced hepatotoxicity
The overall objectives in the project are to identify novel mechanisms, novel mediators, novel biomarkers and novel drugs for the development and treatment of steatosis, NASH, fibrosis and drug-induced hepatotoxicity
The project focused on the use of a novel in vitro 3D hepatic spheroid system for predictions and mechanistic evaluation of chronic of drug hepatotoxicity, liver diseases like NAFLD, NASH and fibrosis as well as studies for liver regeneration and hepatocytes plasticity.
The development of new therapies for chronic liver diseases is currently hindered by a poor understanding of liver regeneration in humans. As a result in the project we report that human hepatocytes acquire a regenerative phenotype upon isolation from the native liver when cultured as 3D spheroids using resolved time-lapse high-throughput –omics analyses. The regenerative process is characterized by rapid depletion of hepatic markers, basic cell machinery readjustments, cell cycle entry and activation of growth factor signalling. In this scenario activation of canonical Wnt signalling. By using this model to study the molecular mechanisms behind liver regeneration, we found an essential role of β-catenin signalling in the expansion of hepatic cells during liver regeneration. The results bring novel information how liver regeneration can be studied in vitro and using this model we will in the future evaluated the premises for liver regeneration and how we can utilize this knowledge for liver repair.
During drug development autoinduction by the drug in question of metabolism places an important role. We started to work with a case there an oncology drug caused induction of its own metabolism by inducing CYP3A4 in vivo leading to its withdrawal from clinical development in phase II. The reason for this failure was the fact that the drug induced induction of metabolism was impossible to mimic in animal studies are the in vitro systems at hand at that time. We did find new indirect mechanisms for drug dependent induction of the important enzyme CYP3A4 whereby the drug interacts with intracellular signal transduction systems that indirectly can activate the nuclear receptor PXR and cause induction of these drug metabolising enzymes. This kind of induction was relevant for at least 4 other drugs and is impossible to mimic in 2D hepatocyte systems. The system as a whole showed 100 % Specificity and sensitivity for prediction of the capability of the drugs tested for induction of CYP3A4 mediated drug metabolism. We propose that this system should be a major tool during future drug development.
In the 3D spheroid system we can induce steatosis under conditions that are causing these changes in vivo in patients. Thus, elevated free fatty acid or higher insulin and glucose induce an important level of steatosis in the spheroids. This condition can be treated with antioxidants as well as by nitrite, an important finding in terms of the effect of vegetables on the liver status. In addition, we examine the role of genetics for the development of steatosis and find that donors carrying mutant TM6SF2 gene are more prone to develop steatosis, a finding that is also seen in vivo in man.
A major achievement within the project has been the development of an HTS compatible system for studying the aetiology, the mechanisms behind and treatment of liver fibrosis. By characterizing liver cells from various donors we were able to develop a unique system by which liver fibrosis develops in response just to elevated amounts of free fatty acids as seen in vivo in man together with somewhat higher concentrations of insulin and glucose. We present the mechanisms behind these events and evaluate the potential of different anti-NASH drugs currently in clinical trials to prevent the fibrosis formation and to reverse fibrosis induced in the spheroids. Most efficient inhibitors were agonists of PPARa and g. This system will now be used for screening of chemical libraries in order to find new candidates for antifibrotic drugs and for treatment of NASH, as well as to in detail investigate the mechanisms behind the FFA induced fibrosis including the identification of biomarkers released like miRNAs and cytokines, that can be novel biomarkers for following the development of NASH and fibrosis in vivo in man.
We evaluated the 3D spheroid system for predictions of drug induced hepatotoxicity. We published that the 3D spheroid system is by far better that e.g. the 2D sandwich system in relevant hepatic phenotype and for prediction 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. One important factor is that we find a long time lag for the hepatotoxicity for some drugs and using knock down of specific genes we could describe the complete mechanisms and reasons for time delay in the action of fialuridine. We propose that this system would be an important addition during drug discovery and development for prediction of drug induced human hepatotoxicity and assist in early decisions regarding the selection of drug candidates going further in the developmental phase.
The development of new therapies for chronic liver diseases is currently hindered by a poor understanding of liver regeneration in humans. As a result in the project we report that human hepatocytes acquire a regenerative phenotype upon isolation from the native liver when cultured as 3D spheroids using resolved time-lapse high-throughput –omics analyses. The regenerative process is characterized by rapid depletion of hepatic markers, basic cell machinery readjustments, cell cycle entry and activation of growth factor signalling. In this scenario activation of canonical Wnt signalling. By using this model to study the molecular mechanisms behind liver regeneration, we found an essential role of β-catenin signalling in the expansion of hepatic cells during liver regeneration. The results bring novel information how liver regeneration can be studied in vitro and using this model we will in the future evaluated the premises for liver regeneration and how we can utilize this knowledge for liver repair.
During drug development autoinduction by the drug in question of metabolism places an important role. We started to work with a case there an oncology drug caused induction of its own metabolism by inducing CYP3A4 in vivo leading to its withdrawal from clinical development in phase II. The reason for this failure was the fact that the drug induced induction of metabolism was impossible to mimic in animal studies are the in vitro systems at hand at that time. We did find new indirect mechanisms for drug dependent induction of the important enzyme CYP3A4 whereby the drug interacts with intracellular signal transduction systems that indirectly can activate the nuclear receptor PXR and cause induction of these drug metabolising enzymes. This kind of induction was relevant for at least 4 other drugs and is impossible to mimic in 2D hepatocyte systems. The system as a whole showed 100 % Specificity and sensitivity for prediction of the capability of the drugs tested for induction of CYP3A4 mediated drug metabolism. We propose that this system should be a major tool during future drug development.
In the 3D spheroid system we can induce steatosis under conditions that are causing these changes in vivo in patients. Thus, elevated free fatty acid or higher insulin and glucose induce an important level of steatosis in the spheroids. This condition can be treated with antioxidants as well as by nitrite, an important finding in terms of the effect of vegetables on the liver status. In addition, we examine the role of genetics for the development of steatosis and find that donors carrying mutant TM6SF2 gene are more prone to develop steatosis, a finding that is also seen in vivo in man.
A major achievement within the project has been the development of an HTS compatible system for studying the aetiology, the mechanisms behind and treatment of liver fibrosis. By characterizing liver cells from various donors we were able to develop a unique system by which liver fibrosis develops in response just to elevated amounts of free fatty acids as seen in vivo in man together with somewhat higher concentrations of insulin and glucose. We present the mechanisms behind these events and evaluate the potential of different anti-NASH drugs currently in clinical trials to prevent the fibrosis formation and to reverse fibrosis induced in the spheroids. Most efficient inhibitors were agonists of PPARa and g. This system will now be used for screening of chemical libraries in order to find new candidates for antifibrotic drugs and for treatment of NASH, as well as to in detail investigate the mechanisms behind the FFA induced fibrosis including the identification of biomarkers released like miRNAs and cytokines, that can be novel biomarkers for following the development of NASH and fibrosis in vivo in man.
We evaluated the 3D spheroid system for predictions of drug induced hepatotoxicity. We published that the 3D spheroid system is by far better that e.g. the 2D sandwich system in relevant hepatic phenotype and for prediction 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. One important factor is that we find a long time lag for the hepatotoxicity for some drugs and using knock down of specific genes we could describe the complete mechanisms and reasons for time delay in the action of fialuridine. We propose that this system would be an important addition during drug discovery and development for prediction of drug induced human hepatotoxicity and assist in early decisions regarding the selection of drug candidates going further in the developmental phase.
Using the advantage of selective gene knock down in an in vitro liver model having an appropriate phenotype for many weeks we can elucidate mechanisms of formation and treatment of several different liver diseases and drug-related alterations. We anticipate the project to result in much novel knowledge about mediators involved, biomarkers to follow the extent of development of liver diseases and drug-induced damage resulting in the identification of novel targets that can effectively be used for screening of the next generation drugs for treatment of liver disease, insulin resistance and drug-induced liver damage.
We expect to identify novel targets to be used for treatment of NASH, new classes of anti NASH active drug candidates and novel biomarkers which can be used to monitor the development of the NASH disease in vivo in man.
We expect to identify novel targets to be used for treatment of NASH, new classes of anti NASH active drug candidates and novel biomarkers which can be used to monitor the development of the NASH disease in vivo in man.