MitoBOOST v2.0 - A Proof of Concept for the Use of Mitochondria-Targeted Polyphenols to Treat Non-Alcoholic Fatty Liver Disease (NAFL)

Overview

Project Summary

Unlike cells of healthy livers, which store little or no fat, fatty liver cells store large amounts of fat, a condition known generically as fatty liver disease. In those cases where the accumulation of fat is not due to excessive alcohol consumption, the condition is known non-alcoholic fatty liver disease. The last few decades have seen a very significant increase in the incidence of non-alcoholic fatty liver disease, in parallel with the increase in cases of hypertension and metabolic comorbidities, including obesity, type 2 diabetes, hyperlipidemia and metabolic syndrome. The prevalence of non-alcoholic fatty liver disease in Europe has already reached 23.7% (26% in Portugal) and is expected to double over the next five decades, in line with the growing consumption of highly processed foods rich in fructose, saturated fat and cholesterol, accompanied by a sedentary lifestyle. In fact, these prevalence figures are likely to be higher, as the disease is not easily diagnosed.

The uncontrolled accumulation of fat in the liver can lead to the progression of non-alcoholic fatty liver disease to a more severe condition with a strong inflammatory component (steatohepatitis). In turn, steatohepatitis can progress to cirrhosis and hepatocellular carcinoma. There is strong evidence that mitochondrial dysfunction plays an important role in this progression, namely through oxidative damage caused by increased levels of reactive oxygen species (ROS), resulting in so-called oxidative stress. In fact, it is in the mitochondria, the cellular organelle responsible for producing almost all metabolic energy, that most ROS are generated. This organelle also plays a key role in cell regulation (e.g., ionic, redox and metabolic regulation) and participates actively in apoptosis, a form of programmed cell death. In recent decades, several conventional antioxidants, such as vitamin E, have been tested for the treatment of non-alcoholic fatty liver disease, with mostly inconclusive or contradictory results. Our project DTP-FTO/2433/2014, funded by the Foundation for Science and Technology (FCT), helped us to understand the reason for these results. In particular, we found that: (i) the concentration of these antioxidants in mitochondria, one of the main sites of ROS damage in this pathology, is insufficient to exert the desired effect; (ii) many of these antioxidants disturb the normal redox balance of other cellular compartments, thereby altering cellular homeostasis.

Main Goals

During the present project, entitled “MitoBOOST v.2.0”, we will carry out a proof of concept aimed at demonstrating in an animal model the efficacy and absence of toxicity of a new molecule (AntiOxBEN2) in the treatment of non-alcoholic fatty liver disease. AntiOxBEN2 is an antioxidant compound of polyphenolic nature with specific targeting for the mitochondria developed by us within the scope of the aforementioned project DTP-FTO/2433/2014, which also included the in vitro validation (in cell cultures) of its antioxidant capacities and degree of safety. The evaluation of the effectiveness and toxicity of AntiOxBEN2 will be carried out in an animal model of simple hepatic steatosis induced by feeding healthy male mice of the C57BL/6J strain, with 6 weeks of age at the beginning of the experiment, with a high fat (30 %) high sugar (30%) (HFHS) diet. The animals will be treated five weeks after the introduction of the HFHS diet and maintained for another 13 weeks (total time of feeding with control diet and HFHS: 18 weeks). Animals fed a so-called normal diet will be used as controls.

The efficacy evaluation will include the determination, by blood marker analysis and/or histopathology, of (i) levels of inflammation; (ii) damage to liver structure and function; (iii) damage to heart, kidney, brain and adipose tissue. A bioenergetic assessment will also be conducted, as well as an analysis of mitochondrial oxidative damage. The present proof of concept is also aimed at extending the physicochemical characterization of AntiOxBEN2 (namely in terms of solubility, stability, lipophilicity, ability to interact with biological membranes and plasma proteins and passive transcellular permeability), as well as to obtain pharmacokinetic and pharmacodynamic data. This information is essential for its progression to the more advanced stages of preclinical studies (e.g., carrying out studies under GLP conditions) and subsequent classification as a drug candidate, which can then be valued for subsequent phase I clinical trials. In terms of maturity, this project is currently in level 3 of the technology readiness level (TRL) scale, as defined at https://euraxess.ec.europa.eu. Upon completion of this project, TRL4/5 level will be reached (TRL4 - pre-clinical studies in model animals to identify safety and efficacy; TRL5 - prototype formulation for pharmaceutical applications). The results of this proof of concept will also increase the value of the patent PCT/IB2017/058508, WO2018122789, US 11,078,222 B2 (HYDROXYBENZOIC DERIVATIVES, METHODS AND USES THEREOF), which protects the structures, production process and applications of the family of compounds that includes AntiOxBEN2 compounds, opening the door to the future submission of new application and/or formulation patents, as well as sub-licensing to pharmaceutical companies. The patent co-ownership belongs to the University of Coimbra (UC) and the partner entity Universidade do Porto (UP) and it is licensed to the company Mitotag for commercial exploitation, with UC and Mitotag being beneficiary entities of these projects and UP being a partner entity.

Project Details