Importance of the topic
Epidemiological data indicate that approximately 180 millions of patients worldwide are affected by a form of chronic liver diseases that can be induced by a number of well defined etiological agents or conditions, as chronic alcohol abuse, chronic HBV and HCV infections, evolution of non-alcoholic steatohepatitis (NASH) in obesity and diabetes. Among diseases of the gastro-intestinal tract, cirrhosis represents the most common non-neoplastic cause of death in Europe and USA and overall represents the 7th most common cause of death in western countries. The process of liver fibrosis can be reversible, whereas cirrhosis, the end-stage consequence of fibrosis, is generally irreversible.
During fibrosis progress, hepatic stelate cells (HSCs) are activated, followed by cytokine and chemokine release, which induces their trans-differentiation into myofibroblast-like cells. This step is marked by the increased expression of smooth muscle α-actin (α-SMA) and procollagen-I, finally resulting in enhanced production and accumulation of ECM components including collagen. Transforming growth factor-β1 (TGF-β1) produced by Kupffer cells, endothelial cells and hepatocytes has been recognized as a key activator of HSCs in the pathogenesis of liver fibrosis and acts by activating Smad signaling pathway.
Therefore, it is important to provide potent anti-fibrotic agents targeted specific citokines or other biomolecules involved in fibrosis progression, able to prevent, retard or reverse liver fibrosis progression before end-stage of cirrhosis.
The aim of this project is to develop, test and validate a new drug delivery system of chrysin (5,7-dihydroxyflavone) based on cyclodextrins targeted liver fibrosis
Oral administration was chosen as route of delivery for the new formulated bioactive product, mainly because it is easily accessible by the patients and allows flexible dosing schedule. As other flavonoids, chrysin have limited water solubility, poor oral bioavailability, and can be easily modified by environmental factors such as temperature, pH and light.
Water-soluble hydroxypropyl-(HPBCD), sulfobutylether-(SBE) and randolmy methylated (RAMEB) β-cyclodextrins compared with parent β-cyclodextrin (BCD) will be used to enhance antifibrotic properties of chrysin, by increasing the solubilization potential and prevention of metabolic degradation within the gastrointestinal tract. The solubility and in vitro intestinal permeability of chrysin and chrysin-cyclodextrin complexes will be compared and the formulations will be ranked.
- Development and characterization of chrysin/cyclodextrin complexes
- Evaluation of in vitro biocompatibility and intestinal absorption for chrysin/cyclodextrin complexes
- Validation of antifibrotic effects of chrysin/cyclodextrin complexes in animal liver fibrosis model
Methods and study design
Due to the complexity of the mechanisms involved in liver fibrogenesis, both in vitro and in vivo model systems will be necessary to be applied.
Aim 1. Development and characterization of chrysin/cyclodextrin complexes. Here we will obtain and characterise the chrysin/cyclodextrin complexes.
Aim 2. Evaluation of in vitro biocompatibility and intestinal absorption for chrysin/cyclodextrin complexes. Here we will investigate the biocompatibility and the permeability anaysis of chrysin/cyclodextrin complexes in vitro.
Aim 3. Validation of antifibrotic effects of chrysin/cyclodextrin complexes in animal liver fibrosis model. Here we will perform the evaluation of antifibrotic activity of chrysin/cyclodextrins complexes by biochemical analysis, histopathology analysis, TGFβ pathway analysis (IHC expresion of proteins and localization; gene expression), extracellular matrix (ECM) analysis, electron microscopy analysis. We will evaluate the in vivo antifibrotic effects mediated by miRNAs involved in hepatic stellate cells activation, proliferation and migration.
Expected results and impact
We expected to select the first two best inclusion complexes of chrysin/cyclodextrins which increase the chrysin’s in vivo bioavailability and anti-fibrotic bio-efficacy. In the finally step, after in vivo histopathological and molecular analysis, we will validate one single chrysin-cyclodextrin complex with high potential to treat liver fibrosis and antifibrotic mechanism will be established.