With aim to deliver vegetal copper-containing amine oxidase (Cu-AO) to the intestine in the presence of catalase as a novel way to control intestinal dysfunctions connected with histamine-related conditions such as allergic/pseudoallergic reactions, colon cancer, Crohn's disease, ulcerative colite, the project here presented will be focus either to gain a better understanding of the mechanisms of the interaction of vegetal Cu-AO, an efficient histaminases, alone or in combination with catalase, with cells or to assess the stability of the enzyme in the intestinal environment. To achieve these goals, the project will be performed in collaboration with Prof. Mircea Alexandru Mateescu of the University of Quebec at Montreal and will be articulated in the following parts:
1) purification of Cu-AO from Lathyrus sativus seedlings and improvement of the procedures to prepare extracts containing Cu-AO and catalase at concentrations appropriate to remove efficiently both histamine and hydrogen peroxide;
2) evaluation of ability of Cu-AO alone or in combination with catalase and histamine to bind to a human colon colorectal adenoma derived cells (CaCo2) or to rat intestinal tissue;
3) analysis of the effect of vegetal Cu-AO alone or in combination with catalase and histamine on the calcium homeostasis in CaCo2 cells;
4) assessment of the stability of Cu-AO and catalase in simulated intestinal fluids.
The study will provide new data to prepare formulations for oral supplementation able to target and deliver Cu-AO and catalase to the intestinal sites of inflammation in a catalytic active forms. The gastric protection against stomach acidity of the enzymes and their controlled release to the colon is the key to treatment and extension to novel forms of interest for nanotechnology, such as nanoparticles, microspheres and tablets will be made. Collaborations with Pharmaqam and BioMed groups (Montreal, Canada) and Galenica Senese (Siena, Italy) will be envisaged.
Allergic/pseudoallergic reactions, CD, UC, colon cancer are worldwide high incidence pathological conditions for which no effective therapies are available so far. Since histamine is an important allergenic, pro-inflammatory and ethiopathological agent of these multifactorial conditions the use of vegetal Cu-AO, which is an efficient histaminases represents a new possible therapeutic tool for their treatment. In these last 15 years Sapienza and UQAM groups have been working together to this novel way to treat such conditions as a tentative to decrease, at least in part, the medication based on antihistaminic and anti-inflammatory drugs that can present undesired side effects such dizziness, nausea, blurred vision, confusion, headache, hemorrhagic risk. Various papers have been published together with prof. Mateescu showing the beneficial effect of vegetal Cu-AO on allergic reaction and ischemia-reperfusion damage in experimental animal systems. In the project here presented, the collaboration will be broaden to collect new information on the effect of vegetal Cu-AO alone or associated to catalase on cellular homeostasis and to better define the therapeutical potential of this new approach to prevent and treat CD, UC , colon cancer. Furthermore, the studies, performed in in vitro and ex vivo systems, will provide data to prepare new formulations able to target appropriate concentrations of Cu-AO and catalase to the intestinal site of inflammation and to deliver them in a catalytic active forms which can remove either the histamine and its catalytic product the hydrogen peroxide. The gastric protection (against stomach acidity) of Cu-AO and catalase and their controlled release to the colon is the key to treatment. The extension to novel forms of interest for nanotechnology, such as nanoparticles, microspheres and tablets will be also made. Collaborations with Pharmaqam and BioMed groups (Montreal, Canada) and with Galenica Senese (Siena, Italy) will be envisaged.