Dissertation committee:
1. Ντότσικα Ελένη (επιβλέπουσα): Διευθύντρια Ερευνών, Τμήμα Μικροβιολογίας,
Εργαστήριο Κυτταρικής Ανοσολογίας, Ελληνικό Ινστιτούτο Παστέρ
2. Σκαλτσούνης Αλέξιος - Λέανδρος: Καθηγητής, Τμήμα Φαρμακευτικής, Τομέας
Φαρμακογνωσίας και Χημείας Φυσικών Προϊόντων, Ε.Κ.Π.Α.
3. Χαλαμπαλάκη Μαρία: Αναπληρώτρια Καθηγήτρια, Τμήμα Φαρμακευτικής, Τομέας
Φαρμακογνωσίας και Χημείας Φυσικών Προϊόντων, Ε.Κ.Π.Α.
4. Μητάκου Σοφία: Καθηγήτρια, Τμήμα Φαρμακευτικής, Τομέας Φαρμακογνωσίας και
Χημείας Φυσικών Προϊόντων, Ε.Κ.Π.Α.
5. Ρούτσιας Ιωάννης: Επίκουρος Καθηγητής, Ιατρική Σχολή, Εργαστήριο Μικροβιολογίας,
Ε.Κ.Π.Α.
6. Παπαδόπουλος Ηλίας: Καθηγητής, Κτηνιατρική Σχολή, Εργαστήριο Παρασιτολογίας &
Παρασιτικών Νοσημάτων, Α.Π.Θ.
7. Σμυρλή Δέσποινα: Κύρια Ερευνήτρια, Τμήμα Μικροβιολογίας, Εργαστήριο Μοριακής
Παρασιτολογίας, Ελληνικό Ινστιτούτο Παστέρ
Summary:
Leishmaniasis is a multisystemic parasitic disease with a wide range of clinical symptoms, including chronic inflammation of skin and internal visceral organs, and can be fatal if left untreated. It affects 12 million people worldwide, while 0.7 - 1.2 million new cases are annually reported from nearly 100 endemic countries. Leishmaniasis is endemic in Southern Europe with a progressively increasing incidence in non-endemic areas, posing a public health problem. It is caused by more than 21 different species of unicellular protozoan parasites of the genus Leishmania (family Trypanosomatidae), which are transmitted to their final vertebrate hosts through the blood meal of infected female insects belonging to the family of Psychodidae (genus Phlebotomus and Lutzomyia), and infect the host's mononuclear phagocytes. The main hosts of the parasite are humans, species of the canine family and rodents. The severity of clinical manifestations of the disease depends on both the species of the parasite and the genetic predisposition and immunocompetence of the infected host, as has been demonstrated in the available experimental animal models. Since no effective vaccine is available, chemotherapy is the only treatment option for the disease. Available chemotherapy and combination therapies include drugs such as pentamidine, paromomycin, azoles, liposomal amphotericin B and miltefosine. However, all these options present severe drawbacks, such as toxicity, high cost and an increasing failure rate, mainly due to the development of protozoan resistance, which necessitates the investigation of new active antileishmanial substances and new effective treatment protocols.
Recent epidemiological studies show that Mediterranean Diet (MD) is associated with a reduced incidence of chronic inflammatory diseases such as cardiovascular diseases, neurological disorders and certain types of cancer. The main ingredient of MD is olive oil, and in particular extra virgin olive oil (EVOO), which comes from the plant Olea europaea L. Numerous studies in in vitro and in vivo models of various diseases, claim that the beneficial effects of olive oil are attributed to monounsaturated fatty acids (MUFAs) and to its polar fraction (total phenolic fraction, TPF), which contains chemical classes of biophenols, such as phenyl alcohols, phenolic acids, secoiridoids, flavonoids and lignans. Thus, the consumption of olive oil, as an indispensable product of MD, is strongly associated with beneficial health benefits, such as antioxidant, anti-inflammatory and anti-cancer properties. Recently, the European Food Safety Committee (EFSA) has issued on the positive contribution of olive oil biophenols to human health. Therefore, the investigation of the role of these bioactive molecules of olive oil in the treatment of endemic infectious diseases in Southern European countries, which are the main oil-producing countries with the highest overall consumption of olive oil, is of special scientific interest.
The aim of this PhD thesis was the comparative study of the antileishmanial properties of two total phenolic fractions, TPF1 and TPF2, with different polyphenolic composition, derived from Greek EVOOs, and their association, as well as their main isolated polyphenolic components association with the commercially available antileishmanial drug miltefosine (HePC), in in vitro and in vivo experimental models of leishmaniasis.
The quantitative analysis revealed that TPF2 was richer in phenolic compounds as it contained hydroxytyrosol (HT) (5 mg/g of extract), tyrosol (T) (12 mg/g), oleacein (OLEA) (144 mg/g) and oleocanthal (OLEO) (301 mg/g), while TPF1 contained HT and T at 7 and 42 mg/g of extract, respectively. Both TPF1 and TPF2, exerted potent antileishmanial activity against two species of the genus Leishmania, L. infantum and L. major, which are the causative agents of the visceral and cutaneous forms of the disease, respectively. However, TPF2 was demonstrated as the most potent antileishmanial compound. Similarly, the isolated polyphenolic compounds oleuropein (OLE), HT, OLEA and OLEO demonstrated significant antileishmanial activity in both models. Also, except OLE, all tested compounds showed low toxicity against macrophages and selectivity against Leishmania spp.
In order to delineate the possible differential mode of action of the two total phenolic fractions, studies aimed to determine the type of induced cell death in Leishmania spp. promastigotes. In particular, various characteristic morphological and biochemical markers of apoptosis were determined in Leishmania spp. parasites.
Due to the parasite's growing resistance to antileishmanial medications that have been used for many decades, new potent therapeutic agents or combination therapy regimens are urgently requested. In recent years, drug combinations have been increasingly advocated as a way to increase efficacy, reduce the duration and the cost of treatment and limit the occurrence of drug resistance. In this thesis, the interactions of each biophenolic compound with HePC were evaluated in vitro, with the ultimate goal of optimizing the effect of HePC, the only oral formulation approved for the treatment of leishmaniasis. This correlation was performed using the modified isobologram method, and the results revealed additivity for HePC-TPF1 and HePC-TPF2 interactions, synergy for HePC-OLEO, indifference for HePC-HT and HePC-OLEA and antagonism for HePC-OLE, in both L. infantum and L. major promastigotes. Among these, the results leading to synergy and additivity were characterized as the most promising, as they induced a significant reduction in the required dose of HePC. These promising interactions were further confirmed by the enhanced intracellular ROS production in extracellular promastigotes. These results led to further investigation of the nature of the interactions of these compounds on intracellular amastigotes, and the results revealed antagonism in both species of the parasite. Furthermore, the three biophenolic compounds TPF1, TPF2, and OLEO were also highlighted for their immunomodulatory action in an in vitro system of Leishmania - infected macrophages, which in most cases was enhanced when combined with HePC.
These results characterized OLEO as the most promising molecule and drove to its further investigation in an in vivo experimental model of cutaneous leishmaniasis, in order to evaluate both its single and combined with HePC therapeutic effect.
Overall, the results obtained from the present PhD thesis verified that total phenolic fractions of olive oil are bioactive extracts with potent antileishmanial and immunomodulatory activity. In addition, they revealed that fractions richer in the phenolic compounds OLEA and OLEO, esters of HT and T, respectively, possess stronger antileishmanial activity. This assumption was further confirmed by the investigation of the leishmanicidal activity of the isolated polyphenols, where OLEO gave the most promising results among the studied pure compounds. Moreover, the association of these bioactive natural components with HePC showed a promising new therapeutic approach against Leishmania parasites, indicating that such approaches can determine potentially effective treatment regimens associated with optimal lower dose of the standard drug. Combination chemotherapy encourages future pursuit of this strategy for treating disease in difficult clinical relapses, in patients who do not respond to the initial available chemotherapy, and in complex situations such as HIV co-infection.
Keywords:
leishmaniasis, Mediterranean diet, olive oil, natural products, total phenolic fraction, biophenols, promastigotes, intracellular amastigotes, combination chemotherapy, isobologram, immune response, BALB/c mice
