Tackling trypanosomiasis: innovative synthetic and natural approaches

Trypanosomiases are a class of pathologies caused by the protozoan parasite Trypanosoma. Among them, the only two affecting humans are Chagas disease and Human African Trypanosomiasis (HAT). Specifically, HAT is a tropical neglected disease caused by the parasite Trypanosoma brucei. It is present mainly in sub-Saharan regions of the African continent and especially in Congo. Two parasites’ subspecies are responsible for two forms of the disease, gambiense and rhodesiense HAT, but the gambiense form is the most widespread. The main vector of the parasite is the tse tse fly, a hematophagous fly that populates wet areas and that infects the hosts through the injection of saliva when biting. HAT manifests itself in two stages; in the hemolymphatic stage more unspecific symptoms appear that are mistakable with malaria; in the late stage, in which the parasite affects the nervous central system, and encephalopathy, mental changes, a constant somnolent state and other more severe symptoms show up. The continuous variation of surface glycoproteins by the parasite poses an overwhelming challenge in developing an effective preventive measure. For this reason, the production of a vaccine remains elusive. On the other hand, the conventional therapy by existing drugs is highly toxic, and impractical for oral administration. Moreover, the local population's reluctance towards synthetic drugs, coupled with the parasite's demonstrated resistance, exacerbates the treatment dilemma. In light of these challenges my PhD research was focused on exploring both natural and synthetic innovative strategies for the development of new and promising alternatives to commercially available drugs. In detail, Chapter 2 reports the study of both essential oils (EOs) and extracts from Anthriscus nemorosa, an aromatic plant from the Apiaceae family which is spread in Europe and in Asia. EOs from aerial parts and roots of A. nemorosa were analyzed through GC-MS analysis, and tested on T. brucei together with their single main constituents, to evaluate their antitrypanosomal activity in vitro. The most interesting data came from the tests of the single components; among all, β-ocimene and farnesene resulted to be strongly active. In order to better understand synergistic effects of the different components in the A. nemorosa EO, a compound elimination assay was carried out, obtaining 11 blends that were subsequently tested on the parasite. Results were compared with Wadley’s formula to assess the synergy among the constituents. Limonene, β-ocimene, p-cymene and farnesene resulted as the EO's major antitrypanosomal constituents and when alternatively present, showed a synergistic effect. Nanoemulsions of the aerial parts EO were also prepared and tested, affording an improved safety profile. The study proceeded by working on extracts. Two different methods of extraction were adopted (digestion with stirring and maceration with ultrasounds) using methanol as solvent for both aerial parts and roots. Resulting extracts were tested on the parasite demonstrating a mild activity for roots, with a very low selectivity index. In order to obtain potentially less toxic fractions, a liquid-liquid partition of the root extract was performed using different solvents at growing polarity and subsequently testing them on T. brucei. Methanolic fraction resulted to be the most active and was characterized through HPLC-MSn analysis. Fourteen compounds were identified and, after performing a preparative HPLC, sub-fractions A, B, C and three pure compounds were obtained. All of them were tested in vitro and anthriscusin emerged as a moderate antitrypanosomal agent, being considerably potentiated by other components of the sub-fraction from which it was isolated. In Chapter 3 my research focused on Cannabis sativa as source of antiparasitic products. Cannabis sativa, belonging to the Cannabaceae family, has lately risen great interest in many fields and in particular in the pharmaceutical one, because of its wide and varied composition. Both EOs and hydrophilic extracts (HEs) from thirteen cultivars of C. sativa developed by crossbreeding selected varieties and provided by Everweed company were evaluated. Nine of these were CBD-rich varieties while the remaining four were CBG-rich ones. EOs were analyzed through GC-MS analysis and their antitrypanosomal potential assessed. HEs were prepared by lyophilization of the residual water used for the hydrodistillation, in order to obtain a product rich in water-soluble compounds, excluding, in this way, all phytocannabinoids. The antiprotozoal activity of these extracts was also evaluated, obtaining interesting results for one of the CBD-rich varieties extracts, Gorilla glue HE. Its composition was investigated performing an HPLC-DAD-MSn analysis that allowed to identify eleven main compounds. Gorilla glue HE was then fractioned in four fractions, and the most active against T. brucei was subsequently purified through preparative HPLC to afford pure Cannflavin A. The flavonoid was characterized and tested against the parasite, providing the first report of the antiprotozoal activity for Cannflavin A. Lastly, Chapter 4 reports the synthesis and the antitrypanosomal evaluation of three series of pyrazolone-based hydrazones (PbH1-36), and metal complexes of the most promising among them. All compounds from the three series (phenyl, thiophene, and benzyl series) were synthesized and preliminary tested on wild type T. brucei cells. The thiophene series resulted practically inactive and cytotoxic; the phenyl series demonstrated good activity with most of the analogs with EC50 values below 10 μM; however, the two most active compounds among all three series resulted to be PbH26 and PbH27 from the benzyl series. These two analogs were selected as ligands for the synthesis of Zn(II) and Cu(II) complexes, affording four complexes, C1-4. Three of them demonstrated good antitrypanosomal activity, with C2 as the most active one (EC50 value = 0.084 μM). PbH26, PbH27, C1, and C2 were selected for the investigation of the mechanism of action. Specifically, cross- resistance with commercially available antitrypanosomal drugs was excluded; then, the trypanocidal or trypanostatic behavior of selected compounds was determined at different concentrations. Subsequently, the reversibility of the antitrypanosomal activity and cell cycle abnormalities were assessed through flow cytometry and fluorescence microscopy. Finally, variations in nucleotide metabolism after treatment were detected through HPLC analysis, allowing to hypothesize an enzymatic target for the drugs.