IN VITRO THERAPEUTIC VALUE OF PLANT EXTRACTS USED IN BURKINA FASO TO TREAT MALARIA AND IN VITRO PHARMACODYNAMIC STUDIES OF ANTIMALARIAL DRUGS

My PhD project has developed in two main research areas, both related to antimalarial drug discovery. The first part was focused on the study of the antimalarial and anti-inflammatory activity of medicinal plants used in Burkina Faso to treat malaria; the second part was dedicated to the development of new tools to measure in vitro the pharmacodynamic characteristics of current or new drugs to predict early on their clinical behavior. The work was conducted in the Laboratory of Parasitology, Department of Public Health- Microbiology -Virology of the University of Milan and at the Liverpool School of Tropical Medicine, Liverpool, UK. The antiplasmodial activity, the inhibition of cytokine production, and the antiproliferative activities of three different plants, Canthium henriquesianum (K. Schum), Gardenia sokotensis (Hutch) and Vernonia colorata (Willd) were investigated. Crude and organic plant extracts were tested in vitro for antimalarial activity against chloroquine susceptible (D10) and resistant (W2) strains of P. falciparum. Cell cytotoxicity was assessed on Human Dermal Fibroblast (HDF) and melanoma cells by the MTT assay. The selectivity index (SI) was used as the ratio of the activity against the parasites compared to the toxicity of the plant extract against HDF. C. henriquesianum aqueous extract had a moderate antimalarial activity (IC50<50\ub5g/ml) with a good selectivity index (SI(HDF/D10)\u2c37). C. henriquesianum diisopropyl ether extract was the most potent inhibitor of parasite growth with an IC50 9.5 \ub5g/ml on W2 and 8.8 \ub5g/ml on D10 and limited toxicity (SI(HDF/D10)<2). On the contrary, G. sokotensis and V. colorata aqueous extracts were shown to be weakly active on the plasmodium parasites (IC50 65 50\ub5g/ml) with limited toxicity. In addition, when the production of IL-1\u3b2 and TNF\u3b1 by human THP1 monocyte was assayed by ELISA, it was found that the extract of C. henriquesianum induced a dose-dependent inhibition of IL-1\u3b2, but not TNF\u3b1 production, thus confirming its traditional use as antipyretic. By NMR analysis, we identified the chromone as the compound mostly represented in the diisopropyl ether extract of C. henriquesianum. Chromone however was less active as antimalarial than the crude extracts and it did not inhibit cytokine production at not toxic doses, indicating that other molecules in the total extracts contribute to the anti-inflammatory activity. In conclusion, out of the three plants examined, only C. henriquesianum seems to possess antimalarial activity in vitro and the capacity to inhibit pyrogenic cytokine production. Gardenia sokotensis extract showed moderate cytotoxic activity on melanoma cell line and it could be further investigated for its antitumor activity. The second part was related to the in vitro study with the objective to develop new tools to measure in vitro the pharmacodynamic features of different antimalarials. Three type of assays were used: dose response curves to measure IC50 and IC90 variables and calculate the slope of the curve; time to kill kinetics using SYBR Green I-based fluorescence (MSF) assay and high content in vitro assays using the Perkin Elmer Operetta High Content Screening microscope to measure the stage specificity of antimalarial action. These tests were first set up with current antimalarials, but the intent is to use them to prioritize the development of new drugs. The results of the dose\u2013response curves confirmed that DHA, atovaquone and CQ are more potent antimalarials compared to pyrimethamine., with a IC50 less than 4 nM for DHA and atovaquone; and 10 nM for CQ. The slope factor, which represents the steepness of the curve, appears to be characteristic of drug class and was >1 for all the drugs suggesting that binding does not follow the law of mass action with a single site and the ligand binds cooperatively to a multivalent receptor. Time to kill studies indicated that DHA has a noticeable effect before 5h as well as CQ and the parasites are irreversibly inhibited after 12hrs of exposure to the drug. The results from the High Content Screening showed that for all the compounds, at the highest dose, the surviving parasites after 48 h are 80-90% young forms and only10-20% mature parasites. No major changes are seen in the distribution of young vs mature forms at doses lower than the IC50. However, at higher doses the percentage of young is increasing and in parallel the percentage of mature forms is decreasing. This indicates that a small number of parasites was able to complete the cycle and re-invade new RBC.The Operetta technique seems to be able to quickly assess the potency as well as the stage specificity of a drug. It should be able to predict the parasite reduction ratios (PRR) without the need to perform the time to kill assays which are more laborious, time consuming and less adaptable to high throughput screenings (HTS). The compounds that inhibit the ring stage would be predicted to have faster time-kill profiles and would be prioritized for further studies in the process of antimalarial drug development