New therapeutic strategies against trypanosomiasis and leishmaniasis

Ibrahim, Hasan Mohamed Saleh (2009) New therapeutic strategies against trypanosomiasis and leishmaniasis. PhD thesis, University of Glasgow.

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Leishmaniasis and African Trypanosomiasis are diseases caused by the Kinetoplastida parasites of Leishmania sp. and Trypanosoma sp. respectively. Control and management of these diseases, which affect a significant number of people in the tropics and subtropical areas of the world, is beset with numerous problems such as drug toxicity, affordability and the emergence and spread of parasites resistance to most of the routinely used drugs. This situation calls for an urgent search for new drugs that would address these concerns. Based on report of excellent antimicrobial activities against other parasites and the possession of other known good values, analogues of choline and curcumin were thoroughly assessed in this study for their potential as antitrypanosomal and antileishmanial drugs.
Standard methods such as the Alamar Blue, propidium iodide and direct microscopy methods were used to determine the susceptibility of the parasites to the different analogues. Toxicity tests were performed to determine the effect of these compounds on Human Embryonic Kidney (HEK) cells. The presence of mediated transport of these compounds across the parasite plasma membrane was investigated using the classical uptake technique. In order to investigate the possible mechanism of antiparasitic action of the compounds, this study employed flow cytometry to assess the mitochondrial membrane potential m, as well as parameters such as production of reactive oxygen species (ROS), the permeability of the plasma membrane and any effects of the test copounds on the parasite’s cell cycle.
Five out of 7 choline compounds tested in this study had EC50 values of 0.13-1.8 µM against T. brucei, 0.14-6.9 µM against L. major, L. mexicana promastigotes and 1.69-12.9 µM against L. mexicana amastigotes. With regard to the curcuminoid compounds, 35 out of 98 tested were observed to exhibit trypanocidal activity better than the original curcumin with EC50 values between 0.05 and 1 µM. Against Leishmania, most of the compounds displayed higher antiparasitic activity than curcumin but lower than observed against trypanosomes. The activity of choline analogues was very similar against L. mexicana and L. major promastigotes (P>0.05), and much higher than against L. mexicana amastigotes. Interestingly, some of the compounds displayed EC50 values below that of pentamidine, the routinely used drug.
Assessment of parasite growth pattern in the presence of choline analogues showed that two of the compounds, T1 and MS1, are fast acting, killing the population of BSF T. b. brucei within 8 h with the onset of cell death at 2-4 hours of treatment. In contrast, the other three choline compounds observed to have antiparasitic activities acted more slowly, completely killing the trypanosome population after more than 30 hours of incubation. However, all the choline compounds appeared to rapidly inhibit trypanosome proliferation.
The choline compounds exhibited low toxic effects against HEK cell line T29, with the selectivity index (S.I.) being high for some of the compounds. The curcumin compounds, too, were observed to have generally similar or lower toxicity against the human cells than the parent curcumin compound (AS-HK001), which in itself is not considered toxic and routinely used in food. Investigations on the toxicological and pharmacological effects of the curcumin compounds on the survival and the glutathione and protein content of primary murine hepatocytes showed no significant difference between hepatocyte cells treated with curcuminoid compounds AS-HK001, AS-HK009, and AS-HK014 compared with controls.
We also investigated how choline and its analogues enter the trypanmosome. Evidence gathered in this study strongly suggests that unlike in Leishmania species and Plasmodium, choline transporters are not expressed in the bloodstream form of T. b. brucei. It was also conclusively shown that the P2, high affinity pentamidine transporter (HAPT) and low affinity pentamidine transporter (LAPT) do not play any significant role in the uptake of this compound. Lacking radiolabeled forms of the choline analogues, this study could not identify a definitive route of uptake of this class of compounds into the parasite.
Analysis of cell cycle progression, by flow cytometry, showed trypanosomes in the G1, S, and G2/M stages. Curcuminoids do not appear to cause any important changes in the proportion of cells in G1, S or G2/M phase of the cell cycle. Cells exposed to various concentrations of some curcumin compounds, such as AS-HK014 and AS-HK096, showed a rapid increase in cell permeability, reaching between 80% and 90% in 4 hours. The permeability was observed to increase with increasing drug concentration and/or the incubation time. Investigations of cell membrane permeability also showed that choline analogues caused plasma membrane defects which could probably lead to cell death.
With regard to the effect of the compound on mitochondrial membrane potential m the dicationic choline compounds, including M38, G25, T4 and MS1, were observed to have pronounced effects on m with an onset as early as 8 h of contact and we believe the mitochondria could be the main target of these compounds rather than indicating the induction of apoptosis, as the action of the test compounds was not associated with the production of reactive oxygen species. Indeed, both choline and curcumin analogues reduced the production of reactive oxygen species in T. b. brucei cultures. Furthermore, there were no major defects in choline phospholipid metabolism upon treatment with the choline compounds, suggesting that phospholipid metabolism is not the target of the anti-trypanocidal activity of these compounds.
Preliminary results with infected ICR mice infected with T. b. brucei did not reveal significant in vivo activity of the three curcumin compounds on blood parasitemia when they were injected intra-peritoneally with two doses of 50 mg/kg body weight.
With reference to evidence obtained in this study, it can firmly be concluded that analogues of choline and curcumin display highly promising antiparasitic activities and are generally non-toxic to human cells. Information provided in this thesis could therefore assist in the further development of these classes of compounds as lead compounds against kinetoplastid diseases. We strongly recommend that further investigation be carried out to understand the full mechanism of action of these compounds in order to facilitate this strategy.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Trypanosoma brucei, Leishmania major, Leishmania mexicana, curcumin, choline
Subjects: Q Science > QL Zoology
Q Science > Q Science (General)
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Supervisor's Name: de Koning, Dr. Harry
Date of Award: 2009
Depositing User: Mr Hasan Ibrahim
Unique ID: glathesis:2009-1158
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 08 Oct 2009
Last Modified: 16 Oct 2014 09:46

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