2023 Fiscal Year Research-status Report
Establishment of novel in vitro assay for evaluation of killing effect of chloroquine against malaria parasite
| Project/Area Number |
20K16238
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| Research Institution | Juntendo University |
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Principal Investigator |
バリカガラ ベテイ 順天堂大学, 大学院医学研究科, 助教 (70805895)
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| Project Period (FY) |
2020-04-01 – 2025-03-31
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| Keywords | Chloroquine / Cytocidal effect / Plasmodium falciparum |
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| Outline of Annual Research Achievements |
The first objective of the research to confirm presence of resistance to the parasiticidal effect of chloroquine using laboratory cultured standard strains according to the method of a previous study (Paguio, Mol Biochem Parasitol 2011) was confirmed. Using laboratory cultured Plasmodium falciparum parasites; chloroquine sensitive (HB3) and resistant parasites: Dd2 (Southeast Asian origin) and 7G8 (South American origin) we compared the cytocidal chloroquine efficacy (lethal dose 50%, LD50) and conventional cytostatic chloroquine efficacy (inhibitory dose 50%, IC50). The difference of cytostatic value between chloroquine-sensitive HB3 and chloroquine-resistant Dd2 was about 10 times [52.4nM versus 501.7nM] and HB3 versus chloroquine-resistant 7G8 was over 5 times [52.4nM versus 331.56nM]. In contrast, 100-fold difference was observed in cytocidal chloroquine efficacy between HB3 and Dd2 [50nM versus 5230nM] and about 10-fold between HB3 versus 7G8 [37.4nM versus 344nM].
The second objective is to verify the chloroquine parasiticidal effect using cryopreserved natural parasites recovered from Uganda field site. Currently a diverse parasite population of 237 fresh natural parasites has been collected from three field surveys: Feb-Mar 2022 (29), Oct-Nov 2022 (106) and May 2023 (102). Parasite recovery and adaptation of these field parasites into continuous culture is now successful for three parasite isolates. More parasites from this diverse pool are expected to get adapted into continuous culture afterwhich the chloroquine parasiticidal assay will be performed.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
The progress of this research is slightly delayed because recovery and culture adaptation of the natural field parasites is not progressing as fast as expected. Parasite recovery from the frozen state and adaptation into culture has been challenging. Although parasites initially recover from the frozen state and growth in culture is good for the first three weeks, parasites eventually die off and adaptation into continuous culture fails. This could be due to individual parasite potential to adapt into continuous culture or probably delayed potential of natural parasites to adapt to environments outside the human body. Nonetheless, parasite adaptation into continuous culture will be continued until a sufficient number of parasites is obtained for the chloroquine drug assays.
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| Strategy for Future Research Activity |
Recovery and adaptation into culture of cryopreserved natural parasites is continuing. All successfully culture adapted natural parasites, will be exposed to the lethal chloroquine dose to evaluate the parasiticidal chloroquine effect in a two step process.
Step 1: Parasites will be cultured for 6 hours at chloroquine concentrations (0.625-2000 μM) covering the maximum chloroquine concentration in the human body, 10 μM, then the drug will be washed off. Parasites will then be incubated for 48 hours without drug and LD50 (lethal dose concentration) determined from surviving parasites. The conventional in-vitro assay will also be performed. Parasites are cultured at chloroquine concentrations of 0.025-1.6 μM for 72 hours and IC50 (50% growth inhibitory concentration) evaluated.
Step 2: The method in step 1 will be improved by developing a new in vitro assay that easily evaluates the parasiticidal chloroquine effect. Specifically, optimize the blood volume used, the chloroquine concentration, drug exposure and culture time, simplify the parasite quantification method, and the objectiveness of the LD50 and IC50 determination methods.
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| Causes of Carryover |
To increase the success of adapting natural parasites in culture and develop the new in-vitro assay that easily evaluates the parasiticidal chloroquine effect, more diverse parasite isolates need to be collected in Uganda. Also, after successfully developing the new assay that evaluates the pasiticidal clinical chloroquine effect, it needs to be applied the Uganda field setting. Therefore, money will be spent to travel to Uganda to perform the experiments. Also money will be allocated to purchasing more reagents and consumables for the experiments.
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