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 Category |
Grant-in-Aid for Early-Career Scientists
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| Allocation Type | Multi-year Fund |
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| Review Section |
Basic Section 49040:Parasitology-related
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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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| Project Status |
Granted (Fiscal Year 2023)
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| Budget Amount *help |
¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000)
Fiscal Year 2023: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2022: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2021: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2020: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
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| Keywords | Chloroquine / Cytocidal effect / Plasmodium falciparum / Plasmodium / Cytociday efficacy / Africa / Cytocidal-efficacy / Novel / In-vitro assay / マラリア / クロロキン |
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| Outline of Research at the Start |
クロロキンの殺原虫効果をin vitroで評価できるアッセイを確立し、ウガンダで、クロロキンによる殺原虫効果が回復しているかについて明らかにする。全流行地で第一選択薬として広く用いられているアルテミシニンへの耐性原虫の出現と拡散、さらに同様の効果を示す代替薬の開発が滞っている現状を鑑みると、将来的な熱帯熱マラリア治療への、クロロキンの再導入に直接貢献することができる本研究のインパクトは極めて高い。
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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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Report
(4 results)
Research Products
(16 results)
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[Journal Article] The impact of sequestration on artemisinin-induced parasite clearance in Plasmodium falciparum malaria in Africa.2022
Author(s)
Fukuda N, Balikagala B, Ueno T, Anywar DA, Kimura E, Palacpac NMQ, Odongo-Aginya EI, Ogwang M, Horii T, Miida T, Mita T.
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Journal Title
Clin Infect Dis.
Volume: 10
Issue: 9
Pages: 1585-1593
DOI
Related Report
Peer Reviewed / Open Access / Int'l Joint Research
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[Journal Article] Circulation of an Artemisinin-Resistant Malaria Lineage in a Traveler Returning from East Africa to France.2022
Author(s)
Coppee R, Bailly J, Sarrasin V, Vianou B, Zinsou BE, Mazars E, Georges H, Hamane S, Lavergne RA, Dannaoui E, Balikagala B, Fukuda N, Odongo-Aginya EI, Mita T, Houze S, Clain J.
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Journal Title
Clin Infect Dis.
Volume: 75
Issue: 7
Pages: 1242-4
DOI
Related Report
Peer Reviewed / Open Access / Int'l Joint Research
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[Journal Article] Evidence of Artemisinin-Resistant Malaria in Africa.2021
Author(s)
Balikagala B, Fukuda N, Ikeda M, Katuro OT, Tachibana SI, Yamauchi M, Opio W, Emoto S, Anywar DA, Kimura E, Palacpac NMQ, Odongo-Aginya EI, Ogwang M, Horii T, Mita T.
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Journal Title
N Engl J Med.
Volume: 385
Issue: 13
Pages: 1163-71
DOI
Related Report
Peer Reviewed / Open Access / Int'l Joint Research
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