クロロキンの殺マラリア原虫効果を評価できる新規in vitroアッセイ法の開発

2020 年度 実施状況報告書

• 研究課題/領域番号: 20K16238
• 研究機関: 順天堂大学
• 研究代表者: バリカガラ ベテイ 順天堂大学, 医学(系)研究科(研究院), 博士研究員 (70805895)
• 研究期間 (年度): 2020-04-01 – 2024-03-31
• キーワード: Plasmodium falciparum / Chloroquine / Cytocidal-efficacy / Novel / In-vitro assay

研究実績の概要

The research was planned to first 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). Secondly, develop a new in vitro assay that can evaluate the parasiticidal effect of chloroquine. Thirdly, verify if parasiticidal chloroquine effect has recovered in Ugandan field parasites.

To achieve the first and second steps of the study, we already have 21 preserved laboratory parasite strains in which the parasiticidal effect of chloroquine at high concentrations as in the human body is to be assessed.
Furthermore, the first step was partially confirmed in a pilot study using laboratory cultured Plasmodium falciparum parasites; chloroquine sensitive (HB3) and resistant parasite (Dd2) by comparing 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. In contrast, 100-fold difference was observed in cytocidal efficacy between two parasites.

現在までの達成度 (区分)

3: やや遅れている

理由

The progress of this research is slightly delayed because there has been some challenge to optimize the study protocol. Initially the suggested medium for maintaining parasite cultures did not support parasite growth and contributed to significant delays in experiments. After overcoming this challenge, the work is beginning to get back on track.

今後の研究の推進方策

Step1: The parasiticidal chloroquine effect will be assessed in additional laboratory parasite strains; 21 preserved in the laboratory and additional 19 to be obtained from MR4. Parasites will be cultured for 6 hours at concentrations (0.625-2000 μM) covering the maximum chloroquine concentration in the human body, 10 μM, then the drug is 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 evaluate IC50 (50% growth inhibitory concentration).

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.

次年度使用額が生じた理由

To initiate the experiments, I used mostly used our laboratory general consumables, so I did not make a lot of purchases for consumables.

I also did not spend on travel costs and perform the field studies because of study restrictions due to the new corona virus pandemic.

研究成果

• [雑誌論文] Ex vivo susceptibility of Plasmodium falciparum to antimalarial drugs in Northern Uganda (2021)
  • 著者名/発表者名: Naoyuki Fukuda, ShinIchiro Tachibana, Mie Ikeda, Miki Sakurai, Betty Balikagala, Osbert Katuro, Toshihiro Horii, Toshihiro Mita and 16 more authors
  • 雑誌名: Parasitology international 巻: 81 ページ: 102277
  • DOI: 10.1016/j.parint.2020.102277
  • 査読あり / オープンアクセス / 国際共著
• [雑誌論文] Recovery and stable persistence of chloroquine sensitivity in Plasmodium falciparum parasites after its discontinued use in Northern Uganda (2020)
  • 著者名/発表者名: Balikagala B, Sakurai-Yatsushiro M, Tachibana SI, Ikeda M, Yamauchi M, Katuro OT, Ntege EH, Sekihara M, Fukuda N, Takahashi N, Yatsushiro S, Mori T, Hirai M, Opio W, Obwoya PS, Anywar DA, Auma MA, Palacpac NMQ, Tsuboi T, Odongo-Aginya EI, Kimura E, Ogwang M, Horii T, Mita T
  • 雑誌名: Malaria Journal 巻: 19 ページ: 76
  • DOI: 10.1186/s12936-020-03157-0
  • 査読あり / オープンアクセス / 国際共著
• [雑誌論文] Global Repertoire of Human Antibodies Against Plasmodium falciparum RIFINs, SURFINs, and STEVORs in a Malaria Exposed Population (2020)
  • 著者名/発表者名: Kanoi, B. N. Nagaoka, H. White, M. T. Morita, M. Palacpac, N. M. Q. Ntege, E. H. Balikagala, B. Yeka, A. Egwang, T. G. Horii, T. Tsuboi, T. Takashima, E.
  • 雑誌名: Frontiers in Immunology 巻: 11 ページ: 893
  • DOI: 10.3389/fimmu.2020.00893
  • 査読あり / オープンアクセス / 国際共著
• [学会発表] In vivo artemisinin resistance has emerged in Uganda (Africa) (2020)
  • 著者名/発表者名: Balikagala Betty
  • 学会等名: The United States-Japan Cooperative Medical Science Program
  • 国際学会