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Experimental Therapeutics

Effect of Therapeutic Chemical Agents In Vitro and on Experimental Meningoencephalitis Due to Naegleria fowleri

Jong-Hyun Kim, Suk-Yul Jung, Yang-Jin Lee, Kyoung-Ju Song, Daeho Kwon, Kyongmin Kim, Sun Park, Kyung-Il Im, Ho-Joon Shin
Jong-Hyun Kim
1Department of Microbiology, and Department of Molecular Science and Technology, Ajou University School of Medicine, Suwon 443-721
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Suk-Yul Jung
1Department of Microbiology, and Department of Molecular Science and Technology, Ajou University School of Medicine, Suwon 443-721
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Yang-Jin Lee
1Department of Microbiology, and Department of Molecular Science and Technology, Ajou University School of Medicine, Suwon 443-721
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Kyoung-Ju Song
2Department of Environmental Medical Biology, Yonsei University College of Medicine, Seoul 121-752, Republic of Korea
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Daeho Kwon
1Department of Microbiology, and Department of Molecular Science and Technology, Ajou University School of Medicine, Suwon 443-721
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Kyongmin Kim
1Department of Microbiology, and Department of Molecular Science and Technology, Ajou University School of Medicine, Suwon 443-721
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Sun Park
1Department of Microbiology, and Department of Molecular Science and Technology, Ajou University School of Medicine, Suwon 443-721
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Kyung-Il Im
2Department of Environmental Medical Biology, Yonsei University College of Medicine, Seoul 121-752, Republic of Korea
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Ho-Joon Shin
1Department of Microbiology, and Department of Molecular Science and Technology, Ajou University School of Medicine, Suwon 443-721
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  • For correspondence: hjshin@ajou.ac.kr
DOI: 10.1128/AAC.00197-08
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  • FIG. 1.
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    FIG. 1.

    In vitro effects of three drugs against N. fowleri. (A) Growth curves of N. fowleri after treatment with each drug. N. fowleri trophozoites were cultured at a density of 104/ml in the absence (control) or presence of each drug. (B) The amoebicidal activity of three drugs against N. fowleri was evaluated by LDH release assay. Amoebae were treated with miltefosine and chlorpromazine at concentrations from 1.56 to 100 μg/ml and with amphotericin B at concentrations from 0.39 to 25 μg/ml. The optical density was measured on days 2, 4, and 6, respectively. Values are means ± the standard errors of three experiments performed in triplicate.

  • FIG. 2.
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    FIG. 2.

    Liver and kidney tissue of mice treated with chlorpromazine. (A and B) Liver tissue from a mouse administered 20 mg of chlorpromazine/kg had hepatocytes of normal size and shape (A, ×100; B, ×400). (C and D) Kidney from a mouse administered 20 mg of chlorpromazine/kg had renal cortical tubules of normal size and shape (C, ×100; D, ×400). The tissues were stained with hematoxylin and eosin.

  • FIG. 3.
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    FIG. 3.

    Survival curve of N. fowleri-infected mice treated with amphotericin B (A), miltefosine (B), or chlorpromazine (C). Mice were inoculated intranasally with 104N. fowleri trophozoites. At 3 days postinoculation, the groups were treated with 10 mg of amphotericin B/kg or 20 mg of miltefosine and chlorpromazine/kg, respectively. Each group (n = 20 mice) was treated with drugs once (day 3), twice (days 3 and 7), or three times (days 3, 7, and 11). The control group received 100 μl of saline.

  • FIG. 4.
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    FIG. 4.

    Brain tissue of mice infected with N. fowleri trophozoites. (A and B) The brain tissue of dead mouse after drug treatment shows N. fowleri trophozoites and inflammatory cells. In the boxed area in panel B, arrows indicate N. fowleri trophozoites surrounded by inflammatory cells (A, ×50; B, ×400). (C and D) The brain tissue of mice that survived during the experimental periods (1 month) after drug treatment had no N. fowleri trophozoites and showed no change (C, ×50; D, ×400). The tissues were stained by hematoxylin and eosin.

Tables

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  • TABLE 1.

    MICs of the drugs tested for N. fowleri

    DrugMIC100 (μg/ml)a
    Day 2Day 4Day 6
    Amphotericin B0.780.780.78
    Miltefosine25.0025.0025.00
    Chlorpromazine12.5012.5012.50
    • ↵ a MIC100, the lowest drug concentration that caused a 100% inhibition of growth. Complete inhibition of trophozoite growth in Nelson's medium was scored on days 2, 4, and 6.

  • TABLE 2.

    BUN analysis in mouse serum after drug treatment

    DrugDose (mg/kg)Mean BUNa level (mg/dl) ± SE
    Amphotericin B527.4 ± 7.18
    1038.6 ± 6.73
    2044.1 ± 6.44
    Miltefosine1029.6 ± 8.28
    2034.6 ± 5.37
    5082.4 ± 5.29
    Chlorpromazine1036.3 ± 2.53
    2038.4 ± 6.86
    5057.5 ± 3.32
    Control (saline)b34.1 ± 2.90
    • ↵ a The reference value of BUN in mice ranges from 15 to 40 mg/dl. Values represent the means of three experiments.

    • ↵ b The control group was treated with 100 μl of saline.

  • TABLE 3.

    Survival and MTD of N. fowleri-infected mice treated with various drugs

    GroupaNo. of miceNo. of surviving miceb (%)MTDc (days)
    Amphotericin B208 (40)17.3
    Miltefosine2011 (55)19.3
    Chlorpromazine2015 (75)17.2
    Controld200 (0)11.2
    • ↵ a All groups were treated with drugs three times at days 3, 7, and 11, respectively. The control group received 100 μl of saline under the same conditions.

    • ↵ b Mice were held for 28 days after inoculation, and the cumulative percentage was recorded on a daily basis.

    • ↵ c The MTD was calculated based only on dead mice.

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Effect of Therapeutic Chemical Agents In Vitro and on Experimental Meningoencephalitis Due to Naegleria fowleri
Jong-Hyun Kim, Suk-Yul Jung, Yang-Jin Lee, Kyoung-Ju Song, Daeho Kwon, Kyongmin Kim, Sun Park, Kyung-Il Im, Ho-Joon Shin
Antimicrobial Agents and Chemotherapy Oct 2008, 52 (11) 4010-4016; DOI: 10.1128/AAC.00197-08

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Effect of Therapeutic Chemical Agents In Vitro and on Experimental Meningoencephalitis Due to Naegleria fowleri
Jong-Hyun Kim, Suk-Yul Jung, Yang-Jin Lee, Kyoung-Ju Song, Daeho Kwon, Kyongmin Kim, Sun Park, Kyung-Il Im, Ho-Joon Shin
Antimicrobial Agents and Chemotherapy Oct 2008, 52 (11) 4010-4016; DOI: 10.1128/AAC.00197-08
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KEYWORDS

Amebiasis
Amebicides
Central Nervous System Protozoal Infections
Chlorpromazine
Naegleria fowleri
Phosphorylcholine

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