AAC Accepted Manuscript Posted Online 16 February 2016 Antimicrob. Agents Chemother. doi:10.1128/AAC.01614-15 Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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Clofazimine inhibits the growth of Babesia and Theileria parasites in vitro and in vivo
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Running Title: Clofazimine inhibits Babesia and Theileria growth
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Bumduuren Tuvshintulga,ac† Mahmoud AbouLaila,ab† Batdorj Davaasuren,ac Aki
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Ishiyama,d Thillaiampalam Sivakumar,a Naoaki Yokoyama,a Masato Iwatsuki,d
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Kazuhiko Otoguro,d Satoshi Ōmura,d and Ikuo Igarashia #
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a
National Research Center for Protozoan Diseases, Obihiro University of Agriculture
and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan b
Department of Parasitology, Faculty of Veterinary Medicine, University of Sadat City,
Sadat City, Menoufiya, Egypt c
Laboratory of Molecular Genetics, Institute of Veterinary Medicine, Ulaanbaatar,
Mongolia d
Kitasato Institute for Life Sciences, Kitasato University, Tokyo, Japan
†
B.T. and M.A. contributed equally to this work.
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#
Correspondence footnote
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Ikuo Igarashi
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National Research Center for Protozoan Diseases, Obihiro University of Agriculture and
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Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan
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Tel.: +81-155-49-5641; Fax: 81-155-49-5643; E-mail address: [email protected]
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ABSTRACT
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The present study evaluated the growth-inhibitory effects of clofazimine, currently used
25
for treating leprosy, against Babesia bovis, B. bigemina, B. caballi, and Theileria equi in
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vitro culture, and against B. microti in mice. The IC50 values of clofazimine against the
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in vitro growth of B. bovis, B. bigemina, B. caballi, and T. equi were 4.5, 3, 4.3, and
28
0.29 μM, respectively. In mice infected with B. microti, treatment with oral
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administration of 20 mg/kg clofazimine resulted in a significant lower peak parasitemia
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(5.3%) as compared to a control group (45.9%), which was comparable to subcutaneous
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administration of 25 mg/kg diminazene aceturate, the most widely used treatment for
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animal piroplasmosis. Although slight anemia was observed in both clofazimine- and
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diminazene aceturate-treated infected mice, the level and duration of anemia were lower
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and shorter than in untreated infected mice. Using blood transfusions and PCR, we also
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examined whether clofazimine completely killed B. microti. On day 40 post-infection,
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when the blood analysis was performed, parasites were not found in the blood smears;
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however, the DNA of B. microti was detected by PCR in the blood of
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clofazimine-treated animals and in several tissues of clofazimine- and diminazene
39
aceturate-treated mice. The growth of parasites was observed in mice after blood 2
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transfusions from clofazimine-treated mice. In conclusion, clofazimine showed
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excellent inhibitory effects against Babesia and Theileria in vitro and in vivo, and
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further study on clofazimine is required for the future development of a novel
43
chemotherapy with high efficacy and safety against animal piroplasmosis and, possibly,
44
human babesiosis.
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INTRODUCTION
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Piroplasmosis in domestic and wild animals is caused by different species of
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the genera Babesia and Theileria, tick-borne hemoprotozoan parasites (1, 2, 3). Among
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the Babesia parasites that infect cattle, B. bovis, B. bigemina, and B. divergens are
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considered to be the most economically significant species (1). In horses, piroplasmosis
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is usually caused by B. caballi and T. equi (formerly known as B. equi) (4). Clinical
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symptoms include malaise, fever, hemolytic anemia, jaundice, hemoglobinuria, and
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edema. In the case of B. bovis infection, animals may die due to cerebral babesiosis. B.
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microti infection is detected worldwide in humans, especially in the USA, with
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malaria-like clinical signs (2). Several drugs, including diminazene aceturate, that have
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been used for years have proven increasingly ineffective due to their toxicity and the
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development of resistance (5, 6). Therefore, the development of new drugs with low
57
toxicity to animal hosts but with high efficacy against parasites is urgently desired.
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Clofazimine
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(2-p-chloroanilino-5-p-chlorophenyl-3,5-dihydro-3-isopropyliminophenazine),
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as B663 and Lamprene, is a phenazine dye that is currently used for treating
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multibacillary leprosy (http://www.who.int/lep/mdt/regimens/en/) and severe erythema 4
known
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nodosum
leprosum
(ENL)
reaction
in
leprosy
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(http://www.who.int/lep/mdt/clofazimine/en/). Clofazimine enhances the activity of
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phospholipase A2 and the release of its enzymatic hydrolysis products, arachidonate and
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lysophosphatidylcholine, from membrane phospholipids (7). Clofazimine has also
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demonstrated antimycobacterial (8, 9), anti-inflammatory (10), antitumor (7),
67
antileishmanial (11), moderate antimalarial (12), and antitrypanosomal (13) properties.
68
This study investigated the impact of clofazimine against Babesia and Theileria
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parasites by evaluating the inhibitory effects of clofazimine on their in vitro growth and
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the in vivo growth of B. microti as well as confirming the compound’s antimalarial
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properties.
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MATERIALS AND METHODS
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Chemical reagents
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Clofazimine was purchased from Sigma-Aldrich (Tokyo, Japan), and
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diminazene aceturate (GANASEG®) was purchased from Novartis Animal Health
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(Tokyo, Japan). A stock solution of 100 mM clofazimine in dimethyl sulfoxide (DMSO)
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was prepared and stored at −30°C until use. For the inhibition of Babesia and Theileria 5
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parasites, a stock solution of 10 mM diminazene aceturate dissolved in Milli-Q water
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was prepared and stored at –30◦C until required for use. For the antimalarial study, stock
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solutions of 8.45 mM clofazimine dissolved in methanol and 10 mM diminazene
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aceturate dissolved in Milli-Q water were prepared and stored at –20◦C until use.
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Chloroquine diphosphate salt (purchased from Sigma-Aldrich) and artesunate
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(purchased from Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) were used as control
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drugs for evaluating diminazene aceturate against Plasmodium falciparum. They were
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dissolved in Milli-Q water and 5% DMSO, respectively. As a negative control, a solvent
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was dissolved at concentrations similar to the highest concentrations of the drug in the
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treated cultures and applied to the in vitro cultures.
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Parasites and mice
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The Munich strain of B. microti was maintained by passage in the blood of
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BALB/c mice (14). Sixty female BALB/c mice were purchased from CLEA Japan
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(Tokyo, Japan) and used for the in vivo studies.
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In vitro cultivation of Babesia and Theileria 6
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In vitro cultures of B. bovis (Texas strain), B. bigemina (Argentina strain), and B.
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caballi and T. equi (both USDA strains) were used for the experiments (15). Parasites
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were grown in bovine or equine red blood cells, using a continuous microaerophilous
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stationary phase culture system (14). Medium 199 (Sigma-Aldrich) was used for B.
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bovis, B. bigemina, and T. equi, whereas RPMI 1640 (Sigma-Aldrich) was used for B.
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caballi. Media were supplemented with 40% normal bovine serum (B. bovis and B.
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bigemina) or horse serum (B. caballi and T. equi), 60 U/ml penicillin G, 60 µg/ml
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streptomycin, and 0.15 µg/ml amphotericin B (all three drugs from Sigma-Aldrich).
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Additionally, 13.6 µg of hypoxanthine (MP Biomedicals, LLC., Santa Ana, CA, USA)
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per ml was added to T. equi culture as a vital supplement. TES-hemisodium salt (229
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mg/ml) N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (Sigma-Aldrich) was
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added to bovine Babesia parasite cultures as a pH stabilizer (pH 7.2) (16). In vitro
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cultures were incubated at 37°C in an atmosphere of 5% CO2, 5% O2, and 90% N2.
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In vitro inhibition assay of Babesia and Theileria
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In vitro experiments were conducted as described previously (14). Briefly, 200
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µl of media, media with various concentrations of clofazimine based on the results of 7
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preliminary investigations, and media with DMSO were added in triplicate in 96-well
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plates. While clofazimine was used at 1, 2, 5, 10, and 25 µM for B. bovis and B. caballi,
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0.05-, 0.1-, 0.5-, 1-, 2-, 5-, and 10-µM concentrations were used for B. bigemina and T.
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equi. Then, 20 µl of parasite-infected (1% parasitemia) bovine (B. bovis and B.
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bigemina) or equine (B. caballi and T. equi) red blood cells (RBCs) was added to the
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respective wells. Culture plates were incubated at 37°C in an atmosphere of 5% CO2,
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5% O2, and 90% N2. Every 24 hours for the next 96 hours, parasitemia was monitored
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with a light microscope using Giemsa-stained thin erythrocyte smears prepared from
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each well; then media with the indicated drug concentrations and media with DMSO
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were replaced with fresh ones. Each experiment was repeated three times. Diminazene
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aceturate (5) was also used for inhibitory assays at 0.001-, 0.005-, 0.01-, 0.05-, 0.1-, 1-,
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and 2-µM concentrations for a comparative evaluation. Changes in the morphology of
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treated Babesia and Theileria parasites were compared with the control using light
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microscopy. The 50% inhibitory concentration (IC50) values were calculated on the third
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day of in vitro culture by interpolation using the curve-fitting technique (16).
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Viability test 8
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After 96 hours of treatment, 6 µL of packed red blood cells from the previously
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drug-treated cultures was added to 14 µL of parasite-free bovine or equine packed red
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blood cells in 200 µL of a fresh growth medium without any drug. The fresh growth
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medium was replaced every day for the next 10 days, and parasite recrudescence was
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monitored daily, in the absence of drugs, by microscopic examination of Giemsa-stained
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thin erythrocyte smears.
136
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Effect of clofazimine on host erythrocytes in vitro
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The effect of clofazimine on host erythrocytes was evaluated as described
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previously (14). Bovine and equine erythrocytes were incubated in the presence of 0.5,
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1, 2, 5, 10, and 25 μM of clofazimine for 3 hours at 37°C, and then erythrocytes were
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washed three times with drug-free media and used for the cultivation of Babesia and
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Theileria parasites for 72 hours. The untreated control cells were handled in the same
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manner as the pretreated cells. The pattern of parasite growth in treated erythrocytes
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was observed and compared with that of untreated control cells.
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In vitro cultivation of Plasmodium falciparum and antimalarial assay 9
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In vitro cultivation and antimalarial evaluation of chemicals using P. falciparum
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have been described previously (17). Briefly, P. falciparum strains Kl (chloroquine
149
resistant) and FCR3 (chloroquine sensitive) were cultured in human erythrocytes in
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RPMI medium supplemented with 10% human plasma at 37°C under 93% N2, 4% CO2,
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and 3% O2 (17). Asynchronous parasites (2% hematocrit and 0.5 or 1% parasitemia)
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were seeded in a 96-well microplate, and serially diluted clofazimine, at concentrations
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from 0.105 µM to 105 µM, or diminazene aceturate, at concentrations from 0.024 µM
154
to 12.5 µM was added. Chloroquine (K1: 0.375 µM–1.5 µM, FCR3: 0.0125 µM–0.05
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µM) and artesunate (0.976 nM–0.125 µM) were added in a similar fashion. After 72
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hours of incubation, parasite lactate dehydrogenase was assayed using a slight
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modification of the procedure reported by Makler et al. (18) and Vivas et al. (19). The
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50% inhibitory concentration (IC50) value was estimated from a dose-response curve.
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This study was approved by the Research Ethics Committee of Kitasato Institute
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Hospital (No12102).
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In vivo inhibitory effect of clofazimine on B. microti As the findings from in vitro experiments were promising, clofazimine was 10
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evaluated for its in vivo efficacy against B. microti in mice (16). Four groups, each
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consisting of 5 female BALB/c mice (8 weeks old), were injected with 1 × 107 B.
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microti-infected RBCs intraperitoneally. Mice in groups 1–3 were treated with
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clofazimine or diminazene aceturate from days 1–5 post-infection. The first and second
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groups were treated with 20 mg/kg of clofazimine by intraperitoneal and oral routes,
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respectively. The third group was treated with 25 mg/kg of diminazene aceturate
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subcutaneously, and the fourth group remained untreated. Each mouse was monitored
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for parasitemia until B. microti was undetectable by microscopy. All animal experiments
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were approved by the Animal Welfare Committee (Approval No. 27-65) and conducted
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in accordance with the standards for the care and management of experimental animals
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stipulated by Obihiro University of Agriculture and Veterinary Medicine, Hokkaido,
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Japan.
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Effect of clofazimine on the development of anemia in treated mice
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To examine possible side effects of clofazimine, the development of anemia was
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monitored in mice treated with clofazimine by measuring hematocrit values as an index.
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Oral treatment with 20 mg/kg of clofazimine was selected for monitoring anemia in 11
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treated mice. Twenty-five female BALB/c mice (8 weeks old) were divided into 5
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groups (5 mice per group). Groups I–II were not infected, and groups III–V were
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intraperitoneally infected with 1 x 107B. microti-infected RBCs. Parasitemia was
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monitored in groups III-V using the Giemsa-staining method every 48 hours. In all
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groups, the hematocrit was monitored every 96 hours using the Celltac α MEK-6450
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automatic hematology analyzer (Nihon Kohden Corporation, Tokyo, Japan). Groups II
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and IV received oral administration of 20 mg/kg clofazimine for the 5 days from days
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3–7 post-infection. Group V received subcutaneous administration of 25 mg/kg of
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diminazene aceturate, while groups I and III received oral administration of 0.2 ml of
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PBS containing DMSO. The experiments were repeated twice.
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Effect of clofazimine on the infectivity of treated parasites
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To examine the infectivity of clofazimine-treated parasites, mice in groups III–V
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were sacrificed on day 40 post-infection, and equal numbers of RBCs (1× 108) from
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these mice were intraperitoneally transferred (reinfected) to normal mice (each group
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consisting of five 8-week-old female BALB/c mice). Parasitemia was monitored by
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microscopic examination of the Giemsa-stained blood smears every 2 days until day 24 12
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post-blood transfusion.
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PCR
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PCR was undertaken to detect parasite DNA in blood and tissues (heart, spleen,
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liver, and kidneys) on day 40 post-infection. DNA was extracted from the blood of B.
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microti-infected groups treated with clofazimine and diminazene aceturate and the
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non-treated control group using a QIAamp DNA Blood Mini Kit (QIAGEN, Tokyo,
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Japan). DNA was also extracted from tissues of these mice using the NucleoSpin®
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Tissue Kit (MACHEREY-NAGEL, Düren, Germany). PCR targeting the B. microti
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small subunit rRNA (ss-rRNA) gene was carried out as described previously (20) using
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nested
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(5'-CTTAGTATAAGCTTTTATACAGC-3')/outer
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(5'-ATAGGTCAGAAACTTGAATGATACA-3')
211
(5'-GTTATAGTTTATTTGATGTTCGTTT-3')/
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(5'-AAGCCATGCGATTCGCTAAT-3'). The expected size of the PCR product was 154
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bp.
PCR
primer
sets:
214 13
outer
forward reverse
and inner
inner
Babl Bab4
forward reverse
Bab2 Bab3
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Statistical analysis
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Differences in parasitemia between the control and treated parasites were
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analyzed by an independent Student’s t test using JMP statistical software (SAS Institute
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Inc., Cary, NC, USA), and P values < 0.05 were considered statistically significant.
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RESULTS
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The in vitro growth inhibitory effect of clofazimine
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The effect of clofazimine was examined using one Theileria and three Babesia
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assays. Clofazimine significantly inhibited (P < 0.05) the growth of B. bovis, B.
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bigemina, B. caballi, and T. equi at 2, 0.1, 5, and 0.1 µM, respectively (Fig. 1). The in
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vitro growth of the four parasites was also significantly inhibited (P < 0.05) by 0.005-
226
µM diminazene aceturate treatment, except that day 1 produced significant impact only
227
with 0.1 µM (data not shown). Lower IC50 values of clofazimine were observed for B.
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bigemina (3 µM) and T. equi (0.29 µM) than for B. bovis (4.5 µM) and B. caballi (4.3
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µM) (Table 1). In particular, clofazimine had a lower IC50 value for T. equi than did
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diminazene aceturate (0.65 µM).
14
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Clofazimine completely cleared B. bovis and B. caballi at 25 µM by days 4 and
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3, respectively, while B. bigemina and T. equi were eliminated at a 10-µM concentration
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on days 4 and 2, respectively (Fig. 1). In addition, B. bovis, B. bigemina, B. caballi, and
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T. equi treated with 25, 5, 5, and 0.05 µM of clofazimine, respectively, failed to grow in
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drug-free media in the viability test. The complete suppression of diminazene aceturate-
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treated parasites was observed at a concentration of 2 µM, while a concentration of 0.05
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µM was required to suppress the growth of B. caballi (data not shown). There was no
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regrowth of the diminazene aceturate-treated parasites in the subsequent viability test at
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concentrations of 0.025 µM (B. caballi) and 0.5 µM (B. bovis, B. bigemina, and T. equi)
240
(data not shown).
241
As clofazimine is reported to have very weak antimalarial activity in vitro (12,
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21), we also evaluated the impact of both clofazimine and diminazene aceturate against
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malaria parasites. Clofazimine exhibited poor antimalarial activity against K1 and FCR3
244
strains, and 50% growth inhibition was not achieved even at the maximum
245
concentration tested. Diminazene aceturate inhibited the growth of P. falciparum more
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strongly than did clofazimine, with IC50 values of 0.046 µM and 0.250 µM for the K1
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and FCR3 strains, respectively (Table 1). As compared to current antimalarial drugs, 15
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artesunate displayed IC50 values of 0.01 µM for K1 and 0.009 µM for FCR3 strains,
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while chloroquine had IC50 values of 0.536 µM for K1 and 0.048 µM for FCR3 strains.
250
251
The morphological and functional effect of clofazimine on parasites and
252
erythrocytes in vitro
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The morphological changes of parasites and erythrocytes were also examined
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in treated cultures. Parasites in clofazimine-treated cultures appeared degenerated with
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the loss of typical parasitic shapes of B. bovis (Fig. 2B), B. bigemina (Fig. 2D), B.
256
caballi (Fig. 3B), and T. equi (Fig. 3D). Clofazimine did not cause morphological
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changes in normal bovine and equine erythrocytes. Furthermore, when erythrocytes
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were pretreated with clofazimine at 0.5-, 1-, 2-, 5-, 10-, and 25-µM concentrations for 3
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hours at 37°C and then used to culture the parasites for 72 h, no differences in the
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morphology of erythrocytes or the growth of parasites were observed as compared to
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non-treated erythrocytes (data not shown).
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263
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In vivo inhibition in mice Based on the growth inhibitory effect of clofazimine against Babesia and 16
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Theileria in vitro, the chemotherapeutic effect was examined for B. microti infection in
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mice, since that parasite is listed in Japan’s Animal Infectious Diseases Control Law. B.
267
microti replication was significantly inhibited by clofazimine and diminazene aceturate,
268
as compared to the untreated infected mice (Fig. 4). Mice treated with 20 mg/kg
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clofazimine through oral and intraperitoneal routes showed peak parasitemias of 5.3 and
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8.8%, respectively, indicating inhibitions of 88.5 and 80.8%, respectively, as compared
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to the control group (45.9% parasitemia). Notably, the parasitemia in mice that received
272
clofazimine orally was comparable to that in diminazene aceturate-treated mice (5.3%)
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(Fig. 4).
274
275
Effect of clofazimine on the development of anemia in uninfected and infected mice
276
To examine the possible side effects of clofazimine, hematocrit changes were
277
monitored as an index for the development of anemia in mice orally administered with
278
20 mg/kg clofazimine for 5 days. Clofazimine-treated uninfected mice (group II) did not
279
show significantly different (P > 0.01) hematocrit values as compared with the
280
DMSO-treated uninfected mice (group I) (Fig. 5A). In addition, there were no apparent
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clinical signs in clofazimine-treated mice, both uninfected and infected. For mice 17
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infected with B. microti, as shown in the previous experiment (Fig. 4), infected mice
283
treated with 20 mg/kg clofazimine indicated 1.6% peak parasitemia, showing 96.5%
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inhibition as compared to untreated control group III (45.1% parasitemia) on day 8 (Fig.
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5B). Significant hematocrit reductions were observed in infected DMSO-treated mice
286
(group III) on days 8–28 after infection as compared to uninfected DMSO-treated mice
287
(group I) (Fig. 5C). While infected clofazimine-treated mice (group IV) showed
288
significantly lower hematocrits (P < 0.01) at days 8 and 12 as compared to the control
289
(group I), the hematocrit reduction level was smaller than that of infected
290
DMSO-treated mice (group III). In addition, infected clofazimine-treated mice quickly
291
recovered from low hematocrit values, as did infected diminazene aceturate-treated
292
mice (Fig. 5C).
293
294
Effect of clofazimine on the infectivity of treated parasites
295
Finally, using PCR and blood transfusions, we examined whether clofazimine
296
completely killed B. microti. Although the parasites were not found in blood smears of
297
clofazimine-treated mice on day 40 after infection, the B. microti ss-rRNA gene was
298
detectable, not only in blood but also in the heart, spleen, kidneys, and liver of these 18
299
mice, as seen in DMSO-treated infected control mice (Fig. 6A, III and IV). PCR
300
amplification also detected B. microti DNA in the heart and spleen, but not in the blood,
301
of diminazene aceturate-treated mice (Fig. 6A, V).
302
The blood from clofazimine- and diminazene aceturate-treated groups was
303
transfused to new mice to determine the infectivity of the treated parasites. B. microti
304
was regrown in recipient mice transfused with blood from both control (untreated) and
305
clofazimine-treated groups, although there was some delay in peak parasitemia in mice
306
transfused with blood from the clofazimine-treated mice. In contrast, the diminazene
307
aceturate-treated parasites did not regrow in mice (Fig. 7).
308
309
DISCUSSION
310
In the present study, clofazimine was shown to inhibit the in vitro growth of
311
three Babesia species and T. equi. As the solvent had no effect on the growth of the
312
parasites, the inhibition was considered to be solely due to clofazimine. Clofazimine
313
also induced severe degenerative changes in the treated parasites as compared to the
314
control (Figs. 2 and 3). After withdrawing the drugs, the growth of parasites could not
315
be observed in the in vitro cultures (Fig. 1). Taken together, the inhibition caused by 19
316
clofazimine at these concentrations was irreversible. The IC50 values of clofazimine to
317
Babesia and Theileria parasites reported in the present study were lower than those for
318
P. falciparum (12, 21, Table 1). In addition, clofazimine was more effective against
319
Babesia and Theileria species than against Leishmania donovani (22). Toxic effects of
320
clofazimine on mammalian cells have been reported. Clofazimine at 42.25 µM (20
321
µg/ml) induced hemolysis of murine RBCs, and the toxic effects were evident when
322
murine macrophages were treated with 10.56 µM (23). On the other hand, clofazimine
323
had no toxic effect on cultured neuronal cells at 211.24 µM (100 µg/ml) (24). In the
324
present study, bovine and equine RBCs were not affected, morphologically or
325
functionally, by pretreatment with clofazimine in vitro. Previous studies have shown
326
that peak concentrations of clofazimine in serum reached 1.48–2.11 µM (0.7–1 µg/ml)
327
and 6.34–8.45 µM (3–4 µg/ml) after the oral administration of 200 and 600 mg,
328
respectively (8, 25). All of the IC50 values recorded in the present study, therefore, were
329
well within the therapeutic range of serum concentration, suggesting that clofazimine
330
may be a potential drug candidate for chemotherapy against piroplasmosis.
331
The low IC50 values of clofazimine in the in vitro inhibition assay encouraged us
332
to further evaluate its in vivo effects on B. microti in a mouse model. Clofazimine 20
333
treatment effectively inhibited the growth of B. microti in mice, especially when it was
334
administered by the oral route, as seen with the subcutaneous administration of
335
diminazene aceturate (Fig. 4). Significant hematocrit reductions were observed in B.
336
microti–infected, DMSO-treated control mice. In a recent study (26), B. microti resulted
337
in low hematocrit values, similar to those obtained in the present study, in 6-week-old
338
BALB/c mice infected with only 1 × 104 parasitized RBCs. Although the different ages
339
of the mice could explain this discrepancy, further studies are necessary to confirm this
340
assumption. Nevertheless, clofazimine, similar to diminazene aceturate, prevented
341
anemia development in the infected mice, although transient reductions of hematocrit
342
were occasionally observed (Fig. 5C). Furthermore, clofazimine treatment had no toxic
343
side effects and did not induce anemia in uninfected mice (Fig. 5A). Clofazimine has
344
also been successfully administered at a daily dose of 200–300 mg for more than 30
345
months for the treatment of drug-resistant tuberculosis in humans (27), indicating the
346
safety of clofazimine for animal and human treatment.
347
Diminazene aceturate is the most widely used antibabesial agent in the field of
348
veterinary medicine. However, diminazene aceturate does not completely eliminate
349
parasites from hosts, which can then lead to a relapse of disease in treated animals and 21
350
produce side effects, including tissue damage at the injection site, restlessness, and colic
351
(5). In the present study, B. microti was not observed on blood smears prepared from
352
clofazimine-treated mice on day 40 post-infection. However, parasite DNA was
353
detected in the blood and other organs by PCR, and the transfusion of blood from
354
clofazimine-treated mice resulted in parasite growth in recipient mice (Figs. 6 and 7),
355
indicating that the drug countered the B. microti infection but the parasites could appear
356
again. Therefore, clofazimine may need to be accompanied by another drug to augment
357
its effect and prevent the regrowth of parasites. Since clofazimine is typically used in
358
combination with other drugs as a multidrug therapy for the treatment of leprosy and
359
tuberculosis, clofazimine efficacy might be improved by combining it with other drugs
360
such as (-)-epigallocatechin-3-gallate (28) or allicin (29), both of which are reported to
361
inhibit Babesia and Theileria in vitro and in vivo. Furthermore, as an example of drug
362
repurposing (30), the oral administration of clofazimine, especially with its lower
363
toxicity as compared with that of diminazene aceturate, might be an alternative
364
treatment for animal babesiosis.
365
Clofazimine might also be an alternative chemotherapeutic agent against human
366
babesiosis caused by B. microti. Several combination therapies, such as azithromycin 22
367
with atovaquone or clindamycin with quinine, are currently used for the treatment of
368
human babesiosis. However, drug-resistant parasites and therapeutic failures have been
369
reported in some severe cases (31), making the development of new drugs and
370
treatments for human babesiosis an urgent priority. In the present study, we have shown
371
the high efficacy of clofazimine against B. microti infection in mice (Fig. 5). Since
372
clofazimine is being used for chemotherapy of leprosy in humans, it has potential for
373
the treatment of B. microti in humans as an alternative to currently used drugs.
374
In conclusion, the present study has demonstrated the in vitro and in vivo
375
inhibitory effect of clofazimine against Babesia and Theileria parasites, suggesting that
376
clofazimine is a potential drug for the treatment of clinical disease caused by Babesia
377
and Theileria in animals and humans. These findings warrant further investigation to
378
evaluate the possible use of this chemical, alone or in combination with other drugs, for
379
animal piroplasmosis and human babesiosis.
380
381
ACKNOWLEDGMENT
382
This study was supported by Cooperative Research Grants (23-joint-3, 24-joint-6,
383
25-joint-17) of the National Research Center for Protozoan Diseases, Obihiro 23
384
University of Agriculture and Veterinary Medicine.
385
386
387
REFERENCES 1.
Parasitology 129:S247–S269.
388
389
2.
Homer MJ, Aguilar-Delfin I, Telford SR, Krause PJ, Persing DH. 2000. Babesiosis. Clin Microbiol Rev 13:451–469.
390
391
Bock R, Jackson L, de Vos A, Jorgensen W. 2004. Babesiosis of cattle.
3.
Bishop R, Musoke A, Morzaria S, Gardner M, Nene V. 2004. Theileria:
392
Intracellular protozoan parasites of wild and domestic ruminants transmitted
393
by ixodid ticks. Parasitology 129:S271–283.
394
4.
piroplasmosis. J Vet Intern Med 27:1334–1346.
395
396
Wise LN, Kappmeyer LS, Mealey RH, Knowles DP. 2013. Review of equine
5.
Mosqueda J, Olvera-Ramirez A, Aguilar-Tipacamu G, Canto GJ. 2012.
397
Current advances in detection and treatment of babesiosis. Curr Med Chem
398
19:1504–1518.
399
400
6.
Vial HJ, Gorenflot A. 2006. Chemotherapy against babesiosis. Vet Parasitol 138:147–160. 24
401
7.
Van Rensburg CE, Van Staden AM, Anderson R. 1993. The riminophenazine
402
agents clofazimine and B669 inhibit the proliferation of cancer cell lines in vitro
403
by phospholipase A2 mediated oxidative and nonoxidative mechanisms. Cancer
404
Res 53:318–323.
405
8.
36:3–7.
406
407
Barry VC, Conalty V. 1965. The antimycobacterial activity of B663. Lepr Rev
9.
Reddy VM, Nadadhur G, Daneluzzi D, O’Sullivan JF, Gangadharam PJ.
408
1996. Antituberculosis activities of clofazimine and its new analogs B4154 and
409
B4157. Antimicrob Agents Chemother 40:633–636.
410
411
10. Bygum A, Toft-Petersen M. 2008. Melkersson-Rosenthal syndrome treated with clofazimine. Ugeskr Laeger 170:159.
412
11. Evans AT, Croft SL, Peters W, Neal RA. 1989. Antileishmanial effects of
413
clofazimine and other antimycobacterial agents. Ann Trop Med Parasitol
414
83:447–454.
415
12. Mahmoudi N, de Julián-Ortiz JV, Ciceron L, Gálvez J, Mazier D, Danis M,
416
Derouin F, Garcıá-Domenech R. 2006. Identification of new antimalarial drugs
417
by linear discriminant analysis and topological virtual screening. J Antimicrob 25
418
Chemother 57:489–497.
419
13. Otoguro K, Ishiyama A, Iwatsuki M, Namatame M, Nishihara-Tukashima
420
A, Nakashima T, Shibahara S, Kondo S, Yamada H, Ōmura S. 2010. In
421
vitro and in vivo anti-Trypanosoma brucei activities of phenazinomycin and
422
related compounds. J Antibiot 63:579–581.
423
14. AbouLaila M, Munkhjargal T, Sivakumar T, Ueno A, Nakano Y, Yokoyama
424
M, Yoshinari T, Nagano D, Katayama K, El-Bahy N, Yokoyama N, Igarashi I.
425
2012. Apicoplast-targeting antibacterials inhibit the growth of Babesia parasites.
426
Antimicrob Agents Chemother 56:3196–3206.
427
15. Igarashi I, Njonge FK, Kaneko Y, Nakamura Y. 1998. Babesia bigemina: In
428
vitro and in vivo effects of curdlan sulfate on growth of parasites. Exp Parasitol
429
90:290–293.
430
16. AbouLaila M, Davaasuren B, Salama A, Ichikawa M, Terkawi MA,
431
Munkhjargal T, Yokoyama N, Igarashi, I. 2014. Evaluation of the inhibitory
432
effects of miltefosine on the growth of Babesia and Theileria parasites. Vet
433
Parasitol 204:104–110.
26
434
17. Otoguro K, Kohana A, Manabe C, Ishiyama A, Ui H, Shiomi K, Yamada H,
435
Ōmura S. 2001. Potent antimalarial activities of polyether antibiotic, X-206. J
436
Antibiot 54:658–663.
437
18. Makler MT, Ries JM, Williams JA, Bancroft JE, Piper RC, Gibbins BL,
438
Hinrichs DJ. 1993. Parasite lactate dehydrogenase as an assay for Plasmodium
439
falciparum drug sensitivity. Am J Trop Med Hyg 48:739–741.
440
19. Vivas L, Easton A, Kendrick H, Cameron A, Lavandera JL, Barros D, de las
441
Heras FG, Brady RL, Croft SL. 2005. Plasmodium falciparum: Stage specific
442
effects of a selective inhibitor of lactate dehydrogenase. Exp Parasitol
443
111:105–114.
444
20. Persing DH, Mathiesen D, Marshall WF, Telford SR, Spielman A, Thomford
445
JW, Conrad PA. 1992. Detection of Babesia microti by polymerase chain
446
reaction. J Clin Microbiol 30:2097–2103.
447
21. Makgatho ME, Anderson R, O’Sullivan JF, Egan TJ, Freese JA, Cornelius N,
448
van Rensburg CEJ. 2000. Tetramethylpiperidine-substituted phenazines as novel
449
anti-plasmodial agents. Drug Dev Res 50:195–202.
450
22. Datta G, Bera T. 2000. The effects of clofazimine, niclosamide and amphotericin 27
451
B, on electron transport of Leishmania donovani promastigotes. Indian J Med Res
452
112:15–20.
453
23. Mehta RT. 1996. Liposome encapsulation of clofazimine reduces toxicity in vitro
454
and in vivo and improves therapeutic efficacy in the beige mouse model of
455
disseminated Mycobacterium avium–M. intracellulare complex infection.
456
Antimicrob Agents Chemother 40:1893–1902.
457
24. Endoh M, Kunishita T, Tabira T. 1999. No effect of anti-leprosy drugs in the
458
prevention of Alzheimer's disease and beta-amyloid neurotoxicity. J Neurol Sci
459
165:28–30.
460
461
462
463
25. Yawalkar SJ, Vischer W. 1979. Lamprene (clofazimine) in leprosy. Basic information. Lepr Rev 50:135–144. 26. Sasaki M, Fujii Y, Iwamoto M, Ikadai H. 2013. Effect of sex steroids on Babesia microti infection in mice. Am J Trop Med Hyg 88:367−375.
464
27. Mitnick CD, Shin SS, Seung KJ, Rich ML, Atwood SS, Furin JJ, Fitzmaurice
465
GM, Alcantara Viru FA, Appleton SC, Bayona JN, Bonilla CA, Chalco K,
466
Choi S, Franke MF, Fraser HS, Guerra D, Hurtado RM, Jazayeri D, Joseph K,
467
Llaro K, Mestanza L, Mukherjee JS, Muñoz M, Palacios E, Sanchez E, 28
468
Sloutsky A, Becerra MC. 2008. Comprehensive treatment of extensively
469
drug-resistant tuberculosis. N Engl J Med 359:563–574.
470
28. AbouLaila M, Yokoyama N, Igarashi I. 2010. Inhibitory effects of
471
(-)-epigallocatechin-3-gallate from green tea on the growth of Babesia parasites.
472
Parasitology 137:785–791.
473
29. Salama AA, AbouLaila M, Terkawi MA, Mousa A, El-Sify A, Allaam M,
474
Zaghawa A, Yokoyama N, Igarashi I. 2014. Inhibitory effect of allicin on the
475
growth of Babesia and Theileria equi parasites. Parasitol Res 113:275–283.
476
30. Andrews KT, Fisher G, Skinner-Adams TS. 2014. Drug repurposing and
477
human parasitic protozoan diseases. Int J Parasitol Drugs Drug Resist 4:95–111.
478
31. Vannier E, Krause PJ. 2012. Human babesiosis. N Engl J Med 366:2397–2407.
479
480
FIGURE LEGENDS
481
Fig. 1. In vitro inhibitory effects of clofazimine on B. bovis (A), B. bigemina (B), B.
482
caballi (C), and T. equi (D). Each value represents the mean ± standard deviation of
483
three separate experiments carried out in triplicate. Asterisks indicate a significant
484
difference (P < 0.05) between the clofazimine-treated and control cultures. Parasite 29
485
viability was indicated as viable (+) and dead (-) in subcultures without clofazimine
486
after 10 days.
487
488
Fig. 2. Light micrographs of clofazimine-treated B. bovis and B. bigemina on day 3 of
489
in vitro cultivation. In vitro cultures of B. bovis treated with 0.75% DMSO (A) or 10
490
µM clofazimine and B. bigemina treated with 0.3% DMSO (C) or 5 µM clofazimine
491
(D) were observed under a light microscope. Drug-treated cultures showed higher
492
numbers of degenerated parasites than did the control cultures. Bars = 10 μm.
493
494
Fig. 3. Light micrographs of clofazimine-treated B. caballi and T. equi on day 3 of in
495
vitro cultivation. In vitro cultures of B. caballi treated with 0.75% DMSO (A) or 10 µM
496
clofazimine and T. equi treated with 0.3% DMSO (C) or 5 µM clofazimine (D) were
497
observed under a light microscope. Drug-treated cultures showed higher numbers of
498
degenerated parasites than did the control cultures. Bars = 10 μm.
499
500
Fig. 4. Inhibitory effects of 20 mg/kg of clofazimine (orally = Oral, intraperitoneally =
501
IP) and 25 mg/kg of diminazene aceturate (subcutaneously = SC) on the growth of B. 30
502
microti in BALB/c mice. Each value represents the mean ± standard deviation of the
503
experiment carried out twice. The double-headed arrow indicates the time of the drug
504
administration. Asterisks indicate a significant difference (P < 0.05) between the
505
clofazimine-treated and DMSO control groups.
506
507
Fig. 5. Changes of hematocrit values in mice treated with clofazimine. (A) Hematocrit
508
changes in uninfected groups treated orally with DMSO and 20 mg/kg of clofazimine
509
(CF). (B) Infectious course of B. microti in mice treated orally with 20 mg/kg of
510
clofazimine (CF), subcutaneously with 25 mg/kg of diminazene aceturate (DA), and
511
with DMSO. (C) Hematocrit changes in B. microti-infected mice treated with CF, DA,
512
and DMSO as well as uninfected-treated with DMSO control. The figure is
513
representative of an experiment that was conducted twice. Asterisks indicate a
514
significant difference (P ≤ 0.01) between the experimental and control groups.
515
516
Fig. 6. Polymerase chain reaction of the ss-rRNA gene in different organs of mice
517
infected with B. microti after DMSO (group III), oral 20-mg/kg clofazimine (group IV),
518
and subcutaneous 25-mg/kg diminazene aceturate (group V) treatment on day 40 31
519
post-infection. B: blood, H: heart, S: spleen, K: kidney, L: liver, NC: negative control.
520
M indicates a 100-bp DNA ladder.
521
522
Fig. 7. Growth of B. microti after transfusion of 1 × 108 RBCs into normal mice from
523
DMSO control (group III), clofazimine-treated (group IV), and diminazene
524
aceturate-treated (group V) animals.
525
526
32
Table 1. IC50 values of clofazimine and diminazene aceturate for Babesia, Theileria, and Plasmodium parasites IC50 (µM)a
Parasites
a
Clofazimine
Diminazene aceturate
B. bovis
4.5 ± 0.3
0.36 ± 0.02
B. bigemina
3 ± 0.2
0.18 ± 0.007
B. caballi
4.3 ± 0.4
0.01 ± 0.001
T. equi
0.29 ± 0.03
0.65 ± 0.03
P. falciparum
> 105b
0.046 ± 0.014, 0.25 ± 0.06c
IC50 values were calculated based on the parasitemia dynamics of treated and untreated parasites in three separate experiments, each conducted in triplicate. b Value for both K1 (chloroquine-resistant) and FCR3 (chloroquine-susceptible) strains. c Values for the K1 (chloroquine-resistant) and FCR3 (chloroquine-susceptible) strains, respectively.
Parasitemia (%)
10 8 6
+ + +
*
*
*
+
4
*
2 0 0
1
2
3
Control DMSO 0.05 M 0.1 M 1 M 2 M 5 M 10 M
4 3
*
2
*
+
1
-
0
*
0
4
1
Days of culture
4
* *
2
*+ + + -
*
0 0
1
2
Days of culture
Fig. 1
3
4
Control DMSO 0.05 M 0.1 M 0.5 M 1 M 2 M 5 M 10 M
6
+ +
Parasitemia (%)
6
7
Viability
Parasitemia (%)
Control DMSO 0.5 M 1 M 2 M 5 M 10 M 25 M
2 Days of culture
3
4
T. equi
B. caballi
8
+ + + * + + +
5 4
+ + * *
3 2
*
*
--
1 0 0
1
-
2
Days of culture
3
4
Viability
Control DMSO 1 M 2 M 5 M 10 M 25 M
B. bigemina
5
Viability Parasitemia (%)
12
Viability
BV sig. at 0.05 by t test at 2um B. bovis
Fig. 2
Fig. 3
60
Parasitemia (%)
DMSO
50
Clofazimine 20 mg/kg Oral
40
Clofazimine 20 mg/kg IP Diminazene aceturate 25 mg/kg SC
30 20 10
*
** * * * **
*
0 0
2
4
6
8
10
12
14
16
Days post-inoculation Fig. 4
18
20
22
Hematocrit %
(A) 60.0 50.0 40.0 30.0 20.0 10.0 0.0
I (DMSO) uninfected
D0
Parasitemia %
(B)
Hematocrit %
D12
60.0
D16
D20
D24
D28
D32
D36
50.0
III (DMSO)
40.0
IV (20mg/kg CF)
30.0
V (25 mg/kg DA)
D40
20.0 10.0 0.0 D4
D6
D8 D10 D12 D14 D16 D18 D20 D22 D24 D26 D28 D30 D32 D34 D36 D38 D40
Days of post-infection 60.0 50.0 40.0 30.0 20.0 10.0 0.0
** *
** *
D8
D12
*
*
D16
D20
*
I (DMSO) uninfected IV (20mg/kg CF) infected
D0
Fig. 5
D8
Days of experimental period
D2
(C)
D4
II (20mg/kg CF) uninfected
D4
* III (DMSO) infected V (25 mg/kg DA) infected
D24
Days of post-infection
D28
D32
D36
D40
M NC B H
1000 bp 500 bp
100 bp
Fig. 6
III S K
L
B
H
IV S K
V L
B
H S
M K
L
25
III (DMSO) IV (20 mg/kg Clofazimine)
Parasitemia (%)
20
V (25 mg/kg Diminazene aceturate) 15
10
5
0
D3
D6
D9
D13
D16
D19
Days of post-blood transfusion Fig. 7
D22
D25
D28
1
Clofazimine inhibits the growth of Babesia and Theileria parasites in vitro and in vivo
2 3
Running Title: Clofazimine inhibits Babesia and Theileria growth
4 5
Bumduuren Tuvshintulga,ac† Mahmoud AbouLaila,ab† Batdorj Davaasuren,ac Aki
6
Ishiyama,d Thillaiampalam Sivakumar,a Naoaki Yokoyama,a Masato Iwatsuki,d
7
Kazuhiko Otoguro,d Satoshi Ōmura,d and Ikuo Igarashia #
8 9 10 11 12 13 14
a
National Research Center for Protozoan Diseases, Obihiro University of Agriculture
and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan b
Department of Parasitology, Faculty of Veterinary Medicine, University of Sadat City,
Sadat City, Menoufiya, Egypt c
Laboratory of Molecular Genetics, Institute of Veterinary Medicine, Ulaanbaatar,
Mongolia d
Kitasato Institute for Life Sciences, Kitasato University, Tokyo, Japan
†
B.T. and M.A. contributed equally to this work.
18
#
Correspondence footnote
19
Ikuo Igarashi
20
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and
21
Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan
22
Tel.: +81-155-49-5641; Fax: 81-155-49-5643; E-mail address: [email protected]
15 16 17
1
23
ABSTRACT
24
The present study evaluated the growth-inhibitory effects of clofazimine, currently used
25
for treating leprosy, against Babesia bovis, B. bigemina, B. caballi, and Theileria equi in
26
vitro culture, and against B. microti in mice. The IC50 values of clofazimine against the
27
in vitro growth of B. bovis, B. bigemina, B. caballi, and T. equi were 4.5, 3, 4.3, and
28
0.29 μM, respectively. In mice infected with B. microti, treatment with oral
29
administration of 20 mg/kg clofazimine resulted in a significant lower peak parasitemia
30
(5.3%) as compared to a control group (45.9%), which was comparable to subcutaneous
31
administration of 25 mg/kg diminazene aceturate, the most widely used treatment for
32
animal piroplasmosis. Although slight anemia was observed in both clofazimine- and
33
diminazene aceturate-treated infected mice, the level and duration of anemia were lower
34
and shorter than in untreated infected mice. Using blood transfusions and PCR, we also
35
examined whether clofazimine completely killed B. microti. On day 40 post-infection,
36
when the blood analysis was performed, parasites were not found in the blood smears;
37
however, the DNA of B. microti was detected by PCR in the blood of
38
clofazimine-treated animals and in several tissues of clofazimine- and diminazene
39
aceturate-treated mice. The growth of parasites was observed in mice after blood 2
40
transfusions from clofazimine-treated mice. In conclusion, clofazimine showed
41
excellent inhibitory effects against Babesia and Theileria in vitro and in vivo, and
42
further study on clofazimine is required for the future development of a novel
43
chemotherapy with high efficacy and safety against animal piroplasmosis and, possibly,
44
human babesiosis.
3
45
INTRODUCTION
46
Piroplasmosis in domestic and wild animals is caused by different species of
47
the genera Babesia and Theileria, tick-borne hemoprotozoan parasites (1, 2, 3). Among
48
the Babesia parasites that infect cattle, B. bovis, B. bigemina, and B. divergens are
49
considered to be the most economically significant species (1). In horses, piroplasmosis
50
is usually caused by B. caballi and T. equi (formerly known as B. equi) (4). Clinical
51
symptoms include malaise, fever, hemolytic anemia, jaundice, hemoglobinuria, and
52
edema. In the case of B. bovis infection, animals may die due to cerebral babesiosis. B.
53
microti infection is detected worldwide in humans, especially in the USA, with
54
malaria-like clinical signs (2). Several drugs, including diminazene aceturate, that have
55
been used for years have proven increasingly ineffective due to their toxicity and the
56
development of resistance (5, 6). Therefore, the development of new drugs with low
57
toxicity to animal hosts but with high efficacy against parasites is urgently desired.
58
Clofazimine
59
(2-p-chloroanilino-5-p-chlorophenyl-3,5-dihydro-3-isopropyliminophenazine),
60
as B663 and Lamprene, is a phenazine dye that is currently used for treating
61
multibacillary leprosy (http://www.who.int/lep/mdt/regimens/en/) and severe erythema 4
known
62
nodosum
leprosum
(ENL)
reaction
in
leprosy
63
(http://www.who.int/lep/mdt/clofazimine/en/). Clofazimine enhances the activity of
64
phospholipase A2 and the release of its enzymatic hydrolysis products, arachidonate and
65
lysophosphatidylcholine, from membrane phospholipids (7). Clofazimine has also
66
demonstrated antimycobacterial (8, 9), anti-inflammatory (10), antitumor (7),
67
antileishmanial (11), moderate antimalarial (12), and antitrypanosomal (13) properties.
68
This study investigated the impact of clofazimine against Babesia and Theileria
69
parasites by evaluating the inhibitory effects of clofazimine on their in vitro growth and
70
the in vivo growth of B. microti as well as confirming the compound’s antimalarial
71
properties.
72
73
MATERIALS AND METHODS
74
Chemical reagents
75
Clofazimine was purchased from Sigma-Aldrich (Tokyo, Japan), and
76
diminazene aceturate (GANASEG®) was purchased from Novartis Animal Health
77
(Tokyo, Japan). A stock solution of 100 mM clofazimine in dimethyl sulfoxide (DMSO)
78
was prepared and stored at −30°C until use. For the inhibition of Babesia and Theileria 5
79
parasites, a stock solution of 10 mM diminazene aceturate dissolved in Milli-Q water
80
was prepared and stored at –30◦C until required for use. For the antimalarial study, stock
81
solutions of 8.45 mM clofazimine dissolved in methanol and 10 mM diminazene
82
aceturate dissolved in Milli-Q water were prepared and stored at –20◦C until use.
83
Chloroquine diphosphate salt (purchased from Sigma-Aldrich) and artesunate
84
(purchased from Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) were used as control
85
drugs for evaluating diminazene aceturate against Plasmodium falciparum. They were
86
dissolved in Milli-Q water and 5% DMSO, respectively. As a negative control, a solvent
87
was dissolved at concentrations similar to the highest concentrations of the drug in the
88
treated cultures and applied to the in vitro cultures.
89
90
Parasites and mice
91
The Munich strain of B. microti was maintained by passage in the blood of
92
BALB/c mice (14). Sixty female BALB/c mice were purchased from CLEA Japan
93
(Tokyo, Japan) and used for the in vivo studies.
94
95
In vitro cultivation of Babesia and Theileria 6
96
In vitro cultures of B. bovis (Texas strain), B. bigemina (Argentina strain), and B.
97
caballi and T. equi (both USDA strains) were used for the experiments (15). Parasites
98
were grown in bovine or equine red blood cells, using a continuous microaerophilous
99
stationary phase culture system (14). Medium 199 (Sigma-Aldrich) was used for B.
100
bovis, B. bigemina, and T. equi, whereas RPMI 1640 (Sigma-Aldrich) was used for B.
101
caballi. Media were supplemented with 40% normal bovine serum (B. bovis and B.
102
bigemina) or horse serum (B. caballi and T. equi), 60 U/ml penicillin G, 60 µg/ml
103
streptomycin, and 0.15 µg/ml amphotericin B (all three drugs from Sigma-Aldrich).
104
Additionally, 13.6 µg of hypoxanthine (MP Biomedicals, LLC., Santa Ana, CA, USA)
105
per ml was added to T. equi culture as a vital supplement. TES-hemisodium salt (229
106
mg/ml) N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (Sigma-Aldrich) was
107
added to bovine Babesia parasite cultures as a pH stabilizer (pH 7.2) (16). In vitro
108
cultures were incubated at 37°C in an atmosphere of 5% CO2, 5% O2, and 90% N2.
109
110
In vitro inhibition assay of Babesia and Theileria
111
In vitro experiments were conducted as described previously (14). Briefly, 200
112
µl of media, media with various concentrations of clofazimine based on the results of 7
113
preliminary investigations, and media with DMSO were added in triplicate in 96-well
114
plates. While clofazimine was used at 1, 2, 5, 10, and 25 µM for B. bovis and B. caballi,
115
0.05-, 0.1-, 0.5-, 1-, 2-, 5-, and 10-µM concentrations were used for B. bigemina and T.
116
equi. Then, 20 µl of parasite-infected (1% parasitemia) bovine (B. bovis and B.
117
bigemina) or equine (B. caballi and T. equi) red blood cells (RBCs) was added to the
118
respective wells. Culture plates were incubated at 37°C in an atmosphere of 5% CO2,
119
5% O2, and 90% N2. Every 24 hours for the next 96 hours, parasitemia was monitored
120
with a light microscope using Giemsa-stained thin erythrocyte smears prepared from
121
each well; then media with the indicated drug concentrations and media with DMSO
122
were replaced with fresh ones. Each experiment was repeated three times. Diminazene
123
aceturate (5) was also used for inhibitory assays at 0.001-, 0.005-, 0.01-, 0.05-, 0.1-, 1-,
124
and 2-µM concentrations for a comparative evaluation. Changes in the morphology of
125
treated Babesia and Theileria parasites were compared with the control using light
126
microscopy. The 50% inhibitory concentration (IC50) values were calculated on the third
127
day of in vitro culture by interpolation using the curve-fitting technique (16).
128
129
Viability test 8
130
After 96 hours of treatment, 6 µL of packed red blood cells from the previously
131
drug-treated cultures was added to 14 µL of parasite-free bovine or equine packed red
132
blood cells in 200 µL of a fresh growth medium without any drug. The fresh growth
133
medium was replaced every day for the next 10 days, and parasite recrudescence was
134
monitored daily, in the absence of drugs, by microscopic examination of Giemsa-stained
135
thin erythrocyte smears.
136
137
Effect of clofazimine on host erythrocytes in vitro
138
The effect of clofazimine on host erythrocytes was evaluated as described
139
previously (14). Bovine and equine erythrocytes were incubated in the presence of 0.5,
140
1, 2, 5, 10, and 25 μM of clofazimine for 3 hours at 37°C, and then erythrocytes were
141
washed three times with drug-free media and used for the cultivation of Babesia and
142
Theileria parasites for 72 hours. The untreated control cells were handled in the same
143
manner as the pretreated cells. The pattern of parasite growth in treated erythrocytes
144
was observed and compared with that of untreated control cells.
145
146
In vitro cultivation of Plasmodium falciparum and antimalarial assay 9
147
In vitro cultivation and antimalarial evaluation of chemicals using P. falciparum
148
have been described previously (17). Briefly, P. falciparum strains Kl (chloroquine
149
resistant) and FCR3 (chloroquine sensitive) were cultured in human erythrocytes in
150
RPMI medium supplemented with 10% human plasma at 37°C under 93% N2, 4% CO2,
151
and 3% O2 (17). Asynchronous parasites (2% hematocrit and 0.5 or 1% parasitemia)
152
were seeded in a 96-well microplate, and serially diluted clofazimine, at concentrations
153
from 0.105 µM to 105 µM, or diminazene aceturate, at concentrations from 0.024 µM
154
to 12.5 µM was added. Chloroquine (K1: 0.375 µM–1.5 µM, FCR3: 0.0125 µM–0.05
155
µM) and artesunate (0.976 nM–0.125 µM) were added in a similar fashion. After 72
156
hours of incubation, parasite lactate dehydrogenase was assayed using a slight
157
modification of the procedure reported by Makler et al. (18) and Vivas et al. (19). The
158
50% inhibitory concentration (IC50) value was estimated from a dose-response curve.
159
This study was approved by the Research Ethics Committee of Kitasato Institute
160
Hospital (No12102).
161
162
163
In vivo inhibitory effect of clofazimine on B. microti As the findings from in vitro experiments were promising, clofazimine was 10
164
evaluated for its in vivo efficacy against B. microti in mice (16). Four groups, each
165
consisting of 5 female BALB/c mice (8 weeks old), were injected with 1 × 107 B.
166
microti-infected RBCs intraperitoneally. Mice in groups 1–3 were treated with
167
clofazimine or diminazene aceturate from days 1–5 post-infection. The first and second
168
groups were treated with 20 mg/kg of clofazimine by intraperitoneal and oral routes,
169
respectively. The third group was treated with 25 mg/kg of diminazene aceturate
170
subcutaneously, and the fourth group remained untreated. Each mouse was monitored
171
for parasitemia until B. microti was undetectable by microscopy. All animal experiments
172
were approved by the Animal Welfare Committee (Approval No. 27-65) and conducted
173
in accordance with the standards for the care and management of experimental animals
174
stipulated by Obihiro University of Agriculture and Veterinary Medicine, Hokkaido,
175
Japan.
176
177
Effect of clofazimine on the development of anemia in treated mice
178
To examine possible side effects of clofazimine, the development of anemia was
179
monitored in mice treated with clofazimine by measuring hematocrit values as an index.
180
Oral treatment with 20 mg/kg of clofazimine was selected for monitoring anemia in 11
181
treated mice. Twenty-five female BALB/c mice (8 weeks old) were divided into 5
182
groups (5 mice per group). Groups I–II were not infected, and groups III–V were
183
intraperitoneally infected with 1 x 107B. microti-infected RBCs. Parasitemia was
184
monitored in groups III-V using the Giemsa-staining method every 48 hours. In all
185
groups, the hematocrit was monitored every 96 hours using the Celltac α MEK-6450
186
automatic hematology analyzer (Nihon Kohden Corporation, Tokyo, Japan). Groups II
187
and IV received oral administration of 20 mg/kg clofazimine for the 5 days from days
188
3–7 post-infection. Group V received subcutaneous administration of 25 mg/kg of
189
diminazene aceturate, while groups I and III received oral administration of 0.2 ml of
190
PBS containing DMSO. The experiments were repeated twice.
191
192
Effect of clofazimine on the infectivity of treated parasites
193
To examine the infectivity of clofazimine-treated parasites, mice in groups III–V
194
were sacrificed on day 40 post-infection, and equal numbers of RBCs (1× 108) from
195
these mice were intraperitoneally transferred (reinfected) to normal mice (each group
196
consisting of five 8-week-old female BALB/c mice). Parasitemia was monitored by
197
microscopic examination of the Giemsa-stained blood smears every 2 days until day 24 12
198
post-blood transfusion.
199
200
PCR
201
PCR was undertaken to detect parasite DNA in blood and tissues (heart, spleen,
202
liver, and kidneys) on day 40 post-infection. DNA was extracted from the blood of B.
203
microti-infected groups treated with clofazimine and diminazene aceturate and the
204
non-treated control group using a QIAamp DNA Blood Mini Kit (QIAGEN, Tokyo,
205
Japan). DNA was also extracted from tissues of these mice using the NucleoSpin®
206
Tissue Kit (MACHEREY-NAGEL, Düren, Germany). PCR targeting the B. microti
207
small subunit rRNA (ss-rRNA) gene was carried out as described previously (20) using
208
nested
209
(5'-CTTAGTATAAGCTTTTATACAGC-3')/outer
210
(5'-ATAGGTCAGAAACTTGAATGATACA-3')
211
(5'-GTTATAGTTTATTTGATGTTCGTTT-3')/
212
(5'-AAGCCATGCGATTCGCTAAT-3'). The expected size of the PCR product was 154
213
bp.
PCR
primer
sets:
214 13
outer
forward reverse
and inner
inner
Babl Bab4
forward reverse
Bab2 Bab3
215
Statistical analysis
216
Differences in parasitemia between the control and treated parasites were
217
analyzed by an independent Student’s t test using JMP statistical software (SAS Institute
218
Inc., Cary, NC, USA), and P values < 0.05 were considered statistically significant.
219
220
RESULTS
221
The in vitro growth inhibitory effect of clofazimine
222
The effect of clofazimine was examined using one Theileria and three Babesia
223
assays. Clofazimine significantly inhibited (P < 0.05) the growth of B. bovis, B.
224
bigemina, B. caballi, and T. equi at 2, 0.1, 5, and 0.1 µM, respectively (Fig. 1). The in
225
vitro growth of the four parasites was also significantly inhibited (P < 0.05) by 0.005-
226
µM diminazene aceturate treatment, except that day 1 produced significant impact only
227
with 0.1 µM (data not shown). Lower IC50 values of clofazimine were observed for B.
228
bigemina (3 µM) and T. equi (0.29 µM) than for B. bovis (4.5 µM) and B. caballi (4.3
229
µM) (Table 1). In particular, clofazimine had a lower IC50 value for T. equi than did
230
diminazene aceturate (0.65 µM).
14
231
Clofazimine completely cleared B. bovis and B. caballi at 25 µM by days 4 and
232
3, respectively, while B. bigemina and T. equi were eliminated at a 10-µM concentration
233
on days 4 and 2, respectively (Fig. 1). In addition, B. bovis, B. bigemina, B. caballi, and
234
T. equi treated with 25, 5, 5, and 0.05 µM of clofazimine, respectively, failed to grow in
235
drug-free media in the viability test. The complete suppression of diminazene aceturate-
236
treated parasites was observed at a concentration of 2 µM, while a concentration of 0.05
237
µM was required to suppress the growth of B. caballi (data not shown). There was no
238
regrowth of the diminazene aceturate-treated parasites in the subsequent viability test at
239
concentrations of 0.025 µM (B. caballi) and 0.5 µM (B. bovis, B. bigemina, and T. equi)
240
(data not shown).
241
As clofazimine is reported to have very weak antimalarial activity in vitro (12,
242
21), we also evaluated the impact of both clofazimine and diminazene aceturate against
243
malaria parasites. Clofazimine exhibited poor antimalarial activity against K1 and FCR3
244
strains, and 50% growth inhibition was not achieved even at the maximum
245
concentration tested. Diminazene aceturate inhibited the growth of P. falciparum more
246
strongly than did clofazimine, with IC50 values of 0.046 µM and 0.250 µM for the K1
247
and FCR3 strains, respectively (Table 1). As compared to current antimalarial drugs, 15
248
artesunate displayed IC50 values of 0.01 µM for K1 and 0.009 µM for FCR3 strains,
249
while chloroquine had IC50 values of 0.536 µM for K1 and 0.048 µM for FCR3 strains.
250
251
The morphological and functional effect of clofazimine on parasites and
252
erythrocytes in vitro
253
The morphological changes of parasites and erythrocytes were also examined
254
in treated cultures. Parasites in clofazimine-treated cultures appeared degenerated with
255
the loss of typical parasitic shapes of B. bovis (Fig. 2B), B. bigemina (Fig. 2D), B.
256
caballi (Fig. 3B), and T. equi (Fig. 3D). Clofazimine did not cause morphological
257
changes in normal bovine and equine erythrocytes. Furthermore, when erythrocytes
258
were pretreated with clofazimine at 0.5-, 1-, 2-, 5-, 10-, and 25-µM concentrations for 3
259
hours at 37°C and then used to culture the parasites for 72 h, no differences in the
260
morphology of erythrocytes or the growth of parasites were observed as compared to
261
non-treated erythrocytes (data not shown).
262
263
264
In vivo inhibition in mice Based on the growth inhibitory effect of clofazimine against Babesia and 16
265
Theileria in vitro, the chemotherapeutic effect was examined for B. microti infection in
266
mice, since that parasite is listed in Japan’s Animal Infectious Diseases Control Law. B.
267
microti replication was significantly inhibited by clofazimine and diminazene aceturate,
268
as compared to the untreated infected mice (Fig. 4). Mice treated with 20 mg/kg
269
clofazimine through oral and intraperitoneal routes showed peak parasitemias of 5.3 and
270
8.8%, respectively, indicating inhibitions of 88.5 and 80.8%, respectively, as compared
271
to the control group (45.9% parasitemia). Notably, the parasitemia in mice that received
272
clofazimine orally was comparable to that in diminazene aceturate-treated mice (5.3%)
273
(Fig. 4).
274
275
Effect of clofazimine on the development of anemia in uninfected and infected mice
276
To examine the possible side effects of clofazimine, hematocrit changes were
277
monitored as an index for the development of anemia in mice orally administered with
278
20 mg/kg clofazimine for 5 days. Clofazimine-treated uninfected mice (group II) did not
279
show significantly different (P > 0.01) hematocrit values as compared with the
280
DMSO-treated uninfected mice (group I) (Fig. 5A). In addition, there were no apparent
281
clinical signs in clofazimine-treated mice, both uninfected and infected. For mice 17
282
infected with B. microti, as shown in the previous experiment (Fig. 4), infected mice
283
treated with 20 mg/kg clofazimine indicated 1.6% peak parasitemia, showing 96.5%
284
inhibition as compared to untreated control group III (45.1% parasitemia) on day 8 (Fig.
285
5B). Significant hematocrit reductions were observed in infected DMSO-treated mice
286
(group III) on days 8–28 after infection as compared to uninfected DMSO-treated mice
287
(group I) (Fig. 5C). While infected clofazimine-treated mice (group IV) showed
288
significantly lower hematocrits (P < 0.01) at days 8 and 12 as compared to the control
289
(group I), the hematocrit reduction level was smaller than that of infected
290
DMSO-treated mice (group III). In addition, infected clofazimine-treated mice quickly
291
recovered from low hematocrit values, as did infected diminazene aceturate-treated
292
mice (Fig. 5C).
293
294
Effect of clofazimine on the infectivity of treated parasites
295
Finally, using PCR and blood transfusions, we examined whether clofazimine
296
completely killed B. microti. Although the parasites were not found in blood smears of
297
clofazimine-treated mice on day 40 after infection, the B. microti ss-rRNA gene was
298
detectable, not only in blood but also in the heart, spleen, kidneys, and liver of these 18
299
mice, as seen in DMSO-treated infected control mice (Fig. 6A, III and IV). PCR
300
amplification also detected B. microti DNA in the heart and spleen, but not in the blood,
301
of diminazene aceturate-treated mice (Fig. 6A, V).
302
The blood from clofazimine- and diminazene aceturate-treated groups was
303
transfused to new mice to determine the infectivity of the treated parasites. B. microti
304
was regrown in recipient mice transfused with blood from both control (untreated) and
305
clofazimine-treated groups, although there was some delay in peak parasitemia in mice
306
transfused with blood from the clofazimine-treated mice. In contrast, the diminazene
307
aceturate-treated parasites did not regrow in mice (Fig. 7).
308
309
DISCUSSION
310
In the present study, clofazimine was shown to inhibit the in vitro growth of
311
three Babesia species and T. equi. As the solvent had no effect on the growth of the
312
parasites, the inhibition was considered to be solely due to clofazimine. Clofazimine
313
also induced severe degenerative changes in the treated parasites as compared to the
314
control (Figs. 2 and 3). After withdrawing the drugs, the growth of parasites could not
315
be observed in the in vitro cultures (Fig. 1). Taken together, the inhibition caused by 19
316
clofazimine at these concentrations was irreversible. The IC50 values of clofazimine to
317
Babesia and Theileria parasites reported in the present study were lower than those for
318
P. falciparum (12, 21, Table 1). In addition, clofazimine was more effective against
319
Babesia and Theileria species than against Leishmania donovani (22). Toxic effects of
320
clofazimine on mammalian cells have been reported. Clofazimine at 42.25 µM (20
321
µg/ml) induced hemolysis of murine RBCs, and the toxic effects were evident when
322
murine macrophages were treated with 10.56 µM (23). On the other hand, clofazimine
323
had no toxic effect on cultured neuronal cells at 211.24 µM (100 µg/ml) (24). In the
324
present study, bovine and equine RBCs were not affected, morphologically or
325
functionally, by pretreatment with clofazimine in vitro. Previous studies have shown
326
that peak concentrations of clofazimine in serum reached 1.48–2.11 µM (0.7–1 µg/ml)
327
and 6.34–8.45 µM (3–4 µg/ml) after the oral administration of 200 and 600 mg,
328
respectively (8, 25). All of the IC50 values recorded in the present study, therefore, were
329
well within the therapeutic range of serum concentration, suggesting that clofazimine
330
may be a potential drug candidate for chemotherapy against piroplasmosis.
331
The low IC50 values of clofazimine in the in vitro inhibition assay encouraged us
332
to further evaluate its in vivo effects on B. microti in a mouse model. Clofazimine 20
333
treatment effectively inhibited the growth of B. microti in mice, especially when it was
334
administered by the oral route, as seen with the subcutaneous administration of
335
diminazene aceturate (Fig. 4). Significant hematocrit reductions were observed in B.
336
microti–infected, DMSO-treated control mice. In a recent study (26), B. microti resulted
337
in low hematocrit values, similar to those obtained in the present study, in 6-week-old
338
BALB/c mice infected with only 1 × 104 parasitized RBCs. Although the different ages
339
of the mice could explain this discrepancy, further studies are necessary to confirm this
340
assumption. Nevertheless, clofazimine, similar to diminazene aceturate, prevented
341
anemia development in the infected mice, although transient reductions of hematocrit
342
were occasionally observed (Fig. 5C). Furthermore, clofazimine treatment had no toxic
343
side effects and did not induce anemia in uninfected mice (Fig. 5A). Clofazimine has
344
also been successfully administered at a daily dose of 200–300 mg for more than 30
345
months for the treatment of drug-resistant tuberculosis in humans (27), indicating the
346
safety of clofazimine for animal and human treatment.
347
Diminazene aceturate is the most widely used antibabesial agent in the field of
348
veterinary medicine. However, diminazene aceturate does not completely eliminate
349
parasites from hosts, which can then lead to a relapse of disease in treated animals and 21
350
produce side effects, including tissue damage at the injection site, restlessness, and colic
351
(5). In the present study, B. microti was not observed on blood smears prepared from
352
clofazimine-treated mice on day 40 post-infection. However, parasite DNA was
353
detected in the blood and other organs by PCR, and the transfusion of blood from
354
clofazimine-treated mice resulted in parasite growth in recipient mice (Figs. 6 and 7),
355
indicating that the drug countered the B. microti infection but the parasites could appear
356
again. Therefore, clofazimine may need to be accompanied by another drug to augment
357
its effect and prevent the regrowth of parasites. Since clofazimine is typically used in
358
combination with other drugs as a multidrug therapy for the treatment of leprosy and
359
tuberculosis, clofazimine efficacy might be improved by combining it with other drugs
360
such as (-)-epigallocatechin-3-gallate (28) or allicin (29), both of which are reported to
361
inhibit Babesia and Theileria in vitro and in vivo. Furthermore, as an example of drug
362
repurposing (30), the oral administration of clofazimine, especially with its lower
363
toxicity as compared with that of diminazene aceturate, might be an alternative
364
treatment for animal babesiosis.
365
Clofazimine might also be an alternative chemotherapeutic agent against human
366
babesiosis caused by B. microti. Several combination therapies, such as azithromycin 22
367
with atovaquone or clindamycin with quinine, are currently used for the treatment of
368
human babesiosis. However, drug-resistant parasites and therapeutic failures have been
369
reported in some severe cases (31), making the development of new drugs and
370
treatments for human babesiosis an urgent priority. In the present study, we have shown
371
the high efficacy of clofazimine against B. microti infection in mice (Fig. 5). Since
372
clofazimine is being used for chemotherapy of leprosy in humans, it has potential for
373
the treatment of B. microti in humans as an alternative to currently used drugs.
374
In conclusion, the present study has demonstrated the in vitro and in vivo
375
inhibitory effect of clofazimine against Babesia and Theileria parasites, suggesting that
376
clofazimine is a potential drug for the treatment of clinical disease caused by Babesia
377
and Theileria in animals and humans. These findings warrant further investigation to
378
evaluate the possible use of this chemical, alone or in combination with other drugs, for
379
animal piroplasmosis and human babesiosis.
380
381
ACKNOWLEDGMENT
382
This study was supported by Cooperative Research Grants (23-joint-3, 24-joint-6,
383
25-joint-17) of the National Research Center for Protozoan Diseases, Obihiro 23
384
University of Agriculture and Veterinary Medicine.
385
386
387
REFERENCES 1.
Parasitology 129:S247–S269.
388
389
2.
Homer MJ, Aguilar-Delfin I, Telford SR, Krause PJ, Persing DH. 2000. Babesiosis. Clin Microbiol Rev 13:451–469.
390
391
Bock R, Jackson L, de Vos A, Jorgensen W. 2004. Babesiosis of cattle.
3.
Bishop R, Musoke A, Morzaria S, Gardner M, Nene V. 2004. Theileria:
392
Intracellular protozoan parasites of wild and domestic ruminants transmitted
393
by ixodid ticks. Parasitology 129:S271–283.
394
4.
piroplasmosis. J Vet Intern Med 27:1334–1346.
395
396
Wise LN, Kappmeyer LS, Mealey RH, Knowles DP. 2013. Review of equine
5.
Mosqueda J, Olvera-Ramirez A, Aguilar-Tipacamu G, Canto GJ. 2012.
397
Current advances in detection and treatment of babesiosis. Curr Med Chem
398
19:1504–1518.
399
400
6.
Vial HJ, Gorenflot A. 2006. Chemotherapy against babesiosis. Vet Parasitol 138:147–160. 24
401
7.
Van Rensburg CE, Van Staden AM, Anderson R. 1993. The riminophenazine
402
agents clofazimine and B669 inhibit the proliferation of cancer cell lines in vitro
403
by phospholipase A2 mediated oxidative and nonoxidative mechanisms. Cancer
404
Res 53:318–323.
405
8.
36:3–7.
406
407
Barry VC, Conalty V. 1965. The antimycobacterial activity of B663. Lepr Rev
9.
Reddy VM, Nadadhur G, Daneluzzi D, O’Sullivan JF, Gangadharam PJ.
408
1996. Antituberculosis activities of clofazimine and its new analogs B4154 and
409
B4157. Antimicrob Agents Chemother 40:633–636.
410
411
10. Bygum A, Toft-Petersen M. 2008. Melkersson-Rosenthal syndrome treated with clofazimine. Ugeskr Laeger 170:159.
412
11. Evans AT, Croft SL, Peters W, Neal RA. 1989. Antileishmanial effects of
413
clofazimine and other antimycobacterial agents. Ann Trop Med Parasitol
414
83:447–454.
415
12. Mahmoudi N, de Julián-Ortiz JV, Ciceron L, Gálvez J, Mazier D, Danis M,
416
Derouin F, Garcıá-Domenech R. 2006. Identification of new antimalarial drugs
417
by linear discriminant analysis and topological virtual screening. J Antimicrob 25
418
Chemother 57:489–497.
419
13. Otoguro K, Ishiyama A, Iwatsuki M, Namatame M, Nishihara-Tukashima
420
A, Nakashima T, Shibahara S, Kondo S, Yamada H, Ōmura S. 2010. In
421
vitro and in vivo anti-Trypanosoma brucei activities of phenazinomycin and
422
related compounds. J Antibiot 63:579–581.
423
14. AbouLaila M, Munkhjargal T, Sivakumar T, Ueno A, Nakano Y, Yokoyama
424
M, Yoshinari T, Nagano D, Katayama K, El-Bahy N, Yokoyama N, Igarashi I.
425
2012. Apicoplast-targeting antibacterials inhibit the growth of Babesia parasites.
426
Antimicrob Agents Chemother 56:3196–3206.
427
15. Igarashi I, Njonge FK, Kaneko Y, Nakamura Y. 1998. Babesia bigemina: In
428
vitro and in vivo effects of curdlan sulfate on growth of parasites. Exp Parasitol
429
90:290–293.
430
16. AbouLaila M, Davaasuren B, Salama A, Ichikawa M, Terkawi MA,
431
Munkhjargal T, Yokoyama N, Igarashi, I. 2014. Evaluation of the inhibitory
432
effects of miltefosine on the growth of Babesia and Theileria parasites. Vet
433
Parasitol 204:104–110.
26
434
17. Otoguro K, Kohana A, Manabe C, Ishiyama A, Ui H, Shiomi K, Yamada H,
435
Ōmura S. 2001. Potent antimalarial activities of polyether antibiotic, X-206. J
436
Antibiot 54:658–663.
437
18. Makler MT, Ries JM, Williams JA, Bancroft JE, Piper RC, Gibbins BL,
438
Hinrichs DJ. 1993. Parasite lactate dehydrogenase as an assay for Plasmodium
439
falciparum drug sensitivity. Am J Trop Med Hyg 48:739–741.
440
19. Vivas L, Easton A, Kendrick H, Cameron A, Lavandera JL, Barros D, de las
441
Heras FG, Brady RL, Croft SL. 2005. Plasmodium falciparum: Stage specific
442
effects of a selective inhibitor of lactate dehydrogenase. Exp Parasitol
443
111:105–114.
444
20. Persing DH, Mathiesen D, Marshall WF, Telford SR, Spielman A, Thomford
445
JW, Conrad PA. 1992. Detection of Babesia microti by polymerase chain
446
reaction. J Clin Microbiol 30:2097–2103.
447
21. Makgatho ME, Anderson R, O’Sullivan JF, Egan TJ, Freese JA, Cornelius N,
448
van Rensburg CEJ. 2000. Tetramethylpiperidine-substituted phenazines as novel
449
anti-plasmodial agents. Drug Dev Res 50:195–202.
450
22. Datta G, Bera T. 2000. The effects of clofazimine, niclosamide and amphotericin 27
451
B, on electron transport of Leishmania donovani promastigotes. Indian J Med Res
452
112:15–20.
453
23. Mehta RT. 1996. Liposome encapsulation of clofazimine reduces toxicity in vitro
454
and in vivo and improves therapeutic efficacy in the beige mouse model of
455
disseminated Mycobacterium avium–M. intracellulare complex infection.
456
Antimicrob Agents Chemother 40:1893–1902.
457
24. Endoh M, Kunishita T, Tabira T. 1999. No effect of anti-leprosy drugs in the
458
prevention of Alzheimer's disease and beta-amyloid neurotoxicity. J Neurol Sci
459
165:28–30.
460
461
462
463
25. Yawalkar SJ, Vischer W. 1979. Lamprene (clofazimine) in leprosy. Basic information. Lepr Rev 50:135–144. 26. Sasaki M, Fujii Y, Iwamoto M, Ikadai H. 2013. Effect of sex steroids on Babesia microti infection in mice. Am J Trop Med Hyg 88:367−375.
464
27. Mitnick CD, Shin SS, Seung KJ, Rich ML, Atwood SS, Furin JJ, Fitzmaurice
465
GM, Alcantara Viru FA, Appleton SC, Bayona JN, Bonilla CA, Chalco K,
466
Choi S, Franke MF, Fraser HS, Guerra D, Hurtado RM, Jazayeri D, Joseph K,
467
Llaro K, Mestanza L, Mukherjee JS, Muñoz M, Palacios E, Sanchez E, 28
468
Sloutsky A, Becerra MC. 2008. Comprehensive treatment of extensively
469
drug-resistant tuberculosis. N Engl J Med 359:563–574.
470
28. AbouLaila M, Yokoyama N, Igarashi I. 2010. Inhibitory effects of
471
(-)-epigallocatechin-3-gallate from green tea on the growth of Babesia parasites.
472
Parasitology 137:785–791.
473
29. Salama AA, AbouLaila M, Terkawi MA, Mousa A, El-Sify A, Allaam M,
474
Zaghawa A, Yokoyama N, Igarashi I. 2014. Inhibitory effect of allicin on the
475
growth of Babesia and Theileria equi parasites. Parasitol Res 113:275–283.
476
30. Andrews KT, Fisher G, Skinner-Adams TS. 2014. Drug repurposing and
477
human parasitic protozoan diseases. Int J Parasitol Drugs Drug Resist 4:95–111.
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31. Vannier E, Krause PJ. 2012. Human babesiosis. N Engl J Med 366:2397–2407.
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FIGURE LEGENDS
481
Fig. 1. In vitro inhibitory effects of clofazimine on B. bovis (A), B. bigemina (B), B.
482
caballi (C), and T. equi (D). Each value represents the mean ± standard deviation of
483
three separate experiments carried out in triplicate. Asterisks indicate a significant
484
difference (P < 0.05) between the clofazimine-treated and control cultures. Parasite 29
485
viability was indicated as viable (+) and dead (-) in subcultures without clofazimine
486
after 10 days.
487
488
Fig. 2. Light micrographs of clofazimine-treated B. bovis and B. bigemina on day 3 of
489
in vitro cultivation. In vitro cultures of B. bovis treated with 0.75% DMSO (A) or 10
490
µM clofazimine and B. bigemina treated with 0.3% DMSO (C) or 5 µM clofazimine
491
(D) were observed under a light microscope. Drug-treated cultures showed higher
492
numbers of degenerated parasites than did the control cultures. Bars = 10 μm.
493
494
Fig. 3. Light micrographs of clofazimine-treated B. caballi and T. equi on day 3 of in
495
vitro cultivation. In vitro cultures of B. caballi treated with 0.75% DMSO (A) or 10 µM
496
clofazimine and T. equi treated with 0.3% DMSO (C) or 5 µM clofazimine (D) were
497
observed under a light microscope. Drug-treated cultures showed higher numbers of
498
degenerated parasites than did the control cultures. Bars = 10 μm.
499
500
Fig. 4. Inhibitory effects of 20 mg/kg of clofazimine (orally = Oral, intraperitoneally =
501
IP) and 25 mg/kg of diminazene aceturate (subcutaneously = SC) on the growth of B. 30
502
microti in BALB/c mice. Each value represents the mean ± standard deviation of the
503
experiment carried out twice. The double-headed arrow indicates the time of the drug
504
administration. Asterisks indicate a significant difference (P < 0.05) between the
505
clofazimine-treated and DMSO control groups.
506
507
Fig. 5. Changes of hematocrit values in mice treated with clofazimine. (A) Hematocrit
508
changes in uninfected groups treated orally with DMSO and 20 mg/kg of clofazimine
509
(CF). (B) Infectious course of B. microti in mice treated orally with 20 mg/kg of
510
clofazimine (CF), subcutaneously with 25 mg/kg of diminazene aceturate (DA), and
511
with DMSO. (C) Hematocrit changes in B. microti-infected mice treated with CF, DA,
512
and DMSO as well as uninfected-treated with DMSO control. The figure is
513
representative of an experiment that was conducted twice. Asterisks indicate a
514
significant difference (P ≤ 0.01) between the experimental and control groups.
515
516
Fig. 6. Polymerase chain reaction of the ss-rRNA gene in different organs of mice
517
infected with B. microti after DMSO (group III), oral 20-mg/kg clofazimine (group IV),
518
and subcutaneous 25-mg/kg diminazene aceturate (group V) treatment on day 40 31
519
post-infection. B: blood, H: heart, S: spleen, K: kidney, L: liver, NC: negative control.
520
M indicates a 100-bp DNA ladder.
521
522
Fig. 7. Growth of B. microti after transfusion of 1 × 108 RBCs into normal mice from
523
DMSO control (group III), clofazimine-treated (group IV), and diminazene
524
aceturate-treated (group V) animals.
525
526
32
Table 1. IC50 values of clofazimine and diminazene aceturate for Babesia, Theileria, and Plasmodium parasites IC50 (µM)a
Parasites
a
Clofazimine
Diminazene aceturate
B. bovis
4.5 ± 0.3
0.36 ± 0.02
B. bigemina
3 ± 0.2
0.18 ± 0.007
B. caballi
4.3 ± 0.4
0.01 ± 0.001
T. equi
0.29 ± 0.03
0.65 ± 0.03
P. falciparum
> 105b
0.046 ± 0.014, 0.25 ± 0.06c
IC50 values were calculated based on the parasitemia dynamics of treated and untreated parasites in three separate experiments, each conducted in triplicate. b Value for both K1 (chloroquine-resistant) and FCR3 (chloroquine-susceptible) strains. c Values for the K1 (chloroquine-resistant) and FCR3 (chloroquine-susceptible) strains, respectively.
Parasitemia (%)
10 8 6
+ + +
*
*
*
+
4
*
2 0 0
1
2
3
Control DMSO 0.05 M 0.1 M 1 M 2 M 5 M 10 M
4 3
*
2
*
+
1
-
0
*
0
4
1
Days of culture
4
* *
2
*+ + + -
*
0 0
1
2
Days of culture
Fig. 1
3
4
Control DMSO 0.05 M 0.1 M 0.5 M 1 M 2 M 5 M 10 M
6
+ +
Parasitemia (%)
6
7
Viability
Parasitemia (%)
Control DMSO 0.5 M 1 M 2 M 5 M 10 M 25 M
2 Days of culture
3
4
T. equi
B. caballi
8
+ + + * + + +
5 4
+ + * *
3 2
*
*
--
1 0 0
1
-
2
Days of culture
3
4
Viability
Control DMSO 1 M 2 M 5 M 10 M 25 M
B. bigemina
5
Viability Parasitemia (%)
12
Viability
BV sig. at 0.05 by t test at 2um B. bovis
Fig. 2
Fig. 3
60
Parasitemia (%)
DMSO
50
Clofazimine 20 mg/kg Oral
40
Clofazimine 20 mg/kg IP Diminazene aceturate 25 mg/kg SC
30 20 10
*
** * * * **
*
0 0
2
4
6
8
10
12
14
16
Days post-inoculation Fig. 4
18
20
22
Hematocrit %
(A) 60.0 50.0 40.0 30.0 20.0 10.0 0.0
I (DMSO) uninfected
D0
Parasitemia %
(B)
Hematocrit %
D12
60.0
D16
D20
D24
D28
D32
D36
50.0
III (DMSO)
40.0
IV (20mg/kg CF)
30.0
V (25 mg/kg DA)
D40
20.0 10.0 0.0 D4
D6
D8 D10 D12 D14 D16 D18 D20 D22 D24 D26 D28 D30 D32 D34 D36 D38 D40
Days of post-infection 60.0 50.0 40.0 30.0 20.0 10.0 0.0
** *
** *
D8
D12
*
*
D16
D20
*
I (DMSO) uninfected IV (20mg/kg CF) infected
D0
Fig. 5
D8
Days of experimental period
D2
(C)
D4
II (20mg/kg CF) uninfected
D4
* III (DMSO) infected V (25 mg/kg DA) infected
D24
Days of post-infection
D28
D32
D36
D40
M NC B H
1000 bp 500 bp
100 bp
Fig. 6
III S K
L
B
H
IV S K
V L
B
H S
M K
L
25
III (DMSO) IV (20 mg/kg Clofazimine)
Parasitemia (%)
20
V (25 mg/kg Diminazene aceturate) 15
10
5
0
D3
D6
D9
D13
D16
D19
Days of post-blood transfusion Fig. 7
D22
D25
D28
