Synergic in vitro combinations of artemisinin, pyrimethamine and methylene blue against Neospora caninum

Mota

Baroni

et al. 2020

Sci Rep

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Neospora caninum is an Apicomplexan parasite related to important losses in livestock, causing abortions and decreased fertility in affected cows. Several chemotherapeutic strategies have been developed for disease control; however, no commercial treatment is available. Among the candidate drugs against neosporosis, phenothiazinium dyes, offer a low cost-efficient approach to parasite control. We report the anti-parasitic effects of the phenothiaziums Methylene Blue (MB), New Methylene Blue (NMB), 1,9-Dimethyl Methylene Blue (DMMB) and Toluidine Blue O (TBO) on N. caninum, using in vitro and in vivo models. The dyes inhibited parasite proliferation at nanomolar concentrations (0.019-1.83 μM) and a synergistic effect was achieved when Methylene Blue was combined with New Methylene Blue (Combination Index = 0.84). Moreover, the phenothiazinium dyes improved parasite clearance when combined with Pyrimethamine (Pyr). Combination of Methylene Blue + 1,9-Dimethyl Methylene Blue demonstrated superior efficacy compared to Pyrimethamine based counterparts in an in vivo model of infection. We also observed that Methylene Blue, New Methylene Blue and 1,9-Dimethyl Methylene Blue increased by 5000% the reactive oxygen species (ROS) levels in N. caninum tachyzoites. Phenothiazinium dyes represent an accessible group of candidates with the potential to compound future formulations for neosporosis control. Neospora caninum, an obligate intracellular protozoan, belongs to Apicomplexa phylum, such as Plasmodium spp and Toxoplasma gondii. It causes neosporosis, a disease strongly correlated to abortion and decreased fertility in cattle, costing billions of dollars worldwide 1-3. There is no effective commercial treatment for neosporosis, despite the efforts of several chemotherapy-focused groups 4-6. For example, Artemisinins 7-10 , Miltefosine 11 , plant extracts 12-14 , Diamidines 15,16 , Buparvaquone 4,17 , organometallic ruthenium complexes 18 , Thiazolides 19,20 were evaluated in in vitro and in vivo models with encouraging results. Moreover, polyether ionophore antibiotics 21 , Triazinones 22-24 , bumped kinase inhibitors 25 have also been tested in farm ruminants 26. Based on the promising effects of Methylene Blue (MB) against Plasmodium spp, the etiologic agent of malaria, our group determined the efficacy of this molecule on N. caninum, either alone or combined with Pyrimethamine (Pyr) 27. MB, a phenothiazinium dye, was the first synthetic drug described to cure a patient affected by malaria in the XIX century, pioneering work performed by Paul Ehrlich 28,29. Indeed, MB was used against Plasmodium until the use of Chloroquine and other drugs (massively utilized after the Second World War), which lack some of the reversible MB side effects (blue urine and sclera) 30,31. During the later 20 th /early 21 st Centuries, the intense use of Chloroquine, Artemisinin, and Pyrimethamine has led to proven cases of Plasmodium resistance, demanding novel therapeutic candidates 32. Interestingly, no reports of Plasmodium resistance hav...

supporting

confidence: 59%

Inhibitory action of phenothiazinium dyes against Neospora caninum

Mota

Baroni

et al. 2020

Sci Rep

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“…Serial dilutions of compounds (starting from 10 μ M) in RPMI were added to the cultures in duplicate and incubated for 72 h, 37°C,and 5% CO 2 . For the drug combination assay, fractioned dilutions (2 × IC 50 , 1.6 × IC 50 , 1.3 × IC 50 , 0.7 × IC 50 , 0.5 × IC 50 , and 0.2 × IC 50 ) of single and combined compounds [11] were added to the infected cultures and incubated under the same conditions. We considered 8 μ M, 1 μ M, 0.05 μ M, 0.7 μ M, and 0.04 μ M as 2 × IC 50 for BZ, MB, NMB, TBO, and DMMB, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…The cultures were washed with PBS and analyzed in a BD FACS-Canto (BD Biosciences) flow cytometer with the FACSDiva (BD) 6.1.3 software. Due to the size, host cells (infected and noninfected) and trypomastigotes were separately analyzed by flow cytometry [11]. The median of fluorescence of oxidized DCF and the populations with active mitochondria were measured with excitation/emission at 488 nm/529 nm and 488 nm/529 nm, respectively.…”

Section: Methodsmentioning

confidence: 99%

Phenothiazinium Dyes Are Active againstTrypanosoma cruziIn Vitro

Portapilla

BioMed Research International

Costa

et al. 2019

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Chagas disease is a tropical illness caused by the protozoan Trypanosoma cruzi. The disease affects populations of the Americas and has been spread to other continents due to the migration process. The disease is partially controlled by two drugs, Benznidazole and Nifurtimox. These molecules are active in the acute phase of the infection but are usually ineffective during the symptomatic chronic phase. Several research groups have developed novel candidates to control Chagas disease; however, no novel commercial formulation is available. In this article, we described the anti-T. cruzi effects of phenothiazinium dyes in amastigote and trypomastigote forms of the parasite. Methylene Blue, New Methylene Blue, Toluidine Blue O, and 1,9-Dimethyl Methylene Blue inhibited the parasite proliferation at nanomolar concentrations and also demonstrated low toxicity in host cells. Moreover, combinations of phenothiazinium dyes indicated a synergic pattern against amastigotes compared to the Benznidazole counterparts. Phenothiazinium dyes levels of reactive oxygen species (ROS) and decreased the mitochondrial potential in trypomastigotes, indicating the mechanism of action of the dyes in T. cruzi. Our article offers a basis for future strategies for the control of Chagas disease using low-cost formulations, an important point for endemic underdeveloped regions.

“…Thus, the application of anti-malarial drugs indicates an interesting source for drug repurposing against N. caninum. For example, methylene blue and analogues, pyrimethamine and artemisinin formulations have been successfully tested on in vitro (Kim et al, 2002;Lindsay & Dubey, 1989;Pereira et al, 2017Pereira et al, , 2018 and in vivo (Pereira et al, 2020) models of N. caninum infection. Likewise, several novel candidates with anti-N. caninum activity were identified from the Malaria Venture (MMV) Pathogen Box, with promising results (Müller et al, 2017(Müller et al, , 2020.…”

Section: Introductionmentioning

confidence: 99%

Atovaquone, chloroquine, primaquine, quinine and tetracycline: antiproliferative effects of relevant antimalarials on Neospora caninum

Rev. Bras. Parasitol. Vet.

Luca

Abichabki

et al. 2021

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Neospora caninum is an apicomplexan parasite that causes abortion in cattle, resulting in significant economic losses. There is no commercial treatment for neosporosis, and drug repositioning is a fast strategy to test possible candidates against N. caninum. In this article, we describe the effects of atovaquone, chloroquine, quinine, primaquine and tetracycline on N. caninum proliferation. The IC50 concentrations in N. caninum were compared to the current information based on previous studies for Plasmodium and Toxoplasma gondii, correlating to the described mechanisms of action of each tested drug. The inhibitory patterns indicate similarities and differences among N. caninum, Plasmodium and T. gondii. For example, atovaquone demonstrates high antiparasitic activity in all the analyzed models, while chloroquine does not inhibit N. caninum. On the other hand, tetracycline is effective against Plasmodium and N. caninum, despite its low activity in T. gondii models. The repurposing of antimalarial drugs in N. caninum is a fast and inexpensive way to develop novel formulations using well-established compounds.