Blood-stage antiplasmodial activity and oocyst formation-blockage of metallo copper-cinchonine complex

Abstract

In the fight against malaria, the key is early treatment with antimalarial chemotherapy, such as artemisinin-based combination treatments (ACTs). However, Plasmodium has acquired multidrug resistance, including the emergence of P. falciparum strains with resistance to ACT. The development of novel antimalarial molecules, that are capable of interfering in the asexual and sexual blood stages, is important to slow down the transmission in endemic areas. In this work, we studied the ability of the mettalo copper-cinchonine complex to interfere in the sexual and asexual stages of Plasmodium. The tested compound in the in vitro assay was a cinchonine derivative, named CinCu (Bis[Cinchoninium Tetrachlorocuprate(II)]trihydrate). Its biological functions were assessed by antiplasmodial activity in vitro against chloroquine-resistant P. falciparum W2 strain. The mice model of P. berghei ANKA infection was used to analyze the antimalarial activity of CinCu and chloroquine and their acute toxicity. The oocyst formation-blocking assay was performed by experimental infection of Anopheles aquasalis with P. vivax infected blood, which was treated with different concentrations of CinCu, cinchonine, and primaquine. We found that CinCu was able to suppress as high as 81.58% of parasitemia in vitro, being considered a molecule with high antiplasmodial activity and low toxicity. The in vivo analysis showed that CinCu suppressed parasitemia at 34% up to 87.19%, being a partially active molecule against the blood-stage forms of P. berghei ANKA, without inducing severe clinical signs in the treated groups. The transmission-blocking assay revealed that both cinchonine and primaquine were able to reduce the infection intensity of P. vivax in A. aquasalis, leading to a decrease in the number of oocysts recovered from the mosquitoes' midgut. Regarding the effect of CinCu, the copper-complex was not able to induce inhibition of P. vivax infection; however, it was able to induce an important reduction in the intensity of oocyst formation by about 2.4 times. It is plausible that the metallo-compound also be able to interfere with the differentiation of parasite stages and/or ookinete-secreted chitinase into the peritrophic matrix of mosquitoes, promoting a reduction in the number of oocysts formed. Taken together, the results suggest that this compound is promising as a prototype for the development of new antimalarial drugs. Furthermore, our study can draw a new pathway for repositioning already-known antimalarial drugs by editing their chemical structure to improve the antimalarial activity against the asexual and sexual stages of the parasite.

Keywords: Plasmodium; antimalarial drugs; cinchonine; malaria treatment; mettalo copper complexes.

Figure 1

Chemical structures of CuCl₄-tetrahedra in Bis[Cinchoninium Tetrachlorocuprate(II)]trihydrate (CinCu). Projection of the unit cell along the c-axis at 100 K. The cages contain single molecules [(cinH₂) ²⁺ (CuCl₄)^2-]₂ ·3H₂O marked as 1-4 with two geometrically inequivalent (CuCl₄) ^2- anions marked by subscripts 1 and 2. The cages 1-2 and 3-4 form two-dimensional planes parallel to the ac-plane coupled by dispersive forces only. Nitrogens assigned numbers are 1 and 13; oxygen, number 12; carbons, the other numbers smaller than 23; and hydrogen, 23–44 Adapted with permission from {Inorg. Chem. 2001, 40, 18, 4526–4533 Publication Date:July 28, 2001 https://doi.org/10.1021/ic001402a}. Copyright {2001} American Chemical Society.

Figure 2

In vitro cytotoxicity analysis of CinCu and chloroquine in RAW264.7 cell line. CQ: chloroquine; CinCu: Copper-coordinated cinchonine. Results are presented as mean ± SD of three replicates. The data displayed here represents the concentrations of compounds that guarantee at least 50% cell viability (CC50). ** p<0.01; * p<0.05.

Figure 3

In vitro antiplasmodial activity of the CinCu and chloroquine against P. falciparum W2 strain. The dose-dependent curve of the parasitemia of CinCu (A) and chloroquine (B). The parasitemia suppression of CinCu (C) and chloroquine (D), respectively. Results are presented as mean ± SD of two replicates in two independent experiments. Suppression of the parasite growth was calculated in relation to control cultures with no drugs.

Figure 4

In vivo analysis of the CinCu and chloroquine effect on parasitemia suppression of P. berghei ANKA infection. The parasitemia suppression activity was evaluated on the fifth and seventh days post-infection, after treatment with 10 mg/Kg (A), 20 mg/Kg (B), 30 mg/Kg (C), and 60 mg/Kg (D) of CinCu and chloroquine. Results are presented as mean ± SD of three independent experiments. * p<0.05; **** p<0.0001.

Figure 5

The survival rate of mice infected with P. berghei ANKA and treated with CinCu or Chloroquine. The survival rates of mice treated with 10 mg/Kg (A), 20 mg/Kg (B), 30 mg/Kg (C), and 60 mg/kg (D) were determined and analyzed by the Kaplan-Meier method with Log-rank (Mantel-Cox) test. (E) Body weight of mice before (day 0) and after (day 14) treatment with CinCu or CQ at 300 mg/Kg in the acute toxicity assay. Nt: non-treated group (control); CinCu: mice treated with CinCu; CQ: mice treated with chloroquine; DPI: days post-infection. **p<0.01; *p<0.05.

Figure 6

Oocyst infection intensity of P. vivax in A. aquasalis after feeding on blood treated with cinchonine, primaquine, and CinCu. (A) Representative image of P. vivax oocysts in the midgut of A. aquasalis; the slide was stained with 2% commercial Mercurochrome (Merbromin); scale bar: 250 µm. (B) Number of oocysts per midgut of A. aquasalis after blood meal. PQ 1: primaquine 1 µg/mL; PQ 10: primaquine 10 µg/mL; Cin 0.23: cinchonine 0.23 µg/mL; Cin 1: cinchonine 1 µg/mL; Cin+Cu 0.06: cinchonine in association with copper at 0.06 µg/mL; Cin+Cu 0,78: cinchonine in association with copper at 0.78 µg/mL NS: non-significant, ** p<0.01, *** p< 0.001.