Effects of Plasmodium falciparum—infected erythrocytes on matrix metalloproteinase-9 regulation in human microvascular endothelial cells

Objective:To investigate the regulation of matrix metalloproteinases(MMPs) and tissue inhibitors of melalloproleinases(TIMPs) in human microvascular endothelium(HMEC-1) exposed to erythrocytes infected by different strains of Plasmodium falciparum(P.falciparum).Methods: HMEC—1 eells were co—incubated for 72 h with erythrocytes infected by late stage trophozoite of D10(chloroquine-sensilive) or W 2(chloroquine-resistant) P.falciparum strains.Cell supernatants were then collected and the levels of pro- or active gelatinases MMP-9 and MMP-2 were evaluated by gelatin zymograpln and densitometry.The release of pro-MMP-9,MMP-3.MMP-1 and TIMP-1 proteins was analyzed by western blotting and densitometry.Results:Infected erythrocytes induced de novo proMMP-9 and MMP-9 release.Neither basal levels of proMMP-2 were altered,nor active MMP-2 was found.MMP-3 and MMP-1 secretion was significant!) enhanced,whereas basal TIMP-1 was unaffected.All effects were similar for both strains. Conclusions:P.falciparum parasites,either chloroquine-sonsitive or -resistant,induce the release of active MMP-9 protein from human microvascular endothelium,by impairing balances between proMMP-9 and its inhibitor,and by enhancing the levels of its activators.This work provides new evidence on MMP involvement in malaria,pointing at MMP-9 as a possible target in adjuvant therapy.

Azacarbazole n-3 and n-6 polyunsaturated fatty acids ethyl esters nanoemulsion with enhanced efficacy against Plasmodium falciparum

Alternative therapies are necessary for the treatment of malaria due to emerging drug resistance.However,many promising antimalarial compounds have poor water solubility and suffer from the lack of suitable delivery systems,which seriously limits their activity.To address this problem,we synthesized a series of azacarbazoles that were evaluated for antimalarial activity against D10(chloroquine-sensitive)and W2(chloroquine-resistant)strains of P.falciparum.The most active compound,9H-3-azacarbazole(3),was encapsulated in a novel o/w nanoemulsion consisting of ethyl esters of polyunsaturated fatty acids n-3 and n-6 obtained from flax oil as the oil phase,Smix(Tween 80 and Transcutol HP)and water.This formulation was further analyzed using transmission electron microscopy,dynamic light scattering and in vitro and in vivo studies.It was shown that droplets of the 3-loaded nanosystem were spherical,with satisfactory stability,without cytotoxicity towards fibroblasts and intestinal cell lines at concentrations corresponding to twice the IC50 for P.falciparum.Moreover,the nanoemulsion with this type of oil phase was internalized by Caco-2 cells.Additionally,pharmacokinetics demonstrated rapid absorption of compound 3(tmax=5.0 min)after intragastric administration of 3-encapsulated nanoemulsion at a dose of 0.02 mg/kg in mice,with penetration of compound 3 to deep compartments.The 3-encapsulated nanoemulsion was found to be 2.8 and 4.2 times more effective in inhibiting the D10 and W2 strains of the parasite,respectively,compared to non-encapsulated 3.Our findings support a role for novel o/w nanoemulsions as delivery vehicles for antimalarial drugs.