Fig 1.
Suppression of L. major upon exposure to azelastine, fexofenadine and the anti-leishmanial drugs amphotericin B and miltefosine using the ex vivo system.
Fig 2.
Anti-leishmanial efficacy (EC50) versus cytotoxicity (CC50) for determining the in vitro therapeutic index of azelastine and fexofenadine.
The EC50 was determined in the ex vivo system while the CC50 was assessed using HepG2 cells.
Table 1.
In vitro therapeutic index of chemically diverse histamine H1 receptor antagonists (H1RAs) determined in the ex vivo lymph node explant system of L. major for anti-leishmanial activity and HepG2 cells for cytotoxicity.
Fig 3.
Decrease of histamine receptor 1 (HR1) and proinflammatory IL-1ẞ expression by addition of azelastine to an ex vivo lymph node explant culture of BALB/c mice infected with Leishmania major.
Addition of histamine increased the expression of IL-1ẞ. Determinations were made using quantitative PCR. (*p = 0.05; **p = 0.01).
Fig 4.
Activity of azelastine (AZ) against L. major.
(A) promastigote numbers of L. major-LUC after 48 h of incubation at 26°C exposed to either AZ or DMSO quantified by luminometry as relative light units (RLU; ****p<0.0001, Tukey’s multiple comparison test). (B), quantification of L. major in ex vivo cultures from infected mice using Leishmania 18s gene expression after 48 h of AZ exposure at 34°C (data expressed with reference to untreated DMSO controls;. *p = 0.02, Mann-Whitney test). (C), anti-leishmanial activity of AZ in L. major-infected mouse peritoneal macrophages co-cultured with either naïve lymphocytes (black bars), Leishmania-primed lymphocytes (gray bars) or without lymphocytes (empty bars). Parasite burden was determined by luminometry (RLU) after 48 h of incubation at 34°C (*p<0.05, Tukey’s multiple comparisons test).
Fig 5.
Evaluation of azelastine efficacy in BALB/c mice infected with L. major.
Mice (n = 7) were infected intradermically (ID) with 107 metacyclic promastigotes of L. major transfected with the luciferase gene. Lesion size (area in mm2 = length x width) was measured using a digital caliper (A). Body weight change was estimated as a major sign of toxicity (B). The parasite load at the infection site (C) and draining lymph nodes (D) was determined in vivo using the IVIS spectrum imager. Animals received 3 ID injections of azelastine or vehicle (control) from day 3 to 10 p.i. Another group of mice was treated orally with miltefosine 50 mg/kg for 10 days as a positive control of parasite suppression. The figures show mean values and their standard deviation (SD). Representative data of 2 independent experiments. P value: *< 0.05; ***< 0.001. The statistical significance of the data was determined using the t test.
Fig 6.
Evaluation of fexofenadine efficacy in BALB/c mice infected with L. major.
Mice (n = 7) were infected ID with 107 metacyclic promastigotes of L. major transfected with the luciferase gene. Lesion size (area in mm2 = length x width) was measured using a digital caliper (A). Body weight change was used as a major sign of toxicity (B). The parasite load at the lesion site (C) and lymph nodes (D) was determined in vivo using the IVIS spectrum. Animals were treated PO with fexofenadine or vehicle (control) from day 3 to 10 p.i. The graphs show mean values and their standard deviation (SD). Representative data of 3 experiments. P values: *< 0.05; **< 0.01; ***< 0.001. The statistical significance of the data was determined using the t test.
Fig 7.
Representative IVIS images of mice treated for 10 days with different schedules and doses of azelastine or fexofenadine and the anti-leishmanial drug miltefosine.