Combination of the Topical Photodynamic Therapy of Chloroaluminum Phthalocyanine Liposomes with Fexinidazole Oral Self-Emulsifying System as a New Strategy for Cutaneous Leishmaniasis Treatment

Abstract

Cutaneous leishmaniasis (CL) is a neglected tropical disease. The treatment is restricted to drugs, such as meglumine antimoniate and amphotericin B, that exhibit toxic effects, high cost, long-term treatment, and limited efficacy. The development of new alternative therapies, including the identification of effective drugs for the topical and oral treatment of CL, is of great interest. In this sense, a combination of topical photodynamic therapy (PDT) with chloroaluminum phthalocyanine liposomes (Lip-ClAlPc) and the oral administration of a self-emulsifying drug delivery system containing fexinidazole (SEDDS-FEX) emerges as a new strategy. The aim of the present study was to prepare, characterize, and evaluate the efficacy of combined therapy with Lip-ClAlPc and SEDDS-FEX in the experimental treatment of Leishmania (Leishmania) major. Lip-ClAlPc and SEDDS-FEX were prepared, and the antileishmanial efficacy study was conducted with the following groups: 1. Lip-ClAlPc (0.05 mL); 2. SEDDS-FEX (50 mg/kg/day); 3. Lip-ClAlPc (0.05 mL)+SEDDS-FEX (50 mg/kg/day) combination; 4. FEX suspension (50 mg/kg/day); and 5. control (untreated). BALB/c mice received 10 sessions of topical Lip-ClAlPc on alternate days and 20 consecutive days of SEDDS-FEX or FEX oral suspension. Therapeutical efficacy was evaluated via the parasite burden (limiting-dilution assay), lesion size (mm), healing of the lesion, and histological analyses. Lip-ClAlPc and SEDDS-FEX presented physicochemical characteristics that are compatible with the administration routes used in the treatments. Lip-ClAlPc+SEDDS-FEX led to a significant reduction in the parasitic burden in the lesion and spleen when compared to the control group (p < 0.05) and the complete healing of the lesion in 43% of animals. The Lip-ClAlPc+SEDDS-FEX combination may be promising for the treatment of CL caused by L. major.

Keywords: chloroaluminum phthalocyanine; combined therapy; cutaneous leishmaniasis; fexinidazole; liposomes; self-emulsifying drug release system.

Figure 1

Timeline course and treatment regimen. Female BALB/c mice were infected with L. (L.) major promastigotes and received 10 sessions of topical Lip-ClAlPc on alternate days and 20 consecutive days of SEDDS-FEX or FEX suspension oral in isolated or combined treatments. Therapeutical efficacy was evaluated by measuring the lesion size (0, 7, 14, and 21 days), the parasite burden, and histological analysis after euthanasia.

Figure 2

The identification of FEX crystals that are insoluble in SEDDS-FEX and FEX suspensions (undiluted samples) was carried out using polarized light microscopy analysis and the Optical Zeiss^® microscope. The analyses were carried out at 100× magnification: (A) SEDDS-Fex and (B) Fex suspension.

Figure 3

In vivo efficacy of different treatments in L. (L.) major. Female BALB/c mice were infected with L (L.) major pomastigotes in the base of the tail. The treatments used were Lip-ClAlPc, SEDDS-FEX, Lip-ClAlPc+SEDDS-FEX, FEX suspension, and control group (untreated). The Lip-ClAlPc and Lip-ClAlPc+SEDDS-FEX groups received 10 sessions of topical Lip-ClAlPc on alternate days and 20 consecutive days of SEDDS-FEX or FEX oral suspension in isolated or combined treatments. One day after the end of the treatments, the parasite burden was determined via the limiting dilution method. (A) Parasite burden in lesions. The letter “a” indicates a statistically significant difference in relation to the control group (p < 0.05). (B) Parasite burden in the spleen. The letters “a” and “b” indicate statistically significant differences in relation to the control and Lip-ClAlPc groups, respectively (p < 0.05). The bars represent the averages and standard deviations (n = 7).

Figure 4

In vivo efficacy of different treatments in L. (L.) major. Female BALB/c mice were infected with L (L.) major pomastigotes in the base of the tail. The treatments used were Lip-ClAlPc, SEDDS-FEX, Lip-ClAlPc+SEDDS-FEX, FEX suspension, and control group (untreated). The Lip-ClAlPc and Lip-ClAlPc+SEDDS-FEX groups received 10 sessions of topical Lip-ClAlPc on alternate days and 20 consecutive days of SEDDS-FEX or FEX suspension in isolated or combined treatments. Images of the macroscopic aspect of the lesions (A–J). Control group: infected and untreated animals at 0 days (A) and 21 days (B). Animal treated with Lip-ClAlPc at 0 days (C) and 21 days (D). Animal treated with SEDDS-FEX at 0 days (E) and 21 days (F). Animal treated with Lip-ClAlPc+SEDDS-FEX at 0 days (G) and 21 days (H). Animal treated with the FEX suspension at 0 days (I) and 21 days (J). (K) Monitoring of average lesion size in response to different treatments. Lesion size is shown as the average and standard error of the mean. * Indicates statistically significant difference in relation to the control group (p < 0.05) on the 21st day (n = 7).

Figure 5

Histopathological analysis (H&E) was performed in the control group (A,B), Lip-ClAlPc (C,D), SEDDS-FEX (E,F), Lip-ClAlPc+SEDDS-FEX (G,H), and FEX suspension (I,J). The first image on the right presents 4× magnification, and the second image on the left the image presents 40× magnification. Arrows indicate the regions with inflammatory processes. (K) Total score of the histopathological analysis. Data presented as median, maximum, and minimum. The groups treated with SEDDS-FEX and Lip-ClAlPc+SEDDS-FEX exhibited statistical differences when compared to the control group (* p < 0.05) (n = 7).

Figure 6

Evaluation of the weight of BALB/c mice infected with L. (L.) major and submitted to different treatments. Female BALB/c mice were infected with L (L.) major promastigotes at the base of the tail. After the development of ulcerated lesions, the animals were treated with the Lip-ClAlPc, SEDDS-FEX, Lip-ClAlPc+SEDDS-FEX, FEX suspension, and Control (untreated). Animals were weighed at baseline (0 days) and on days 7, 14, and 21 after starting treatment. The bars represent means and standard deviations. Comparisons between the weights at all times were not statistically significant (p > 0.05).