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

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.

In vitro activity and in vivo efficacy of fexinidazole against New World Leishmania species.

OBJECTIVES: To evaluate the in vitro activity and in vivo efficacy of fexinidazole against the main species that cause visceral and cutaneous New World leishmaniasis. METHODS: The inhibitory concentrations of fexinidazole against Leishmania (Leishmania) infantum chagasi, Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis in amastigotes were determined by in vitro activity assays. For the in vivo evaluation, animals were infected with L. (L.) infantum chagasi, L. (L.) amazonensis, L. (V.) braziliensis or Leishmania (Viannia) guyanensis and divided into groups: (i) control; and (ii) treated with oral fexinidazole, from 50 to 300 mg/kg/day. For cutaneous leishmaniasis, the size of the lesion was determined weekly after the beginning of the treatment. Upon completion, parasites were recovered from the spleen and liver, or skin lesion and spleen, and evaluated by a limiting dilution assay. RESULTS: All Leishmania isolates were susceptible to fexinidazole in the in vitro assays. The viable parasites in the liver and spleen were reduced with 100 and 300 mg/kg/day, respectively, for L. (L.) infantum chagasi. For the species causing cutaneous leishmaniasis, the viable parasites in lesions and the size of the lesions were reduced, starting from 200 mg/kg/day. The viable parasites in the spleen were also reduced with 200 and 300 mg/kg/day for L. (V.) braziliensis and L. (L.) amazonensis. CONCLUSIONS: Considering the defined parameters, fexinidazole showed in vitro and in vivo activity against all tested species. This drug may represent an alternative treatment for the New World species.

Efficacy of Meglumine Antimoniate in a Low Polymerization State Orally Administered in a Murine Model of Visceral Leishmaniasis.

Progress toward the improvement of meglumine antimoniate (MA), commercially known as Glucantime, a highly effective but also toxic antileishmanial drug, has been hindered by the lack of knowledge and control of its chemical composition. Here, MA was manipulated chemically with the aim of achieving an orally effective drug. MA compounds were synthesized from either antimony pentachloride (MA-SbCl5) or potassium hexahydroxyantimonate [MA-KSb(OH)6] and prepared under a low polymerization state. These compounds were compared to Glucantime regarding chemical composition, permeation properties across a cellulose membrane and Caco-2 cell monolayer, and uptake by peritoneal macrophages. MA-SbCl5 and MA-KSb(OH)6 were characterized as less polymerized and more permeative 2:2 Sb-meglumine complexes than Glucantime, which consisted of a mixture of 2:3 and 3:3 Sb-meglumine complexes. The antileishmanial activities and hepatic uptake of all compounds were evaluated after oral administration in BALB/c mice infected with Leishmania infantum chagasi, as a model of visceral leishmaniasis (VL). The synthetic MA compounds given at 300 mg Sb/kg of body weight/12 h for 30 days significantly reduced spleen and liver parasite burdens, in contrast to those for Glucantime at the same dose. The greater activity of synthetic compounds could be attributed to their higher intestinal absorption and accumulation efficiency in the liver. MA-SbCl5 given orally was as efficacious as Glucantime by the parenteral route (80 mg Sb/kg/24 h intraperitoneally). These data taken together suggest that treatment with a less-polymerized form of MA by the oral route may be effective for the treatment of VL.

Combined suboptimal schedules of topical paromomycin, meglumine antimoniate and miltefosine to treat experimental infection caused by Leishmania (Viannia) braziliensis.

OBJECTIVES: This study aimed to evaluate the efficacy of binary combinations of suboptimal schedules of drugs with different administration routes (topical paromomycin, intramuscular meglumine antimoniate and oral miltefosine) to treat animals infected with Leishmania (Viannia) braziliensis. METHODS: Hamsters were inoculated with L. (V.) braziliensis and after ulceration of lesions, divided into seven groups: untreated control, paromomycin, miltefosine, meglumine antimoniate, meglumine antimoniate + paromomycin, miltefosine + paromomycin and meglumine antimoniate + miltefosine. Meglumine antimoniate and miltefosine were administered at low doses and topical paromomycin at a single daily application regimen. The animals were treated for 20 consecutive days (meglumine antimoniate and/or paromomycin) and/or 10 alternate days (miltefosine). Lesion sizes were determined weekly. Upon completion of treatment, parasites were recovered from skin lesions and spleens and evaluated by limiting dilution assay. RESULTS: The combinations of a once daily application of paromomycin with low doses of miltefosine or meglumine antimoniate yielded higher efficacies in reducing the parasite load as well as lesion size when compared with any of these drugs administered as monotherapy regimens at the same suboptimal schedules. CONCLUSIONS: Considering the parameters evaluated, the combinations of a systemic therapy with topical treatment were more effective than monotherapy with each of these drugs. These combinations may represent an alternative combination strategy for the treatment of leishmaniasis caused by L. (V.) braziliensis.

In vitro interaction between paromomycin sulphate and four drugs with leishmanicidal activity against three New World Leishmania species.

OBJECTIVES: To evaluate in vitro interactions between paromomycin sulphate and the antileishmanial drugs meglumine antimoniate, amphotericin B, miltefosine and azithromycin against intracellular Leishmania (Leishmania) infantum chagasi, Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis amastigotes in peritoneal mouse macrophages. METHODS: First, drug susceptibility was assessed in 3, 5 and 7 day assays, followed by drug interaction assays with a modified fixed-ratio method. An overall mean sum fractional inhibitory concentration (∑FIC) was calculated for each combination and each Leishmania species. The nature of the interactions was classified as synergistic if the mean ∑FIC was ≤ 0.5, indifferent if the mean ∑FIC was >0.5-4.0 and antagonistic if the mean ∑FIC was >4.0. RESULTS: In vitro synergism was observed for the combinations of paromomycin plus miltefosine [at 50% and 90% inhibitory concentrations (IC50 and IC90, respectively)] and paromomycin plus amphotericin B (at the IC90 level) against L. (L.) amazonensis, paromomycin plus meglumine antimoniate (at the IC50 and IC90 levels) and paromomycin plus amphotericin B (at the IC50 level) against L. (V.) braziliensis, and paromomycin plus miltefosine, paromomycin plus amphotericin B (both at the IC90 level) and paromomycin plus azithromycin (at the IC50 level) against L. (L) infantum chagasi. CONCLUSIONS: This work provides a preclinical dataset that supports future studies on multidrug treatment schedules against New World leishmaniasis.

Validation of quantitative real-time PCR for the in vitro assessment of antileishmanial drug activity.

In vitro assays play an important role in the discovery and development of new antileishmanial drugs. The classic macrophage-amastigote models using murine peritoneal macrophages or human-monocyte derived macrophages as host cells are useful for drug screening. A major limitation of these models is the dependence on microscopic counting, a time-consuming and subjective method of analysis. The present study describes a detailed protocol for applying quantitative real-time PCR (qPCR) as an accurate and sensitive tool to assess parasite load in an amastigote-macrophage model. This assay can be performed in a standardized medium-to-high throughput procedure, replacing traditional readout of number of amastigote per macrophages by DNA load measurement.

Antileishmanial activity of azithromycin against Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, and Leishmania (Leishmania) chagasi.

Azithromycin, an azalide antibiotic, is highly concentrated within different phagocytic cells, especially macrophages. The potential antileishmanial activity of azithromycin against three species of Leishmania from the New World was assessed using in vitro models. Azithromycin decreased viability of promastigote cultures of Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, and Leishmania (Leishmania) chagasi as determined by the colorimetric Alamar blue assay. In amastigote intracellular cultures, a significant decrease in infected macrophages counts was observed for all three species with IC(50) of 20.83 (27 micromol/L), 2.18 (2.7 micromol/L), and 6.12 (7.8 micromol/L) microg/mL, respectively. Azithromycin showed in vitro activity against L. (L.) amazonensis, L. (V.) braziliensis, and L. (L.) chagasi and may offer an alternative to current leishmaniasis treatment.