In Vivo Safety and Efficacy of Chalcone-Loaded Microparticles with Modified Polymeric Matrix against Cutaneous Leishmaniasis.

Current chemotherapy of cutaneous leishmaniasis (CL) is based on repeated systemic or intralesional administration of drugs that often cause severe toxicity. Previously, we demonstrated the therapeutic potential of biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) loaded with 8% of the nitrochalcone CH8 (CH8/PLGA) prepared by a conventional bench method. Aiming at an industrially scalable process and increased drug loading, new MPs were prepared by spray drying: CH8/PDE with PLGA matrix and CH8/PVDE with PLGA + polyvinylpyrrolidone (PVP) matrix, both with narrower size distribution and higher drug loading (18%) than CH8/PLGA. Animal studies were conducted to evaluate their clinical feasibility. Both MP types induced transient local swelling and inflammation, peaking at 1−2 days, following a single intralesional injection. Different from CH8/PDE that released 90% of the drug in the ear tissue in 60 days, CH8/PVDE achieved that in 30 days. The therapeutic efficacy of a single intralesional injection was evaluated in BALB/c mice infected with Leishmania (Leishmania) amazonensis and golden hamsters infected with L. (Viannia) braziliensis. CH8/PVDE promoted greater reduction in parasite burden than CH8/PDE or CH8/PLGA, measured at one month and two months after the treatment. Thus, addition of PVP to PLGA MP matrix accelerates drug release in vivo and increases its therapeutic effect against CL.

Leishmanicidal activity of Piper marginatum Jacq. from Santarém-PA against Leishmania amazonensis.

Leishmaniasis is an infectious disease that has high endemicity and is among the six parasitic diseases of higher occurrence in the world. The current treatments are limited due to their toxicity, treatment resistance and high cost which have increased the search for new substances of natural origin for its therapy. Based on this, an in vitro biological and chemical investigation was carried out to evaluate the potential of Piper marginatum against Leishmania amazonesis. P. marginatum leaves were collected to obtain the essential oil (EO) and the ethanolic extract (CE). The chemical profile of the CE and fractions was obtained by 1H NMR. The analysis of the EO chemical composition was performed by GC-MS. EO, CE and fractions were submitted to antileishmanial and cytotoxicity assays against macrophages. The chromatographic profiles of EO, CE and fractions showed the presence of phenolic compounds and terpenoids, having 3,4-Methylenedioxypropiophenone as a major compound. All P. marginatum samples showed low toxicity to macrophages. The CE and the methanolic, hexane and ethyl acetate fractions had low cytotoxicity when compared to Pentamidine. All tested samples inhibited growth of L. amazonensis promastigotes. The antileishmanial activity of EO, CE and fractions were evaluated in macrophages infected with L. (L.) amazonensis and treated with the concentrations 1, 10 and 100 µg/mL for 48 h. All samples were active, but EO and CE showed superior activity against amastigote forms when compared to the promastigote forms of L. amazonensis. This work describes for the first time the antileishmanial activity of the species P. marginatum and its cytotoxicity against macrophages, suggesting that it can be an alternative source of natural products in the phytotherapeutic treatment of leishmaniasis.

Novel and safe single-dose treatment of cutaneous leishmaniasis with implantable amphotericin B-loaded microparticles.

The development of an effective amphotericin B (AmB) topical formulation to replace the systemically toxic injections currently used in cutaneous leishmaniasis (CL) treatment is challenging due to poor absorption through the skin. Aiming at an effective local chemotherapy, we designed PLGA (poly(lactide-co-glycolide acid) microparticles loaded with deoxycholate amphotericin B (d-AmB) for both macrophage intracellular targeting and sustained extracellular release. For that, d-AmB/PLGA microparticles with sizes ranging from 0.5 µm to 20 µm were synthesized and tested both in vitro and in vivo. In vitro, d-AmB/PLGA was more selectively active against intracellular amastigotes of Leishmania amazonensis than free d-AmB (selectivity index = 50 and 25, respectively). In vivo, the efficacy of a single intralesional (i.l) injection with d-AmB/PLGA was determined in early and established BALB/c mouse ear lesions. In early lesions, a single injection given on day 10 of infection was more effective in controlling parasite growth than eight i.l. injections with free d-AmB, as measured on day 120. Such d-AmB/PLGA injection was also effective in established lesions (day 30), leading to 97% parasite burden reduction, as compared with d-AmB or liposomal AmB (Ambisome®) i.l. injection containing the same AmB dose. Pharmacokinetic studies showed that following d-AmB/PLGA injection, AmB leaked slower from non-infected than infected ears, yet remaining in the ear tissue for as long as 30 days. Of interest, AmB was not detectable in the circulating plasma for at least two weeks of d-AmB/PLGA injection, contrasting with the rapid and durable (2 days) detection after free d-AmB injection. Despite the transient ear swelling and local cell infiltration, no alterations in AST, ALT and creatinine serum levels was induced by d-AmB/PLGA. For its approved components, local efficacy, and single-dose applicability, this novel and safe AmB microparticle depot formulation has strong potential as a new therapy for human CL.

Depot Subcutaneous Injection with Chalcone CH8-Loaded Poly(Lactic-Co-Glycolic Acid) Microspheres as a Single-Dose Treatment of Cutaneous Leishmaniasis.

Conventional chemotherapy of cutaneous leishmaniasis (CL) is based on multiple parenteral or intralesional injections with systemically toxic drugs. Aiming at a single-dose localized therapy, biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles loaded with 7.8% of an antileishmanial nitrochalcone named CH8 (CH8/PLGA) were constructed to promote sustained subcutaneous release. In vitro, murine macrophages avidly phagocytosed CH8/PLGA smaller than 6 µm without triggering oxidative mechanisms. Upon 48 h of incubation, both CH8 and CH8/PLGA were 40 times more toxic to intracellular Leishmania amazonensis than to macrophages. In vivo, BALB/c were given one or three subcutaneous injections in the infected ear with 1.2 mg/kg of CH8 in free or CH8/PLGA forms, whereas controls received three CH8-equivalent doses of naked PLGA microparticles or meglumine antimoniate (Glucantime; Sanofi-Aventis). Although a single injection with CH8/PLGA reduced the parasite loads by 91%, triple injections with free CH8 or CH8/PLGA caused 80 and 97% reductions, respectively, in relation to saline controls. Meglumine antimoniate treatment was the least effective (only 36% reduction) and the most toxic, as indicated by elevated alanine aminotransferase serum levels. Together, these findings show that CH8/PLGA microparticles can be effectively and safely used for single-dose treatment of CL.

Effectiveness of the local or oral delivery of the novel naphthopterocarpanquinone LQB-118 against cutaneous leishmaniasis.

OBJECTIVES: This paper describes the antileishmanial properties of LQB-118, a new compound designed by molecular hybridization, orally active in Leishmania amazonensis-infected BALB/c mice. METHODS: In vitro antileishmanial activity was determined in L. amazonensis-infected macrophages. For in vivo studies, LQB-118 was administered intralesionally (15 µg/kg/day, five times a week), intraperitoneally (4.5 mg/kg/day, five times a week) or orally (4.5 mg/kg/day, five times a week) to L. amazonensis-infected BALB/c mice throughout experiments lasting 85 or 105 days. At the end of the experiments, serum levels of alanine aminotransferase, aspartate aminotransferase and creatinine were measured as toxicological parameters. RESULTS: LQB-118 was active against intracellular amastigotes of L. amazonensis [50% inhibitory concentration (IC(50)) 1.4 µM] and significantly less so against macrophages (IC(50) 18.5 µM). LQB-118 administered intralesionally, intraperitoneally or orally was found to control both lesion and parasite growth in L. amazonensis-infected BALB/c mice, without altering serological markers of toxicity. CONCLUSIONS: These results demonstrate that the molecular hybridization of a naphthoquinone core to pterocarpan yielded a novel antileishmanial compound that was locally and orally active in an experimental cutaneous leishmaniasis model.

Antileishmanial sesquiterpenes from the Brazilian red alga Laurencia dendroidea.

Investigation of the bioactive crude extracts from two populations of the red alga Laurencia dendroidea from the southeastern Brazilian coast led to the identification of five sesquiterpenes: (+)-obtusane (1), a triquinane derivative (2), (-)-elatol (3), obtusol (4), and cartilagineol (5). An antileishmanial bioassay against Leishmania amazonensis was conducted for crude lipophilic extracts and for sesquiterpenes 2, 3, and 4. Compounds 3 and 4 displayed in vitro and in vivo leishmanicidal activity and very low cytotoxicity.