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.

Chalcones identify cTXNPx as a potential antileishmanial drug target.

With current drug treatments failing due to toxicity, low efficacy and resistance; leishmaniasis is a major global health challenge that desperately needs new validated drug targets. Inspired by activity of the natural chalcone 2',6'-dihydroxy-4'-methoxychalcone (DMC), the nitro-analogue, 3-nitro-2',4',6'- trimethoxychalcone (NAT22, 1c) was identified as potent broad spectrum antileishmanial drug lead. Structural modification provided an alkyne containing chemical probe that labelled a protein within the parasite that was confirmed as cytosolic tryparedoxin peroxidase (cTXNPx). Crucially, labelling is observed in both promastigote and intramacrophage amastigote life forms, with no evidence of host macrophage toxicity. Incubation of the chalcone in the parasite leads to ROS accumulation and parasite death. Deletion of cTXNPx, by CRISPR-Cas9, dramatically impacts upon the parasite phenotype and reduces the antileishmanial activity of the chalcone analogue. Molecular docking studies with a homology model of in-silico cTXNPx suggest that the chalcone is able to bind in the putative active site hindering access to the crucial cysteine residue. Collectively, this work identifies cTXNPx as an important target for antileishmanial chalcones.

Single-dose treatment for cutaneous leishmaniasis with an easily synthesized chalcone entrapped in polymeric microparticles.

Cutaneous leishmaniasis (CL) is a major health problem in many countries and its current treatment involves multiple parenteral injections with toxic drugs and requires intensive health services. Previously, the efficacy of a single subcutaneous injection with a slow-release formulation consisting of poly(lactide-co-glycolide) (PLGA) microparticles loaded with an antileishmanial 3-nitro-2-hydroxy-4,6-dimethoxychalcone (CH8) was demonstrated in mice model. In the search for more easily synthesized active chalcone derivatives, and improved microparticle loading, CH8 analogues were synthesized and tested for antileishmanial activity in vitro and in vivo. The 3-nitro-2',4',6'-trimethoxychalcone (NAT22) analogue was chosen for its higher selectivity against intracellular amastigotes (selectivity index = 1489, as compared with 317 for CH8) and more efficient synthesis (89% yield, as compared with 18% for CH8). NAT22 was loaded into PLGA / polyvinylpyrrolidone (PVP) polymeric blend microspheres (NAT22-PLGAk) with average diameter of 1.9 µm. Although NAT22-PLGAk showed similar activity to free NAT22 in killing intracellular parasites in vitro (IC50 ~ 0.2 µm), in vivo studies in Leishmania amazonensis - infected mice demonstrated the significant superior efficacy of NAT22-PLGAk to reduce the parasite load. A single intralesional injection with NAT22-PLGAk was more effective than eight injections with free NAT22. Together, these results show that NAT22-PLGAk is a promising alternative for single-dose localized treatment of CL.

Improved drug loading via spray drying of a chalcone implant for local treatment of cutaneous leishmaniasis.

Current chemotherapy of cutaneous leishmaniasis (CL), even the mildest forms, encompasses multiple and painful injections with toxic drugs that cause systemic adverse effects. Recently, we showed the promising use of poly(lactic-co-glycolic acid) (PLGA) microparticles loaded with an antileishmanial nitrosylated chalcone (CH8) for effective, safe, local, and single-dose treatment of CL. Here, we proposed to optimize the delivery system by increasing the CH8 loading in PLGA-microparticles using spray drying instead of emulsification-solvent evaporation. The effect of solvent composition and polymeric matrix changes on thermal properties, loading efficiency, particle size, morphology, and spatial drug distribution of the CH8-loaded microparticles was evaluated. The results showed that spray drying allowed a higher CH8 content (18% w/w), as contrasting with the previous solvent evaporation technique that maximally incorporated 7.8% of CH8. In vitro studies on 96-hour incubation with L. amazonensis-infected macrophages showed that entrapment in spray-dried PLGA microparticles rendered CH8 safer, preserved its antileishmanial activity, and did not affect its antioxidant properties.

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.