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Development of lipid nanoparticles based on solid matrix for Benznidazole oral delivery targeting to the lymphatic system to treat Chagas disease

Periodic Reporting for period 1 - LYMPHVECs (Development of lipid nanoparticles based on solid matrix for Benznidazole oral delivery targeting to the lymphatic system to treat Chagas disease)

Reporting period: 2019-03-01 to 2021-02-28

There are 7 million people chronically infected by Chagas disease resulting in 10,000 deaths reported annually according to the World Health Organization. This disease is one of the most important health problems in Latin America. However, today it has been reported all over the world. So far, there is only one drug approved by the FDA to treat this disease, Benznidazole.

Benznidazole has limited efficacy in the chronic phase of this disease. Besides, people infected with Chagas and treated with Benznidazole develop skin rashes, fever, nausea, headache, allergic dermatitis, digestive intolerance (anorexia), peripheral neuropathy, and insomnia. These serious side effects limit its use and encourage non-compliance. In addition, the reservoirs of parasites that cause the disease have been located in the lymphatic system. Therefore, the maldistribution of compounds in this system could be the reason why prolonged treatment with Benznidazole is required and why reactivation of parasites is commonly found in the chronic stage.

Thus, developing Benznidazole formulations that reduce the exposure of the compound in the circulatory system while facilitating distribution in the lymphatic system could reduce the effective dose (currently 5-7 mg/kg/day), reduce side effects and possibly help to shorten the treatment. In this project, lipid matrix-based Benznidazole formulations have been investigated and developed to achieve these three goals.
A wide range of excipients, such as solid and liquid lipids, surfactants and co-surfactants, have been evaluated by solubility studies to identify the best components to develop a Benznidazole formulation. Additionally, compatibility studies have been carried out to ensure its stability so that there is no phase separation in the formulation.

In this project Solid Lipid Nanoparticles (SLNs), Nanostructurated Lipid Carriers (NLCs) and Lipid Nanoemulsions (LNEs) has been investigated to identify the best lipid matrix (solid, liquid and/or hybrid) for Benznidazole formulation. Several nanoparticle preparation approaches, including high energy (sonication, high-amplitude ultrasonication and jet dispersers) and low energy (spontaneous emulsification and phase inversion temperature - PIT) methodologies, have been explored to identify the most suitable one for this case.

Most of the nanoparticles prepared have been physically characterized by Dynamic Light Scattering (DLS) to determine their size, polydispersity index and zeta potential. Also, Entrapment Efficiency studies has been carried out and Drug Load have been calculated . Some nanoparticles have been purified by dialysis cassettes and others also lyophilized with or without cryoprotectant. Besides, reconstitution studies have been carried out using DLS, based on the evaluation of different solvents and dilutions, of the lyophilized nanoparticles to identify the best conditions to recover their physical properties once they are in solution again. Furthermore, crystallinity studies have been performed for these lyophilized nanoparticles through Differential Scanning Calorimetry. In addition, stability studies have been develop evaluating its size and polydispersity index again in different periods of time.

To date, more than 300 nanoparticles have been developed and, specifically, the LNE prepared using the PIT method have shown most of the physical properties necessary to reach the lymphatic system. Furthermore, to identify the best ratio of excipients in the formulation, ternary phase diagrams have been obtained, showing clear and fairly homogeneous lipid nanoemulsions. In vivo tests will soon be carried out to confirm the project hypothesis.
The final results of this project will be published in high-impact journals and will be presented at conferences specialized in drug delivery systems and medical chemistry.
Lipid formulation have been successfully described as vectors to deliver hydrophobic drugs, such as Benznidazole, into the lymphatic system through different administration routes. However, their main drawback is drug loading. So far, very few lipid nanoparticles have been described and all showed a poor loading of Benznidazole due to the weak interaction of the drug with the investigated oils and/or faster initial burst release. In this project, several hitherto unexplored oils have been evaluated for the formulation of Benznidazole and 3 have been identified, which showed greater solubility of Benizdazole compared to the oils previously used. Therefore, it is expected that the main problems encountered in previous attempts to formulate Benznidazole will be overcome, as well as that the drug is distributed in both lymphatic and circulatory systems in order to kill all the parasites that cause this disease.

This project has a translational approach to take advantage of available technology that allows access to the lymphatic system, as a way to effectively reach parasites. In addition, it tries to show that this novel strategy could be the way to end this disease that mainly affects low- and middle-income countries. If the hypothesis is confirmed, this strategy could be implemented in other pathologies where viruses, bacteria or parasites are present or have reached the lymphatic system, such as tuberculosis and leishmaniasis, among others.