Encapsulation of the HSP-90 Chaperone Inhibitor 17-AAG in Stable Liposome Allow Increasing the Therapeutic Index as Assessed, in vitro, on Leishmania (L) amazonensis Amastigotes-Hosted in Mouse CBA Macrophages

The current long-term treatment for leishmaniasis causes severe side effects and resistance in some cases. An evaluation of the anti-leishmanial potential of an HSP90-inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), demonstrated its potent effect against Leishmania spp. in vitro and in vivo. We have previously shown that 17-AAG can kill L. (L) amazonensis promastigotes with an IC50 of 65 nM and intracellular amastigote at concentrations as low as 125 nM. As this compound presents low solubility and high toxicity in human clinical trials, we prepared an inclusion complex containing hydroxypropyl-β-cyclodextrin and 17-AAG (17-AAG:HPβCD) to improve its solubility. This complex was characterized by scanning electron microscopy, and X-ray diffraction. Liposomes-containing 17-AAG:HPβCD was prepared and evaluated for encapsulation efficiency (EE%), particle size, polydispersity index (PDI), pH, and zeta potential, before and after accelerated and long-term stability testing. An evaluation of leishmanicidal activity against promastigotes and intracellular amastigotes of L. (L) amazonensis was also performed. The characterization techniques utilized confirmed the formation of the inclusion complex, HPβCD:17-AAG, with a resulting 33-fold-enhancement in compound water solubility. Stability studies revealed that 17-AAG:HPβCD-loaded liposomes were smaller than 200 nm, with 99% EE. Stability testing detected no alterations in PDI that was 0.295, pH 7.63, and zeta potential +22.6, suggesting liposome stability, and suitability for evaluating leishmanicidal activity. Treatment of infected macrophages with 0.006 nM of 17-AAG:HPβCD or 17-AAG:HPβCD-loaded liposomes resulted in almost complete amastigote clearance inside macrophages after 48 h. This reduction is similar to the one observed in infected macrophages treated with 2 μM amphotericin B. Our results showed that nanotechnology and drug delivery systems could be used to increase the antileishmanial efficacy and potency of 17-AAG in vitro, while also resulting in reduced toxicity that indicates these formulations may represent a potential therapeutic strategy against leishmaniasis

Encapsulation of the HSP-90 Chaperone Inhibitor 17-AAG in Stable Liposome Allow Increasing the Therapeutic Index as Assessed, in vitro, on Leishmania (L) amazonensis Amastigotes-Hosted in Mouse CBA Macrophages

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2018-09-26T14:28:51Z No. of bitstreams: 1 Petersen AL Encapsulation of the HSP-90 Chaperone .....pdf: 2608327 bytes, checksum: 2db9c22cabfff8dec5cd2bfdc63df658 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2018-09-26T17:13:47Z (GMT) No. of bitstreams: 1 Petersen AL Encapsulation of the HSP-90 Chaperone .....pdf: 2608327 bytes, checksum: 2db9c22cabfff8dec5cd2bfdc63df658 (MD5)Made available in DSpace on 2018-09-26T17:13:47Z (GMT). No. of bitstreams: 1 Petersen AL Encapsulation of the HSP-90 Chaperone .....pdf: 2608327 bytes, checksum: 2db9c22cabfff8dec5cd2bfdc63df658 (MD5) Previous issue date: 2018Fundação de Amparo à Pesquisa do Estado da Bahia and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (PV http:// www.fapesb.ba.gov.br, FAPESB.CAPES-PET 039.2013), Conselho Nacional de Pesquisa e Desenvolvimento Científico (PV http://www.cnpq.br, CNPq Universal 14/2013, Programa de Excelência em Pesquisa—PROEP/CPqGM). PV holds a grant from CNPq for productivity in research (307832/2015-5).Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Laboratório de Interação Parasito-Hospedeiro e Epidemiologia. Salvador, BA, BrasilFederal University of Pernambuco. Center of Biological Sciences. Graduate Program in Biological Sciences. Recife, PE, Brazil / Center of Strategical Technologies. Laboratory of Electron Microscopy and Microanalysis.Recife, PE, BrazilFundação Oswaldo Cruz. Instituto Gonçalo Moniz. Laboratório de Interação Parasito-Hospedeiro e Epidemiologia. Salvador, BA, BrasilFundação Oswaldo Cruz. Instituto Gonçalo Moniz. Laboratório de Interação Parasito-Hospedeiro e Epidemiologia. Salvador, BA, Brasil / University of Pernambuco. Institute of Biological Sciences. Recife, PE, BrazilCenter of Strategical Technologies. Laboratory of Electron Microscopy and Microanalysis.Recife, PE, BrazilFundação Oswaldo Cruz. Instituto Gonçalo Moniz. Laboratório de Interação Parasito-Hospedeiro e Epidemiologia. Salvador, BA, BrasilUniversity of Pernambuco. Institute of Biological Sciences. Postgraduate Program in Applied Cellular and Molecular Biology. Recife, Brazil / Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Laborátorio de Enfermedades Infecciosas Transmitidas por Vetores. Salvador, BA, BrasilCenter of Strategical Technologies. Laboratory of Electron Microscopy and Microanalysis.Recife, PE, BrazilCenter of Strategical Technologies. Laboratory of Electron Microscopy and Microanalysis.Recife, PE, BrazilFundação Oswaldo Cruz. Instituto Gonçalo Moniz. Laboratório de Interação Parasito-Hospedeiro e Epidemiologia. Salvador, BA, Brasil / National Institute of Technology in Tropical Diseases-National Council for Scientific and Technological Development. Brasilia, DF, BrazilThe current long-term treatment for leishmaniasis causes severe side effects and resistance in some cases. An evaluation of the anti-leishmanial potential of an HSP90-inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), demonstrated its potent effect against Leishmania spp. in vitro and in vivo. We have previously shown that 17-AAG can kill L. (L) amazonensis promastigotes with an IC50 of 65 nM and intracellular amastigote at concentrations as low as 125 nM. As this compound presents low solubility and high toxicity in human clinical trials, we prepared an inclusion complex containing hydroxypropyl-β-cyclodextrin and 17-AAG (17-AAG:HPβCD) to improve its solubility. This complex was characterized by scanning electron microscopy, and X-ray diffraction. Liposomes-containing 17-AAG:HPβCD was prepared and evaluated for encapsulation efficiency (EE%), particle size, polydispersity index (PDI), pH, and zeta potential, before and after accelerated and long-term stability testing. An evaluation of leishmanicidal activity against promastigotes and intracellular amastigotes of L. (L) amazonensis was also performed. The characterization techniques utilized confirmed the formation of the inclusion complex, HPβCD:17-AAG, with a resulting 33-fold-enhancement in compound water solubility. Stability studies revealed that 17-AAG:HPβCD-loaded liposomes were smaller than 200 nm, with 99% EE. Stability testing detected no alterations in PDI that was 0.295, pH 7.63, and zeta potential +22.6, suggesting liposome stability, and suitability for evaluating leishmanicidal activity. Treatment of infected macrophages with 0.006 nM of 17-AAG:HPβCD or 17-AAG:HPβCD-loaded liposomes resulted in almost complete amastigote clearance inside macrophages after 48 h. This reduction is similar to the one observed in infected macrophages treated with 2 μM amphotericin B. Our results showed that nanotechnology and drug delivery systems could be used to increase the antileishmanial efficacy and potency of 17-AAG in vitro, while also resulting in reduced toxicity that indicates these formulations may represent a potential therapeutic strategy against leishmaniasis

Micro-emultocrit technique: A valuable tool for determination of critical HLB value of emulsions

The aim of this work was to develop a methodology for rapid determination of the critical hydrophilic-lipophilic balance (HLB) value of lipophilic fractions of emulsions. The emulsions were prepared by the spontaneous emulsification process with HLB value from 4.3 to 16.7. The preparations were stored at 2 different temperatures (25°C and 4°C) and their physicochemical behavior was evaluated by the micro-emultocrit technique and the long-term stability study. The experimental data show a reverse relationship between HLB values of the surfactant mixtures and emulsion stability. A close correlation between the results for both stability procedures was observed, suggesting the use of micro-emultocrit to predict stabilities of such systems. In addition, it was found that the critical HLB of the Mygliol 812 was 15.367