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Abstract
There is a great need for orally active drugs for the treatment of the neglected tropical disease leishmaniasis. Amphiphilic Sb(V) complexes, such as 1:3 Sb–N-octanoyl-N-methylglucamide complex (SbL8), are promising drug candidates. It has been previously reported that SbL8 forms kinetically stabilized nanoassemblies in water and that this simple dispersion exhibits antileishmanial activity when given by oral route to a murine model of visceral leishmaniasis. The main objective of the present work was to interfere in the structural organization of these nanoassemblies so as to investigate their influence on the oral bioavailability of Sb, and ultimately, optimize an oral formulation of SbL8 for the treatment of cutaneous leishmaniasis. The structural organization of SbL8 nanoassemblies was manipulated through addition of propylene glycol (PG) to the aqueous dispersion of SbL8. The presence of 50% (v/v) PG resulted in the loss of hydrophobic microenvironment, as evidenced by fluorescence probing. However, nanostructures were still present, as demonstrated by dynamic light scattering, small-angle X-ray scattering, and atomic force microscopy (AFM). A remarkable property of these nanoassemblies, as revealed by AFM analysis, is the flexibility of their supramolecular organization, which showed changes as a function of the solvent and substrate polarities. The formulation of SbL8 in 1:1 water:PG given orally to mice promoted significantly higher and more sustained serum levels of Sb, when compared to SbL8 in water. The new formulation, when given as repeated doses (200 mg Sb/kg/day) to BALB/c mice infected with Leishmania amazonensis, was significantly more effective in reducing the lesion parasite burden, compared to SbL8 in water, and even, the conventional drug Glucantime® given intraperitoneally at the same dose. In conclusion, this work introduces a new concept of polarity-sensitive nanocarrier that was successfully applied to optimize an oral formulation of Sb(V) for treating cutaneous leishmaniasis.
Supplementary materials
Figure S1 Time course of fluorescence intensity of DPH probe in 1:1 W:PG after addition of EPC liposomes (at 50 seconds).
Abbreviations: DPH, 1,6-diphenylhexatriene; EPC, egg yolk phosphatidylcholine; W:PG, water:propylene glycol; au, arbitrary units.
Figure S2 Particle size analysis of SbL8 dispersion in water by the NTA technique.
Notes: Particle concentration vs diameter. The red bars indicate standard error.
Abbreviations: SbL8, 1:3 Sb–N-octanoyl-N-methylglucamide complex; NTA, Nanoparticle Tracking Analysis.
Figure S3 AFM image of SbL8 in water applied onto cleaved mica and analysis.
Notes: The dimensions of the nanoparticles, shown in the top image, support the formation of bicelles rather than micelles and the aggregation of them. Magnification in the bottom image: scan size 200×130 nm.
Abbreviations: AFM, atomic force microscopy; SbL8, 1:3 Sb–N-octanoyl-N-methylglucamide complex.
Figure S4 CD spectra of SbL8 and L8 in water or 1:1 W:PG.
Notes: Temperature =25°C and L8 concentration of 7.5 mM. KSb(OH)6 salt used in SbL8 synthesis is not chiral. Therefore, only the free ligand (L8) and SbL8 contribute to the CD spectra.
Abbreviations: CD, circular dichroism; SbL8, 1:3 Sb–N-octanoyl-N-methylglucamide complex; L8, N-octanoyl-N-methylglucamide; W:PG, water:propylene glycol.
Figure S5 Effect of the presence of 50% PG in Glucantime® on its antileishmanial efficacy, given by oral route, in a murine model of cutaneous leishmaniasis.
Notes: The graph shows the parasite load in the lesion of BALB/c mice infected with Leishmania amazonensis following oral treatment with Glucantime® (200 mg Sb/kg/day for 30 days) dissolved either in water or 1:1 W:PG, in comparison with nontreated control. Parasite load was determined by qPCR, using DNA polymerase primers specific to Leishmania spp.
Abbreviations: PG, propylene glycol; W:PG, water:propylene glycol; qPCR, quantitative polymerase chain reaction; NT, nontreated control; Glu, Glucantime®.
Figure S6 Effect of the presence of PG in SbL8 oral formulation on its antileishmanial efficacy in a murine model of visceral leishmaniasis.
Notes: The graph shows the parasite load in the liver of BALB/c mice infected with Leishmania infantum following treatment with SbL8 (150 mg Sb/kg/day for 30 days) dissolved in water or 1:1 W:PG, in comparison with saline control and positive control treated with intraperitoneal Glucantime® (Glu, 80 mg Sb/kg/day for 30 days). Parasite load was determined by limiting dilution assay. **P<0.01, ***P<0.001, and ****P<0.0001, in comparison with saline; one-way ANOVA and Tukey’s multiple comparison test.
Abbreviations: PG, propylene glycol; SbL8, 1:3 Sb–N-octanoyl-N-methylglucamide complex; W:PG, water:propylene glycol; ANOVA, analysis of variance; Glu, Glucantime®.
Acknowledgments
The authors would like to specially thank Diógenes de Sousa Neto, Mateus Borba Cardoso, Nayara Kesia Lima Mendes Moura, and Larissa Procópio Carvalho for technical support. This work was supported by the following Brazilian agencies: Conselho Nacional de Desenvolvimento Científico e Tecnológico (303227/2013-3, 472468/2013-8), Fundação de Amparo a Pesquisa do Estado de Minas Gerais (RED-00007-14, APQ-01373-14 PRONEX, APQ-01542-13), Coordenação de Aperfeicoamento de Pessoal de Nível Superior (studentship), and the LNLS – Brazilian Synchrotron Light Laboratory/MCT (SAXS1-14258).
Author contributions
FRF, JSL, LAMF, CD, and MNM conceived and designed the study. JSL carried out the experiments. FRF, JSL, and FF carried out the SAXS measurements. RM-P contributed to the analysis and interpretation of SAXS curves. JDC-J carried out TEM analysis. JMCV carried out AFM analysis. JMCV and MSA contributed to the analysis of AFM images. FF and JSL drafted the manuscript. All authors have contributed to writing and the critical revision of the manuscript during all stages of submission. All authors read and approved the final manuscript.
Disclosure
The authors report no conflicts of interest in this work.