Intravenous liposomal benznidazole as trypanocidal agent: increasing drug delivery to liver is not enough.

With the aim of investigating if delivery of benznidazole (BNZ) to liver could be increased by incorporating the drug in multilamellar liposomes, single bolus of free BNZ or liposomal BNZ formulations (MLV-BNZ) composed of HSPC:DSPG:Chol 2:1:2 (mol/mol/mol) at 0.7% (w/w) drug/total lipid ratio, were injected by intramuscular (i.m.), subcutaneous (s.c.) and intravenous (i.v.) routes, at 0.2 mg BNZ/kg, in rats. The resulting blood concentrations were followed along 9 h post-injection (p.i.) and drug accumulation in liver was determined after 4 and 9 h p.i. Only upon i.v. injection of MLV-BNZ, a threefold higher BNZ accumulation in liver was obtained, together with blood BNZ concentrations of 1.1 microg/ml (30% lower than the blood BNZ concentration achieved upon i.v. administration of free drug) occurred 4 h p.i. However, such increased liver uptake of BNZ, raised twice a week had no effect on parasitaemia levels of mice infected with the RA strain of Trypanosoma cruzi. Our results indicate that the relationship between increased selectivity for an infected tissue and therapeutic effect is not always straightforward, at least for the MLV-BNZ regimen used in the present study.

Liposomal benznidazole: a high-performance liquid chromatographic determination for biodistribution studies.

In this work, an isocratic high-performance liquid chromatographic method for quantitation of liposomal benznidazole (BNZ) in biological tissues is presented. The method comprises protein precipitation together with an efficient extraction of bulk or liposomal BNZ with acetonitrile-dimethylsulfoxide (1:1, v/v) at a 2:1 (extraction solvent-tissue matrix, v/v or /vw) ratio; the process is completed by a final precipitation with trichloroacetic acid. The resultant supernatants are assayed chromatographically using a Kromasil C18 (25- x 0.4-cm i.d., 100 A, 5- microm particle size), with an isocratic mobil phase consisting of acetonitrile-water (40:60, v/v), a flow rate of 0.9 mL/min, and detected at 324 nm. Bulk BNZ is used as a reference standard for the analysis of samples containing liposomal BNZ. The assay is linear over a concentration range of 0.75 (the lowest quantity of analyte determined with precision and accuracy of >or= 20%) to 25 microg/mL-g in all liquid and solid matrices. Within-day precision is better than 6.4% in plasma and 8.6% in liver, the same for the two assayed concentrations. Between-day precision is 5.4% and 12.3% in plasma and 9% and 6.9% in liver for the two assayed concentrations, respectively. The absolute recoveries range between 70% and 97%. Therefore, the method is accurate and precise to be employed for detection of minor quantities of liposomal BNZ in biological tissues.

Development and in vitro characterisation of a benznidazole liposomal formulation.

The purpose of this study was to find a multilamellar liposomal formulation for the antichagasic drug Benznidazole (BNZ). Different lipid matrices and organic solvents for BNZ were tested in order to obtain the liposomes with the highest g BNZ/100 g total lipid (D/TL) ratio. The best lipid matrices resulted from hydrogenated phosphatidylcholine from soybean (HSPC): Cholesterol (Chol): distearoyl-phosphatidylglycerol (DSPG) (molar ratio 2:2:1) prepared with BNZ dissolved in DMSO. Drug loading of 2 g BNZ/100 g total lipids at a total lipid concentration of 20-30 mM was obtained. Two in vitro assays on the HSPC:Chol:DSPG formulation to predict its in vivo behaviour were performed. In the first experiments, after 60 min at 1-450-fold dilution in buffer at 37 degrees C, the amount of drug associated to liposomes was reduced from 2 to 0.25 g BNZ/100 g total lipids at a rate of 65% (drug lost) min(-1) at the first minute followed by 0.4% (drug lost) min(-1) during the next hour. When incubated in plasma at 37 degrees C, the HSPC:Chol:DSPG formulations bounded a high amount of plasma proteins: r=2400 microg plasma protein per micromol total lipid.