No abstract

A promising strategy to improve the therapeutic efficiency of antimicrobial agents is targeted therapy. Although vancomycin has been considered a gold standard for the therapy of MRSA pneumonia, clinical failure rates have also been reported owing to its slow, time-dependent bactericidal activity, variable lung tissue penetration and poor intracellular penetration into macrophages. Liposomal encapsulation has been established as an alternative for antimicrobial delivery to infected tissue macrophages and offers enhanced pharmacodynamics, pharmacokinetics and decreased toxicity compared to standard preparations. The aim of the present work is to prepare vancomycin in two different liposomal formulations, conventional and PEGylated liposomes using different methods. The prepared formulations were optimized for their particle size, encapsulation efficiency and physical stability. The dehydration-rehydration was found to be the best preparation method. Both the conventional and PEGylated liposomal formulations were successfully formulated with a narrow particle size and size distribution and % encapsulation efficiency of 9 ± 2 and 12 ± 3, respectively. Both the formulations were stable at 4°C for 3 months. These formulations were successfully used to evaluate for their intracellular killing of MRSA and in vivo pharmacokinetic and bio-distribution studies.

Pneumonia due to methicillin-resistant

A rapid high-performance liquid chromatography (HPLC) method for the quantification of vancomycin in mouse plasma samples was developed and validated. Norvancomycin was used as the internal standard. Chromatographic separation was achieved on a Vydac C18 column (4.6×50 mm, 3 µm particle size) and the detection was made at 214 nm. A gradient elution was programmed with the mobile phases of 0.1% v/v trifluoroacetic acid (A) and 95:5 v/v acetonitrile: 0.1% TFA (B) and a flow rate of 1 ml/min. The total run time was 15 min. The calibration curve was linear over the range of 0.1-20 µg/ml, with a correlation coefficient (r) higher than 0.997 and the lower limit of quantitation (LLOQ) of 0.1 µg /ml. The intra-day accuracy values were between 90 and 112% and the inter-day ones ranged from 96 to 104%. Precision values ranged from 1.7 to 9.5% for intra-day and 6.3 to 9.4% for inter-day. Stability studies indicated that the mouse plasma samples containing vancomycin could be stored in freezer at -80°C and handled under normal laboratory conditions without significant loss of the drug. The assay was successfully applied to the pharmacokinetics and bio-distribution study of novel formulations of vancomycin in mice.The method was validated according to the International Conference on Harmonization (ICH) guidelines [15] for validation of analytical procedures with respect to specificity, linearity, lower limit of detection (LLOD), LLOQ, accuracy and precision, recovery and stability. The LLOD was selected at a signal/noise (S/N) ratio of 5. The LLOQ was selected at an S/N ratio of 10.Linearity: Linearity of the method was evaluated by a calibration curve in the range of 0.1 to 20 µg/ml of vancomycin. The calibration curve was obtained by least-squares linear regression analysis.Precision and accuracy: Calibration curves, with triplicates of QCs at each concentration level (0.2, 1 and 20 µg/ml) were performed in three consecutive days to determine intra-day and inter-day precision and accuracy. The intra-day variation was determined by evaluating triplicate measurements of low, medium and high QC samples on one single day whereas the inter-day variation was assayed for triplicate measurements of each QC sample for three consecutive days. The percent co-efficient of variation (% CV) of the regressed (measured) concentrations was used to report precision. Precision for all concentrations was accepted if the % CV fell within ± 15%. The accuracy was determined by comparing the calculated concentration from the standard curves to the theoretical concentration. The limits for the accuracy values were set as the range of 85-115%. Intra-day vancomycin in 1 ml of water (1 mg/ml). The vancomycin stock solution was diluted with water to working solutions ranging from 1-100 µg/ml. Norvancomycin stock solution was also prepared by dissolving 1 mg of the drug in 1 ml of water. Internal standard working solution (100 µg/ml) was prepared by diluting the stock solution with water. All samples were freshly prepared.