Formulation And Characterization Of Novel Pegylated Selfnanoemulsifying Formulations (Snefs) For Oral Delivery Of Gentamicin And Its Possible Use In The Treatment Of Pneumococcal Meningitis.

ABSTRACT

The solubility of gentamicin in various formulation vehicles (oils, surfactants and co-surfactants)

was determined using the shake flask method. Emulsifying ability of the surfactants for the

selected oil was screened. Different SNEF prototypes were developed following the construction

of ternary phase diagrams using water titration method. The effect of drug and other additives on

the ternary phase diagrams were studied and the selected formulations were optimized.

Optimized formulations were characterized by weight uniformity of their capsule shells and

visual assessment of their self nanoemulsification. The optical clarity and robustness to dilution

of the SNEFs were evaluated. Emulsification time, droplet size, zeta potential, polydispersity

index and Fourier transform-infrared spectroscopy were measured. SNEFs were also

characterized by scanning electron microscopy (SEM). Apparent viscosities, absolute drug

content and drug content efficiencies were determined. Stability studies and octanol/water

partition coefficient were evaluated, while in vitro antibacterial studies of the SNEFs and

permeation studies were carried out. In vitro anti-pneumococcal study of the SNEFs against S.

pneumoniae was done followed by extrapolation of their minimum inhibitory concentrations

(MICs). In vivo anti-pneumococcal studies of gentamicin released in the sera and CSF of white

albino rats were evaluated. Haematological studies were carried out to measure packed cell

volume (PCV), total white blood cells (WBC), red blood cells (RBC) and haemoglobin (Hb)

concentrations. Biochemical studies were performed to evaluate alanine aminotransferase (ALT),

aspartate aminotransferase (AST), alkaline phosphatase (ALP), bilirubin and creatinine

concentrations. This was followed by histopathological examinations of the brain, kidney and

liver of the animals. Results were expressed as mean ± SD. ANOVA and student t-tests were

performed on the data sets. Ternary phase plots were analyzed using SigmaPlot® 11.0.

Permeation calculations were performed with a special Microsoft excel programme. Differences

were considered significant for p values < 0.05. Gentamicin produced maximum solubility in

soyabean oil, Kolliphor® EL, Kolliphor® P188, and in Transcutol® HP. PEG 4000 and

gentamicin reduced the area of nanoemulsion formation in the ternary phase diagrams for the

selected systems. The emulsion droplet size was in the nanometer scale. The SNEF capsules had

uniform average weight of 300 mg ± 0.7. The SNEFs had good optical clarity with percentage

transmittances above 50 % and showed low propensity to drug precipitation, and exhibited rapid

emulsification rate (17 – 40 s). FTIR revealed that the structure of gentamicin remained

completely intact in all the formulations. SEM micrographs showed smooth and spherical

globules. Rheological studies showed decrease in apparent viscosities with increase in shearing

speed. Drug content ranged from 36.3 – 99.8 %. Stability studies suggest that the SNEFs were

relatively stable over 4 months. Octanol/water partition coefficient ranged from 0.38 – 1.46. In

vitro antibacterial studies showed susceptibility in the order: K. pneumonia > E. coli > S. aureus

> B. subtilis. In vitro permeation studies of showed overall extended permeation of gentamicin.

In vitro anti-pneumococcal study showed MICs of 2.5 – 5 mg/ml. In vivo anti-pneumococcal

study of gentamicin in sera suggests that sera from animals administered with batch C (3:1 w/w)

gentamicin SNEFs at 7 mg/kg produced good inhibition of the bacteria. The in vivo antipneumococcal

activity of gentamicin in CSF showed rapid establishment of a biocidal

concentration after 30 min. Haematological studies showed increase in PCV, RBC and Hb

counts while WBC count and its differentials decreased. Biochemical studies showed decreased

ALP and varying AST, ALT, bilirubin and creatinine concentrations. Histopathological findings

showed dominant astrocytosis of the brain for gentamicin-loaded SNEFs indicating a breach of

the integrity of the BBB.