Abstract:
The current study was aimed to develop stable and reproducible liposomal
formulations of Diclofenac sodium (DFS) and Diclofenac potassium (DFP)
using purified soya lecithin (PSL) and purified egg lecithin (PEL) for oral
delivery.
For the accomplishment of analysis task of DFS and DFP in In-vitro/In-vivo
evaluation as well as for entrapment studies, two simultaneous methods were
developed and validated. In one study for analysis of DFS, an isocratic system
was employed for the flow of mobile phase consisting of 10 mM sodium
dihydrogen phosphate buffer and acetonitrile in molar ratio of 67: 33 with
adjusted pH of 3.2. The stationary phase was hypersil ODS column (C18,
250×4.6 mm i.d., 5 μm) with controlled temperature of 30 ̊C. DFS in
liposomes, microcapsules and marketed drug products was determined in range
of 99.76-99.84%. FLP and TMD in microcapsules and brands formulation were
99.78 - 99.94 % and 99.80 - 99.82 %, respectively. Single step liquid-liquid
extraction procedure using combination of acetonitrile and trichloroacetic acid
(TCA) as protein precipitating agent was employed. The detection limits (at
S/N ratio 3) of quality control solutions and plasma samples were 10, 20, and
20 ng.mL-1 for DFS, FLP and TMD, respectively. The Assay was acceptable in
linear dynamic range. All other validation parameters were found in limits of
FDA and ICH method validation guidelines. The proposed method for DFS
analysis was found as sensitive, accurate and precise and applied for
dissolution studies as well as in human plasma samples for bioequivalence and
pharmacokinetics studies.
For analysis of DFP, a new, easy and consistent reversed-phase high-
performance liquid chromatographic method with diode array detection was
developed and validated for DFP and MLX (Meloxicam. The optimized mobile
phase was used in the molar ratio of 20:20:60 (v/v/v) mixture of acetonitrile,
methanol and 20 x 10-3 M potassium dihydrogen phosphate buffer (pH 3.7),
pumped at an optimized flow rate of 1.0 mL.min-1. The linearity was performed
in the concentration range of 15 ng.mL−1 to 10μg.mL-1 with r2 values of
0.9989 ± 0.13 and 0.9979 ± 0.11 (n = 6) for DP and MLX, respectively. The
assay was repeatable at concentration levels of 10 ng.mL-1, 1 μg.mL-1 and 10
μg.mL-1 with coefficient of variation of 0.168 - 0.603% for 10 ng.mL-1 (DP),
15 ng.mL-1 (MLX) and 1 μg.mL-1 &10 μg.mL-1 for DP and MLX. The LOD
values were 0.3 and 0.5 ng.mL−1, while values of LOQ were 10 ng.mL-1 and
15 ng.mL-1, for DP and MLX. The present method was applied in advanced
drug delivery formulations (Liposomes), In-vitro and In-vivo studies.
An important part of study was development of an optimized liposomal
formulation of diclofenac sodium (DFS) of most suitable concentration of
formulating ingredients, soya lecithin (SL) and Cholesterol (CH) with
maximum entrapment efficiency. For this purpose, response surface
methodology (RSM) was used to optimize formulation variable. Cholesterol
was selected as independent variable 1 and designated as X1 while soya lecithin
was independent variable 2 designated as X2. The response was the entrapment
of drug and designated as dependent variable Y. The two formulation
ingredients were ranged with central composite rotatable design (CCD) and
quadratic model at five levels (α=1.267) was followed for blending the
liposomal formulation. It was observed that cholesterol (variable 1) may
decrease the entrapment of DFS in the order of increasing concentration while
soya lecithin (variable 2) was found to increase entrapment (dependant
variable, Y) with increasing concentration. The central composite design has
resulted in an optimized formulation (Formulation No. 9) with an optimum
concentration of cholesterol and soya lecithin (ratio of 25:75) with maximum
of entrapment of 82.56%. The study was also extended to compare different
methods employed for the preparation of liposomes using optimized
formulation by RSM. It was concluded that formulation prepared by micro-
emulsification evaporation (MEE) followed by freeze drying method showed
maximum entrapment of DFS.
A comprehensive study was conducted for development of liposomal
formulations of DFS and DFP with variable concentrations of purified soya
lecithin (PSL) and purified egg lecithin (PEL) employing micro-emulsification
evaporation method (MEE) followed by freeze drying. The prepared liposomes
were free flowing and of uniform particle size distribution in the rage of 1.01 ±
0.011 to 1.80 ± 0.008 μm for DFS liposomal formulations while the mean size
of (mean ± SEM) 1.94 ± 0.008 μm for diclofenac potassium (DFP). The
selected liposomal formulations of DFS and DFP were also characterized by
using scanning electron microscopic studies (SEM), differential scanning
calorimetry (DSC), x-ray diffractometry (XRD) and fourier transform infra-red
spectroscopy (FT-IR). Drug entrapment efficiency was above 82%. The
entrapping efficiency and in-vitro release of DFS and DFP of all liposomal
formulations were determined by reversed phase high-performance liquid
chromatography (RP-HPLC). Different kinetics models of in-vitro were
applied and release of DFS and DFP from liposomal formulations of DFS and
DFP and it was concluded that release followed higuchi model and relatively
zero order release, calculated on the basis of r2 value of straight line fit
equation. A sustained release was observed for 16-24 hours from all range of
liposomal formulations. The selected formulations after similarity factor (f-2)
were subjected to in-vivo evaluation in eighteen healthy human subjects.
Present study results in new formulation of DFS and DFP using PSL and PSL
for oral delivery, which was found stable, reproducible and sustained release by
using modified micro-emulsification evaporation method (MEE) followed by
freeze drying which was found a probable and better to produce liposomes for
oral drug delivery system (ODDS).
Keywords: Liposomes; Phospholipids; Diclofenac sodium; Diclofenac
potassium; Validation; Response surface Methodology (RSM), micro-
encapsulation vesicle method (MEE); In-vitro Release; Kinetics Models;
Higuchi Model; In-vivo studies.