الفهرس 
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Abstract
The primary aim of this thesis was to improve absorption and bioavailability of orally administered drugs by the aid of vesicular systems for oral delivery. The main goal was to select model drugs that suffer from poor solubility and/or low bioavailability after oral administration. Atorvastatin, a lipid lowering agent, and lopinavir, an HIV-protease inhibitor, were carefully chosen for this work. Both drugs exhibit low aqueous solubility and poor bioavailability after oral administration. In addition, premature degradation by enterocytic and/or hepatic pre-systemic metabolism and due to P-gp efflux pump action. Several strategies were previously conducted to enhance water solubility, dissolution rate and intestinal absorption and decrease pre-systemic degradation. Some of these strategies include nanoparticles, solid dispersions, solid self-emulsifying drug, and proliposomes. Thus, the objective of this study was to fabricate and evaluate niosomal vesicular system incorporating the drug. Excipients were added to modify the vesicular membrane rigidity such as cholesterol or alter vesicular surface charge to allow more prolonged vesicular muco-adhesion with intestinal membrane such as chitosan, and addition of absorption enhancers within the vesicular bilayers such as menthol. characterization of the prepared vesicles included recording particle shape and size using transmission electron microscope. Furthermore, drug loading efficiency and entrapment efficiency were calculated. In-vitro release rate of the entrapped drug was monitored using the dialysis sac method. Mice were selected for the in-vivo evaluation of the therapeutic effect of atorvastatin chitosan coated and uncoated niosomes. Rabbit in-situ intestinal perfusion method was employed to evaluate the enhancement of intestinal absorption of lopinavir from niosomes containing Abstract . 2 Pharmaceutical Technology Department, Faculty of Pharmacy, Tanta University, Egypt menthol and the standard niosomes. The following sections summarize the studies performed to achieve this objective. 1. Chitosan-encapsulated niosomes for enhanced oral delivery of atorvastatin (a) High Pressure Liquid chromatography HPLC method was established for the analysis of atorvastatin. The method was validated for its linearity, accuracy, precision, limit of detection and limit of quantification. The drug was at a retention time of 3.54 minutes. The assay was able to quantify atorvastatin with a linearity range of 1.25 to 40 μg/ml. The lower limit of detection was calculated to be 0.00297 μg/ml with the lower limit of quantification being 0.009 μg/ml. The accuracy of the method was indicated from the percent recovery, which was in the range of 98-102% for intraday and was in the range of 99-106% for interday. The precision of the method was reflected from the computed relative standard deviation, which was in the range of 1.2 to 4.7%. (b) Morphology and size of niosomes The TEM micrographs reflected the spherical nature of the vesicles and revealed the existence of niosomes in the nano size range. The average vesicle size was calculated to be 143.2 + 60 nm and 96.9 + 21 nm for the chitosan-encapsulated and standard niosomes, respectively. The difference between the vesicle size was found to be statistically significant (P < 0.05). The recorded significant reduction in the vesicle size in case of chitosanencapsulated niosomes can be attributed to the fact that chitosan encapsulation will provide a rigid film on the surface of the vesicles. This will hinder the expected vesicle swelling which takes place upon Abstract . 3 Pharmaceutical Technology Department, Faculty of Pharmacy, Tanta University, Egypt incubation. Noteworthy, the recorded large value of standard deviation reflects the heterogeneous nature of the vesicular population, which is expected, with the method of preparation, which depends on mechanical dispersion followed by bath sonication. (c) Entrapment efficiency and drug loading The entrapment efficiency (EE) values were 57 + 1.85% and 55.5 +2.1 % for the chitosan-encapsulated and standard niosomes, respectively. There was no significant difference in the recorded EE in the chitosanencapsulated and standard vesicles. This is expected taking into consideration the fact that the coating operation is conducted after entrapment. The drug loading was calculated as 11.5 + 0.37 and 11.2 + 0.43 mg atorvastatin per gram of lipid for chitosan-encapsulated and standard niosomes, respectively. The results correlate with the entrapment efficiency data with no significant difference between coated and uncoated vesicles. The entrapment efficiency and drug loading were not significantly affected after coating lipid nanocarrier with chitosan. (d) In Vitro Release Studies The regression line fitting reflected that the release pattern of atorvastatin from niosomal formulations follows Higuchi release kinetics. This finding suggests the existence of matrix diffusion-based release, which is unexpected with such fluid formulation. The reason for such behavior despite of the fluidity of the system under test can be attributed to the multi-lamellar structure of the prepared niosomes with the lipophilic drug being entrapped within the lipid bilayer. The drug will undergo initial rapid release from the surface layer followed by diffusion-controlled release from the interior layers of the multi-lamellar structure. Comparing the release profiles of atorvastatin from chitosan-encapsulated and Abstract . 4 Pharmaceutical Technology Department, Faculty of Pharmacy, Tanta University, Egypt standard niosomes, the similarity factor test revealed no significant difference between both formulations. This can suggest the existence of the coat as very thin layer on the vesicular surface. (e) In vivo evaluation of antihyperlipidemic effect Oral delivery of atorvastatin formulations resulted in significant decrease in the total cholesterol level compared with the reference group. This effect was evident irrespective to the administered formulation. Comparing the tested formulations of atorvastatin with respect to their anti-hyperlipedemic effect, the formulations were ranked as chitosanencapsulated niosomes > unencapsulated niosomes > drug suspension. For the chitosan-encapsulated vesicles, there was statistically significant increase in the efficacy compared with the suspension form (P < 0.05). Chitosan-encapsulated niosomes were ranked even better than the corresponding standard vesicles. The assessment was extended to histopathological examination of liver samples. This was conducted to monitor the damaging effect of cholesterol on liver tissues. Oral administration of atorvastatin alone resulted in moderate improvement with the liver histology showing focal hepatic vacuolation. Administration of atorvastatin standard niosomes showed slight decrease in hepatic vacuolation. Administration of atorvastatin encapsulated in chitosan-encapsulated niosomes decreased the signs of both glycogen and fat hepatic vacuolation. Abstract . 5 Pharmaceutical Technology Department, Faculty of Pharmacy, Tanta University, Egypt 2. Lopinavir-menthol co-crystals for enhanced dissolution rate and intestinal absorption (a) characterization of the Prepared Formulations The lopinavir drug content values were determined to be 104.7% + 1.3, 105.6% + 1.2, 103.3% + 1.3 and 115.4% + 3.2 respectively for formulations containing lopinavir with menthol at molar ratios of 1:1, 1:1.5, 1:2, and 1:3. The closeness of the calculated drug content values to 100% in addition to the small SD reflects the homogeneity of the prepared mixtures. The prepared formulations were characterized using Fourier transform infra-red spectroscopy (FTIR), differential scanning calorimetry (DSC), and X-ray diffraction pattern (XRD). The FTIR spectrum of the prepared formulations showed a summation of the main peaks that were recorded for lopinavir and menthol except for the absorption band of the NH group which showed broadening and shifting to lower wave number. This suggests possible hydrogen bonding. This effect might imply possible transformation in the crystalline structure. Wet co-grinding of lopinavir with menthol resulted in significant modulation in the recorded thermal pattern regardless the molar ratio used. The thermograms showed sharp symmetric endothermic peak at 119.8oC. This sharp endotherm was recorded in all tested molar ratios of lopinavir to menthol but with different enthalpy. The enthalpy of the recorded endotherm increases with increasing menthol concentration in the system up to 1:2 molar ratio (lopinavir to menthol). Further increase in menthol reduced the enthalpy of this endotherm and was associated with the appearance of an endothermic peak at 40oC which can be attributed to excess menthol which underwent phase separation in formulation Abstract . 6 Pharmaceutical Technology Department, Faculty of Pharmacy, Tanta University, Egypt containing lopinavir with menthol at molar ratio of 1:3. This thermal behavior suggests development of new crystalline species with mixture containing lopinavir and menthol at 1:2 molar ratio being optimum for this transformation. Wet co-grinding of lopinavir with menthol at increasing molar ratios produced crystalline product with XRD pattern which is different from the sum of diffraction pattern lopinavir and menthol highlighting the development of new crystalline species. The recorded changes in the X-ray diffraction pattern supports the supposition of co-crystallization between menthol and lopinavir. To assess the impact of lopinavir wet co-processing with menthol on its dissolution rate, the dissolution studies were performed. Wet cogrinding of lopinavir and menthol resulted in significant enhancement in lopinavir dissolution rate (P < 0.01). This enhancement depended on the molar ratio of lopinavir to menthol. This enhancement in lopinavir dissolution rate in case of the formulations containing lopinavir and menthol at molar ratios of 1:1, 1:1.5, and 1:2 can be accredited to formation of a new crystalline species with weaker crystallinity. The in-situ rabbit intestinal perfusion was adopted to assess the intestinal permeability of lopinavir. Co-perfusion of lopinavir with menthol increased the intestinal absorption of lopinavir compared with the corresponding aqueous solution. The absorptive clearance normalized to segment length was increased by 1.6 and 1.3-fold after co-perfusion of lopinavir and menthol at molar ratio of 1:2 in case of duodenum and jejuoileum respectively. This increase in the absorptive clearance was reflected as an increase in the % fraction absorbed (%Fa) in addition to reduction in length required for complete drug absorption (L95%). The recorded Abstract . 7 Pharmaceutical Technology Department, Faculty of Pharmacy, Tanta University, Egypt enhancement of intestinal permeation of can be explained on the base of the intestinal membrane fluidizing effect of menthol. 3. Lopinavir-menthol niosomal delivery for enhanced oral delivery (a) Morphology and size of niosomes The transmission electron micrographs of menthol containing niosomes confirmed the spherical morphology of the vesicles. The vesicles were in the nano scale with an average size of 148.2 + 27 nm. (b) Entrapment efficiency and drug loading The EE values were 94.4 + 1.7% and 96.3 +0.47 % for the niosomes containing menthol and niosomes without menthol, respectively. There was no significant difference between the recorded EE in both vesicles (P > 0.05). Lopinavir loading was expressed as mg lopinavir per gram of the lipids. The drug loading was 33.46 + 0.37 and 34.02 + 0.12 mg lopinavir per gram of lipids in case of menthol containing niosomes and menthol free vesicles, respectively. The recorded high entrapment efficiency values are expected for the lipophilic lopinavir which is expected to be intercalated between the lipid molecules of the vesicles and a low affinity to the continuous aqueous compartment. (c) In Vitro Release Studies The release data reflected slow-release rate of lopinavir from niosomes. This was evident both in presence and absence of menthol in vesicles. The significant increase (p < 0.05) in the amount of lopinavir release from niosomes after increasing the pH from 6.8 to 7.4 is expected taking into consideration the acidic nature of lopinavir which will allow more drug to dissolve in the continuous aqueous phase at higher pH value. Interestingly, the amount of lopinavir release Abstract . 8 Pharmaceutical Technology Department, Faculty of Pharmacy, Tanta University, Egypt was significantly higher in case of menthol containing vesicles. This can be attributed to the fluidizing effect of menthol on the vesicular membrane which can augment the drug release. (d) In-situ intestinal perfusion of lopinavir Perfusion of lopinavir in a standard or menthol containing niosomes resulted in significant increase (P < 0.05) in the intestinal absorption of lopinavir compared with its absorption from simple drug solution. The magnitude of enhancement in intestinal absorption was greater in case of menthol containing niosomes. The absorption does not depend on drug release. The independence of intestinal absorption from niosomes on drug release rate was further confirmed by the lack of correlation between the drug release and fraction absorbed as indicated from Pearson correlation test. This poor correlation highlights the possibility of direct vesicular absorption.