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Abstract Alzheimer’s disease (AD) is a neurodegenerative disease. Many theories were proposed for the etiology of AD including amyloid theory and acetylcholine theory. AD is characterized by amyloid-β plaques, neurofibrillary tangles, neuro-inflammation and decrease of acetylcholine in the brain. There’s no cure for AD, just symptomatic treatment that can improve the patient’s life or at least slow the progression of the disease. Those treatment options include acetylcholine esterase (AChE) inhibitors, antiinflammatory drugs and anti-oxidant drugs but the brain delivery of many molecules is still a challenge due to the blood brain barrier. Glycyrretinic acid (GA) is the aglycone of glycyrrhizin that is a component of Glycyrrhiza, also called licorice root, and one of the most common drugs used clinically. GA increases the synaptic acetylcholine that plays a great role in improving memory and learning skills, reduces the reactive oxygen species(ROS) produced due to oxidative stress in amyloid cascade and inhibits glutamate-mediated excitotoxicity which make it an excellent candidate for many nerurodegenerative diseases including AD. If taken orally, its resulting bioavailability is very low due to its high lipophilicity and high molecular weight. Thus, reticuloendothelial system (RES) can capture GA easily and moreover it gets expelled by P-glycoprotein efflux pumps in the brain capillaries. Therefore, exploiting lipid-based carriers that can deliver GA to the brain and using the nose-to-brain direct delivery as route of administration will probably be exceptionally beneficial in the field of neurodegenerative diseases. Lipid based carriers developed through this work include: lipid nanocapsules (LNCs) and microemulsions (MEs). LNCs represent smart drug Summary 134 delivery systems that consist of an oil core (in which lipophilic drugs can be dispersed) and a PEGylated surfactant shell together with lecithin. The choice of these nano-carriers was based on several merits such as being biodegradable and biocompatible, avoidance of the use of organic solvents, being stable up to one year and their ability of encapsulating hydrophilic and lipophilic drugs. Its PEGylated structure enables its bypassing the engulfment of LNCs by macrophages was feasible. MEs are nano-colloidal systems composed of oily and aqueous phases, together with surfactants and co-surfactants. MEs are isotropic, macroscopically homogenous, kinetically thermodynamically stable, translucent and with low energy input method of preparation and long-term storage. Lipophilic drugs can be integrated in the oily phase and surfactants/co-surfactants used can enhance the penetration through nasal mucosa. Thus using MEs as a delivery system can improve the efficacy of a drug allowing the total dose to be reduced and minimize the possible side effects. Hence the aim of the work in this thesis was the development of Lipidbased nano-systems capable of enhancing the delivery of GA to the brain. This was planned to be achieved by encapsulating GA in LNCs and MEs and using the intranasal route for brain delivery. Accordingly, the work in this thesis is divided into three chapters: Chapter One: Preparation and evaluation of GA loaded LNCs. Chapter Two: Preparation and evaluation of GA loaded MEs. Chapter three: In-vivo assessment of selected GA loaded LNCs and MEs. Summary 135 Chapter I: Preparation and evaluation of GA loaded LNCs In this chapter, GA loaded LNCs were successfully prepared using the phase inversion method followed by a shock introduced to the system by cold water to break the produced ME and enhance the formation of nanocapsules. LNCs was composed of Labrafac PG®, Solutol®, salted aqueous medium in addition to Epikuron®. Epikuron® acted as a stabilizer to the LNC shells, increasing the biocompatibility to the biological membranes. The favorable stealth properties of LNCs and its prolonged circulation were imparted by the PEGylated surfactant; Solutol®. Labrafac® PG represented the oily phase. The salted aqueous medium enhanced the phase inversion temperature of Solutol® to be easily achieved. LNCs were optimized using the D-optimal mixture design. The percentages of the three independent variables; Labrafac® PG, Solutol®, aqueous phase concentrations were varied to generate mathematical models for the two responses: particle size (PS) and polydispersity index (PDI). Validation of the model was performed utilizing 4 different points and the % Bias was calculated. The model was evaluated according to its significance using ANOVA, its R-squared value, adjusted R-squared, Predicted R-squared and adequate precision. Moreover, the actual runs were compared to the predicted ones. Effect of aging on selected GA loaded LNCs was studied after storing the formulae at 40 C for 3 months. PH of the selected formulae was measured. The selected GA loaded LNCs were visualized using transmission electron microscopy (HR-TEM). Drug release profile over 24 h was obtained for the Summary 136 selected GA-LNCs compared to GA solution. An ex-vivo permeation study was performed on the selected GA-LNCs compared to GA suspension. from the obtained results, it was found that: 1) All the prepared GA loaded LNCs scored sizes between 20 - 100 nm which was significantly affected by the ratio of Solutol® to oil. Increasing this ratio greatly resulted in the reduction of particle size (PS) owing to its effect on the interfacial tension of the oily droplets of nanocapsules. 2) The entire prepared GA loaded LNCs were characterized by a uniform particle size distribution (PDI) less than 0.499. This could be attributed to the insertion of Solutol® at the water–oil interface enhancing the incorporation of GA in the oily core and reducing the interfacial tension of oily droplets. These results also reflect the high efficiency of the adopted method; phase-inversion method in producing the lipid nanocapsules. 3) Based on the D-optimal mixture design results: a. The suggested model for PS response was a special cubic one while that for PDI was a quadratic counterpart. b. Analysis of the models was performed using ANOVA where the generated models were extremely significant at P<0.05. In case of PS, the P-value was less than 0.0001, while the P-value for PDI was 0.0007. Summary 137 c. High correlation was indicated between the actual and predicted runs for the two investigated models. This was explicated in the graphical presentation of the actual versus the predicted runs. This was in good agreement with the R2 values which were 0.9987 and 0.9515 corresponding to PS and PDI, respectively. d. Adequate precision measuring the signal to noise ratio greater than 4 was desirable. The ratio was found to be 78.744 and 13.624 for the two models; PS and PDI, respectively, indicating high adequacy of the model. e. Experimental validation of the model and its feasibility for navigation through the model was investigated by comparing other 4 actual runs vs their predicted values to calculate the % Bias for the two responses. The two responses scored less than 7 % confirming the model validation and its sufficiency to navigate the experimental spaces. 4) After storage for 3 months at 40 C, it was found that GA loaded LNCs showed slight non-significant changes in PS indicating the stability of the selected formulae. 5) pH values of the selected formulae were compatible with nasal mucosa non-irritant pH range. 6) TEM micrographs confirmed that all the particles were spherical in shape possessing smooth surface with no obvious particle aggregation. Summary 138 7) In-vitro drug release evaluation of the selected LNC formulae (LNC6 and LNC9) showed sustained release behavior which was enhanced by the presence of Solutol®. Being a surface active agent, Solutol® was inserted at the water-oil interface forming a coherent shell completely entrapping GA at the oily core hindering its release from oily core owing to its bulky structure. Its solubilizing nature as well may have entrapped the drug at the interface retarding its release. 8) Ex-vivo permeation studies, across nasal mucosa, of the selected formulae showed that LNC9 significantly scored the highest steady state permeation flux (Jss) and permeability coefficient (Kp) with values of 2.85 ± 0.19 (µg/cm2 /h) and 0.00166 ± 0.0001 (µm/h), respectively, indicating a generally better permeation results than LNC6 and GA suspension. Chapter II: Preparation and evaluation of GA loaded MEs This chapter deals with the preparation of GA loaded ME formulations. Accordingly, pseudo-ternary phase diagrams were constructed using several combinations of oils, surfactants and co-surfactants and following the water titration method. Twenty GA loaded MEs formulae were prepared according to Simplex Lattice Mixture design. GA was dispersed in the oil-surfactant/cosurfactant (Smix) mixture followed by the addition of the required weight of water, and stirring to form a clear and transparent liquid. Stability of the formulae based on droplet size was assessed at 4o C and room temperature over a period of 3 months. The selection of the representative formulae was based on droplet size (DS), PDI and stability Summary 139 studies. The selected formulae were then characterized by high resolution transmission electron microscope (HR-TEM), viscosity and PH measurement. In-vitro release and ex-vivo permeation of the selected formulae were evaluated. from the obtained results, it was found that: 1. Solubility study was done to select oil of highest solubilizing power for GA, two oils were selected for formulating the MEs; Lauroglycol® and Labrafac® PG. 2. Two systems were selected for further work, after constructing the pseudoternary phase diagrams, namely; ME LG and ME LabPG, because of their largest ME domain formed and the highest GA solubility in those oils (lauroglycol and labrafac PG). 3. The data obtained from system ME LG showed that the droplets sizes ranged from ٥٫٦ nm to 405.85 nm and polydispersity indices ranged from 0.357 to 0.713 except for ME LG 6 & 8 which had a relatively high PDI value of 1. 4. The data obtained from system ME LabPG showed that the droplet size ranged from 8.5 nm to 389.95 nm and polydispersity indices ranged from 0.165 to 0.99 except for ME LabPG 4 which had a relatively high PDI value of 1. 5. Increasing the water percentage in both systems lead to reduction in the recorded microemulsion droplet size. Summary 140 6. Simplex lattice mixture design was used to mathematically model the resultant DS and PDI of the obtained MEs in both systems. Twenty formulae were suggested by the design according to different percentages of the oily phase, surfactant/co-surfactant and water. a. Analysis of the models was performed using ANOVA where the generated model for DS was significant (P<0.05) for both ME LG and ME LabPG, while PDI model was not significant (p>0.05). The suggested model for DS in case of ME LG was linear with P-value 0.0023 and quadratic in case of ME LabPG with P-value 0.0023 as well. b. High correlation was indicated between the actual and predicted runs for both ME LG and ME LabPG DS models. This was explicated in the graphical presentation of the actual versus the predicted runs. This was in good agreement with the R2 values which were 0.8239 and 0.9769 corresponding to DS model of ME LG and ME LabPG, respectively. c. Adequate precision measuring the signal to noise ratio greater than 4 was desirable. The ratio was found to be 10.919 and 14.238 for ME LG and ME LabPG DS models, respectively, indicating high adequacy of the models and sufficiency of the model to navigate the whole experiment space. Summary 141 7. Stability of the MEs showed good results on room temperature and in refrigerator (4o c), minimal changes in DS were recorded with no phase separation or creaming all over the 3 months. 8. TEM images of the selected MEs confirmed the homogenous spherical and elongated ME droplets with size results complying with DLS. 9. pH results showed that the selected formulae were in the non-irritantrange for nasal mucosa and a viscosity of 50 cp could ensure easy nasal application and optimum residence time. 10.In-vitro drug release studies of the selected formulae showed sustained release behavior compared to the drug solution. 11. Ex-vivo nasal permeation studies of the selected formulae showed that ME LG1 scored the highest steady state permeation flux (Jss) and permeability coefficient (Kp) of values 2.78 ± 0.97 (µg/cm2/h) and 5.6*10-4 ± 0.0002 (µm/h), respectively, indicating a general better permeation results than LabPG5 and GA suspension. Chapter III: In-vivo assessment of selected GA-loaded LNCs and MEs. This chapter deals with in-vivo investigation of the selected LNC9 from LNCs and LG1 from MEs systems. A pharmacodynamics study was conducted to assess the improvements in memory in scopolamine-induced AD model relative to negative and positive control groups and to oral treatment. Summary 142 Thirty-six rats were divided into six groups, each of six rats. group I was negative control, group II was positive control with memory deficits induced using scopolamine dose of 1 mg/kg given through intraperitoneal route. Groups III to VI, received the same dose of scopolamine as group II in addition to treatment. group III received GA-LNC9, while group IV received ME LG1, both taken in dose of 1 mg/kg intranasally. group V and VI received GA suspension through oral route in dose of 50 mg/kg and intranasal route in dose of 1mg/kg, respectively. The treatment continued for 2 weeks after which behavioral memory tests, including Moris water maze, Y-maze and light/dark tests, were conducted. Afterwards, animals were sacrificedand their brains were taken. A part of the brains was used for biochemical markers evaluation, including catalase and superoxide dismutase (SOD). Sections of the hippocampus of the other part were used for histopathological study. from the obtained results, it was found that: group III, that received LNC9 intranasally, showed the best results in the behavioral tests assessing the memory function improvements of the diseased rats. group 4, that received ME LG1 intranasally, and group 5, that received oral GA suspension in dose of 50 times greater than the intranasal formulae, showed comparable results. group 6, that received intranasal GA suspension, scored the lowest. These results were further confirmed by histological examination. Summary 143 Oxidative stress markers complied with the behavioral tests and the histopathological study. group 3 was better than group 4 and 5 that showed comparable results. These results confirmed that LNC9 showed the best memory improvements upon intranasal administration with a dose of 50 times less that the oral suspension that showed comparable results to ME LG1 used in the same small dose as LNC9 |