الفهرس 
GePreparation and evaluation of GA loaded LNCsneral IntroductionOnly 14 pages are availabe for public view
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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
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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.
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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
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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.
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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.
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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
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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.
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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.
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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.
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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.
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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