الفهرس 
General IntroductionOnly 14 pages are availabe for public view
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Abstract
Parkinson’s disease (PD) is the second most prevalent central nervous system
(CNS) disorder worldwide after Alzheimer disease. There is no radical cure for PD
as it results from the death of dopaminergic neurons in the nigrostriatal pathway in
the brain which can’t be regenerated leading to both motor and non-motor
symptoms. Hence, drug treatment is only symptomatic to alleviate symptoms and
delay the progress from one stage to another. Levodopa (l-DOPA), the corner stone
of the treatment, has reported many severe highly frequent side effects such as
motor fluctuations and dyskinesia (on-off phenomenon), athetosis, hallucinations,
delusions, intraocular pressure elevation, nausea, vomiting, peptic ulcer,
tachycardia, arrhythmia and postural hypotension. l-DOPA suffers from very short
half-life (t1/2) which leads to pulsatic stimulation of the dopaminergic (DA)
receptors. DA agonists are a smart class for PD treatment and they are more
advantageous than l-DOPA in having longer t1/2 (no on-off phenomenon), more
specificity on D2 receptors and they don’t need synthesizing enzymes.
Rotigotine HCl (Ro.HCl) is classified as the most powerful DA agonist.
However, it suffers from poor water solubility and severe first path effect which
limits its oral bioavailability. Moreover, Ro.HCl is highly distributed in the body
leading to high incidence of side effects with small amount of the drug reaching
the brain. It is available in the market in the form of Neupro® transdermal patch.
Actually, the patch is able to solve the challenge of the extensive first path effect of
Ro.HCl, however, it can’t overcome the challenge of the high volume of
distribution of the drug. Meanwhile, the patch reported severe side effects which
limited its worldwide use such as skin reactions, high risk of melanoma, change in
bioavailability by changing application site and heat application, interference with
daily activities, lack of dose flexibility and high drug loading. Further, magnetic
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resonance imaging (MRI) or cardioversion can cause skin burn at the area of
application. Hence, in order to deliver Ro.HCl efficiently to the brain with minimal
side effects, it was aimed to encapsulate it in a certain nano-sized drug delivery
system (DDS). The superiority of these nano-carriers over the conventional drug
therapy for brain delivery lies in their ability to transport the drug molecule across
the BBB efficiently, hence, increasing the brain bioavailability, increasing the
efficiency and the safety, and decreasing the dose and the side effects.
Polymeric micelles (PMs) were chosen as promising nanocarriers for brain
delivery. PMs are nanoscopic carriers of amphiphilic copolymers formed by their
self-assembly in an aqueous medium at a concentration above their critical micelle
concentration (CMC), presenting their hydrophilic shell or corona to the medium,
while the core is composed of their hydrophobic portion. PMs are more
advantageous than other nanocarriers due to small size (10-100 nm), which is in
the ideal range to pass BBB, ease of preparation and handling (just by selfassembly),
ease of incorporation of hydrophobic drugs with high encapsulation
efficiency (EE %), ease of sterilization by filtration due to small size and
possibility of surface modification by various ligands for efficient brain targeting.
Additionally, the unique inherit stealth-properties of PMs can guarantee the long
circulation time.
On the other side, the concept of active brain targeting via surface decoration by
a certain ligand that targets a certain pathway in the brain is very beneficial.
Lactoferrin (Lf) is an iron-binding multifunctional mammalian cationic
glycoprotein with a molecular weight of about 80 kDa. Lf proved to be more
advantageous than other ligands for brain targeting due to its much lower plasma
concentration than the saturation value of its receptors, ability to allow more
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chemicals across the BBB and participation in the brain lesions pathogenesis.
Besides, its receptors (LfR) expression was higher in patients with PD.
Consequently, the aim of this thesis was to increase the brain bioavailability of
Ro.HCl. Both unmodified and Lf-modified Ro.HCl loaded neutral PMs were
prepared for direct/active brain targeting, respectively. IV administration of
sustained release drug loaded PMs would confirm rapid and continuous stimulation
of DA receptors, more efficiency, less side effects and high patient compliance
with accurate and flexible dosing.
The work in this thesis was divided into three chapters:
Chapter (I): Preparation and optimization of plain and Rotigotine HCl loaded
polymeric micelles
Chapter (II): Preparation and optimization of Rotigotine HCl loaded lactoferrinmodified
polymeric micelles
Chapter (III): Biological study of optimized Rotigotine HCl loaded polymeric
micelles
Chapter (I): Preparation and optimization of plain and Rotigotine
HCl loaded polymeric micelles
In this Chapter plain and Ro.HCl loaded PMs were prepared using thin film
hydration method. The design of experiment (DOE), the generation of a response
surface methodology (RSM) set of experimental runs and data analysis were
performed using Design-Expert® v. 11.0 applying User Defined model. Three
different independent variables namely, (i) copolymer type (PEG2000-PLA2000,
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PEG2000-PLGA(75/25) 2000 and PEG2000-PLGA(50/50) 2000), (ii) copolymer amount
(10, 20, 30 mg) and (iii) drug amount (0, 2, 4, 6 mg) were studied. RSM helped to
evaluate the effect of those variables on particle size (P.S), polydispersity index
(PDI), zeta (ξ) potential, encapsulation efficiency (EE%) and drug loading (DD%),
which were the chosen responses for the RSM giving rise to 36 formulae. The
selected formulae after optimization of several factors were subjected to further
characterization viz serum stability, physical stability under refrigeration, effect of
freeze drying, in-vitro drug release and morphological examination. The selected
optimized Ro.HCl loaded PMs formula was subjected to further surface
modification with Lf in Chapter II and in-vivo study in Chapter III.
The following conclusions can be abstracted from this chapter:
1- DSC thermograms revealed Tg values of 42.83, 46.72 and 47.15ºC which
corresponded to PEG2000-PLA2000, PEG2000-PLGA (75/25) 2000 and PEG2000-
PLGA(50/50) 2000, respectively. Meanwhile, Tm of Ro.HCl was found to be
187.98 ºC.
2- CMC values of PEG2000-PLA2000, PEG2000-PLGA (75/25) 2000 and PEG2000-
PLGA (50/50) 2000 as determined by fluorescence spectroscopy were 0.89, 0.98
and 1.41μg/ml which confirmed the inverse relationship between the
hydrophobicity of the core and the CMC value. Moreover, the small CMC
values would guarantee high thermodynamic stability and high resistance
against dilution upon IV administration of the PMs.
3- ξ potential investigations of the prepared PMs revealed that all the prepared
PMs were negatively charged with values ranging from -9.23 to -4.12 mV,
which were expected to impart a good serum stability of the prepared PMs. No
further mathematical modeling was required for ξ potential.
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4- The four constructed mathematical models were valid and can be used in the
detection of P.S, PDI, EE% and DL% of PMs.
5- P.S investigations of the prepared PMs revealed that:
a- All the obtained P.S values were in the optimum range for brain targeting as
they were less than 100 nm and more than 10 nm.
b- Copolymer type was the most significant factor affecting P.S. There was a
slight significant increase in P.S between PEG2000-PLA2000 and PEG2000-
PLGA(75/25) 2000 PMs. However, there was a sharp significant increase in
P.S in case of PEG2000-PLGA (50/50) 2000 PMs.
c- Drug amount starting from 4 mg significantly affected P.S of Ro.HCl loaded
PMs compared to plain PMs due to increase drug loading and expansion of
the core.
d- Copolymer amount didn’t affect significantly the P.S, while there were some
significant interactions between copolymer amount and copolymer type and
between copolymer amount and drug amount.
6- PDI investigations of the prepared PMs revealed that:
a- PEG2000-PLA2000 and PEG2000-PLGA(75/25)2000 could form almost
monodisperse PMs, while aggregation occurred in case of PEG2000-
PLGA(50/50)2000 PMs
b- Copolymer amount and drug amount didn’t significantly affect the PDI
values, while there was a significant interaction between the copolymer
amount and copolymer type
7- EE% investigations of the prepared PMs revealed that:
a- The hydrophobic cores of PMs could efficiently encapsulate the highly
hydrophobic Ro.HCl with high EE% values from 39.4±0.2 to 87.58±0.26%
in case of PEG2000-PLA2000 PMs, 38.44±0.19 to 89.47±0.23% in case of
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PEG2000-PLGA(75/25)2000 PMs and 33.98±0.47 to 73.51±0.27% in case of
PEG2000-PLGA(50/50)2000 PMs.
b- Copolymer amount and drug amount affected significantly the EE% with the
copolymer amount had a direct relationship, while drug amount had an
inverse relationship with EE%.
c- There was no significant difference between the EE% values of PEG2000-
PLA2000 and PEG2000-PLGA(75/25)2000 PMs, while there was a significant
decrease in EE% in case of PEG2000-PLGA(50/50)2000 PMs. Meanwhile,
there was a significant interaction between copolymer type and drug amount.
8- DL% investigations of the prepared PMs revealed that:
a- Copolymer amount and drug amount affected significantly the DL% with
copolymer amount had an inverse relationship, while drug amount had a
direct relationship with DL%.
b- There was no significant difference between the DL% values of PEG2000-
PLA2000 and PEG2000-PLGA(75/25)2000 PMs which showed respective
ranges of 5.49-14.78% and 5.58-14.44%, while there was a significant
decrease in DL% in case of PEG2000-PLGA(50/50)2000 PMs (4.61-
12.83%). Meanwhile, there was a significant interaction between
copolymer amount and drug amount.
9- Three PMs formulae were selected as they had the highest EE% values with
optimum P.S, PDI, ξ potential values viz F10, F22 and F34 which had the
following compositions:
Rotigotine HCl………...2mg Rotigotine HCl………..........2mg
PEG2000-PLA2000 ………30mg PEG2000-PLGA(75/25)2000 …30mg
F
F34
Rotigotine HCl………..............2mg
PEG2000-PLGA(50/50)2000 …… 30mg
F10 F22
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10- F10 showed the highest serum stability followed by F22, while F34 showed
poor serum stability at 37 ºC over 24 h in presence of 50% v/v serum, where the
order of serum stability came in line with the order of the core hydrophobicity.
F34 was excluded from the study.
11- High serum stability of PMs may be a result of their small P.S and small ξ
potential values. Additionally, the PEG shell prevents the opsonization which is
followed by phagocytosis.
12- F10 possessed the highest physical stability at 4ºC for 60 days followed by
F22.
13- Physical stability of PMs was attributed to the presence of PEG moiety on
the surface that created steric hindrance, which prevented the aggregation of
particles. Meanwhile, the hydrophobic core could entrap Ro.HCl for a long time
and prevent its leakage. However, the order of physical stability came again in
line with the order of the core hydrophobicity.
14- Lyophilization was an efficient method to increase the physical stability of
PMs, where F10 showed the highest stability against lyophilization stresses
without any cryoprotection. In contrast, F22 showed significant increase in P.S,
hence, an urgent need for cryoprotection.
15- F10 and F22 could achieve a sustained release of Ro.HCl over 4 days with
initial burst release over the first 8 h. F10 and F22 released 64.34±0.98%, and
78.87±0.49% of Ro.HCl released over 4 days respectively, with F10 achieving
the highest sustained release of Ro.HCl.
16- F10 was chosen for further active targeting study in Chapter II and in-vivo
study in Chapter III, as it had the highest physical stability, serum stability and
stability against lyophilization stresses. Further, it showed the highest in-vitro
sustained release of Ro.HCl. Meanwhile, similar to F22, F10 had P.S, PDI, ξ
potential and EE% values suitable for brain targeting of Ro.HCl.
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17- TEM confirmed the spherical shape of F10 and the absence of any
aggregates, while SEM, which was performed on the freeze dried sample,
showed the formation of irregular aggregates upon lyophilization.
18- Finally, F10 is Ro.HCl PMs formulation that owned P.S, PDI and ξ potential
values suitable for IV administration for brain targeting with expected high
serum stability in terms of minimal phagocytosis and renal clearance. It can be
dispensed as a lyophilized cake that is readily redisperserd with PBS (pH 7.4)
giving a clear colloidal dispersion that is stable for 2 weeks.
Chapter (II): Preparation and optimization of Rotigotine HCl
loaded lactoferrin-modified polymeric micelles
In this chapter surface modification of Ro.HCl loaded optimized PMs with Lf
for active targeting of the brain was performed. The pre-conjugation strategy and
the amine-N-hydroxysuccinimide (NH2-NHS) coupling method were applied.
Three ratios of PEG2000-PLA2000: Lf-PEG2000-PLA2000 mg/mg viz 9:1, 7:3 and 5:5
mg/mg were studied giving rise to three formulae namely: LP1, LP2 and LP3,
respectively. The prepared formulae underwent the same characterization
parameters as in Chapter I. The formulae with optimum properties were selected
and subjected to further in-vivo study in Chapter III.