New Particulate System for Ocular Glaucoma Therapy / Thesis Presented by Engi Ahmed Ahmed Abdelazim; Under the Supervision of Professor Seham Abdel khalek Elkheshen, Professor Rania Mohammed Hathout, Doctor Nada Mohamed El Hoffy.

Thesis (M.Sc.)-Future university in Egypt, faculty of pharmacy, 2021.

Includes bibliographical references

Glaucoma, being asymptomatic for relatively late stage, is recognized as a
worldwide cause of irreversible vision loss. The eye is an impervious organ that
exhibits natural anatomical and physiological barriers which renders the design of
an efficient ocular delivery system a formidable task and challenges scientists to find
alternative formulation approaches. In the field of glaucoma treatment, the advanced
delivery systems have aroused interest in the topical ocular delivery field owing to
its potentiality to oppress many treatment challenges associated with many of
glaucoma types. The current momentum of nano-pharmaceuticals in the
development of advanced drug delivery systems, hold promises for much improved
therapies for glaucoma to reduce its impact on vision loss.
Liposomes are structured bi-layered system composed of phospholipids and
cholesterol which are utilized for the encapsulation of nutrients and drugs. These
concentric lipid bilayers incorporate internal aqueous volume. This unique structure
of liposomes permits both hydrophilic and lipophilic drugs to be effectively
entrapped. The lipophilic drugs are ensnared inside the lipid bilayer while the
hydrophilic drugs are captured in the focal watery core of the vesicles.
As a consequence, to the low entrapment efficiency of the hydrophilic drugs
into the liposomal core, modifications have been introduced into the conventional
liposomes. Gelatinized core liposomes, as a new liposomal modification, was
intended to overcome some of these problems such as low drug loading, poor
physical stability and poor corneal penetration of the water-soluble molecules.
Gelatin is collagen-derived in nature that is often prepared from porcine skin,
bovine bone or fish scale by means of acid or alkaline hydrolysis. It has a triple helix
structure having ampholytic nature (have both cationic and anionic charges). It
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displays superb attributes as a raw material for pharmaceutical application because
of many characteristics including its biocompatibility, biodegradability, low cost and
being an FDA-approved biomaterial. The terminal amino deposits of gelatin that are
created during the collagen hydrolysis are responsible for its enzymatic
degradability.
Brimonidine tartrate, is an alpha-adrenergic agonist which work by
stimulation of alpha-1 receptors that prompts vasoconstriction as well as expansion
in the pupil while the initiation of alpha-2 receptors outcomes in vasoconstriction,
restrains the norepinephine discharge and insulin release. Alpha-2 agonists abates
IOP though obstruction of the afferent ciliary vasculature prompting diminished
aqueous humor secretions in addition to expanding uveoscleral outflow, affirmed for
the treatment of open-angle glaucoma as monotherapy or in combination with
timolol. Brimonidine has been reported to have neuroprotective action on retinal
ganglion cells (RGC) situated close to the inner layers of the cornea. It expands the
survival of RGC cells in post-glaucoma treatment by its neuroprotective
mechanisms. The concerns regarding the topical brimonidine delivery include
significant localized side effects, medication compliance and suspected insufficient
penetration into the posterior segment to consistently affect neuronal targets.
Due to this limitation in the ocular delivery of drugs, the pharmaceutical
companies have recently designed alternatives dosage forms to the conventional eye
drops. These delivery systems may exist in the form of gel forming solution,
microemulsion, nano-suspension, and nano-emulsion. In addition, advanced drug
delivery systems comprising liposomes and their structural modifications are being
extensively investigated as promising platform for ocular delivery of drugs.
In this work, a newly introduced drug delivery system; namely gelatinized
core liposomes encapsulating brimonidine tartrate has been prepared and optimized
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utilizing response surface methodology adopting, D-Optimal Design using Design©
Expert 9 software. Thee factors were evaluated as independent variables, namely;
A) phosphatidylcholine percentage (PC%) per the total lipids weight (200 mg), B)
amount of gelatin per the formula (Gel) in mg and C) the volume of organic solvent
(Org) in mL used for dispersion, each at two levels, while the dependent variables
were the particle size of the obtained vesicles, their polydispersity index, their zeta
potential values and the drug entrapment efficiency. The program suggested twelve
formulations which were prepared with and without the incorporation of 1%
glycerol.
The vesicles were prepared by the thin-film hydration method and the effect
of the independent formulation factors on the dependent counterparts was studied.
Applying the desirability function as well as some logic judgement, thee selected
formulae were further investigated for in vitro drug release, morphological
examination using transmission electron microscopy, possible interactions using
DSC and FTIR, rheological behavior and stability studies. The biological
performance of the selected formulations was studied using glaucomatous Albino
rabbits where the intra-ocular pressure (IOP) reduction of the rabbits’ eyes was
evaluated applying simple randomized parallel design. The safety profile was
assessed using histopathological examinations. Moreover, Draize test was
performed to evaluate the irritancy effect of the tested formulations. The obtained
results can be summarized as follow:
• The wavelength of maximum absorbance (λmax) for brimonidine tartrate was
249 nm in phosphate buffer solution of pH 7.4.
• Brimonidine tartrate loaded gelatinized core liposomes were successfully
prepared by the thin film hydration method.
• Characterization of the glycerol-free formulations (GL1:GL12) revealed that:
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o The particles showed decrease in their size from day 1 till day 10
starting from micrometer reaching nanometer ranges for all
formulations. GL9 exhibited the largest initial particle size (PS) of
5,611.75 ± 162.19 nm which matured into 1,010.43 ± 82.89 nm after
10 days. While the smallest value of PS was initially observed with
formula GL11 measuring 905.93 ± 174.10 nm and ended up with
729.60 ± 100.86 nm after 10 days.
o The matured glycerol free GLs obtained were mostly in the nano-meter
range except for GL6, GL7, GL9 and GL12 which were in the
micrometer range. The maturation PS ranged from 413.03 ± 24.85 to
1,308.68 ± 125.33 nm. Formula GL12 showed the largest final PS
1,308.68 ± 125.33 nm while formula GL1 exhibited the smallest mature
PS of 413.03 ± 24.85 nm.
o Regression model and ANOVA results for glycerol-free formulation
GL1:GL12 revealed the significance (p=0.0057) of the quadratic
models adopted for the study of the influence of formulation factors on
the particle size diameter of the obtained GLs.
o Results showed that all three independent factors namely; gelatin mass
(A), organic solvent volume (B) phosphatidyl choline concentration
(C), had significant (p=0.0015, p=0.0361 and p=0.0059, respectively)
effect on particle size of the obtained liposomes. Additionally, the
factor combination AB, and the second level of all independent
variables; A2
, B2
and C2
significantly (p=0.0216, p=0.0067, p=0.0069
and p=0.0170, respectively) affected the PS of glycerol-free
formulations (GLs).
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o Regression model and ANOVA of PDI, zeta potential and
encapsulation efficiency were insignificant (p>0.05) and better
presented by the mean effect.
• Characterization of the glycerol-containing formulations (GLG1:GLG12)
revealed that:
o Throughout the maturation process which extended for 7 days, GLG4
exhibited the largest initial PS of 1,867.50 ± 880.35 nm that decreased
to 468.38 ± 51.61 nm upon maturation. Whereas the smallest initial
particle size of 193.70 ± 131.95 nm was observed for GLG5, with an
increase in the PS upon maturation to 494.96 ± 31.49 nm.
o Formula GLG8 showed the largest final PS of 567.01 ± 113.01 nm
while formula GLG1 exhibited the smallest mature PS of 140.75 ±
23.59 nm.
o The regression model and ANOVA results for the glycerol containing
formulations GLG1:GLG12, revealed the significance (p=0.0022) of
the quadratic models adopted for the study of the influence of
formulation factors on the particle size diameter of the obtained GLGs
formulations.
o Results showed that all three independent factors namely; gelatin mass
(A), organic solvent volume (B) phosphatidyl choline concentration
(C), had significant (p=0.0017, p=0.0015 and p=0.0057, respectively)
effect on particle size of the obtained liposomes. Additionally, the
factor combination AB, and BC as well as the second level of all
independent variables; A2
, B2
and C2
significantly (p=0.0013,
p=0.0017, p=0.0178, p=0.0014, and p=0.001, respectively) affected
the PS of glycerol-Containing formulations (GLGs).
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o Regression model and ANOVA of PDI revealed the insignificance
(p=0.0685) of the linear model adopted for the study of the influence
of formulation factors on the PDI of the obtained GLGs while zeta
potential revealed the insignificance (p=0.3125) of the quadratic model
adopted for the study of the influence of formulation factors on the zeta
potential of the obtained GLGs. Similarly, the adopted encapsulation
efficiency model (two factor interaction model) was insignificant
(p>0.05). They were all better presented by the mean effect.
• The addition of glycerol to the formulations, GLG1:GLG12, significantly
(p<0.0001) decreased the particle size reaching the nanometer range with only
7 days maturation period.
• The presence of the glycerol in GLG1:GLG12 significantly (p=0.0001)
decreased the entrapment effieceincy compared to the formulation lacking
glycerol; GL1:GL12.
• Based on applying the desirability function on Design Expert® software
regarding the PS as well as some logical assessment, three formulations;
namely GLG1, GLG4 and GL12 were chosen for further in vitro and in vivo
evaluation.
• All investigated formulations exhibited pH values within the accepTable
range for ocular administration.
• All investigated formulations exhibited nearly Newtonian behavior (N=1, 1,
0.98 for GLG1, GLG4 and GL12, respectively) with slight thixotropy.
• The obtained transmission electron micrograph showed a particle size of
133.85 nm, 426.03 nm and 0.74 μm for GLG1, GLG4 and GL12, respectively.
All investigated formulations revealed a slightly deformed spherical shape
with a dense core.
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• DSC showed that the peak of BRT disappeared from the thermograms of
GLG1, GLG4 and GL12 formulations, indicative of amorphization of BRT in
the gelatinized core liposomes formulations. Upon comparing the DSC
thermograms of blank cholesterol, phosphatidylcholine, and gelatin, it can be
confirmed that an interaction has taken place between phosphatidylcholine
and gelatin which suggests the formation of gelatinized core liposomes.
• FTIR thermograms showed that similar peaks of pure components were
identified in the spectrum of the selected formulation and the physical mixture
of their components with minor differences in frequencies which confirms
that the drug had no interaction with excipients of the vesicles.
• The release profile of the drug from the selected formulae fitted the Higuchi
diffusion model and was confirmed by Koresmayer Peppas diffusion model
resulting in a Fickian transport mechanism where the diffusion exponent “n”
was 0.349, 0.357 and 0.427 for GLG1, GLG4 and GL12, respectively.
• Applying statistical analysis (one-way ANOVA) to the physical stability data
of the selected formulae comparing the particle size, zeta potential, PDI and
entrapment efficiency after storage for thee months to those freshly prepared
showed insignificantly (p>0.05) different parameters (except for the Zeta
potential of GLG4 which significantly increased) which assures the stability
of the prepared formulations.
• The re-evaluation of the particle size, the PDI, the zeta potential and the
entrapment efficiency of the sterilized formulations which received a radiation
dose of 10 Kgy for the purpose of sterilization revealed that formulations were
sTable after gamma irradiation.
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• The sterility testing after sterilization revealed the absence of both bacterial
and fungal growth which confirms the efficiency of the gamma irradiation
process for sterilizing the gelatinized core liposomal formulations.
• The IOP showed a sudden reduction in the group of animals received the drug
solution from 37.20 mmHg to 29.47 ± 1.88 mmHg in the first hour followed
by decreasing in IOP till it reached the lowest reduced IOP of 19.73 ± 2.70
mmHg after 6 hours.
• GLG1, GLG4 and GL12 caused much prolonged efficacy intraocular
reduction profile trend over 12 hours with less fluctuations and insignificantly
(p>0.05) different (among each other) average lowest IOP reduction level of
17.28 ± 1.34 mmHg, 15.83 ± 1.17 mmHg and 17.53 ± 1.46 mmHg, for
formulations GLG1, GLG4 and GL12, respectively.
• The eye irritancy testing adopting modified Draize sensitivity test was
performed on GLG1, GLG4 and GL12 as well as the drug solution and
revealed that no irritation was caused by all investigated formulations or drug
solution based on simplified scoring system abstracted from the Draize scale.
• Microscopic examination of eye’s tissue, including the cornea, filtration
apparatus, choroid and retina for histopathological changes after the
application of all thee selected formulations as well as the drug solution every
12 hours for one week revealed normal histology for all examined tissues in
all animals’ groups.
The above-mentioned outcomes would, definitely, point out the potentiality
of formulating BRT in the form of gelatinized core liposomes, with or without the
incorporation of glycerol, for better entrapment of the hydrophilic drug with
sustainment in the release profile leading to more prolonged IOP reduction and
hence better patient compliance