الفهرس 
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Abstract
SUMMARY
Glaucoma is a retinal neuropathic disease considered as the silent thief of
sight. It is the leading cause of irreversible blindness worldwide and is ranked
number two among all blindness causing diseases. The high intraocular pressure
(IOP) is the major risk factor for induction of glaucoma.
Acetazolamide (ACZ) is a highly effective systemic drug used in
management of both types of glaucoma (open and closed angle). It belongs to
the carbonic anhydrase inhibitors (CAIs) class known to decrease the aqueous
humor production from the ciliary body lowering IOP. Unfortunately, ACZ is
available only as oral tablets, capsules and intravenous injection. Its
administration in large doses is usually accompanied by several side effects
including metabolic, respiratory acidosis and diuresis therefore, patients tend to
be incompliant and often discontinue treatment.
Ocular administration of ACZ could achieve a great advance in
glaucoma treatment excluding several side effects besides enhancing patient
compliance. Nevertheless, ocular ACZ delivery is constrained by its low
aqueous solubility and permeability being categorized as class IV according to
BCS, in addition to the ocular biological barriers that should be surmounted.
Nanosuspension/nanocrystals (NS) is a leading technology capable of
enhancing saturation solubility and dissolution rate of poorly soluble drugs
besides increasing adhesiveness and permeation to the cornea. Composed
mainly of drug particles along with small proportions of stabilizers authorized
for ocular delivery, NS was selected for the ocular delivery of ACZ in this work.
To overcome the short term stability of liquid NS, solidification via spray drying
technique and conversion into redispersible spray dried NS (SDN) maintaining
original NS properties was undertaken in this work.
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Hence, the ultimate goal of this work was to prepare and characterize
ACZ-NS with the highest possible loading efficiency and the dispersed particles
should possess uniform particle size (PS) in the range of 100-300nm eligible for
corneal permeability with a target zeta potential (ζ) i.e. higher than ±25 mV to
maintain system stability. For further stabilization, ACZ-NS was converted into
ACZ-SDN via spray drying technique using suitable excipients. Finally, the
prepared ACZ-SDN was assessed for its safety and biological activity via the
modified Draize test and the ocular hypertensive model, respectively.
Accordingly, the work in this thesis was divided into three chapters namely:
Chapter I: Preparation and characterization of acetazolamide nanosuspensions.
Chapter II: Preparation and characterization of spray dried acetazolamide
nanosuspensions.
Chapter III: Safety and biological activity of ocular spray dried acetazolamide
nanosuspensions.
Chapter I: Preparation and characterization of acetazolamide
nanosuspensions
The purpose of this chapter was to prepare acetazolamide
nanosuspensions (ACZ-NS) using antisolvent precipitation followed by
sonication (AS-PT) method. Treatment of the prepared NS with mucoadhesive
cationic and anionic polymers was also endeavored; hence this chapter
comprised three consecutive studies.
- Optimization of ACZ-NS using a single stabilizer: The various
experimental and formulation factors were studied using one factor at time
(OFAT). The experimental factors included: stirring rate and time as well as
sonication type and time, while the formulation factors included:
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solvent/antisolvent ratio, ACZ concentration and finally type and
concentration of stabilizer.
- Optimization of ACZ-NS using a binary stabilizer: Various combinations
with PVA were tried including soya lecithin phospholipid (SL) as a natural
SAA, chitosan (CS) as a cationic polymer and poly-γ-glutamic acid (PG)
and hyaluronic acid (Y) as anionic polymers. Modulation of sonication was
also performed with the combined stabilizers.
- Optimization of polymer treated ACZ-NS prepared with binary stabilizer:
ACZ-NS prepared with PVA/SL binary combination was further treated
with cationic (CS) /anionic (Y and PG) polymers then increasing ACZ
loading was the secondary target of this study.
The prepared ACZ-NS was characterized by measurement of particle
size, polydispersity index and zeta potential. The prepared particles were also
visualized using transmission electron microscope (TEM). ACZ solid state was
evaluated using differential scanning calorimetry (DSC) and X- ray powder
diffraction (XRPD) and finally saturation solubility was determined.
The results of this chapter showed that, using PVA as steric stabilizer,
the optimized conditions for the preparation of ACZ NS by the AS-PT method
were found to be: stirring rate of 1000rpm for 15min, operation temperature
(5ºC), bath sonication for 45min, S/AS ratio of 1:50, ACZ concentration of
50mg/mL in DMSO and 0.2%w/v of PVA. Implying previous parameters
yielded NS of PS and PDI 117.4±11.1nm and 0.29±0.04, respectively. However,
the particles exhibited a bimodal distribution. Hence, combination of PVA with
SAA or polymeric stabilizers was the adopted technique.
Preparation of ACZ-NS using a combination of PVA with natural lipid
SL (SAA) showed concentration dependent increase in PS, PDI and ζ of the
prepared NS, 0.05%w/v SL represented the optimum concentration. Modulation
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of probe sonication for 3min had produced NS with optimum properties with
respect to PS 105±1.7nm, PDI 0.23±0.005and ζ of -31.5±2.8mV with unimodal
PSD. Treating the NS, prepared with PVA/SL as stabilizer, with negatively and
positively charged polymers CS, Y or PG resulted in production of NS
possessing PS, PDI and ζ within optimum range. All had achieved maximum
drug loading using ACZ concentration of 100mg/mL in DMSO.
Spherical well defined non aggregated particles were visualized using
TEM imaging. DSC and XRPD revealed that both crystalline and amorphous
forms of ACZ existed during nanoprecipitation process.
ACZ saturation solubility showed a seven fold increase following its
formulation in NS. Trials of DMSO elimination implying dialysis,
centrifugation and lyophilization had failed to maintain system integrity as either
drug loss or poor redispersibility had occurred. Therefore, colloidal stability
study was implemented and revealed that PS, PDI and ζ of ACZ-NS did not
significantly change for 4days except TPH (poly-γ-glutamic acid treated) that
exhibited significant increase in PS.
Finally, the selected ACZ-NS formulae, for further drying study, were
(untreated) BH, (CS-treated) TCH, (Y-treated) TYH and (PG-treated) TPH. These
formulae showed the highest ACZ loading of 0.2%w/v, except for TCH with
0.1%w/v, optimum PS in the range of 100-300nm and ζ within stability range,
more than ±25mV.
Chapter II: Preparation and characterization of spray dried acetazolamide
nanosuspensions
In this chapter, the spray drying technique was used to convert ACZ-NS
into ACZ-SDN. The optimized NS formula obtained from chapter I (formula
B2) was selected for optimization of spray drying process and formulation
variables. The process variables included inlet temperature and pump rate, while
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formulation variables included selection of optimum carrier composition
(leucine and mannitol) and nanoparticles (NPs) to carrier (C) weight ratio. The
effect of formulation composition was also studied using the selected SD
parameters with optimum carrier composition. Consequently, this chapter
comprised four consecutive studies according to the scope of preparation.
The prepared ACZ-SDN was characterized by evaluating yield%, drug
content and association efficiency. PS, PDI and ζ were determined after
redispersion in water. The spray dried powders were visualized using scanning
electron microscope (SEM). ACZ solid state was also evaluated using
differential scanning calorimetry (DSC) and X- ray powder diffraction (XRPD).
The moisture content and redispersibility index in different isotonicity adjusting
agents were also determined. The redispersed particles were visualized using
transmission electron microscope (TEM). Sterilization of the selected formulae
using gamma radiation was also tried. Finally, in-vitro ACZ release and stability
were investigated.
The results of the work in this chapter revealed that spray drying of
ACZ-NS was successfully achieved using inlet temperature of 110ºC, pump rate
of 3% and aspiration of 80% at constant air flow rate of 40mmHg. Optimum
carrier composition of Leu and Man in 1:1 ratio and NPs: C ratio of 1:2 resulted
in the preparation of SDN exhibiting high yield (72.6±0.79%w/w), small PS
(177.9±12.6nm) after redipsersion in deionized water and high AE
(96.6±3.2%w/w). Applying the optimized conditions, no traces of DMSO could
be detected in the SDN using 1H NMR technique and the moisture content
evaluated by TGA was less than 4%.
The SEM micrographs of SDN showed non agglomerated collapsed,
doughnut shaped particles decorated with minute fine crystals on the surface.
The obtained particles were spherical, non agglomerated with a PS in the
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desirable range of 100-300nm as revealed by TEM of redispersed SDN. DSC
thermograms and XRPD of SDN formulae revealed crystallinity in ACZ-SDN.
Glycerol at 2.6%w/v concentration represented the best isotonicity
adjusting agent that maintained PS, PDI and ζ in their optimum values with a
desirable resultant NS pH was in the range of 5.65- 5.89. Release of ACZ from
ACZ-SDN lasted for 480min with non-significant difference between polymer
treated and untreated formulae.
Sterilization was successfully achieved using gamma radiation of 5KGY
dose. The physicochemical properties including PS, PDI, ζ and AE were
maintained unchanged after sterilization. Stability of Y-treated SDN (SY) was
maintained upon storage in silica gel desiccator at ambient conditions for 180
days without significant changes in PS, PDI and ζ. Colloidal stability of
redispersed Y-treated SDN (SY) was maintained for 10days at 4˚C without
significant changes in PS, PDI and ζ.
Chapter III: Safety and biological activity of ocular spray dried
acetazolamide nanosuspensions.
The ocular tolerance of Y-treated ACZ-SDN selected formulae viz (SY),
untreated (SA), blank formula of SY and ACZ solution was evaluated as per
modified Draize test. The in vivo therapeutic activity of the selected formulae
compared to ACZ solution and its ability to decrease intra ocular pressure (IOP),
the main therapeutic index in glaucoma treatment, was evaluated using
hypertensive animal model by glaucoma induction in rabbits using steroids.
The results of this chapter showed that the modified Draize test revealed
no conjunctival discharge, redness or chemosis in any of the rabbits following
instillation of ACZ solution, SY and it’s blank with the exception of slight
redness that occurred with instillation of SA formula in one rabbit. Hence, the
ocular tolerance and safety of the prepared ACZ-SDN were verified.
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The histopathological examination of all parts of the eye (cornea, iris,
retina, choroid and sclera) after instillation of all tested preparations revealed
normal tissue composition without any alterations except for SA that showed
mild congestion and dilated blood vessels in iris and sclera.
Glaucoma model in rabbits was successfully induced by sub conjunctival
injection of mixture of rapid and long acting corticosteroids for three
consecutive weeks. The ocular hypotensive efficacy of ACZ-SDN formulae was
significantly higher than ACZ solution with non-significant difference among
them. ACZ solution and SYL showed the fastest therapeutic effect with a tmax of
3h; while SY and SA produced significant delayed IOP reduction with a tmax