000			15513nam a22003017i 4500
008			210404s2021 ua a|||| bm|| 00| 0 eng d
040			_aEG-NcFUE _erda
082	0	4	_222 _a617.74106 _bA.E.N
100	1		_aAbdelazim, Engi Ahmed Ahmed _eauthor
245	1	0	_aNew Particulate System for Ocular Glaucoma Therapy / _cThesis Presented by Engi Ahmed Ahmed Abdelazim; Under the Supervision of Professor Seham Abdel khalek Elkheshen, Professor Rania Mohammed Hathout, Doctor Nada Mohamed El Hoffy.
246	1	5	_aأنظمة جزيئية مستحدثة لعالج المياه الزرقاء
264		1	_c2021
300			_aviii, 197 pages : _billustrations ; _c25 cm.
336			_atext _2rdacontent
337			_aunmediated _2rdamedia
338			_avolume _2rdacarrier
502			_aThesis (M.Sc.)-Future university in Egypt, faculty of pharmacy, 2021.
504			_aIncludes bibliographical references
520	3		_aGlaucoma, 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 ii 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 iii 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: iv 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). v 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). vi 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. vii • 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. viii • 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
650		4	_aGlaucoma
650		4	_aGlaucoma _xTreatment.
650		4	_aGlaucoma _xdrug therapy.
700	1		_aElkheshen, Seham Abdel khalek _eSupervisor
700	1		_aHathout, Rania Mohammed _eSupervisor
700	1		_aEl Hoffy, Nada Mohamed _eSupervisor
856	4	0	_3DSpace electronic resource _uhttp://repository.fue.edu.eg/xmlui/handle/123456789/5782
942			_cTHESIS
999			_c12953 _d12953