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