الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
Abstract
Dry Eye Disease (DED) or Kerataconjunctivitis sicca is a common condition affecting millions of patients worldwide.
Its management ranges from tear replacement or lubricants for milder cases to topical anti-inflammatory agents such as corticosteroids and cyclosporine A (CsA) for more resistant cases. CsA is used as a last line therapy for severe cases of DED, it works by inhibiting the T cell-mediated inflammations that may affect the tear glands.
Due to the highly lipophilic nature of the drug, low solubility and low permeability (class IV drug in the BCS), formulating an aqueous eye DROP preferably for once daily administration is challenging, instead of the frequent administrations needed with lubricants or tear substituents.
Aside from the specific issues facing delivery of CsA to the eye, ocular delivery in general is challenging. Management of DED using CsA requires uptake and accumulation of the drug in the anterior layers of the cornea.
The barriers presented include thick impermeable corneal layer, reflex blinking that removes any foreign objects entering the eye as well as elimination through the naso-lacrimal duct.
Developing an aqueous eye DROP formulation of CsA continues to attract attention of many researchers. A marketed product Restasis® microemulsion eye drops was approved by the FDA in 2004 and recently, Ikervis® eye drops appeared on the European market.
An extemporaneous oily eye drops prepared locally using Neoral® soft capsules content dispersed in castor oil is also available. Restasis® formulation has been reported to elicit side effects including ocular irritation and discomfort in addition to its high cost.
The use of castor oil has also been controversial; instilling oil in the eye is not patient-friendly and may cause temporary blurring of vision.
Concerning a possible CsA carrier needed to formulate an aqueous eye drops formulation, lipid nanocapsules (LNCs) appear as a potential candidate for ocular delivery of a lipophilic water insoluble drug.
A part from providing a suitable carrier for lipophilic drugs such as CsA, the particle size can be controlled by changing the ratio of ingredients to achieve a particle size suitable for ocular administration.
<It has the added benefit over other lipid nanocarriers of ease of preparation and relatively low cost of ingredients, in addition to absence of organic solvents, higher loading efficiency, good monodispersion and storage stability.
<The present thesis aimed to develop and characterize an aqueous CsA eye drops formulation for the management of DED.
<The formulation approach attempted aimed to achieve specific performance characteristic suitable for this medical condition, such as ease of administration being an aqueous liquid and rapidly gelling upon instillation providing relatively prolonged residence in addition to mucoadhesion potential to the surface of the cornea to resist clearance by tears.
<Among the targeted formulation features, encapsulation of CsA in LNCs aims to enhance drug uptake and accumulation in the outer layers of the cornea where inhibition of inflammation is required.
< The LNCs also achieve high drug solubility in the oily core.
<<In vitro, ex vivo and in vivo characterization studies are planned. Approval of the Institute Ethics
Committee was obtained prior to studies involving experimental animals.
The work in this thesis is divided into two chapters:
<Chapter One:
<Thermoresponsive gel containing lipid nanocapsules loaded with Cyclosporine A for delivery to the eye: Development and in vitro characterization
Different CsA eye drops formulations (0.10% w/w) were prepared and characterized.
<In situ blank gels were first prepared on the cold using poloxamer 407.
The formulation was modified by the addition chitosan; mechanical strength and mucoadhesion of the gel were improved. The prepared in situ gels were characterized for gelling temperatures, gelling times, viscosity and rheological properties.
< Gelling temperatures of optimized formulations were below but close to physiological temperature, with gelling time of around 11 seconds, viscosity showed a major increase after gelation and the gel showed pseudoplastic flow favorable for ocular administration.
Secondly, LNCs (standard and modified formulations) were loaded with CsA using phase inversion technique for the preparation; ingredients used were all generally regarded as safe (GRAS).
Colloidal properties of the prepared LNC dispersions were characterized, as well as drug loading and entrapment efficiency (using a validated HPLC method of assay).
< The same HPLC method was used for determining drug content. High loading capacity were obtained. LNCs prepared were monodisperse with a small particle size as well as bearing a small negative charge.
TEM images showed dispersed spherical LNCs. Modified LNCs had either a positive or negative charge depending on the additive used.
<Finally, dual system formulations were prepared combining both LNCs and in-situ gels to obtain LNC-in situ gels that utilize the advantages of both a nanocarrier and the increased precorneal retention potential of in-situ gels.
<To ensure the compatibility between LNCs and poloxamer 407, FTIR analysis was performed. The effect of LNCs on poloxamer properties was investigated.
Similarly, the effect of poloxamer on the colloidalproperties of the LNCs was also studied. Further performance characterization including in vitro release and effects of storage of the five CsA-loaded formulations developed (poloxamer in situ gel, chitosan-poloxamer in situ gel, LNC dispersion, LNC-poloxamer in situ gel and LNC-chitosan poloxamer in situ gel) was carried out.
<The release properties compared to CsA solution in ethanol as control were determined by dialysis, over 24-hour period.
<Choice of release medium to provide sink condition was guided by a CsA solubility study in different media. Release samples were assayed by HPLC.
Release results were confounded by diffusion of release medium into the dialysis bags.
< CsA release followed Weibull model. A 6-month stability study was carried out on the LNC formulations at 4℃; results showed an overall good storage stability of the preparations. Formulations were further tested in Chapter two>
<Chapter 2:
<Thermoresponsive gel containing lipid nanocapsules loaded with Cyclosporine A: Ex vivo corneal adhesion, corneal penetration and preclinical in vivo pharmacodynamics in dry eye rabbit model
This chapter aimed to further characterize the drug-loaded formulations and to compare them with a marketed product (Restasis® eye drops) and with CsA dissolved in castor oil.
Ex vivo bioadhesion strength of the formulations to isolated bovine cornea was studied.
< Ex vivo corneal uptake studies were carried out also on isolated bovine cornea. An in vivo corneal biodistribution study was carried out.
<In vivo efficacy studies and safety studies were performed on male New Zealand rabbits in which the dry eye condition was induced by repeated instillation of atropine sulphate eye drops.
<Mucodhesive properties were characterized for the different LNC formulations using the modified balance method to determine the force required to separate two freshly obtained bovine corneas enclosing a layer of formulation between them.
<Lowest mucoadhesive strength was seen with the LNCs dispersion.
< The LNC-P in situ gel showed a significant increase in mucoadhesive strength when compared to LNCs dispersion. The inclusion of chitosan achieved a higher mucoadhesive strength.
<This property is important with ocular delivery as it can potentially increase precorneal residence time.
<A comparative ex-vivo corneal uptake study (permeation across and accumulation in bovine cornea) was conducted on the developed formulations and compared with marketed CsA eye drops using modified Franz diffusion cells.
Corneal accumulation was significant for the LNC formulations and served as a parameter to differentiate between the formulations. Among the LNC formulations, highest retention in corneas was achieved with LNC-CP in situ gel.
The results of the ex vivo studies led to the selection of this formulation as the optimal formulation to undergo further in vivo testing.
An in vivo efficacy study was performed on male New Zealand rabbits.
Dry eye was induced using multiple topical administration of 1% Atropine sulphate eye drops. Once the dry eye condition was proven, the rabbits were treated with the optimum formulation prepared (CsA-LNC-CPin situ gels, 0.05% and 0.1%) and two products (CsA in castor oil and Restasis® eye drops).
Tears production was measured using Schirmer Tear Test strips.
Atropine sulphate was able to rapidly and efficiently induce dry eye as indicated by decreased tear production.
The LNC formulation showed the higher efficacy among the 4 tested formulations, where the treated animals regained baseline tear level in a shorter time of treatment, followed by Restasis® eye drops.
CsA in castor oil showed the lowest efficacy where after a week of treatment, it failed to achieve return to baseline tear production level.
Safety of the LNC formulation upon ocular administration was determined using Draize ocular irritation test on male New Zealand rabbits, in comparison with CsA in castor oil and Restasis® eye drops.
Draize score was recorded at the end of a two-week period where twice daily administration was implemented. Ocular irritation study showed no visible signs of irritation following CsA-LNC-CPin situ gel and CsA in castor oil with some signs of corneal opacity occurring in one rabbit following Restasis® microemulsion administration.
To further validate these results, histological examination of the anterior segment of the eye was carried out after sacrificing the animals and extracting the eyeballs.
This confirmed the results obtained from the Draize test where no microscopical changes in the cornea, conjunctiva, sclera and iris were observed compared to the control animal (receiving saline drops).
Based on the results of the first and second chapters, it is suggested that the optimum developed CsA formulation (CsA-LNC-CP in situ gel) is ready for future clinical study in patients suffering from DED.
A suitable sterilization method for the optimum formulation has been identified in preparation for future clinical study.