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Design and Characterisation of Niosomes for Ocular Delivery of Naltrexone Hydrochloride

Hamdy Abdelkader Mohamed Abdelkader

A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy, School of Pharmacy, The University of Auckland




Background: Recent reports have demonstrated that topical ocular administration of naltrexone (an opioid growth factor antagonist) is able to reverse the diabetic complications on the cornea (diabetic keratopathy). The topical administration of naltrexone accelerates corneal wound healing, restores corneal sensitivity and enhances corneal epithelialisation in diabetic rats, rabbits and humans. Naltrexone can be considered as a new therapeutic agent for treatment of diabetic keratopathy. To our best knowledge, an ophthalmic formulation has not yet been developed for naltrexone hydrochloride (NTX) nor has it been properly formulated in a liquid dosage form. Niosomes are non-ionic surfactant vesicles and have the same closed bilayer structures and properties of the well-known phospholipid vesicles, liposomes. Niosomes are considered more chemically stable and more economically viable alternatives to liposomes. At the same time, they could offer the same advantages as vesicular ocular delivery systems such as prolonged precorneal residence time and enhanced corneal penetration. Objective: The aim of this work is to design and characterise niosomal formulations for ocular delivery of NTX having the convenience of being delivered as eye drops. Methods: An HPLC method was developed and validated for NTX; preformulation studies were performed to study the physicochemical properties of NTX, such as aqueous solubility, lipid solubility, melting behaviour, spectrometric identification and construction of the Arrhenius plots for predicting NTX stability. Two classes of non-ionic surfactants, sorbitan fatty acid esters (Span ® ) and polyoxyethylene alkyl ethers (Brij ® ), were selected and investigated for their ability to form niosomes and encapsulate NTX using different levels of cholesterol (a bilayer membrane stabiliser). Also, different membrane additives [dicetyl phosphate (DCP), Solulan C24 (C24) and sodium cholate (CH)] were studied to modify the physical properties of niosomes, such as their percentage entrapment efficiency (EE %), size, morphology, rheology and spreading ability characteristics. Four different niosomes; F-S60, F- DCP, F-C24 and F-CH were prepared and evaluated for ocular irritation, using the hen’s egg chorioallantoic membranes (HET-CAM) test, bovine eye test and histological corneal examination. Finally, they were studied for in vitro NTX release; transcorneal permeation of NTX; and physical stability. Results and discussion: The preformulation studies showed that NTX is a hydrophilic drug with a log P value of 1.61 at 35 o C. The main degradation pathway of NTX in aqueous solutions was found to be autoxidation. Span 60-based niosomes, consisting of Span 60: cholesterol 7:3 mol/mol (F-S60), demonstrated superior EE % for NTX. Cryogenic scanning electron microscope (Cryo-SEM) images of niosomes showed onion-like structures, indicating multilamellarity. Incorporation of the membrane additives into bilayer membranes produced both spherical niosomes and discomes (giant disc-like niosomes), as confirmed by confocal laser scanning microscope (CLSM). The vesicular incorporation of NTX imparted a protective effect against light-induced oxidation, and the prepared niosomes demonstrated minimal to no irritation potential; significantly controlled NTX release; enhanced transcorneal permeation of NTX through excised cow corneas compared with the aqueous NTX solution; and were physically stable at least for three months at 4 o C. Conclusion: The developed niosomes were able to protect NTX chemically, impart a penetration-enhancing effect and control NTX corneal permeation through bovine corneas, and demonstrate good ocular tolerability, suggesting that they are potential ocular delivery systems for NTX.


Dedicated to

My parents, brother Mohamed Abdelkader,

My wife Zeinab, my son Omar, my daughter Malak

and the 1000 young people who gave up their lives for Egypt’s freedom in 25 th January revolution



It is with a great moment of pride and relief that I come to the end of a challenging and exciting project of my PhD Research. The completion of this thesis could not have been possible without the help, support, encouragement and input of many people.

Foremost, I would like to express my gratitude to the main supervisor Dr Raid Alany for his thorough guidance, mentorship, editing my manuscripts and financial support of the third year. I would also like to cordially congratulate him on the post of professor at Kingston University, London, UK.

I also greatly appreciate the expertise and guidance provided by my co-supervisors Dr Zimei Wu and Dr Raida Al-Kassas for their keen interest, valuable input and encouragement.

I would like to thank my advisors Prof. Sayed Ismail (Department of Pharmaceutics, Faculty of

Pharmacy, Assiut University, Assiut, Egypt) and Dr Amal Kamal (Department of Pharmaceutics,

Faculty of Pharmacy, Minia University, Minia, Egypt) for their help with scholarship application

to the Egyptian government.

My sincere thanks go to Mr Ahmed Abu Moussa and Dr Moustafa Ibrahim at the Egyptian Embassy in Canberra for connecting me to the Culture and Mission Bureau, Ministry of Higher Education, Cairo, Egypt.

I would like to pay my immense gratitude to Professor John Show and the School of Pharmacy

for providing me the travel grant for international conference which was an important part of my PhD process and the professional career.

A big thank you goes to Mrs Satya Amirapu from Histology Lab. for preparing histological

sections of the bovine corneas for my research project. Another big thank you also goes to Hilary

Holloway (the Biological Imaging Research Unit), who helped me to get niosome images using confocal laser scanning microscopy. A big thank you also goes to Associate Professor Brian

Palmer (Auckland Cancer Society Research Centre) for help interpret H 1 -NMR and mass spectra

of naltrexone hydrochloride.

I would like to acknowledge the Association for Research in Vision and Ophthalmology as the

copyright holder to grant me a permission to reprint the figure “the Architecture of human


corneal nerves” in my PhD thesis. I also would like thank the head of the English Language Self- Access Centre Dr Penny Hacker for her great help and assistance and Mr. Ben Jackson who helped with proofreading.

I have been fortunate to have some of my dearest colleagues during my studies. Thank you guys Alvin, Darren, Akhlaq, Thilini, Sara and James.

Finally, I would like to take this opportunity to acknowledge my family who have always been supportive and have never given up on me.


Publications to date arising from this thesis

Abdelkader, H., Kamal, A., Ismail, S., & Alany, R. (2010). Preparation of niosomes as an ocular delivery system for naltrexone hydrochloride: Physicochemical characterization. Die Pharmazie, 65, 811-817.


approaches in treatment of diabetic keratopathy. Clinical and Experimental

Ophthalomology, 39, 259-270.












Abdelkader, H., Wu, Z., Al-Kassas, R., Brown, J. E., & Alany, R. (2011). Preformulation

characteristics of the opioid growth factor antagonist- naltrexone hydrochloride: stability and

lipophilicity studies. Journal of Drug Delivery Science and Technology, 21, 157-163.

Abdelkader, H., Kamal, A., Ismail, S., & Alany, R. (2011). Design and evaluation of niosomes and discomes for controlled release ocular delivery of naltrexone hydrochloride. Journal of Pharmaceutical sciences, 100, 1833-1846.

Conference papers to date arising from this thesis

Abdelkader, H., McGhee, C., & Alany, R. (2009). Naltrexone from alcoholism to corneal

wound healing: a formulation approach. Paper presented at Australasian Pharmaceutical Science Association (APSA) conference, Hobart, Australia. (Poster).

Abdelkader, H., Kamal, A., Ismail, S., & Alany, R. (2010). Naltrexone hydrochloride a new

ophthalmic pharmaceutical: physichochemical characterisation. Paper presented at the 12 th Conference on Formulation and Delivery of Bioactives, the University of Otago, Dunedin, New Zealand (Podium).

Abdelkader, H., Kamal, A., Ismail, S., & Alany, R. (2010). Development of a validated

HPLC-method for naltrexone hydrochloride-encapsulated in niosomal formulations. Paper presented at the 12th conference on formulation and delivery of bioactives, The University of Otago, Dunedin, New Zealand. (Poster).

Abdelkader, H., Kamal, A., Ismail, S., & Alany, R. (2010). Effect of selected ionic and non-

ionic membrane additives on the entrapment efficiency of naltrexone hydrochloride in niosomes. (2010). Paper presented at the 37 th Annual Meeting and Exposition of the controlled release Society (CRS), Portland, USA. (Poster).


Abdelkader, H., Kamal, A., Ismail, S., & Alany, R. (2010). Characterization of ocular

naltrexone niosomes using differential scanning calorimetry (DSC) and cryo-scanning electron microscopy (Cryo-SEM). Paper presented at the 37 th Annual Meeting and Exposition of the CRS CRS, Portland, USA. (Poster).

Abdelkader, H., Wu, Z., Al-Kassas, R., & Alany, R. (2010). Evaluation of niosomes and




delivery of






Paper presented











Abdelkader, H., Wu, Z., Al-Kassas, R., & Alany, R. (2011). In vitro release, transcorneal

permeation and stability studies of an opioid growth factor antagonist (naltrexoneHCl) niosomes and discomes. Paper presented at the 38 th Annual Meeting & Exposition of the CRS, National Harbor, Maryland, USA. (Poster)

Abdelkader, H., Wu, Z., Al-Kassas, R., & Alany, R. (2011). Bovine corneal opacity,

permeability (BCOP) and histological examination for evaluating ocular irritation and toxicity of non-ionic surfactant vesicles (niosomes). Paper presented at the 38 th Annual Meeting & Exposition of the CRS, National Harbor, Maryland, USA. (Poster).

Abdelkader, H., Wu, Z., Al-Kassas, R., & Alany, R. (2011). Niosomes as a potential ocular

delivery system for the opioid growth factor antagonist-naltrexone hydrochloride: influence of

vesicular encapsulation on photoxidation. Paper presented at the 3 rd PharmSciFair, Prague, Czech Republic. (Poster).








Publications to date arising from this thesis


Conference papers to date arising from this thesis





List of Figures


List of Tables


List of Abbreviations



General introduction



Ocular bioavailability from ophthalmic dosage forms


1.1.1. Anatomical and physiological considerations


1.1.2. Pharmacokinetic considerations


1.1.3. Drug and formulations factors



Drug delivery systems providing controlled and continuous ocular delivery


1.2.1. Ophthalmic inserts


1.2.2. Collagen shields


1.2.3. Polymeric gels


1.2.4. Colloidal delivery systems



Diabetic keratopathy and the available treatment options


1.3.1. Physiology of the cornea


1.3.2. Diabetes mellitus and the cornea


1.3.3. Corneal innervations


1.3.4. Mechanisms of corneal maintenance and healing


1.3.5. Treatment of diabetic keratopathy



Thesis aims and structure




Preformulation studies of naltrexone hydrochloride





2.1.1. Physical properties of naltrexone hydrochloride


2.1.2. Pharmacological uses and dosage for systemic administration of NTX


2.1.3. Pharmacokinetics of NTX


2.1.4. Adverse reactions of systemic administration of NTX


2.1.5. Warnings on the systemic administration of NTX


2.1.6. Naltrexone as a new ophthalmic pharmaceutical


2.1.7. Chemical stability of NTX


2.1.8. Oxidation


2.1.9. Arrhenius plot and predicting drug stability


2.2. Chapter aims


2.3. Materials and methods


2.3.1. Materials


2.3.2. Methods


2.3.3. Statistical analysis



Results and discussion


2.4.1. DSC study


2.4.2. Spectrometric analyses of NTX powder


2.4.3. UV-spectrophotometric assay


2.4.4. HPLC method validation


2.4.5. Forced degradation studies


2.4.6. Degradation products identification using ESI-MS


2.4.7. pH-degradation rate of NTX


2.4.8. Accelerated stability and chemical kinetic studies


2.4.9. Solubility studies of NTX












coefficient (P) of NTX





3. Preparation of niosomes using biocompatible surfactants






3.1.1. Niosomes



3.1.2. Non-conventional niosomes



3.1.3. Niosomes as drug delivery systems


3.2. Chapter aims



3.3. Materials and methods



3.3.1. Materials



3.3.2. Methods


3.3.3. Statistical analysis




Results and discussion


3.4.1. Niosome formation and imaging using plane /polarised microscopy



3.4.2. Niosome imaging using Cryo-SEM



3.4.3. Niosome size measurements


3.4.4. NTX EE%



3.4.5. DSC studies




4. Effect of membrane additives on the physical properties of niosomes






4.1.1. Bilayer membrane additives



4.1.2. Surfactant and ocular delivery


4.2. Chapter aims



4.3. Materials and methods




4.3.1. Materials


4.3.2. Methods


4.3.3. Statistical analysis



Results and discussion


4.4.1. Effect of selected membrane additives on NTX EE%


4.4.2. Effect of preparation methods on NTX EE%


4.4.3. Niosome size and distribution measurements


4.4.4. Morphology and lamellarity


4.4.5. DSC studies


4.4.6. Surface tension measurements


4.4.7. Contact angle and spreading coefficient measurements


4.4.8. Rheological properties measurements


4.4.9. Effect of niosomal encapsulation on NTX oxidation




5. Evaluation of niosomal formulations for ocular delivery of naltrexone hydrochloride






5.1.1. Eye irritation tests


5.1.2. Toxicity of niosomes


5.1.3. Physical stability of niosomes


5.2. Chapter aims


5.3. Materials and methods


5.3.1. Materials


5.3.2. Methods


5.3.3. Statistical analysis



Results and discussion




5.4.1. Conjunctival (HET-CAM) test


5.4.2. Cornea (bovine eye) test


5.4.3. Histopathological evaluation of the bovine corneas


5.4.4. In vitro release studies


5.4.5. In vitro release kinetic studies


5.4.6. Ex-vivo corneal permeation studies


5.4.7. Physical stability of the prepared niosomes





General discussion, conclusion and direction for future research


6.1. General discussion and conclusion


6.2. Limitations and future direction




Appendix 1


List of Figures

List of Figures

Figure 1.1 Gross anatomy of the anterior segment of the human eye

Figure 1.2 Factors limiting ocular bioavailability of an ophthalmic formulation following topical


administration to the eye surface


Figure 1.3 Structure of the precornal tear film


Figure 1.4 Photomicrograph of H & E stained bovine cornea


Figure 1.5 Human corneal nerves penetration and distribution


Figure 2.1 Chemical structure of NTX


Figure 2.2 Schematic of morphine autoxidation in aqueous solutions


Figure 2.3 DSC thermogram of NTX


Figure 2.4 APCI mass spectra of NTX


Figure 2.5 FT-IR spectrum of NTX


Figure 2.6 1 H-NMR spectrum of NTX in CD 3 SO, 400 MHz


Figure 2.7 UV-Spectrum of NTX (100 µg/ml) in PBS pH 7.4


Figure 2.8 Standard UV-spectrophotometric calibration curve of NTX in PBS at pH 7.4


Figure 2.9 Representative HPLC chromatograms of standard NTX (0.4 mg/ml) solution and NTX

resolved from Span 60: cholesterol: DCP-niosomal formulation

Figure 2.10 PDA- spectra of the NTX peak acquired from 4 different positions throughout the


Figure 2.11 Percentage (%) remaining of NTX after 24 h exposure to various stress conditions 75

Figure 2.12 HPLC chromatograms of the blank solution and aquous NTX solution (0.4 mg/ml)


Figure 2.13 PDA spectra of NTX eluted at 4.16 min (A), the degradation product resolved at 3.7 min (B) and the second degradation product resolved at 3.3 min (C) after exposure to pH 10 at 60


pH 10 after 24 h exposure to heat at 60 o C

eluted peak for peak purity assessment




Figure 2.14 HPLC chromatograms of the blank solution and aquous NTX solution (0.4 mg/ml)

of pH 1.2 after 24 h exposure to heat at 60 o C

Figure 2.15 HPLC chromatograms of the blank solution and aquous NTX solution (0.4 mg/ml)


of PBS pH 7.4 after 24 h exposure to heat at 60 o C

Figure 2.16 HPLC chromatograms of the blank solution and aquous NTX solution (0.4 mg/ml) of

PBS pH 7.4 after 24 h exposure to H 2 O 2 (2% v/v)



List of Figures

Figure 2.17 HPLC chromatograms of the blank and aquous NTX solution (0.4 mg/ml) of PBS

pH 7.4 after 24 h exposure to artificial daylight illumination (10,000 lux) at 40 o C


Figure 2.18 ESI mass spectra of product I and Product II of NTX oxidation


Figure 2.19 Schematic of the proposed oxidative degradation pathway for NTX


Figure 2.20 First-order degradation plots for NTX at 60 o C in phosphate buffers of various pH



Figure 2.21 pH-rate profile for NTX at 60 o C


Figure 2.22 First-order degradation plots for NTX in PBS pH 7.4 at various temperatures


Figure 3.1 Locations of entrapped hydrophilic, lipophilic and amphipathic drugs in niosomes


Figure 3.2 Major types of niosomes, MLV (multilamellar vesicles), OLV (oligolamellar vesicles)

and ULV (unilamellar vesicles)


Figure 3.3 Schematic of a single-chain surfactant


Figure 3.4 Chemical structure of cholesterol


Figure 3.5 Suitable niosome sizes for particular routes of administration


Figure 3.6 Representative micrographs of Brij 72:cholesterol 7:3 mol/mol-based niosomes under


plane light and polarised light microscope

Figure 3.7 Niosomal dispersions containing various molar ratios of Brij 52:cholesterol and Span



Figure 3.8 Cryo-SEM micrographs of Span 60: cholesterol 7:3 mol/mol and Brij 72: cholesterol

7:3 mol/mol niosomes

Figure 3.9 Effect of HLB on Span-based niosomes composed of surfactant:cholesterol at 7:3

molar ratio

Figure 3.10 Effect of gel/liquid transition temperature on EE% for niosomes composed of



surfactant:cholesterol at 7:3 molar ratio


Figure 3.11 Effect of cholesterol concentration on EE% for Span-based niosomes


Figure 3.12 Effect of the total surfactant/lipid content on EE % for Span 60: cholesterol


Figure 3.13 Effect of the total surfactant/lipid content on DL % for Span 60: cholesterol


Figure 3.14 Effect of the initial amount of NTX on EE % for Span 60: cholesterol


Figure 3.15 Effect of the initial amount of NTX on DL % for Span 60: cholesterol


Figure 3.16 DSC thermograms of Span 60-based niosomes containing different concentrations of



List of Figures

Figure 3.17 Effect of NTX on gel/liquid transition temperature of (Span 60: cholesterol 7:3

mol/mol) niosomes


Figure 4.1 Equilibrium between forces acting on a drop of liquid on a solid surface


Figure 4.2 KSV-CAM 101 goniometer setup for measuring contact angle


Figure 4.3 Effect of selected membrane additives concentration on NTX EE% for niosomes

prepared using the TFH method


Figure 4.4 Effect of the preparation method on NTX EE%


Figure 4.5 Size-frequency distribution curves of the prepared niosomes


Figure 4.6 Cryo-SEM micrographs of F-S60 and F-DCP prepared using the REV method


Figure 4.7 Cryo-SEM micrographs of F-C24 and F-CH prepared using the REVmethod


Figure 4.8 CLS micrographs of F-S60 niosomal formulation loaded with CF produced by the

REV method

Figure 4.9 CLS micrographs of F-DCP niosomal formulation loaded with CF produced by the



Figure 4.10 CLS micrographs of F-C24 discomes loaded with CF produced by the REV method



Figure 4.11 CLS micrographs of F-CH niosomal formulation loaded with CF produced by the


Figure 4.12 Effects of different membrane additives on gel/liquid transition temperatures of the

prepared niosomes

Figure 4.13 Hypothetical position occupied by DCP in the bilayer membrane of F-DCP based on


Figure 4.14 Hypothetical position occupied by C24 in the bilayer membrane of F-C24 based on


Figure 4.15 Hypothetical position occupied by CH in the bilayer membrane of F-CH based on


Figure 4.16 Surface tension measurements for the prepared niosomes compared with NTX





REV method


aqueous solution

Figure 4.17 Representative rheograms for the aqueous vehicle (PBS) and the prepared niosomes



Figure 4.18 Viscosity values for the aqueous solution (PBS) compared with the prepared


niosomes at two different temperatures 25 o C and 35 o C

o C

List of Figures

Figure 4.19 Effect of niosomes encapsulation on the chemical stability of NTX against

oxidation and daylight illumination

Figure 4.20 First-order degradation kinetics for PBS solution of NTX and NTX encapsulated


Figure 4.21 First-order degradation rate constants for PBS solution of NTX and NTX

niosomes under artificial daylight illumination at 40 o C in PBS pH 7.4


encapsulated niosomes under artificial daylight illumination at 40 o C in PBS pH 7.4


Figure 5.1 Development stages of the growing embryos


Figure 5.2 Temperature, percentage relative humidity (% RH) and dew point inside the egg

incubator over the 10-day incubation period


Figure 5.3 Vascular responses used to score the test substances


Figure 5.4 Excised cow eyes immersed in normal saline with silicone O-rings centered on top of

the cornea and incubated a water bath thermostatically equilibrated at 37 o C ± 0.5 o C

Figure 5.5 Degree of corneal opacity and fluorescein permeability used to score the test


Figure 5.6 Degree of corneal opacity and fluorescein permeability used to score the test


Figure 5.7 Excised bovine cornea, modified parts and top view of the final assembly of the Franz-

diffusion cell

Figure 5.8 Cumulative HET-CAM scores for the controls and representative examples of the test


Figure 5.9 Cumulative bovine eye scores for the controls and representative examples of the test


Figure 5.10 Photomicrographs of H&E stained corneal sections of negative control treated with

saline for 30 s

Figure 5.11 Photomicrographs of H&E stained corneal sections of acetone-treated cornea for 30 s









Figure 5.12 Photomicrographs of H&E stained corneal sections of NaOH (0.5 M)-treated cornea

for 30 s

Figure 5.13 Photomicrographs of H&E stained corneal sections treated with CH Powder, C24

powder and the prepared niosomes for 30 s

Figure 5.14 Photomicrographs of H&E stained corneal sections treated with the prepared



niosomes for 1 h


List of Figures

Figure 5.15 Photomicrographs of H&E stained corneal sections treated with the prepared

niosomes for 3 h

Figure 5.16 Photomicrographs of H&E stained corneal sections treated with the prepared


niosomes for 8 h

Figure 5.17 In vitro release profiles of NTX from the NTX solution and the prepared niosomes




Figure 5.18 Effect of sample volume on the diffusion of NTX from the aqueous solution


Figure 5.19 Transcorneal permeation profiles of NTX from the NTX solution and the prepared

niosomes using excised cow corneas

Figure 5.20 Relationship between NTX release rate and apparent permeability coefficient (P app )

for NTX from the NTX solution and the prepared niosomes

Figure 5.21 Relationship between contact angle and lag time from the NTX solution and four

niosomal formulations

Figure 5.22 Effect of storage temperature on NTX retention in the prepared niosomes over three






Figure 5.23 Effect of niosome composition on NTX retention over three months


List of Tables

List of Tables

Table 1.1 Causes of decreased corneal sensitivity


Table 1.2 Major attributes of insulin


Table 1.3 Major attributes of murine NGF


Table 1.4 Major attributes of naltrexone


Table 2.1 Summary for HPLC conditions of NTX determination


Table 2.2 Assignments of 1 H-NMR spectra for NTX


Table 2.3 Reverse predicted concentrations, % recovery and regression coefficient (R 2 )


Table 2.4 Precision and accuracy data of the QC samples


Table 2.5 First–order kinetic parameters and correlation coefficient of NTX degradation in

aqueous solution of PBS pH 7.4 at different temperatures

Table 2.6 n-octanol-buffer distribution coefficient (D) and partition coefficient (P) and calculated


Table 3.1 Composition of the prepared niosomes using various surfactant: cholesterol molar


pKa at different temperatures



Table 3.2 Chemical structure, phase transition temperature, HLB and vesicle-forming ability of

the used surfactants

Table 3.3 Effect of surfactant type and cholesterol level on the D [4,3] and EE % of the prepared




Table 3.4 DSC parameters of Span 60-based niosome dispersions containing different

concentrations of cholesterol


Table 4.1 Codes and composition of the prepared niosomal formulations


Table 4.2 Chemical structure, molecular weight, CMC and phase transition temperature of the


investigated additives

Table 4.3 Effect of selected membrane additives on D [4,3] and span values for niosomes

prepared by the REV method


Table 4.4 Contact angle and spreading coefficient measurements for the prepared niosomes


Table 4.5 Tukey’s pair wise comparison of viscosity values for the prepared niosomes at 25 o C

and 35 o C


Table 4.6 Tukey’s pair wise comparison of oxidation and photolysis for the prepared niosomes



Table 5.1 Ocular tissue and score for assessing ocular irritation using the Draize test


List of Tables

Table 5.2 HET-CAM scoring system


Table 5.3 Classification of cumulative scores in HET-CAM


Table 5.4 Average dimensions of human and cow corneas


Table 5.5 Bovine eye scoring system


Table 5.6 Classification of cumulative scores in bovine eye test


Table 5.7 Summary of HET-CAM and bovine eye test interpretations


Table 5.8 Histological corneal lesions scores for controls and the prepared niosomes at different

time points

Table 5.9 In vitro release parameters for NTX from the prepared niosomes compared with NTX


aqueous solution

Table 5.10 Tukey’s pair wise comparison of Q 2h , Q 6h and DE%-12 for NTX from the aqueous


PBS solution and the prepared niosomal formulations


Table 5.11 In vitro release kinetic parameters for NTX from the prepared niosomes


Table 5.12 Steady state flux, apparent permeability coefficient (P app ) and t L for NTX from the


aqueous PBS solution and the prepared niosomes though excised cow corneas

Table 5.13 Tukey’s pair wise comparison of steady state flux, P app and t L for NTX from the

aqueous PBS solution and the prepared niosomal formulations


Table 5.14 Effect of ageing on the volume diameter (D [4,3]) for the prepared niosomes over


three months at three different temperatures

List of Abbreviations

List of Abbreviations



Optimal polar head group area of an amphiphile




Analysis of variance


Area under the curve


o C

Degree Celsius




Sodium cholate


Critical micelle concentration





Cryogenic scanning electron microscope


Solulan C24


Distribution coefficient





Chemical shift


Dicetyl phosphate


Differential scanning calorimetry

D [4,3]

Volume diameter


Entrapment efficiency %


Freeze and thaw




Surface tension


Haematoxylin and eosin stain


Hen’s egg test chorioallanoic membrane




International Conference on Harmonisation

List of Abbreviations



o K

Degree Kelvin


Potassium chloride

KH 2 PO 4

Potassium dihydrogen phosphate

λ max

Wave length of maximum drug absorption

l c

critical chain length of an amphiphile


Loading efficiency %

Lipid vesicles

Liposomes and niosomes


Phospholipid vesicles


Limit of detection


Limit of quantitation


Large unilamellar vesicles






Multilamellar vesicles














Mole per mole

Mol wt

Molecular weight


MilliPascal-second = 1centipoise


Release exponent


Sodium chloride

List of Abbreviations

Na 2 HPO 4







P app




R 2














Disodium phosphate

Non-ionic-surfactant vesicles


Naltrexone hydrochloride

Oil in water

Partition coefficient

Apparent permeability coefficient

Phosphate buffer saline

Photodiode array

Negative log hydrogen ion concentration

Determination (regression) coefficient

Registered trade mark

Reverse phase evaporation

Rotation (s) per minute


Small unilamellar vesicles

Thin film hydration

Contact angle

Lag time

Time required for 50% drug degradation

Time at which drug decomposed to 90% of its original concentration


1 unit of insulin




Volume of the hydrocarbon chains of an amphiphile


Volume per volume


Water in oil

List of Abbreviations


Weight per volume


Weight per weight

Chapter 1….Literature Review

1. General introduction: challenges in ocular delivery, controlled ocular drug delivery systems and new therapeutic approaches in diabetic keratopathy

and new therapeutic approaches in diabetic keratopathy 1.1. Ocular bioavailability from ophthalmic dosage forms

1.1. Ocular bioavailability from ophthalmic dosage forms

Topical ocular drug delivery is a commonly used and preferred route for treating disorders that affect the anterior segment of the eye (the cornea, conjunctiva, sclera, iris and lens) (Figure 1.1). This is because of the rapid and local effects, accessibility to the ocular tissue, relative safety and patient acceptability, as well as the relatively lower risk compared with systemic routes of drug administration (Macha et al., 2003).

routes of drug administration (Macha et al . , 2003). Figure 1.1 Gross anatomy of the

Figure 1.1 Gross anatomy of the anterior segment of the human eye

However, there are many anatomical and physiological barriers forming part of the eye’s natural defence (Lang et al., 2002). These barriers and protective mechanisms prevent the administered ophthalmic formulations from residing on the eye surface for enough time to allow complete drug absorption. This prevents a significant portion of the instilled dose from being absorbed by the ocular tissue (ocular bioavailability). Therefore, the ocular bioavailability of topically applied drugs from simple solutions is often less than 1% (Urtti, 2006b). The protective barriers and constraints limiting ocular bioavailability of topically administered drugs are outlined in Figure 1.2 and are discussed in this chapter.

Chapter 1….Literature Review

Chapter 1….Literature Review Figure 1.2 Factors limiting ocular bioavailability of an ophthalmic formulation following

Figure 1.2 Factors limiting ocular bioavailability of an ophthalmic formulation following topical administration to the eye surface

1.1.1. Anatomical and physiological considerations

The corneal and the conjuctival-scleral routes are the two major routes for ocular drug absorption from topically administered ophthalmic dosage forms (Ahmed & Patton, 1987, 1989). Corneal route

The cornea is the transparent avascular dome-shaped structure at the anterior part of the eye (Figure 1.1). The anterior corneal surface is covered by the tear film and the posterior surface is bathed by the aqueous humour. The surface area of the cornea is approximately 1.3 cm 2 , forming one-sixth of the surface area of the globe. The cornea is thinnest at the centre and its thickness gradually increases towards the periphery, with mean thickness of 0.5 mm and 0.7 mm respectively. Anatomically, it is divided into five layers (Klyce & Beuerman, 1998) (Figure 1.4):

Chapter 1….Literature Review

1. Epithelium

2. Bowman’s layer

3. Stroma

4. Descemet’s membrane

5. Endothelium

Although it is not a part of the cornea, the precorneal tear film is intimately associated with the cornea both anatomically and functionally (Macha et al., 2003) (Figure 1.3).

Precorneal Tear film

The surface of the cornea must be kept moist to prevent damage to the corneal epithelium and to maintain the optical quality of the cornea. Moisture and smoothing are provided to the ocular surface by the preocular tear film in conjunction with the spreading function of the eyelids during blinking. The tear film is approximately 7 µm thick and 6.5 µl in volume. It consists of three structurally identifiable layers (Figure 1.3) (Ehlers & Hjortdal, 2006; Klyce & Beuerman, 1998):

1. A thin superficial lipid layer (0.1 to 0.5 µm) secreted by meibomian glands retards evaporation from the preocular tear film, which prevents drying between blinks.

2. A thick (approximately 6 µm) middle aqueous layer supplied by lacrimal glands, into which a mucin-rich glycocalyx extends.

3. A thin (0.02 to 0.05 µm) inner mucous layer composed mainly of glycoprotein and produced by the conjuctival goblet cells and the lacrimal gland. The mucin layer ensures the wetting of the corneal and conjunctival epithelia.

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Chapter 1….Literature Review Figure 1.3 Structure of the precorneal tear film Figure 1.4 Photomicrograph of H

Figure 1.3 Structure of the precorneal tear film

Review Figure 1.3 Structure of the precorneal tear film Figure 1.4 Photomicrograph of H & E

Figure 1.4 Photomicrograph of H & E stained bovine cornea showing five layers [epithelium (EP), Bowman’s membrane (BM), stroma (ST), Descemet’s membrane (DM) and endothelium (EN)]

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Corneal epithelium

The entire epithelium is about 4 to 6 cell layers thick and represents 10 % of the corneal thickness. The epithelial cells are non-keratinised, stratified squamous and divided morphologically into three layers (Klyce & Beuerman, 1998):

The outermost layer comprises non-keratinised, stratified squamous epithelial cells. The innermost layer is a basal cell layer. The basal cells are the only epithelial cells that undergo mitosis. The daughter cells thus formed push anteriorly and change their shape conforming to the middle epithelial layer, which is called the wing cell layer. The wing cells are a transition stage between basal cells and superficial squamous cells. The superficial cells are polygonal in shape and 40-60 µm in diameter and have unique specialisations that maintain the tear film and the barriers that separate the extracellular space of the cornea from the tears. The surface membranes of superficial cells are distinctive in two respects:

1. They have microscopic projections (microvilli, reticulations and microplicae).

2. The outer leaflet is thickened and supports an extensive fibrillar glycocalyx, also called the buffy coat.

The cells of all epithelial layers interdigitate and are separated by a 10-20 nm intercellular space. Important properties of the superficial cells include adherent junctions and junctional complexes formed with laterally adjacent cells. These complexes consist of tight junctions (zonulae occludens) that surround the entire cell and resist the flow of fluid through the epithelial surface. If the aqueous humour passes into the stroma due to a defect in the tight epithelial junction, it is trapped within the epithelium, resulting in epithelial oedema (Ehlers & Hjortdal, 2006).

Basal lamina and bowman’s layer

The basal lamina is an extracellular secretory product of the basal epithelial cells. It forms a scaffold for the organisation of the epithelium and separates the epithelium from the stroma.

Bowman’s layer is an acellular region lying just under the basal epithelial membrane. It composed of randomly organised collagen fibrils that merge into more organised anterior stroma. Bowman’s layer is very thin and functions as a dome-shaped structure anchored to the limbus. Bowman’s layer is a rigid structure and it shapes the anterior corneal curvature. Once damaged, its architecture many not be restored, leading to abnormalities in the corneal thickness and optical properties that could result in permanent vision deficit (Klyce & Beuerman, 1998).

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Corneal stroma

The stroma constitutes approximately 90% of corneal thickness and it is composed of collagen fibrils in parallel arrays which make up the 300 to 500 lamellae of the stroma. The lamellae extend from limbus to limbus. Collagen constitutes approximately 71% of the dry weight of the cornea and is the structural macromolecule providing tissue transparency and mechanical resistance to intraocular pressure. Nerve axons and their Schwann cells are found in the anterior and middle third of the stroma. The stroma can be considered a comparatively open structure allowing diffusion of solute molecules weighing about 500 kilo Dalton (kDa). However, it acts as a diffusion barrier to all lipophilic drugs (Klyce & Beuerman, 1998).

Descemet’s membrane

Descemet’s membrane is the thick basal lamina (~5 µm thick) secreted by the endothelium. It is loosely attached to the stroma. Descemet’s membrane increases in thickness as an inevitable consequence of ageing. On the other hand, many diseases might damage the endothelial cells (e.g. interstitial keratitis) and consequently this damage can result in overproduction of Descemet’s membrane materials (Klyce & Beuerman, 1998). Also, wrinkling of the Descemet’s membrane is a diabetic complication of the anterior segment of the eye (Schultz et al., 1981 ).

Corneal endothelium

The corneal endothelium forms a single layer, of approximately 4 to 6 µm thick, on the posterior corneal surface. These cells play a key role in maintaining corneal transparency through their transporting, synthetic and secretory functions. The endothelium maintains tight apposition with neighbouring cells, preventing excessive seepage of aqueous humour into the stroma (Klyce & Beuerman, 1998).

As previously outlined, the corneal route has exclusive tight junctional complexes of epithelial cells which endow the cornea with tight barriers against chemical and microbial insults. This makes the corneal epithelium a significant barrier to the penetration of many drugs. However, paracellular (small ions and hydrophilic molecules) and transcellular (hydrophobic) diffusion is reported. Most topically applied drugs permeate via the transcellular route (Lee & Li, 1989).

The conjunctival-scleral route is thought to be an alternative portal gate for ocular permeation of hydrophilic large molecules such as peptides and proteins and its anatomical and physiological features are discussed in the following section.

Chapter 1….Literature Review Conjunctival-scleral route

The conjunctival-scleral route is the major pathway for polar and large-molecular weight drugs to reach the intraocular tissues (Ahmed & Patton, 1989). For example, high molecular weight drugs such as inulin (mol wt 5500) can gain access to the iris and ciliary body by diffusion via the conjuctival-sclera route. Further, drugs permeated through this route gain access directly to the posterior segment of the eye (choroid, vitreous humour and retina) (Ahmed & Patton, 1987).


Conjunctiva is a thin and vascularised mucus membrane. It covers the anterior surface of the eye globe with the exception of the cornea, where the conjunctiva ends at the limbus bordering the cornea. The conjunctiva consists of 2-3 layers of non-keratinised epithelial cells but conjunctival epithelial cells are not tight as corneal epithelial cells. The conjunctiva is permeable to molecules up to 20 kDa, in contrast to the cornea which is impermeable to molecules larger than 5 kDa (Greaves & Wilson, 1993). Anatomically, the conjunctiva is divided into three areas: conjunctiva covering the interior of eyelids, called palpebral conjunctiva; conjunctiva which is folded to cover the fornix, called fornix conjunctiva; and conjunctiva covering the anterior surface of the globe, called bulbar conjunctiva. The conjunctiva of the eyelid and globe forms a continuous surface area of 18 cm 2 , 17 times larger than the cornea1 surface area (Watsky et al., 1988). The bulbar conjunctiva, combined with the subsequent layer (sclera), represents the non-corneal barrier to drugs topically applied to the surface of the eye.


The sclera (the white part of the eye) is the outermost layer that covers the anterior surface of the eye. It protects the sensitive inner parts of the eye. The sclera is a firm and resistant structure (0.5-1 mm thick) composed of the same collagen fibrils as the corneal stroma. However, the sclera’s collagen fibres are arranged in an irregular network rather than a lattice structure. This makes the sclera appear opaque compared with the transparent cornea. The sclera constitutes the posterior five-sixths of the globe, whereas the cornea comprises the remaining one-sixth (Macha et al., 2003).

Generally, the sclera is significantly more permeable to solutes than the cornea. Solute size has a more pronounced effect on the sclera than the cornea whereas drug lipophilicty predominantly controls drug corneal permeability (Ahmed & Patton, 1989). The conjunctival-scleral route can be a promising avenue for intra-ocular drug delivery of hydrophilic and/or large molecules. These

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molecules can benefit more from the conjunctival-scleral route than the corneal route (Fattal & Bochot, 2006; Geroski & Edelhauser, 2001). Therefore, large bio-organic compounds such as proteins and bioactive oligonucleotides drugs can be absorbed via this route and gain access to the intraocular tissues for safer and more convenient topical treatment of posterior segment diseases (e.g. diabetic retinopathy) than invasive intravitreous injection route.

1.1.2. Pharmacokinetic considerations

Topical ocular pharmacokinetics studies the main pathways of drug administration, elimination and ocular bioavailability such as tears dynamics, non-productive absorption and enzymatic degradation (Urtti, 2006a). Tears and tears dynamics

The precorneal tear film structure, composition and function were previously discussed and shown in Figure 1.3. Human tears also contain around 0.6 to 2% w/v of proteins. The proteins in the tear fluid are mainly albumin, globulin, lysozyme and lactoferrin (Mikkelson et al., 1973a; Nishida, 2005). Emotional stress, irritation and inflammatory diseases greatly influence the protein content in the tear fluid. The relatively high concentration of proteins coupled with a relatively rapid tear turnover leads to a considerable loss in the drug activity. For example, the mitotic activity of pilocarpine hydrochloride was tested in the presence and absence of added rabbit serum albumin. A 3-fold increase in the mitotic activity was obtained in absence of the protein compared with that in the presence of 3% w/v albumin. The mitotic activity decreased gradually depending on the available protein concentration (Mikkelson et al., 1973a).

The normal volume of tears, present in the human eye is approximately 7-10 µl. The average basal secretion is 1.2-1.5 µl/min and the physiological turnover rate is 0.1-0.15 min -1 . Upon instillation of fluid into the eye, the drainage of instilled solution proceeds at a rapid rate until the volume is back to normal volume, about 7 µl. Thus as the volume of instilled dose increases, the volume of solution lost by spillage on the cheek and drainage increases (Chrai et al., 1974; Chrai et al., 1973). The drop size is responsible for considerable loss of the drug and hence affects the ocular bioavailability. Furthermore, this drainage contributes to non-productive absorption and leads to unwanted side effects. Therefore, it is recommended that the size of the eye drop should be reduced from 50-75 µl/drop to 5-10 µl/drop to maximise drug activity from ophthalmic ocular dosage forms (Chrai et al., 1973). In support of previous findings, it has been found that the ocular bioavailability, estimated as the area under the miosis–time curve, in rabbits did not show

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any significant differences after the treatment using a 30 µl instillate of 1% w/v pilocarpine hydrochloride solution and the spray delivery of 5 µl volumes of the 1% w/v solution of pilocarpine hydrochloride. This study demonstrated that a 6-fold reduction in the administered volume achieved an equivalent miotic response (Martini et al., 1997). Non-productive absorption

Non-productive absorption is the absorption of topically applied ophthalmic drugs into the systemic circulation due to absorption from conjunctival blood vessels and dominantly from nasal mucosa due to the naso-lacrimal drainage (Chang & Lee, 1987; Shell, 1982). Most of the instilled dose (approx. 70%) is lost due to drainage via the naso-lacrimal duct within the first 15 to 30 s after instillation (Shell, 1982).

Non-productive absorption following ocular drug administration not only leads to decreasing the ocular bioavailability, but is also seriously responsible for systemic side effects. Systemic side effects were reported with many drugs topically applied to the eye surface. For instance, topical ocular administration of atropine (German & Siddiqui, 1970), timolol (Kaila et al., 1986), betaxolol (Polansky & Alvarado, 1985), epinephrine (Anderson, 1980), oxymetazoline (Duzman et al., 1983), pilocarpine (Urtti et al., 1985), flurbiprofen (Tang-Liu et al., 1984) and cyclosporine (Mosteller et al., 1985) has been associated with systemic side effects.

In addition, serious cardiovascular and pulmonary side effects were observed with topical ocular administration of timolol (a widely used anti-glaucomic drug) (Nelson et al., 1986). It has been reported that the nasal mucosa accounts for 70% of the timolol systemically absorbed (Chang & Lee, 1987). This is probably due to the consequence of rapid drainage of the instilled solution into the naso-lacrimal duct and then the nasal cavity.

The conjunctival systemic absorption of ocularly applied drugs is increased by the relative leakiness of the membrane, rich blood flow, and large surface area (Urtti & Salminen, 1993). The nasal cavity has a large surface area of 150-200 cm 2 and a total volume of 20 ml in humans. The nasal cavity surface area is 13 times that of the conjunctiva and almost 200 times that of the cornea (Lee et al., 2002; Pontiroli et al., 1989; Urtti & Salminen, 1993). The contact area between the instilled drug solution and nasal mucosa increases with increasing the instilled volume of the eye drop. The drug can gain access to the systemic circulation via absorption from nasal mucosa lining the mucosal cavity and/or via the gastrointestinal tract. Therefore, the absorption of the drug from the nasal region is reproducible and comparable to parenteral drug administration. This

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efficient systemic absorption has been utilised as a non-invasive route of systemic drug delivery of proteins and peptides (Lee et al., 2002).

Punctal occlusion and eyelid closure following topical solution instillation are the only measure recommended to the patients to minimise systemic drug absorption via the nasolacrimal apparatus (Kaila et al., 1986; Passo et al., 1984; Zimmerman et al., 1984). However, neither approach is a satisfactory long-term solution due to patient incompliance. The formulation approach could be a more effective solution. For example, both charged and neutral multilamellar liposomes have been shown to decrease the ocular drainage of [ 111 In]indium chloride and [ 99 Tc] sodium pertechnate compared with the buffer solution (Fitzgerald et al., 1987b). The two isotopes encapsulated in liposomes demonstrated prolonged precorneal residence time. The suspending medium drains away independently of the liposomes. As the size increases the nasolacrimal drainage decreases due to the larger liposomes restricted at the inner canthus region. In another study, they concluded that multilamellar liposomes have been found to reduce effectively the precorneal clearance rate and offer an advantage as a delivery system compared with small unilamellar vesicles, as the lower entrapment efficiency of the latter negate any advantages gained due to higher surface area (Fitzgerald et al., 1987a). Enzymatic degradation

A number of enzymes are secreted and detected in the precorneal tear film. These include carbonic anhydrases (Lonnerholm, 1974), esterases (Lee et al., 1982), peptidases (Stratford & Lee, 1985), ketone reductase (Lee et al., 1988) and glutathione-conjugating enzyme (Saneto et al., 1982). Depending on the nature of the applied drugs, these enzymes can adversely affect the biological activity of the applied active drugs or can activate topically applied prodrugs (Lee & Li, 1989).


Drug and formulations factors

Physicochemical properties of the drug

The three main drug properties governing corneal drug absorption are:

1. Lipophilicity as reflected by n-octanol/buffer partition coefficient.

2. Dissociation constant (pK a ) which determines the proportion of drug in its preferentially absorbed form at a given pH.

3. Drug molecular size.

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Firstly, drug lipid solubility (lipophilicity) is an essential property for drug permeation via the lipidic epithelial corneal membrane (Schoenwald & Ward, 1978). Drug lipophilicity is related to its chemical structure and can be assessed by measuring the partition coefficient (P). According to drug partition coefficients, β-adrenoreceptor blockers are classified into three classes: very lipophilic (P ranged from 1640 to 14200), lipophilic (P ranged from 76 to 249) and hydrophilic (P ranged from 1.46 to 59) (Schoenwald & Huang, 1983). Lipophilic β-blockers, such as timolol, propranolol, atenolol, metoprolol and practolol, are able to lower the intraocular pressure and are used successfully as antiglaucomic agents, whereas other β-blockers, such as nadolol and sotatol, appear not to. This could be ascribed to the marked difference in lipid solubility and their ability to cross corneal barriers. The corneal epithelium is the rate-determining barrier for hydrophilic drugs whereas it is not for lipophilic drugs. Lipophilic drugs penetrate the cornea more rapidly (Huang et al., 1983). For instance, dipivalyl epinephrine ester (apparent P = 4.89) has been found to be about 100 times more effective than epinephrine (apparent P = 0.0081) in the management of glaucoma and about 100-400 times weaker than epinephrine in affecting the cardiovascular system in dogs and cats (McClure, 1975; Wei et al., 1978). This is due to enhancement of lipophilicity of epinephrine through the use of dipivalyl ester which can facilitate penetration through the lipoidal layers of the cornea (Lee & Li, 1989). Topically administered drugs, however, must possess adequate aqueous solubility in tear fluid in order to achieve an adequate penetration rate, to diffuse across the water-filled corneal stroma and to gain access to deeper ocular tissues (Schoenwald & Huang, 1983). In another report, the optimal P for corneal drug absorption was found to be in the range of 10-100 (Kaur & Smitha, 2002).

Secondly, ionised drugs, such as weak acids and weak bases, can exist in both ionised and unionised forms at the pH of the lacrimal fluid. Unionised species usually penetrate more easily lipophilic barriers of the epithelial layer than ionised ones. Therefore, the degree of drug ionisation (expressed in pKa) in lacrimal fluid can determine how readily the drug passes epithelial corneal barriers. For example, the in vitro corneal permeability of pilocarpine base was 2-3 times greater than that of the ionized form (Francouer et al., 1983) and the miotic response of pilocarpine eye drops was greater at pH 7.0 than at pH 4.5 (Jarvinen et al., 1994). Timolol (weak base, pKa 9.2) concentrations in ocular tissues increase with increasing the pH of the instilled solution. pH values ranging from 6.2 to 7.5 are found to be optimum for ocular bioavailability (Kyyrönen & Urtti, 1990). Also, the charge of the drug molecule may influence its corneal

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permeability. Cationic drugs are expected to permeate the cornea more easily than anionic drugs where the latter encounters electrostatic repulsion from the negatively charged mucin layer on the eye surface as well as the negatively charged pores present in the corneal epithelium (Jarvinen et al., 1995; Le Bourlais et al., 1998; Loftssona & Jarvinen, 1999). However, the permeability of the positively charged drugs can be hampered by ionic interactions with the negatively charged carboxylic groups of the tight junction proteins (Hornof et al., 2005; Palmgren et al., 2002).

Thirdly, drug molecular size can also affect the amount of drug permeated through the tight junctions of the corneal epithelium. Drugs of medium molecular weight such as inulin (mol wt 5500) can cross the cornea solely by paracellular mechanism rather than transcellular and in smaller amounts than uncharged compounds of lower molecular weight (Lee et al., 1986). However, larger molecules such as insulin (mol wt ~6000) can only cross the cornea in the presence of permeation enhancers, such as saponin or Brij 78, which loosen the epithelial tight junctions (Pillion et al., 1991). Formulation factors

Some excipients were found to alter drugs’ ocular permeability, and consequently their ocular bioavailability from the used ophthalmic dosage forms. For instance, benzalkonium chloride is a cationic surfactant and is widely used as a preservative in ocular preparations. Benzalkonium chloride and other cationic surfactants have been found to enhance the ocular absorption of a number of drugs varying in molecular size and lipophilicity, including pilocarpine (Mikkelson et al., 1973b), carbachol (Smolen et al., 1973) and prednisolone (Green & Downs, 1974). This might be attributed to the amphiphilic nature of benzalkonium chloride, which imparts drug molecules with some penetration-enhancing properties through the corneal epithelium. Other formulation factors such as drop size, pH and the tonicity of the solution instilled into the surface of the eye must be physiologically compatible to minimise tissue irritation, reflex tearing and blinking, and consequently, this may lead to a substantial loss of the administered dose.

As mentioned above, the cul-de-sac of the eye normally holds 7-10 µl, but it can accommodate

up to 30 µl without overflowing. However, most commercially available eye droppers deliver a

drainage to naso-lacrimal duct and

drop of approximately 50 µl. This leads to reflex blinking,

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spillage of a significant portion of the instilled dose to the cheek until the tear film returns to the normal volume (Chrai et al., 1974; Chrai et al., 1973; Martini et al., 1997).

The normal pH of the tear fluid is 7.4. Increased blinking rate and lacrimation are associated with any disturbance of the physiological pH of the tear fluid. Therefore, the pH of the ophthalmic preparations should be formulated in pH 7 to 7.7 to minimise irritation to the eye surface (Ludwig, 2005). In some cases, however, the ideal pH for drug solubility and/or stability is outside this range. Therefore, it is recommended in this case to use the pH which can offer maximum stability, permeability and solubility of the administered drug with minimal buffer capacity, in order to allow the tear fluid to regain its normal pH rapidly (Ali & Lehmussaari, 2006; Van Ooteghem, 1993).

Parallel to the ophthalmic formulations’ pH, adjustment of the osmolality of the instilled formulations is an issue. Lacrimal fluid contains various dissolved electrolytes (Na, K, Ca, Cl, and HCO 3 ions) constituting about 310-350 mOsm/kg (Ludwig, 2005). Therefore, the instilled formulation must be isotonic with the tear fluid to minimise ocular irritation. The normal iso- osmolar range is 270–330 mOsm/kg (Waymouth, 1970). Generally, hypotonic solutions are more tolerable than hypertonic solutions. Osmolality lower than 260 mOsm/kg or higher than 480 mOsm/kg is considered irritating to the eye (Lang et al., 2002; Ludwig, 2005).

From the previous discussion, there seems to be a need for the development of an effective ocular delivery system for topically applied drugs, in order to overcome the aforementioned hurdles. The properties of the proposed ocular delivery system should be tailored to fit the major anatomical and physiological barriers of the eye.

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1.2. Drug delivery systems providing controlled and continuous ocular


Conventional ocular dosage forms such as simple solutions, suspensions and ointments account for approximately 90 % of currently marketed ophthalmic pharmaceuticals (Lang, 1995). This wide-spread use is due mainly to simplicity and patient acceptability. However, the ocular bioavailability of drugs topically applied from eye drops is typically less than 5%. This is due to the very short contact time with the ocular surface, normal tear turnover, conjunctival absorption and rapid drainage through the nasolacrimal duct. This prompts the clinician to recommend a frequent drug instillation at an extremely high concentration. This typical pulse-type dosing might pose safety concerns, by subjecting the ocular tissues to a relatively high local drug concentration and causing several systemic side effects. Therefore, conventional ocular dosage forms no longer constitute optimal therapy (Bochot et al., 1998; Urtti, 2006a). On the other hand, controlled release ocular drug delivery systems can offer many advantages compared with the conventional ones. These include:

Reducing the dose regimen with the goal of improving the patient compliance (Kaur et al., 2004; Kaur & Kanwar, 2002).

Decreasing the effects of washout when instilling multiple drops and hence decreasing systemic side effects (Kaur & Kanwar, 2002).

Minimising the preservative burden by decreasing the number of drops administered per day (Furrer et al., 2002a).

Formulation of topical ocular delivery system with consistent bioavailability and minimal adverse effects is still a challenging endeavour facing the pharmaceutical formulator (Kaur et al., 2004; White & Byrne, 2010). The two options currently available to improve ocular drug bioavailability are firstly, prolonging precorneal residence time and minimising precorneal loss and secondly, maximising ocular permeability (du Toit et al., 2011; Kaur et al., 2004). There are many formulation approaches which have been investigated. These formulation approaches are discussed below.

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1.2.1. Ophthalmic inserts

Ocular inserts are solid or semi-solid sterile preparations to be placed in the cul-de-sac of the eye or on the cornea, in order to prolong residence time (Gurtler & Gurny, 1995; Rathore & Nema, 2009). The size and shape of the inserts are tailored for ophthalmic application. They are composed of a polymeric support with or without drug(s). The drug is incorporated as dispersion or solution in the polymeric support. The inserts can be used for topical or systemic therapy (Gurtler & Gurny, 1995; Rathore & Nema, 2009). Ocular inserts are mostly inserted in the lower fornix and less frequently in the upper fornix or on the cornea. These solid ocular devices require minor surgery by which the polymeric system (into which the drug has been incorporated) is implanted inside the eye (Gurtler & Gurny, 1995). Based on solubility behaviour, ophthalmic inserts can be classified as insoluble, soluble and bioerodible inserts. Insoluble inserts

Insoluble inserts can be classified into three groups: diffusional, osmotic systems and contact lenses (Rathore & Nema, 2009). The first two systems include an insoluble membrane surrounding a drug reservoir to control the drug release rate. The reservoir contains a liquid, gel, colloid, semisolid or solid matrix containing a drug homogeneously or heterogeneously dispersed or dissolved therein. Drug-controlling membranes are composed of a wide range of polymers including hydrophobic, hydrophilic, organic, inorganic, naturally occurring or synthetic materials (Gurtler & Gurny, 1995). The third class includes contact lenses which are fabricated using insoluble ophthalmic devices. These classes are likely associated with a high rate of patient incompliance resulting from foreign body sensation due to insolubility of these ocular devices (Rathore & Nema, 2009). Soluble inserts

Ocular soluble inserts are completely soluble or biodegradable. This is likely to be an advantage over insoluble ocular inserts, because the former do not need to be removed from their site of application. Ocular soluble inserts can be formulated from natural polymers, synthetic or semi- synthetic polymers (Gurtler & Gurny, 1995; Rathore & Nema, 2009). For example, a soluble insert loaded with gentamicin sulfate and dexamethasone phosphate was prepared (Baeyens et al., 1998). The system not only provided a sustained drug release but also showed a concomitant release of the two drugs for the first 10 h, followed by a sustained release of gentamicin over a period of 50 h. However, blurred vision was experienced by patients during treatment. In addition, bioerodible inserts are composed of a drug homogenously dispersed in monolithic

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matrix devices to control the drug release rate. The main components used for the production of this type of inserts are bioerodible polymers. Bioerodible inserts-based polymers undergo hydrolysis of chemical bonds and hence dissolve over a prolonged period of time, in response to the environment in the eye. Further, a soluble bioadhesive ophthalmic drug insert (BODI) contained 25% w/w gentamicin sulphate was inserted into the lower cul de sac of dogs’ eyes. The clinical efficacy of the treatment of keratoconjunctivitis was compared with that of the aqueous gentamicin eye drops. The total clinical recovery outcomes from the BODI and gentamicin eye drops were obtained after 3 and 7 days respectively. The BODI significantly reduced the treatment period and improved the patient compliance when compared with the aqueous drug solution (Baeyens et al., 2002). Acyclovir water-soluble inserts were fabricated from polyvinyl alcohol and methylcellulose. Both the rate and acyclovir release profile were modified depending on the additives used. The prepared inserts were stored for 6 months at 25 o C and the aged inserts demonstrated no change in the drug release profiles indicating good physical stability (El Gamal et al., 2008).

1.2.2. Collagen shields

Collagen shields are ocular devices fabricated from porcine or bovine scleral tissues, which bear a collagen composition similar to that of the human cornea and sclera. These ocular devices should be hydrated before insertion to the eye (Lee, 1990; Willoughby et al., 2002). Typically, the drug is loaded into the collagen shield by soaking it in the drug solution for a period of time prior to application. Once in the eye, shields are hydrated by tear fluids, soften and form a clear, pliable, thin film approximately 0.1 mm in thickness with a diameter of 14.5 mm and a base curve of 9 mm that conforms to the corneal surface and is designed to dissolve slowly within 12, 24, or 72 h (Lee, 1990).

Collagen shields have been utilised as ocular delivery systems for many pharmacological classes of drugs such as antibiotics, antifungal agents, steroids and immunosuppressive agents (Lee et al., 1992; Schwartz et al., 1990; Unterman et al., 1988; Vasantha et al., 1988; Willoughby et al., 2002). Collagen shields pre-soaked in tobramycin produced significantly higher concentrations of antibiotic in the cornea at one hour than subconjunctival injections of tobramycin (Unterman et al., 1988). The authors claimed that collagen shields containing antibiotics can serve as a vehicle for drug delivery and may prove superior to current methods for preoperative and postoperative antibiotic prophylaxis and the initial treatment of bacterial keratitis.

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In addition, amphotericin B (an antifungal agent) was formulated in collagen shields. The prepared ocular devices were able to maintain ocular drug levels in the anterior segment of rabbit eyes, as well as frequent-drop delivery with the potential benefit of added convenience and compliance (Schwartz et al., 1990). Further, a combination of gentamicin and methypredinsolone, commonly used drugs in treatment of ocular problems, was formulated in a collagen shield as prolonged drug release ocular devices. However, severe corneal toxicity was reported due to sudden rapid drug release from the device (Lee et al., 1992).

In other two reports, collagen shields pre-soaked in aqueous antibiotic solutions were studied for a potential topical ocular treatment for bacterial endophthalmitis (one of serious complications after intraocular surgery) (Hariprasad et al., 2005; Haugen et al., 2008). In one study, both 0.3% w/v gatifoxacin eye drops and collagen shield pre-soaked in 1% w/v gatifloxacin solution achieved significantly lower endophalmitis incidences than balanced salt solution controls in rabbits. However, no statistically significant difference was obtained between the presoaked collagen shields and the eye drops (Haugen et al., 2008). In the other study, collagen shields pre- soaked in 0.5% aqueous solution of moxifloxacin were studied as a topical ocular treatment option for prevention of bacterial endophthalmitis in 10 patients prior to ocular surgery (Hariprasad et al., 2005). The aqueous humour moxifloxacin levels estimated for collagen shields were lower than those for topical drops. However, the ability to leave the eye batch undisturbed after surgery and avoid patient manipulation of the eye post surgery, so as to instill topical drops could be a potential advantage for this route, especially in the immediate post-operative period (Hariprasad et al., 2005).

1.2.3. Polymeric gels

Polymeric gels can be divided into two groups: preformed hydrogels and in situ forming gels (Le Bourlais et al., 1998). Preformed or bio-adhesive hydrogels

Bioadhesive hydrogels (hydrocolloids) are hydrophilic polymers which tend to swell, viscolise, gel and adhere upon contact with physiological mucus membranes by virtue of hydrophilic functional groups and structures. Bioadhesive polymers are capable of forming strong non- covalent bonds with the mucin coating mucus membranes and reside in place as long as the

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mucin is present (Le Bourlais et al., 1998). Over the past three decades bioadhesive hydrogels have been utilised in topical ocular drug delivery to prolong the ocular residence time and minimise the frequency of the instilled drops (Robinson & Mlynek, 1995). For instance, betaxolol, a β-adrenoreceptor antagonist, polyacrylic acid (PAA) gel showed significantly higher bioavailability than a 0.5% w/v solution of betaxolol in rabbits (Weinreb & Jani, 1992). Also, acetazolamide (an antiglaucomic agent) formulated in carboxymethylcellulose hydrogel showed a prolonged effect when compared with the drug solution (Tous & El Nasser, 1992). Moreover, polycarbophil gel enhanced the ocular delivery of topically applied gentamicin in rabbits. The polymeric formulation increased the uptake of gentamicin by the bulbar conjunctiva twice, compared with an aqueous control formulation (Lehr et al., 1994). A three-fold increase of the precorneal residence time of tobramycin was achieved when formulated in a chitosan gel compared with the commercial drug solution of the drug (Ludwig, 2005). Xyloglucan [tamarind seed polysaccharide (TSP)] was tested as a novel vehicle for ophthalmic delivery of timolol (Burgalassi et al., 2000). The polymer used, in spite of a comparatively low viscosity, showed high timolol concentrations in the ocular tissues and a low systemic absorption compared with the commercially available product based on in situ gelling (Timoptic XE ® ). The results presented TSP as a potentially useful carrier for ophthalmic delivery systems.

One of the most successful applications of hydrogels in ophthalmology is their use as tear substitutes due to their commercial availability. For example, celluloses (Lacril ® ), polyvinyl alcohol (Liquifilm ® ), polyacrylic acid (Lacrigel ® , Lubrithal ® , Gel-Larm ® ), and hyaluronic acid (Hy-Drop ® ) are frequently used as tear substitutes for the treatment of dry eye disease (Tsubota & Dogru, 2006; Zignani et al., 1995). Bioadhesive polymers, however, are associated with some pitfalls as an ocular drug delivery system. The drop size or volume of commercial ocular medication formulated using bioadhesive polymers is not as uniform as conventional ocular solutions. The amount delivered to the eye, consequently, is generally incorrect. Moreover, the presence of a viscous vehicle can cause blurred vision and formation of a veil in the corneal area leading to loss of eyesight (Winfield et al., 1990). In situ activated gel forming systems

The abovementioned problems related to the accuracy of the instilled dose and blurred vision from the preformed hydrogels could be overcome by in situ activated gel delivery systems. In situ activated gel-forming systems can be described as viscous liquids that upon exposure to

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physiological conditions will shift to a gel phase (Le Bourlais et al., 1998). Three methods causing phase transition on the eye surface have been reported in the literature: a change in temperature (Miller & Donovan, 1982), pH (Gurny, 1981; Gurny et al., 1987) or electrolyte composition (Rozier et al., 1989). Other methods can induce in situ gel activation have been recently reported in the literature such as UV/visible light-irradiation (photopolymerisable hydrogels) and enzyme mediated gelation, however, they have not been investigated for ocular drug delivery (Van Tomme et al., 2008). Here are some applications of various methods used to induce in situ sol/gel transition on the surface of the eye.

Poloxamers or pluronics are block co-polymers consisting of poly (oxyethylene) and poly (oxypropylene) units. They rapidly undergo thermal gelation when the temperature is raised to that of the ocular surface, while they remain liquid at refrigerator temperature. A mixture of pluronic F127 and pluronic F68 was prepared as an in situ gelling system for ocular delivery of sparfloxacin (antibacterial agent) for treatment of bacterial keratitis. The developed formulations provided sustained release of the drug over a 24-h period and better improvement in artificially induced bacterial conjunctivitis in rats' corneas (Nesseem, 2011). However, Poloxamers exhibit thermal gelation at high concentrations (usually between 20 and 30% w/v); Pluronic F127 has been found to be more damaging to the cornea than a physiological saline solution (Furrer et al., 2000). The relatively high polymer concentration (20-30% w/v) required for in situ thermal gelation can pose toxicological concerns (Miyazaki et al., 2001).

Cellulose acetophthalate (CAP) latex is defined as a highly unstable system which coagulates when its native pH of 4.5 is raised by the tear fluid to pH 7.4(Gurny, 1981). This pH-induced coagulation of CAP occurs at a high polymer concentration (30% w/v) (Gurny, 1981; Kumar et al., 1994). Also, carbopol is a polyacrylic acid (PAA) polymer, which shows sol-gel transition in an aqueous solution as the pH is raised above its pKa of about 5.5. As the concentration of carbopol increases in the vehicle in order to improve its rheological properties, the acidity of the vehicle increases. Hence, the inherent acidity of the vehicle could induce ocular tissue irritation and induced lacrimation (Nanjawade et al., 2007).

An example of the ion-activated gelation is gellan gum (an anionic polysaccharide) which exhibits phase transition with increased ionic strength. Also, it has been found that the extent of

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gel-formation of gellan gum increases proportionally with the amount of mono- or divalent cations present in the tear fluid. Hence, the main triggering effect inducing phase transition is due to the generous availability of mono- and divalent cations associated with reflex tearing (Greaves et al., 1990). The dependency of the gel transition of some in situ gelling systems on the uptake of cations present in the tear film may be disrupted in the presence of dry eye syndromes (especially with diabetic patients). Moreover, the dependency on electrolytes for gel transition is likely to hinder the tonicity adjustment and consequently, the final product is not iso-osmotic with the tear fluid.


Colloidal delivery systems


Microemulsions (MEs) have been utilised as topical ocular drug delivery systems because of their inherent properties and structures (Vandamme, 2002). The use of MEs for ocular drug delivery is advantageous because the presence of surfactant and co-surfactant increases membrane permeability to the applied drug (Lawrence & Rees, 2000). Moreover, these systems offer the additional advantage of having low viscosity and the convenience of eye drops.

Pilocarpine-based MEs have shown more sustained drug activity than the drug solution where twice daily instillations of these systems were equivalent to four instillations of conventional eye drops (Naveh et al., 1994). Recently, ME-based phase transition systems have been prepared and evaluated as ocular delivery systems using pilocarpine hydrochloride as a model water-soluble drug (Chan et al., 2007). These systems underwent phase transition from ME to liquid crystalline (LC) and to coarse emulsion (EM) with a change in viscosity depending on water content. ME and LC showed higher drug release retarding efficiency than EM. However, toxicity and tissue irritancy have been observed with microemulsion formulations. For example, ocular application of ME and LC disrupted the precorneal tear film and the tear evaporation rate increased compared with the aqueous solution. This is due to the use of relatively high concentrations of surfactants and co-surfactants within the formulations (Alany et al., 2006; Chan et al., 2008). Nanosuspensions

The ocular formulation of poorly water-soluble drugs is not an easy task. These drugs suffer from erratic ocular absorption and reduced availability (Kayser et al., 2005). This class of drugs can be formulated as a nanosuspension in an appropriate dispersing vehicle, in an attempt to reduce the

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aforementioned problems. Drug compounds that form crystals with high energy content, rendering them insoluble in either organic (lipophilic) or hydrophilic media, are good candidates for formulation as a nanosuspension (Kayser et al., 2005). Thus, the use of nanosuspensions in ophthalmic pharmaceutical formulations is likely to be an attractive area offering a great possibility to overcome the inherent difficulties associated with the ocular delivery of poorly water-soluble drugs. For instance, methyl prednisolone was prepared in a nanosuspension. The prepared formulations showed localised and controlled ocular anti-inflammatory activity in rabbits (Adibkia et al., 2007). Polymeric microspheres and nanoparticles

Polymeric microspheres and nanoparticles are polymer-based particulate drug delivery systems. The difference between these systems is based on their size. Nanoparticles can be defined as particles with a diameter of less than 1 µm, whereas particles in the micrometre size (> 1 µm) are called microspheres (Ali & Lehmussaari, 2006). They can be fabricated from various biodegradable polymers such as natural and synthetic polymers. Drugs can either be dispersed in the polymeric matrix or physically bound to the surface. Polylactides (PLAs), polycyanoacrylate, chitosan, gelatin, sodium alginate and albumin were studied for efficient drug delivery to the ocular tissues (Sahoo et al., 2008).

For instance, Polylactide-based nanoparticles (NPs) encapsulating two flourochromes (Rhodamine-6G and Nile red) were tested as drug delivery systems targeting retina and retinal pigment epithelium (RPE) and applied intravitreally (Bourges et al., 2003). NPs were able to give a high local concentration of the tested compounds at the RPE suggesting the feasibility of targeting the posterior segment of the eye by NPs. Moreover, chitosan is a cationic polysaccharide and is obtained by deacetylation of chitin. Chitosan has been shown to promote intraocular drug penetration with good ocular tolerability (Zambito & D i Colo, 2010). Chitosan- based nanoparticles have been prepared to enhance the ocular bioavailability of indomethacin, an anti-inflammatory drug used to minimise ocular inflammation after cataract surgeries. The prepared ocular carriers showed controlled and higher bioavailability than that of the drug solution (Badawi et al., 2008). Solid lipid nanopraticles

Solid lipid nanoparticles (SLNs) are used as a colloidal carrier system for controlled drug delivery. SLNs represent an alternative carrier system to emulsions and polymeric nanoparticles.

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The main reasons for their development is more stable alternatives to liposomes and the combination of advantages from different carriers systems like liposomes and polmeric nanoparticles (Muller et al., 2000).

SLNs have been evaluated for ocular controlled drug delivery (Cavalli et al., 2002; Cavalli et al., 1995; Gonzalez-Mira et al., 2011). For instance, tobramcin-loaded SLNs produced significantly higher bioavailability in aqueous humour when compared with an equal dose of tobramycin administered using standard commercial eye drops (Cavalli et al., 2002). Physical instability and encapsulating water-soluble drugs into the lipidic core are still unresolved issues. It has been found that SLN formulations gelled after a relatively short period of storage. Although this can be prevented by altering the composition and concentration of stabilising surfactants, these stabilising additives are hampered by toxicological considerations and incompatibilities with the incorporated drugs due to chemical degradation (Freitas & Muller, 1999). Recently, flurbiprofen- loaded SLNs (FB-SLNs) have been prepared from stearic acid, miglyol 812 and castor oil. The prepared FB-SLNs were evaluated for ocular toxicity using the ocular Draize test. The tested formulations were found to be minimally irritant (Gonzalez-Mira et al., 2011).


Dendrimers are artificial nano-constructs and as the name suggests, have a tree-like structure. Their basic units are active chemical moieties, which construct around small molecules or the core using connectors and branching units. Poly(amidoamine) (PAMAM), poly-lysine, or poly(oropylenimine)-based dendrimers are amongst the preferred classes of these (Jain & Asthana, 2007; Tomalia et al., 1985). The final shape of the dendrimers is globular, with hollow internal cavities and a number of terminal groups. By virtue of the highly branched terminals, a number of drugs can be encapsulated or attached to the surface or peripheral group and hence, dendrimers can be utilised as a potential drug delivery system. For instance, many poorly soluble drugs can be properly encapsulated in the internal cavities of dendritic constructs (Cheng et al., 2008; Jain & Asthana, 2007; Quintana et al., 2002; Tomalia et al., 1985).

PAMAM dendrimers were prepared for controlled ocular delivery of pilocarpine nitrate and tropicamide (Vandamme & Brobeck, 2005). The macromolecular carriers showed a prolonged ocular residence time compared with 0.2% w/v carbopol 980 NF solution. The residence time of the dendrimers was markedly dependent on the size and molecular weight of the prepared

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carriers. The main drawback which hampers the wide use of dendrimers as a drug delivery system is the lack of sufficient evidence of their safety and toxicity (Jain & Asthana, 2007). Recent evidence has shown that fatal complication such as disseminated intravascular coagulopathy was reported after iv injection of small doses (> 10 mg/kg) of positively charged PAMAM dendrimers generations 3.5 to 7 in sizes of 50 nm in mice. Also, hemobilia (haemorrhage of the biliary tract) and a preferential toxicity on the intestinal wall in terms of haemorrhage and intraluminal bleeding were recorded with oral administration of the PAMAM dendrimers (Greish et al., 2010). Cubosomes

Cubosomes are submicron lipid particles of bicontinous cubic liquid crystalline phase. They have been proposed as a colloidal delivery system for lipophilic drugs for better solubilisation and a sustained drug release effect (Boyd, 2003; Spicer, 2005). Research on cubosomes, as drug carriers for ocular delivery, has been scarce (du Toit et al., 2011).

Cubosomes were prepared from a mixture of monoolein (glycerol mono-oleate) and water by high pressure emulsification using poloxamer 407 as a dispersing agent (Gan et al., 2010). These cubosomes were formulated as an ocular delivery system for dexamethasone (DEX). Ex vivo permeation studies, using excised rabbit corneas showed that the apparent permeability coefficient calculated for the prepared DEX cubosomes was 3.5-4.5 times greater than the DEX aqueous solution. Also, the ocular bioavailability for DEX cubosomes was 1.8 times greater than that for the DEX aqueous solution. Histological examination of the excised rabbit corneas incubated in DEX cubosomes for 2 h did not show any harmful signs. These findings might present cubosomes as a new ocular delivery system for water-insoluble drugs (Gan et al., 2010). Vesicular delivery systems

The abovementioned ocular delivery devices can achieve more controlled and effective therapeutic actions with minimal systemic and ocular side effects associated with pulse-type conventional dosage forms. However, these systems are still not devoid of pitfalls, including:

Poor patient compliance during insertion, as in ocular inserts.

Tissue irritation and damage induced by penetration enhancers and co-surfactants commonly used to stabilise microemulsion and collagen shields (Alany et al., 2006; Kaur et al., 2004).

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Ocular toxicity of some polymers used to fabricate microspheres and nanoparticles such as albumin and polybutylcyanoacrylate (Zimmer & Kreuter, 1991).

Toxicological concerns due to relatively high polymer concentrations required for some in situ thermal gelation (Miyazaki et al., 2001).

Failure of drug delivery devices. For example, some in situ gelling systems rely on a generous supply of cations from the resident tears for sol-to-gel phase transition. Alteration of the composition of the tear fluid and/or the presence of dry eye symptoms (common with diabetic patients) (Akinci et al., 2007; Dogru et al., 2001; Kaiserman et al., 2005) might compromise the sol-to-gel phase transition of these systems. Hence, these systems might not be optimal for drug delivery in diabetic patients.

Surfactant vesicles (liposomes and niosomes) could overcome the aforementioned hurdles (du Toit et al., 2011; El-Gazayerly & Hikal, 1997; Gregoriadis & Florence, 1993; Le Bourlias et al., 1995; Uchegbu & Vyas, 1998). They can offer a prolonged and controlled action at the corneal surface and a substantial increase of ocular bioavailability, by providing an intimate contact of the drug with the lipidic corneal barrier and preventing the metabolism of the drug by the enzyme present at the precorneal tear film (Azeem et al., 2009; Kaur et al., 2004). Moreover, lipid vesicles offer a promising approach to meet the need for an ophthalmic drug delivery system that has the convenience of being in the form of eye drops, but will localise and maintain drug activity at its site of action (Azeem et al., 2009; Kaur et al., 2004). Furthermore, plain liposomes have been used in a form of spray for topical ocular administration for treatment of dry eye symptoms. Two commercially available liposome products, clinically indicated for the management of dry eye, are ClaryMist ® manufactured by Savant, UK and Ocusoft ® manufactured by Ocusoft, USA. They are sprayed on the eyelids to improve the stability of the lipid layer of the tear film and treatment of dry eye symptoms (Craig et al., 2010).


Liposomes are closed bilayer lipid vesicles that can be produced basically from natural non-toxic phospholipids and a membrane stabilising lipid, most notably cholesterol. Liposomes offer many advantages as a unique drug delivery system. They are biodegradable and biocompatible; they can encapsulate both hydrophilic drug moieties in their aqueous milieu and lipophilic moieties between hydrocarbon chain bilayer membranes; and their properties can be tuned with lipid

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composition, size, surface charge and the method of preparation (Kaur et al., 2004; Schaeffer & Krohn, 1982).

Liposomes encapsulating gancyclovir and iododeoxyuridine (antiviral drugs) were prepared for more efficient treatment of herpes simplex virus eye infections (Norley et al., 1986). It was found that these delivery systems could act as improved vehicles for drug delivery in treatment of ocular herpes simplex virus infection. Also, antisense oligonucleotides which can be used efficiently to treat ocular diseases like cytomegalovirus retinitis can be encapsulated in the liposomes and efficiently targeted at the retina (Bochot et al., 2002). Moreover, liposomes encapsulating mitomycin C (MMC) were prepared in an attempt to minimise the toxicity of mitomycin on ocular tissues. Liposomes containing 0.2 mg/ml of MMC were tested against aqueous solutions and tamarind seed polysaccharide (TSP)-based viscous solutions. The results showed reduced cytotoxicity of MMC in TSP viscous vehicles and liposomes compared with the drug solution. Only liposomes encapsulating MMC vehicles produced both lower toxicity for a rabbit corneal epithelial cell line culture and a marked reduction of the corneal healing rate in vivo (Chetoni et al., 2007). Recently, fluconazole liposomes have been found to be more effective than the fluconazole aqueous solution in treating Candida keratitis (corneal inflammation due to fungal infection) in rabbits. The fluconazole liposome-treated group showed complete corneal healing for 86.4% of the infected rabbits compared with 50% with the fluconazole solution treated group. Liposomes offer more intimate contact with corneal barriers and can enhance corneal drug absorption (Habib et al., 2010).


Over the last three decades, liposomes of different compositions used for topical ophthalmic drug delivery have increased/prolonged the therapeutic effect while minimising toxic effects (Chetoni et al., 2007; du Toit et al., 2011; Gregoriadis & Florence, 1993; Kaur et al., 2004; Treblay et al.,


Vesicles consisting of one or more surfactant bilayers enclosing aqueous spaces are called non- ionic surfactant vesicles or simply niosomes. Niosomes are considered of particular interest as they offer several advantages over liposomes:

Niosomes are more stable chemically (Kaur et al., 2004; Uchegbu & Florence, 1995).

Niosomes incur lower production cost due to the availability of starting materials (Sahin,


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Niosomes are biodegradable and non-immunogenic (Kaur et al., 2004).

Niosomes do not require expensive handling (storing in a freezer and preparation under nitrogen gas).

Nevertheless, the formulation of niosomes can pose challenges due to possible physical instability and poor ability to encapsulate a considerable amount of water-soluble drug molecules (Essa, 2010). Niosomes have been evaluated as ocular vehicles for a wide range of therapeutic classes such as anticholinergic (cyclopentolate), anti glaucomic (acetazolamide and timolol maleate) and antibiotics (gentamicin) with minimal signs of ocular irritation as well (Abdelbary & El-gendy, 2008; Aggarwal et al., 2004; Saettone et al., 1996b; Vyas et al., 1998). Tween 20- based niosomes significantly improved the ocular bioavailability of cyclopentolate, with respect to the reference buffer drug solution and micellar solution, suggesting that it can be used as an efficient vehicle for ocular drug delivery (Saettone et al., 1996b). Also, niosomes encapsulating timolol maleate (anti-gluacomic agent) were prepared using the reverse phase evaporation method and coated with 0.5% w/v chitosan solution. The timolol niosomes achieved a significantly (1.7 times) higher timolol concentration in the aqueous humour than that of the drug solution. This was attributed to better improvement of corneal penetration and prolonged precorneal residence time compared with the timolol aqueous solution (Kaur et al., 2010). Niosomes should have certain attributes in relation to their potential use as vehicles for ocular drug delivery. These include:

The size of the vesicle should be large enough to resist drainage by reflex tearing and eye blinking. For example, large multilamellar vesicles (MLV) were found to reside for a longer period on the ocular surface than small unilamellar liposomes. This was attributed to the ability of MLV to entrap a relatively higher amount of the drug and resist nasolacrimal drainage (Fitzgerald et al., 1987a; Hathout et al., 2007). The same argument applies to niosomes. Ideally, it has been reported that niosome sizes > 10 µm are suitable for ocular delivery (Sahin, 2006; Uchegbu & Vyas, 1998).

The shape of the prepared niosomes should show some irregularities to fit properly into the cul-de-sac of the eye and lodge on the eye surface (Uchegbu et al., 1992; Uchegbu et al., 1997; Uchegbu & Vyas, 1998).

Ocular niosomes should ideally be thermo-responsive in order to release drug content in a controlled, yet timely manner before being flushed by blinking and nasolacrimal drainage

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(Uchegbu et al., 1997; Uchegbu & Vyas, 1998). Complete abolishment of gel/liquid transition of surfactant forming niosomes could produce niosomes with an extremely low release rate to water soluble drugs, which does not suit the short residence time of ocularly administered ophthalmic products.

The abovementioned criteria have typically been attributed to discomes and polyhedral niosomes. These are modified vesicular structures and are different from conventional spherical niosomes (Azeem et al., 2009; Uchegbu & Florence, 1995; Uchegbu & Vyas, 1998). Discomes containing a low cholesterol concentration less than 30% mol/mol were prepared by incubating the preformed spherical niosomes in cholesteryl poly-24-oxyethylene ether (Solulan C24) at 75 o C for 1 h. This resulted in the formation of large (11-60 µm) and multifaceted vesicular systems (Uchegbu et al., 1992). Since then, there has been only one report on discomes as an ocular delivery system for timolol maleate (Vyas et al., 1998). The prepared discomes have been found to entrap a relatively large quantity of timolol and improve the ocular bioavailability compared with a timolol maleate solution (Vyas et al., 1998). One possible reason for such scarcity is the need for a relatively high temperature during discome preparation which might affect the chemical stability of some thermo-labile therapeutic agents. On the other hand, spherical niosomes have been shown to possess more stable membranes than polyhedral ones due to the presence of cholesterol (Arunothayanun et al., 1999).

In conclusion, ophthalmic niosomes could offer prolonged drug release and enhance drug permeation. Thus, the potential of niosomes as a vehicle for ocular delivery of naltrexone hydrochloride are studied in this thesis. Naltrexone hydrochloride has been found to be a promising treatment for diabetic keratopathy. Causes, prevalence and other treatment options of diabetic keratopathy are discussed in the following sections.

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1.3. Diabetic keratopathy and the available treatment options

1.3.1. Physiology of the cornea

The physiological functions of the cornea are (Klyce & Beuerman, 1998; Nishida, 2005):

Refractive: it transmits and focuses light onto the retina.

Protective: it forms a mechanical and chemical barrier to the posterior segment of the eye.

Regulatory: it contains the intraocular pressure.

1.3.2. Diabetes mellitus and the cornea

Diabetes mellitus has increasingly attracted the attention of scientists, especially since the WHO’s announcement that the worldwide burden of diabetes in adults was around 173 million in the year 2002”. The incidence of diabetes has risen dramatically in recent decades and a two- fold, or greater, increase is expected to occur in the coming decades (Wild et al., 2004).

Diabetes mellitus has a serious impact on the tissues of the entire eye. For instance, diabetic retinopathy (damage of the retina from diabetes) and cataracts (opacity of the eye lens) are major ocular complications arising from diabetes. Diabetic retinopathy and cataracts are the leading causes of vision loss in the industrial world. These disorders account for approximately 6% of the US population and 17% of Americans ≥ 40 years, respectively (Frank, 2004; Kenny et al., 1995). However, in the last four decades, diabetic retinopathy has been investigated fully. Advances in treatment over the past 40 years have greatly reduced the risk of blindness from this disease (Frank, 2004; McLaughlin et al., 2010).

On the other hand, diabetic effects on the anterior segment of the eye have been reported for many decades (Herse, 1988; Hyndiuk et al., 1977; McLaughlin et al., 2010; Schwartz, 1974). Observed diabetic abnormalities in the anterior segment of the eye include:

Decreased tear production (Herse, 1988).

Presence of glucose in tears (Gasset et al., 1968).

Increased risk of infection (Herse, 1988; Schultz et al., 1981 ).

Corneal epithelial fragility (Schultz et al., 1981 ).

Wrinkling of Descemet’s membrane (Henkind & Wise, 1961).

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Reduced corneal sensation (Ishida et al., 1984; Schwartz, 1974).

Delayed wound repair (Lambiase et al., 2000; Lambiase et al., 1998b).

Neurotrophic corneal ulcers (Hyndiuk et al., 1977).

Corneal disorders secondary to diabetes (diabetic keratopathy) are increasingly recognized as a cause of the morbidity associated with diabetes (Cisarik-Fredenburg, 2001; Kaji, 2005). Indeed, diabetic keratopathy has been estimated to occur in 47-64% of diabetic patients during the course of their disease (Schultz et al., 1981 ) and diabetics have an increased risk of developing corneal epithelial fragility, epithelial defects, recurrent epithelial erosions, decreased sensitivity, abnormal wound healing, increased susceptibility to injury and non-healing or infected corneal ulceration. Ultimately such corneal complications may lead to visual impairment. Diabetic keratopathy is mainly attributed to corneal nerve tortuosity and degeneration as part of general neuropathy of diabetes mellitus (Rosenberg et al., 2000; Ruben, 1994; Schwartz, 1974). Nevertheless, the available treatment options for diabetic keratopathy are tear substitutes and antibiotics. Such treatments are symptomatic and conservative to keep the cornea hydrated with minimal microbial burden. There has not yet been a causative treatment developed for this serious diabetic condition. However, damage of the corneal nerves due to diabetes has been thought to be the main culprit (Feman, 2000; McLaughlin et al., 2010; Schultz et al., 1981 ). The following section sheds light on corneal nerve role and structure.

1.3.3. Corneal innervations

Innervations of the cornea are essential for pain sensation and for tissue repair (Nishida, 2005). The cornea is the most densely innervated mammalian tissue (Figure 1.5). Human cornea has a nerve density of 300-600 times greater than that of the skin and 20-40 times greater than that of the tooth pulp (Muller et al., 1997). Most of the sensory nerves in the cornea are derived from the ciliary nerves of the ophthalmic branch of the trigeminal nerve (Nishida, 2005). Nerve bundles enter the corneal mid stroma at the periphery, each containing a few myelinated fibres and a number of finer unmyelinated fibres. The unmyelinated nerve endings are sensitive to touch, temperature and chemical stimulation (Muller et al., 1997). Because transparency is of prime importance, the cornea is avascular and receives necessary nutrients via diffusion from the tear film, the aqueous humour and from neurotrophins supplied by nerve fibres (Gobbels et al., 1989). Axons from the trigeminal ganglion and sympathetic ganglion terminate in delicate endings among the epithelial cells of the cornea.

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Chapter 1….Literature Review Figure 1.5 Human corneal nerves penetration and distribution, modified from (Muller et al.,

Figure 1.5 Human corneal nerves penetration and distribution, modified from (Muller et al., 1997) and reproduced with permission of the copyright owner.

Many ocular, neurological and systemic diseases can alter the typical corneal nerve architecture and impair corneal sensitivity (Table 1.1) (Lambiase et al., 1999). A major complication of that is the neurotrophic corneal ulcer. It is a degenerative corneal disease characterised by the impairment of corneal sensitivity associated with epithelial breakdown, a deficiency in the healing process leading to corneal ulceration, and subsequent vision loss. Neurotrophic corneal ulcers are classified into three stages (Mackie, 1995):

Stage 1 is characterised by rose bengal staining of the inferior palpebral conjunctival surface, a decrease in tear breakup time, and punctate keratopathy. If these changes become chronic, superficial vascularisation, stromal scarring, epithelial hyperplasia and irregularity may develop.

Stage 2 is characterised by epithelial breakdown where cells assume an oval or circular shape that is often localised in the superior half of the cornea. The area of epithelial deficit is usually

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surrounded by loose epithelium that, in the absence of a healing process, becomes hazy, oedematous, and poorly adherent to Bowman’s layer. The edges of the defect become smooth and rolled as the defect ages without appreciable epithelial growth (Cavanagh et al., 1976).

Stage 3 is characterised by stromal involvement with a corneal ulcer that may progress to melting and perforation. Secondary infection or topical treatment with corticosteroids increases the risk of perforation (Groos, 1997).

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Table 1.1 Causes of decreased corneal sensitivity




Riley-Day syndrome (familial dysautonomia) Goldenhar-Gorlin syndrome Mobius syndrome Familial corneal hypoesthesia


Diabetes Leprosy Vitamin A deficiency

Central nervous system

Neoplasm Aneurysm Ischaemia Neurosurgical procedures for acoustic neuroma or trigeminal neuralgia Other surgical injury to the trigeminal nerve


Herpes infection (herpes simplex or herpes zoster) Chemical and physical burns Abuse of topical anaesthetics Topical toxicity (timolol, betoxolol, diclofenac sodium, sulfacetamide) Surgical or laser treatment to cornea Corneal dystrophies (lattice, granular) Chronic ocular surface injury or inflammation Contact lens wear

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Over the past few years, there has been growing interest in exploring the mechanisms by which various growth factors and therapeutic agents promote healing of corneal epithelial cells in order to establish any significant correlations between topical administration and accelerated corneal healing (Kaji, 2005; Micera et al., 2004; Schultz et al., 1981 ; Woo et al., 2005). Before discussing the treatment options, different factors ameliorating altered corneal healing will be mentioned.

1.3.4. Mechanisms of corneal maintenance and healing

The corneal epithelium is composed of a squamous epithelium of 5-7 layers of stratified cells attached to a basement membrane and is separated from the stroma by Bowman’s layer. Trauma, disease and corneal surgery, including the increasingly popular refractive laser surgery, all cause the cornea to enter a phase of wound healing. Upon wounding, the cornea, which has less immune protection than other tissues due to the lack of blood vessels, needs to re-epithelialise urgently in order to restore its barrier function.

The healing of epithelial wounds can be divided into several distinct but continuous phases:

sliding of superficial cells to cover the denuded surface, cell proliferation, and stratification for re-establishment of multicellular layers (Lu et al., 2001). Although several layers participate in cell migration, the majority of the defect is initially covered by the sheet-like movement of a monolayer of epithelial cells, followed by a landslide-like mass movement of the epithelium (Suzuki et al., 2003).

Based on the XYZ hypothesis for corneal epithelial maintenance put forward by (Thoft & Friend, 1983), researchers have assumed that limbal stem cells and transient amplifying cells are induced after acute wounding and that these cells always migrate centripetally to repair the wound. However, it has been recently reported that after corneal wounding, the capacity for epithelial cell proliferation and migration appears to be as active in the central cornea as in the peripheral/limbal areas (Chang et al., 2008). Indeed, central and peripheral epithelial recovery remains equal even after ablation of the limbus. Thus corneal epithelial recovery may be independent of limbal stem cells (the mother cells of the Y component in the previous hypothesis) in a human corneal organotypic model. Therefore, central human corneal epithelial cells are fully capable of corneal epithelial regeneration, at least in the first 12 h after wounding. This observation may lead to new insights into the pathogenesis and therapy of corneal epithelial diseases.

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Corneal epithelial cell migration is modulated by various humoral and extracellular matrix (ECM) proteins (Lu et al., 2001). Growth factors, such as insulin-like growth factor (IGF)-1 (Nagano et al., 2003), epidermal growth factor (EGF)(Maldonado & Furcht, 1995a), interleukin 6 (Nishida et al., 1992), fibroblast growth factor-2 (FGF-2) (David et al., 1995), transforming growth factor-β (TGF-β) (Saika et al., 2004), keratinocyte growth factor (KGF), and hepatocyte growth factor (HGF) (Sharma et al., 2003; Wilson et al., 1993) have been shown to stimulate corneal epithelial cell migration both in vivo and in vitro. Glycoproteins of the ECM, such as fibronectin (Fn), laminin (Ln), and collagen IV, also facilitate cell migration (Maldonado & Furcht, 1995b; Ohji et al., 1993). However, the precise relationship between these cytokines and ECM protein production in the corneal epithelium is not well understood.

Once the wound area is covered the cells are further induced to proliferate to restore the stratified nature of the epithelium. Corneal wounding induces an increased release of several growth modulating cytokines and many of these cytokines have been linked to the regulation of epithelial cell proliferation (Lu et al., 2001). The presence of TGF-β in the later stages prevents over proliferation of the epithelial cells (Joyce & Zieske, 1997).

Whilst the above data seems to indicate specific roles for specific cytokines in corneal wound healing, the interaction between growth factors and cells, and growth factor to growth factor, inevitably means that the right combination of factors needs to be expressed with spatial and temporal coordination to ensure correct wound healing.


Treatment of diabetic keratopathy

Standard treatment

The standard treatment of neurotrophic corneal ulcers consists of a number of components including: maximising preservative free topical lubricants, minimising evaporative tear loss, use of topical antibiotics, protecting the corneal surface with a bandage contact lens, covering the ocular surface via lid closure by patch, tarsorrhaphy or induced ptosis, and more permanent solutions such as construction of a conjunctival flap. However, even in combination these measures may be ineffective, and the outcome is often severe impairment or loss of vision (Lambiase et al., 1998b).

Chapter 1….Literature Review Growth factors

A number of growth factors has been identified in the corneal epithelium and their gene

expression unravelled (Hongo et al., 1992; Kiritoshi et al., 1991; Schultz et al., 1990). The role of

growth factors in maintaining the normal structure and function of the cornea, and in corneal epithelial healing has emerged over the past three decades (Schultz et al., 1990; Tripathi et al., 1990). Autologous serum has been shown to be beneficial in epithelial wound healing both in vitro and in clinical studies (Schulze et al., 2006). Although the exact mechanism for these effects is not entirely clear, growth factors such as EGF and TGF may play a role.

A brief overview of some of the key growth factors involved in corneal epithelial healing


Insulin-like growth factor-1 (IGF-1)

IGF-1 is a multifunctional regulatory peptide that shares structural homology with proinsulin. It has been shown to mediate proliferation, differentiation, and survival effects, depending on the target cell and the presence of other hormones and growth factors (Gockerman et al., 1995). IGF- 1 is secreted by the liver. Like other growth factors, it is present in high concentrations in serum, produced locally, and may act in an autocrine or paracrine manner (McAvoy & Chamberlain, 1990). Accumulated evidence indicates that IGF-1 promotes cell motility in a variety of normal and malignant cell types. However, the effect of IGF-1 on corneal epithelial cell motility and migration is controversial (Nishida et al., 1996). It has been reported that substance P (SP) and IGF-1 synergistically stimulated corneal epithelial migration in an organ culture of the cornea. Accordingly, it was found that addition of SP or IGF-1 separately did not affect epithelial cell migration, yet co-treatment with SP and IGF-1 significantly stimulated epithelial cell migration (Nakamura et al., 1997). Also, it was noted that IGF-1 significantly increased migration and expression of laminin-5 in cultured human corneal epithelial cells (Lee et al., 2006).

In the clinical setting, the administration of eye drops containing both SP and IGF-1 has been

shown to be an effective treatment in the case of a child with neurotrophic and anhidrotic keratopathy (Brown et al., 1997). Moreover, eye drops containing peptides based on SP and IGF- 1 have been reported to be effective in the prevention of superficial punctate keratopathy in diabetic patients after cataract surgery and have also successfully induced rapid epithelial resurfacing in patients with persistent corneal epithelial defects (Yamada et al., 2008).

Chapter 1….Literature Review


Insulin, an anabolic peptide hormone closely related to IGF, is another growth factor implicated in wound repair, but its role is not well documented (McAvoy & Chamberlain, 1990). Insulin stimulates hepatotactic migration of human epidermal keratinocytes (Benoliel et al., 1987) and topical insulin therapy has been shown to aid the healing of ulcerations (Rosenthal, 1968; Van Ort & Gerber, 1976) and burns (Pierre et al., 1998). More interestingly, it has been shown that insulin is present in human tear fluid, and receptors to insulin have been detected on the human ocular surface (Rocha et al., 2002), cornea (Naeser, 1997) and neuronal and vascular tissues of the retina (Im et al., 1986; Thomopoulos & Pessac, 1979). The functions of insulin receptors within structures of the eye are not yet known, but diabetes is the major cause of blindness in people of working age and is often associated with disorders of the corneal epithelium (Rosenberg et al., 2000; Zagon et al., 2002a).

Recently, intensive systemic therapy with insulin which establishes normoglycemia in rats with diabetes was found to prevent the delay in wound healing of the ocular surface epithelium in poorly controlled diabetic animals (Zagon et al., 2006c). Normoglycemia in diabetic animals, induced by systemic treatment with insulin, restores decreased levels of DNA synthesis in the ocular surface epithelium to normal values when examined three weeks after wounding (Zagon et al., 2006c). Also, it has been reported that treatment with topical insulin significantly accelerates wound healing in diabetic rats compared with the untreated diabetic group. However, topical insulin had no effect on corneal re-epithelialisation of corneal wounds in healthy rats (non- diabetic) (Zagon et al., 2007a). These results were attributed to the ability of topical insulin to restore the decreased levels of DNA synthesis in basal epithelial cells to normal values, seen 48 h after wounding.

These observations present a new and promising therapeutic indication for insulin in the treatment of corneal ulcers in diabetics. Topical application of insulin to the surface of the eye will inevitably result in some systemic absorption. It has been shown previously monitoring serum glucose levels up to 14 h of topical exposures to 1 U of insulin had no effect on plasma glucose values in diabetic or healthy rats (Zagon et al., 2007a). However, the insulin used in this study was compounded as a simple solution. This is different from the commercially available insulin dosage forms. Commercial insulin contains pharmaceutical additives that are added to

Chapter 1….Literature Review

maintain and prolong the physical and microbial stability of the formulation. These may include benzalkonium chloride (preservative) and sodium editate (antioxidant and chelating agent). These additives have well-established penetration enhancing effects. This might increase systemic insulin absorption and as such might have an effect on blood glucose levels. Therefore, the aforementioned therapeutic benefits might be offset by possible systemic absorption of insulin via the ocular route, hence potentiating the effect of co-administered hypoglycaemic drugs (Lee et al., 2002). Table 2.1 presents the major attributes of insulin.

Table 1.2 Major attributes of insulin


Successfully enhanced corneal healing rate in diabetic rodent


Limited clinical trials (unknown mechanism) Insulin (6 kDa protein) cannot easily cross intact corneal barrier Long term safety on ocular tissue is controversial

Nerve growth factor (NGF)

NGF is a non-covalently linked dimer consisting of two 118-residue polypeptides, each of which contains three intramolecular disulphide bridges. NGF is a prototypical member of the neurotrophin family of growth factors that promote survival and growth of sympathetic and sensory neurons and differentiation of neurons in the central nervous system (Levi-Montalcini, 1987). Its biological action is not restricted to cells of neuronal origin but extends to cells of the immune system. NGF receptors (TrKA) were identified on the human corneal surface (Lambiase et al., 1998a). NGF is expressed constitutively in normal human and rat corneas. Published data supports the possibility that NGF modulates ocular inflammation and corneal epithelial proliferation and differentiation through its receptors. Also, recent evidence has demonstrated that NGF is identified in the cornea, conjunctiva, tear fluid, and lacrimal glands (Ghinelli et al., 2003; Ríos et al., 2007; Touhami et al., 2002; You et al., 2000).

Chapter 1….Literature Review

Many reports describe the relationship between NGF and the ocular surface health and well- being: one in vitro study showed that NGF induces proliferation and differentiation of rabbit corneal epithelial cells (Kruse & Tseng, 1993). A clinical report showed an increase in NGF plasma levels in vernal keratoconjunctivitis (VKC) with a direct correlation between mast cell conjunctival infiltration and NGF levels (Lambiase et al., 1995).

Topical murine NGF eye drops was applied to 14 eyes with non-infectious corneal ulcers caused by essential neurotrophic keratitis (5 eyes), chemical burns (3 eyes), abuse of topical anaesthetics (2 eyes), orbital tumour surgery (1eye), surgery of acoustic neuroma (1 eye), penetrating keratoplasty of unknown reason (1 eye) and a lamellar keratoplast for a herpatic vascularized scar (1 eye) (Lambiase et al., 1998b). Remarkably, considering the diverse aetiology, all patients had complete corneal healing after 10 days to 6 weeks of NGF treatment. However, all patients experienced mild to moderate conjunctival hyperaemia accompanied by pain, photophobia, and superficial or deep corneal neovascularisation occurred in 9 of the 14 treated eyes. All ocular symptoms disappeared once the corneal ulcers had completely healed.

In another study, it has been reported that murine NGF (dosage: one drop of 200 µg/ml NGF solution every 2 h for 2 days, followed by one drop six times daily until the ulcer showed signs of healing, followed by one drop four times daily of 200 µg/ml NGF solution) improved corneal sensitivity and promoted corneal epithelial healing in 45 eyes with both moderate and severe neurotrophic keratitis within 12 to 42 days of NGF treatment (Bonini et al., 2000). Visual functions such as corneal sensitivity and visual acuity were improved, and healing of the corneal ulcers was promoted in NGF-treated eyes. Interestingly, except in a few cases, no relapse of the disease occurred during the follow-up period. However, transient adverse effects such as hyperaemia and pain in the eye were observed on instigation of NGF treatment. Supporting to that, a report has suggested NGF as a future treatment in several pathologies of both the ocular surface and the retina (Micera et al., 2004).

Therefore, NGF has attracted the attention of pharmaceutical scientists to develop specialised drug delivery systems aimed at inhibiting NGF’s degradation, prolonging its biological half-life and enhancing its biological activity (Pfister et al., 2008; Sakiyama-Elbert & Hubbell, 2000; Xie

Chapter 1….Literature Review

et al., 2005). All of the aforementioned evidence augments the clinical interest in NGF as a potential therapeutic agent to promote corneal wound healing. However, most of the experimental evidence in relation to the clinical efficacy of NGF has been obtained with the 2.5S murine NGF which has several limitations (Table 1.3).

Several attempts have been made to produce recombinant human nerve growth factor (rhNGF) using different micro-organisms, including Saccharomyces cerevisiae (Kanaya et al., 1989; Nishizawa et al., 1993) and Escherichia coli (Negro et al., 1992; Rattenholl et al., 2001) as well as insect (Allen et al., 2001; Barnett et al., 1991) and mammalian cells (Iwane et al., 1990). However, most of these studies have been carried out in vitro, and the available evidence in vivo indicates that the action of rhNGFs in human peripheral neuropathies was not comparable with the effect of murine NGF (Apfel, 2002). The technology of rhNGF preparation is not complex; however the biological activity of β-NGF relies on the formation of three disulfide bonds and a cysteine knot within two β-chains of 120 amino acids each after cleavage of pro-peptide sequences from a larger precursor molecule (Edwards et al., 1988; Shooter, 2001; Ullrich et al., 1983). Recently, it has been reported that the production (on a laboratory scale) of large amounts of rhNGF was shown to be effective both in vitro and most importantly in vivo (Colangelo et al.,


Table 1.3 Major attributes of murine NGF


Successfully used in many preclinical and clinical trials


High cost Limited sources and insufficient quantity (adult male mouse submandibular glands, snake venom) Allodynia (hyperalgesia induced upon topical administration)

Chapter 1….Literature Review

Hepatocyte growth factor (HGF), epidermal growth factor (EGF), keratinocyte growth factor (KGF), transforming growth factor-β (TGF-β)

HGF, EGF, KGF, and TGF-β are expressed in keratinocytes, corneal endothelial cells and up- regulated in rabbit keratocytes after wound healing (Li & Tseng, 1996; Wilson et al., 1993). However, conflicting results have been reported in human trials, such as those described for EGF (Daniele et al., 1992; Hosotani et al., 1995). Moreover, TGF-β has been implicated as a potent stimulant of the scarring process in the eye. Corneal scarring is a major problem in influencing results of photorefractive surgery, giving rise to symptoms of haze and resulting in a reduction of the best corrected visual acuity (Jester et al., 1997; Mayers et al., 1997).

Opioid growth factor (OGF)

Endogenous opioids and their receptors were originally thought to be related to neural modulation (Akil et al., 1984). Although there are numerous endogenous opioids and respective receptors, only the native opioid peptide, [Met 5 ]enkephalin, is classified as an opioid growth factor (OGF). OGF is a pentapeptide with the sequence Tyr-Gly-Gly-Phe-Met. OGF has been determined to serve a growth regulatory role and its binding receptor is not homologous to classic opioid receptors. OGFs act as growth regulators in many normal and malignant tissues (Zagon & McLaughlin, 1991b, 1993a). It has been identified in both eukaryotes and prokaryotes as a potent inhibitor of cell replication (Zagon & McLaughlin, 1991a, 1991b, 1993a). In vertebrates, OGF is also thought to be important in cellular maturation and survival (Meriney et al., 1991; Zagon & McLaughlin, 1993a). OGF has been documented as being produced in an autocrine and/or paracrine, manner (Zagon et al., 1994a). OGF activity is mediated by the zeta (ζ)-opioid receptor (Zagon & McLaughlin, 1993a, 1993b).

Endogenous opioid systems influence proliferation in a wide variety of cells and tissues both in vivo and in vitro. These include developing neurons and galia of the mammalian nervous system (including the retina), glial cells in culture, myocardial and epicardial cells in neonatal heart, neuronal and non-neuronal cancer cells, and cellular renewal in the epithelium of the adult tongue (McLaughlin, 1994; Zagon & McLaughlin, 1991a, 1991b, 1993a; Zagon et al., 1994b). OGF has been detected in the bovine corneal epithelium (Tinsley et al., 1990; Tinsley et al., 1989). OGF and the ζ-receptor are also present in the normal rabbit corneal epithelium (Zagon et al., 1995).

Chapter 1….Literature Review

A study was performed to show the physiological role of endogenous opioid systems (opioid peptides and receptors) in the mammalian corneal epithelium in vitro using rabbit corneal explants (Zagon et al., 1995). The following strategies were adopted; firstly, naltrexone (opioid antagonist) was administered to explant cultures of rabbit corneal epithelium to examine whether this approach modulates epithelial outgrowth; secondly, OGF was administered to cultures of corneal explants to investigate the effects of this opioid peptide on the extent and organization of epithelial outgrowth. Immunocytochemistry experiments using antibodies specific to OGF and ζ- receptor were performed to confirm that OGF systems are present in normal corneal epithelium in culture. The study demonstrated that OGF and ζ-receptor are present in the normal corneal epithelium and, more surprisingly, OGF exerted an inhibitory effect on cellular proliferation and growth in the corneal epithelium. This tonically active growth regulatory inhibitory pathway is involved in cell replication. OGF targets cyclin-dependent kinase inhibitors (P16 and/or P21) (McLaughlin et al., 2010). The regulation of cells proliferation by OGF-OGF receptor (OGFr), as aforementioned, has been termed OGF-OGFr axis for cellular renewal and proliferation (McLaughlin et al., 2010).

Fluorescence imaging of OGFr by green fluorescent protein-labelled OGFr revealed that OGFr is assembled into outer nuclear envelope. This complex is shuttled to the nucleus. The OGF-OGFr complex unregulated p16 and/or p21 leading to a marked delay in the G1-S phase of the cell cycle. Naltrexone (a long acting potent opioid antagonist) administration blocks OGF-OGFr interactions. Hence, this increases cell proliferation (Cheng et al., 2009). Blockade of opioid receptors by naltrexone (a long acting potent opioid antagonist) for seven days significantly increased the extent of proliferation and outgrowth of the corneal epithelium in vitro. This study suggested, for the first time, an inhibitory role for an endogenous growth factor. Moreover, the study demonstrated that blocking of the corresponding receptor by naltrexone significantly increased DNA synthesis, cell migration and proliferation of corneal epithelium.

Other reports have demonstrated that topical or systemic application of naltrexone markedly accelerates DNA synthesis and wound healing of corneas in humans (Zagon et al., 2000), rats (Zagon et al., 1998b) and rabbits (Zagon et al., 1998a) as well as in diabetic rodents (Zagon et al., 2002a). Indeed, topical application of naltrexone healed corneal epithelial wounds in uncontrolled Type 1 diabetic rodents at rates equal to or surpassing that in normal controls (Klocek et al.,

Chapter 1….Literature Review

2007). However, concomitant application of topical naltrexone and topical insulin does not have an additive or synergistic effect on corneal re-epithelialisation (Klocek et al., 2009).

In another study, the same group used a gene gun to introduce sense and antisense complementary DNA (cDNA) for OGFr to establish that the effects of OGF on corneal reepithelialization are specifically dependent on the OGFr. This study establishes, for the first time, that the OGF-OGFr axis is an autocrine loop that serves as a crucial regulatory system for corneal epithelial wound healing. The plasmid pcDNA3.1-OGFr, carrying the rat OGFr- complementary DNA in both the sense and antisense orientations, and control, were delivered by gene gun to the rat cornea. After 24 h, corneas were abraded and re-epithelialisation was monitored by fluorescein photography. Corneal cells exposed to sense OGFr exhibited corneal defects that were up to 52% larger than the control group. On the other hand, corneas subjected to the antisense constructs of OGFr exhibited defects that were up to 56% smaller than the control group (Zagon et al., 2006b).

Naltrexone has been found to enter human cells by passive diffusion. Fluorescent-tagged naltrexone was found to accumulate in the cytoplasm of the human head and neck squamous cell carcinoma cell line as little as 60 s after incubation. Neither fluorescein dye nor fluorescein- tagged dextrans were found to enter the cells (Cheng et al., 2009). In the same study, untagged naltrexone and fluorescent-tagged naltrexone were found with an increase of up to 70% in cell proliferation markers compared with the vehicle-treated group (Cheng et al., 2009). Table 1.4 summarises the major features of naltrexone.

Chapter 1….Literature Review

Table 1.4 Major attributes of naltrexone



Successfully reversed most signs of diabetic keratopathy (corneal sensation, delayed wound repair, dry eye)


Well-defined mode of action Synthetic chemical entity (commercially available) Safe on ocular tissues

Extremely bitter taste (nasolacrimal drainage) Substance P

Substance P (SP) (11-amino acid polypeptide) is a neurotransmitter of the trigeminal nerve and a member of the tachykinin family. SP is present in physiologically relevant concentrations in the normal cornea (Nishida et al., 1996). SP concentrations in tears are thought to reflect the neuropeptide levels in ocular tissues. In patients with unilateral corneal hypesthesia, substance P concentrations in tears from the affected eye were significantly lower than those in tears from the unaffected eye (Yamada et al., 2000). Topical application of SP alone has no detectable effect on the rate of corneal epithelial wound closure in rabbits (Kingsley & Marfurt, 1997). However, SP in combination with IGF-1 has been demonstrated to facilitate synergistically corneal epithelial migration in rats with trigeminal denervation (Nagano et al., 2003) and in organ cultured corneas (Nishida et al., 1996). As discussed above, this combination has been shown to be effective in healing corneal epithelium in humans in the clinical setting. Unfortunately, SP is not available commercially. Aldose reductase inhibitors

Aldose reductase (AR) is the rate-limiting enzyme in the polyol pathway in which glucose is converted to sorbitol (Herse, 1988). AR activity is implicated in diabetic corneal epithelial disease primarily because of the effect that AR inhibitors have on these documented diabetic epithelial abnormalities (Herse, 1988). The AR inhibitor was applied topically to treat a non-

Chapter 1….Literature Review

healing corneal ulcer in a patient with diabetes. The corneal ulcer healing was dramatically improved (Ohashi et al., 1988). Topical CT-112 (an AR inhibitor) was used to treat two patients with diabetes with non-resolving corneal epithelial lesions (Ohashi et al., 1988). Not only did the lesions resolve, but they returned when the CT112 therapy was discontinued and resolved again when CT-112 therapy was reinstituted. The patients remained lesion-free on long-term maintenance therapy with CT112. Also, topical CT-112 was applied to a group of galactosaemic rats, vehicle alone to a second group of galactosaemic rats and vehicle alone to a third group of normal rats (Yokoi et al., 1997). After 3 weeks, fluorescein uptake (as an indicator of corneal permeability) was measured by fluorophotometry in the three groups. The galactosaemic rats that received vehicle alone displayed a significantly higher sodium fluorescein uptake than the normal rats and the CT-112 treated galactosaemic rats showed no significant difference in the fluorescein uptake between the vehicle-treated normal rats and the CT-112-treated galactosaemic rats. Treatment with AR inhibitors has been shown to reduce corneal changes after they occurred, and limit the development of these corneal changes when compared to untreated controls in both animals and humans. The mechanism by which increased AR activity contributes to diabetic corneal pathology remains unknown (Herse, 1988).


Nicergoline is an ergoline derivative known to cross the blood-brain barrier and is now widely and safely used to treat cognitive impairment from stroke and degenerative dementia. In vivo studies have shown that nicergoline treatment induces significant increase in the NGF levels within the frontal region of the brain and supports cholinergic neurons, increasing the content of NGF and brain-derived neurotrophic factor in the brain of aged rats (Giardino et al., 2002; Nishio et al., 1998). Recently, it has been found that oral nicergoline (10 mg/kg per day) for 2 weeks increased the rate of corneal wound healing in 50 rat eyes (Kim et al., 2009). The promoting effect of nicergoline in corneal wound healing is attributed to increased NGF in corneas and/or lacrimal glands. However, the exact ocular tissue responsible for increasing NGF levels as a response to nicergoline has not yet been identified. Recently, oral treatment of nicergoline at least for 4 weeks has been found to restore corneal sensation in rats after photorefractive keratectomy (Kim et al., 2011).

Chapter 1….Literature Review

1.4. Thesis aims and structure

The cornea is the most densely innervated mammalian tissue and the integrity of corneal nerves is of paramount importance for the refractive and protective functions of the cornea. With the advent of modern refractive surgeries and the increasing number of diabetic patients worldwide, the cornea is routinely subjected to injury and this damage may cause transient or chronic neurotrophic deficits (corneal scarring). If this damage does not receive proper medical attention loss of vision can ensue.

The corneas of diabetic patients show significant and characteristic signs known collectively as diabetic keratopathy. These characteristic diabetic signs include: delayed wound repair, reduced tear production, reduced corneal sensation and neurotrophic corneal ulcers. The prevalence of diabetic keratopathy is approximately 47% to 64% during the course of diabetes mellitus (Schultz et al., 1981 ).

The available treatment options are topical antibiotics, tear substitutes and eye patches. This treatment is in most cases insufficient because it does not treat the underlying causes (McLaughlin et al., 2010).

Naltrexone (a potent opioid antagonist) has recently been found to reverse the signs and symptoms of diabetic keratopathy. Naltrexone successfully accelerates corneal wound healing, both systemically and topically, in normal and diabetic rats, rabbits and humans (McLaughlin et al., 2010; Zagon et al., 2009; Zagon et al., 1997; Zagon et al., 1998a, 1998b; Zagon et al., 2007b). Recently, it has been found that naltrexone can restore impaired corneal sensation in diabetic rats (McLaughlin et al., 2010; Zagon et al., 2009). These effects are attributed to blocking opioid growth factor receptors in corneal tissues and consequently accelerate DNA synthesis in the cornea and re-epithelialisation. Whilst there is substantial clinical evidence on the effectiveness of naltrexone in diabetic keratopathy, there are hardly any reports on its formulation or incorporation into an appropriate ocular delivery system.

Naltrexone has an extremely bitter taste. This bitterness can be tasted upon ocular administration of the drug from simple solution due to rapid and extensive nasolacrimal drainage. There are also limited reports on the chemical stability of naltrexone in aqueous solution. Parallel to that, frequent instillation of a simple drug solution to the eye is associated with pulse-type entry and may prompt patient incompliance.

Chapter 1….Literature Review

As previously outlined, niosomes lend themselves to potential ocular drug delivery systems. They have the convenience of being delivered as eye drops, the ability to prolong precorneal drug residence, a localised effect, complete biodegradability and minimal irritancy.

This work aims to formulate and characterise niosomal formulations for the ocular delivery of naltrexone hydrochloride. To achieve this aim, this thesis will explore the following objectives:

Develop and validate an analytical method for accurate and selective determination of naltrexone hydrochloride in the prepared formulations. In addition, forced degradation studies will be conducted under different stress conditions to demonstrate that the developed method is a stability-indicating method.

Study the chemical stability of naltrexone hydrochloride in aqueous solutions.

Study the fundamental physicochemical properties of naltrexone hydrochloride such as melting behaviour, aqueous solubility and lipophilicity (octanol-buffer partition coefficient determination).

Perform formulation studies on niosomes that includes:


Investigating the effect of surfactant type (sorbitan esters and ployoxyethylene alkyl ethers) and cholesterol (membrane stabiliser) on vesicle sizes, entrapment efficiency and vesicle membrane fluidity (gel/liquid phase transition temperatures). Also, the effect of total lipid and initial drug concentration on the entrapment efficiency of the prepared niosomes.


Determining whether some selected membrane additives could alter the physical properties such as drug entrapment efficiency, morphology, size and gel/liquid transition temperatures of the prepared niosomes with emphasis on topical application to the eye surface.


Determining the physical and chemical properties of the prepared niosomes such as contact angle, spreading coefficient, viscosity measurements and influence of vesicular encapsulation on oxidative degradation of naltrexone hydrochloride.

Evaluate the prepared niosomes for ocular naltrexone hydrochloride delivery; ocular toxicity using suitable in vitro models; in vitro drug release; ex vivo drug permeation through excised bovine corneas; and physical stability of the selected formulations at different temperatures.

Chapter 2….Preformulation of Naltrexone hydrochloride

2. Preformulation studies of naltrexone hydrochloride

2. Preformulation studies of naltrexone hydrochloride 2.1. Introduction Preformulation characteristics are certain

2.1. Introduction

Preformulation characteristics are certain fundamental physical and chemical properties of a drug that can eventually help in selecting the suitable formulation approach to develop an effective, stable and economic dosage form (Wells & Aulton, 2007). The selection of particular preformulation properties to study depends on the proposed dosage form. For example, powder flow, compressibility, intrinsic solubility and dissolution studies are essential for developing solid dosage forms. Generally, fundamental preformulation experiments include: development of a suitable analytical method; aqueous solubility studies; melting point measurement; partition coefficient and dissociation constant determination. Studying these preformulation characteristics for the drug under investigation is essential especially at the early stages of designing and developing a new dosage form.

Up to the early seventies, the available compounds for treatment of narcotic addiction were cyclazocine and naloxone. These two drugs proved to be excellent opioid receptors antagonists but both had drawbacks (Fink et al., 1968; Freedman et al., 1968; Renault, 1981). Cyclazocine was limited because of dysphoric side effects due to weak agonistic activity (not pure antagonist). Naloxone is a pure antagonist and thus free of side effects, but lack potency when administered orally (Blumberg & Dayton, 1973; Freedman et al., 1968; Renault, 1981). Naltrexone has been developed to fulfil the need for a long-acting, potent and pure antagonist with minimal unwanted side effects. These requirements are met by naltrexone; it is about twice as active as naloxone as an antagonist in mice. Furthermore, it is longer acting than naloxone in human (Blumberg & Dayton, 1973). Currently, naltrexone is a widely used opioid antagonist for treatment of heroin dependence (Renault, 1981) and alcoholism (Sweetman, 2007). Naltrexone is marketed commercially as hydrochloride salt. Naltrexone hydrochloride is administered orally either in liquid (extemporaneously prepared) or solid (tablets and capsules) form. The capsule form is preferable, because naltrexone has an extremely bitter taste (Renault, 1981).

Chapter 2….Preformulation of Naltrexone hydrochloride

2.1.1. Physical properties of naltrexone hydrochloride

Naltrexone hydrochloride (NTX) is a hydrochloride salt of naltrexone. It is chemically named N- cyclopropylmethyl-14-hydroxydihydromorphinone. NTX is a white crystalline solid, soluble in water. NTX has an empirical formula of C 20 H 23 NO 4 . HCl (mol wt 377.87) and a melting point of 275 o C (crystals from methanol)(O’Neil et al., 2006). It has an extremely bitter taste (Crabtree, 1984); has a low partition coefficient (P), a log P (octanol/water) value of 1.9 (Galichet et al., 2006); it is a weak basic drug, a pKa value of 8.38 at 20 o C (Kaufman et al., 1975). Figure 2.1 shows a schematic of the chemical structure of NTX.

2.1 shows a schematic of the chemical structure of NTX. Figure 2.1 Chemical structure of NTX

Figure 2.1 Chemical structure of NTX

2.1.2. Pharmacological uses and dosage for systemic administration of NTX

NTX is widely used in treatment of alcohol dependence and rescue medications to reverse the side effects of opioid agonists (respiratory depression overdose) (Goodman et al., 2007; Kastrup et al., 2009). Off-labelled uses of NTX include treatment of obesity, severe pruritus, psychosis and Parkinson’s disease (Goodman et al., 2007). The usual dosage of NTX is 50 mg once daily or 350 mg per week in three divided doses (Crabtree, 1984; Goodman et al., 2007).

2.1.3. Pharmacokinetics of NTX

Absorption: NTX is a pure opioid receptor antagonist. Although it is absorbed well orally, naltrexone is subject to significant first-pass metabolism. Its oral bioavailability ranges from 5% to 40% (Kastrup et al., 2009).

Chapter 2….Preformulation of Naltrexone hydrochloride

Distribution: The volume of distribution for NTX following iv injection is estimated to be 1350 litres. In vitro tests of human plasma show NTX to be 21% bound to plasma proteins over the therapeutic dose range (Kastrup et al., 2009). Metabolism: The systemic clearance (after iv administration) of naltrexone is 3.5 ml/min. The major metabolic site is the liver and the major metabolite is 6-β-naltrexol. The primary metabolic pathway is glucuronide conjugation (Misra, 1981).

Excretion: Urinary excretion is the major excretion route of NTX and its metabolites (> 95%). Less than 5% of an orally administered dose is found in faeces after 48 h (Crabtree, 1984; Misra, 1981).

2.1.4. Adverse reactions of systemic administration of NTX

Combined reporting of adverse events from oral and injectable formulations includes:

Cardiovascular syncope (13%), headache (25%), insomnia (14%), anxiety (12%), nausea (33%), vomiting (14%), abdominal pain and cramps (11%) (Fuller & Sajatovic, 2009; Kastrup et al.,


Contrary to expectation, injectable forms of NTX seem to induce more adverse reactions than oral forms, leading to a higher rate of treatment discontinuation by patients. Amongst these adverse reactions associated with injectable NTX administrations are pain, bruising, induration, angioedema and nodules at the injection site (Roozen et al., 2007).

2.1.5. Warnings on the systemic administration of NTX

The following warnings are reported with oral administration of NTX (Kastrup et al., 2009):

NTX has the capacity to cause hepatocellular injury when given in excessive doses.

NTX is contraindicated in acute hepatitis or liver failure, and its use in patients with active liver disease must be carefully considered in light of its hepatotoxic effects.

Patients should be warned of the risk of hepatic injury and advised to stop the use of NTX and seek medical attention if they experience symptoms of acute hepatitis.

NTX and its primary metabolite are excreted primarily in urine and caution is recommended in administering the drug to patients with renal impairment.

Chapter 2….Preformulation of Naltrexone hydrochloride

2.1.6. Naltrexone as a new ophthalmic pharmaceutical

Topical ocular application of NTX in doses of up to 0.4 mg/ml has been found to accelerate markedly the wound healing of cornea in humans (Zagon et al., 2000), rats (Zagon et al., 1998b) and rabbits (Zagon et al., 1998a), as well as diabetic rodents (Zagon et al., 2002a). Moreover, topical application of NTX can enhance diabetic corneal epithelial healing without causing morphologic abnormalities in the reassembly of adhesion structures (Zagon et al., 2007b). Furthermore, topical treatment with NTX has been found to normalise tear production and corneal sensitivity in type 1 diabetic rats (Zagon et al., 2009).

The mechanism of NTX for accelerating corneal wound healing is the blockade of opioid growth factor (OGF) interaction with the OGF receptor (Zagon et al., 2002b). Consequently, it can enhance DNA synthesis and corneal epithelialisation. However, the exact mechanism for normalising tear production and restoring corneal sensation in diabetes mellitus is still unknown (Zagon et al., 2009). It is worth mentioning that the corneal safety of topically applied NTX has been studied (Zagon et al., 2006a). The results showed that concentrations of naltrexone ranging from 10 -3 -10 -7 M are not toxic when applied topically to the cornea.

2.1.7. Chemical stability of NTX

There are few reports on the chemical stability of NTX and its major degradation pathways in aqueous solutions (Fawcett et al., 1997; Gupta, 2008). Table 2.1 summarises some of the reported HPLC methods for NTX analysis.

The physicochemical stability of NTX oral liquid formulations prepared from commercially available tablets and powder at two different concentrations (1 mg/ml and 5 mg/ml) was studied (Fawcett et al., 1997). A vehicle of pH 3.5 was used and contained ascorbic acid (0.5%), sodium benzoate (0.1%), glycerol (20%) and distilled water (to 100%).

The prepared formulations were stored in the dark at three temperatures 4, 25, and 70 o C and monitored for chemical and physical stability of NTX up to 90 days. The samples were assayed for NTX concentration to assess decomposition over 90 days. The results showed that decomposition of both NTX from drug powder and tablet stored at 4 and 25 o C was not significant over 90 days. However, the percentage of NTX remaining after storage for 90 days at 70 o C was 51.1 % and 64.1% for NTX liquid formulations prepared from tablet and powder respectively. In

Chapter 2….Preformulation of Naltrexone hydrochloride

terms of physical stability, powder formulations stored at 4 o C developed a pale yellow colour after 90 days. Both solutions prepared from powder and tablets stored at 25 and 70 o C developed a pale yellow colour by day 30 and day 2 respectively.

In another study, the chemical stability of a NTX injection (extemporaneously prepared) was studied. NTX was dissolved in sufficient sterile water for injection under aseptic conditions to form a concentration of 1.4 mg/ml. The solution was stable for at least 42 days when stored in clear glass vials at room temperature. Also, the physical appearance of the injection did not change during the study period (Gupta, 2008).

Having a morphinan structure, oxidation could be the main culprit for the loss of potency and degradation of NTX. Furthermore, oxidation is the main degradation pathway for phenolic compounds such as opioids and catecholamines (Florence & Attwood, 1998a; Lachman et al.,


Chapter 2….Preformulation of Naltrexone hydrochloride

Table 2.1 Summary for HPLC conditions of NTX determination


(Fawcett et al., 1997)

(Iyer et al., 2007)

(Gupta, 2008)

Chromatographic conditions

Chromatographic conditions


C18: Supelcosil TM LC-18-DB (250 mm × 4.6mm, 5µm)

C18: Supelcosil TM LC-18-DB (150 mm × 4.6mm, 5µm)

C18: Beckman Ultrasphere TM LC-18-DB (150 mm × 4.6mm,


Mobile phase

A : 0.06% triethylamine solution in Phosphate buffer (40mM) pH 5 (50%v/v) B:

Acetonitrile (50% v/v)

A : 0.06% triethylamine solution in Phosphate buffer (40mM) pH 4.75 (88 parts)B:

Acetonitrile (12 parts), A and B were premixed together

A : 0.1% of 85% phosphoric acid and 0.1% glacial acetic acid solution (90% v/v) pH 2.6 B: Acetonitrile (10% v/v)

Injection volume (µl)




Elution type




Temperature ( o C)




Flow rate (ml/min)





UV at 214 nm

UV at 204 nm

UV at 285 nm

Retention time (min)

Linearity range (µg/ml)




Chapter 2….Preformulation of Naltrexone Hydrochloride

2.1.8. Oxidation

Oxidation is one of the most common causes of the chemical degradation of a considerable number of pharmaceutical compounds (Florence & Attwood, 1998a; Lachman et al., 1986). For instance, the degradation of steroids, vitamins, antibiotics and morphine is due to oxidation reactions (Lachman et al., 1986). Oxidation involves the removal of electrons, radicals or electropositive atoms (Florence & Attwood, 1998a).

The most common form of oxidation occurring in pharmaceutical dosage forms is autoxidation, which is the reaction between molecular oxygen and organic compounds under mild conditions (Johnson & Gu, 1988). Apart from the loss of drug potency due to autoxidation, other instability problems can occur such as discolouration, changes in dissolution rate, precipitation, loss of preservative efficacy, evolving of off-odours and sometimes generation of toxic compounds (Johnson & Gu, 1988). It is well accepted that most liquid-phase autoxidations are free-radical chain reactions. A free radical is an atom or molecule with one or more unpaired valence electrons. Three well-distinct steps are identified in autoxidation reactions (Johnson & Gu, 1988):


autoxidation reactions (Johnson & Gu, 1988): Initiation: equation 2.1 This stage is initiated once free radicals

equation 2.1

This stage is initiated once free radicals are generated. This inevitably takes place over a certain period of time but the generation of free radicals can be accelerated by the action of heat, light and traces of heavy metals (Florence & Attwood, 1998a; Johnson & Gu, 1988). The length of the initiation is called the induction period (Stewart & Tucker, 1984).


induction period (Stewart & Tucker, 1984). Propagation: equation 2.2 In the propagation stage, the generated free

equation 2.2

In the propagation stage, the generated free radicals in the initiation stage react with molecular oxygen to yield peroxy radicals. Therefore, the oxygen concentration is of importance in the autoxidation process, especially when determining the reaction rate of oxidation at different temperatures due to the effect of temperature on the oxygen content (concentration of oxygen) of the liquid (Lachman et al., 1986); the higher the temperature, the lower the oxygen concentration. The hydroperoxide reacts with a new available free radical to propagate the reaction.

Chapter 2….Preformulation of Naltrexone Hydrochloride


2….Preformulation of Naltrexone Hydrochloride Termination: equation 2.3 The oxidation reaction proceeds until the free

equation 2.3

The oxidation reaction proceeds until the free radicals are scavenged by inhibitors or halted by side reactions which eventually break the chain reactions.

Figure 2.2 is a schematic showing a typical example of autoxidation reactions of morphine (M) with atmospheric oxygen in aqueous solution to morphine N-oxide (MNO) (Yeh & Lach, 1961).

Chapter 2….Preformulation of Naltrexone Hydrochloride

Chapter 2….Preformulation of Naltrexone Hydrochloride Figure 2.2 Schematic of morphine autoxidation in aqueous solutions

Figure 2.2 Schematic of morphine autoxidation in aqueous solutions

Chapter 2….Preformulation of Naltrexone Hydrochloride

2.1.9. Arrhenius plot and predicting drug stability

Temperature is a key factor in determining the velocity of the degradation reaction of pharmaceutical products. It is necessary to evaluate the temperature dependency of the reaction. This allows prediction of the stability of the product (Lachman et al., 1986). The speed of many degradation reactions doubles for each 10 o rise in temperature (Lachman et al., 1986). The most satisfactory method for quantitative determination of the influence of temperature on the reaction velocity is that proposed by the Arrhenius equation which will be described later in the method section in this chapter. The most important parameter which can be calculated from the Arrhenius equation is the heat of activation (∆Ea), which represents the energy the reaction molecules must acquire to undergo reaction. Plotting reaction rate constant values obtained at several elevated temperatures versus the inverse of absolute temperature gives rise to what is known as the Arrhenius plot. Since the plot is linear, the prediction of stability at shelf temperature is possible by extrapolating the curve to the lower temperature (Lachman et al., 1986). The use of the Arrhenius equation to predict drug stability at shelf temperature is most useful when the reaction is too slow to be monitored conveniently and when∆Ea is relatively high. For example, for a reaction with a ∆Ea value of 25 kcal/mol, a temperature increase from 25 to 45 o C brings about a 14-fold increase in the reaction rate constant (Guillory & Poust, 2002). Limitations of the Arrenius plot for predicting oxidative degradation

There are some issues related to accelerated thermal degradation methods hindering the ability of the Arrhenius equation to predict the shelf-life of dosage forms at lower temperatures (Lachman et al., 1986; Waterman et al., 2002):

Higher temperatures may evaporate solvents, thus producing unequal moisture concentration.

At higher temperatures, there is less oxygen solubility, thus hindering the predictability of the room temperature stability of drugs sensitive to oxidation.

The permeability of oxygen through packaging is temperature dependent.

If the generation of radicals is rate limiting, the kinetics can show autocatalysis, the rate of reaction increases as the radical concentration increases.

Instability may be related to the amount of peroxide impurities present in a particular lot of excipient. This is related to age of the excipient and the conditions under which the material was stored.

Chapter 2….Preformulation of Naltrexone Hydrochloride

2.2. Chapter aims

Designing a new ophthalmic liquid dosage form for NTX compels performing preformulation

This chapter aims at carrying out preformulation studies of NTX which can

help select subsequent approaches during the formulation development. Specific objectives include:

Development and validation of a sensitive and standard HPLC method for NTX analysis in the prepared formulation.

studies for NTX.

Studying various stress conditions which can affect the chemical stability of NTX in accordance to International Conference on Harmonization (ICH) guidelines.

Determining the following fundamental physicochemical properties of NTX:


Melting behaviour.


Spectrometric fingerprints (infrared, mass and 1 H-NMR spectra).


Aqueous solubility.


Ionisation constants (pKa values) at different temperatures.


Lipid solubility (distribution and partition coefficients).

Chapter 2….Preformulation of Naltrexone Hydrochloride

2.3. Materials and methods

2.3.1. Materials

NTX was purchased from Mallinckrodt Inc., St. Louis, MO, USA; n-octanol was purchased from Schalau Chemie, Sentmenat, Spain. All other solvents and buffer salts were of analytical grade and used as received.



Melting behaviour study of NTX using differential scanning calorimetry (DSC)

A small amount (typically 2 mg) of NTX powder was weighed in an aluminium pan and covered

with an aluminium lid and hermitically sealed using a pan press (Thermal Science, USA). The temperature of the pan gradually increased from 50 to 300 o C at a rate of 10 o C/min using a differential scanning calorimeter (Q 1000 series, Thermal Analysis, USA) precalibrated with indium. The purging gas was nitrogen at a flow rate of 45 ml/min. Spectrometric identification of NTX

Mass spectroscopy (MS)

A sample weight (approx. 1 mg) was dissolved in 1 ml methanol and injected directly into the

ionisation chamber. The atmospheric pressure chemical ionisation (APCI) technique was used to

ionise the sample and generate ions using a LC-M spectrometer (Surveyor MSQ, ThermoFinnigan, New Jersey, USA).

Fourier transform infrared (FT-IR) spectroscopy

A FT-IR spectrometer (Bruker Miracle Micro ATR, Tensor 37, GmbH, Germany) was used to

record the FT-IR spectrum. A clean diamond window was used to measure the background spectrum. A sufficient amount (approx. 1mg) of NTX was placed to form a thin film covering the diamond window. The data was acquired and analysed using OPUS software (OPUS 6.5, Germany).The FTIR spectrum was recorded at spectral resolution of 2 cm -1 with an average of 120 scans.

Proton nuclear magnetic resonance ( 1 H-NMR) spectroscopy

1 H-NMR spectrum of NTX was determined in deuterated dimethyl sulfoxide (CD 3 ) 2 SO on a 1 H- NMR spectrometer (Bruker Avance 400 Spectrometer, GmbH, Germany) at 400 MHz. Chemical shifts are recorded in ppm.

Chapter 2….Preformulation of Naltrexone Hydrochloride Analytical methods

Ultraviolet (UV) spectrophotometry

An amount of NTX (10 mg) was accurately weighed and dissolved in 10 ml of phosphate buffer saline (PBS) pH 7.4 to produce a concentration of 1mg/ml. Aliquots of 0.3-1ml of the stock solution were transferred in triplicate to a series of 10-ml volumetric flasks and diluted to 10 ml using PBS to obtain final concentrations of 30-100 µg/ml. Then, absorbance values of these dilutions were measured at 281.5 nm using a UV-spectrophotometer (Thermospectronic, Helios Gamma, England). A plot of absorbance vs. concentrations (µg/ml) was constructed.

High performance liquid chromatography (HPLC) method development

A modification of the method described by (Fawcett et al., 1997) was performed. In brief, an HPLC system (Agilent 1200, Agilent Corporation, Germany) comprising a quaternary pump, an automatic sampler and a photodiode array (PDA) detector was used with data acquisition by ChemStation ® software (Agilent Corporation, Germany). The chromatographic separation was achieved using a Supelcosil C18 column (5 µm; 250 mm×4.6 mm, Supelco Corporation, PA, USA) maintained at 25 C. The mobile phase was prepared and premixed. Seventy parts (by volume) of monobasic potassium dihydrogen phosphate solution (40 mM) pH 4 were mixed with thirty parts (by volume) of acetonitrile; and the isocratic flow rate was 0.85 ml/min. A water:

acetonitrile (70:30 v/v) mixture was used as a rinse solution for the injector, and the injection volume was fixed at 5 µl. Detection was carried out using a wavelength of 214 nm. The peak purity for NTX was also determined.

Validation of the HPLC analytical method

The developed method was validated according to International Conference on Harmonisation (ICH) guidelines (Shah et al., 1992).

Approximately 10 mg of NTX were accurately weighed, transferred into a 10 ml volumetric flask and dissolved in triple-distilled water obtained in-house by reverse osmosis and commercially known as Milli-Q water (MilliQ, Millipore, USA) to yield a stock solution of 1mg/ml. From the stock solution, a serial dilution was performed using the water:acetonitrile (70:30 v/v) mixture to yield a standard calibration curve with a concentration range from 5 to 50 µg/ml.

Chapter 2….Preformulation of Naltrexone Hydrochloride

Precision and accuracy

Intra-day and inter-day variabilities were determined by repeated injections of quality control (QC) samples. The QC samples were prepared at 8, 18 and 40 µg/ml representing low, middle and high controls respectively. Accuracy was assessed by comparing the predicted concentrations of the QC samples with the nominal 8, 18 and 40 µg/ml concentrations.

Limit of detection (LOD) and limit of quantitation (LOQ)

LOD is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value, calculated using equation 2.1.




equation 2.1

Where σ is the standard deviation of the intercept, and S is the slope of the calibration curve

LOQ is the lowest amount of analyte in a sample, which can be quantitatively determined in suitable precision and accuracy calculated using equation 2.2.




equation 2.2

Specificity (peak purity determination)

The HPLC-PDA detector with the Agilent ChemStation software allowed on-line data acquisition of UV-spectra between 190-400 nm during peak elution. The PDA detector provides more information on sample composition than single-wavelength detection (Krull & Szulc, 1997; Sinha et al., 2007). The UV spectra were obtained at four different points across the NTX peak; two points before the peak apex (leading front), one point at the apex and one point after the apex (tailing front). The peak purity was assessed by examining the similarity of the UV spectra obtained at the five points. If an impurity or degradation product coelutes with the NTX peak, the five UV spectra obtained across the peak are different. The peak purity analysis was carried out for all NTX samples throughout the study. Forced degradation studies

NTX was subjected to common stress conditions according to ICH guidelines to force NTX degradation. The stress conditions were acid (0.1 M HCl) at 60 o C, base (0.1 M NaOH) at 60 o C, oxidative (2% v/v H 2 O 2 ) at room temperature, thermal (PBS pH 7.4) at 60 o C and photolysis (exposure to artificial daylight illumination of 10,000 lux) at 40 o C for 24 h (Alsante et al., 2003;

Chapter 2….Preformulation of Naltrexone Hydrochloride

Reynolds et al., 2002). The samples were allowed to cool, neutralise (acid and base samples), be filtered and be diluted before injection into the chromatographic system. All experiments were performed in triplicate. Degradation product identification using electron spray ionisation-mass spectroscopy


To identify peaks of the resultant degradation products, samples were run on a Bruker MicroOTOFQ mass spectrometer (Bruker Daltronics, Bremen, Germany) coupled with a KD Scientific syringe pump (KD Scientific, Holliston, Massachusetts, USA). Samples were diluted to 1-10 µg/ml and were introduced by direct infusion at 180 µl/h into an ESI source in a positive ionisation mode (capillary voltage -4500V) using 99.999% nitrogen gas as drying nebuliser gas (180 o C, 5 L/min and pressure of 0.5 bar). External mass calibration was performed using sodium formate clusters and an enhanced cubic fit for the mass curve. Data was acquired for 3 min over a 50 to 1000 m/z range. Spectra were averaged over the 3 min and processed using Compass software 1.3 (Bruker Daltonics, Bremen, Germany). pH-degradation rate studies

NTX solutions were prepared at a concentration of 0.4 mg/ml in phosphate buffers of pH 4.5-9.5. The buffer solutions were prepared by dissolving a suitable amount of NaH 2 PO 4 or Na 2 HPO 4 to make up final concentrations of 40 mM with MilliQ water. The pH of the final solutions was adjusted using H 3 PO 4 (1M) or NaOH (1M) solutions to the desired pH values at room temperature using a pH-meter (SevenEasy, Mettler-Toledo, Schwerzenbach, Switzerland) precalibrated using standard buffers at pH 4 and pH 7 (BDH, England). The prepared NTX solutions at pH 4.5-9.5 were incubated in a stability chamber (BINDER, Tuttlingen, Germany) at

60 o C. Accelerated stability and chemical kinetic studies

Screw-capped glass vials containing NTX solutions (0.4mg/ml) in PBS pH 7.4 were stored at 40, 50, 60 and 70 o C to force degradation of NTX. Aliquots were withdrawn at suitable time intervals depending on the decomposition rate, and analysed immediately using the HPLC method described above. The observed first order degradation rate constant (k obs ) was calculated from the slope of log C versus time (t) plot according to equation 2.3 (Lachman et al., 1986).

Chapter 2….Preformulation of Naltrexone Hydrochloride