Conception d'un vecteur de médicaments à base de nanoparticules d'or pour la thérapie du glaucome

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Conception d’un vecteur de médicaments à base de

nanoparticules d’or pour la thérapie du glaucome

Mémoire

Mahmoud Omar

Maîtrise en sciences cliniques et biomédicales

Maître ès sciences (M. Sc.)

Québec, Canada

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Conception d’un vecteur de médicaments à base de

nanoparticules d’or pour la thérapie du glaucome

Mémoire

Mahmoud Omar

Sous la direction de :

Élodie Boisselier, directrice de recherche

Vincent Pernet, co-directeur de recherche

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RÉSUMÉ

Introduction : Le glaucome est la seconde cause de perte de vision dans le monde. L’augmentation de la pression intraoculaire constitue un important facteur de risque associé au glaucome. Actuellement, des médicaments permettant de réduire la pression intra-oculaire sont administrés chez les patients présentant une pression intraoculaire élevée. De plus, seule une petite fraction des médicaments (<0,06%) peuvent atteindre leurs sites d’action dans la chambre antérieure de l’œil. En effet, le film précornéen ainsi que le drainage lacrymal rapide empêchent la pénétration du médicament dans l’œil. Pour résoudre ce problème majeur en ophtalmologie, nous proposons d’utiliser des nanoparticules d’or. Notre hypothèse est que les nanoparticules d’or sont mucoadhésives et relarguerons plus lentement leur contenu médicamenteux dans la cornée, augmentant ainsi le temps de résidence des molécules administrées. Cela permettrait d’augmenter l’efficacité du traitement. Objectifs : Dans un premier temps, plusieurs nanoparticules d’or stables et biocompatibles ont été synthétisées. Ensuite, leurs propriétés mucoadhésives et d’encapsulation ont été étudiées. Finalement, les nanoparticules produites ont été testées dans un modèle de souris in vivo en comparaison avec le travoprost. Matériels et méthodes : Plusieurs différents types de nanoparticules ont été synthétisées en utilisant une méthode de Brust modifiée dans laquelle un mélange de solvants contenant de l’acétonitrile permet au polymère de bien stabiliser la nanoparticule en formation. Les nanoparticules sont stabilisées par des polyéthylènes glycol (PEG) de différents poids moléculaires : 800, 2000 et 6000 Da. Les nanoparticules sont caractérisées par spectroscopie UV visible, diffusion dynamique de la lumière, microscopie électronique en transmission et analyse élémentaire. Un médicament couramment utilisé dans le traitement du glaucome, le travoprost (TV), a été encapsulé dans les nanoparticules d’or (GNP) en chauffant le mélange pour évaporer le solvant contenant le travoprost et forcer le médicament à s’insérer dans les cavités à la fois hydrophiles et hydrophobes formées par la couronne de PEG. L’encapsulation a ensuite été évaluée par spectroscopie UV-visible et chromatographie en phase liquide à haute performance. La mucoadhésion a été évaluée par spectrométrie de fluorescence. Enfin, un modèle de glaucome utilisant des injections de microbilles chez la souris a été utilisé pour comparer le temps d’action du travoprost utilisé en clinique et notre solution de GNP médicalisée. Résultats : Le diamètre du cœur métallique des GNP est de 2,2 nm à 5,2 nm et le diamètre hydrodynamique a été mesuré entre

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8,5 et 18,9 nm, selon la méthode de synthèse et le PEG qui a été utilisé. Il a aussi été observé qu’une petite quantité de GNP (77 pmol) peut encapsuler 0,004% travoprost (concentration utilisée sur le marché). De plus, 77 pmol de GNP réduisent de 17% à 27% la fluorescence des mucines en fonction du PEG utilisé, suggérant la possibilité de l’adhésion aux muqueuses comme la cornée. En fonction du protocole développé, le travoprost du marché semble avoir une durée d’action supérieure à celle des solutions médicamenteuses de GNP préparées. Conclusion : Nos résultats montrent que les GNP peuvent efficacement encapsuler le TV et interagir avec les mucines. Plus d’analyses sont nécessaires pour comprendre les résultats inattendus obtenus in vivo. Les propriétés de mucoadhésion des GNP pourraient éventuellement mener à l’amélioration de l’efficacité des traitements pharmacologiques dans une gamme étendue de maladies ophtalmiques.

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ABSTRACT

Introduction: Glaucoma is the second cause of blindness worldwide. Elevated intraocular pressure (IOP) is the highest risk factor associated with glaucoma. Currently, hypotensive drugs can be administered topically to decrease the elevated IOP in patients. However, only a small fraction of the drugs (<0.06%) can reach their site of action in the anterior chamber of the eye. The corneal barrier and rapid drainage prevent drug penetration in the eyeball. To solve this major issue in ophthalmology, we propose to use gold nanoparticles (GNP). Our hypothesis suggests that GNP can be mucoadhesive and thus increase the residence time of glaucoma medication on the cornea, leading to the improvement of the drug efficacy. Objective: Firstly, several biocompatible and stable GNP will be synthesized. Secondly, their mucoadhesion and encapsulation properties will be studied. Lastly, the prepared medicated GNP will be analyzed in vivo with a mouse model, in comparison with marketed travoprost formulation. Methodology: Several GNP were synthesized using a modified one-step Brust method. The modification involves the use of acetonitrile to stabilize GNP during polymer capping. GNP were capped with thiolated polyethylene glycol (PEG) of different lengths: 800, 2000 and 6000 Da. GNP were then characterized by UV-visible spectroscopy, dynamic light scattering, transmission electron microscopy and elemental analysis. A commonly used glaucoma medication, travoprost (TV), was encapsulated in the GNP by heating the mixture to remove TV solvent and entrapping the drug in the PEG hydrophobic pockets. The encapsulation was thus evaluated by UV and high-performance liquid chromatography. Mucoadhesion was evaluated by spectrofluorimetry. A microbead injection glaucoma model was used to compare the duration of action of the marketed travoprost and the medicated GNP solution. Results: The core diameter of GNPs were found to be 2.2 nm to 5.2 nm whereas the hydrodynamic size was found to be between 8.5 and 18.9 nm depending on the PEG used. It was also found that as little as 77 pmol of GNP can encapsulate 0.004% travoprost (marketed concentration). Moreover, 77 pmol of GNPs quenched by 17% to 27% mucins fluorescence depending on the PEG used, thus suggesting their possible binding to mucosae such as the cornea. According to the developed protocol, marketed travoprost appears to have a longer duration of action than the prepared medicated GNP. Conclusion: Our results show that GNP can efficiently encapsulate TV and interact with mucins. More analysis is needed to understand

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the unexpected in vivo results. The mucoadhesive properties of GNP could lead to the improvement of the efficacy of pharmacological treatments in a large spectrum of ophthalmic diseases.

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LIST OF TABLES

Table 1. Topical ocular hypotensive agents. ... 11

Table 2. Gold nanoparticles size.. ... 56

Table 3. Elemental analysis of gold nanoparticles synthesis. ... 56

Table 4. The UV absorption of different gold nanoparticles at different wavelengths. ... 70

Table 5. The ratio of the absorbance of gold nanoparticles at the surface plasmon resonance peak (Aspr) to the absorbance at 450 nm (A450) ... 71

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LIST OF FIGURES

Figure 1. The structure of the eye ... 1

Figure 2. The aqueous humor drainage mechanism ... 3

Figure 3.The structure of the retina ... 4

Figure 4. The global causes of blindness ... 5

Figure 5. Progression of glaucoma ... 7

Figure 6. Classification of glaucoma. ... 8

Figure 7. The mechanism of hypertensive glaucoma ... 9

Figure 8. The different routes for drug penetration into the eye ... 20

Figure 9. Various ocular barriers for drug delivery. ... 23

Figure 10. Examples of different scenarios through which mucoadhesion can occur ... 27

Figure 11. The structure of a gold nanoparticle... 33

Figure 12. The uses of gold nanoparticles as drug delivery vectors... 36

Figure 13. The proposed hypothesis for the project ... 41

Figure 14. The travoprost encapsulation procedure. ... 51

Figure 15. Gold nanoparticles UV surface absorbance plasmon peak. ... 54

Figure 16. TEM images and size distribution of different gold nanoparticles ... 55

Figure 17. The volume-weighted size distribution of gold nanoparticles. ... 55

Figure 18. UV/visible spectra of gold nanoparticles after freeze-drying and heating overnight... 58

Figure 19. Gold nanoparticles UV surface plasmon absorbance spectra after centrifugation ... 58

Figure 20. The average quenching of mucins static fluorescence ... 60

Figure 21. The average percentage loss of mucins static fluorescence ... 60

Figure 22. The Stern-Volmer equation plot calculated from the mucin quenching. ... 61

Figure 23. The UV/visible absorption spectrum for travoprost in ethanol and in water ... 62

Figure 24. UV spectra of gold nanoparticles encapsulating several concentrations of travoprost. ... 62

Figure 25. The absorbance of different concentrations of travoprost at 280nm... 63

Figure 26. The IOP increase after injection of 15 µm beadsand 10 µm beads. ... 64

Figure 27. The IOP after injection of 10 µm beads at baseline and at day 7. ... 64

Figure 28. The screening experiment ... 65

Figure 29. The IOP-lowering effect of marketed travoprost versus medicated gold nanoparticles... 66

Figure 30. The HPLC chromatogram of travoprost. ... 67

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LIST OF EQUATIONS

Equation 1. The volume (V) of gold nanoparticles core ... 47

Equation 2. The number of gold atoms present in the gold core (n) ... 47

Equation 3. The ratio of gold to sulfur ... 47

Equation 4. The molecular weight of the gold nanoparticles (MWGNP) ... 47

Equation 5. The correction factor for excitation... 49

Equation 6. The correction factor for emission. ... 49

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LIST OF ABBREVIATIONS

ARVO: Association for Research in Vision and Ophthalmology BCRP : Breast cancer resistance protein

HAuCl4: Chloroauric acid CYPs: Cytochromes P450 DNA: Deoxyribonucleic acid DLS: Dynamic light scattering GMP: Good manufacture procedure HAuCl4∙3H2O: Gold chloride trihydrate

HPLC: High-performance liquid chromatography HCl: Hydrochloric acid

IOP: Intraocular pressure

ICP-AES: Inductively coupled plasma atomic emission spectroscopy HNO3: Nitric acid

OCT: Optical coherence tomography PEG: Polyethylene glycol

PVP: Polyvinylpyrrolidone F2: Prostaglandin F2 receptor RGCs: Retinal ganglion cells

ROCK: Rho-associated protein kinase MRP: Resistance-associated proteins NaBH4: Sodium borohydride

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Acknowledgment

I would like to thank Dr. Élodie Boisselier for giving me the opportunity to pursue this research in her lab and under her supervision and for giving me the chance to participate in several congresses in Quebec and Montreal, that helped me to evolve as a scientist. I would also want to thank the amazing team at Dr. Boisselier lab: Quentin, Mathieu, Xiaolin and Mélissa. Their presence in the lab created an excellent environment for work and learning.

I would also want to thank Dr. Vincent Pernet for his dedication and effort. Dr. Pernet is truly an exquisite professor with unique skills and knowledge that mesmerized me throughout my study. I would also want to thank Sandrine Joly for her support and eagerness to transfer her experience to me. I consider myself privileged to be able to work with her. I would also want to thank the team of Dr Vincent Pernet: Baya and Léa for their support during my project.

I would also want to thank all the members of the CUO-Recherche, students, research professionals, and professors, who make this place an excellent learning environment.

Finally, I would like to thank my family, who has always believed and supported me in pursuing my dream.

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CHAPTER 1- INTRODUCTION

1.1. The Eye

The eye is the part of the central nervous system that is responsible for vision (Masland 2012, Choplin and Traverso 2014, Ansari and Nadeem 2016 a). Anatomically, the eye is composed of two segments: an anterior and a posterior chamber. The anterior chamber protects and seals the eye from the surrounding environment and allows light to access the retina. The posterior segment transforms light waves into electrical impulses that pass through the brain via the optic nerve. The structure of the eye is shown in Figure 1.

1.1.1. Physiology of the Eye

The eye is divided into two fluid-filled chambers the anterior chamber is filled with the aqueous humor, and the posterior chamber is filled with vitreous humor. The tissues outlining the anterior chamber are in the eye’s anterior segment (tissues in front of the vitreous humor). The tissues outlining the posterior chamber are known as the eye’s posterior segment (Ansari and Nadeem 2016 a).

Figure 1. The structure of the eye, showing the eye’s anterior and posterior segments with different tissues.

Blood Vessels

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1.1.1.1. The Anterior Segment

Several tissues are found in the anterior segment, including the cornea, conjunctiva, iris, ciliary bodies, trabecular meshwork and the linked aqueous humor drainage system. The lens’s actual location is debatable because it is found in the posterior chamber and is in front of the vitreous humor. The cornea generally seals the eye from the external environment while the conjunctiva lubricates the eye and contributes to forming the tear film. In addition, the conjunctive blood network removes drug molecules from the eye’s surface. The iris maintains the pupil size, thus governing the amount of light entering the eye (Snell, Lemp et al. 1997, Yellepeddi and Palakurthi 2016, Ansari and Nadeem 2016 a). This review will focus mainly on the cornea and the aqueous humor drainage.

The cornea is the uppermost layer on the eye surface. The corneal epithelium forms the corneal surface. The corneal epithelium is a hydrophobic layer usually composed of five layers of tightly joined cells. According to the review done by Deepta Ghate and Henry Edelhauser (2008), It is estimated that the cornea prevents the entrance of 90% of hydrophilic drugs and 10% of hydrophobic drugs into the eye. The corneal epithelium is laid on the Bowman’s membrane. Below the corneal epithelium lies the stroma, an aqueous environment containing glycosaminoglycans and collagen fibrils. The stroma has a function of storing water and electrolyte, thus maintaining the corneal dome shape. It is only considered a barrier against lipophilic xenobiotics. The Descemet’s membrane separates the stroma from the endothelium, a single layer of cells that minimally controls the entrance of xenobiotics. It does however, play a role in maintaining the corneal homeostasis (Snell, Lemp et al. 1997, Snell, Lemp et al. 1997, Zimmerman 1997, Ghate and Edelhauser 2008).

The shape of the eye is maintained by producing a liquid known as the aqueous humor from the ciliary bodies. This liquid circulates in the eye, thereby maintaining its globular shape. The aqueous humor mainly exits the eye through a series of mesh like structures like the trabecular meshwork. This liquid is eventually drained into the Schlemm’s canal and the blood. This drainage pathway is called the trabecular outflow, accounting for approximately 95% of aqueous humor drainage in healthy adults. The remaining 5% exits the eye via inter-cellular spaces between the ciliary muscles. It is drained through channels and lymphatic

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vessels between the sclera and the choroid, representing the uveoscleral outflow (Toris 2014, Ansari and Nadeem 2016 a). Figure 2 shows the many routes for aqueous humor drainage.

Figure 2. The aqueous humor drainage mechanism. The yellow line represents the trabecular outflow corresponding to 95% of aqueous humor drainage. The green line represents the uveoscleral outflow. The red line represents the production and circulation of aqueous humor in the eye.

1.1.1.2. The Posterior Segment

The posterior segment contains several tissues, such as the sclera, choroid, optic nerve head and retina. The sclera is the eye’s outermost layer and acts as a support for the eye structure and underlying tissues. The choroid is a vascular tissue involved in several eye functions: namely, providing nutrients and oxygen to the retina. This review will focus mainly on the retina and optic nerve formation.

The retina is the neural part of the eye composed of three layers: the outer nuclear layer containing photoreceptors; the inner nuclear layer (bipolar cells); and the retinal ganglion cells (RGCs and displaced amacrine cells) (Masland 2012, Choplin and Traverso

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2014, Ansari and Nadeem 2016 a) (Figure 3). The RGC axons combine to form bundles; these bundles exit the eye by passing through a sieve-like structure in the sclera known as lamina cribrosa. The lamina cribrosa is a porous meshwork of connective and elastic tissues. The prelaminar, laminar and retrolaminar parts of the optic nerve are known as the optic nerve head (Holló 2014). As the light falls onto the retina, the phototransduction process occurs in the photoreceptors. The change of light electromagnetic waves through photoreceptors and neuronal impulse is the result of this process. The signal then travels from the photoreceptors to the bipolar cells, finally reaching the RGCs. Many other pathways can allow the signal to reach the RGCs. The signal passes through the brain along the optic nerve (Craven 2014, Holló 2014) (Craven 2014, Holló 2014, Ansari and Nadeem 2016 a, Ansari and Nadeem 2016 b). Another type of that is cells present in the retina are Müller cells, which are specialized glial cells involved in several neuroprotective mechanisms in the retina.

Figure 3.The structure of the retina showing the many cellular layers and cell populations. The image also shows the light path (red arrows) and the neuronal impulse path (green arrows).

1.1.2. Common Eye Pathologies

Figure 4 shows the epidemiology and causes of global blindness. The eye is prone to several conditions that affect normal vision, which in turn impact patients’ quality of life. The main causes of global blindness include cataracts, age-related macular degeneration, and

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glaucoma (Zarbin 2016). Although this review will focus on glaucoma, a short summary of other causes of blindness will be discussed.

Cataracts are the first cause of global blindness. Cataracts refer to a pathological condition where lens opacification occurs. Most patients experience blurred vision that progressively worsens until no more light can pass through the lens and reach the retina. Ultimately, patients become blind. Age and diabetes are the highest risk factors in cataracts. Other risk factors include trauma or eye perforation (Nicholas, Peter et al. 2006).

Age-related macular degeneration is another common cause of blindness. It is the third and most common cause of global blindness among patients over 65. Patients usually experience a loss of central vision that could be progressive and lead to blindness if left untreated. Age-related macular degeneration has two forms: dry and wet. On one hand, dry age-related macular degeneration is caused by the accumulation of waste products and debris under the retinal pigmented epithelium. On the other hand, wet age-related macular degeneration is caused by neovascularization of choroidal blood capillaries into the retina. Both forms cause loss of retinal neuronal cells in the macula, thus causing central vision loss. As the condition specifies, age is the main risk factor in age-related macular degeneration. Other factors such as genetic disposition and the presence of miscellaneous ocular conditions could contribute to disease development (Nicholas, Peter et al. 2006, Ambati and Fowler 2012).

Figure 4. The global causes of blindness represented as percentages in 2010 (Pascolini and Mariotti 2012).

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1.2. Glaucoma

1.2.1. Background

Glaucoma is a group of asymptomatic diseases. It is the second cause of global blindness after cataracts. Approximately 10% of patients will still develop blindness with treatment (Choplin and Traverso 2014, GRF 2016, WHO 2016). Glaucoma diagnoses are usually based on a variety of clinical findings. Glaucoma does not describe a single disease, instead, it describes a variety of diseases with common characteristics. Optic nerve head damage is the main characteristic of glaucoma, it leads to a progressive loss of vision (Choplin and Traverso 2014). Because glaucoma is not a single disease, no one condition could be used to describe it. Consequently, no one treatment is ideal for all patients. Classifications of glaucoma and the various diseases involved will be discussed in the following section.

Glaucoma is usually an asymptomatic disease until significant visual impairment is observed. The disease progression spectrum differs from one patient to the next, since the causative agent for glaucoma varies. Glaucoma normally manifests itself in an individual with full functional retinal nerve fibers. It starts as subcellular and undetectable axonal dysfunction, progressing to clear axonal dysfunction and an acceleration in axonal loss that could not be explained by factors such as age. Axonal loss may be initially undetectable, but becomes detectable with the help of diagnostic tests (axon subsets and standard white-on-white perimetry). Visual dysfunction occurs as axonal loss continually progresses. Only initial subsets of RGC axons—mainly superior and inferior nerve fibers—appear damaged through diagnostic tests. Ultimately, however, all axons will be affected, and visual impairment will continue to progress until vision loss becomes permanent (Figure 5) (Choplin and Traverso 2014).

In early glaucoma, mild diffused visual field defects start to appear. The defects usually are more visible in the periphery of the visual field. Isolated visual defects tend to appear in the superior half of the visual field. However, central vision is usually preserved in the early stages of the disease. As the glaucoma progress, visual field loss can occur in many different ways. The more common scenario is that the affected shallow defects in the visual

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field will start to merge, extend, deepen and enlarge until blind patchs are formed in the patient visual field. These blind patches are usually referred to as arcuate scotomas. In advanced cases, arcuate scotomas can appear in the superiorly and inferiorly quadrants of the retina. At this point, only separated islands of functional retinal are left. Further progression of the disease will cause loss of the central and temporal vision. Finally loss of nasal island will occur, causing a complete functional loss and blindness (Adler, Huang et al. 2008, Choplin and Traverso 2014, Chris. Johnson 2014).

Many risk factors are associated with the development of glaucoma. Age is one of the key risk factor, because the trabecular meshwork extracellular matrix changes as age advances. In particular, morphological changes to the trabecular meshwork occur when age progresses (Cavallotti 2008). Furthermore, age affects other eye tissues such as the iris and cornea, tissues that were found to contribute to glaucoma development. Genetic disposition and family history appear to be leading risk factors (Choplin and Traverso 2014). In addition, the presence of other ocular conditions is considered a disposition factor for glaucoma.

Figure 5. Progression of glaucoma, starting with normal retina leading to blindness.

Increased intraocular pressure (IOP) is the major risk factor. The average human IOP is between 12 and 22 mm Hg. Maintained high levels of IOP (22 mm Hg) are usually associated with optic nerve damage (Choplin 2014). The mechanism of how IOP affects the optic nerve is discussed below.

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1.2.2. Classifications

It is not easy to develop glaucoma into one classification, since glaucoma is a group of diseases. Glaucoma could be classified according to the age of disease onset, IOP, mechanism of glaucoma development, and the stage of the disease (Choplin 2014). Other less common classifications exist as well.

There are two common classifications of glaucoma: based on the mechanism of action and based on the intraocular pressure. According to the mechanism of action, glaucoma could be divided into four main pathways: open angle, angle closure, developmental, or mixed (Figure 6). Another easier classification is based on IOP. It could be either normotensive or hypertensive glaucoma.

Figure 6. Classification of glaucoma by the mechanism of development.

Normotensive glaucoma appears when patients’ IOP remains within normal range, but a decrease or fluctuation in the ocular perfusion pressure—the amount of blood perfused into the eye—can cause retinal damage by a series of ischemia-reperfusion to the retina and the optic nerve (Caprioli and Coleman , Leske 2009). Although the specific prevalence and frequency of normotensive glaucoma is different among populations, it is less frequent than hypertensive glaucoma (Bonomi, Marchini et al. 1998). Hypertensive glaucoma usually manifests as an asymptomatic buildup of aqueous humor in the eye, which leads to a higher IOP that damaging the retina and the optic nerve over time (Figure 7) (Choplin 2014).

This review will focus on the more frequent hypertensive glaucoma. The elevated IOP causes optic nerve damage by several mechanisms. Firstly, an elevated IOP affects the production and turnover of the extracellular matrix in the lamina cribrosa, leading to changes

Classification of Glaucoma by Mechanism

Open Angle

Primary Secondary

Mixed

Mechanism Angle Closure

Primary Secondary

Developmental

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in its mechanical and elastic properties. This results in mechanical stress on the nerve bundles passing through the lamina cribrosa and causes optic nerve head damage and partial optic nerve crush injuries (Downs, Roberts et al. 2008, Holló 2014). Secondly, the difference between the pressure inside the eye and behind the lamina cribrosa causes an axonal transport blockage. Thus, cellular organelles such as the mitochondria produced in the cell body of the RGCs, cannot pass through its axon to supply energy. In addition, the brain-derived neurotrophic factor released from the brain is unable to reach the RGC cell body (Downs, Roberts et al. 2008, Holló 2014). The combination of these conditions leads to apoptosis mediated damage to the RGCs (Downs, Roberts et al. 2008, Holló 2014). Lastly, an elevated IOP causes dysregulation in the arterial blood supply to the optic nerve head, resulting in a series of ischemia-reperfusion damage that causes RGC apoptosis over a period of several years (Downs, Roberts et al. 2008, Holló 2014). The exact mechanism of RGCs deaths however, remains mostly unknown; the discussed events could only describe a portion of the full mechanism for optic nerve damage.

Figure 7. The mechanism of hypertensive glaucoma. A) Normal eye (aqueous humor is produced by the ciliary bodies and drains from the trabecular meshwork). B) In hypertensive glaucoma, the drainage of the aqueous humor is blocked. C) The blockage in aqueous humor causes IOP elevation. D) IOP elevation causes optic nerve head damage.

1.2.3. Current Treatments

Many treatment options have been proposed to fight glaucoma over the past few years. Reducing progression of this disease and preserving quality of life are the main

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treatment goals (Weinreb, Aung et al. 2014). Lowering the IOP is the only proven factor that can treat glaucoma (McKean-Cowdin, Wang et al. 2008, Boland, Ervin et al. 2013). Several clinical trials have demonstrated that lowering the intraocular pressure can delay the disease’s progression and maintain the patient’s quality of life (Paul, Fred et al. 2000, Lichter, Musch et al. 2001, Kass, Heuer et al. 2002). Hence, pharmacological treatments are the first line of treatment for glaucoma instead of surgery.

Several pharmacological treatments are available to reduce intraocular pressure (Johnson and Tomarev 2016). Current management guidelines developed by the American Academy of Ophthalmology Preferred Practice Pattern require clinicians to set a target IOP for each patient upon diagnosis. The chosen ideal pressure is based on the severity of glaucoma and the patient’s retinal damage. In general, an IOP reduction between 25% and 50% is set. However, the target IOP may change with the progress of patient follow-up (Danias and Podos 1988, Weinreb, Aung et al. 2014, Prum, Rosenberg et al. 2016). Many classes of drugs are used to treat glaucoma; they either reduce aqueous humor production or increase its drainage. A list of these medications is shown in Table 1.

Drug choices will depend on the cost, adverse effect, and administration schedule. Prostaglandins agonists are usually one of the most effective drugs, and many clinicians use it as the first line of treatment. The average IOP reduction with these drugs is between 23% and 34% (Stewart, Konstas et al. 2008, Williamson and Serle 2014). More details about this drug class will be discussed in the following section.

Other less effective drug classes are β-adrenergic receptors blockers—the most used drug from this class being timolol. These drugs reduce the production of aqueous humor, thus reducing intraocular pressure in the eye (Ellis, Wu et al. 1991). Due to the high possibility of the drugs’ systemic absorption, they are not often prescribed these days (Weinreb, Aung et al. 2014). Other classes involve the use of α-adrenergic agonists such as brimonidine or carbonic anhydrase inhibitors such as dorzolamide. Both of these drugs act by reducing the production of aqueous humor. However, there are several contraindications involved with them. The biggest disadvantage with this medication is that they are given three times daily, which requires a lot of patient commitment (Brusini 2014).

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Table 1. Topical ocular hypotensive agents. Information obtained from (Williamson and Serle 2014).

Drug Class Primary Mechanism Secondary Mechanism IOP Reduction (%)

Prostaglandins Agonists Increasing uveoscleral outflow Increasing trabecular meshwork outflow 25-35

Beta-blockers Decreasing aqueous

production 17-29 Carbonic Anhydrase Inhibitors Decreasing aqueous production 15-24 Alpha-adrenergic Agonists Decreasing aqueous production Increasing uveoscleral outflow 14-28

Finally cholinergic agonists, such as pilocarpine, could be used to decrease production of aqueous humor. Unfortunately, pilocarpine has a muscarinic action, thus stimulating the lacrimal gland to increase tear production (Ellis, Wu et al. 1991). This fact is crucial: an increase in tear fluid reduces the potential for drug absorption into the eye. This could explain why pilocarpine is prescribed four times daily and requires more commitment on the patients’ behalf (Weinreb, Aung et al. 2014).

Aside from pharmacological treatment, surgical options are available if drugs have adverse effects or glaucoma is acute. Laser trabeculoplasty is the least invasive surgery for glaucoma: a laser beam is used to induce biological changes in the trabecular meshwork, thus increasing the aqueous humor outflow. This procedure is easier than other techniques discussed later on (Odberg and Sandvik 1999). Trabeculectomy is another highly performed surgical option chosen to lower the IOP. It is, however, more invasive than laser trabeculoplasty. Trabeculectomy removes a small part of the trabecular meshwork to provide another route of drainage for the aqueous humor. Another introduced surgical option for glaucoma is the nonpenetrating glaucoma surgery (Rulli, Biagioli et al. 2013). Although the mechanism through which the surgery is performed differs, this surgery does not penetrate the eye ball. As a result, there are less risk involved in the surgery. The principle involves

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creating a deep scleral flap to expose the canal of Schlemm’s. Then striping off some of the inner canal wall thereby enhancing natural out flow (Chan and Ahmed 2014). Another used technique is using aqueous shunts. Nowadays there are several devices. Generally, the device is made of several plates connected to a tube. The device is then placed in the eye to maintain aqueous humor outflow through the exiting tube (Song, Giaconi et al. 2014).

1.2.4. Treatments under Development

Several treatment options are under development for glaucoma. Though many new surgeries and implantation devices are being developed, the scope of this review will focus only on medical treatment (Weinreb and Kaufman 2011).

Many of the newly developed treatments still aim to reduce IOP. Since prostaglandins agonists are the most effective class, most of the new treatments work like them. Latanoprostene is one of the anticipated glaucoma treatments . It had recently passed phase III clinical trials and will be marketed soon. Latanoprostene is a combination of two drugs: latanoprost, a powerful prostaglandin agonist, and butanediol mononitrate, a nitric oxide donor (Dikopf, Vajaranant et al. 2017).

Latanoprostene breaks down and releases its components once absorbed. Latanoprost acts like other prostaglandins agonists, which will be discussed in the next section. Butanediol mononitrate will undergo several changes to release nitric oxide. The released nitric oxide will alter the actin cytoskeletons and actomyosin contractility, causing a decreased resistance to trabecular outflow (Weinreb, Ong et al. 2015). In addition, animal studies suggest that released nitric oxide could relax ciliary muscles, thus increasing the uveoscleral outflow (Gabelt, Kaufman et al. 2011).

The use of Rho-associated protein kinase (ROCK) inhibitors is another promising treatment. ROCK have been found to have several receptors in the trabecular meshwork and Schlemm’s canal. Additionally, they are involved in several cellular processes, such as growth, shape, apoptosis, adhesion, smooth muscle contraction, and they negatively regulate endothelial nitric oxide levels in the body (Liao, Seto et al. 2007, Rao and Epstein 2007). Animal studies first suggested that ROCK inhibitors caused cellular separation. In fact, ROCK inhibitors separated the trabecular meshwork’s inner wall from the Schlemm’s canal

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and separated the Schlemm’s canal cells. This tissue remodeling can explain why intraocular pressure decreased (Waki, Yoshida et al. 2001, Nishio, Fukunaga et al. 2009, Dikopf, Vajaranant et al. 2017). Ripasudil recently passed phase III clinical trials, and is a common drug in this class. Other drugs under development include SNJ-1656 and verusodil; both have passed phase II clinical trials (Dikopf, Vajaranant et al. 2017).

In addition, adenosine receptor (A1) agonists reduce intraocular pressure. Firstly, animal studies have suggested that selective activation of adenosine receptor (A1) agonists cause a reduction in aqueous humor formation in the ciliary bodies. Secondly, it was also proven that adenosine receptor (A1) agonists cause an increase in metalloproteinases production in the trabecular meshwork and the Schlemm’s canal. Finally, an increase in metalloproteinases will result in tissue remodeling and improved aqueous humor outflow (Avila, Stone et al. 2001, Zhong, Yang et al. 2013, Dikopf, Vajaranant et al. 2017).

1.2.5. Animal Models of Glaucoma

There are various animal models in place for studying glaucoma in preclinical settings. Aside from genetic models, the inducible glaucoma models currently available could be divided into three main categories: pre-trabecular, trabecular, and post-trabecular. Pre-trabecular models inject substances such as microbeads or magnetic beads into the eye’s anterior chamber. These injected materials will restrict aqueous humor drainage. Trabecular models induce direct damage to the rodent’s trabecular meshwork, such as the use of laser trabeculoplasty. Unfortunately, using lasers on a rodent’s eye is difficult and impractical for drug screening. Lastly, post- trabecular models involve a variety of surgical procedures aimed at inflicting damage to the episcleral veins. Examples of these models include laser episcleral coagulation, cauterization, and hypertonic saline injection (Bron 2014, Smedowski, Pietrucha-Dutczak et al. 2014). Trabecular and post-trabecular methods are usually invasive and dramatically alter many biochemical reactions and anatomical structures in the eye. Thus, the use of trabecular and post-trabecular models is limited to drug testing (Goldblum and Mittag 2002, Pang and Clark 2007).

Microbead injection is one of the relatively new inducible glaucoma models. Unlike trabecular and post-trabecular methods, microbead injection does not involve complicated

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surgical procedure. Moreover, microbead injection does not damage the trabecular meshwork and surrounding tissues, making it easy to undergo drug evaluations. Lastly, microbead injection induces a more chronic form of glaucoma, that is often observed in patients (Vidal, Díaz et al. 2010, Samsel, Kisiswa et al. 2011, Yang, Cho et al. 2012, Bron 2014, Matsumoto, Kanamori et al. 2014, Smedowski, Pietrucha-Dutczak et al. 2014, Lambert, Carlson et al. 2015).

1.3. Travoprost

1.3.1. Mechanism of Action and Pharmacokinetics

Travoprost is a prostaglandin agonist; it is one of the most popular drugs because, unlike other available medications, it reduces IOP with minimal side effects (Quaranta, Riva et al. 2015). This drug class is designed to work on the prostaglandin F2 receptor (F2), which lowers IOP by three mechanisms (Lindén 2002).

Firstly, a short-term effect by acting on the F2 receptor in the smooth muscle tissues, causing ciliary muscle relaxation, thus facilitating aqueous humor exit through the uveoscleral pathway (Doucette and Walter 2016). Secondly, a principal long-term effect due to the expansion of intercellular spaces in the ciliary bodies and trabecular meshwork through remodeling of the extracellular matrix (Pradhan, Dalvi et al. 2014). F2 receptor activation was found to induce upregulation of matrix metalloproteinases, which breaks down collagen in the extracellular matrix. The result is an enhancement of the uveoscleral outflow of aqueous humor. This effect persists after the treatment is stopped (Pradhan, Dalvi et al. 2014).

Finally, an indirect effect F2 activation was also found to induce prostaglandin E2 and E3, both are playing a role in tissue remodeling and reduction of IOP as well (Ota, Aihara et al. 2006). However, clinical pharmacokinetics following administration of 0.004% travoprost showed that its plasma half-life is between 30-45 minutes, reaching maximum concentration after 30 min of application. The drug concentration then falls below detection limit after one hour of application (Lindén 2002, Robin, Faulkner et al. 2002, Quaranta, Riva et al. 2015). The short kinetics time might be related to its low solubility and difficulties to cross the optical barriers.

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1.3.2. Current Travoprost Formulation

Many travoprost formulations are currently on the market. The brand name Travatan® is given to travoprost 0.004%, produced by Alcon. Initially, the Food and Drug Administration (FDA) and European marketing authorization approved travoprost as an eye drop solution (40 μg/mL/0.004%) preserved with benzalkonium chloride in 2001 (Houda, Angelica et al. 2014).

This formulation has produced many side effects because of the preservative benzalkonium chloride. Ocular surface toxicity, such as tear film hyperosmolarity and corneal epithelium inflammation, was demonstrated with long- term exposure that reduced drug tolerability (Ammar, Noecker et al. 2010, Actis and Rolle 2014). This gave way to developing two newer formulations. The first formulation was travoprost 0.004%, using sofZia® as a preservative. By 2006, this formula was approved for marketing in the United States, Canada, and Japan under the brand name Travatan Z ®. Travatan Z ® is the currently used formulation in Canada. The second was travoprost 0.004%, preserved by polyquaternium-1, which was approved and marketed in Europe and worldwide in 2010 under the brand name Travatan PQ ®. Both new formulas produced fewer side effects, such as conjunctival inflammation, during chronic usage (Mirza and Johnson 2010, Houda, Angelica et al. 2014).

In 2006, Alcon started to market a new travoprost formula named DuoTrav PQ®. This formula contains two drugs: travoprost 0.004% w/v and timolol 0.5% w/v. In addition, polyquaternium-1 was used as the preservative. This formula shows a stronger IOP control than travoprost alone (Alcon 2013).

Shortly after this formula was on the market, drug companies realized that the travoprost molecule was still causing side effects, with eye redness being the most common. About one in every three patients will develop this condition. Clinical trials have demonstrated that redness was mild and would gradually decrease in intensity. Other less common side effects included stings, itchy eyes, iris pigmentation, dry eyes, eyelid inflammation, and blurred vision (Mirza and Johnson 2010, Houda, Angelica et al. 2014).

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Efforts were made to optimize the used travoprost concentration versus efficacy. Consequently, a new formula was proposed containing only 0.003% of travoprost. Further to this proposal, several clinical studies have tried to demonstrate the advantage of this smaller dose. Although the 0.003% and 0.004% travoprost had the same efficacy, the difference in side effects was not significant: only less corneal hyperemia was observed. This clinical study, however, lasted only three months (Houda, Angelica et al. 2014, Alcon 2016). Nowadays, many countries are shifting to smaller medication doses. For example, travoprost 0.003%, preserved by polyquaternium-1, is the approved formula in Europe. Although several generic formulations are expected to appear for travoprost 0.003%, the current brand name for the new formula developed by Alcon is IZBA ®. The product was approved by the FDA, European marketing authorization in 2016 (Houda, Angelica et al. 2014, Alcon 2016).

1.3.3. Advances in Travoprost Delivery

As suggested from the fast elimination pharmacokinetic data of travoprost, the main way to enhance travoprost action is to sustain the delivery of travoprost for a longer period. For that reason, many of the research is aimed to overcome the ocular barriers (discussed in the coming section) and sustain the delivery for prolonged periods. It seems that most of the research and clinical trials are more directed toward ocular implants for travoprost.

Lambert et al had developed a nanosponge made from cross-linked polyethylene glycol. The nanosponge was administrated by an intravitreal injection in a mouse based glaucoma model. Results have shown that the nanosponge was able to deliver travoprost for more than 14s days. The nanosponge formulation showed slightly better retinal ganglion survival than marketed topical travoprost (Lambert, Carlson et al. 2015). Although the nanosponge has an impressive elongated of travoprost action, the route of administration of this sponge is much too invasive to be used in chronic disorders. In addition, in the case of side effects removing the injected sponge from the vitreous humor is difficult (Pharma Tips 2011).

In a study done by Trevino et al (2014), a biodegradable implant formulation containing travoprost (25 to 45 µg) was developed. The implant was made by a patent PRINT® technology (Trevino, Navratil et al. 2014). The PRINT® technology was developed

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by a pharmaceutical company called Envisia Therapeutics. This is a newly developed company in 2013 with an aim to improve ocular drug delivery. The developed method allows the designing and printing of nanoparticles with high precision. The designed medicated particles were injected into the anterior chamber in dogs, and the drug concentration remained within the detectable limit for a period longer than 60 days post administration (Trevino, Navratil et al. 2014). Nonetheless, such delivery system will have the disadvantages of the nanosponge discussed above. The company is currently in phase 2 clinical trial with a new formulation based on this technology under the name of ENV515. Unfortunately no details about the composition of this new formula is known at this point (Envisia Therapeutics 2017).

Another study by Miller et al (2013) showed travoprost could be encapsulated in polymeric microspheres. Spheres were suspended in a polyethylene glycol-based hydrogel that was used as a punctual plug (tear duct plug) on dogs. Results revealed that the travoprost concentration was visible three months after post-insertion (Miller, Blizzard et al. 2013). Such a device would be less invasive than implants and could be removed if patients suffer from side effects. This system, however, requires special training and cannot be applied by patients themselves.

Avery, R. L et. Al. (2010) had formulated a mini-drug pump. The pump is composed of a drug reservoir, an electrolysischamber, battery, and electronics for controlling drug delivery. The fluid is injected into the eye via a cannula. The system can work remotely, and the rate of drug infusion could be controlled. Through a simple clinic visit, the device reservoir could be refilled, and the battery could be recharged (Avery, Saati et al. 2010). The device implanted in the temporal conjunctiva, with a cannula inserted into the anterior chamber, was tested in dogs. The device was tested for the delivery of travoprost 0.004%. Results showed that the device could control IOP as topical formulations. In addition, no side effects were observed during three months of treatment (Avery, Saati et al. 2010). Unfortunately, such a device is complicated and requires special care that may be unsuitable for some patients.

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1.4. Ocular Drug Delivery

1.4.1. Drug Administration in the Eye

Drug delivery to the eye has a simple goal: deliver the correct pharmacological agents to their site of action. The delivery route taken will depend entirely on the drug action site. Because the eye is mainly divided into the anterior and posterior segments, the drug route for administration will differ.

Topical administration is the preferred method for drug delivery to the anterior segment, and the most common topical administration—making up 90% of all ophthalmic formulations—is eye drops. They are easy to use and require minimal patient training. Topical eye drops also come in a variety of different formulations, which allow them to deliver most of the ophthalmic drugs available (Cholkar, Patel et al. 2013, Patel, Cholkar et al. 2013). More details about various formulations will be provided in section 4.3. Furthermore, most topical eye drops have low viscosity and are not retained on the eye surface for long periods. For this reason, other topical drug formulations like ointment and cream exist (Cholkar, Patel et al. 2013, Patel, Cholkar et al. 2013).

Recent advances in formulating drug-eluting contact lenses or inserts could formulate dosage forms which retains the drug for four weeks on the eye surface. In addition, drug therapy could be stopped at any time by removing the medicated lens or insert. An example of this medication is Ocusert®, a marketed ocular insert of pilocarpine that produces a sustained and controlled dosage with almost zero order kinetics. It is claimed that this dosage form can work for at least one-week after post administration (Edwards 1997).

Nonetheless, drugs with low permeation, such as charged protein and peptide, can be administered topically with special delivery methods like iontophoresis. This technique uses a small electrical current (the same charge as the drug) to create a repulsive force that coerces the drug through the cornea, thus improving its permeation. Many clinical and preclinical studies have shown that iontophoresis could increase drug permeation 100 times more than topical eye drops. This delivery method, however, is more complicated than eye drops and must be applied by a trained professional (Eljarrat-Binstock and Domb 2006, Yavuz and Kompella 2016, Yellepeddi and Palakurthi 2016).

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Microneedles are more extreme and are widely discussed in drug delivery—most recently in ophthalmology. Small microneedles are used to disturb the corneal barrier by creating holes in the corneal epithelium, thus allowing better drug permeation. Animal studies suggested that microneedles can improve drug delivery 60 times more than topical eye drops. Unlike other topical delivery methods, microneedles have less patient compliance (Yellepeddi and Palakurthi 2016, Raghu, Ismaiel et al. 2017).

With respect to drug delivery to the posterior segment, topical drug application is usually insufficient for optimum drug bioavailability. Upon installing a drug drop on the cornea, it reaches the posterior segment via several routes. In most cases, the drug concentration is insignificant. Posterior segment delivery can be made in a few ways. The first is an intravitreal injection; 100% of the drug is assured to reach the back of the eye without any loss. Many risk factors are associated with the operation; only trained ophthalmologists can perform it. Further, treatment is invasive and uncomfortable for patients (Shah, Denham et al. 2010, Yavuz and Kompella 2016). The other two methods include subretinal and suprachoroidal injections. Although both methods provide unique advantages compared to intravitreal injection, they are more complicated because of a high risk of hemorrhage and tissue damage, causing retinal damage and blindness (Yavuz and Kompella 2016).

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Figure 8. The different routes for drug penetration into the eye. The green arrows represent the trans-corneal transport, which is the most important route for ocular delivery. The purple and red arrows represent the less common route for ocular delivery: noncorneal route and systemic recirculation, respectively.

1.4.2. Challenges for Topical Drug Delivery

There are several challenges for drug delivery. The challenges for non-topical methods for ocular drug delivery systems, such as injection, could be simply explained by their inherent potential side effects and low patient compliance. In addition, other systems aimed at targeting the eye through systemic circulation are usually less effective due to the eye’s special anatomy. The scope of this review will focus mainly on challenges for topical drug delivery.

Once an eye drop is applied to the eye, the drug can reach the eye through various routes. The most important route for topical ophthalmic drug delivery is the trans-corneal route. Drug molecules pass through the cornea to the aqueous humor where it can reach its site of action (predominately in the anterior segment). Alternatively, the drug can pass

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through the noncorneal route, which involves drug drainage to the conjunctiva and the systemic circulation. The drug could also enter the eye by passing from the conjunctiva to the sclera, either draining outside the eye or reaching the posterior segment through the choroidal blood vessels. Drug molecules normally passing through the noncorneal route is considered lost, for drug concentration reaching the eye is usually low and unnoticeable (Cholkar, Patel et al. 2013, Yavuz and Kompella 2016). Figure 8 outlines all the various routes for drug absorption following topical application.

A better understanding of the challenges facing optimum topical drug delivery through the trans-corneal route is needed. It was reported that less than 0.0006% to 0.02% of a topically administered drug will be absorbed into the eye (Keβler, Bleckmann et al. 1991, Joshi, Maurice et al. 1996, Maurice 2002, Loftsson, Jansook et al. 2012). We can divide the challenge for drug delivery into three barriers. The first is patient related, since many of the ophthalmic conditions are asymptomatic for most of the course, such as in glaucoma. Hence, many patients are unmotivated to undergo a rigorous daily schedule of medication for a disease they cannot feel (Williamson and Serle 2014). In addition, several antiglaucoma medications have side effects, especially if they are used for an extended period. In a clinical trial, about 28% to 58% of patients admitted they missed their daily prescribed dose of medication. This discontinuation of treatment could be due to low patient's compliance related to demanding schedules or increased side effects (Zimmermann and Zalta 1983).

The second barrier for optimum drug delivery could be related to the drug’s own physicochemical properties. Many of the drugs used to treat glaucoma are hydrophobic with minimal water absorption, making drug formulation difficult. Worse, many of the drugs have low permeation if they are not formulated with the correct delivery vehicle (Yee 2007, Souza, Dias et al. 2014).

The final drug delivery barrier is biochemical and biological. The eye’s cornea is covered with several transporter proteins. About 29% of transporter proteins in the eye are present in the cornea; many proteins cause drug efflux outside the cornea, thus preventing its absorption (Nakano, Lockhart et al. 2014). Examples of such proteins include members of the ATP-binding cassette transporters, especially multidrug resistance-associated proteins (MRP) 1, 2, 3, 5, and 6. Breast cancer resistance protein (BCRP) was also found in the cornea

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(Dey, Patel et al. 2003, Karla, Pal et al. 2007, Karla, Pal et al. 2007, Dahlin, Geier et al. 2013, Nakano, Lockhart et al. 2014). In an animal study, prostaglandins agonists used to treat glaucoma were effluxed out of the rabbit eye. The degree of drug efflux was found to depend on the vehicle used with the drug (Hariharan, Minocha et al. 2009).

Another biochemical barrier includes the drug metabolism in the cornea. Many drug metabolizing enzymes are present in the cornea. The most common are members of the Cytochromes P450 (CYPs), in particular CYP4B1, CYP3A4, CYP2A6, CYP2C8, CYP2D5, CYP2E1, and CYP3A4 (Nakano, Lockhart et al. 2014). CYPs were reported to metabolize drugs in the liver; it could be assumed that they do the same in the cornea, thus reducing the amount of drug permeating to the eye. Another important metabolizing enzyme includes ocular esterases. Several drugs are reported to be metabolized by corneal esterases such as acyclovir and prostaglandins agonists (McCue, Cason et al. 2002, Anand, Katragadda et al. 2004, Nakano, Lockhart et al. 2014).

Regarding the biological barriers, the eye is designed as a closed environment to prevent the entry of any xenobiotics as discussed in section (1.1.1). Firstly, the conjunctival capillary network quickly drains any drug that comes in its contact. Secondly, the tear film on the eye is a mixture of lipids and secreted mucins that quickly binds drug molecules and prevents them from reaching the corneal surface (Chrai and Robinson 1976, Gunda, Hariharan et al. 2008). Thirdly, nasolacrimal drainage and blinking swipe the drug away from the cornea. Fourthly, the transmembrane mucins and corneal epithelium are a challenge for drug permeation into the aqueous humor (Chrai and Robinson 1976, Gunda, Hariharan et al. 2008). Figure 9 shows the predominant biological barriers for drug delivery.

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Figure 9. Various ocular barriers for drug delivery.

1.4.3. Topical Ocular Drug Formulations

There are several topical formulations available for topical drug delivery. The conventional delivery method used is ophthalmic eye drops. Eye drops could be either solutions, emulsions, or suspensions. Solution eye drops are an easily formulated dosage form of water-soluble drugs. Eye drop solutions usually contain a variety of additives, such as viscosity modifiers (to increase drug residence time onto the eye), preservatives, permeation enhancers, and sometimes solubility enhancers. Modern solutions usually contain carrier molecules and polymers, such as cyclodextrins, that can act as a viscosity enhancer and drug encapsulating agent (Patel, Cholkar et al. 2013).

Emulsions could be made with eye drops, oil in water, or water in oil. The emulsion type depends on the emulsifying agent used and the amount of each phase. Emulsions could be used to deliver hydrophilic and hydrophobic drugs; many of the oils used in emulsions may have an anti-inflammatory property crucial for medications used during extended periods. In addition, the lipophilic components in emulsions can act as a supplement for the tear film lipids in patients with dry eye syndrome (Patel, Cholkar et al. 2013). Clinical studies have demonstrated that emulsions can improve precorneal residence time and drug corneal permeation, ultimately providing sustained drug release and enhancing ocular bioavailability

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(Stevenson, Tauber et al. 2000, Tamilvanan and Benita 2004, Liang, Brignole-Baudouin et al. 2008).

Suspensions are another form of eye drops on the market. They are usually a finely divided insoluble drug (dispersive phase) suspended in an aqueous phase (continuous phase). In many cases, suitable suspending and dispersive agents are used to maintain drug flocculation after shaking for a suitable period, allowing uniform dosage (Patel, Cholkar et al. 2013). Aside from delivering insoluble drugs, suspended particles were found to remain in the precorneal pocket. When compared to solutions, these particles can improve drug contact time and duration of drug action, the latter being related to the particle size. Small particle size can provide faster dissolution and drug release, while larger particles can remain for a longer period in the precorneal pockets (Lang, Roehrs et al. 2006).

Other ophthalmic topical formulations include ointments and gels. They offer the same advantages as liquid topical formulations and eye drops, but semisolid consistency enables them to be retained for a longer period on the eye surface, thereby improving drug bioavailability. They are particularly useful in applying antibiotic ointments that will last on the eye surface until morning hours (Patel, Cholkar et al. 2013).

Modern advancement in polymer and material science has allowed the formulation of liquid and semisolid with improved qualities. Nano-ophthalmology is a growing field under development with the use of nanosuspensions such as liposomes, nanoemulsions, polymeric microspheres, nanomicelles, and dendrimers. Such unconventional formulations, are now widely investigated and are on the market. Still, many other strategies have been devised to enhance topical formulation, including iontophoresis, ocular inserts, contact lenses, permeating polymers, and mucoadhesive polymers (Ghate and Edelhauser 2008, Patel, Cholkar et al. 2013, Yellepeddi and Palakurthi 2016). Sections 1.4.4 and 1.5 will focus on mucoadhesion and nanoparticles (gold nanoparticles) used to enhance drug delivery.

1.4.4. Mucoadhesion

Mucoadhesion is a phenomenon of some substances binding to the mucosal surfaces. When used as a vehicle for drug delivery, such substances can prolong the residence time of drug molecules onto mucosal surfaces such as the cornea. The corneal epithelium contains

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various transmembrane mucins. Mucoadhesion drug formulations applied topically to the cornea will have an increased residence time, thus achieving a higher concentration onto the cornea that favors their permeation (Dhawan, Singla et al. 2004). There are several mechanisms through which mucoadhesion can occur as entrapment of a large polymeric substance within the transmembranal mucin (Robinson and Mlynek 1995, Ludwig 2005, Nagarwal, Kant et al. 2009).

The human corneal surface contains several species of mucins that could be divided into secretory mucins and membrane-associated mucins (Ruponen and Urtti 2015, Ablamowicz and Nichols 2016, Leonard, Yanez-Soto et al. 2016). Secretory mucins are either soluble or gel-forming, and are considered part of the lacrimal fluid. In the human cornea, MUC2, MUC5AC, MUC5B, MUC6, and MUC19 are the most present. MUC5AC has a low molecular weight and is the most expressed on the ocular surface preventing the formation of viscous mucus on the eye (Gipson, Spurr-Michaud et al. 2014, Uchino, Woodward et al. 2016).

Membrane-associated mucins are more challenging for drug delivery. In the human cornea, MUC1, MUC4, MUC16, and MUC20 are expressed on the corneal surface. Of these mucins, MUC16 is the most abundant and has the largest molecular weight. A fully glycosylated MUC16 molecule has a molecular weight of 20 MDa. Given this reality, it was reported that MUC16 prevented corneal staining when a topically applied dye was administered on a mouse’s eye (Gipson, Spurr-Michaud et al. 2014, Uchino, Woodward et al. 2016).

1.4.4.1. Theories of Mucoadhesion

Numerous theories explain how mucoadhesion occurs. The first is wetting, usually referring to the formulation’s ability to spread on a mucosal surface. It provides an understanding of lipophilic or low-viscosity system mucoadhesion properties (Boddupalli, Mohammed et al. 2010, Yu, Andrews et al. 2014), and is examined by adding a drop of a mucoadhesive substance onto a mucosal surface. If the solid-liquid angle is small, the drop spreads on the surface. If, however, the angle is large, and the drop remains spherical, the

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formulation is not mucoadhesive (Shafrin and Zisman 1960, Boddupalli, Mohammed et al. 2010, Yu, Andrews et al. 2014).

The second theory is interpenetration, which takes two forms. Mechanical interpenetration refers to an entangled mucosal surface with a rough or an uneven dosage form surface (Yu, Andrews et al. 2014), usually suggesting that mucoadhesion can occur between the buccal mucosa and tablet. Mechanical interpenetration can also be achieved through diffusion, where a large molecular weight polymer is entangled with a mucin molecule, anchoring it in place (Vasir, Tambwekar et al. 2003).

The third theory is electronic transfer. Here, mucoadhesion is the result of electron transfers between mucins and a mucoadhesive system with opposite charges. These transfers give way to the formation of a double-layered electronic charge at the interface (Dodou, Breedveld et al. 2005, Boddupalli, Mohammed et al. 2010, Yu, Andrews et al. 2014).

The final mucoadhesion theory is absorption. Various interactions occur at the mucosal surface that results in adhesion. These can be divided into two sub-interactions. On one hand, primary interactions, such as ionic and covalent bonds, are strong and offer good adhesion, but are undesirable because they form a strong, permanent interaction with mucins that may be toxic. On the other hand, secondary interactions, such as Van der Waals forces, hydrogen bonds, and hydrophobic interactions, are more desirable, because they form a less permanent bond and are less toxic (Boddupalli, Mohammed et al. 2010, Yu, Andrews et al. 2014). Figure 10 shows a summary of the mucoadhesion theories discussed above (hydrophobic interaction, molecular entanglement, electronic transfer and primary mucin interaction).

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Figure 10. Examples of different scenarios through which mucoadhesion can occur. The first picture represents the hydrophobic interaction theory. The second picture represents molecular entanglement theory. The third picture shows the electronic transfer theory and lastly the fourth picture shows an example of primary mucin interaction theory.

1.4.4.2. Mucoadhesion Substances in Drug Delivery

One of the main problems with topical ocular drug delivery is the medication’s short residence time on the corneal surface. Mucoadhesion increases the drug’s residence time on the ocular surface. A small increase in the liquid formulation viscosity is satisfactory to achieve mucoadhesion. Such a formulation, however, would have low patient compliance because of its potential for blurring patient’s vision. Therefore, topical ophthalmic ointments and gels are usually prescribed before patients go to sleep. But if a delivery system can achieve satisfactory mucoadhesion without patient discomfort, drug retention time can be increased, and the prescribed drug concentration and volume may be reduced, hence decreasing the administrative frequency and side effects (Lorentz and Sheardown 2014).

This may be achieved by incorporating mucoadhesive substances such as polysaccharides in the topical formulations. Polysaccharides include polymers such as chitosan, cellulose and its derivatives, alginate, and hyaluronic acid.

Chitosan has excellent mucoadhesion properties that are mostly attributed to their positively charged surfaces, enabling them to interact with negatively charged mucins. Methyl cellulose and cationic cellulose derivatives are widely used in ophthalmic dosage forms because they are not irritant to the eye. On top of it all, these polymers are usually large and branched; consequently, molecular entanglement could explain why they are mucoadhesive. In addition, cationic cellulose derivatives could act directly with mucins like

Conception d'un vecteur de médicaments à base de nanoparticules d'or pour la thérapie du glaucome

Conception d’un vecteur de médicaments à base de

nanoparticules d’or pour la thérapie du glaucome

Mémoire

Mahmoud Omar

Maîtrise en sciences cliniques et biomédicales

Maître ès sciences (M. Sc.)

Québec, Canada

Conception d’un vecteur de médicaments à base de

nanoparticules d’or pour la thérapie du glaucome

Mémoire

Mahmoud Omar

Sous la direction de :

Élodie Boisselier, directrice de recherche

Vincent Pernet, co-directeur de recherche

RÉSUMÉ

ABSTRACT

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

LIST OF EQUATIONS

LIST OF ABBREVIATIONS

Acknowledgment

CHAPTER 1- INTRODUCTION

1.1. The Eye

1.2. Glaucoma

1.3. Travoprost

1.4. Ocular Drug Delivery