ALL 2015
`MYLAN PHARMACEUTICALS V. ALLERGAN
`IPR2016-01128
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`1
`
`ALL 2015
`MYLAN PHARMACEUTICALS V. ALLERGAN
`IPR2016-01128
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`Mucosal Delivery of Biopharmaceuticals
`
`2
`
`

`

`José das Neves • Bruno Sarmento
`Editors
`
`Mucosal Delivery
`of Biopharmaceuticals
`
`Biology, Challenges and Strategies
`
`2123
`
`3
`
`

`

`Editors
`José das Neves
`IINFACTS – Instituto de Investigação e Formação
`Avançada em Ciências e Tecnologias da Saúde
`Instituto Superior de Ciências da
`Saúde-Norte, CESPU
`Gandra, Portugal
`
`Bruno Sarmento
`NEWTherapies Group
`INEB – Instituto de Engenharia
`Biomédica
`Porto, Portugal
`
`ISBN 978-1-4614-9523-9
`DOI 10.1007/978-1-4614-9524-6
`Springer New York Heidelberg Dordrecht London
`
`ISBN 978-1-4614-9524-6 (eBook)
`
`Library of Congress Control Number: 2013958086
`© Springer Science+Business Media New York 2014
`This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the
`material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,
`broadcasting, reproduction on microfilms or in any other physical way, and transmission or information
`storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology
`now known or hereafter developed. Exempted from this legal reservation are brief excerpts in connection
`with reviews or scholarly analysis or material supplied specifically for the purpose of being entered and
`executed on a computer system, for exclusive use by the purchaser of the work. Duplication of this
`publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s
`location, in its current version, and permission for use must always be obtained from Springer. Permissions
`for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to
`prosecution under the respective Copyright Law.
`The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication
`does not imply, even in the absence of a specific statement, that such names are exempt from the relevant
`protective laws and regulations and therefore free for general use.
`While the advice and information in this book are believed to be true and accurate at the date of publication,
`neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or
`omissions that may be made. The publisher makes no warranty, express or implied, with respect to the
`material contained herein.
`
`Printed on acid-free paper
`Springer is part of Springer Science+Business Media (www.springer.com)
`
`4
`
`

`

`Contents
`
`Part I Biology of Mucosal Sites
`
`1 Concepts in Mucosal Immunity and Mucosal Vaccines . . . . . . . . . . . . .
`Simona Gallorini, Derek T. O’Hagan and Barbara C. Baudner
`
`2 Mucoadhesion and Characterization of Mucoadhesive Properties . . . .
`Tao Yu, Gavin P. Andrews and David S. Jones
`
`3 Mucus as a Barrier for Biopharmaceuticals and Drug
`Delivery Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Hongbo Zhang, Mohammed-Ali Shahbazi, Patrick V. Almeida
`and Hélder A. Santos
`
`4 Epithelial Permeation and Absorption Mechanisms
`of Biopharmaceuticals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Hanne Mørck Nielsen
`
`3
`
`35
`
`59
`
`99
`
`Part II Delivery Strategies for Specific Mucosal Sites
`
`5 Oral Delivery of Biopharmaceuticals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
`Catarina Oliveira Silva, Bruno Sarmento and Catarina Pinto Reis
`
`6 Buccal Delivery of Biopharmaceuticals: Vaccines and Allergens . . . . . 149
`Sevda ¸Senel, Merve Cansız and Michael J. Rathbone
`
`7 Pulmonary Delivery of Biopharmaceuticals . . . . . . . . . . . . . . . . . . . . . . . 169
`Fernanda Andrade, Catarina Moura and Bruno Sarmento
`
`8 Nasal Delivery of Biopharmaceuticals . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
`Eiji Yuba and Kenji Kono
`
`9 Ocular Delivery of Biopharmaceuticals . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
`Holly Lorentz and Heather Sheardown
`
`ix
`
`5
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`

`

`x
`
`Contents
`
`10 Vaginal Delivery of Biopharmaceuticals . . . . . . . . . . . . . . . . . . . . . . . . . . 261
`José das Neves
`
`Part III Case Studies of Mucosal Delivery of Biopharmaceuticals
`
`11 Nanoparticles-in-Microsphere Oral Delivery Systems (NiMOS)
`for Nucleic Acid Therapy in the Gastrointestinal Tract . . . . . . . . . . . . . 283
`Shardool Jain and Mansoor Amiji
`
`12 Bacteria-Based Vectors for Oral Gene Therapy . . . . . . . . . . . . . . . . . . . . 313
`Yong Bai, Rachael Burchfield, Sangwei Lu and Fenyong Liu
`
`13 Self-Assembled Polysaccharide Nanogels for Nasal Delivery
`of Biopharmaceuticals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
`Tomonori Nochi, Yoshikazu Yuki, Kazunari Akiyoshi and Hiroshi Kiyono
`
`14 PheroidTM Vesicles and Microsponges for Nasal Delivery
`of Biopharmaceuticals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
`Lissinda H. du Plessis and Awie F. Kotzé
`
`15 Delivery Strategies for Developing siRNA-Based
`Vaginal Microbicides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
`Joseph A. Katakowski and Deborah Palliser
`
`16 Delivery Strategies for Developing Vaginal DNA Vaccine Combining
`Cell-Penetrating Peptide and Jet Injection . . . . . . . . . . . . . . . . . . . . . . . . 367
`Takanori Kanazawa and Hiroaki Okada
`
`17 Vaccine Delivery Systems for Veterinary Immunization . . . . . . . . . . . . . 379
`Juan M. Irache, Ana I. Camacho and Carlos Gamazo
`
`18 Eligen® Technology for Oral Delivery of Proteins and Peptides . . . . . . 407
`Sunita Prem Victor, Willi Paul and Chandra P. Sharma
`
`19 The RapidMistTM System for Buccal Delivery of Insulin . . . . . . . . . . . . 423
`Meena Bansal, Sanjay Bansal and Rachna Kumria
`
`20 The Pharmaceutical Development of rhDNase (Dornase Alpha)
`for the Treatment of Cystic Fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 437
`Steven J. Shire and Thomas M. Scherer
`
`21 Development of the Exubera® Insulin Pulmonary Delivery System . . . 461
`Cynthia L. Stevenson and David B. Bennett
`
`22 Technosphere®: An Inhalation System for Pulmonary Delivery
`of Biopharmaceuticals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483
`António J. Almeida and Ana Grenha
`
`6
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`Contents
`
`xi
`
`23 ChiSys® as a Chitosan-Based Delivery Platform
`for Nasal Vaccination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499
`Peter Watts, Alan Smith and Michael Hinchcliffe
`
`24 Development of a Cationic Nanoemulsion Platform (Novasorb®)
`for Ocular Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517
`Frédéric Lallemand, Philippe Daull and Jean-Sébastien Garrigue
`
`Part IV Regulatory, Toxicological and Market Issues
`
`25 Regulatory Aspects and Approval of Biopharmaceuticals
`for Mucosal Delivery: Quality, Toxicology, and Clinical Aspects . . . . . 539
`Karen Brigitta Goetz, Yuansheng Sun, Katrin Féchir,
`Evelyne Kretzschmar and Isabel Buettel
`
`Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591
`
`7
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`

`Chapter 24
`Development of a Cationic Nanoemulsion
`Platform (Novasorb®) for Ocular Delivery
`
`Frédéric Lallemand, Philippe Daull and Jean-Sébastien Garrigue
`
`24.1 Introduction
`
`Drug delivery across mucosal barriers has always been a challenge, and crossing the
`eye mucosa is no exception. The eye surface is a unique and complex mucosa with
`its own physiology and mechanisms of protection. This chapter illustrates how, from
`a clear understanding of the eye mucosal barrier structure, a new delivery system
`was designed to better treat ocular surface diseases. This case study describes the
`development of a new drug delivery system, the Novasorb® platform, designed to
`overcome ocular barriers to improve ophthalmic drug delivery. This technology is
`based on cationic emulsions primarily developed in the late 1990s by University of
`Jerusalem professor Simon Benita. Several years later, and after the creation of a spin-
`off company, Novagali Pharma (Evry, France) in 2001, the Novasorb® technology
`was successfully transferred to clinical use. The main steps of the development are
`briefly presented; from concept formulations to preclinical pharmacokinetics (PK)
`and toxicity studies to the clinic.
`
`24.1.1 Eye Protection Systems
`
`Of the sensory organs, the eye is probably the most precious and the organ upon which
`our daily activities depend most. As an extension of the central nervous system, the
`eye needs to be well protected although continuously exposed to and threatened by
`an external and aggressive environment. As a consequence, the body has developed
`several effective protection mechanisms to preserve the eye’s structure and function.
`However, these protection mechanisms make it particularly difficult to access the
`eye’s inner tissues, even the different corneal layers, when treating the eye becomes
`necessary.
`J.-S. Garrigue ((cid:2)) · F. Lallemand · P. Daull
`Novagali Pharma, 1 rue Pierre Fontaine, 91058 Evry Cedex, France
`e-mail: jean-sebastien.garrigue@santen.fr
`
`J. das Neves, B. Sarmento (eds.), Mucosal Delivery
`of Biopharmaceuticals, DOI 10.1007/978-1-4614-9524-6_24,
`© Springer Science+Business Media New York 2014
`
`517
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`518
`
`F. Lallemand et al.
`
`For other mucosal tissues (pulmonary, nasal, intestine, etc.), mucus production
`and excretion is one of the main protection mechanisms, primarily by preventing
`pathogen adherence and tissue infection through their entrapment in a thick external
`mucus layer. For example, the thickness of the mucus layer varies from 100 μm in the
`large intestine to only 0.5 μm on the ocular surface. Hence, the membrane-tethered
`mucins (the mucus layer) of the ocular surface cannot be the only mechanism of
`protection.
`The ocular mucosa is protected by both physical and dynamic mechanisms. The
`eye is firstly protected by the eyelid, which blinks every 4–5 s. The eyelids possess
`several important ocular functions, with the primary objective of protecting both the
`anterior globe (cornea) from injury and the retina from excessive incoming light.
`Eyelid blinking also helps spread and maintain the ocular tear film. Eyelid behaviors
`achieving these functions include blinking (voluntary, spontaneous, or reflexive) and
`voluntary eye closure (gentle or forced) [1]. Blinking swipes away the overloads of
`tears as well as xenobiotic or solid particles present on the ocular surface, including
`any active ingredients administered topically.
`The precorneal tear film is also of major importance in the protection and health of
`the eye. The tear film is a nourishing, lubricating, and protecting layer that bathes the
`ocular surface. It is continuously replenished through cycles of production and elim-
`ination via evaporation, absorption, and drainage. These processes are often referred
`to as tear-film dynamics [2]. Tears fight desiccation, microbial contamination, and
`the effects of xenobiotic and solid particles. This film also maintains surface humid-
`ity to provide transparency and optical quality of the cornea as a refracting surface
`[3]. The tear film comprises three layers: a thin superficial layer of meibomian lipid;
`an intermediate aqueous layer containing dissolved mucins, salts and proteins; and
`an internal layer of mucus network, secreted mainly from conjunctival goblet cells
`whose chemical structure has now been fully described [4]. These ocular mucins are
`highly negatively charged, with the majority terminated by sialic acid, while those
`from rabbits are mainly neutral and terminated by alpha 1-2 fucose and/or alpha
`1-3 N-acetylgalactosamine [3]. In addition, these O-glycan mucins prevent bacte-
`rial adhesion and endocytic activity and maintain epithelial barrier function through
`interactions with galectins [5]. However, in addition to their traditional protective
`functions (selective barrier to the penetration of xenobiotics, antiadhesive that pre-
`vent pathogen adherence, and lubrication), the membrane mucins are also signaling
`molecules via their cytoplasmic tails and epidermal growth factor (EGF) domains [6].
`While providing lubrication and protection of the ocular surface, this layer reduces
`the efficacy of pharmaceutical treatment by limiting tissue penetration. However, as
`we will see below, this negatively charged mucosa may also be diverted from its
`original function and may be a critical player in the improvement of ocular drugs
`formulated in cationic emulsions.
`The presence of a constant tear flow of approximately 1.2 μL/min (0.5–2.2
`μL/min) is also an important protection system. This constant flow results in a tear
`turnover rate of 16 % per minute during waking hours, and the reflex lachrymation
`may increase this rate up to 100-fold, to 300 μL/min. The high turnover rate of
`this precorneal tear film contributes to the protection of the eye, but also to the low
`availability of topically administered drugs [7].
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`24 Development of a Cationic Nanoemulsion Platform (Novasorb®) for Ocular Delivery
`
`519
`
`The inner part of the eye is protected by the cornea, which has a complex structure
`of three different layers with varying physiological properties alternating between
`lipophilicity and hydrophilicity [8]. This layered and alternated construction makes
`corneal crossing of most drugs very difficult. Optimal permeant molecules should
`have a log D of 2–3 [9]. Examining in greater detail the corneal structure, first the
`outermost layer of the cornea is distinguished, i.e., lipophilic epithelium, which
`is formed by epithelial cells linked by tight junctions providing a strong barrier
`to the molecules present in the tear fluid. The corneal epithelium is consequently
`almost impermeable to any substance larger than 500 Da [10]. The next layer is
`made up of the stroma, a hydrophilic layer composed of fibrous tissue made of large
`collagen fibers and proteoglycans that form the major part of the cornea. Finally,
`the endothelium is a monolayer of hexagonal cell interfaces, which is also quite
`lipophilic [11].
`In addition, it should be noted that the cornea is innervated by sensory nerve ter-
`minals of the trigeminal ganglion. Physical and chemical agents acting on the ocular
`surface (extreme environmental temperatures, wind, foreign bodies, and chemicals)
`induce conscious sensations and reflex motor and autonomic responses (blinking,
`lacrimation, conjunctival vasodilation) aimed at protecting the eye from further injury
`and drug penetration [12].
`These successive physical and biological protections result in less than10 % of
`an instilled drug being absorbed by ocular tissues [13], leading to poor efficacy and
`the need for repeated instillations for the vast majority of eye drops. As a result of
`extending and maintaining the efficacy of eye drop solutions, the drug concentration
`needs to be increased but with potential exacerbation of local and/or systemic side
`effects with potent drugs such as timolol (a beta-blocker). Consequently, there is a
`need for new formulations that will improve efficacy while also limiting the risk of
`local side effects.
`
`24.1.2 Options to Overcome Physiological Barriers
`
`Ocular drug absorption from the lacrimal fluid to the anterior ocular tissues via trans-
`corneal absorption is determined by two major factors: drug permeability through
`the cornea and contact time of the product with ocular tissues. Based on these
`two principles, scientists have created several valuable approaches to overcome the
`barriers.
`
`24.1.3 Enhancing Penetration
`
`Firstly, to promote drug permeability, penetration enhancers have been added to
`aqueous eye drops with some success [14]. These excipients, based on their surface-
`active property, are able to open corneal epithelium tight junctions and desmosomes
`leading to penetration toward the anterior chamber. The literature is very rich in
`examples of studies testing penetration enhancers, their effect toward hydrophilic
`
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`520
`
`F. Lallemand et al.
`
`and lipophilic drugs, the size and charge of molecules, etc. [15]. However, all authors
`agree on one fact: Although penetration enhancers can be good adjuvants for ocular
`penetration, as regards their surfactant nature they are intrinsically deleterious for
`the ocular surface [16]. Long-term use of penetration enhancers might result in poor
`patient compliance due to chronic discomfort, thus limiting the use of penetration
`enhancers in ophthalmology.
`Another way to increase corneal penetration is to increase the specific uptake
`of the drug through the use of vectors, such as nanoparticles or liposomes, which
`have been described as being specifically taken up by corneal cells, as shown and
`discussed by Calvo et al. [17] and Diebold et al. [18]. However, the exact mechanism
`is not yet fully elucidated, therefore, limiting the use of this promising approach.
`Corneal penetration can also be influenced by the specific contact surface created
`by the colloidal system with the cornea. Emulsions are a typical example of such
`systems. An oil-in-water emulsion is a dispersion of oil into a water phase. This
`dosage form has been used in pharmacy for decades but only recently in ophthal-
`mology as eye drops. Ophthalmic emulsions have shown promise in topical ocular
`delivery as they are nontoxic systems, easy and inexpensive to manufacture, and able
`to deliver a lipophilic active agent with enhanced corneal penetration [19]. In addi-
`tion, ophthalmic emulsions are able to protect unstable active agents from chemical
`degradation (such as latanoprost) and to mask the irritation potential of some drugs.
`The first ophthalmic emulsion to be approved in the USA was Restasis® in 2002.
`This dosage form is now routinely used to treat dry eye conditions. A few years
`later, Durezol® was marketed to treat ocular inflammation. The exact mechanistic
`processes regarding enhanced corneal penetration has still not been fully elucidated
`[20] but they most likely involve several physicochemical and biological mechanisms
`whose increased specific surface of exchange with the cornea has major importance.
`
`24.1.4 Increasing Retention Time
`
`Increased retention time by enhanced viscosity or bioadhesion has been widely used
`and described during the past 20 years [8], leading to a number of ophthalmic prod-
`ucts. Mucoadhesion is based on noncovalent bonds (hydrogen or electrostatic bonds)
`between polymers and mucus or physical entanglement. In spite of their current wide
`use to decrease the frequency of administration and/or concentration in the solution,
`hydrogels present only a limited value because such aqueous formulations are elim-
`inated by the usual routes in the ocular domain and cause blurred vision and patient
`discomfort.
`
`24.1.5 Enhancing Penetration and Increasing Retention Time
`
`Improving the eye surface’s drug retention alone is inadequate in bringing a signif-
`icant improvement of drug bioavailability. It should be combined with penetration
`
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`24 Development of a Cationic Nanoemulsion Platform (Novasorb®) for Ocular Delivery
`
`521
`
`Drop of emulsion
`
`Ca(cid:415)onic nanodroplet of oil
`loaded with ac(cid:415)ve ingredient
`
`+
`
`+
`
`+
`
`+
`
`+
`+
`
`+
`
`+
`
`+
`
`+
`
`+
`
`+
`+
`+
`
`+
`+
`
`+
`
`Nega(cid:415)vely charged
`mucins layer
`
`Cornea
`
`Corneal epithelium
`
`•
`
`•
`
`•
`
`•
`
`Stroma
`
`+ + + + + + + + +
`• • • • •
`
`• • • •
`
`•
`
`•
`
`•
`
`•
`
`•
`
`•
`
`•
`
`• •
`
`•
`
`•
`
`•
`
`Fig. 24.1 Novasorb® enhanced the spread of a drop emulsion on the corneal surface and increased
`the contact surface of oily drops on the ocular surface
`
`enhancement. Several examples of combined systems (penetration and retention)
`have been described in the literature, such as liposomes combined with hydrogels
`[21], liposomes combined with a collagen shield [22], and even more solid lipid
`nanoparticles associated with a bioadhesive hydrogel such as chitosan [23]. Although
`promising, these examples all remain today at the prototype stage. Nevertheless, a
`new and innovative system has come to the market combining the effect of a very
`large specific surface of colloidal systems with improved bioadhesion properties. This
`drug-delivery system is the cationic nanoemulsion registered under the trademark
`Novasorb® technology (Fig. 24.1).
`
`24.2 Cationic Emulsions
`
`24.2.1 Cationic Agent
`
`While ophthalmic emulsions are becoming an essential tool for topical delivery,
`cationic emulsions combining intrinsic advantages of emulsions with a bioadhesive
`mechanism have provided enhanced efficacy of emulsions [24, 25]. This innovative
`approach uses the physiological barrier of mucus as a tool to increase retention
`
`12
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`

`522
`
`Fig. 24.2 Benzalkonium
`chloride is a mixture of
`various aliphatic chain
`lengths starting from C8 to
`C18 with C12, C14, and C16
`representing the major
`entities of the mixture
`
`F. Lallemand et al.
`
`time on the ocular surface. As discussed above, the last protection layer of the tears
`is composed of mucins that have a negative charge. Cationic nanoemulsions use
`this negative charge to interact with the ocular surface via a strong electrostatic
`interaction leading to a prolonged residence time of the cationic nanodroplets on the
`ocular surface [26].
`Basically, cationic emulsions are composed of oil that is dispersed in ultrafine
`droplets into a physiologically acceptable aqueous external phase (pH and osmot-
`ically adjusted). These droplets are stabilized by an interfacial film of surfactants
`such as cremophors, polysorbates, poloxamers, and tyloxapol in which a cationic
`charge is included. The cationic agent can be chosen over those commonly used
`and described in the literature. One can cite the primary amines stearylamine and
`oleylamine, the cationic phospholipid DOTAP and the polymers poly-L-lysine and
`polyethylenimine. Yet, these cationic agents are not suitable for use in pharmaceu-
`tical products in terms of regulatory requirements, stability, or toxicity issues. The
`alternative is to use registered excipients such as quaternary amines usually used
`as preservative agents in ophthalmic aqueous solutions. The most widely used are
`cetylpyridinium chloride, benzalkonium chloride (BAK), and benzethonium chlo-
`ride. However, the current tendency is to withdraw these preservatives from eye drops,
`due to long-term intolerance to them [27, 28], and to replace them either with soft
`preservatives (sodium perborate, SofziaTM) or single-use containers or preservative-
`free multidose containers. Nonetheless, BAK has several significant advantages over
`the other cationic agents: it is listed in all pharmacopeias, used in more than 80 %
`of eye drops at a concentration of 0.02 %, and has excellent interfacial properties,
`making this excipient a potential cationic agent candidate.
`Ten years ago, Sznitowskaat et al. [29] noted that when used in combination with
`oil-in-water emulsions, the preservative efficacy of BAK was drastically decreased.
`This observation was explained by the inclusion of part of BAK in the oily phase of
`the emulsion, leading to a lower molecular concentration available in the aqueous
`phase. Only the freely soluble molecules of BAK present in the aqueous phase can
`exert their antimicrobial effects on bacteria. Concretely, BAK is a mixture of sev-
`eral quaternary ammoniums (Fig. 24.2) with varying lipophilicity. According to US
`and European pharmacopeias, three main entities should be present in the mixture:
`benzododecinium chloride (C12-substituted alkyl chain), myristalkonium chloride
`(C14), and cetalkonium chloride (C16), as presented in Fig. 24.3. In presence of oil
`droplets, the most lipophilic entity of BAK, cetalkonium chloride, is rearranged at the
`oil or water interface, providing a cationic charge on the droplets while hydrophilic
`
`13
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`

`24 Development of a Cationic Nanoemulsion Platform (Novasorb®) for Ocular Delivery
`
`523
`
`Fig. 24.3 Benzalkonium
`chloride made of three
`different entities of increasing
`lipophilicity (cetalkonium
`chloride, myristalkonium
`chloride, and benzododecin-
`ium chloride) and cetalkoni-
`um chloride schematic
`developed formulae
`
`entities remain in the aqueous phase. Based on this observation, it was decided to use
`only cetalkonium chloride at a very low concentration (0.002–0.005 %) as a cationic
`agent to make cationic nanoemulsions [30].
`
`24.2.2 Physicochemical Considerations Regarding Novasorb®
`
`Cationic nanoemulsions are characterized by two main physicochemical properties.
`First, the zeta potential, which is defined as the electrical potential difference between
`the dispersion medium (i.e., water) and the stationary layer of fluid attached to the
`dispersed oil nanodroplets [31]. This surface charge will provide the system with its
`bioadhesion property on the ocular surface as well as the stability of the emulsion by
`providing electric repulsion between droplets. For optimal electrostatic interaction
`between cornea and product, a zeta potential of about + 20 mV is sufficient [30].
`The second main property is the oil droplet size. As described by the Stokes
`law (Fig. 24.4), the smaller the droplet size, the slower the dispersed system will
`separate, thus providing greater stability to the system. Even more importantly, the
`size of the droplets significantly participates in the penetration rate of the drug in the
`ocular tissue. With the active ingredient being solubilized in the lipid nanodroplets,
`a smaller particle size should provide a greater contact surface, hence a higher tissue
`concentration. In the case of the Novasorb® technology, a size between 100 and
`200 nm was demonstrated to be small enough to provide a stable emulsion.
`Surface bioadhesion was demonstrated by measurement of the spreading prop-
`erties of the emulsion in contact angle studies. On excised rabbit eyes, a drop of
`cationic emulsion is applied and compared to anionic emulsion and hyaluronic gel
`in terms of contact angle (Fig. 24.5). Immediately after drop deposit, the cationic
`
`Fig. 24.4 Stokes law equation where vs is the particle creaming velocity, g is the gravitational
`acceleration, ρp is the mass density of the particles, ρf is the mass density of the fluid and μ is the
`viscosity of the continuous phase
`
`14
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`

`524
`
`F. Lallemand et al.
`
`Fig. 24.5 Dynamic contact angle measurements on rabbit eyes confirm optimal and immediate
`spreading of cationic emulsions compared to anionic emulsions and hyaluronic acid-based product
`(adapted from Lallemand et al. [30])
`
`emulsion based on Novasorb® spread all over the cornea while other tested products
`remained with high contact angles.
`Other physicochemical parameters are important for the development and use of
`Novasorb®. It should be noted that to avoid reflex tearing and blinking the emulsions
`should be compatible with biological parameters (pH and osmolality). pH is adjusted
`to physiological pH (about 7–7.2). Osmolality should be adjusted to avoid osmotic
`stress to the epithelial cells. Neutral molecules should be used to avoid charge mask-
`ing and emulsion destabilization such as mannitol, sorbitol or, glycerol. Glycerol is
`favored because this compound possesses an intrinsic beneficial demulcent property
`on the eye surface that is particularly useful in dry eye disease (DED) [32].
`Finally, several lipophilic active ingredients were added to the cationic emulsion
`either to address unmet medical needs or to improve existing products: cyclosporin
`A (CsA), latanoprost, antihistaminics, anti-inflammatories, antibiotics, and antifun-
`gals. For example, latanoprost, the active ingredient of the antiglaucoma blockbuster
`product Xalatan®, was included in the emulsion providing new properties. La-
`tanoprost is an unstable molecule in presence of water due to ester hydrolysis. When
`encapsulated in oil, the concerned ester function is masked in the oily phase, thus
`avoiding hydrolysis and providing an advantage over Xalatan®. In addition, as dis-
`cussed below, the beneficial effect of Novasorb® on the ocular surface should be
`beneficial to patients with combined glaucoma and ocular surface disease.
`Another example is CsA. This molecule has always been a huge challenge to
`formulate in an adequate and efficient product [33] due to its high lipophilicity.
`Novasorb® seems the most appropriate technology to administer this molecule to
`patients.
`
`24.3 Nonclinical Evaluation
`
`24.3.1 Pharmacokinetics and Efficacy of Cationic Emulsions
`
`The ocular delivery of Novasorb® cationic emulsions loaded CsA and latanoprost
`was evaluated in rabbits. The first indications that cationic emulsions were effective
`ocular drug-delivery vehicles with prolonged precorneal residence time come from
`
`15
`
`

`

`24 Development of a Cationic Nanoemulsion Platform (Novasorb®) for Ocular Delivery
`
`525
`
`the work of Benita et al., who used delta 8-tetrahydrocannabinol and pilocarpine as
`model drugs [34, 35]. These results were confirmed later with CsA cationic emul-
`sions, which have an increased ocular absorption, especially in the conjunctiva and
`cornea, when compared to anionic emulsions [20, 36]. However, these first cationic
`emulsions all used noncompendial excipients, or primary amines, such as steary-
`lamine and oleylamine as the cationic agent, which are not devoid of toxicity. Based
`upon these results, the Novasorb® technology was developed to create cationic emul-
`sions of CsA and latanoprost with improved ocular tolerance safety profiles. CsA
`cationic emulsions were compared to Restasis®, a commercially available unpre-
`served BAK-free anionic emulsion of 0.05 % CsA, in single- and multiple-dose
`PK studies [37]. The corneal absorption of CsA following a single instillation of a
`0.05 % CsA cationic emulsion was approximately twice that observed with Restasis®
`(Fig. 24.6a), and a 0.025 % CsA cationic emulsion was as effective as Restasis® at
`delivering CsA to the cornea. The area under the curve (AUC) in the cornea following
`a single instillation was 14,210, 14,476, and 26,476 ng h/g for Restasis®, 0.025 %
`CsA cationic emulsion, and 0.05 % CsA cationic emulsion, respectively (Fig. 24.6b).
`Interestingly, a second peak is observed 4 h post instillation with the 0.05 % CsA
`cationic emulsion, which is not present with the Restasis® anionic emulsion. This
`suggests that the cationic emulsion does indeed possess a prolonged precorneal res-
`idence time. With the 0.1 % CsA cationic emulsion, this second peak was observed
`12 h post instillation [37], confirming that for such a peak to be present, a prolonged
`residence time in the precorneal space was necessary. This prolonged residence time
`can be explained by the presence of the positive charge on the oil droplets of the
`emulsion, which can interact with the negatively charged corneal epithelium. In the
`multiple-dose PK studies, bis in die (BID) (twice daily) instillations for 7 days of
`the 0.05 % CsA cationic emulsion or once daily instillation for 7 days of the 0.1 %
`CsA cationic emulsion were not accompanied by an increased systemic absorption
`of CsA.
`Confirmation of the good absorption following instillations of the CsA cationic
`emulsions was obtained with a 0.005 % latanoprost cationic emulsion. In a monkey
`model of laser-induced elevated intraocular pressure (IOP), the 0.005 % latanoprost
`cationic emulsion was as effective as Xalatan® (0.005 % latanoprost) at reducing
`elevated IOP [38], suggesting an equivalence in the latanoprost-delivered dose be-
`tween the two 0.005 % latanoprost formulations. Xalatan® contains 0.02 % BAK, a
`quaternary ammonium that at this high concentration acts both as a preservative for
`the eye drop solution and a permeation enhancer for latanoprost. For example, Al-
`lergan increased the BAK concentration, from 0.005 to 0.02 %, while decreasing the
`concentration of bimatoprost (a prostaglandin analog, PGA) from 0.03 to 0.01 % in
`order for the new Lumigan® 0.01 % bimatoprost eye drop solution to have the same
`efficacy as the original 0.03 % bimatoprost formulation. In this regard, increasing the
`BAK concentration potentiated the absorption of bimatoprost and helped in main-
`taining its efficacy. This formulation change was motivated by the side effect of the
`PGA class: hyperemia, i.e., conjunctival redness, which results from PGA-induced
`conjunctival vein vasodilation. Hyperemia is the major cause of antiglaucoma PGA
`treatment cessation and poor compliance.
`
`16
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`

`

`526
`
`F. Lallemand et al.
`
`CsA concentra(cid:415)on in the cornea
`
`Restasis®
`(0.05% CsA)
`0.025% CsA
`Ca(cid:415)onic EM
`0.05% CsA
`Ca(cid:415)onic EM
`
`1600
`
`1400
`
`1200
`
`1000
`
`800
`
`600
`
`400
`
`200
`
`0
`
`0
`
`4
`
`8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72
`Time (hour)
`AUC
`
`30000
`25000
`2000