EVA RAMSAY TOOLS FOR PRECLINICAL RESEARCH ON OCULAR DRUG DELIVERY AND PHARMACOKINETICS

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1 PUBLICATIONS OF THE UNIVERSITY OF EASTERN FINLAND Dissertations in Health Sciences EVA RAMSAY TOOLS FOR PRECLINICAL RESEARCH ON OCULAR DRUG DELIVERY AND PHARMACOKINETICS

2 EVA RAMSAY Tools for Preclinical Research on Ocular Drug Delivery and Pharmacokinetics To be presented by permission of the Faculty of Health Sciences, University of Eastern Finland for public examination in auditorium 2, Infokeskus Korona, Helsinki, on Saturday, May 4 th 2019, at 12 noon Publications of the University of Eastern Finland Dissertations in Health Sciences Number 505 School of Pharmacy Faculty of Health Sciences University of Eastern Finland KUOPIO 2019

3 Grano Oy Jyväskylä, 2019 Series Editors: Professor Tomi Laitinen, M.D., Ph.D. Institute of Clinical Medicine, Clinical Physiology and Nuclear Medicine Faculty of Health Sciences Associate Professor Tarja Kvist, Ph.D. Department of Nursing Science Faculty of Health Sciences Professor Kai Kaarniranta, M.D., Ph.D. Institute of Clinical Medicine, Ophthalmology Faculty of Health Sciences Associate Professor (Tenure Track) Tarja Malm, Ph.D. A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences Lecturer Veli-Pekka Ranta, Ph.D. (pharmacy) School of Pharmacy Faculty of Health Sciences Distributor: University of Eastern Finland Kuopio Campus Library P.O.Box 1627 FI Kuopio, Finland ISBN (print): ISBN (pdf): ISSN (print): ISSN (pdf): ISSN-L:

4 III Author s address: Supervisors: School of Pharmacy Faculty of Health Sciences University of Eastern Finland KUOPIO FINLAND Professor Arto Urtti, Ph.D. School of Pharmacy Faculty of Health Sciences University of Eastern Finland KUOPIO FINLAND Docent Marika Ruponen, Ph.D. School of Pharmacy Faculty of Health Sciences University of Eastern Finland KUOPIO FINLAND Docent Sanjay Sarkhel, PhD. Faculty of Pharmacy University of Helsinki HELSINKI FINLAND Reviewers: Associate Professor Ilva Rupenthal, Ph.D. Director of the Buchanan Ocular Therapeutics Unit Department of Ophthalmology New Zealand National Eye Centre Faculty of Medical and Health Sciences The University of Auckland AUCKLAND NEW ZEALAND Professor Yogeshvar N. Kalia, Ph.D. School of Pharmaceutical Sciences University of Geneva GENEVA SWITZERLAND Opponent: Associate Professor Sara Nicoli, Ph.D. Department of Food and Drug University of Parma PARMA ITALY

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6 V Ramsay, Eva Tools for Preclinical Research on Ocular Drug Delivery and Pharmacokinetics University of Eastern Finland, Faculty of Health Sciences Publications of the University of Eastern Finland. Dissertations in Health Sciences p. ISBN (print): ISBN (pdf): ISSN (print): ISSN (pdf): ISSN-L: ABSTRACT Globally, age-related vision disorders, such as glaucoma and age-related macular degeneration (AMD), are an increasing cause of concern. The currently available treatments for AMD and glaucoma are far from perfect, often resulting in poor patient compliance e.g. in glaucoma patients who use eye drops to reduce their intraocular pressure. The neovascular form of AMD requires monthly intravitreal injections; these are a heavy burden on patients and a major expense on the health care budget. Clearly, new drugs and patient-friendly drug delivery systems, with controlled and sustained delivery, would be advantageous in ophthalmology. Ocular drug development requires overcoming problems currently associated with drug delivery, such as low bioavailability, short duration of action, and invasive drug administration. New tools and novel approaches would facilitate ocular drug development. In this thesis project, we aimed to devise new tools for topical and intravitreal drug development. Our first aim was to determine the impact of physicochemical properties of a drug on its conjunctival and corneal permeability. Secondly, corneal and conjunctival permeabilities of the drugs were systematically compared to generate computational tools for the prediction of ocular bioavailability after topical administration. Our third aim was to develop a high-throughput approach for drug release studies and the extension of release data to the intravitreal drug concentrations. Fourthly, new tools were devised to predict the effects of the polymer degradation rate on intravitreal concentrations of polymer-borne fragments. Fifth, drug permeability in the retinal pigment epithelium (RPE) was assessed and the role of RPE in intravitreal drug elimination was estimated. Sixth, we evaluated the in vitro cellular efficacy of a small interfering ribonucleic acid (sirna) against interleukin-6 (IL-6) after its delivery in a new complexing block-copolymer. Predictive quantitative structure-property relationship (QSPR) models were generated to estimate conjunctival and corneal permeability; polar surface area and hydrogen bond donor groups were the most valuable descriptors in the prediction of permeability. Conjunctival drug permeability sets limits on ocular drug bioavailability, due to drug loss from the lacrimal fluid into the systemic circulation across the conjunctiva. A potential high-throughput screening tool was used to assess the release of different sized molecules from alginate-based microspheres. Kinetic modeling was further used for simulating intravitreal concentrations. Simulations on intravitreal polymer dissolution and hydrolysis revealed that degradation can significantly speed up the elimination of polymer-borne molecules and reduce ocular exposure. Permeability studies with isolated RPE specimens indicate that the assessment of RPE permeability makes it possible to estimate the intravitreal drug clearance. Finally, the use of IL-6 silencing sirna may represent a potential anti-inflammatory therapy for ailments affecting the back of the eye, but the studied block copolymer-sirna complex did not achieve effective sirna mediated gene silencing. This thesis provides insights into ocular drug delivery and pharmacokinetics. New approaches and tools may be useful in the preclinical development of novel ocular drugs. National Library of Medicine Classification: QV 38, QV 745, QV 771, WB 340, WW 166 Medical Subject Headings: Drug Delivery Systems; Eye; Eye Diseases/drug therapy; Drug Development; Administration, Ophthalmic; Ocular Absorption; Cornea; Conjunctiva; Retinal Pigment Epithelium; Permeability; Quantitative Structure-Activity Relationship; Pharmacokinetics; Polymers; RNA, Small Interfering; Interleukin-6; Drug Liberation; Microspheres; Intravitreal Injections; Administration, Topical; Drug Evaluation, Preclinical

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8 VII Ramsay, Eva Silmälääkkeiden prekliinisen farmakokinetiikan ja lääkkeiden saaton uudet menetelmät Itä-Suomen yliopisto, Terveystieteiden tiedekunta Publications of the University of Eastern Finland. Dissertations in Health Sciences s. ISBN (nid.): ISBN (pdf): ISSN (nid.): ISSN (pdf): ISSN-L: TIIVISTELMÄ Ikääntymiseen liittyvät silmäsairaudet, kuten glaukooma ja silmänpohjan ikärappeuma, ovat kasvava kansanterveydellinen ongelma. Sairauksien nykyiset hoidot ovat ongelmallisia. Glaukooman hoidossa silmänpainetta alennetaan päivittäin annettavilla silmätipoilla, mutta valitettavasti glaukoomapotilaiden hoitomyöntyvyys on heikko, ja vain puolet potilaista käyttää silmätippoja oikein. Kosteaa silmänpohjan ikärappeumaa hoidetaan kuukausittaisilla lasiaisinjektioilla, mikä on raskas taakka potilaille ja hoitohenkilökunnalle. Silmäsairauksien hoitoon tarvitaan uusia lääkeaineita ja lääkkeen saattomenetelmiä, jotka vapauttavat lääkeainetta kontrolloidusti ja pitkäkestoisesti. Silmälääkkeiden kehitystyössä tarvitaan ratkaisuja lääkkeen saaton ongelmiin, joita ovat alhainen biologinen hyötyosuus, vaikutuksen lyhyt kesto ja tarve käyttää silmänsisäisiä injektioita lääkkeen annossa. Uusia menetelmiä tarvitaan silmälääkkeiden kehityksen tueksi. Tässä väitösprojektissa on kehitetty uusia menetelmiä silmän pintaan ja lasiaisinjektiona annettavien lääkkeiden kehitykseen. Ensimmäiseksi työssä määritettiin lääkeaineiden fysikaaliskemiallisten ominaisuuksien vaikutus niiden kykyyn läpäistä sidekalvoa ja sarveiskalvoa. Toisessa kokonaisuudessa kehitettiin lääkeaineiden sarveiskalvon ja sidekalvon läpäisevyyden systemaattisen analyysin perusteella laskennallinen menetelmä silmän pintaan annettujen lääkeaineiden biologisen hyötyosuuden ennustamiseksi. Seuraavana tavoitteena oli kehittää suuren kapasiteetin menetelmät lääkeaineiden vapautumisen analyysiin mikropartikkeleista ja vapautumistulosten käyttöön lääkeainepitoisuuksien ennustamiseen silmän lasiaisessa lasiaisinjektion jälkeen. Neljänneksi kehitettiin ennustavia laskennallisia menetelmiä biohajoavien polymeerien eliminaation arviontiin silmän lasiaisessa. Viidentenä tavoitteena oli määrittää lääkeaineiden läpäisevyys verkkokalvon pigmenttiepiteelissä ja arvioida sen merkitys lääkeaineiden eliminoitumiselle lasiaisesta. Kuudenneksi tutkittiin interleukiini-6 proteiinin ilmentymistä estävän sirna:n tehoa verkkokalvon pigmenttiepiteelin soluissa, kun sirna annettiin kompleksoituna uuden kopolymeerin kanssa. Väitöskirjassa kehitetyn kvantitatiivisen rakenneominaisuus mallin perusteella polaarinen pintaala ja vetysidosten luovuttajaryhmät olivat tärkeimmät lääkeaineiden ominaisuudet, joilla niiden läpäisevyys sidekalvossa ja sarveiskalvossa voidaan ennustaa. Läpäisevyys näissä kalvoissa edesauttaa lääkeaineen imeytymistä silmään, mutta toisaalta sidekalvon läpäisevyys rajoittaa imeytymistä, koska suuri osa lääkeannoksesta silmän pinnassa imeytyy silmän asemesta verenkiertoon sidekalvon läpi. Lääkeaineen vapautumisen seulontamenetelmä kehitettiin onnistuneesti alginaatin mikropartikkeleille, joista useiden erikokoisten molekyylien vapautumista seulottiin ja simuloitiin yhdisteiden pitoisuuksia lasiaisessa. Simulaatiot osoittivat, että polymeerin hajoaminen nopeuttaa merkittävästi polymeerin hajoamistuotteiden eliminoitumista silmästä ja vähentää näin altistusta silmässä. Yhdisteiden läpäisevyys verkkokalvon pigmenttiepiteelissä vaikuttaa keskeisesti lääkeaineiden lasiaispuhdistumaan. IL-6 proteiinin ilmentymistä estävä sirna on mahdollinen tulehdusta hillitsevä hoito silmän takaosan tauteihin, mutta tutkittu sirnapolymeeri ei johtanut tehokkaaseen IL-6:n vaimentamiseen. Tämä väitöskirja lisää tietoa silmälääkkeiden saattomenetelmistä ja farmakokinetiikasta. Uusi tieto ja uudet menetelmät ovat hyödyllisiä silmälääkkeiden kehitystyössä prekliinisessä vaiheessa. Luokitus: QV 38, QV 745, QV 771, WB 340, WW 166 Yleinen Suomalainen asiasanasto: silmät; silmätaudit; lääkkeet; lääkehoito; annostelu; lääkesuunnittelu; kehittäminen; imeytyminen; läpäisevyys; vapautuminen; sarveiskalvo; verkkokalvo; farmakokinetiikka; polymeerit

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10 IX Acknowledgements This thesis work was started in 2012 in the Centre for Drug Research and the Division of Biopharmacy and Pharmacokinetics, Faculty of Pharmacy, University of Helsinki, and in 2014, the work continued in the School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland (years 2014, ). The work was financially supported by Panoptes and Alexander projects within the 7 th Framework Programme for Research of the European Union; UEF doctoral student position; Finnish Cultural Foundation; FinPharmaNet; Leo, Mary and Mary-Ann Hackmann Foundation; Orion Research Foundation; The Finnish Pharmaceutical Society. I wish to acknowledge several people who have helped and supported my journey to grow as a scientist and person, and to complete this work. First of all, I want to express my deepest gratitude to my supervisors Professor Arto Urtti, Docent Marika Ruponen, PhD, and Docent Sanjay Sarkhel, PhD. Arto gave me the freedom to grow as a scientist and to learn new exciting things. In those moments when I felt lost, he was always optimistic and helped me to see the most important issues and questions. Your ideas, calmness and way of thinking have always inspired and encouraged me. I also wish to thank you for giving me the opportunity to work with great people! I am grateful to Marika for her support and encouragement. We have had some very nice discussions and it has always been easy to turn to you. Furthermore, you have always taken the time to answer my questions, no matter how busy you have been. Your laughter, optimism, and way of thinking and seeing science have helped me a lot. Sanjay was my supervisor at the beginning of my PhD studies; you were an excellent teacher! I wish to thank you for teaching me valuable skills in the lab and giving me scientific advice that I will carry with me on my future journeys. I wish to acknowledge my co-authors for their hard work. With their contributions and help, we were able to publish the work presented in this thesis. I wish to thank especially, Eva del Amo, PhD; Elisa Toropainen, PhD; Veli-Pekka Ranta, PhD; Marja Hagström, MSc; Théo Picardat, MSc; Unni Tengvall-Unadike, PhD; Professor Seppo Auriola; Leena-Stiina Kontturi, PhD. Eva taught me and supported me so much, especially during the writing of our first manuscript; I truly value your opinions. It was a long road, but it has been worth it! Thank you also for the pleasant discussions not only about science but also on life! I got to know Elisa during conjunctival dissection from the porcine eyes. We enjoyed many good times in the lab, discussing all kinds of things, and it has been a privilege to have had the opportunity to work and learn from you. Veli-Pekka is thanked for valuable advice and help. Marja, Théo, Unni and Seppo are acknowledged for skillful LC-MS/MS analyses. The sirna delivery work is not published, but these experiments taught me the most during my journey. I wish to express my appreciation to Manuela Raviña, PhD, who took the time to discuss matters related with sirna delivery remotely from Spain by Skype. Professor Neil Cameron and Sarah Hehir, PhD, are thanked for excellent collaboration and providing the polymer carrier needed for the sirna work. Heidi Kidron, PhD, is thanked for encouraging and helpful discussions. Professor Marjo Yliperttula is acknowledged for giving me the opportunity to work in the lab during my master studies, and giving me the opportunity to participate in the Biopharmacy teaching during my PhD studies.

11 X I wish to thank Professor Ilva Rupenthal, PhD, and Professor Yogeshvar N. Kalia, Ph.D for taking their time to review this thesis. I am honored to have Professor Sara Nicoli from the University of Parma, Italy to act as my opponent, and for giving up her time to come to Finland. Docent Maija Lahtela-Kakkonen, PhD, Soile Nymark, PhD, and Mikko Gynther, PhD, are acknowledged for valuable discussion and advice on my research plans. Additionally, I wish to thank Maija and Susanna Boman, veterinary, for helping with the logistics of the bovine eyes. During my years as a doctoral student I have had the honor to meet wonderful people who have filled my days with ideas, discussions, laughter, and fun. Without you, all of this would not have been worth it. I wish to thank past and present colleagues from Helsinki! Especially Ansku, Otto, Alma, Shirin, Sina, Vijay, and Polina are thanked for nice discussions and lunchbreaks. Special thanks go to Madhu for her friendship and support. From Kuopio I wish to thank Laura, Mika, Kati-Sisko, Emma, and Karoliina. In 2012, when I had just started my doctoral studies, I made my first trip to a scientific conference in Australia. I gave my first poster presentation at GPEN, Melbourne. Afterwards, I went on a journey with Noora, Astrid, and Melina that I will never forget! I overcame my fears; we sailed along the Great Barrier Reef, and walked in the jungle (infested by spiders!). Thank you for making the trip and for your friendship! Astrid is also acknowledged for teaching me valuable skills in the lab and introducing me to scientific work. Leena Pietilä, Timo Oksanen, Erja Piitulainen, and Lea Pirskanen have done tremendous work in maintaining and helping in the lab. Work could not have been done without you! I wish to thank especially Lea for her valuable and hard work! Colleagues in the Panoptes ( ) consortium are also acknowledged for the great collaboration between people from different scientific backgrounds. They were the best three years of my life! Science and life would not feel as good without family and friends! I have had the honor to play basketball in a great team, Topolan Mamit! I wish to thank all the members for the fun times we had, it has really given me the time to switch off and think of something else! Thanks are expressed to my closest friends Annika, Ane, Pinja, and Jonna for your unconditional friendship and for being there! Goda och nära vänner är värt guld! I wish to send my warmest thank to my in-laws, Tepa and Wollie. Your presence and help have meant a lot. I wish to thank my brother Marcus and his wife Wilma, and my brother in-law Macke for all their support during all these years! I can t thank my mother and father enough for always being there for me. Without your support and love I would not have made it as far as I have. Tack Mamma! Kiitos Pappa! The most important thanks go to my lovely husband Martin. Thank you for your unconditional love and support during our years together. You have always encouraged me to pursue the things in my life that I enjoy and are important for me. You have also made it possible for me to work remotely from Kuopio and attend conferences, while taking care of our home. Our lives would feel meaningless without our lovely son Benjamin. Tack älskling för att du visat mamma och pappa vad som är viktigast i livet! Den, som äger tålamod, äger allt! Eva Ramsay December 2018, Helsinki

12 XI List of the original publications This dissertation is based on the following original publications: I II III Eva Ramsay, Marika Ruponen, Théo Picardat, Unni Tengvall, Marjo Tuomainen, Seppo Auriola, Elisa Toropainen, Arto Urtti and Eva M. del Amo: Impact of Chemical Structure on Conjunctival Drug Permeability: Adopting Porcine Conjunctiva and Cassette Dosing for Construction of In Silico Model. J Pharm Sci, 106: , Eva Ramsay, Eva M. del Amo, Elisa Toropainen, Unni Tengvall-Unadike, Veli- Pekka Ranta, Arto Urtti and Marika Ruponen: Corneal and conjunctival drug permeability: Systematic comparison and pharmacokinetic impact in the eye. Eur J Pharm Sci, 119: 83 89, Sanjay Sarkhel, Eva Ramsay, Leena-Stiina Kontturi, Jonne Peltoniemi and Arto Urtti: High-throughput in vitro drug release and pharmacokinetic simulation as a tool for drug delivery system development: Application to intravitreal ocular administration. Int J Pharm, 477: , Additional published material: IV Chapter Degradation rate of polymers in: Eva M. del Amo, Anna-Kaisa Rimpelä, Emma Heikkinen, Otto K. Kari, Eva Ramsay, Tatu Lajunen, Mechthild Schmitt, Laura Pelkonen, Madhushree Bhattacharya, Dominique Richardson, Astrid Subrizi, Tiina Turunen, Mika Reinisalo, Jaakko Itkonen, Elisa Toropainen, Marco Casteleijn, Heidi Kidron, Maxim Antopolsky, Kati-Sisko Vellonen, Marika Ruponen and Arto Urtti. Pharmacokinetic aspects of retinal drug delivery. Prog Retin Eye Res, 57: , The publications were adapted with the permission of the copyright owners. This thesis contains the following unpublished material: V VI The role of RPE permeability in intravitreal clearance of small molecular drugs sirna delivery to the RPE cells The published and unpublished data are referred to in the text by their Roman numerals.

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14 XIII Contents 1 INTRODUCTION REVIEW OF THE LITERATURE Ocular drug administration routes and barriers Barriers in topical drug delivery Barriers in intravitreal drug delivery Ocular pharmacokinetics and improved drug delivery Drug movement by passive diffusion Topical administration Intravitreal administration Pharmacokinetic models Top-down approach Bottom-up approach AIM OF THE STUDY OVERVIEW OF THE MATERIALS AND METHODS Cell culture and ocular tissue preparation Drugs and delivery systems Drug permeability Drug release from microspheres sirna delivery Characterization of sirna polyplex Cytotoxicity Silencing efficacy Cell uptake Intracellular distribution Computational methods QSPR modeling Ocular pharmacokinetic simulations and calculations RESULTS Topical drug permability and QSPR modeling Ocular pharmacokinetic predictions for topically applied drugs Intravitreal drug release and pharmacokinetic predictions Degradation rate of polymer carriers RPE permeability and clearance sirna delivery to the RPE DISCUSSION AND FUTURE DIRECTIONS Bottom-up approaches for topical drug delivery Intravitreal drug release and carrier degradation The role of RPE permeability in intravitreal clearance sirna delivery to the RPE... 38

15 XIV 7 CONCLUSIONS REFERENCES... 43

16 XV Abbreviations AMD BAB BRB CL CLIVT CLRPE F HBD higg age-related macular degeneration blood-aqueous barrier blood-retinal barrier clearance intravitreal clearance clearance across the retinal pigment epithelium bioavailability hydrogen bond donor human immunoglobulin G IL-6 interleukin 6 ILM inner limiting membrane LogD7.4 logarithm of the octanol-water distribution coefficient at ph 7.4 LPS MW N/P OLM Papp Papp, CJ Papp, CO Papp, RPE-choroid P-gp PCA PBPK PLS lipopolysaccharide molecular weight amine/phosphate outer limiting membrane apparent permeability coefficient conjunctival permeability corneal permeability retinal pigment epithelium-choroid permeability P-glycoprotein principal component analysis physiologically based pharmacokinetics partial least square PBE30-b-PK30 poly (benzyl-glutamate) 30-b-poly-l-lysine 30 PSA QSPR RPE sirna polar surface area quantitative structure-property relationship retinal pigment epithelium small interfering ribonucleic acid

17 XVI t1/2 half-life TER Vd Vd, IVT VEGF transepithelial electrical resistance volume of distribution intravitreal volume of distribution vascular endothelial growth factor

18 1 Introduction The number of individuals with impaired vision continues to grow. In 2010, approximately 733 million people suffered from visual impairment but by the year 2020, that number is predicted to increase to 929 million and of these, almost every fifth i.e. 200 million will be blind (Gordois et al., 2012). Age-related vision disorders, such as cataract, glaucoma, diabetic retinopathy, and age-related macular degeneration (AMD) are the leading causes of blindness (Pascolini and Mariotti, 2012). Thus, the burden of impaired vision will not only increase patient suffering but also represent a major financial burden on health care programmes (Gordois et al., 2012). In global terms, glaucoma is the leading cause of irreversible blindness (Pascolini and Mariotti, 2012). In glaucoma, the optic nerve is progressively damaged; the disease is associated with increased intraocular pressure. Untreated glaucoma may lead to blindness. There is no cure for glaucoma, there are only drugs that can slow the progress of the disease. Antiglaucoma drugs (e.g. prostaglandins, beta-blocking agents, carbonic anhydrase inhibitors) decrease the intraocular pressure during chronic by daily administration as eye drops. Unfortunately, patient compliance with this kind of glaucoma treatment is only about 50% (Reardon et al., 2011). AMD is the leading cause of blindness in the industrial countries (Gordois et al., 2012; Ojamo, 2017). This disease is associated with abnormalities in the retinal pigment epithelium (RPE), causing degeneration of the overlying photoreceptors and loss of central vision. Only the neovascular form of AMD can be treated with monthly intravitreal injections of inhibitors of vascular endothelial growth factor (VEGF) (e.g. ranibizumab, aflibercept, bevacizumab). Repeated anti-vegf injections are a burden to the patients and they must be administered by healthcare professionals. It is evident that not only new drugs but also improved drug delivery systems capable of achieving sustained delivery, are needed in the treatment of eye diseases. Ocular drug delivery is a challenge due to the unique anatomy and physiology of the eye (Gaudana et al., 2010). Ocular barriers are intended to maintain a desirable homeostasis and visual function of the eye, but they also restrict drug delivery. Ocular drugs must be delivered to the eye so that sufficient drug concentrations will be achieved at the site of action. Route of administration, ocular barriers, and physicochemical properties of the drug and formulation play critical roles in ocular drug delivery. The knowledge of ocular barrier permeability is a key factor in successful ocular delivery, but it also important to be aware of the interplay of several pharmacokinetic factors. New tools are needed to improve our understanding and prediction of ocular pharmacokinetics in early drug development; these could facilitate drug development, decrease development costs, and refine animal testing. For example, the U.S. Food and Drug Administration (FDA) has emphasized and encouraged the development of new preclinical predictive methods to accelerate the development of new drugs (FDA, 2004). The use of biologics, such as antibodies, for the treatment of the posterior eye segment, has increased. In addition, other macromolecules hold potential as ocular drugs, for instance oligonucleotides (e.g. sirna, aptamers). These compounds cannot be delivered as topical eye drops, but intravitreal injections are possible. Unfortunately these injections must be given frequently and therefore controlled release systems would be beneficial. Furthermore, oligonucleotides with intracellular targets would need to be administered in an appropriate delivery system (e.g. incorporated into nanoparticles) to allow effective cellular delivery. This thesis work sheds new light on tools for use in preclinical research on ocular drug delivery and pharmacokinetics, in terms of drug release and delivery, as well as the impact of membrane permeability on ocular absorption and elimination.

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20 3 2 Review of the literature 2.1 OCULAR DRUG ADMINISTRATION ROUTES AND BARRIERS Anatomically, the eye can be divided into the anterior and the posterior segments. The anterior segment of the eye includes the cornea, conjunctiva, aqueous humour, ciliary body, iris, and lens (Fig. 1). The posterior segment of the eye refers to the back of the eye, i.e. the vitreous humour, retina, choroid, and sclera. Depending on the ocular disease and the treatment s target tissue, drugs can be administered topically, systemically, or intravitreally (Fig. 1). There are other routes of administration e.g. intracameral and periocular, such as sub-conjunctival injection. The administration route depends mostly on the drug s properties and the ocular barriers that must be overcome to allow the drug to reach a therapeutic drug concentration at its site of action. The most commonly used administration routes in ophthalmology, topical and intravitreal, and the relevant barriers will be presented in the following sections. Figure 1. The structure of the eye with the different administration routes: topical, systemic, and intravitreal administration. The figure is modified from (del Amo et al., 2015), permission for original figure from Elsevier.

21 Barriers in topical drug delivery In most cases, drugs are delivered to the eye using topical administration of eye drops and ointments (Fig. 1). Topical administration is non-invasive with the drug being self-administered by the patient. Topical administration can only be used in the treatment of anterior segment diseases, such as conjunctivitis, keratitis, dry eye syndrome, and glaucoma (Urtti, 2006). A topically applied drug comes into contact with the tear film on the ocular surface (Dartt, 2002). The volume of tear fluid is about 7 µl and its turnover rates are in rabbit 0.53 μl/min but higher, i.e. 1.2 μl/min, in humans (Chrai et al., 1973; Mishima et al., 1966). The tear film is composed of three layers: the outer lipid layer, the middle aqueous layer, and the inner mucous layer (Dartt, 2002). The role of mucus as a barrier for ocular drug delivery is still unclear (Ruponen and Urtti, 2015); it may reduce ocular penetration of large molecules (e.g. proteins), but it does not affect the passage of small molecules. Nevertheless, the mucus may even be a beneficial component in drug delivery, since it enables mucoadhesion and prolonged surface retention of some formulations. The cornea is the outermost anterior tissue in the eye (Fig. 1) that allows light to penetrate into the eye. The cornea has three main parts: epithelium, stroma, and endothelium (Huang et al., 1983; Prausnitz and Noonan, 1998). The cells in multilayered epithelium are joined by tight junctions at the most outer cell layers and these represent the main diffusional barrier of the cornea (Table 1; Huang et al., 1989, 1983; Prausnitz and Noonan, 1998). The transcellular route across the epithelial cells is the main pathway for drug delivery that allows permeation of small and lipophilic compounds. The paracellular route between the cells is hindered, due to the narrow pore size (Table 1); this restricts the permeation of large and hydrophilic molecules in the epithelium (Ahmed et al., 1987; Huang et al., 1989; Hämäläinen et al., 1997). The stroma and endothelium are not considered to be significant barriers to trans-corneal drug delivery (Huang et al., 1989, 1983; Prausnitz and Noonan, 1998), even though in some cases, the partition of highly lipophilic drugs may be restricted from the epithelium to the hydrophilic stroma. The conjunctiva is a thin transparent mucous membrane that covers the anterior part of the sclera (bulbar) and the inner side of the eyelids (palpebral) (Fig. 1). Conjunctiva has a 9 times larger surface area than the cornea in rabbit eyes and 17 times larger in human eyes (Watsky et al., 1988). The epithelium of the conjunctiva, which is 2-3 cell layers thick, is composed of stratified cells with inter-cellular tight junctions that provide the barrier properties of the conjunctiva (Table 1; Huang et al., 1989). Beneath the conjunctival epithelium is a layer of connective tissue, composed of lymphatics, blood vessels and nerves. The connective tissue is loosely associated to the underlying sclera (Hosoya et al., 2005). Molecular size does not affect conjunctival permeability as strictly as it influences the corneal permeability. The conjunctiva is a rather permeable route for hydrophilic and large molecules, which permeate mainly through the paracellular route (Table 1) (Ahmed and Patton, 1985; Hämäläinen et al., 1997). Macromolecules up to 20 kda have been reported to be able to cross the conjunctiva, whereas the permeability of macromolecules of 40 kda is completely restricted (Huang et al., 1989). The sclera extends posteriorly from the corneal limbus, covering 95 % of the total surface area of the eye globe (Fig. 1; (Geroski and Edelhauser, 2000). It has an elastic and microporous structure, composed of collagen fibrils in a glycosaminoglycan matrix (Ambati and Adamis, 2002) that is essential for the protection of inner ocular tissues and the maintenance of the shape of the eyeball. The matrix is poorly vascularized, but it is perforated by some blood vessels leading to and from the eye.

22 5 Drug permeability in the sclera resembles that of the corneal stroma (Prausnitz and Noonan, 1998). Permeability of macromolecules in the sclera was ten times higher than in the rabbit cornea and two times higher than in the conjunctiva (Hämäläinen et al., 1997). Scleral permeation seems to depend on molecular size, but not on molecular lipophilicity (Ahmed et al., 1987; Ambati et al., 2000; Pitkänen et al., 2005; Prausnitz and Noonan, 1998). For example, a large macromolecule of 150 kda was able to permeate across the rabbit sclera (Ambati et al., 2000). Table 1. Transepithelial electrical resistances (TER) and pore diameters of ocular tissues. Tissue Origin TER ( x cm 2 ) Reference Cornea Rabbit 7500 ± 200 (Marshall and Klyce, 1983) Conjunctiva Rabbit 1400 ± 100 (Kompella et al., 1993) RPE-Choroid Rabbit 348 (Frambach et al., 1988) RPE-Choroid Bovine 109 ± ± ± ± 35 (Miller and Edelman, 1990; Pitkänen et al., 2005) RPE-Choroid Human 79 ± 48 (Quinn and Miller, 1992) Choroid Bovine 9 (Miller and Edelman, 1990) Choroid-Sclera Rabbit 2.3 ± 0.8 (Frambach et al., 1988) Tissue Origin Pore diameter (nm) Reference Cornea Rabbit (Huang et al., 1989; Hämäläinen et al., 1997) Conjunctiva bulbar palpebral Rabbit (Hämäläinen et al., 1997) BRB* & BAB** Monkey 2 (Smith and Rudt, 1975) Choroid Rabbit (Bill et al., 1980) ILM*** 10 (Jackson et al., 2003) Vitreous Bovine 550 (Xu et al., 2013) *, ** Blood-retinal barrier and blood-aqueous barrier *** Inner limiting membrane Barriers in intravitreal drug delivery Diseases of the posterior eye segment, such as AMD, macular edema and diabetic retinopathy, are treated with intravitreally injected drugs that exert their effects in the retina and choroid. (Fig. 1; Zhang et al., 2012) Vitreous humour (Fig. 1) is a transparent gel, composed of unbranched collagen fibrils, hyaluronan, and water (> 98%) (Bishop, 2000). The vitreous does not significantly restrict the diffusion of most solutes, but diffusion of macromolecules and particles depends on their size, charge and hydrophobicity (Peeters et al., 2005; Pitkänen et al., 2003). The mesh size of bovine vitreous was estimated to be 550 ± 50 nm (Xu et al., 2013) allowing diffusion of smaller species (Peeters et al., 2005; Xu et al., 2013). The diffusion of positively charged nanoparticles and

23 6 polymers was hindered due to their interaction with the polyanionic vitreous gel (Pitkänen et al., 2003). Retina is the innermost tissue layer in the posterior eye segments (Fig. 1 and Fig. 2) that is essential for vision, since it detects light signals and converts them to electric stimuli that are transmitted to the brain via the optic nerve. The inner limiting membrane (ILM) and outer limiting membrane (OLM) of the neural retina may limit the diffusion of macromolecules and particles across the retina (Table 1; Jackson et al., 2003; Omri et al., 2010). Macromolecules with molecular weights less than kda diffuse freely across the ILM in rabbit, porcine, and human eyes (Jackson et al., 2003; Kamei et al., 1999; Kwan et al., 2006). The negative charge of the ILM (originating from glycosaminoglycans) may hinder the permeation of positively charged polymers and nanoparticles (Pitkänen et al., 2004). Similarly, the molecular weight (MW) cut-off of the OLM was estimated to be between kda, because peroxidase, but not albumin, was able to cross the membrane (Bunt-Milam et al., 1985). The retinal pigment epithelium (RPE) between the choroid and photoreceptors of the neural retina, is a pigmented monolayer of polarized epithelial cells with inter-cellular tight junctions (Fig. 2; Hornof et al., 2005). The RPE has a crucial role in the function of the neural retina by providing essential nutrition, phagocytosing outer segments from photoreceptors, and restricting foreign substances in the circulation from accessing the retina. The RPE permeability depends on the molecular size, because the paracellular permeation is limited by the tight junctions (Table 1). Increased lipophilicity improved the trans-cellular permeation in the RPE (Pitkänen et al., 2005) but in addition, affinity to the transporters in the RPE could also contribute to drug permeation (Mannermaa et al., 2006). Permeability decreased as the molecular size increased; for example, permeation of FITC-dextran 77 kda was 35 and 619 times slower than that of carboxyfluorescein (376 Da) and lipophilic β-blocker betaxolol (307 Da), respectively (Pitkänen et al., 2005). The RPE and the endothelia of the retinal capillaries make up the blood-retinal barrier (BRB) that limits solute transport between retina and the blood circulation (Table 1; Hornof et al., 2005). The BRB is also known for expressing both influx and efflux transporters, such as monocarboxylate transporters (MCT) and multidrug resistance associated proteins (MRP), respectively (Vellonen et al., 2018). Figure 2. The structure of the retina. ILM: inner limiting membrane; NFL: nerve fiber layer; GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; OLM: outer limiting membrane; POS: photoreceptor outer segment; RPE: retinal pigment epithelium; BM: Bruch s membrane; CHR: choroid. The figure is modified from (Mannermaa et al., 2006), permission for original figure from Elsevier.

24 7 2.2 OCULAR PHARMACOKINETICS AND IMPROVED DRUG DELIVERY Drug movement by passive diffusion Drugs can cross ocular barriers by either passive diffusion or active transport. Passive diffusion does not require energy and is dependent on the physicochemical properties and tightness of the membrane, whereas active transport depends on the affinity of the drug for the transporter, and the expression of the transporter in the barrier (Vellonen et al., 2018). Often, drug flux across the membranes may utilize both basic mechanisms simultaneously. Obviously, the overall flux of the drug depends on the drug concentration. Diffusional flux can be described by Fick s first law, where flux (J) is proportional to a concentration gradient (C) in one dimension (x), with a diffusion coefficient of D: Eq. 1 The study of flux is common in biopharmaceutical studies, utilizing an in vitro cell layer or ex vivo animal/human tissue models. If the concentration gradient in the barrier is kept constant, the concentration gradient becomes equal to the donor site concentration. Then, flux is dependent on the concentration gradient (Cdonor) and drug permeability (P) in the membrane: Eq. 2 Permeability depends on the diffusion coefficient of the drug, the barrier thickness and the partition coefficient of the molecule to the barrier. Permeability can be estimated by measuring drug flux across the membrane in a diffusion chamber, considering donor concentration (Cdonor), membrane area (A), and drug flux across the barrier (dm/dt). Apparent permeability (Papp) can be determined using Eq. 3: Eq. 3 Ocular tissues, and their barrier properties and their role in ocular pharmacokinetics can be studied by permeability studies. Permeability values can be used for prediction of drug bioavailability (F) and clearance (CL) in the eye, or for selecting the compounds with the desired permeability values. The following sections will provide a general overview of the pharmacokinetics of the two most commonly used routes of ocular drug delivery, topical and intravitreal Topical administration Topically administered eye drops have a short contact time i.e. less than 5 minutes, with the ocular surface (Lee and Robinson, 1979; Thombre and Himmelstein, 1984; Urtti et al., 1990). This is all the time that is available for corneal drug absorption to the eye (Fig. 3) (Ahmed and Patton, 1987, 1985; Doane et al., 1978). Pre-corneal factors, such as solution drainage, tear turnover, induced lacrimation, and conjunctival absorption (Fig. 3), and the barrier of the cornea limit ocular drug absorption. Therefore, the ocular bioavailability of topically applied drugs in the aqueous humour is less than 5% (Chrai and Robinson, 1974; Lazare and Horlington, 1975; Maurice and Mishima, 1984; Urtti et al., 1990). On the other hand, the bioavailability in the systemic blood circulation is often higher than 70% (Urtti and Salminen, 1993).

25 8 After instillation of an eye drop (volume of μl), the solution will become diluted in the tear fluid (Lederer and Harold, 1986). Drainage of the eye drop (Fig. 3) takes place after instillation, depending on the volume and viscosity of the eye drop (Chrai et al., 1973; Thombre and Himmelstein, 1984). Lacrimation may be evoked, depending on osmotic pressure, ph, drug properties, and excipients (Conrad et al., 1978). After the pre-corneal elimination, the drug solution enters the lacrimal drainage system and nasal cavity (Chang and Lee, 1987; Urtti and Salminen, 1993), where the drug may be absorbed to the systemic circulation or flow further to the gastrointestinal (GI) tract. When present on the ocular surface, the drug may be absorbed across the cornea and conjunctiva. Trans-conjunctival absorption delivers drug to the systemic circulation, because the conjunctiva is vascularized and its epithelium is leakier and has a larger surface area than the cornea (Fig. 3; Urtti et al., 1985). Intraocular drug delivery through the conjunctiva may take place, bypassing the anterior chamber, to the ciliary body and iris (Ahmed and Patton, 1987, 1985; Ranta et al., 2010; Shikamura et al., 2016). Conjunctival permeability, which is known to depend mostly on the molecular weight of the drug, varies between cm/sec in the rabbit (Fig. 4). A few studies with only a narrow set of drug molecules have described the impact of physicochemical factors on the conjunctival permeability, with limited success (Prausnitz and Noonan, 1998; Wang et al., 1991). As shown in Figure 4, there is no clear correlation between the conjunctival permeability and simple molecular descriptors, such as molecular weight and octanol/water distribution coefficient (LogD7.4). Nonetheless, systematic studies on conjunctival drug permeability with a large set of molecules are still lacking. Passive permeability across the cornea depends on the molecular properties of the drug; for example, molecular weight, lipophilicity, and charge (Ahmed et al., 1987; Brechue and Maren, 1993; Chien et al., 1990; Huang et al., 1983; Liaw et al., 1992; Pescina et al., 2015; Schoenwald and Huang, 1983; Wang et al., 1991). In particular, lipophilicity is an important descriptor of transcorneal permeability (Fig. 4; Ahmed et al., 1987; Huang et al., 1983; Wang et al., 1991). An optimal LogD7.4 for corneal permeation was 2-3 (Järvinen et al., 1995). Lipophilic betaxolol (LogD ) permeates through the rabbit cornea 25 times faster than the more hydrophilic compound, atenolol (LogD ) (Prausnitz and Noonan, 1998; Wang et al., 1991). Due to the tight barrier properties of the cornea, the permeability across ex vivo rabbit cornea is in the range of cm/sec (Fig. 4). The peak drug concentration in the aqueous humour is typically reached within minutes after instillation, but the peak concentrations are usually two orders of magnitude lower than the instilled drug concentration (Schoenwald, 2003; Urtti et al., 1990). In the aqueous humour, the drug becomes distributed to the surrounding tissues, such as the iris and ciliary body (Urtti, 2006). In these tissues, the drug may bind to melanin. Drug distribution to the lens and vitreous is insignificant and therefore topical instillation is not suitable for the treatment of diseases of the posterior segment (Ahmed and Patton, 1987, 1985). Depending on the properties of the drug and its binding capacity to tissues, the ocular volume of distribution (Vd) may vary from 210 μl to 3 ml in the rabbit (Schoenwald, 2003; Urtti, 2006). The drug is eliminated from the aqueous humour across the blood-aqueous barrier (BAB) to the blood flow of ciliary-body and iris, and with the aqueous humour outflow via the trabecular meshwork to the Schlemm s canal. Lipophilic drugs may cross the endothelial walls of the BAB and be cleared faster from the aqueous humor than hydrophilic and/or large molecules. For example, a hydrophilic compound, inulin, was reported to have a clearance (4.7 μl/min) that was close to the aqueous humour turnover rate of μl/min in the rabbit (Conrad and Robinson, 1977). Small molecular ophthalmic drugs have exhibited clearance values of μl/min, which is due to their ability to pass across the BAB (Schoenwald, 2003). The elimination half-lives (t1/2) of ophthalmic drugs in the aqueous humour are usually short, i.e hours.

26 9 Figure 3. Delivery routes for topically instilled drugs and processes, which may take place at the ocular surface. Trans-corneal permeability is the main route to the aqueous humour, from where the bioavailability of the ocular drug is mostly determined. GI-tract (gastrointestinal tract) Figure 4. Permeability across ex vivo cornea, conjunctiva, sclera, and RPE versus MW (A) and LogD 7.4 (B). Cornea and conjunctiva are from rabbit specimens (Ahmed et al., 1987; Hämäläinen et al., 1997; Kidron et al., 2010; Prausnitz and Noonan, 1998; Wang et al., 1991); sclera from rabbit, bovine, and human specimens (Ahmed et al., 1987; Ambati et al., 2000; Hämäläinen et al., 1997; Olsen et al., 1995; Prausnitz and Noonan, 1998); and RPE from bovine specimens (Pitkänen et al., 2005). Ocular bioavailability after topical administration can be improved by either enhancing corneal permeability or prolonging the retention on the ocular surface (Chang et al., 1988, 1987; Kaur et al., 2004; Urtti et al., 1990, 1985; Zhang et al., 2004). Polymers with increased viscosity or mucoadhesive properties have been used in eye drop formulations, to prolong the contact with the ocular surface. Prodrug technology has been applied for promoting corneal permeability and thereby reducing a required dose (Schoenwald, 1990). For example, an anti-glaucoma drug,

27 10 latanoprost, is an isopropyl ester prodrug of a prostaglandin that penetrates through the corneal epithelium, where it undergoes hydrolysis and releases the active drug. In ocular drug research, the emphasis has been directed on the corneal permeability, but that does not alone determine ocular bioavailability. A more systematic approach is needed to take into account also the role of conjunctiva Intravitreal administration An intravitreal injection is the most direct route if one needs to deliver ophthalmic drugs to the posterior eye segment (Fig. 1 and Fig. 5). The vitreal drug concentration profile depends on the intravitreal volume of distribution (Vd,IVT) and clearance (CLIVT). Vd,IVT depends on the ability of drug molecules to permeate into the surrounding tissues and on its binding to tissue components (del Amo and Urtti, 2015). The Vd,IVT is relatively constant in rabbits, as 80% of the drugs had a Vd,IVT within ml, which is close to the anatomical vitreous volume of 1.15 ml in the rabbit (del Amo et al., 2015). Intravitreally administered drugs are mainly eliminated to the systemic blood circulation. There are two main pathways of vitreal drug elimination (Fig. 5): the anterior route to the aqueous humour and the posterior route across the retina to the choroid (Maurice and Mishima, 1984). The route of elimination depends on the physicochemical properties of the drug (del Amo et al., 2015; Kidron et al., 2012). Small and lipophilic drugs are mainly eliminated via the posterior route across the retina to the choroid, leading to short half-lives of 1-10 hours (del Amo et al., 2015; Kidron et al., 2012; Maurice and Mishima, 1984). On the contrary, large molecules, e.g. proteins, are cleared mainly through the anterior route, which means that they have longer half-lives of 2-5 days (del Amo et al., 2015; del Amo and Urtti, 2015). For anterior elimination, the drug needs to diffuse in the vitreous, across the hyaloid membrane, and further to the posterior and anterior chambers. From the anterior chamber, the drug is cleared with the aqueous humour flow to the Schlemm s canal. Macromolecules are unable to cross the blood-retinal barrier (Pitkänen et al., 2005) and therefore, they are cleared anteriorly (CLIVT = ml/h) (del Amo and Urtti, 2015). The intravitreal clearance of proteins is lower than the aqueous humour turnover rate (0.18 ml/h), which is due to their restricted access to the aqueous humour (del Amo and Urtti, 2015). The posterior elimination across the blood-retinal barrier takes place mainly across the large surface area of the RPE (Reichenbach et al., 1994). Small molecular drugs show a 50-fold range of CLIVT ( ml/h) into the choroidal blood flow, due to their varying permeabilities in the RPE. Some of these values are higher than the aqueous humour turnover rate, which accounts for their ability to be eliminated across the blood-retina barrier to the systemic circulation (del Amo et al., 2015). However, the CLIVT values are much smaller than the choroidal blood flow (62 ml/h) (del Amo and Urtti, 2015), which indicates that the RPE acts as a rate limiting barrier in the ocular elimination process (del Amo et al., 2015; Pitkänen et al., 2005). RPE permeability depends on lipophilicity and molecular weight of the molecule (Pitkänen et al., 2005). RPE showed permeability values between 1-19 x 10-6 cm/sec for a range of β- blockers with varying values of lipophilicity (LogD7.4: ) (included in Fig. 4). In view of the surface area, the permeability values could be converted to RPE clearance (CLRPE) values of ml/h (Pitkänen et al., 2005). These values represent a large fraction of the likely CLIVT, and highlight the important role of the RPE in drug elimination. The literature still lacks experimentally-derived information on the role of RPE permeability on vitreal drug clearance.

28 11 Figure 5. Intravitreal injection and the elimination pathways from the vitreous. The blood-retinal barrier includes the retinal capillaries and the RPE. The blood-aqueous barrier includes the endothelia of the iris and ciliary blood vessels, as well as, the epithelium of the posterior iris and non-pigmented ciliary body. Improved intravitreal drug delivery: sustained, controlled and targeted delivery Intravitreal drugs are usually administered as solutions, suspensions or implants. Repeated injections are needed and they may cause adverse effects and represent a burden to healthcare (Jager et al., 2004). There is a need for sustained and controlled intraocular drug delivery systems that can prolong the injection intervals. In addition, new biologics (e.g. sirna) must be formulated with smart carriers capable of delivering these compounds to their intracellular targets. Extensive research on novel drug carriers, such as nano- and micro-particles, implants and encapsulated cells is ongoing (Bourges et al., 2006, 2003; del Amo and Urtti, 2008; Herrero- Vanrell et al., 2014; Tao et al., 2002). Small molecular weight drugs are rapidly eliminated from the vitreous; their half-lives are typically a few hours. Without long acting delivery system prolonged injection intervals cannot be reached. For example, Ozurdex (Allergan Inc.) is a clinically used biodegradable poly(lactide) based implant that releases dexamethasone for up to 6 months after insertion into the vitreous (Chang-Lin et al., 2011). Protein drugs, such as anti-vegf compounds, bevacizumab and ranibizumab, are administered intravitreally as solutions. Repeated monthly injections are required, because the half-lives of these agents are only a few days (Bakri et al., 2007a, 2007b). Their administration intervals could be prolonged if they were incorporated into a drug delivery system, for example microparticles, which would release the protein drug to the vitreous in controlled manner. Microparticles have been investigated as prolonged release systems for small molecular drugs and proteins (Andrés-Guerrero et al., 2015; Moritera et al., 1991; Nieto et al., 2015). Due to their large size and the vitreous humour mesh, these particles are mainly retained in the vitreous (Andrés-Guerrero et al., 2015). Drug release from the microparticles is a complex phenomenon that depends on the carrier, the drug, and the environment. In the preclinical drug development phase, drug release is tested usually in a buffer solution, as single formulation in each test. In vitro screening methods that allow drug release testing from many formulations simultaneously could speed up drug development. The development could also be improved by using the release rate data as input in pharmacokinetic models for prediction of drug concentrations in the vitreous in vivo. In recent years, cellular and targeted delivery of biologics has been widely investigated. These therapeutic modalities include gene therapy, small interfering RNA (sirna), and aptamers (Fattal and Bochot, 2008, 2006). These compounds must be delivered intracellularly to specific cell types in the retina, for example, the photoreceptors and RPE cells (Fattal and Bochot, 2006; Guzman-Aranguez et al., 2013; Wittrup and Lieberman, 2015). Due to their large molecular weight, negative charge, and lability in the presence of nucleases, delivery systems

29 12 are needed for cell specific targeting, shuttling into the cells, and escaping from the endosomes into the cytosol or nucleus (Guzman-Aranguez et al., 2013; Wittrup and Lieberman, 2015). The use of nano-sized carriers or conjugate moieties may facilitate in achieving these goals. 2.3 PHARMACOKINETIC MODELS Ocular pharmacokinetic research involves measuring drug concentrations from ocular tissues and fluids, usually after topical, systemic or intravitreal administration and usually performed in rabbits (Maurice and Mishima, 1984). There are only a few studies where ocular pharmacokinetics from the human eye has been measured using sampling from the aqueous humor or vitreous. Obviously, tissue sampling from human eyes is not feasible. The primary aim in pharmacokinetic studies has been to assess whether therapeutic drug concentrations can be reached in the target tissues (Schoenwald, 2003). However, this information alone does not allow the prediction of how changes of formulation, chemical structure or dosage will affect pharmacokinetics. Pharmacokinetic models are valuable in preclinical and clinical drug development, because the models can be used to predict the effects of formulation and dosage, or patient related factors on pharmacokinetics. Ocular pharmacokinetic models are divided into top-down and bottom-up approaches (Fig. 6). The top-down approach is based on clinical or in vivo data. Pharmacokinetic parameters are solved using a defined model structure and then the model with the parameter values can be constructed. The bottom-up approach integrates information from in vitro and in silico data, which are then combined into a model (Jamei, 2016; Jamei et al., 2009; Singh Badhan, 2015; Tsamandouras et al., 2013). However, bottom-up and top-down approaches can also be used in combination to strengthen the reliability of the model. Figure 6. Bottom-up and top-down approaches in pharmacokinetic modeling. In the bottom-up approach, the predictive physiologically based pharmacokinetic model is built on data based on properties of the drug and formulation, physiology and anatomy. Prior in vivo data is not necessarily required, but may be used to verify the reliability of the model. The model can then be used for predicting the kinetics related to new formulations, drugs, disease states or other species. The topdown approach is based on in vivo data (i.e. concentrations in the ocular tissues). Mechanistic predictions cannot be made with the top-down approach, and it has limitations in extending the predictions to other species, disease states or chemical drug structures. The figure is modified from (Schmitt and Willmann, 2005), permission for original figure from Elsevier.

30 Top-down approach Pharmacokinetic top-down models include the classical empirical compartmental modeling, non-compartmental modeling, and population based pharmacokinetic modeling. The aim of the top-down approach is to use in vivo data to solve pharmacokinetic parameters. The parameters can be used to simulate different dosing schedules. The modeling does not require prior knowledge of the system, but an assumed compartment structure is often used in the fitting procedure. Additionally, the approach lacks mechanistic insights, which are possible in the bottom-up version of modeling (Rowland et al., 2011). Models for topical and intravitreal administration In the 1970s and 1980s, extensive research of ocular pharmacokinetics in rabbits helped to provide an understanding of the ocular pharmacokinetics after topical eye drop instillation (Himmelstein et al., 1978; Lee and Robinson, 1979; Makoid and Robinson, 1979; Oh et al., 1995; Sieg and Robinson, 1981, 1976). Classical compartmental models were built, in order to describe pre-corneal, corneal, and intraocular dispositions. In addition to the topical instillation, also a topical infusion method and intracameral injections were used for data generation (Chastain, 2003). The pharmacokinetic parameters were solved from the concentration vs time curves by curve fitting (Chiang and Schoenwald, 1986; Tang-Liu et al., 1987, 1984). The top-down models have helped to solve important pharmacokinetic parameters, such as rates of drug elimination from the tear fluid, absorption rate constants, ocular bioavailability, volume of distribution, and clearance from the anterior chamber. Furthermore, these models helped to build relevant compartmental models in which tear fluid, corneal epithelium and aqueous humor constituted the compartments. It is noteworthy that the corneal stroma and iris-ciliary body were considered to be in the same compartment with aqueous humor. However, non-corneal routes were rarely included in these models. Population based pharmacokinetic modeling have been adopted recently for the comparisons of topical formulations in both preclinical and clinical settings (Abduljalil et al., 2008; Djebli et al., 2017). The advantage of this method is that even a limited amount of sample points from different individuals is enough for model generation and concentration-time predictions. In contrast, the traditional compartmental models require many sampling times for proper curve fitting and solving of the pharmacokinetic parameters. Population kinetics is especially valuable in clinical ocular pharmacokinetic, because the repeated sampling from the human eye compartments is not feasible. Simple pharmacokinetic models were also developed for intravitreal injections. The most simple, and usually adequate approach, is to assume vitreous as a single compartment (i.e. mixed tank approach). Intravitreal pharmacokinetic parameters (CLIVT, Vd,IVT, t1/2) were determined using this approach and curve fitting of intravitreal drug concentration data (del Amo et al., 2015; Kidron et al., 2012). These data proposed the route of drug elimination from the vitreous; macromolecules eliminating via anterior route and small molecules primarily posteriorly, but also anteriorly (Araie and Maurice, 1991; del Amo et al., 2015; Maurice and Mishima, 1984; Yoshida et al., 1992). Population kinetics has also been used for the analysis of the intravitreal injections. Ranibizumab and pegaptanib were intravitreally injected in patients with different posterior eye diseases or in the situation where there was also renal failure (Audren et al., 2004; Basile et al., 2015, 2012; Zhang et al., 2014). Systemic exposure of the protein drug was studied by using the non-linear mixed effect approach. Furthermore, this approach has been extended to the drug responses with pharmacodynamics/pharmacokinetic (PD/PK) modeling. Audren et al., (2004) studied the effect of triamcinolone acetonide on the thickness of central macular in patients, using population PD/PK modeling.

31 Bottom-up approach The most common bottom-up approach is physiologically based pharmacokinetic modeling (PBPK), which combines the properties of the system and the drug into a structural model (Singh Badhan, 2015). System properties are basically the anatomical and physiological factors. Drug specific properties include the physicochemical features and descriptive properties, such as drug permeability and its affinity for transporters, enzymes and other proteins. The model is generated on data that originates mainly from in vitro and in silico experiments (Jamei, 2016; Jamei et al., 2009; Ruiz-Garcia et al., 2008; Singh Badhan, 2015; Tsamandouras et al., 2013). The performance of the model is later verified experimentally. The bottom-up approach makes it possible to obtain a broader pharmacokinetic understanding of the human or animal body. Prior in vivo experiments are not necessarily needed, unlike in the top-down approach (Jamei, 2016). A PBPK model can be used to scale-up the pharmacokinetics from animal models to humans. Typically, a model is generated and tested in a laboratory animal, and thereafter, the physiological parameters are scaled-up to generate the human model. The inter-species translation between animals and man cannot be done with the compartmental top-down approach. Several investigators have applied simple or more complex physiological and anatomical parameters in ocular pharmacokinetic modeling. Models for topical and intravitreal administration The first physiologically based compartmental models were devised to estimate the disposition of topical pilocarpine (Himmelstein et al., 1978; Miller et al., 1981) and timolol (Francoeur et al., 1985) in the rabbit eye. They combined physiological data and experimentally determined parameters, such as in vitro ocular tissue binding results, to predict the intraocular tissue levels of the drugs. The overall performance of the physiological models was in relatively good agreement with the in vivo data (Francoeur et al., 1985; Miller et al., 1981). More accurate models have been developed for trans-corneal permeation, where the ultrastructure of the corneal membranes and the physicochemical properties of the drug solutes were considered (Edwards and Prausnitz, 2001; Zhang et al., 2004). For example, Zhang et al., 2004 generated a model which incorporated the ultrastructure of the cornea, the molecular size and lipophilicity of the drug, the tear flow rate, and Vd and CL in the anterior chamber. Compared to the simple mixed-tank models for intravitreal injection (Araie and Maurice, 1991; Yoshida et al., 1992), Ohtori and Tojo (Ohtori and Tojo, 1994; Tojo and Ohtori, 1994) developed a more complex model, which considered the geometry and boundaries of the vitreous, and simulated more precisely the elimination of the drug through the anterior part in the vitreous. The model was based on Fick s second law of diffusion, and it modelled vitreous as a cylindrical container with three elimination pathways: retina-choroid-sclera, posterior chamber, and lens. Model parameters, such as diffusion coefficients and tissue partition coefficients were determined by in vitro experiments using isolated rabbit tissues. The model was successfully used to describe the in vivo pharmacokinetics of intravitreally injected dexamethasone sodium m-sulfobenzoate (Ohtori and Tojo, 1994). Finite element modeling can also be classified as a bottom-up approach, where a virtual eye is built using specialized computer software, such as COMSOL Multiphysics. These more complex models takes into account detailed geometry and boundary conditions of the vitreous, and accurately predicts concentration gradients within the vitreous (Friedrich et al., 1997a, 1997b). Finite element models of the rabbit and human eye have shown that the rate of drug elimination from the vitreous depends greatly on the diffusivity of the molecules in the vitreous and their retinal permeability (Friedrich et al. 1997a, 1997b). Finite element models for clearance predictions have been built for human, rabbit, and monkey eye, but the performance of the

32 15 human and monkey models is uncertain, because the performance of these models has not been compared to experimental data (Lamminsalo et al., 2018; Missel, 2012). As was discussed in chapter 2.2.3, drug delivery systems, with sustained and controlled drug delivery are needed for intravitreal delivery of small molecular weight drugs and protein drugs. Pharmacokinetic models can speed up the development of ocular formulations and lower the costs of in vivo experiments. Simple models based on differential equations can be generated, for example, with Stella (ISEE systems) or Berkeley Madonna software. Integration of in vitro data into PBPK models can provide scientists with predictions of drug and carrier formulation concentrations in the vitreous; these simulations can be used also for biologicals as well as taking into account degradation products of the polymer carriers. In silico approaches for supporting ocular bottom-up modeling Drug specific data, which can be incorporated in PBPK models, can be collated already during the drug discovery and development stage from in vitro and in silico experiments (Ruiz-Garcia et al., 2008). In vitro studies can be used to gain information on drug permeability, protein binding, toxicity, and metabolism, by using cell lines and isolated tissues. Furthermore in silico experiments i.e. computational studies, may involve quantitative structure-activity/property relationship (QSAR/QSPR) modeling, comparative molecular field analysis (CoMFA), and correlation approaches for the prediction of ADME (absorption, distribution, metabolism, and elimination) properties. A quantitative structure-property relationship model (QSPR) describes the relationship between physicochemical descriptors and pharmacokinetic properties of the compounds (Eriksson et al., 2003). The goal of these models is to obtain an equation that can be used in predicting the pharmacokinetic properties of new compounds. The QSPR model may be generated by regression analysis, neural nets, and classification approaches. Regression techniques include multiple linear regression and partial least square (PLS) analysis. The multivariate projection method, such as principal component analysis (PCA), including also PLS, is also used for model generation. Multivariate tools can be used for model building, for example by Simca (Umetrics, Sartorius-stedim). The QSPR approach has been used previously in ocular pharmacokinetics to predict CLIVT, t1/2 in the vitreous, as well as for estimating a drug s corneal permeability (del Amo et al., 2015; Kidron et al., 2012, 2010). A QSPR model was generated to describe corneal permeability across ex vivo rabbit cornea (Kidron et al., 2010). The model indicated that lipophilicity (LogD) and the ability to form hydrogen bonds were important and adequate descriptors in predicting a compound s permeability. The same descriptors were evident in the prediction of intravitreal t1/2 in albino and pigmented rabbits (Kidron et al., 2012). The data for these models had been collected from different experimental studies in the literature, which generated some uncertainty due to the differences in the experimental conditions. Del Amo et al., 2015 devised a QSPR model for intravitreal CLIVT predictions, where the in vivo data was gathered from different sources, but the pharmacokinetic parameters were estimated using the same method. Firstly, in vivo data of intravitreally studied proteins and small molecular drugs were collected and fitted for CLIVT and Vd,IVT estimations. Secondly, CLIVT values were correlated with a set of physicochemical descriptors of each drug to generate the QSPR models. The most important descriptors for CLIVT prediction were the ability to donate hydrogen bonds and LogD7.4. Thirdly, CLIVT and Vd,IVT parameters were used as components in a PBPK model to predict intravitreal drug concentration profiles. These kinds of QSPR models can be of great value for parameter estimation in PBPK and devising finite element models both of which may help in the development of ocular drugs.

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34 17 3 Aim of the Study The aim of this thesis was to develop in vitro, ex vivo, and in silico tools that may be applicable in ocular drug development. The more specific aims of the study were: 1. To determine the impact of physicochemical drug properties on drug permeability through the cornea and conjunctiva with experimental studies and QSPR modeling. 2. To predict the impact of corneal and conjunctival drug permeability on ocular pharmacokinetics. 3. To develop a high-throughput screening tool for drug release studies and to combine this approach with intravitreal pharmacokinetics. 4. To simulate the impact of the polymer degradation rate on the intravitreal concentrations of the polymer-borne materials. 5. To estimate the role of RPE permeability on intravitreal clearance of small molecular weight drugs. 6. To study the delivery and cellular level effects of an anti-inflammatory IL-6 silencing sirna, coupled to a block copolymer carrier.

35 18

36 4 Overview of the materials and methods 19 The materials and methods relating to the original publications I-IV are presented shortly below; more detailed information regarding the published work can be found in the publications. The unpublished material including RPE permeability and sirna delivery studies are presented more in detail (Unpub. V-VI). 4.1 CELL CULTURE AND OCULAR TISSUE PREPARATION Table 2 represents the test models in the cellular and ex vivo ocular studies. Table 2. Cell line and ocular tissues used in the studies. Ex vivo model Ocular tissue Source Pub. / Unpub. Porcine eye Conjunctiva Local slaughterhouse I Cornea II Bovine eye RPE-choroid V Cell line ARPE-19 cell Undifferentiated RPE cells ATCC (CRL-2302) VI Ocular tissue preparation. The conjunctiva and cornea were isolated from porcine eyes, as described in publications I and II. The RPE-choroid tissues were isolated from freshly enucleated bovine eyes (Unpub. V). Bovine eyes were chosen due to easier isolation of the RPEchoroid than from porcine eyes. Firstly, extraocular tissue was removed with scissors, then the anterior part of the eye was removed by cutting circumferentially 10 mm behind the limbus. The vitreous was gently removed from the eyecup that was further cut into three parts. The neural retina was gently peeled from the underlying RPE. The RPE-choroid tissue samples were detached gently from the underlying sclera using forceps and scissors, avoiding the optic nerve area. Balanced salt solution (BSS Plus, Alcon Laboratories, TX, USA) supplemented with 10 mm Hepes (ph 7.4) was applied on the tissues to avoid drying during the dissection procedures. Cell culture. The human retinal pigment epithelial cells (ARPE-19) (Dunn et al., 1996) were maintained in Dulbecco s modified Eagle s medium (DMEM): Nutrient Mixture F12, 1:1 mixture, supplemented with 10% fetal bovine serum, 2 mm L-glutamine, 50 U/ml streptomycin and 50 U/ml penicillin. The cells were maintained at 37 C in a humidified atmosphere containing 7% CO2. The cells were sub-cultured (1:3 or 1:5) and medium was renewed once a week.

37 DRUGS AND DELIVERY SYSTEMS The drugs and delivery systems used in the different studies are presented in Table 3. Table 3. Delivery systems, drugs, and their working concentrations or amount of loading. Tissue/ cell line Conjunctiva Delivery system Drug/Biologic Working concentration Cassette dose: 32 small 1 or 10 µg/ml molecular drug compounds Pub. / Unpub. I Cornea Cassette dose: 32 small molecular drug compounds 2 or 20 µg/ml II RPE-choroid ARPE-19 Microspheres: Alginate solution, 1% and 1.5%, cross-linked with: - CaCl 2, uncoated - BaCl 2, uncoated - 60:40 CaCl 2/BaCl 2, uncoated - 60:40 BaCl 2/CaCl 2, uncoated - CaCl 2, coated with poly-l-lysine - BaCl 2, coated with poly-l-lysine = 12 formulations -Block copolymer of poly(benzyl-glutamate) and poly-l-lysine (PBE30-b- PK30)*: Cassette dose**: aztreonam, ciprofloxacin, fluconazole, ganciclovir, ketorolac, methotrexate, quinidine, and voriconazole - dexamethasone phosphate - vancomycin - α-lactalbumin - lysozyme - myoglobin - bovine serum albumin (BSA) - lactoferrin - human IgG (higg) -sirna (siil6) and carboxyfluorescein-sirna (FAM-siIL6) for silencing IL-6 expression (Eurofins MWG Operon, Ebersberg, Germany): 2 or 20 µg/ml Amount of loading 300 µg/ml 200 ng 7.5 µg 7.5 µg 7.5 µg 5 µg 5 µg 5 µg V III Working concentration nm VI Sense strand: 5 - GAACGAAUUGACAAACAAAtt Antisense strand: 3 - tgcuugcuuaacuguuuguuu -Lipofectamine 2000 (Invitrogen Corporation, CA, USA) -non-specific sirna, sins (Silencer Negative Control #1 sirna, Ambion ) * (Stukenkemper et al., 2014) ** Information on manufacturer and preparation of stock solutions in Pub. I and II

38 DRUG PERMEABILITY Porcine conjunctiva and cornea, and bovine RPE-choroid were isolated to study the permeability of drugs in a cassette mix (or dose) consisting of either 32 or clinically relevant small molecular drug compounds (Table 3), by using the Ussing permeability chambers (Harvard Apparatus, MA, USA). The reader is referred to publications I and II for more detailed information regarding the conjunctiva and cornea permeability studies, including the TER measurements. The RPE-choroid permeability studies (Unpub. V) followed the same protocol as in the published articles, with some minor differences. Permeability studies were performed in both outward (retina-to-choroid) and inward (choroid-to-retina) directions using BSS Plus (Alcon Laboratories, TX, USA) buffer, supplemented with 10 mm Hepes (ph 7.4). Both the apical and basolateral sides of the chamber contained 5 ml buffer, which was maintained at 37 C and supplied with gas flow (5% CO2, 10% O2 and 85% N2). The experiment was started by removing 500 µl of the donor side and replacing it with the same volume of the cassette dose. Samples were then withdrawn from the receiver side at specific time points up to 6 h. Additionally 40 µl was withdrawn from the donor side at the beginning and at the end of the experiment. The quantities of the drugs that permeated through the ex vivo tissues were analyzed by a LC-MS/MS technique modified from publication I (Pub. I; Rimpelä et al., 2018). Internal standards were included in the analysis, to confirm stability of the cassette mixture drugs. The apparent permeability coefficients (Papp) were calculated based on the results (Eq. 3). Additionally, the mass balance was calculated for the small molecular drugs, in order to evaluate whether drug would be missing and stuck to tissue. 4.4 DRUG RELEASE FROM MICROSPHERES A 96-well plate set-up combined with robotic sampling was used to study the release of eight model drugs and biologics, with varying physicochemical properties, from 12 different alginate microsphere formulations (Table 3). Blank alginate microspheres were prepared using a custom-built device (Kontturi et al., 2011), where the alginate solution was dispensed to a crosslinking solution. Freshly prepared microspheres were soaked overnight with the model drugs, followed by washing and the start of the release experiment. Drug release from the microspheres was measured at specific time intervals for over 90 hours, using either photometric or fluorometric analytical methods. (Pub. III)

39 SIRNA DELIVERY Characterization of sirna polyplex A block copolymer based on poly(benzyl-glutamate) and poly-l-lysine (PBE30-b-PK30) was studied as a carrier for sirna (Table 3) (Stukenkemper et al., 2014). First, a micellar block copolymer stock solution was prepared in distilled water at 1 mg/ml concentration. Then sirna (Table 3) and the micellar solutions were diluted separately in 10 mm Hepes (ph 7.2) and finally mixed together. This was followed by at least 20 minutes incubation at room temperature to allow polyplex formation. The block copolymer and sirna were complexed at different charge ratios (N/P), calculated based on the molar ratio of positive/negative charges, which originate from the amines of the block copolymer and the phosphate of the sirna strand, respectively. Lipofectamine 2000 was used as a positive control sirna carrier and the lipoplex preparation was performed according to the instructions of the manufacturer (Invitrogen Corporation, CA, USA). The sirna binding to micellar block copolymer was assayed by agarose gel electrophoresis. The agarose gels were prepared as a 2 % (w/v) concentration in UltraPure Tris-acetate-EDTA running buffer (TAE, Gibco, NY, USA), containing 0.5 µg/ml ethidium bromide (Sigma- Aldrich, MO, USA). Polyplexes with varying charge ratios (N/P) as well as free sirna were mixed with Orange DNA loading dye (Thermo Scientific, CA, USA). Then, the samples were loaded into the wells of the gel (0.6 µg sirna/well), with the first well being reserved for the DNA ladder (O GeneRuler Ultra Low Range DNA Ladder Thermo Scientific, CA, USA). The gel was run in TAE running buffer for 30 minutes, at 60 V in an electrophoresis chamber (BioRad, CA, USA). Afterwards the gel was illuminated with UV light and photographed (BioRad, CA, USA). The size and zeta-potential of the polyplexes were studied by dynamic light scattering (DLS) and electrophoretic light scattering (ELS), respectively. In the hydrodynamic diameter measurements, polyplexes were prepared similarly as in the gel retardation assay and measured by Zetasizer APS (Malvern Instruments, UK), using a nominal 5 mv He-Ne laser operating at 633 nm wavelength. The refractive index of the polyplexes was and viscosity was cp at 25 C. For each sample, three separate measurements were conducted with 20 runs each. In the zeta-potential measurements, 8 µg of sirna were dissolved in 10 µl 10 mm Hepes (ph 7.2) and combined with 90 µl of the micellar block copolymer solution. Just before the measurement, Hepes buffer (700 µl) was added to the sample, and the zeta-potential was determined with Zetasizer Nano ZS (Malvern instruments, UK), adopting similar settings as in the size measurements. For each sample, five separate measurements were conducted each with approximately 20 runs Cytotoxicity The cytotoxicity of the polyplexes was determined by the MTT assay (Mosmann, 1983). One day prior to the experiment, ARPE-19 cells were seeded onto a 24-well plate at a density of cells/well in 500 µl of supplemented growth medium. On the experimental day, the cells were washed with PBS (1x, ph 7.2), 350 µl of Opti-MEM (Gibco, NY, USA) were added to the cells, followed by 100 µl of the formulations and controls (Table 4). After a 5 h incubation, the cells were washed once with PBS and then 400 µl of 5 mg/ml thiazolyl blue tetrazolium bromide (MTT, Sigma-Aldrich, MO, USA) were added to the cells. During the 4 h incubation, the mitochondria of living cells form formazan crystals, which are then solubilized by addition of 100 µl of 10% sodium dodecyl sulphate M hydrochloric acid. On the next day, the amount of formazan was quantified by measuring the absorbance at 570 nm (Varioskan Flash, Thermo

40 23 Scientific). The cell viability was calculated as a percentage of the non-treated cells. The reliability of the MTT assay had been confirmed earlier by testing the cytotoxicity of polyethyleneimine (positive control), polyvinyl alcohol (negative control) and PBE30-b-PK30 at a concentration range of mg/ml. Table 4. Formulations and controls used in the cytotoxicity, transfection, cell uptake, and intracellular distribution experiments. Formulations/ Controls Non-treated cells (Opti-MEM ) Cytotoxicity Negative control for cytotoxicity Silencing efficacy Negative control for IL-6 secretion Cell Uptake Cell specific adjustments Intracellular distribution Lipopolysaccharide (LPS) Positive control for IL-6 secretion Lipoplex with siil6 Negative control for cytotoxicity (50 nm siil6) Positive control for IL-6 silencing (50 nm siil6) Positive control for cell uptake (100 nm FAM-siIL6) Lipoplex with sins Negative control for IL-6 silencing (50 nm sins) Polyplex with siil6 Study formulation: 8/1 (N/P) (50 nm siil6) Study formulation: 8/1 (N/P) (50 nm siil6) Study formulation: 8/1 (N/P) (100 nm FAM-siIL6) Study formulation: 8/1 (N/P) (50 nm FAM-siIL6) Polyplex with sins Negative control for IL-6 silencing (50 nm sins) Free sirna (siil6 or FAM-siIL6) Negative control for cytotoxicity Negative control for IL-6 silencing Negative control for cell uptake Silencing efficacy The silencing efficacy study followed the same procedure as that applied in the cytotoxicity test, but after 5 h incubation with the different formulations and controls (Table 4), the cells were first washed with PBS and then 1 ml of supplemented growth medium containing 10 μg/ml lipopolysaccharide (LPS, Escherichia coli O55:B5 Sigma-Aldrich, MO, USA) was added. LPS was used to induce IL-6 secretion in the ARPE-19 cells (Leung et al., 2009). Samples of 500 µl were collected from the supernatant at 24 h, 48 h, and 72 h after the end of the incubation. The removed volume was replaced with fresh LPS containing medium. The amount of IL-6 was quantified from supernatant samples according to the instructions in the human IL-6 ELISA kit (Gen-Probe Diaclone SAS, France). The percentage of IL-6 secretion was determined by comparing the samples to the LPS treated cells, which was set to 100% IL-6 secretion Cell uptake ARPE-19 cells were seeded one day prior to the cell uptake study on 6-well plates at a density of cells/ well in 2 ml of growth medium. On the next day, the cells were washed once with PBS, followed by the addition of 1.75 ml of Opti-MEM and 500 µl of the different formulations or controls containing fluorescently labeled sirna (FAM-siIL6) (Table 4). After a 4 h incubation, the cells were washed 3 times with Hanks Balanced Salt Solution (HBSS) (Gibco, Invitrogen, NY, USA) supplemented with 10 mm Hepes (ph 7.4). Then, the cells were detached

41 24 with trypsin and re-suspended in the aforementioned buffer. Cells were centrifuged at 1200 rpm for 10 minutes, after which the supernatant was removed and cells in buffer were analyzed by flow cytometry (BD LSR II, NJ, BD Biosciences, USA). The collection of data and analysis of the results were controlled by FACSDiva software (BD Biosciences). For each sample, events were counted and the cells were visualized on a forward scatter versus a side scatter display Intracellular distribution ARPE-19 cells were seeded 48 hours prior to the experimental day on Petri dishes (36.2 mm, Nunclon Surface, Nunc, Denmark) at a density of cells/dish in 2 ml growth medium. On the experimental day, the cells were rinsed once with PBS. Then 1.75 ml of Opti-MEM and 500 µl of the polyplex solution (Table 4) was added to the dishes. The cells were exposed to the polyplexes for 4 hours, but 60 minutes and 30 minutes before the end of the incubation LysoTracker Red (working concentration: 50 nm) and Hoechst (working concentration: 5 µg/ml) were added to the cells, respectively. After the incubation, the cells were rinsed twice with HBSS buffer supplemented with 10 mm Hepes (ph 7.4). The Leica TCS SP5 Confocal Microscopy System (Leica, Germany) was employed for the observation of the living cell samples in the HBSS buffer. An upright microscope stand equipped with a 63x/0.90 W water-dipping objective was used for imaging. The following lasers were used as excitation sources for Hoechst, FAM-siIL6, and LysoTracker Red: UV laser (405 nm), Argon laser (488 nm) and DPSS laser (561 nm), respectively. Sequential scanning was performed because the emission spectra of FAM-siIL6 and Hoechst were too close to each other. The fluorescent probes were detected by three-dimensional detection, as stacks of varying thickness; and the image capturing was performed at 37 C. IMARIS software (Bitplane AG, Switzerland) was used in analyzing 3D multichannel images.

42 COMPUTATIONAL METHODS QSPR modeling Pub. I and II: QSPR models for predicting corneal or conjunctival permeability were generated by combining the drug permeability data of the cassette dose drugs with over 30 molecular descriptors. A more detailed description of model generation can be found in publications I and II Ocular pharmacokinetic simulations and calculations Pub. II: Data from the conjunctival and corneal permeability studies were used for simulating drug bioavailability in the aqueous humour and trans-conjunctival systemic bioavailability. Pub. III: Data from the drug release study (section 4.4) was used in predicting drug concentrations in vivo. The drug release data was used as the input in the model while drug elimination was simulated by using intravitreal clearance for each model drug in order to simulate intravitreal drug concentrations. Stella software was used (ISEE systems 10.0). Pub. IV: Two models for simulating the impact of polymer degradation rate and molecular weight dependent intravitreal clearance (CLIVT) on vitreous concentrations of polymer fragments were built by employing Stella (ISEE systems 10.0). In the first model, dissolution was followed by hydrolysis. In the second model, both dissolution and hydrolysis took place at the same time. The clearance values for the degraded fragments were associated with known intravitreal clearance values of drugs with varying molecular weights (del Amo et al., 2015). The route of elimination (anterior or anterior and posterior) was also considered and was dependent on the size of the degraded fragments. Unpub. V: The RPE clearance (CLRPE) was calculated by multiplying the outward RPEchoroid permeability (Papp, RPE-choroid) values (section 4.3) with the surface area of the rabbit RPE (5.2 cm 2 ) (Reichenbach et al., 1994), assuming that the RPE-choroid permeability is the same in both bovine and rabbit species. Then the CLRPE values were compared with experimental in vivo CLIVT values of the same drugs, reported in the literature (del Amo et al., 2015).

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44 27 5 Results 5.1 TOPICAL DRUG PERMABILITY AND QSPR MODELING Conjunctival and corneal permeability of various drugs were studied using isolated ex vivo ocular tissues and a cassette dose, for the generation of predictive QSPR models (Pub. I and II). The main results of the studies are presented in Tables 5 and 6. Table 5. Porcine conjunctival, corneal, and bovine RPE-choroid permeability range, and their transepithelial electrical resistance (TER). Ocular tissue Permeability (P app) a TER Pub. / Unpub. Conjunctiva P app, CJ: x 10-6 cm/s 86 ± 29 Ω x cm 2 (start) I 17-fold range (32 drugs) 477 ± 224 Ω x cm 2 (end) Cornea P app, CO: x 10-6 cm/s 52-fold range (25 drugs) 372 ± 54 Ω x cm 2 (start) 917 ± 362 Ω x cm 2 (end) II RPE-choroid P app, RPE-choroid: Outward: x 10-6 cm/s Inward: x 10-6 cm/s (7 drugs) 93 ± 53 Ω x cm 2 (start) 26 ± 20 Ω x cm 2 (end) VI a in parenthesis, the number of drugs in the cassette dose with reliable and calculated P app values Table 6. The linear partial least square (PLS) model for conjunctival and corneal permeability, including validation results. Polar surface area (PSA) and hydrogen bond donor (HBD). (Pub. I and II) Permeability models for Descriptors R 2 X a R 2 Y a Q 2 Y b Qi 2c Qe 2c Conjunctiva LogP app, CJ = (logPSA) (HBD) (Halogen ratio) PSA, HBD & Halogen ratio Cornea LogP app, CO = (logPSA) (HBD) (Halogen ratio) PSA, HBD & Halogen ratio a R 2 X and R 2 Y describe the goodness of fit of the training set b Q 2 Y describe the goodness of prediction of the model c Qi 2 (internal test set) and Qe 2 (external test set) is the regression coefficient of predicted versus experimental values

45 OCULAR PHARMACOKINETIC PREDICTIONS FOR TOPICALLY APPLIED DRUGS Conjunctival and corneal permeability data was used for predicting aqueous humour bioavailability (F) of the cassette dose drugs (Pub. II). Based on the calculation, aqueous humor bioavailability due to trans-corneal permeability was % (Fig. 7) and trans-conjunctival systemic bioavailability was 34-79%. Figure 7. Aqueous humour bioavailability (F) versus corneal permeability of 25 model drugs of the cassette dose (Pub. II). 5.3 INTRAVITREAL DRUG RELEASE AND PHARMACOKINETIC PREDICTIONS The cumulative release profiles of the small molecular drugs and proteins from one of the microsphere formulations (1% alginate, BaCl2 cross-linked and uncoated) can be seen in Figure 8. The rest of the release profiles with the other formulations can be found in the supplementary material of the publication (Pub. III). Simulated drug concentrations in the vitreous were affected by drug release and vitreal clearance. Figure 9 suggests that drug release from the alginate microspheres can lead to prolonged drug concentrations in the vitreous (Fig. 9a) as compared to simple intravitreal injections of drug solutions (Fig. 9b). For example, intravitreally injected human immunoglobulin G (higg), due to its large size and slow vitreal elimination show higher and more sustained vitreal concentration than the other compounds after simulated injection of the solution (Fig. 9b). Additionally, the slow vitreal elimination of higg combined with its release profile from the microspheres contributes to an even more sustained concentration profile in the vitreous (Fig. 9a).

46 29 Figure 8. The release profile of the small molecular drugs and proteins from one of the microsphere formulations (1% alginate, BaCl 2 cross-linked and uncoated). Each release curve is labeled with the model drug name, charge at ph 7.4, and in parenthesis the hydrodynamic diameter. (Pub. III) Figure 9. Simulated drug quantity in the vitreous after release from alginate microspheres (different formulations) (a) and after injected as a solution to the vitreous (b). (Pub. III) 5.4 DEGRADATION RATE OF POLYMER CARRIERS The degradation of polymer carriers was simulated using hypothetical values for dissolution, hydrolysis and clearance. In the first model, polymer degradation occurred by dissolution followed by hydrolysis. The vitreous concentrations of the polymer-borne species were dependent on both dissolution and hydrolysis. Prolonged exposure of polymer degradation fragments at low concentrations was obtained with a slower dissolution rate (200 days) (Fig. 10). Additionally, hydrolysis lowered the concentrations compared to no hydrolysis, especially when the posterior elimination route was present. In the second model, dissolution and hydrolysis took place at the same time. Fast hydrolysis eliminated the polymer-borne fragments more rapidly, independently of the dissolution rate. On the other hand, in cases where polymer degradation only depended on dissolution (200 days), the exposure time to the polymer fragments increased drastically (Fig. 11).

47 30 Figure 10. The simulated concentrations of dissolved polymer and its fragments in the vitreous over time, using model 1. Three durations of polymer dissolution were simulated (2, 20 and 200 days). The figures above represent the duration of dissolution of 2 days (upper figures) and 200 days (lower figures) (20 days in Pub. IV). The polymer hydrolysis were 20 days (red lines), 2 days (green lines), and 5 hours (purple lines), and without hydrolysis (blue line). The clearance values of the anterior elimination route were: 0.01, 0.02, 0.03 and 0.04 ml/h (figures on left) and for the anterior and posterior route were: 0.01, 0.04, and ml/h (figures on right). (Pub. IV) Figure 11. The simulated concentrations of dissolved polymer and its fragments in the vitreous over time, using model 2. Three durations of polymer dissolution were simulated (2, 20 and 200 days). The figures above represent the duration of dissolution of 2 days (upper figures) and 200 days (lower figures) (20 days in Pub. IV). The polymer hydrolysis were 20 days (red lines), 2 days (green lines), and 5 hours (purple lines), and without hydrolysis (blue line). The clearance values of the anterior elimination route were: 0.01, 0.02, 0.03 and 0.04 ml/h (figures on left) and for the anterior and posterior route were: 0.01, 0.04, and ml/h (figures on right). (Pub. IV)

48 RPE PERMEABILITY AND CLEARANCE Isolated bovine RPE-choroid was used for studying the bidirectional permeability of eight small molecular weight drugs. Most of the drugs had similar permeabilities in both directions, which indicates that passive permeability dominated over active transport (Fig. 12). Only ciprofloxacin and ketorolac displayed significant directional differences in the permeability values: over 6.7 and 14.5 times higher outward permeability than inward permeability, respectively. Outward permeability values were used for the calculation of RPE clearance (CLRPE) values, which were further compared to the intravitreal clearance (CLIVT) found in the literature (del Amo et al., 2015) (Fig. 13). CLRPE could explain the CLIVT of all the drugs, with the exception of ketorolac. The results of quinidine are not presented due to significantly lower drug flux compare to the rest of the small molecular drugs, and the mass balance was incomplete. Figure 12. Outward versus inward RPE-choroid permeability. The dashed line represents a 1:1 slope and the dotted line represents a 3-fold deviation range. The number of RPE-choroid tissue samples tested/drug=8-9. Figure 13. Intravitreal clearance (CL IVT) (del Amo et al., 2015) versus RPE clearance (CL RPE). The dashed line represents a 1:1 slope and the dotted line represents a 3-fold deviation range.

49 SIRNA DELIVERY TO THE RPE The micellar block copolymer PBE30-b-PK30 started to bind sirna at a charge ratio (N/P) of 4/1, and complete binding was observed at a charge ratio of 8/1 (Fig. 14 A). These polyplexes were less than 50 nm in size, displaying a positive surface charge (+31 mv) (Fig. 14 C). Experiments were conducted with polyplexes with various charge ratios (N/P), but mainly the results of charge ratio 8/1 are presented here. In the cytotoxicity assay, the polyplexes and the control formulations did not evoke cell death, as over 80% of the ARPE-19 cells were viable (Fig. 14 B). The polyplexes silenced approximately 20% of IL-6 secretion, while the lipoplexes (50 nm siil6) resulted in 60% silencing (Fig. 14 E). In the cell uptake studies, the polyplexes of different charge ratios were efficiently taken up by the ARPE-19 cells, as over 70-90% of the cells were positive for fluorescence (Fig. 14 D). This was confirmed by the intracellular distribution study using confocal imaging (Fig. 14 F). In Figure 14 F, the polyplexes seem to be intracellularly delivered (green), but it is difficult to determine whether the complexes are present in the cytoplasm or trapped inside the lysosomes (red).

50 33 Figure 14. sirna complexation and cellular delivery. A: Complexation was visualized with gel electrophoresis, first band with free sirna followed by polyplex formulations with increasing charge ratios (N/P). B: Cytotoxicity: viable ARPE-19 cells relative to control after 5 h incubation (n: 3 wells treated at three different days/sample). C: Size and zeta-potential of polyplex formulations with increasing charge ratio (N/P). The polyplex formulations were polydispersed (index not shown), which is also observed as two size peaks for N/P 8/1. D: Cell uptake of lipoplex and polyplex of increasing charge ratios, after 4 h of incubation (n: 3 wells treated at one day/sample). E: Silencing efficacy: percentage IL-6 secretion of ARPE-19 cells relative to only LPS treated cells 48 h after 5 h incubation of the different formulations (n: 3 wells treated at five different days /sample). si-il6: sirna for silencing IL-6 secretion; si-ns: non-specific sirna. F: Intracellular delivery of the polyplex (N/P: 8/1) in green after 4 h of incubation (n: 1 well); nuclei and lysosomes are colored in blue and red, respectively.

51 34

52 35 6 Discussion and Future Directions Drug research and development generate a substantial amount of in vitro, and in silico data of new and old drugs. The interpretation of these data in the context of in vivo studies and clinical application is important in drug development. The use of predictive models would be beneficial for improving the success rate in drug and formulation development. This thesis work focused on the development of in vitro, ex vivo, and in silico tools for the improvement of ocular drug development. 6.1 BOTTOM-UP APPROACHES FOR TOPICAL DRUG DELIVERY Topical instillation of eye drops is the most widely used mode of administration in ophthalmic medicinal treatment. Although topical drugs and drug delivery systems have been studied extensively preclinically, their further development to effective products could be improved with predictive and quantitative modeling tools. The ocular permeability of topical drugs was studied ex vivo with porcine cornea and conjunctiva. A cassette mix of clinically used drugs was used to construct QSPR models for corneal and conjunctival permeability. Furthermore, pharmacokinetic calculations were performed for predicting drug bioavailability in the aqueous humour and systemic bloodstream. The first conjunctival QSPR model was generated successfully. The most essential descriptors for drug permeability were polar surface area (PSA), hydrogen bond donor (HBD) and the halogen ratio. In addition, a reliable QSPR model for corneal permeability was generated with the same descriptors. Previously, PSA has been described as an important permeability predictor in the intestinal wall (Linnankoski et al., 2006; Winiwarter et al., 1998), blood-brain barrier (Norinder et al., 1998), and cornea (Kidron et al., 2010). The QSPR models were successfully built based on the physicochemical parameters that are related to passive diffusion across lipoidal cell membranes. The outcomes of these models, without outlier compounds, suggests that permeation of the compounds in the cornea and conjunctiva is primarily determined by passive diffusion, not active transport. This conclusion is in line with earlier experimental results that were recently reviewed (Vellonen et al., 2018). Recent simulations conducted by Vellonen et al., (2018) suggest that the role of transporters is low also in the in vivo corneal drug absorption. These results do not rule out the possibility that active transport may be important for some individual hydrophilic drugs that have poor passive permeability. Previously, most corneal and conjunctival permeability experiments have been conducted with ex vivo rabbit tissues (Ahmed et al., 1987; Huang et al., 1989; Hämäläinen et al., 1997; Kidron et al., 2010; Prausnitz and Noonan, 1998; Wang et al., 1991). The results from our experiments show that the permeability in the rabbit cornea and conjunctiva is higher than in the corresponding porcine tissues. This is in line with the conclusion of Loch et al., (2012). These differences can probably be explained by anatomical differences. Due to the limited permeability data in humans, it is not possible to have a widely applicable translation to the clinical situation. In this study, on average, the corneal drug permeability was 9 times slower than the conjunctival permeability. Additionally, the permeability range was broader in the cornea (50 fold) than in the conjunctiva (17 fold). This emphasizes the tight barrier properties of the cornea,

53 36 the main barrier to ocular drug absorption (Ahmed and Patton, 1987, 1985). Increasing corneal permeability, however, does not increase the ocular bioavailability to the same extent. For the compounds in the cassette mix, the calculated bioavailability in the aqueous humour was less than 4.4%, while trans-conjunctival systemic bioavailability was over 34%. An extensive loss of drug to the systemic circulation due to trans-conjunctival permeability sets limits on ocular bioavailability. Frequent instillation of eye drops, typically once or several times daily, is needed in the treatment of ocular diseases. This is due to the tissue barriers (cornea and conjunctiva) and rapid drug loss from the ocular surface. In order to achieve a longer drug action, controlled release formulations with prolonged retention on the ocular surface are needed. The conjunctival route may offer a potential pathway for drug delivery to the back of the eye, especially for highly potent compounds, but the retinal bioavailability is only about 0.1% (Ranta et al., 2010). In such a case, some kind of drug delivery system would be required to prolong the duration of drug action. The combination of predicting topical drug permeability with the QSPR models and the follow-up estimation of ocular bioavailability is a relatively simple approach for producing estimates for ocular drug absorption. These estimations can be obtained even before the compound has been synthesized, because the QSPR models are based on the molecular descriptors that can be generated from the chemical structure in silico. Additionally, the permeability information can be incorporated into ocular PBPK or finite element models. In silico predictions can refine and reduce animal experimentation and furthermore, reduce the required time and cost of drug development. 6.2 INTRAVITREAL DRUG RELEASE AND CARRIER DEGRADATION Monthly intravitreal injections are required in the treatment of neovascular AMD that affects the posterior segment of the eye. The injections, which need to be administered by healthcare professionals, are a burden to both the health care system and patients, and occasionally cause adverse effects in the patients (del Amo and Urtti, 2008; Gordois et al., 2012). Drug delivery systems with sustained and controlled delivery are needed not only for biologicals, but also for the small molecular drugs that have short half-lives in the vitreous (del Amo et al., 2015). The rate of drug release is a key parameter in the development of a formulation. Usually, these experiments are conducted with one drug and one formulation at a time using dialysis bags or other approaches to maintain sink conditions i.e. in vivo screening of the release kinetics is both tedious and expensive. Therefore, we developed a tool to support the development of intraocular drug carriers: a high-throughput release method was combined with pharmacokinetic predictions. The miniaturized 96-well set up allowed us to test 12 different polymeric microsphere formulations at the same time, each one with eight different model drugs (two small molecules and six proteins). The extensive release data was further processed with pharmacokinetic simulations to produce predictions of drug concentrations in the vitreous. The method made it possible to compare the different formulations, thereby guiding the development work. The release tests and follow-up kinetic simulations facilitate the choice of which formulations should proceed to in vivo tests. The alginate microsphere formulations and drug loading could be improved, but for proving the concept they worked well. Thus, this approach may facilitate drug development and reduce the required time and costs. Similar tools could be developed also for other types of formulations at a time when automated pipetting robots are becoming more affordable and common in laboratories. It is important to note that the drug release rate depends also on the release medium. Unfortunately, in vitro drug release in the vitreous and buffer solutions have not been compared. The next step

54 37 in the development of this high-throughput method would be exploitation of vitreous humour as the release medium. Drug release from the controlled release systems is governed by the carrier materials, typically polymers (Herrero-Vanrell and Refojo, 2001). Usually, drug release is controlled by drug diffusion in the polymer, but also material degradation may play a role if the polymer is biodegradable. Often the materials exploited in drug delivery are new and the degradation rate and the appearance of polymer-borne fragments may be unknown. Therefore, we simulated the impact of polymer dissolution, hydrolysis, and clearance on the ocular exposure to the polymer fragments. Depending on the polymer properties, the rates of dissolution and hydrolysis could possibly affect the resulting concentrations, and exposure times of the polymer-borne species in the vitreous. The molecular sizes of the fragments were expected to have an impact on the clearance, because a small molecular size increases the elimination rate from the vitreous. In our simulations, from a toxicity point of view, it was observed that a slow polymer dissolution (20 to 200 days) in the vitreous followed by high hydrolysis (5 h or 2 days) of the dissolved polymer resulted in the lowest possible ocular concentrations of polymer-borne species. Furthermore, if dissolution and hydrolysis occurred at the same time, rapid hydrolysis decreased drastically the area under the concentration-time curve (AUC) of the polymer-borne species. Low exposure would be expected to minimize the ocular toxicity of the polymer and its fragments. The simulation tool for polymer degradation and exposed concentrations of dissolved compounds can be coupled with in vitro assays on cellular toxicity to guide the development of drug delivery carriers. 6.3 THE ROLE OF RPE PERMEABILITY IN INTRAVITREAL CLEARANCE Diseases at the back of the eye are most commonly treated with intravitreal injections of small molecular drugs (suspensions, implants) or protein drugs (solutions) (del Amo and Urtti, 2008; Zhang et al., 2012). Depending on the drug properties, intravitreal drug elimination may take place across the blood-ocular barriers (BAB and BRB) or via the flow of aqueous humour in the anterior chamber (del Amo et al., 2015). The half-life of drugs in the vitreous is governed by the clearance; this displays about a 100 fold range of values, whereas the range of values of the volume of distribution is very narrow (only about 2 fold) (del Amo et a., 2015). Small molecular drugs are cleared from the vitreous through various parts of the blood-ocular barriers in the ciliary body and retina, as well as via the aqueous humour flow. We evaluated the role of RPE permeability in the intravitreal clearance of several small molecular weight drugs. Bidirectional ex vivo permeability studies were conducted with bovine RPE-choroid specimens. The choroid is a leaky tissue with TER values of 9 x cm 2 ; therefore, it is not considered to represent a significant permeation barrier (Table 1). The outward and inward permeability values were similar for most compounds and comparable with previous results utilizing ex vivo bovine RPE-choroid (Pitkänen et al., 2005). The RPE clearance (CLRPE ) values were calculated and compared to the intravitreal clearance (CLIVT) values of the same drugs in the rabbit (del Amo et al., 2015). Thus, most of the drug elimination from the rabbit vitreous seemed to occur through the RPE, instead of the anterior chamber or other parts of the bloodocular barriers (ciliary epithelia and endothelia, retinal capillaries). It should be noted that there are fewer retinal capillaries in the rabbit eye as compared to the human eye. Ciprofloxacin had a 6.7 times greater outward than inward permeability suggesting involvement of active transport. CLIVT of ciprofloxacin was 1.9 times higher than the CLRPE. Similarly, a higher CLIVT than CLRPE was obtained for fluconazole. These data may be explained by the possible presence of additional elimination routes in the rabbit in vivo, for example permeability across the barriers in the ciliary body. The low inward permeability for

55 38 ciprofloxacin compared to the rest of the small molecular weight drugs could be attributable to its affinity for efflux transporters at the basolateral side of the RPE (Mannermaa et al., 2006; Vellonen et al., 2018). Similarly, ketorolac had a 14.5 times higher value of outward permeability than inward permeability. The CLIVT was 5 times smaller than the CLRPE. These results indicate that active transport might be involved in the high outward permeability compared to the rest of the small molecular weight drugs. There may also be species differences in the transporters, because the ketorolac CLIVT in the rabbit is smaller than the CLRPE. The permeability results of quinidine were not included, as they varied significantly from the other drugs. The mass balance of quinidine was incomplete, suggesting accumulation into cells. Quinidine is known to bind avidly to melanin (Pelkonen et al., 2017; Rimpelä et al., 2018a), as well as having affinity for P-gp (Duvvuri et al., 2003; Vellonen et al., 2018). The RPE-choroid contains more than 60% of the total ocular melanin and melanin binding has been associated with prolonged drug retention in melanosomes (Rimpelä et al., 2018a). Melanin binding should not affect the permeability coefficient, but it may prolong the lag-time before achieving steadystate permeation. Due to these results in this study, reliable CLRPE value could not be calculated for quinidine. However, based on the in vivo CLIVT of quinidine (0.448 ml/h), its RPE permeability should be similar to the rest of the drugs (del Amo et al., 2015; Duvvuri et al., 2003). The difference between our ex vivo and the in vivo results may be explained by the experimental set up and the used animal models. First of all, our permeability experiments where conducted using isolated bovine RPE-choroid where melanin was present, compared to the in vivo study that was conducted using albino rabbits lacking melanin. Second, the RPE clearance was calculated based on the outward permeability, where the drug first crosses the pigmented tissue and is then measured from the choroidal side, compared to the in vivo study where the intravitreal clearance was determined by a microdialysis technique, which only measures drug concentration from the vitreous, not from the systemic blood circulation after crossing the RPE. In drug development, melanin binding and transporter affinity are important factors to be considered. Their impact depends on the drug concentrations, transporter affinities, as well as the expression levels of the transporters. Drugs with low passive permeability are more likely to be affected by active transporters in the RPE, and this can impact on drug elimination from the vitreous (Vellonen et al., 2018). However, the role of active transporters on drug distribution at the tissue level (vitreous, retina) is probably small, but they may have a greater effect on the drug exposure at the cellular level, for example in the RPE (Vellonen et al., 2018). This is the first time that the role of RPE permeability in CLIVT has been determined experimentally. It was found that in the drug development phase of small molecular weight drugs, the intravitreal pharmacokinetics, especially the CLIVT, could be predicted by estimating a drug s RPE permeability. 6.4 SIRNA DELIVERY TO THE RPE Retinal disorders, such as age-related macular degeneration, have been associated with inflammation (Rodrigues, 2007). Elevated secretion of cytokines and chemokines by the RPE has been reported to disrupt the retinal barrier and contribute to immunological and inflammatory responses (Holtkamp et al., 2001; Leung et al., 2009). IL-6 is a pro-inflammatory cytokine that is secreted by the RPE; it plays a critical role in the immune processes (Holtkamp et al., 2001). The silencing of the production of IL-6 with sirna could be a potential therapeutic antiinflammatory approach. The micellar block copolymer of poly (benzyl-glutamate) and poly-l-lysine was evaluated as a potential carrier for IL-6 silencing sirna. Small polyplexes with diameters below 50 nm were obtained. The small nanoparticle size is beneficial in the retinal drug delivery after intravitreal

56 39 or subretinal delivery. After intravitreal delivery, the particles must diffuse in the vitreous and permeate across the retinal layers, particularly across the inner limiting membrane at the vitreous-retinal surface. Particles of less than 500 nm have been observed to diffuse freely in the vitreous (Peeters et al., 2005; Xu et al., 2013), but the ILM seems to restrict the access of at least positively charged nanoparticles to the retina (Koo et al., 2012). The positive surface charge of these polyplexes (+ 31 mv) is optimal for cell uptake, but may hinder diffusion in the vitreous and across the ILM, both presenting negatively charged properties (Pitkänen et al., 2004, 2003). This could possibly be overcome either by further modification of the formulation to a more neutral form or the sirna formulation could be injected in the subretinal space between the photoreceptors and the RPE. The polyplexes evoked no cytotoxicity and were taken up sufficiently by the RPE cells, as was expected based on their small size and positive surface charge. In addition, the RPE cells have a strong phagocytosing capacity. However, the silencing effect was minor with the polyplexes, even though the lipoplex caused a silencing effect. Although the nanoparticles were taken into the cells, their silencing efficacy may have been blocked if they are trapped within the lysosomes (Wittrup and Lieberman, 2015). From the confocal images, it is difficult to draw any conclusions about whether the polyplexes had been trapped inside the endosomes and lysosomes or if they were present in the cytosol. It has been reported that Lipofectamine bound sirna is released from the endosomes by membrane fusion in which endosomal lipids displace the sirna from Lipofectamine (Xu and Szoka, 1996). Polyethylene imine (PEI) and poly-l-lysine (PLL) have been studied as carriers for nucleic acids (Van Gaal et al., 2011). PEI escapes from the endosomes by a proton sponge effect (Behr, 1997), but PLL lacks ph buffering capacity, which may be the reason for its poor transfection efficacy (Ruponen et al., 1999). The micellar block copolymer contains poly-l-lysine, and it is likely that the polyplex is trapped in the endosomes or alternatively the sirna is not released from the carrier. The block copolymers showed good properties in terms of cell uptake, but further modifications of the polymer will be needed to promote sirna release and escape from the endosomes. Such developments might result in effective IL-6 silencing in the retina. The eye is an optimal target for the delivery of nucleic acids, compared to other organs in the human body that needs to be targeted through the systemic blood circulation. The eye has well defined compartments in an immune privileged environment (Guzman-Aranguez et al., 2013). This enables good local delivery with potentially higher bioavailability and reduced adverse effects. In addition, the RPE cells at the back of the eye undergo minimal or no proliferation throughout normal life. The duration of the gene silencing effect of sirna has been observed to depend on cell division and not on the intracellular half-life of the sirna (Bartlett and Davis, 2006). The sirna silencing effect diminishes faster in rapidly dividing cells than in nonproliferating cells. This could mean that the silencing effect of the RPE cells could be maintained for a longer time and repeated injections would be avoided.

57 40 7 Conclusions In conclusion, new experimental and computational tools were developed to be exploited in ocular drug development. The specific conclusions are presented below and in Figure A predictive QSPR model for conjunctival permeability was generated with good accuracy based on ex vivo permeability experiments. The most relevant molecular descriptors for predicting the conjunctival permeability were polar surface area, hydrogen bond donor, and halogen ratio. 2. A predictive QSPR model was generated for corneal permeability using ex vivo permeation data. Polar surface area, hydrogen bond donor, and halogen ratio were adequate descriptors for predicting corneal permeability. 3. On average, the conjunctival permeability was about 9-fold higher than the corneal permeability. 4. Pharmacokinetic calculations resulted in the production of generalized estimates for the ocular drug bioavailability, demonstrating the limiting role of systemic conjunctival drug absorption on ocular bioavailability. 5. In vitro high-throughput method of drug release and follow-up kinetic simulations for intravitreal drug concentration predictions were developed. 6. Simulations based on polymer degradation and vitreal clearance revealed the role of polymer degradation rate in reducing the ocular material exposure. 7. Clearance across the RPE has a central role in the intravitreal clearance of small molecular weight drugs. The RPE permeability may be used for the estimation of vitreal drug clearance. 8. The use of IL-6 silencing sirna for the treatment of inflammation in the RPE may be a potential therapy when coupled to a proper delivery system, but the block copolymer based polyplex in this study did not result in effective sirna mediated gene silencing.

58 Figure 15. The specific conclusions presented with numbers referring to the text, and in colors. 41