Melanin Binding and Drug Transporters in the Retinal Pigment Epithelium: Insights to Retinal Drug Delivery

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DISSERTATIONS | LAURA HELLINEN | MELANIN BINDING AND DRUG TRANSPORTERS IN THE RETINAL... | No 428

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THE UNIVERSITY OF EASTERN FINLAND Dissertations in Health Sciences

ISBN 978-952-61-2566-4 ISSN 1798-5706

Dissertations in Health Sciences

THE UNIVERSITY OF EASTERN FINLAND

LAURA HELLINEN

MELANIN BINDING AND DRUG TRANSPORTERS IN THE RETINAL PIGMENT EPITHELIUM: INSIGHTS

INTO RETINAL DRUG DELIVERY

Retinal pigment epithelium (RPE) serves as a barrier in the posterior eye affecting the ocular pharmacokinetics (PK). RPE surface transporters and melanin inside the cells are

expected to affect the drug distribution. In this thesis, 33 drugs were categorized by the

extend of melanin binding and RPE surface transporters were quantitated. Computer- aided simulations showed that transporters

may restrict the drug access to its target inside the RPE clarifying the role of RPE

transporters on the PK.

LAURA HELLINEN

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LAURA HELLINEN

Melanin Binding and Drug Transporters in the Retinal Pigment Epithelium: Insights

into Retinal Drug Delivery

To be presented by permission of the Faculty of Health Sciences, University of Eastern Finland for public examination in lecture hall MS302, Medistudia building, Kuopio, on Saturday, August 19^th

2017, at 12 noon.

Publications of the University of Eastern Finland Dissertations in Health Sciences

Number 428

School of Pharmacy Faculty of Health Sciences, University of Eastern Finland

Kuopio 2017

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Grano Oy Kuopio, 2017

Series Editors:

Professor Tomi Laitinen, M.D., Ph.D.

Institute of Clinical Medicine, Clinical Physiology and Nuclear Medicine Faculty of Health Sciences

Professor Hannele Turunen, 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-70211 Kuopio, Finland http://www.uef.fi/kirjasto

ISBN (print): 978-952-61-2566-4 ISBN (pdf): 978-952-61-2567-1

ISSN (print): 1798-5706 ISSN (pdf): 1798-5714

ISSN-L: 1798-5706

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Author’s address: School of Pharmacy Faculty of Health Sciences University of Eastern Finland KUOPIO

FINLAND

Supervisors: Professor Arto Urtti, Ph.D.

School of Pharmacy

University of Eastern Finland KUOPIO

FINLAND

Mika Reinisalo, Ph.D.

Department of Ophthalmology Institute of Clinical Medicine University of Eastern Finland KUOPIO

FINLAND

Heidi Kidron, Ph.D.

Centre for Drug Research

Division of Pharmaceutical Biosciences Faculty of Pharmacy

University of Helsinki HELSINKI

FINLAND

Reviewers: Professor Bente Steffansen, Ph.D.

Department of Physics, Chemistry and Pharmacy University of Southern Denmark

ODENSE DENMARK

Associate Professor Birger Brodin, Ph.D.

Department of Pharmacy University of Copenhagen COPENHAGEN

DENMARK

Opponent: Sibylle Neuhoff, Ph.D.

Senior Principal Scientist & Deputy Head of Translational Science Simcyp

SHEFFIELD

UNITED KINGDOM

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Hellinen, Laura

Melanin Binding and Drug Transporters in the Retinal Pigment Epithelium: Insights into Retinal Drug Delivery University of Eastern Finland, Faculty of Health Sciences

Publications of the University of Eastern Finland. Dissertations in Health Sciences 428. 2017. 51 p.

ISBN (print): 978-952-61-2566-4 ISBN (pdf): 978-952-61-2567-1 ISSN (print): 1798-5706 ISSN (pdf): 1798-5714 ISSN-L: 1798-5706

ABSTRACT

Diseases affecting the posterior of the eye (retina, choroid) are difficult to treat since there are protective ocular barriers. As the population ages, the number of patients with sight threatening diseases is expected to grow rapidly. Currently, the treatment of retinal diseases involves frequent intravitreal injections, but many patients suffer from diseases for which even today there is no effective treatment. The retinal pigment epithelium (RPE) is a single cell layer in the posterior of the eye. The RPE protects the eye from xenobiotics and therefore it affects drug entry into the ocular tissues. It also represents an important drug target since its functions are compromised in some retinal diseases, such as age-related macular degeneration.

One aim of this study was to quantify the drug transporting proteins on the RPE surface. These transporters might significantly influence ocular pharmacokinetics (PK), but the published literature is based on qualitative data, complicating the prediction of their functionality. In addition, the localization of the transporters in the RPE is mostly unknown. Thus, another aim was to quantify the transporters separately from the apical and basolateral surfaces of the RPE. The localization is important when predicting PK parameters since a drug can enter the RPE from either side, depending on the administration route. The clinical significance of the efflux transporters was evaluated with simulation models.

RPE is heavily pigmented, i.e. it contains melanin polymer packaged inside intracellular organelles, melanosomes. Melanin binds many drugs, thereby affecting their ocular PK. Since melanin binding can prolong a drug’s action, which would be desirable for many retinal drugs, melanin targeting is an interesting targeting approach. However, many aspects of melanin binding are far from clear. One aim of this study was to develop an isolation method for RPE melanosomes so that they could be used in in vitro pigment binding assays. An additional aim was to assay melanin binding with a small compound library to compare melanin binding under similar study conditions.

These experiments revealed that multidrug-resistance associated proteins (MRPs) are most likely involved in drug efflux in the RPE. Most of the studied drug transporters remained below the detection limit, indicating that passive permeation may be the most prominent permeation mechanism by which many drugs cross the RPE. The majority of the detected transporters were expressed on both surfaces of the RPE. The simulations indicated that efflux proteins hindered the intracellular drug accumulation in the RPE regardless of the localization of the efflux proteins. This finding highlights the importance of RPE efflux proteins for modifying a drug’s PK properties. Therefore, substrates of efflux proteins should be avoided when the RPE is targeted in drug discovery programs. We devised an isolation method that isolated intact and functional melanosomes for further in vitro studies. We also classified 33 compounds based on their melanin binding into low, intermediate and high binders. It was demonstrated that melanin binding and plasma protein binding do not correlate. These two parameters can be used as selection criteria in drug discovery programs aiming to target the pigmented ocular tissues from the systemic circulation. This study provided insights into the role of transporters and melanin binding for retinal drug delivery.

National Library of Medicine Classification:QS 532.5.E7, QU 55.7, QU 110, QU 300, QU 350, QV 38, QV 745, QV 785, WW 140, WW 270

Medical Subject Headings:Eye Diseases; Retina; Retinal Diseases; Cells; Epithelial Cells; Cells, Cultured; Organelles; Retinal Pigments; Melanins; Cell Membrane; Membrane Transport Proteins; Pharmacokinetics; Drug Design; Drug Delivery Systems

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Hellinen, Laura

Melaniinisitoutumisen ja kuljetinproteiinien rooli verkkokalvon pigmenttiepiteelissä: näkökulmia lääkeaineiden kohdentamiseen verkkokalvolle

Itä-Suomen yliopisto, terveystieteiden tiedekunta

Publications of the University of Eastern Finland. Dissertations in Health Sciences 428. 2017. 51 s.

ISBN (nid.): 978-952-61-2566-4 ISBN (pdf): 978-952-61-2567-1 ISSN (nid.): 1798-5706 ISSN (pdf): 1798-5714 ISSN-L: 1798-5706

TIIVISTELMÄ

Silmän takaosan (verkkokalvo, suonikalvo) lääkitseminen on haasteellista silmää suojaavien anatomisten esteiden vuoksi. Näkökykyä uhkaavien sairauksien oletetaan lisääntyvän nopeasti väestön ikääntyessä.

Verkkokalvon sairauksien hoidossa käytetään usein annosteltavia lasiaisinjektioita, mutta suuri osa potilaista kärsii sairausmuodoista, joihin ei nykyisin ole ollenkaan tehokasta hoitoa. Verkkokalvon pigmenttiepiteeli (RPE) on silmän takaosassa sijaitseva yksisolukerros. Se suojaa silmää vierasaineilta vaikuttaen myös lääkeaineiden kulkeutumiseen systeemisestä verenkierrosta silmään. RPE on myös tärkeä lääkehoidon kohde, sillä sen toiminnot häiriintyvät verkkokalvon sairauksissa, kuten ikääntymiseen liittyvässä verkkokalvorappeumassa.

Tässä tutkimuksessa oli tavoitteena määrittää RPE:n pinnalla sijaitsevien kuljetinproteiinien ilmentymistä proteiinitasolla käyttäen kvantitatiivista massaspektrometriaan perustuvaa mentelmää. Nämä lääkeaineita kuljettavat proteiinit saattavat vaikuttaa olennaisesti silmän farmakokinetiikkaan, mutta koska aiemmat tutkimukset ovat olleet laadullisia, on kuljetinproteiinien toiminnan arvioiminen haasteellista. Lisäksi kuljetinproteiinien sijainti RPE:n pinnalla haluttiin määrittää. Lääkeaine voidaan annostella solukerroksen kummaltakin puolelta, joten kuljetinproteiinien sijainti (solujen apikaali- tai basolateraalimembraani) on tärkeä tietää, jotta farmakokinetiikkaa voidaan ennustaa. Kuljetinproteiinien kliinistä merkitystä arvioitiin simulaatiomallien avulla.

RPE on voimakkaasti pigmentoitunut: RPE-soluissa on runsaasti melaniinia, joka sijaitsee solujen sisällä melanosomeissa. Melaniini sitoo monia lääkeaineita, ja siten vaikuttaa lääkeaineiden kinetiikkaan silmässä.

Melaniinisitoutuminen voi aiheuttaa pidentyneen lääkevasteen, mikä on hyvä ominaisuus verkkokalvon lääkinnän kannalta. Siksi melaniinisitoutumisen hyödyntäminen verkkokalvolääkinnässä on mielenkiintoinen kohdentamisvaihtoehto. Melaniinisitoutumisen yksityiskohtia ei kuitenkaan kunnolla tunneta. Tässä tutkimuksessa oli tavoitteena kehittää menetelmä, jolla voidaan eristää RPE:sta melanosomeja pigmenttisitoutumistutkimuksia varten. Lisäksi tavoitteena oli tutkia melaniinisitoutumista pienellä molekyylikirjastolla, jotta eri lääkeaineiden melaniinisitoutumista voitaisiin paremmin vertailla.

Väitöskirjatyössä havaittiin, että MRP-proteiinit poistavat lääkeaineita RPE-solujen sisältä todennäköisimmin. Suurin osa tutkituista kuljetinproteiineista jäi analyyseissa alle detektiorajan, mikä tarkoittaa, että monen lääkeaineen kulkeutuminen RPE-solukerroksen läpi tapahtuu todennäköisimmin passiivisella kuljetuksella. Suurin osa havaituista kuljetinproteiineista löydettiin RPE:n molemmilta puolilta.

Simulaatiot osoittivat, että lääkeaineita solun sisältä poistavat kuljetinproteiinit alentavat RPE:n solun sisäistä lääkeainealtistusta merkittävästi: tästä syystä kuljetinproteiinien substraatteja tulisi välttää lääkekehityksessä, mikäli RPE on kohdekudos. Väitöskirjatutkimuksessa kehitettiin menetelmä, jolla voidaan eristää RPE:sta melanosomeja. Lisäksi 33 lääkeainetta luokiteltiin melaniinisitoutumisen suhteen heikosti, keskimääräisesti tai voimakkaasti sitoutuviksi. Tutkimuksessa osoitettiin, että melaniinisitoutuminen ja plasman proteiineihin sitoutuminen eivät ole yhteydessä toisiinsa. Näitä parametreja voidaan hyödyntää valintakriteereinä kehitettäessä kohdentamissysteemejä silmän pigmentoituneisiin kudoksiin systeemisen annostelun avulla.

Väitöskirjatutkimus loi uusia näkökulmia melaniinisitoutumisen ja kuljetinproteiinien vaikutuksesta lääkeaineiden kulkeutumiseen verkkokalvolle.

Luokitus: QS 532.5.E7, QU 55.7, QU 110, QU 300, QU 350, QV 38, QV 745, QV 785, WW 140, WW 270

Yleinen suomalainen asiasanasto: silmätaudit; verkkokalvo; verkkokalvorappeuma; solut; epiteelisolut; soluviljely;

soluelimet; pigmentti; solukalvot; farmakokinetiikka; lääkkeet; annostelu; lääkesuunnittelu

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Acknowledgements

The present study was carried out in the School of Pharmacy, University of Eastern Finland during 2013-2017. The study was conducted as a part of the Doctoral Programme in Drug Research and funded by the Doctoral School of the University of Eastern Finland and Academy of Finland.

Firstly, I wish to express my deepest gratitude to the main supervisor of my thesis, Professor Arto Urtti. It has been a privilege to work in your research group; your enthusiasm towards science is amazing and the group has an encouraging and warm atmosphere.

Discussions with you are always inspiring. I also want to thank my other supervisors, Mika Reinisalo, PhD, and Heidi Kidron, PhD. Mika - I’ve learned so much from you, and you are truly a great teacher with endless patience and optimism. Working with you has also been a lot of fun! Heidi - I’m very thankful for your guidance during these years, you’ve always been there for my questions. You were the one to remind me of our main goals, whenever I was getting side-tracked.

Secondly, I want to thank Professor Bente Steffansen and Adjunct Professor Birger Brodin for reviewing this thesis. I’m honored that Sibylle Neuhoff, PhD, has agreed to act as the opponent during the public examination. I also want to thank Ewen MacDonald, PhD, for revising the language of this thesis.

I’m very grateful to our collaborators, Professor Tetsuya Terasaki and his research group in the Tohoku University: Kazuki Sato, MSc; Masanori Tachikawa, PhD; Michitoshi Watanabe, PhD; Yasuo Uchida, PhD. I’m very honored to have worked in this international collaboration project during my PhD work. Our collaboration taught me a lot.

I also want to thank my other co-authors Eva del Amo, PhD; Unni Tengvall-Unandike, PhD; Emmanuelle Morin-Picardat, PhD and Docent Marika Ruponen, PhD. I also thank Kati- Sisko Vellonen, PhD, who helped a lot with the un-published simulation model.

From our research group, Marika deserves a special thank you. Even though you have not been my assigned supervisor, your door has always been open for me and other PhD students with a variety of science- as well as non-scientific-related problems. I also want to thank my “office-mate” Eva, who has helped me a lot to resolve all sorts of practical issues during these years. In addition, I owe my thanks to Lea Pirskanen and Jaana Leskinen who have kept the labs in order, and for their valuable help during these years. Thank you, Lea, for the countless cell plates scraped – without your help I’d probably still be scraping.

I want to thank the other personnel of the UEF School of Pharmacy: Marja Lappalainen, Anne Roivainen, Markku Taskinen and Marko Antikainen. Whether I’ve needed a help with my computer, centrifuge, sending packages or ordering reagents, the help was always there.

I want to thank the friends I made in my high school (Enni and Kata) and from my undergrad times (Riikka, Laura and Elina). I’m glad that we’ve managed to stay in touch and can always pick up where we left off. The time has flown but you’ve always been there to support me. I also want to thank my friends in Kuopio Roller Derby who have spent the last 4 years beside me. I can’t imagine my life without this insane sport and our community. I’m grateful for all the blood, sweat and tears, hits and bruises, and of course all the parties we’ve had during these years. A special thanks to Emma whom I got to know as my fellow PhD student, but later became also my team mate. You’ve literally made my life healthier by showing up to work with nutritious lunches that made me change my lunch routine (from ready-made miso soups to actual food) and dragging me to the crossfit gym. Your friendship means a lot to me.

Last, but not least, I want to thank my family. You’ve always supported me and shown interest towards my work even though I’m sometimes struggling to explain what I actually do. Finally, I want to thank my husband Tuomas for all the love, laughter and support you bring to my life. You’ve also rescued me countless times when my computer was giving me

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troubles during these last 9 years I’ve spent in the university. Thank you for reminding that life outside the lab (or roller derby track) is pretty amazing. You’re my wonderwall.

Kuopio, July 2017

Laura Hellinen

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List of the original publications

This dissertation is based on the following original publications:

I Pelkonen L, Reinisalo M, Morin-Picardat E, Kidron H and Urtti A. Isolation of Intact and Functional Melanosomes from the Retinal Pigment Epithelium.

PLOS One 11(8): pp. e0160352, 2016

II Pelkonen L, Tengvall-Unadike U, Ruponen M, Kidron H, del Amo E. M, Reinisalo M and Urtti A. Melanin binding study of clinical drugs with cassette dosing and rapid equilibrium dialysis inserts.

Manuscript accepted for publication in European Journal of Pharmaceutical Sciences.

DOI:10.1016/j.ejps.2017.07.027

III Pelkonen L*, Sato K*, Reinisalo M, Kidron H, Tachikawa M, Watanabe M, Uchida Y, Urtti A and Terasaki T. LC-MS/MS Based Quantitation of ABC and SLC Transporter Proteins in Plasma Membranes of Cultured Primary Human Retinal Pigment Epithelium Cells and Immortalized ARPE19 Cell Line.

Molecular Pharmaceutics, 2017.DOI: 10.1021/acs.molpharmaceut.6b00782

*equal contribution

The publications were adapted with the permission of the copyright owners.

This thesis contains the following unpublished material:

Transporter protein expression in apical and basolateral plasma membranes of human primary RPE cells

The effect of efflux protein expression on the simulated drug exposure in the RPE

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Abbreviations

ABC ATP-binding cassette transporter

ADME absorption; distribution; metabolism; excretion AMD age-related macular degeneration

ATP adenosine 5'-triphosphate AQ1 aquaporin channel 1

BCRP breast cancer resistance protein BEST1 bestrophin-1

EMA European Medicines Agency FDA Food and Drug Administration GLUT glucose transporter

HSP60 heat-shock protein 60 kDa

LAT large neutral amino acids transporter MATE multidrug and extrusion proteins

MCT monocarboxylate transporter; lactate transporter

MDCK Madin-Darby canine kidney; kidney epithelial cell model MITF-A microphthalmia-associated transcription factor A MRP multidrug-resistance associated protein

OAT organic anion transporter

OATP organic anion transporting polypeptide OCT organic cation transporter

Papp apparent permeability coefficient PBS phosphate buffered saline

P-gp P-glycoprotein

PEPT peptide transporter family

QTAP quantitative targeted absolute proteomics RPE retinal pigment epithelium

SLC solute carrier transporter family TYRP1 tyrosinase-related protein 1 ZO-1 zonula occludens 1

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1 Introduction

Retinal and choroidal disorders, such as diabetic retinopathy and age-related macular degeneration (AMD) are the leading cause of vision loss in the industrialized countries. Some of these diseases, e.g. diabetic retinopathy, affect also the working age population. In Finland, AMD is the main cause of sight impairment: 41 % of all patients and 59 % of older patients (i.e. over 65- years-old) with vision impairment are diagnosed with AMD (Ojamo 2014). It is estimated that by the year 2020, the global prevalence of AMD will be 196 million patients and that number is estimated to increase to 288 million by the year 2040 (Wong, Su et al. 2014).

Drug delivery to the posterior ocular targets (retina, choroid, Fig. 1) is a major problem in drug development owing to the presence of the several barriers limiting the drug permeation (Urtti 2006). Topically administered eye drops can be used to treat the anterior part of the eye (tissues surrounding the anterior chamber, Fig. 1), but only a small fragment of the administered dose reaches the posterior tissues, resulting in insufficient drug concentrations to treat retinal or choroidal diseases (Del Amo, Rimpela et al. 2016). Blood-ocular barriers limit drug permeation from the systemic circulation to the various tissues present in the eye (Fig. 1). The blood-retinal barrier limits drug access from the systemic bloodstream to the posterior eye. It consists of two separate barriers; the inner part consists of retinal vessels and the outer blood-retinal barrier is formed by the retinal pigment epithelium (RPE) cell layer (Fig. 1B). The RPE is not a true barrier, instead it is selective, providing nutrients from the choroidal bloodstream to the photoreceptors, while protecting the photoreceptors from harmful substances by preventing the access of xenobiotics to the neural retina. Therefore, systemic drug administration is usually not feasible in ocular drug treatment: only drugs with broad therapeutic windows and/or selective targeting to the posterior eye tissues can be used (Del Amo, Urtti 2008). In addition, the RPE itself is an important drug target since its functions are compromised in certain degenerative retinal diseases, such AMD (Zajac-Pytrus, Pilecka et al. 2015, La Cour, Kiilgaard et al. 2002).

Currently, intravitreal injections are used to treat retinal diseases, however this approach is not feasible with small molecular weight drugs that have short elimination half-lives in the vitreous. Biological drugs (monoclonal antibodies, soluble receptors) are widely used in the treatment of the neovascularization related to wet AMD (Haller 2013, Yang, McKeage 2014). Drug administration needs to be performed by a specialized nurse or ophthalmologist at 1-3 month intervals representing a significant cost burden on the health care system. The injections are invasive, thus potentially risky (Geck, Pustolla et al. 2013), and often uncomfortable for the patients. The number of patients suffering from the dry form is significantly greater (90 % of all patients) than the number of wet-AMD patients (10 % of patients) (Zajac-Pytrus, Pilecka et al.

2015). The dry form of AMD is currently without any effective treatment, and in fact, the etiology of the disease is unclear.

Many drug transporter proteins are expressed in the RPE, thus they might have a significant role in the posterior eye pharmacokinetics (Mannermaa, Vellonen et al. 2006). However, their clinical significance is currently unclear: the published expression data originates mainly from investigations using RPE cell models rather than in human tissue and the expression has usually only been described at the mRNA level not in terms of actual proteins present. Therefore, quantitative protein expression and activity profiling of the drug transporters in the RPE are needed if one wishes to evaluate their impact on the pharmacokinetics in the posterior eye.

The RPE and choroid in the posterior eye, as well as the iris and ciliary body in the anterior eye (Fig. 1) are pigmented: they contain melanin pigment inside specialized organelles - melanosomes. Melanin can influence the drug distribution in the eye: it binds several ocular drugs and other compounds, and melanin binding can lead either to prolonged drug action or it may prevent the drug’s efficacy (Leblanc, Jezequel et al. 1998, Larsson 1993, Rimpela, Schmitt et al. 2016, Nagata, Mishima et al. 1993, Urtti, Salminen et al. 1984, Salazar, Shimada et al. 1976,

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Salazar, Patil 1976). However, details of melanin binding are still shrouded in mystery. Many different assay procedures have been applied when investigating the extent of melanin binding of different compounds, thus the comparison of their melanin binding properties between different studies is complicated.

Since there are many challenges in the treatment of posterior eye diseases, new non-invasive delivery methods targeting the retina and longer acting injections are needed. In addition, more detailed knowledge of the pharmacokinetic characteristics of the posterior eye would support retinal drug discovery: detailed in silico models (pharmacokinetic simulation models, finite element models) can be advantageous in compound selection in the early stages of drug discovery before embarking on laborious in vivo assays. Decision criteria with their foundations based on in vitro assays and predictive models are needed to guide in lead compound selection in retinal drug design. The aims of this study were to evaluate the role of transporter protein expression for the pharmacokinetics in the RPE and to assess the melanin binding of a clinically relevant compound library. These properties can be utilized to support lead compound selection in the development of efficacious retinal drugs.

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Figure 1.Ocular structure, blood-ocular barriers and drug kinetics in the eye. A) (1) Drug permeation through the cornea from the lacrimal fluid into the anterior chamber. (2) Drug permeation across the conjunctiva and sclera into the anterior uvea. (3) Distribution of the drug through the blood-aqueous barrier into the anterior chamber. (4) Elimination of drugs from the anterior chamber by the aqueous humor turnover. (5) Elimination of drugs into the systemic uveoscleral circulation from the aqueous humor (6) Drug permeation into the posterior segment across the retinal pigment epithelium. (7) Intravitreal drug administration. (8) Elimination of drugs from the vitreous across the blood-retinal barrier (9) Drug elimination from the vitreous via the anterior route to the posterior chamber. Image adapted from (Hornof, Toropainen et al. 2005) B) Blood-ocular barriers. Original image modified from (Del Amo, Rimpela et al. 2016).

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2 Review of the Literature

2.1 TRANSPORTER PROTEINS IN THE RETINAL PIGMENT EPITHELIUM Currently, more than 400 membrane transporters have been recognized in the human genome, and the transporter genes have been divided into two major families: ABC and SLC families (The International Transporter Consortium 2010). ABC - ATP-binding cassette – transporter family proteins are ATP-driven efflux pumps, whereas SLC family proteins function by exchanging ions or small charged molecules with their substrates. For instance, organic anion transporting polypeptides, OATPs, take up organic anions in exchange for endogenous intracellular components, such as bicarbonate. SLC proteins can also be involved in facilitated diffusion (e.g.

glucose transport via GLUT1). With respect to the ABC family transporters, P-glycoprotein (P- gp), breast cancer resistance protein (BCRP) and multidrug-resistance associated proteins (MRPs) are widely recognized as being involved in the pharmacokinetics of many drugs due to the expression of MRPs in various tissues, including intestine, liver, kidneys and blood-brain barrier (The International Transporter Consortium 2010). These efflux proteins are usually expressed in a polarized manner, either preventing the xenobiotic entry from the extracellular space into the tissue or enhancing their elimination, thus protecting the tissues from harmful substances.

According to the International Transporter Consortium, a total of 7 ABC and 12 SLC transporters have been recognized as possessing clinical relevance in the systemic absorption and disposition of drugs (The International Transporter Consortium 2010). Subsequently, additional proteins, such as multidrug and extrusion proteins (MATEs) were included (Hillgren, Keppler et al. 2013).

The importance of drug transporting proteins in drug development has been recognized by regulatory agencies: FDA (The U.S. Food and Drug Administration) recommends clarification of whether new chemical entities are substrates of P-gp and BCRP, and in certain situations, also substrates of OATP1B1 and OATP1B3; organic anion transporters OAT1 and OAT3; and organic cation transporter OCT2 (FDA 2012). European Medicines Agency (EMA) has also issued a guideline for transporter interaction studies: in addition to the proteins listed by the FDA, OCT1 and the bile salt export pump BSEP are included in the EMA’s guideline (EMA 2010). Transporter interactions are a major factor affecting a drug’s pharmacokinetics and need to be considered during drug development.

2.1.1 Transport functions in the RPE

RPE displays apical microvilli towards the neural retina, and the basolateral membrane faces the choroid (Fig. 2). The key function played by RPEs is to provide nutrients to the photoreceptors and maintain photoreceptor renewal (Strauss 2005). One third of the retina is nourished by the choroidal blood flow, thus the RPE filters 1/3 of the blood supplied to retina, whereas retinal vessels provide the nutrients for the inner two thirds of the tissue. To provide these functions, many transporter proteins and channels are expressed in the RPE in a polarized manner (Fig. 2B) (Strauss 2005, Lehmann, Benedicto et al. 2014). RPE removes excess water from the sub-retinal space via aquaporin channels (e.g. aquaporin 1 AQ1, apical surface of the RPE, Fig. 2), and transports glucose and other nutrients from the choroidal bloodstream to the retina via nutrient transporters (e.g. glucose transporter GLUT1, expressed at high levels on both surfaces of the RPE). In addition, the RPE maintains an optimal concentration of ions and pH in the sub-retinal space. This involves exchange of potassium and sodium (Na⁺K⁺ATPase expression on the apical side) and lactate removal (expression of monocarboxylate transporters MCT1 and MCT3 on the apical and basolateral membranes of the RPE, respectively) (Fig. 2).

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Figure 2. RPE functions and examples of the transport processes in the RPE. A) A schematic presentation of RPE’s physiological functions, adapted according to (Lehmann, Benedicto et al. 2014). B) Nutrients are transported from the choroidal blood flow to the RPE via specific transport systems, such as glucose via GLUT1. Metabolic waste product lactate is removed from the subretinal space by lactate transporters (MCT1, MCT3). Excess water is removed from the sub-retinal space via aquaporin channels (e.g. AQ1). RPE stringently maintains the K⁺ ion concentration in the sub-retinal space (e.g. via Na⁺K⁺ATPase function).

2.1.2 Drug-related transporter protein expression and function in the RPE

Tight junctions between the RPE cells restrict the non-specific diffusion of compounds from the choroidal bloodstream to the subretinal space, thus forming the outer blood-retinal barrier (Fig.

2). Furthermore, the efflux transporter proteins are thought to provide a functional barrier restricting the access of xenobiotics to the retina (Fig. 3). In fact, transport directionality of one MRP substrate, fluorescein, was estimated to be 31-times higher in the outward direction (retina- to-choroid) compared to that in the opposite direction in humans, i.e. evidence of its active transport out of the eye (Oguro, Tsukahara et al. 1985). A more recent ex vivo study with porcine RPE-choroid also determined that fluorescein transport was 11.3 times higher in the apical-to- basolateral direction (Steuer, Jaworski et al. 2005). The differences in directional permeability were lost in the presence of the MRP inhibitor, probenecid indicating that RPE possesses MRP activity, and it is likely that the efflux proteins are located on the choroidal surface of the RPE, thus protecting neural retina from MRP substrates. In addition, the permeabilities of two P-gp substrates, rhodamine 123 and verapamil, were higher in the apical-to-basolateral direction (2.6 and 3.5-fold, respectively). Furthermore, the uptake of calcein, another P-gp substrate, was seen to increase in the presence of verapamil, a competing P-gp substrate, indicating P-gp activity in the ex vivo model, probably in the choroidal surface of the RPE.

In 2006, Mannermaa et al. reviewed the literature regarding the transporter expression in ocular tissues (cornea and blood-retinal barrier) (Mannermaa, Vellonen et al. 2006). The main conclusion was that many transporter proteins that have ocular drugs (and some other drugs) as substrates are expressed in the RPE. These proteins included both efflux proteins (e.g. P-gp, MRPs) and SLC family transporters with known drug substrates (e.g. MCTs, OATPs). However, in most cases, the studies had described the expression at the mRNA level, and many had been conducted in cell models rather than in human or animal tissue. However, mRNA levels do not necessarily correlate with the protein expression levels in the plasma membrane (Ohtsuki, Schaefer et al. 2012), i.e. the mRNA levels may not correlate to transporter expression or activity in the cells. This was demonstrated in Caco-2 cells with BCRP expression: quantitative protein expression, but not mRNA expression, correlated with transporter functionality in vitro (Harwood, Neuhoff et al. 2016). Since 2006, many new reports have been published in this field.

Table 1 summarizes the key literature regarding drug transporter expression in the RPE, focusing onthose studies providing the evidence of protein expression. Protein expression in cell lines (e.g.

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ARPE19, D407, RPE-J) are not included: only cultured primary or differentiated stem cells are displayed in addition to expression in human or animal tissue. Figure 3 summarizes the current knowledge regarding the direction of function and localization of these transporters. Importantly, the protein expression studies reveal only qualitative information, thus the effect of the transporter protein expression to pharmacokinetics is difficult to evaluate.

Table 1. Transporter protein expression in outer blood-retinal barrier – key literature.

Species Tissue/cells Detection Localization/comments Reference EFFLUX PROTEINS

P-glycoprotein

Human RPE tissue + IC apical and basolateral membranes (Kennedy, Mangini 2002)

Porcine RPE-choroid tissue + IC (Steuer, Jaworski et al.

2005) BCRP

Human hfRPE cells - WB (Mannermaa, Vellonen et al.

2009)

Bovine RPE tissue - WB human ab used (Mannermaa, Vellonen et al.

2009)

Mouse RPE + IC basolateral membrane of the RPE (Gnana-Prakasam, Reddy et al. 2011)

MRP1

Porcine RPE-choroid tissue + IC MRP protein not specified with number

(Steuer, Jaworski et al.

2005)

Human hfRPE cells + WB (Mannermaa, Vellonen et al.

2009)

Bovine RPE cells + WB (Mannermaa, Vellonen et al.

2009)

Human hESC-RPE + WB + IC apical plasma membrane (Juuti-Uusitalo, Vaajasaari et al. 2012)

Human cultured RPE cells + WB (Aukunuru, Sunkara et al.

2001)

Human retina -IC not detected in retina (Dahlin, Geier et al. 2013)

Horse RPE tissue + MS (Szober, Hauck et al. 2012)

MRP2

Human hfRPE cells - WB (Mannermaa, Vellonen et al.

2009)

Bovine RPE cells - WB human ab used (Mannermaa, Vellonen et al.

2009) MRP4

2009)

Bovine RPE cells + WB (Mannermaa, Vellonen et al.

2009)

Mouse RPE + IC basolateral membrane (Chapy, Saubamea et al.

2016) MRP5

2009)

Bovine RPE cells - WB (Mannermaa, Vellonen et al.

2009)

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INFLUX PROTEINS

MCT1

Human RPE tissue + IC apical membrane (Philp, Wang et al. 2003)

Mouse RPE + IC apical membrane of the RPE (Gnana-Prakasam, Reddy et al. 2011)

Mouse RPE apical microvilli + MS

(Bonilha, Bhattacharya et al.

2004) Mouse RPE AV and CB + MS detected in both fractions and in

young and old mice

(Gu, Neric et al. 2012) Rat retina + WB + IC apical membrane of the RPE (Philp, Yoon et al. 1998)

Rat RPE + WB (Chidlow, Wood et al. 2005)

Rat retina + IC IEM: apical membrane of the RPE (Gerhart, Leino et al. 1999) MCT2

Rat retina and choroid -IC IEM: not detected in the RPE cells (Gerhart, Leino et al. 1999) Rat RPE + WB + IC faint expression throughout the

RPE

(Chidlow, Wood et al. 2005) MCT3

Human RPE tissue + IC basolateral membrane (Philp, Wang et al. 2003) Human hfRPE cells + WB + IC basolateral membrane (Gallagher-Colombo,

Maminishkis et al. 2010) Mouse RPE AV and CB + MS detected in both fractions in

young mice, only in cell bodies in old mice

(Gu, Neric et al. 2012) Rat retina + WB + IC basolateral membrane of the RPE (Philp, Yoon et al. 1998) Rat RPE + WB + IC basolateral membrane (Chidlow, Wood et al. 2005)

MCT4

Human hfRPE cells + WB + IC basolateral membrane (Gallagher-Colombo, Maminishkis et al. 2010)

Rat RPE - WB - IC (Chidlow, Wood et al. 2005)

OCT3

Human retina + IC expression in the RPE (Dahlin, Geier et al. 2013) OATP1A2

Human RPE tissue + IC apical membrane (Chan, Zhu et al. 2015)

Human cultured adult RPE cells

+ WB (Chan, Zhu et al. 2015)

OATP1A4

Rat retina + WB + IC apical membrane (Akanuma, Hirose et al.

2013) OATP1C1

Rat retina + WB + IC apical and basolateral membranes (Akanuma, Hirose et al.

2013) OATP2A1

Human RPE tissue + IC (Kraft, Glaeser et al. 2010)

OATP1B3

Human retina -IC not detected in retina (Dahlin, Geier et al. 2013) OATP2B1

Human RPE tissue + IC (Kraft, Glaeser et al. 2010)

RPE = retinal pigment epithelium; IC=immunochemistry; WB=Western blot; MS=mass spectrometry; IEM = immuno electron microscopy; hfRPE cells=human fetal RPE cells; horse RPE tissue: membrane fraction (Szober et al. 2012); AV=apical microvilli, CB= cell bodies (Gu et al. 2012)

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Figure 3. Transporter protein expression in the outer blood-retinal barrier. The proteins are localized on the cell surfaces, if the localization is known. The proteins inside the cells represent situations where the localization (apical, basolateral cell surface) is not known, or when the localization in cell model differs from expression or direction of function in vivo. The direction of function is based on in vivo or ex vivo studies. Dotted lines represent situations where the function in inner and outer blood-retinal barrier could not be distinguished. Image modified from (Mannermaa, Vellonen et al. 2006).

In addition to protein expression studies, the role of transporter proteins in retinal uptake has been investigated with in vivo methods in recent years. Several rodent studies have shown that P-gp has a more modest role in the blood-retinal than in the blood-brain barrier (Toda, Kawazu et al. 2011, Chapy, Saubamea et al. 2016, Fujii, Setoguchi et al. 2015). A rabbit study also suggested that P-gp does not have a major role in retinal drug uptake (Senthilkumari, Velpandian et al.

2008). Similar results were seen in humans with a PET scan: verapamil distribution in the human retina was increased (1.4-fold) in the presence of an inhibitor, tariquidar (Bauer, Karch et al. 2016), however, as in rodent studies, the effect of the P-gp inhibitor on drug distribution was less marked in the retina than in the brain. In contrast, Chapy et al. demonstrated that MRPs had a similar effect on zidovudine distribution in retina as in brain, indicating that MRPs are more likely responsible for the drug efflux in the retina than P-gp (Chapy, Saubamea et al. 2016). In vivo studies have also pointed to the involvement of OCT transport in rabbit retina (Nirmal, Velpandian et al. 2012), and Oatp1a4 function in rats (Fujii, Setoguchi et al. 2015), but the clinical significance of retinal transporters is still open.

Importantly, these in vivo studies cannot distinguish the role of the outer and inner blood- retinal barrier: the effect observed is the sum of the barriers. Thus, RPE’s role in drug uptake cannot be estimated with these in vivo methods, even though they provide valuable insights into the overall role of the active transport in the blood-retinal barriers. Furthermore, influx and efflux proteins often share same substrates, meaning that active transport may occur in both directions simultaneously complicating the interpretation of the function of a single transporter (Chapy, Saubamea et al. 2016). For instance, verapamil is recognized to be substrate not only to P-gp, but also of two influx proteins, OCT3 and OCTN1-2 (Dahlin, Geier et al. 2013, Morrissey, Wen et al.

2012).

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2.2 RPE PIGMENTATION AND ITS SIGNIFICANCE FOR OCULAR PHARMACOKINETICS

2.2.1 Melanin in the RPE

Pigmented tissues throughout the human body such as skin and hair but also iris, choroid and the RPE in the eye, contain melanin. Melanin is a large, polyanionic polymer, which contains many carboxylic groups (Figure 4) (Ito 1986). There are two melanin types with distinct structures and colors: eumelanin is black whereas pheomelanin is yellow-brown (Fig. 4 displays eumelanin).

Both melanin types are synthetized from tyrosine and require the action of the melanosomal enzyme, tyrosinase. Even though some details of melanin sub-units in the eumelanin and pheomelanin structure are recognized, the detailed overall structure of melanin is currently not known. In the RPE, eumelanin is the major melanin type (Hu, Simon et al. 2008), whereas the eumelanin/pheomelanin ratio in the iris melanocytes varies; this is responsible for the differences in eye color (Prota, Hu et al. 1998, Wakamatsu, Hu et al. 2008). Both melanin types are present also in the choroid. Most natural melanins consist of a mixture of the two melanin types – at varying ratios (Ito, Wakamatsu 2008).

In the RPE, melanin absorbs scattered light, enables sharp vision and protects the neural cells in the retina (rods and cones) from excessive light exposure (Fig. 2A). Inside cells, melanin is located inside specialized organelles, melanosomes, where it is synthetized (Fig. 4). These organelles share some similarities with lysosomes, and their developmental origin is thought to be the same (Orlow 1995). Proteomic study on porcine RPE melanosomes showed that the organelles expressed some lysosomal proteins, such as cathepsin D and V-type H⁺ATPase (Azarian, McLeod et al. 2006). Cathepsin D has been confirmed to localize on the melanosomal membrane suggesting that melanosomes participate in the RPE’s degradative processes.

Interestingly, the efflux transporter, MRP4, was detected by LC-MS in the isolated melanosomal fraction. Since the methodology required isolation of the melanosomes, cross-contamination of other organelles cannot be completely ruled out.

Melanosome maturation can be divided into four stages (I-IV), and each stage displays differences in the melanin amount (Seiji, Shimao et al. 1963). At the final stage, melanin is packed into protein fibrils covering completely the intraorganellar space: the organelles are displayed as completely black, round or ellipsoidal organelles in electron microscopic images (Seiji, Shimao et al. 1963). In the RPE, the melanosome maturation takes place during fetal development and in early childhood, thus in the adult RPE, the melanosomes are only present in their mature forms (stage IV) (Simon, Hong et al. 2008).

Figure 4. Melanin and melanosomes in the RPE (Del Amo, Rimpela et al. 2016).

2.2.2 Drug-melanin interactions

In addition to melanin’s role in light absorption in the eye, RPE melanin is postulated to act as an antioxidant (Różanowska 2011). It participates in metal homeostasis in the cells by binding metals (Hong, Simon 2007, Kaczara, Zareba et al. 2012). In addition, melanin can bind many drug

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molecules and other xenobiotics (Leblanc, Jezequel et al. 1998, Larsson 1993, Dayhaw-Barker 2002). The physiological basis of melanin binding has been argued to protect the cells from harmful substances i.e. after the melanin-compound interaction, the free fraction of the xenobiotic inside the cells is reduced and therefore, toxic levels are not reached. However, melanin binding can lead to the accumulation of compounds into pigmented tissues, and cause adverse reactions if the compound has a toxic nature. The interest toward drug binding to ocular melanin was proposed to provide an explanation for the toxic effects of phenothiazines in the eye (Potts 1964).

However, a literature review by Leblanc et al. in 1998 concluded that melanin binding does not necessarily lead to toxicity: several compounds with affinity towards melanin (e.g. pilocarpine, atropine) do not cause ocular adverse effects (Leblanc, Jezequel et al. 1998). For instance, timolol binds to melanin (Salminen, Imre et al. 1985, Nagata, Mishima et al. 1993), but timolol eye drops are in clinical use to treat glaucoma.

Melanin binding can alter the pharmacokinetics of some drugs in ocular tissues. Already in the 1970’s, Salazar et al. found that topically administered atropine exerted a longer mydriatic effect in the pigmented than in albino rabbits (half-life 96 h and 43 h in pigmented and albino animals, respectively) (Salazar, Patil 1976). The retention studies using excised irides in vitro and in vivo biodistribution studies confirmed that the longer duration of action was caused by the drug retention in pigmented tissues (Salazar, Patil 1976, Salazar, Shimada et al. 1976). Similar effects have been observed with pilocarpine: albino animals had less prolonged miosis after pilocarpine administration compared to their pigmented counterparts (Urtti, Salminen et al.

1984). In addition, melanin binding can prevent the drug response: timolol was noted to lower the intraocular pressure of albino animals, but this response was not achieved in pigmented animals (Nagata, Mishima et al. 1993). In addition, recent simulation models have indicated that melanin binding has a significant role in the drug uptake and distribution in the RPE (Rimpela, Schmitt et al. 2016, Del Amo, Rimpela et al. 2016). Thus, melanin binding is an important aspect that needs to be taken into account in ocular drug discovery.

2.2.3 Pigment targeting

As described above, melanin binding can cause prolonged drug action, but does not necessarily lead to adverse reactions (chapter 2.2.2). Since a long duration of action in the retina is needed in degenerative retinal diseases, melanin binding is an interesting option for achieving a natural depot effect in the retina. Recently, small molecular weight anti-VEGF-agents panzopanib and GW771806 were recognized to have long retention in the uveas of pigmented rats after single oral dose (Robbie, Lundh von Leithner et al. 2013). These compounds bound to melanin in vitro. The drugs were still present in the uveal tract 35 days after dosing, and both compounds had long ocular half-lives (approximately 440 h). A comparison between albino and pigmented rats showed that the concentrations were higher in the ocular tissues of pigmented animals at 3 days after dosing. In addition, the compounds exerted a pharmacological effect in the choroidal neovascularization rat model. A single oral panzopanib dose resulted in smaller lesions in the treated compared to control animals and GW771806 administered topically had a similar effect.

The pharmacodynamics was not compared between albino and pigmented animals, thus the effect of pigment binding on the drug response remained undetermined. However, the biodistribution studies showing retention in the pigmented tissues suggest that melanin binding could be utilized as a targeting approach in retinal drug treatment.

Currently, anti-VEGF therapy is performed via intravitreal injection with 1-3 months dosing intervals. Pigment targeting drug delivery systems could provide longer dosing intervals or even the need for less invasive administration routes as demonstrated in the rodent study conducted by Robbie et al. 2013. However, further investigations will be needed to design pigment targeting drug delivery systems: melanin binding mechanisms and physicochemical properties driving the binding are not well understood. It has been suggested that ionic interactions, van der Waals forces, hydrophobic interactions and charge-transfer interactions are all involved in the binding (Larsson 1993). Recently, a QSPR model was built with a large compound library (263 molecules)

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to describe the melanin binding (Reilly, Williams et al. 2015). That study concluded that basicity, hydrophobicity, and charge–transfer properties were important for melanin binding, in confirmation of earlier work conducted with significantly less extensive compound sets (less than 30 molecules) (Zane, Brindle et al. 1990, Radwanska, Frackowiak et al. 1995). Reilly et al. (2015) also highlighted that the number of rigid bonds and rings as being the most important contributing factors for melanin binding. However, the study provided the information only on the methodology and did not provide any detailed assessment of the physicochemical properties of melanin binding: the QSPR equation was not published. Thus, the details of melanin binding are still unclear. In addition, the information regarding the association and dissociation kinetics of melanin binding is currently limited.

2.3 IN VITRO METHODS TO STUDY TRANSPORTER EXPRESSION AND MELANIN BINDING

2.3.1 Protein quantification and subcellular fractionation

Protein quantitation with LC-MS/MS technology has many applications in the ADME (absorption, distribution, metabolism, excretion) research. For example, with quantitative targeted absolute proteomics (QTAP), the absolute protein expression in biological samples can be determined (Uchida, Tachikawa et al. 2013). Compared to antibody-based protein detection techniques (ELISA, Western blot, immunofluorescent techniques), QTAP is more specific: LC- MS/MS recognizes single amino acid changes in peptide structures, whereas the identification of different isoforms e.g. in CYP enzymes, is difficult with antibodies due to their high sequence similarities (Ohtsuki, Uchida et al. 2011). However, the method is currently less sensitive, and therefore, subcellular fractionation is often used to ensure that the protein expression is enriched in the studied fraction and within the quantification limits (Harwood, Russell et al. 2014).

Secondly, subcellular fractionation enables the quantification of proteins specifically in certain cellular compartments, such as plasma membranes, providing more specific information for ADME prediction. However, the loss of plasma membrane proteins during the fractionation should be considered, otherwise the expression data loses accuracy and the quantification of membrane protein abundances in modeling approaches might be misleading (Harwood, Russell et al. 2014, Harwood, Neuhoff et al. 2013).

Protein quantification of plasma membrane samples has been conducted with several different tissues and cell types, including blood-brain barrier (Ohtsuki, Ikeda et al. 2013), hepatic (Schaefer, Ohtsuki et al. 2012), lung cells (Sakamoto, Suzuki et al. 2016) and the intestinal cell model, Caco- 2 cells (Uchida, Ohtsuki et al. 2015). Purified plasma membrane fractions are required for the analysis and therefore the cell lysate or homogenate needs to be purified; traditionally differential centrifugation and sucrose density gradient centrifugation are exploited (Fig. 5, Method A) (Harwood, Neuhoff et al. 2013). In addition, methods consisting of simpler fractionation procedures by commercial extraction kits can be used (Fig. 5, method B). However, these methods result in crude membrane fractions containing not only the plasma membrane, but also intracellular membranes. Thus, intracellular expression of the target transporter protein may interfere with the interpretation of the results. In contrast, sucrose gradient centrifugation produces a plasma membrane specific fraction (Fig. 5, method A).

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Figure 5. Plasma membrane isolation methods. (Harwood, Neuhoff et al. 2013). Sucrose density gradient centrifugation results in a membrane fraction that consists of plasma membrane specific proteins (method A), whereas kit-based extraction provides a membrane fraction with both intracellular and plasma membrane proteins (method B).

The quantitative expression values of membrane transporters and their activity profiling is needed to build relevant pharmacokinetic models that take into account the active transport (Kamiie, Ohtsuki et al. 2008). In vivo activity can be extrapolated from in vitro activity data, if the expression levels of transporter proteins in both systems are quantified. However, the information on the protein abundance and its relation to transporter function in human is currently very limited, for example, there are no reports describing the quantitative protein expression in ocular tissues in the literature.

2.3.2 RPE cell models

Since RPE is a polarized monolayer in vivo, the RPE cells are typically cultured on supportive filters enabling their polarization (Vellonen, Malinen et al. 2014, Hornof, Toropainen et al. 2005).

However, simple efflux and uptake experiments can be conducted in regular culture dishes e.g.

24-well plates.

ARPE19 cell line. ARPE19 cell line has been widely used in the ophthalmic research since the 1990s. Even though the cell line seemed to be a promising RPE model when it was first introduced (Dunn, Aotaki-Keen et al. 1996), recently it has been criticized as lacking important RPE characteristics, such as pigmentation, down-regulation of RPE-specific gene expression (Strunnikova, Maminishkis et al. 2010) and also to display shortcomings in the formation of tight junctions (Rizzolo 2014). However, Mannermaa et al. (2009) showed that the ARPE19 cell line

Melanin Binding and Drug Transporters in the Retinal Pigment Epithelium: Insights to Retinal Drug Delivery

uef.fi

Dissertations in Health Sciences

LAURA HELLINEN

MELANIN BINDING AND DRUG TRANSPORTERS IN THE RETINAL PIGMENT EPITHELIUM: INSIGHTS

INTO RETINAL DRUG DELIVERY

LAURA HELLINEN

Melanin Binding and Drug Transporters in the Retinal Pigment Epithelium: Insights

into Retinal Drug Delivery

Acknowledgements

List of the original publications

Contents

Abbreviations

1 Introduction

2 Review of the Literature