DISSERTATIONS | ELENA PURIS | PHARMACOKINETIC CONCEPTS IN DRUG DELIVERY TO THE BRAIN... | No 518
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THE UNIVERSITY OF EASTERN FINLAND Dissertations in Health Sciences
ISBN 978-952-61-3139-9 ISSN 1798-5706
Dissertations in Health Sciences
PUBLICATIONS OF
THE UNIVERSITY OF EASTERN FINLAND
ELENA PURIS
PHARMACOKINETIC CONCEPTS IN DRUG DELIVERY TO THE BRAIN VIA TRANSPORTER-MEDIATED PRODRUG APPROACH
The doctoral thesis is aimed to investigate pharmacokinetics, BBB transport and intra- brain distribution of LAT1-utilizing prodrugs.
A stepwise methodology for early stage development of the transporter-utilizing prodrugs to improve the brain delivery of the drugs with targets located in the intracellular
compartment of the brain parenchyma is presented.
ELENA PURIS
PHARMACOKINETIC CONCEPTS IN DRUG DELIVERY TO THE BRAIN VIA
TRANSPORTER-MEDIATED PRODRUG
APPROACH
Elena Puris
PHARMACOKINETIC CONCEPTS IN DRUG DELIVERY TO THE BRAIN VIA
TRANSPORTER-MEDIATED PRODRUG APPROACH
Publications of the University of Eastern Finland Dissertations in Health Sciences
Number 518
University of Eastern Finland Kuopio
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 (Tenure Track) 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.
School of Pharmacy Faculty of Health Sciences
Distributor:
University of Eastern Finland Kuopio Campus Library
P.O.Box 1627 FI-70211 Kuopio, Finland
www.uef.fi/kirjasto
GRANO KUOPIO, 2019
ISBN: 978-952-61-3139-9 (print/nid.) ISBN: 978-952-61-3140-5 (PDF)
ISSNL: 1798-5706 ISSN: 1798-5706 ISSN: 1798-5714 (PDF)
Author’s address: School of Pharmacy
University of Eastern Finland KUOPIO
FINLAND
Doctoral programme: Doctoral programme in Drug Research Supervisors: Kristiina Huttunen, Ph.D.
School of Pharmacy
University of Eastern Finland KUOPIO
FINLAND
Professor Seppo Auriola, Ph.D.
Department/Institute/Name of the School University of Eastern Finland
KUOPIO FINLAND
Aleksanteri Petsalo, Ph.D.
School of Pharmacy
University of Eastern Finland KUOPIO
FINLAND
Reviewers: Professor Peter Swaan, Ph.D.
Department of Pharmaceutical Sciences University of Maryland
BALTIMORE MARYLAND, USA
Professor Jari Yli-Kauhaluoma, Ph.D.
Faculty of Pharmacy University of Helsinki HELSINKI
FINLAND
Opponent: Professor Mikko Niemi, Ph.D.
Department of Clinical Pharmacology University of Helsinki
HELSINKI FINLAND
Puris, Elena
Pharmacokinetic concepts in drug delivery to the brain via transporter-mediated prodrug approach
Kuopio: University of Eastern Finland
Publications of the University of Eastern Finland Dissertations in Health Sciences 518. 2019, 100 p.
ISBN: 978-952-61-3139-9 (print) ISSNL: 1798-5706
ISSN: 1798-5706
ISBN: 978-952-61-3140-5 (PDF) ISSN: 1798-5714 (PDF)
ABSTRACT
Limited delivery of drugs into the brain is one of the reasons of high failure rate of the CNS drug candidates. The blood-brain barrier (BBB) separating brain from blood is a key player in brain delivery restricting the entrance of the drugs and xenobiotics.
The transporter-mediated prodrug approach is promising method for improving the CNS delivery of drugs. In this respect, L-type amino acid transporter 1 (LAT1), selectively expressed at the BBB and brain parenchymal cell membrane, is of high interest and mediates the brain delivery of the antiparkinsonian drug L-Dopa.
Moreover, several prodrugs of CNS agents have been developed to utilize this transporter for brain delivery. However, there is a lack of systematic knowledge about pharmacokinetics, BBB transport and intra-brain distribution of LAT1- utilizing prodrugs.
The purpose of the study was to apply a stepwise approach based on pharmacokinetic (PK) principles to preclinical development of LAT1-utilizing prodrugs of ketoprofen and ferulic acid, as two potential agents for treatment of Alzheimer’s disease. The structure-PK relationship study of five LAT1-utilizing prodrugs of ketoprofen confirmed the previous findings that the aromatic promoiety in the prodrug's structure plays important role in binding to LAT1 and utilization of the transporter for prodrug cellular uptake. A single dose PK study in mice revealed that the meta- or para-conjugation of phenylalanine directly to ketoprofen can be a vital feature providing targeted brain delivery and reduced systemic exposure. The aliphatic amino acids promoieties and additional methylene linker in phenylalanine conjugate structure may not ensure these properties. In addition, the meta- substituted phenylalanine prodrug of ketoprofen demonstrated predominant distribution into the brain parenchymal cells, where the target and metabolizing enzymes are located. Moreover, the estimated plasma to brain delivery efficacy of ketoprofen released from the prodrug was significantly higher than after ketoprofen dosing. Importantly, the distribution of the prodrug did not affect the LAT1 protein expression and function at the brain cellular barrier. The findings from the structure-
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PK relationship study regarding LAT1 binding and brain delivery were confirmed by developed prodrugs of ferulic acid. Furthermore, it was shown that the selection between amide or ester linker connecting the promoiety and parent drug is important, as it may alter the transporter utilization ability and bioconversion properties in different species. Finally, the optimized methodological strategy for preclinical development of transporter-utilizing prodrugs as exemplified by LAT1 is presented in the thesis.
In conclusion, this present work provides essential information for development of the transporter-utilizing prodrugs improving the brain delivery of drugs with targets located in the intracellular compartment of the brain parenchyma.
National Library of Medicine Classification: QV 38, QV 785, WL 200, WL 300
Medical Subject Headings: Drug Delivery Systems; Brain; Parenchymal Tissue; Blood-Brain Barrier; Pharmacokinetics; Prodrugs; Large Neutral Amino Acid-Transporter 1; Ketoprofen
Puris, Elena
Aivoihin kohdennettujen kuljettajaproteiinia hyödyntävien aihiolääkkeiden farmakokinetiikka.
Kuopio: Itä-Suomen yliopisto
Publications of the University of Eastern Finland Dissertations in Health Sciences 518. 2019, 100 s.
ISBN: 978-952-61-3139-9 (nid.) ISSNL: 1798-5706
ISSN: 1798-5706
ISBN: 978-952-61-3140-5 (PDF) ISSN: 1798-5714 (PDF)
TIIVISTELMÄ
Lääkeaineiden vähäinen kulkeutuminen aivoihin on yksi syy miksi keskushermostoon vaikuttavien lääkeaineiden kehitys epäonnistuu. Veri-aivoeste erottaa aivot verenkierrosta ja on siten tärkeässä roolissa säädellessä lääkeaineiden ja vierasmolekyylien (ksenobioottien) pääsyä aivoihin. Kuljetinproteiineja hyödyntävä aihiolääketeknologia on lupaava menetelmä, jolla voidaan lisätä keskushermostolääkkeiden pääsyä aivoihin. Aminohappokuljetinproteiini LAT1 (L- type amino acid transporter 1), joka ekspressoituu selektiivisesti veri-aivoesteessä on mielenkiintoinen kuljetinproteiini lääkeaineiden aivoihin kohdentamisen kannalta.
Kyseinen kuljetinproteiini kuljettaa aivoihin esimerkiksi Parkinsonin taudin hoidossa käytettyä levodopa-lääkettä. Lisäksi tätä kuljetinproteiinia on käytetty hyödyksi monien muiden keskushermostossa vaikuttavien aihiolääkkeiden kehityksessä. LAT1-proteiinia hyödyntävien aihiolääkkeiden farmakokinetiikasta, kuljetuksesta veri-aivoesteen läpi tai jakautumisesta aivoissa ei kuitenkaan ole systemaattista tietoa.
Tämän tutkimuksen tarkoituksena oli soveltaa farmakokineettisiä periaatteita kahden potentiaalisesti Alzheimerin taudin hoitoon soveltuvan molekyylin, ketoprofeenin ja ferulahapon, LAT1-proteiinia hyödyntävien aihiolääkkeiden kehityksessä. Ketoprofeenin viiden LAT1-proteiinia hyödyntävän aihiolääkkeen rakenne-farmakokinetiikka-suhteiden tutkimus vahvisti aikaisemmat havainnot, että aromaattinen aihio-osa aihiolääkkeen rakenteessa on tärkeä lääkeaineen LAT1- sitoutumisen kannalta. Hiirillä tehdyn yhden annoksen farmakokineettinen tutkimus paljasti myös, että fenyylialaniin meta- tai para-konjugaatio suoraan ketoprofeenin rakenteeseen on tärkeä ominaisuus, joka mahdollistaa kohdistetun kulkeutumisen aivoihin ja samalla pienemmän systeemisen altistuksen, kun taas alifaattiset aminohappo-aihio-osat tai metyleenilinkkeri fenyylialaniinikonjugaatti- rakenteessa eivät tuoneet näitä ominaisuuksia. Lisäksi meta-substituoidun ketoprofeenin fenyylialaniini-aihiolääkkeen jakautuminen kohdistui aivojen parenkymaalisiin soluihin, missä aihiolääkettä metaboloivat entsyymit ja
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vapautuneen lääkeaineen kohdeproteiini sijaitsevat. Tämän lisäksi aihiolääkkeestä vapautuneen ketoprofeenin aivo- ja plasmapitoisuuksien suhde oli merkittävästi suurempi kuin vastaavan ketoprofeenin annostelun jälkeen. Tämä aihiolääke ei myöskään vaikuttanut LAT1-proteiinin ilmentymiseen tai sen toimintaan solukalvoilla. Ketoprofeiinin aihiolääkkeiden LAT1-proteiiniin sitoutumisesta ja aivoihin kulkeutumisesta saadut tulokset pystyttiin todistamaan myös toisella vastaavasti kehitetyllä ferulahapon LAT1-aihiolääkkeillä. Lisäksi ferulahapon aihiolääkkeen tutkimuksessa osoitettiin, että aihio-osan ja lääkeaineen välisen amidi- tai esterisidoksen valinnalla on tärkeä merkitys niin aihiolääkkeen kyvyssä hyödyntää kuljettajaproteiinia kuin vapauttaa vaikuttava lääkeaine.
Lopuksi opinnäytetyössä esitetään optimoitu metodologinen strategia LAT1- kuljetinproteiinia hyödyntävien aihiolääkkeiden prekliiniselle kehitykselle. Näin ollen tämä väitöskirjatutkimus tarjoaa olennaista tietoa aivoihin kohdennettujen kuljetinproteiinia hyödyntävien aihiolääkkeiden kehitykselle.
Luokitus: QV 38, QV 785, WL 200, WL 300
Yleinen suomalainen ontologia: lääkkeet; lääkeaineet; kulkeutuminen; kohdentaminen;
jakautuminen; aivot; farmakokinetiikka; aihiolääkkeet; ketoprofeeni
“If you see it there, darling, then it’s there”
– Freddie Mercury
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ACKNOWLEDGEMENTS
The present study was conducted at Drug Research Program, Faculty of Health Sciences, University of Eastern Finland during the years 2016-2019. The study described in publication II was performed in collaboration with the Translational PKPD group, Department of Pharmaceutical Biosciences, Uppsala University and the Predictive Pharmacology Group, Division of Systems Biomedicine and Pharmacology, Leiden Academic Centre for Drug Research, Leiden University. The work was funded by the Finnish Cultural Foundation (grant of Elena Puris), Alfred Kordelinin Foundation (grant of Elena Puris), Graduate School of University of Eastern Finland, the Academy of Finland (grant of Kristiina Huttunen), Sigrid Juselius Foundation (grant of Kristiina Huttunen), Emil Aaltonen Foundation (grant of Kristiina Huttunen), the Biocenter, Finland. In addition, the travel grants for the conference meetings were provided by the University of Eastern Finland.
I am grateful to all of you who have supported and helped me so much throughout the entire thesis work, and encouraged me during my time as a PhD student and a special thanks to:
My main supervisor Dr. Kristiina Huttunen and co-supervisors Prof. Seppo Auriola and Dr. Aleksanteri Petsalo for introducing me to a new field of the research, for the guidance, support, accepting my ideas and providing the resources and help for their realization. I am thankful for the invaluable experience I got while working together.
My scientific mentor and office-mate Mikko Gynther for everything you have done for me and my projects during these years. Thank you for your knowledge, ideas, support, comprehensive feedback and endless help in developing my scientific skills.
Dr. Irena Loryan, my supervisor and “navigator in neuropharmacokinetics” for the opportunity to work with you, for your endless support, ideas, constructive criticism and motivating me to be a good scientist.
My “pharmacokinetic instigators”, Prof. Elizabeth de Lange and Prof. Margareta Hammarlund-Udenaes for opening my eyes to my research after communication with you and very interesting course on the BBB. I could only dream to work with you, and I am proud that I had this experience.
Prof. Markku Pasanen for being my supervisor in “obesity” project and motivating me to continue with the Ph.D. studies. Thank you for your support in daily situations, positiveness and always “opened door” for scientific discussions.
Prof. Jussi Pihlajamäki for the possibility to work in your interesting project, your guidance and support. Our “obesity” study gave me a better understanding of the present work.
Dr. Veli-Pekka Ranta for your guidance in the field of pharmacokinetics, help, support and possibility to learn how to look at the data from the different angles.
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Prof. Anna-Liisa Levonen, Prof. Jarkko Rautio and Mrs. Arja Afflekt for the professional help at the final steps of my PhD studies.
Prof. Antti Poso for your professional advices in work and life situations.
My colleagues at the Drug Targeting research group and Pharmaceutical Medicinal Chemistry (PMC) group for creating a positive environment. Special thanks to my co-author M.Sc. Johanna Huttunen for conducting the in vitro cellular uptake experiments in publication I and III; Dr. Marko Lehtonen for the constant professional assistance in analytics related questions. I am thankful to Dr. Tuomo Laitinen for the calculations of clogD and clogP of the investigated compounds in publication I and III. My office-mate, Juulia Järvinen, for a short, but pleasant time in our office.
My co-authors and collaborators in different projects, Dr. Ville Männistö, Dr. Pirjo Käkelä, Dr. Merja Häkkinen, Dr. Katja Kanninen, Dr. Tarja Malm, Prof. Tapio Keränen, Prof. Paavo Honkakoski, Prof. Hannu Raunio, Dr. Aaro Jalkanen, Dr. Kati- Sisko Vellonen, Dr. Marika Ruponen. It has been a great pleasure to work with such professionals as you are.
Ms. Helly Risanen, Ms. Miia Reponen, Ms. Tiina Koivunen and Ms. Jessica Dunhall for their excellent technical assistance in the laboratory.
Prof. William Elmquist for asking important questions about my work during the GRC 2018 and interesting discussions which helped me to write the thesis.
My official reviewers of the dissertation, Prof. Peter Swaan and Prof. Jari Yli- Kauhaluoma, for the valuable and expert comments during the final preparation of this thesis. I am proud that my dissertation was highly assessed by the leading experts. I am honoured to have Prof. Mikko Niemi as my opponent. I am grateful to Dr. Ewen MacDonald for revising the language of the original articles.
My parents Irina and Ivan Puris for the endless love and support. My warmest thanks go to my close friends Tajus, Zinok, Tonya, Vikas, Kirya, Natasha, Ksyu and Kuopio friends Vikus, Natasha, Boris, Oksana, Ville, Tatjana, Tatu and Ennska for the support and all the great moments we shared together.
Mrs. Anna-Maija Gynther and Prof. Jukka Gynther for support and help in preparation for the defense, discussions and relaxing time we spent together.
My spiritual advisers Ms. Viktoria Cherdakova, Prof. Mikhail Litvak, Dr. Igor Naumenko and his project Vmeste, Mr. Boris Litvak for the valuable knowledge and motivating me throughout the entire process, both by keeping me harmonious and helping me putting pieces together.
My son Timur for turning my life upside down and helping me to understand my way, for your love, support and assistance in lab.
Last but not least , I want to express my sincere gratitude to my husband Mikko for the constant support, motivation, rational view on different things and your love, which does wonders.
Kuopio, 23 August 2019 Elena Puris
LIST OF ORIGINAL PUBLICATIONS
This dissertation is based on the following original publications:
I Puris E, Gynther M, Huttunen J, Petsalo A and Huttunen K M. L-type amino acid transporter 1 utilizing prodrugs: how to achieve effective brain delivery and low systemic exposure of drugs. Journal of Controlled Release 10: 93-104, 2017.
II Puris E, Gynther M, de Lange E.C.M, Auriola S, Hammarlund-Udenaes M, Huttunen K M and Loryan I. Mechanistic study on the use of the L-type amino acid transporter 1 for brain intracellular delivery of ketoprofen via prodrug: a novel approach supporting the development of prodrugs for intracellular targets. Molecular Pharmaceutics 16(7): 3261-3274, 2019
III Puris E, Gynther M, Huttunen J, Auriola S and Huttunen K M. L-type amino acid transporter 1 utilizing prodrugs of ferulic acid revealed structural features supporting the design of prodrugs for brain delivery. European Journal of Pharmaceutical Sciences 129: 99-109, 2019.
The publications were adapted with the permission of the copyright owners.
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CONTENTS
ABSTRACT ... 7
TIIVISTELMÄ ... 9
ACKNOWLEDGEMENTS ...13
1 INTRODUCTION ...25
2 REVIEW OF THE LITERATURE ...27
2.1 The barriers of the central nervous system ...27
2.2 The blood-brain barrier and the neurovascular unit ...27
2.3 Transport across the blood-brain barrier ...28
2.3.1 Passive diffusion ...29
2.3.2 Carrier-mediated transport ...31
2.3.3 Vesicular trafficking ...32
2.4 Drug metabolism in the brain ...33
2.5 General principles and methods in neuropharmacokinetics ...34
2.5.1 Plasma protein binding ...35
2.5.2 Rate of permeation ...36
2.5.3 Extent of brain delivery ...37
2.5.4 Intra-brain distribution ...38
2.6 Prodrug approach for brain delivery ...40
2.6.1 Changes in drug physicochemical properties via prodrug approach .40 2.6.2 Carrier-mediated prodrug approach ...41
2.6.3 Bioconversion of prodrugs in the brain ...43
2.7 LAT1 as an effective tool for targeted brain delivery ...45
2.7.1 Physiological function of the transporter ...46
2.7.2 Transporter expression in tissues ...47
2.7.3 LAT1 in central nervous system diseases ...48
2.7.4 Drug delivery via LAT1 ...48
3 AIMS OF THE STUDY ...52
4 MATERIALS AND METHODS ...53
4.1 Design and synthesis of prodrugs ...53
4.2 LAT1-mediated uptake of prodrugs in vitro ...54
4.3 Bioconversion of prodrugs in vitro ...55
4.4 Animals ...56
4.5 Plasma and nonspecific tissue binding ...56
4.6 Blood-brain barrier permeation using in situ perfusion ...57
4.7 Single dose pharmacokinetic study in mice ...57
4.8 Determination of intra-brain distribution ...58
4.8.1 Evaluation of intra-brain distribution ...58
4.8.2 LAT1 contribution to intra-brain distribution of prodrug ...59
4.8.3 Estimation of neuroPK parameters of prodrug ...59
4.9 Impact of prodrug distribution on LAT1 function and protein expression ....61
4.10 Bioanalytical methods ...61
4.10.1Sample preparation ...61
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4.10.2Instrumentation and analytical methods ... 61
4.11 Data analysis ... 63
5 RESULTS AND DISCUSSION ... 65
5.1 Development of LAT1-utilizing prodrugs of ketoprofen ... 65
5.1.1 Structure-PK relationship analysis ... 66
5.1.2 Intra-brain distribution study of LAT1-utilizing prodrug of ketoprofen 71 5.1.3 Effect of ketoprofen prodrug on LAT1 function and protein expression ………73
5.2 Development of LAT1-utilizing prodrugs of ferulic acid ... 74
5.2.1 Confirmation of the findings from structure-PK analysis ... 74
5.2.2 Interspecies difference in bioconversion of ester-based prodrug ... 78
5.3 Optimal strategy for development of transporter-utilizing prodrugs exemplified by LAT1 ... 78
5.3.1 Parent drug ... 78
5.3.2 Design and synthesis of LAT1-utilizing prodrugs………..79
5.3.3 Selection of prodrugs utilizing LAT1 ... 79
5.3.4 Selection of prodrugs crossing the BBB ... 82
5.3.5 Selection of prodrugs releasing the parent drug in the brain………..82
5.3.6 Selection of prodrugs releasing the parent drug in the brain intracellular compartment………..83
5.3.7 Effect of the prodrug on transporter protein expression and function83 5.3.8 Translation to human and neuropathological conditions ... 84
6 CONCLUSIONS ... 85
REFERENCES ... 87
ABBREVIATIONS
ABC ATP binding cassette AD Alzheimer’s disease ADA Adenosine deaminase
ADME Absorption, distribution, metabolism, and excretion ANOVA Analysis of variance
ARPE-19 Human retinal pigmented epithelial cells Atot,brain Total drug amount per g brain
AUCtotal,plasma Area under the total plasma drug concentration−time curve
AUCtotal,tissue Area under the total tissue concentration–time curve
AUCu,brainECF Area under the unbound brain ECF concentration–time curve
AUCu,brainICF Area under the unbound brain ICF concentration–time curve
AUCu,brainISF Area under the unbound brain ISF concentration–time curve
AUCu,plasma Area under the unbound plasma drug concentration−time curve
AUCu,tissue Area under the unbound tissue drug concentration−time curve
aECF Artificial extracellular fluid AJ Adherens junction
AMT Absorptive-mediated transcytosis ATP Adenosine triphosphate
APP Amyloid precursor protein BBB Blood-brain barrier
BCSFB Blood-cerebrospinal fluid barrier
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BCRP Breast cancer-resistance protein BUI Brain uptake index
Caco-2 Human epithelial colorectal adenocarcinoma cells CES Carboxylesterases
Clin Net influx clearance from blood to brain Clout Net efflux clearance from the brain CMA Combinatory mapping approach CMT Carrier-mediated transport CNS Central nervous system COMT Catechol O-methyltransferase COX Cyclooxygenase
Cpf Drug concentration in the perfusion buffer during the in situ brain perfusion CSF Cerebrospinal fluid
Css,brain Total concentration of the drug in brain at steady state conditions Css,plasma Total concentrations of the drug in plasma at steady state conditions Ctot,blood Total drug concentration in blood
Cu,cell Average concentration of unbound drug in brain parenchymal cells CYP Cytochrome P450
D Dilution factor ECF Extracellular fluid
EPT Enzyme Prodrug Therapy
fu,plasma Fraction of unbound drug in plasma fu,tissue Fraction of unbound drug in tissue GLUT Glucose transporter
GST Glutathione S-transferases HBSS Hank’s balance salt solution HNMT Histamine N-methyltransferase
HPLC-UV High-performance liquid chromatography with ultraviolet detection ICF Intracellular fluid
IC50 Concentration of an inhibitor at which the response (or binding) is reduced by half
i.p. Intraperitoneal route of administration i.v. Intravenous route of administration ISF Interstitial fluid
JAM Junctional adhesion molecules Kin Unidirectional transfer constant Km Constant of Michaelis-Menten kinetics
Kp Ratio of total brain to total plasma drug concentrations Kp,uu Ratio of brain ISF to plasma unbound drug concentrations Kp,uu,cell Ratio of brain ICF to ISF unbound drug concentrations LAT1 L-type amino acid transporter 1
LC-MS/MS Liquid chromatography-tandem mass spectrometry logD Distribution coefficient
LPS Lipopolysaccharide
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MCF-7 Human breast adenocarcinoma cell line (acronym of Michigan Cancer Foundation-7)
MCT Monocarboxylic acid transporter MDR Multidrug resistance protein MDSK Madin-Darby canine kidney cells mRNA Messenger Ribonucleic acid
MRP Multidrug resistance-associated protein NAT N-acetyltransferases
neuroPK Neuropharmacokinetics NVU Neurovascular unit OAT Organic anion transporter OCT Organic cation transporter
OATP Organic anion transporting polypeptide PBS Phosphate-buffered saline
PD Pharmacodynamics
PET Positron emission tomography P-gp P-glycoprotein
PK Pharmacokinetics PSEN Presenilin
PS Permeability surface
QSAR Quantitative structure-activity relationship
Qtotal Drug concentration in the brain measured during in situ brain perfusion
RMT Receptor-mediated transport SD Standard deviation
SEM Standard error of the mean SLC Solute carrier
SPECT Single-photon emission computed tomography SVCT Sodium-dependent vitamin C transporter TJ Tight junction
T3 Triiodothyronine T4 Thyroxine
TPMT Thiopurine S-methyltransferase TPSA Topological polar surface area UGT UDP-glucuronosyltransferases Vblood Blood volume per g brain
Vcell Physiological fractional volume of brain parenchymal cells Vbrain,ISF Physiological fractional volume of ISF
Vmax Maximum transport velocity
Vu,brain Unbound volume of distribution in the brain
Vu,cell Unbound volume of distribution in the brain parenchymal cells
3D Three-dimensional space
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1 INTRODUCTION
The diseases of the central nervous system (CNS), such as neurological disorders and brain cancers remain one of the most important reasons for disability and death globally (Group, 2017). Worldwide, the burden of CNS diseases has increased significantly over the past 25 years due to aging and growing population, which leads to the rising demand for development of CNS drugs (Group, 2017). However, since 1990, there has been a deсrease in development of CNS drugs, which has been reflected in substantial slowdown of both preclinical and clinical trials due to the lack of efficacy and safety issues (Kesselheim et al., 2015).
What are the main obstacles to the development of CNS drugs? First, the targets to affect the cause of some diseases for example Alzheimer’s disease (AD) are still uncertain. Second, animal models resembling the disease pathogenesis in humans, as well as clinical end-points or validated biomarkers, as a measure of drug effects on disease course, are limited. Finally, the poor drug delivery across the blood-brain barrier (BBB) and the lack of adequate information about the concentration-time profile of unbound drug at the brain target-site hinders the development of CNS drugs.
To interact with a target and produce the subsequent therapeutic effect, a drug needs to be diffused or transported from blood to the brain across the BBB. However, the passage of the molecules via the brain capillary endothelial cells is highly protected and regulated in order to avoid entrance of harfmul xenobiotics to the brain. Therefore, the defensive function of the BBB represents one of the main obstacles in the development of CNS drugs.
Nevertheless, the BBB allows entering of endogenous compounds and nutrients from blood and eliminating metabolites and other potentially harmful molecules to maintain CNS function. Many compounds such as glucose and amino acids can cross the BBB via transporters expressed at the membrane of the brain capillary (Pardridge and Oldendorf, 1977). The development of the drugs or prodrugs resembling the substrates of the transporters has been succesfully used for drug delivery (Rautio et al., 2008a). However, the development of the transporter-utilizing prodrugs for brain delivery is a complex process involving both the delivery of prodrug across the BBB and cellular barrier of brain parenchyma as well as bioconversion of the parent drug at the site of action.
Several methods, starting from simple uptake experiments in BBB in vitro models to complex cerebral microdialysis technique, enabling investigation of BBB transport of drugs and distribution inside the brain has been developed. These methods together with application of pharmacokinetic (PK) principles can aid in estimation of the concentration of the parent drug released from the transporter-utilizing prodrug at the target site inside the brain. This provides important information about the efficacy of brain delivery via transporter-mediated prodrug approach.
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The aim of this doctoral thesis was to investigate the efficacy of drug delivery to the brain, in particular into the brain intracellular compartment via prodrugs utilizing influx transporters at the BBB exemplified by L-type amino acid transporter 1 (LAT1). In order to achieve this goal, PK principles and different in vitro, in situ and in vivo methods were used. The second aim was to propose the strategy for preclinical development of transporter-utilizing prodrugs for brain delivery of drugs with the brain intracellular targets.
2 REVIEW OF THE LITERATURE
2.1 THE BARRIERS OF THE CENTRAL NERVOUS SYSTEM
The CNS comprised the brain and spinal cord is a central player controlling normal functioning of the body via integrating and coordinating the input signals and providing an appropriate response. Due to the complex physiology and function, it is crucial to maintain homeostasis of the microenvironment in the CNS (Abbott et al., 2010). There are three main cellular barriers at the blood-CNS interfaces regulating nutrient flux, waste product removal, access for immune cells and restricting the entry of toxicants and pathogens (Abbott, 2013).
These three barriers include the BBB, the blood-cerebrospinal fluid barrier (BCSFB) and the arachnoid barrier. The BBB separating blood and the brain interstitial fluid (ISF) is formed by the brain capillary endothelial cells in combination with extracellular basement membrane, astrocytes, microglia and pericytes (Hawkins and Davis, 2005). The BCSFB between the blood and the cerebrospinal fluid (CSF) is formed by the epithelium of the choroid plexus responsible for the secretion of CSF. The arachnoid barrier consisting of tight-junctioned cells constitutes the interface between the CSF in the subarachnoid space from the bones and extracellular fluids of dura mater (Vandenabeele et al., 1996).
In terms of drug delivery, the BBB plays the most important role due to extensive blood capillary network. The surface area of the cerebral microvessels accounts an average of 100–200 cm2 per gram of brain tissue in adult human (Crone, 1963; Gross et al., 1986), which is available for the exchange of compounds between the blood and brain parenchyma. Moreover, the neuronal cells are located about 10–20 μm from the nearest cerebral microvessel (Schlageter et al., 1999). The restrictive properties of this barrier are attributed to the presence of tight junctions (TJ) between the brain capillary endothelial cells and lack of fenestrations (Wolburg and Lippoldt, 2002, Fenstermacher and Joseph, 1998). In addition, metabolizing enzymes and specific efflux transporters and receptors play important role in barrier functioning.
The ISF bulk flow to the CSF facilitates the elimination of substances from the brain providing the maintenance of the microenvironment (Rosenberg et al., 1980).
2.2 THE BLOOD-BRAIN BARRIER AND THE NEUROVASCULAR UNIT
For nearly 100 years since the first mentioning by Stern and Gautier, the BBB has been known as a main protector of the brain from toxic compounds and other xenobiotics as well as the provider and exchanger of essential nutrients (Stern and Gautier, 1921).
As it has been mentioned before, the main features of the BBB as a ‘physical’ barrier are lack of fenestrations and the precense of TJs. The latter composed of a 3D complex morphology of transmembrane proteins such as occludin, claudins and junctional
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adhesion molecules (JAM) (Cording et al., 2013). These proteins interact with their counterparts on adjacent cell plasma membranes and form apposition spanning the intercellular cleft to restrict paracellular diffusion (Farquhar and Palade, 1963). TJs are in close interaction with the adherens junctions (AJs), actin skeleton and perivascular cells such as astrocytes and pericytes (Luissint et al., 2012). In addition, TJs establish and regulate the polarization of the BBB endothelial cells (McCaffrey et al., 2007). AJs, in turn, play important role in development, maturation and stabilization of contacts between cells (Paolinelli et al., 2011).
The BBB is a dynamic barrier, which functionality is dependent on the presence of several transporters, receptors and metabolizing enzymes as well as interaction with group of cells. These cells form “neurovascular unit” (NVU) and consist of pericytes, perivascular astrocytic end-feet, mast cells, microglia and immune cells (Fig. 1)(Hawkins and Davis, 2005). The close communication between the NVU cells is regulated by multiple signalling system providing protective function of the BBB.
The disturbance of this microenvironment can lead to impairment of the barrier properties and CNS diseases (Zhao et al., 2015, Banks et al., 2018).
Figure 1. Schematic representation of the BBB endothelial cells surrounded by the NVU cells.
2.3 TRANSPORT ACROSS THE BLOOD-BRAIN BARRIER
Despite the presence of paracellular TJs and AJs at the BBB, the glycocalyx, bulk flow and metabolic enzymes restricting the brain exposure to compounds, there are several routes by which essential nutrients and other molecules can be transported across the BBB (Fig. 2). In general, these routes include passive diffusion, carrier-
mediated transport and vesicular trafficking (see more detailes in chapter 2.3.1, 2.3.2, 2.3.3, respectively). These routes facilitate not only entry of endogenous compounds, but also can be used for drug delivery into the brain. In addition, immune cells such as macrophages and monocytes can be recruited to the brain in disease conditions by means of transcytosis (Davoust et al., 2008). Importantly, neuropathological conditions can affect the integrity and functioning of the BBB and alter transport of molecules.
Figure 2. Different routes of transport across the BBB. Passive diffusion and facilitated carrier- mediated transport across the lipid bilayer occur according to the concentration gradient in both directions from blood to brain or from brain to blood. ABC - ATP binding cassette transporters; AMT - adsorptive-mediated transcytosis; RMT - receptor-mediated transport;
SLC - Solute carrier transporters.
2.3.1 Passive diffusion
During long time, transcellular passive diffusion has been considered as the only way the compounds can cross the BBB and most of the brain delivery strategies were based on improvement of lipophilicity of the drugs. Passive diffusion is an energy- independent process occuring according to the concentration gradient of the unbound compounds on both sides of the cell membrane, which is directly proportional to the diffusion rate (Levin, 1980). The passive diffusion can be either paracellular (between the endothelial cells) or transcellular (through the endothelial cells)(Fig. 2). The diffusion between cells is considered negligible due to the presence of TJs between the endothelial cells. In contrast, highly lipophilic compounds with small molecular size such as heroin and diazepam can be transported across the BBB via transcellular diffusion.
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In terms of passive diffusion, there is a correlation between BBB permeability and physicochemical properties of the compounds with some exceptions (Table 1). A strong correlation between the drug lipophilicity and its BBB permeation rate was observed, where increase in lipophilicity resulted in higher permeability (Neuwelt et al., 2008). It can be explained by the fact, that increased lipophilicity of compounds leads to enhanced nonspecific brain tissue binding in the brain parenchyma and drives the transcellular diffusion according to the concentration gradient (Summerfield et al., 2007). However, one should remember, that only unbound drug can interact with the target within the brain. Therefore, higher brain nonspecific tissue binding and BBB permeation due to lipophilicity should be considered with caution. In addition, Waring (2009) investigated the relationship between permeability of structurally diverse compounds in human epithelial colorectal adenocarcinoma cells (Caco-2) and suggested the limits for the molecular weight and logD in order to achieve the BBB permeation (Table 1) (Waring, 2009). The drugs with high potential to form hydrogen bond decreases the permeation across the BBB (Pajouhesh and Lenz, 2005). Molecular topological polar surface area (TPSA) has been used to predict BBB penetration of drugs with the upper limit of 90 Å2 (Pajouhesh and Lenz, 2005; van de Waterbeemd et al., 1998). In general, weak basic and acidic compounds in uncharged form can pass across the membrane, therefore the negative logarithm of the acid dissociation constant, pKa, of the drug at the physiological pH 7.4 will also show effect on the BBB permeation (Fischer et al., 1998).
Table 1. Physicochemical properties associated with the BBB permeation of drugs.
Parameter Limit values Reference
Molecular weight vs. logD
<300 >0.5 300–350 >1.1 350–400 >1.7 400–450 >3.1
Waring, 2009
TPSA < 90 Å2 van de Waterbeemd et
al.,1998
H-bond donor atoms < 3 Pajouhesh and Lenz,
2005
H-bond acceptor atoms < 7 Pajouhesh and Lenz,
2005
pKa 4-10 Fischer et al., 1998
2.3.2 Carrier-mediated transport
The carrier-mediated transport (CMT) occurs after the interaction between transport protein expressed in the membrane of the cerebral endothelial cells and endogenous compounds or xenobiotics, which can be both lipid soluble or hydrophilic molecules.
The transporters can be expressed in either luminal or abluminal membrane or both sides of the endothelial cells (Table 2). Depending on transporter expression and function, the carrier-mediated passage of molecules (Fig. 2) can be unidirectional and denote either influx or efflux across the BBB or bidirectional meaning the transport of solutes in both directions across the BBB according to the concentration gradient (Begley, 2004). In addition, the CMT can be active or facilitated (also known as equilibrative transport).
The facilitated transport is employed by concentration gradient without any energy consumption to drive the process. Active transporters refer to primary or secondary carriers and can deliver molecules against the concentration gradient.
Primary transporters (e.g. ABC transporters) use the energy of the hydrolytic reaction of adenosine triphosphate (ATP) and efflux the substrates from a cell against the concentration gradient, while secondary transporters deliver molecules across the BBB in antiport or symport of ions such as H+, Na+, K+, Cl-, etc. (O'Kane et al., 2004, Dallas et al., 2006). The uptake of molecules via transporters is a temperature- dependent and saturated process following Michaelis-Menten kinetics. In addition, the process can be also affected by competitive (competing for substrate binding), non-competitive (modulating binding to substrate allosterically), uncompetitive (hindering conformational modification of the complex “substrate-transporter”) or mixed-type inhibitors (Krupka, 1983).
The carrier-mediated uptake of different essential nutrients such as amino acids, vitamins, glucose and nucleosides occur across the BBB to keep the brain homeostasis. The same transporters can facilitate the influx of therapeutic compounds. In addition, the efflux transporters can facilitate transport of the drugs from the brain to the plasma, which results in low brain uptake of the drugs, substrates of the efflux transporters. The examples of the transporters expressed at the BBB, their location and substrate specificity are presented in Table 2. The BBB transporters include solute carrier (SLC) family represented by facilitative glucose transporter 1 (GLUT1/SLC2A1), L-type amino acid transporter 1 (LAT1/SLC7A5), monocarboxylic acid transporter 1 (MCT1/SLC16A1), organic anion transporter 3 (OAT3/ SLC22A8), organic anion transporting polypeptide 1A2 (OATP1A2/
SLCO1A2). The efflux transporters are represented by important family of ATP binding cassette (ABC) transporters including P-glycoprotein (P-gp, MDR1/ABCB1), breast cancer-resistance protein (BCRP/ABCG2), and multidrug resistance- associated protein 4 (MRP4/ABCC4), which are considered to be a major obstacle for brain delivery of drugs (Uchida et al., 2011). In addition, it is important to remember that transporters expressed not only at the BBB, but also at the membrane of brain parenchymal cells and BCSFB and can influence elimination and distribution of the drugs in the brain.
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Table 2. The example of transporters, which protein expression has been quantified at the human BBB and their substrates (Ohtsuki and Terasaki, 2007; Uchida et al., 2011)
Gene name (alternative transporter name)
Localization in
plasma membrane Substrates
Protein expression
fmol/µg
Efflux transport
ABCG2 (BCRP) Luminal site glutathione, folic acid, mitoxantrone, topotecan,
dantrolene
8.14 ± 2.26
ABCB1 (P-gp, MDR1) Luminal site vincristine, quinidine,
verapamil 6.06 ± 1.69
ABCC4 (MRP4) Luminal site E217βG, methotrexate,
topotecan 0.195 ± 0.069
Influx transport
SLC2A1 (GLUT1) Luminal, abluminal
sites D-glucose 139 ± 46
SLC7A5 (LAT1) Luminal, abluminal sites
Large neutral amino acids,
L-Dopa, gabapentin 0.431 ± 0.091 SLC16A1 (MCT1) Luminal, abluminal
sites L-Lactate, monocarboxylates 2.27 ± 0.85
2.3.3 Vesicular trafficking
For normal brain functioning, the transport of large molecular weight compounds such as proteins and peptides is required. These large-molecular cargos can be transported across the BBB via receptor-mediated transport (RMT) or non-specific routes such as cationization and adsorptive-mediated transcytosis (AMT) (Fig. 2).
The RMT involves the formation of the vesicles carrying the molecules across the BBB exemplified by nutrients such as insulin and leptin transported via insulin and leptin receptors, respectively (Duffy and Pardridge, 1987; Golden et al., 1997). The large molecule interacts with a corresponding receptor at the apical side of the BBB cell plasma membrane followed by initiation of the endocytosis process by invagination of receptor-molecule complex and formation of the intracellular trafficing vesicles (Brown and Greene, 1991). The process is finalized by sorting and transporting the vesicles containing the receptor-molecule complex to the basolateral side of the polarized membrane of the endothelial cell, where the release occurs
without disturbance of the barrier properties. In addition, the vesicle can be reversed back to the luminal membrane of the endothelial cells followed by release of molecule into the blood. Alternatively, the vesicle containing the receptor-molecule complex can be transported to lysosomes where it will be degraded. The RMT is a selective process as it requires the initial binding of a molecule to specific receptor present in the plasma membrane of the endothelial cells.
In contrast, the AMT is based on nonspecific electrostatic interactions providing penetration of different cationic proteins such as histone and albumin (Bickel et al., 2001; Herve et al., 2008). The initiation of the process starts when positively charged macromolecular moieties bind to negatively charged plasma membrane of the endothelial cells (Pardridge, 1991). The disadvantage of this transport route is a lack of targeting specificity and possible distribution of compounds to other organs (Bickel et al., 2001).
2.4 DRUG METABOLISM IN THE BRAIN
The elimination of drugs from the brain can occure not only by efflux transport across the BBB and ISF bulk flow, but also via metabolism in the endothelial cells of the brain capillary and inside the brain. Both phase I and phase II metabolizing enzymes are expressed in the BBB endothelial cells and brain parenchymal cells. The enzymatic biotransformation facilitates protection of the brain from the drugs, toxic compounds and neurotransmitters circulating in the blood. The biotransformation at the BBB is likely to change the response to drug and may lead to the therapeutic failure if the drug is a substrate of the specific enzyme(s) expressed at the BBB. This can be a result of chemical inactivation of the drug due to metabolism or alteration of drug polarity, which in turn will affect the ability of drug to cross the BBB. In addition, metabolites can be substrates of efflux transporter(s). The presence of metabolizing enzymes in glial cells such as astrocytes and microglia constitute a second defence line to protect the CNS from entering of drugs and xenobiotics.
Importantly, several cytochrome P450 (CYP) isoforms expressed in the brain microvessels are different to those expressed in hepatocytes. Thus, the main isoforms detected at the BBB were CYP1B1 and CYP2U1 (Shawahna et al., 2011, Decleves et al., 2011). In addition, the protein expression in the brain microvessels was found for the following drug-metabolizing enzymes: CYP1B1, CYP2U1, glutathione S- transferase P1 (GSTP1), glutathione S-transferases M (GSTM2-3, GSTM5) and glutathione S-transferase omega 1 (GSTO1) (Shawahna et al., 2011). The mRNA levels of CYP2D6, CYP2J2, CYP2E1, CYP2R1 were detected (Shawahna et al., 2013).
These enzymes mediate metabolism of several drugs exemplified by amodiaquine, melatonin (CYP1B1); ergocalciferol, ebastine, astemizole (CYP2J2), ethanol (CYP2E1); ergocalciferol and colecalciferol (CYP2R1)(Schuster, 2011; Zakhari, 2006;
Ma et al., 2005; Shimada et al., 1997; Aiba et al., 2006). The expression of GSTs, phase II enzymes, is highly abundant at the BBB as it is responsible for detoxification of oxidative substances (Shawahna et al., 2011). The enzymes mediate
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biotransformation of several antiepileptic drugs (Shang et al., 2008) Other phase II enzymes identified in the brain microvessels includes the histamine N- methyltransferase (HNMT), catechol O-methyltransferase (COMT), and thiopurine S-methyltransferase (TPMT) (Shawahna et al., 2013). In contrast to liver enzyme expression profile, UDP-glucuronosyltransferases (UGTs) and N-acetyltransferases (NATs) enzymes were not detected at the BBB (Shawahna et al., 2013).
In neurons and glial cells, several CYP isoforms were detected with different distribution in various regions of the brain (Dutheil et al., 2009). Those include CYP1B1 with highest expression in dura mater and CYP2D6, CYP2E1, CYP2J2, CYP2U1, CYP46A1 expressed at smaller extent (Dutheil et al., 2009). Interestingly, in the brain, CYP46A1 (which is virtually absent in the liver and highly expressed in the brain), CYP1B1 and CYP2U1 possess higher expression than in liver. CYP2R1 has similar expression profile in the brain and liver, while CYP2J2 and CYP2D6 account about 10% and 2%, of the liver levels, respectively (Petryszak et al., 2016). Other CYPs highly expressed in the liver (CYP1A1, CYP2C8, CYP2C9, CYP2C19, CYP3A4, CYP3A5, and CYP2E1) showed negligible expression in the human brain (Petryszak et al., 2016). Other enzymes, such as carboxylesterases (CESs) have been identified in human brain extracts (Hojring and Svensmark, 1976). The high expression of phase II enzymes such as GST, COMT and SULT1A4 was detected, while other important drug-metabolizing enzymes (SULT1A1, UGT1A6, UGT2B7, NAT1, and NAT2) were not expressed at any meaningful extent (Petryszak et al., 2016). However, the expression of the most drug-metabolizing enzymes can be altered due to disease.
2.5 GENERAL PRINCIPLES AND METHODS IN NEUROPHARMACOKINETICS
After administration by specific route (Fig. 3) and before reaching the brain, a drug generally undergoes several PK processess including absorption (except intravenous or intraarterial routes), distribution, metabolism, and excretion (ADME). These processes can influence the bioavailability of the drug and its access to the site of action inside the brain. In addition, the drug can distribute to other organs than brain and cause side effects. Therefore, along with determination of parameters decribing brain delivery of drugs, the knowledge of their ADME properties is essential in development of the effective CNS treatments.
The investigation of drug delivery across the BBB and within the brain known as neuropharmacokinetics (neuroPK) is important for understanding of pharmacodynamic (PD) responses. The study of neuroPK parameters is based on the main PK postulates, that only unbound drug can cross the cellular membrane, be metabolised and interact with a target inside the brain. However, there are specific aspects of neuroPK caused by the unique composition and physiology of the brain and CNS barrier properties. In general, there are three main determinants of the drug delivery to the brain, such as rate and extent of the BBB permeation as well as intra- brain distribution. They will be discussed in this chapter.
Figure 3. A schematic illustration of the main pharmacokinetic processes after the drug administration including distribution across the blood-brain barrier (BBB) and within the brain.
CB – brain parenchymal cellular barrier, CSF – cerebrospinal fluid, GI tract – gastrointestinal tract, ICF – intracellular fluid, ISF – interstitial fluid.
2.5.1 Plasma protein binding
As unbound drug concentration in plasma determines the level of the drug available for the transport across the BBB, the measurement of drug binding to plasma proteins is required for estimation of the BBB transport. The introduction of high-throughput equilibrium dialysis method has provided the opportunity for simple and fast measuring of unbound fraction in plasma (fu,plasma) and other tissues (fu,tissue) (Kalvass and Maurer, 2002, Kariv et al., 2001). The equilibrium dialysis chambers are separated by a semipermeable membrane preventing the mixture of the plasma or investigated tissue matrix and dialysis buffer. The plasma or tissue homogenate containing the drug are supplied to the “sample” chamber, while dialysis buffer is added to “buffer” chamber. The drug diffuses into the buffer according to its concentration gradient. The method is based on the assumption of reversible interactions between drug and plasma/tissue matrix with the subsequent equilibration of unbound and bound drug molecules. Thus, the ratio of the drug concentration in the buffer and the sample chambers (taking into account dilution) at equilibrium provides the estimate of the unbound fractions (Eq. 1) (Kalvass and Maurer, 2002). Importantly, the drug concentration used in equilibrium dialysis should be much higher than saturation limit of a specific target binding. Thus, being negligible to the determined total binding, it enables estimating nonspecific tissue binding.
36
𝑓𝑢,𝑡𝑖𝑠𝑠𝑢𝑒= 𝑓𝑢,ℎ𝑜𝑚𝑜𝑔𝑒𝑛𝑎𝑡𝑒
𝐷 − (𝐷 − 1) × 𝑓𝑢,ℎ𝑜𝑚𝑜𝑔𝑒𝑛𝑎𝑡𝑒 (1) where D is the dilution factor of the media; fu,homogenate is the ratio of the investigated compound concentration measured in the sample chamber and buffer chamber after the equilibrium dialysis.
The determined fu,plasma is used for calculation of the unbound plasma concentration over time (AUCu,plasma) according to Eq. 2:
𝐴𝑈𝐶𝑢,𝑝𝑙𝑎𝑠𝑚𝑎 = 𝐴𝑈𝐶𝑡𝑜𝑡,𝑝𝑙𝑎𝑠𝑚𝑎× 𝑓𝑢,𝑝𝑙𝑎𝑠𝑚𝑎 (2) where AUCtotal,plasma is an area under the concentration−time curve for the total (both unbound and bound) drug in plasma.
2.5.2 Rate of permeation
The measurement of the BBB permeation rate provides an estimate of the unidirectional transport of a drug across the BBB. Importantly, the rate of permeation does not provide information about the extent of equilibrated drug across the BBB at steady state, but it shows how fast is the process of drug delivery from blood to brain across the BBB. There are several techniques developed for the assessment of the permeation rate from blood to brain also known as influx clearance (Clin): in vivo brain uptake index (BUI) method (Oldendorf, 1970), cerebral microdialysis technique (de Lange et al., 1997; Hammarlund-Udenaes, 2000), in situ brain perfusion (Takasato et al., 1984) or in vitro cell uptake studies in the models of the BBB as well as Madin- Darby canine kidney cells (MDSK) and the human epithelial Caco-2 cells (Abbott, 2004). The efflux clearance (Clout) describes the net passage out of the brain and includes passive and active BBB transport as well as metabolism in the brain and elimination to the CSF by bulk flow (Hammarlund-Udenaes, 2000). Clout can be evaluated using the brain efflux index method (Kakee et al., 1996).
In the current project, in situ brain perfusion technique was selected for investigation of the BBB permeation rate of the compounds, as it provides a rapid and sensitive evaluation of the permeation across the BBB at its physiological state without influence of the peripheral metabolism. Moreover, the method enables controlling the composition and flow rate of the perfusate fluid containing investigated drug. In this method, the determination of the Clin is based on evaluation of unidirectional transfer constant (Kin) and permeability surface area product (PS product). Both parameters describe measurements of clearances, rather than rates per se (Hammarlund-Udenaes et al., 2008). Kin in µL/s/g brain is calculated using Eq. 3:
𝐾𝑖𝑛 = 𝑄𝑡𝑜𝑡𝑎𝑙
𝐶𝑝𝑓× 𝑡 (3)
where Qtotal is the drug concentration in the brain, Cpf is the drug concentration in the perfusion buffer and t is the time of perfusion.
The Kin can be transformed to the PS product using the model introduced by Crone and Renkin (Crone and Levit, 1984) according to Eq. 4 (Killian et al., 2007):
𝑃𝑆 = −𝐹 × ln[1 − (𝐾𝑖𝑛⁄ )]𝐹 (4) where F is the flow rate investigated with lipophilic marker for cerebral blood flow such as diazepam.
The rate of BBB transport is dependent on the drug physiochemical properties and route of the permeation such as passive permeability for lipophilic drugs, or active CMT for drugs - transporter substrates. In chronic treatment, the rate of BBB permeation is not as vital as the extent of transport across the BBB.
2.5.3 Extent of brain delivery
The extent of the BBB transport is an important characteristic of drug delivery to the brain. It is described by the drug amount or concentration in the brain at steady state in relation to blood levels of the drug. In general, the extent of brain delivery is estimated by measuring ratio of total concentrations in the brain and plasma (Kp) or the ratio of unbound concentrations in the brain ISF and plasma (Kp,uu) according to Eq. 5 and 6, respectively (Hammarlund-Udenaes et al., 2008):
𝐾𝑝= 𝐴𝑈𝐶𝑡𝑜𝑡𝑎𝑙,𝑏𝑟𝑎𝑖𝑛
𝐴𝑈𝐶𝑡𝑜𝑡𝑎𝑙,𝑝𝑙𝑎𝑠𝑚𝑎
= 𝐶𝑠𝑠,𝑏𝑟𝑎𝑖𝑛
𝐶𝑠𝑠,𝑝𝑙𝑎𝑠𝑚𝑎
(5)
𝐾𝑝,𝑢𝑢=𝐴𝑈𝐶𝑢,𝑏𝑟𝑎𝑖𝑛𝐼𝑆𝐹
𝐴𝑈𝐶𝑢,𝑝𝑙𝑎𝑠𝑚𝑎 = 𝐶𝑢,𝑠𝑠,𝑏𝑟𝑎𝑖𝑛 𝐶𝑢,𝑠𝑠,𝑝𝑙𝑎𝑠𝑚𝑎
= 𝐶𝑙𝑖𝑛
𝐶𝑙𝑜𝑢𝑡= 𝐾𝑝× 𝑓𝑢,𝑏𝑟𝑎𝑖𝑛 𝑓𝑢,𝑝𝑙𝑎𝑠𝑚𝑎
(6)
where Css,brain and Css,plasma are total concentrations in the brain and plasma at steady state conditions, respectively, e.g. during a constant rate drug infusion; AUCtotal,brain
and AUCtotal,plasma are areas under the concentration−time curve for total (both unbound and bound) drug in the brain and plasma, respectively; Cu,ss,brain and Cu,ss,plasma are unbound concentrations in the brain and plasma at steady state conditions, respectively, AUCu,brainISF and AUCu,plasma are the areas under the concentration−time curve for unbound drug in the brain ISF and plasma, respectively; fu,plasma and fu,brain are the fractions of unbound drug in plasma or brain homogenate, respectively, estimated using in vitro equilibrium dialysis technique (Kalvass and Maurer, 2002, Banker et al., 2003). The calculation of Kp,uu using unbound fractions in brain (Eq. 6) is possible only in case of equal intra- and extracellular unbound concentrations of drug in the brain.
The values of Kp are affected by both the transport across the BBB as well as plasma and brain tissue binding. As only unbound drug can cross the BBB, Kp,uu provides more relevant information in terms of the net flux across the BBB. Thus,
Kp,uu is the combination of the influx and efflux transport across the BBB and can be
therefore defined as the ratio of Clin and Clout (Eq .6). When Clin and Clout are equal,
38
Kp,uu is close to unity, thus indicating that drug BBB delivery is dominated by passive
processes (Hammarlund-Udenaes, 2000). If to assume the low impact of metabolism in the brain and low ISF bulk flow, Kp,uu larger than unity (e.g. oxycodone, nicotine) will indicate the dominating an active influx of the drug to the brain ISF, while Kp,uu lower than unity (e.g. atenolol, morphine) will show that an active efflux of the drug is dominating (Bostrom et al., 2006, Tega et al., 2013, Bostrom et al., 2008, Friden et al., 2009b).
The extent of brain delivery can be measured after an intravenous (i.v.) or intraperitoneal (i.p.) injection of investigated drug followed by monitoring its concentration in blood and brain. The method is fast and simple, and therefore can be used for the screening of the drug candidates. The limitation of the method is the restriction to one individual brain and blood concentration measurement at terminal sampling. The obtained AUCtotal in plasma and brain are used for calculation of Kp
(Eq. 5) or can be combined with unbound fraction determined by equilibrium dialysis to calculate Kp,uu (Eq. 6).
The gold standard method for evaluation of the BBB permeation is cerebral microdialysis technique, which is used for continuous monitoring of unbound concentrations of the drug in the brain ISF and blood over time (de Lange et al., 1997) via inserted probes into the brain parenchyma. Thus, the method provides direct estimation of Kp,uu. Microdialysis is a complex technique, which requires adjustment of the experimental design to methodological aspects and can be restricted to lipophilic compounds.
In addition, imaging methods such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) have been developed for evaluation of drug distribution to the brain over time in vivo (Syvanen and Hammarlund-Udenaes, 2010). However, the application of imaging techniques for investigation of the BBB transport of compounds has been restricted by the availability and high costs of relevant radiotracers.
2.5.4 Intra-brain distribution
After crossing the BBB, a drug distributes within the brain. The drug can be transported from the brain ISF into the brain parenchymal cells and bind to the cell constituents, e.g. phospholipids or cellular proteins. The parameter describing the drug distribution to the brain parenchyma is the unbound volume of distribution in the brain (Vu,brain,mL/g brain), which can me evaluated using a brain slice method in vitro (Wang and Welty, 1996, Hammarlund-Udenaes et al., 2008, Kakee et al., 1996, Loryan et al., 2013). The Vu,brain implies the relationship between the total brain concentration of the drug and its unbound concentration in the brain ISF as described in Eq. 7 (Hammarlund-Udenaes et al., 2008):
𝑉𝑢,𝑏𝑟𝑎𝑖𝑛 =𝐴𝑡𝑜𝑡𝑎𝑙,𝑏𝑟𝑎𝑖𝑛− 𝑉𝑏𝑙𝑜𝑜𝑑× 𝐶𝑡𝑜𝑡𝑎𝑙,𝑏𝑙𝑜𝑜𝑑
𝐶𝑢,𝑏𝑟𝑎𝑖𝑛𝐼𝑆𝐹 (7)
where Atot,brain is the quantified total drug amount per g brain, which includes blood present in the brain, Vblood is the blood volume per g brain, and Ctot,blood is the total drug concentration in blood.
In terms of physiological volumes of the brain fluids, the lowest possible value of Vu,brain is around 0.2 mL/g brain which is equal to volume of ISF, Vbrain,ISF (Nicholson and Sykova, 1998). The Vu,brain close to the 0.8 mL/g brain representing the water volume in the brain indicates an even distribution within the brain (Reinoso et al., 1997). In contrast, Vu,brain greater than 0.8 mL/g brain represents the distribution to the brain parenchymal cells. The inverse value of Vu,brain is considered to be equal to unbound fraction in the brain (fu,brain), only if the drug distribution within the brain parenchymal cell is primarily governed by the tissue binding, and not by active transport (Hammarlund-Udenaes et al., 2008).
As the drugs can be further distributed in and out of the brain parenchymal cells via transporters expressed at cellular membranes, the ISF concentration of unbound compound is not necessarily equal to that in the brain intracellular compartment (Wang and Zuo, 2018). The direct measurement of unbound concentrations of drugs
(Cu,cell) inside the brain parenchymal cells currently cannot be achieved in practice.
Therefore, indirect methods combining the brain slice experiments and brain homogenate method have been established to estimate Cu,cell (Friden et al., 2007). The estimation is based on several assumptions. First, the binding of the drug occurs mainly intracellularly, as the brain ISF protein content is very low and only small part of membrane surface area faces the brain ISF. Second, the estimation of Cu,cell
indicates the overall unbound drug concentration in the brain intracellular compartment, while transporter expression in parenchymal cells can vary and result in different distribution to the cells and subcellular structures.
The brain homogenate method using equilibrium dialysis technique measures the brain tissue binding related to intracellular compartment and provides estimation of unbound volume of distribution in the brain parenchymal cells (Vu,cell). The latter can be combined with Vu,brain, measured using the brain slice method to estimate unbound drug concentration ratio in intra- and extracellular compartments of the brain
(Kp,uu,cell) according to Eq. 8 (Hammarlund-Udenaes et al., 2008):
𝐾𝑝,𝑢𝑢,𝑐𝑒𝑙𝑙 = 𝐶𝑢,𝑐𝑒𝑙𝑙
𝐶𝑢,𝑏𝑟𝑎𝑖𝑛𝐼𝑆𝐹 =𝑉𝑢,𝑏𝑟𝑎𝑖𝑛− 𝑉𝑏𝑟𝑎𝑖𝑛,𝐼𝑆𝐹
𝑉𝑐𝑒𝑙𝑙× 𝑉𝑢,𝑐𝑒𝑙𝑙
(8)
where Vcell is the physiologic fractional volumes of the brain cells.
Kp,uu,cell describes the unbound drug distribution between the brain ISF and
extracellular compartment, which represents an average ratio for all parenchymal cells at steady-state. The estimation of Kp,uu,cell assumes that drugs are unbound in the brain ISF. Kp,uu,cell higher than unity characterises the intracellular distribution of the drugs, while Kp,uu,cell lower than unity represents preferably the extracellular distribution in the brain.
