ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2018

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 254

Translational Aspects of Blood-Brain Barrier Transport and BrainDistribution of Drugs in Healthand Disease

SOFIA GUSTAFSSON

ISSN 1651-6192ISBN 978-91-513-0294-2urn:nbn:se:uu:diva-347204

Dissertation presented at Uppsala University to be publicly examined in B21, Biomedicinsktcentrum (BMC), Husargatan 3, Uppsala, Friday, 18 May 2018 at 09:15 for the degree ofDoctor of Philosophy (Faculty of Pharmacy). The examination will be conducted in English.Faculty examiner: Danica Stanimirovic (Department of Cellular and Molecular Medicine,University of Ottawa).

AbstractGustafsson, S. 2018. Translational Aspects of Blood-Brain Barrier Transport and BrainDistribution of Drugs in Health and Disease. Digital Comprehensive Summaries of UppsalaDissertations from the Faculty of Pharmacy 254. 75 pp. Uppsala: Acta UniversitatisUpsaliensis. ISBN 978-91-513-0294-2.

A high unmet medical need in the area of CNS diseases coincides with high failure rates inCNS drug development. Efficient treatment of CNS disease is constrained by limited entrance ofdrugs into the brain owing to the blood-brain barrier (BBB), which separates brain from blood.Insufficient inter-species translation and lack of methods to evaluate therapeutic, unbound,drug concentrations in human brain also contribute to development failure. Further diseaserelated changes in BBB properties and tissue composition raise a concern of altered drugneuropharmacokinetics (neuroPK) during disease. This calls for the evaluation of translationalaspects of neuroPK parameters in health and disease, and exploration of strategies for neuroPKtranslations between rodents and humans.

Positron emission tomography (PET) enables corresponding PK analysis in various species,although being restricted to measuring total, i.e. both unbound and nonspecifically bound, drugconcentrations. However, the current work shows that PET can be used for the estimationof unbound, active, brain concentrations and for assessment of drug BBB transport, ifcompensation is made for intra-brain drug distribution and binding. Adapted PET designs couldbe applied in humans where rat estimates of drug intra-brain distribution may be used withreasonable accuracy for concentration conversions in healthy humans, but preferably not inAlzheimer’s disease (AD) patients. As shown in this thesis, a high variability in nonspecificdrug tissue binding was observed in AD compared to rats and human controls that might lead tounacceptable bias of outcome values if used in PET. Furthermore, heterogeneity in drug tissuebinding among brain regions in both rodents and humans was detected and must be considered inregional investigations of neuroPK. By the use of transgenic animal models of amyloid beta andalpha-synuclein pathology, the work further suggests that the BBB is able to uphold sufficientcapacity for the transport of small molecular drugs and integrity towards large molecules despitethe presence of hallmarks representative of neurodegenerative diseases.

This thesis work provides insight into neurodegenerative disease impact on neuroPK andcontributes with translational strategies for neuroPK evaluation from preclinical investigationsto the clinic, aimed to aid drug development and optimal disease management.

Keywords: Blood-brain barrier, Neurovascular unit, Pharmacokinetics, Neurodegenerativedisease, Drug transport, Brain tissue binding, Positron emission tomography, Brain regions

Sofia Gustafsson, Department of Pharmaceutical Biosciences, Box 591, Uppsala University,SE-75124 Uppsala, Sweden.

© Sofia Gustafsson 2018

ISSN 1651-6192ISBN 978-91-513-0294-2urn:nbn:se:uu:diva-347204 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-347204)

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Gustafsson, S., Eriksson, J., Syvänen, S., Eriksson, O., Hammar-

lund-Udenaes, M., Antoni, G. (2017) Combined PET and Micro-dialysis for In Vivo Estimation of Drug Blood-Brain Barrier transport and Brain Unbound Concentrations. Neuroimage, 155:177-186

II Gustafsson, S., Lindström, V., Ingelsson, M., Hammarlund-Ude-naes, M., Syvänen, S. (2018) Intact Blood-Brain Barrier Transport of Small Molecular Drugs in Animal Models of Amy-loid Beta and Alpha-Synuclein Pathology. Neuropharmacology, 128:482-491

III Gustafsson, S., Gustavsson, T., Roshanbin, S., Hultqvist, G., Hammarlund-Udenaes, M., Sehlin, D., Syvänen, S. (2018) Blood-brain barrier integrity in a mouse model of Alzheimer’s disease with or without acute 3D6 immunotherapy. In manu-script

IV Gustafsson, S., Sehlin, D., Lampa, E., Hammarlund-Udenaes, M., Loryan, I. (2018) Heterogeneous drug tissue binding in brain regions of rats, Alzheimer’s disease patients and controls: impli-cations for translational drug development and pharmacotherapy. In manuscript

Reprints were made with permission from the respective publishers.

Contents

Introduction ..................................................................................................... 9 The blood-brain barrier and the neurovascular unit ................................. 10 Transport at the BBB ................................................................................ 11 

Passive permeability ............................................................................ 11 Carrier-mediated transport ................................................................... 12 Vesicular transport ............................................................................... 13 

The BBB and the NVU in neurodegenerative disease ............................. 14 CNS drug development and the BBB ....................................................... 15 In vivo models of disease ......................................................................... 16 

Mouse model of amyloid beta pathology ............................................ 17 Mouse model of α-synuclein pathology .............................................. 17 

PK concepts .............................................................................................. 18 Rate and extent of BBB drug transport ................................................ 18 Intra-brain distribution ......................................................................... 19 

Methods to investigate BBB drug transport in health and disease ........... 21 Microdialysis ....................................................................................... 21 Positron emission tomography ............................................................ 22 Plasma and tissue binding of drugs ..................................................... 23 Molecules to study BBB integrity ....................................................... 24 Cassette dosing .................................................................................... 25 

Aim ............................................................................................................... 26 

Materials and methods .................................................................................. 27 Study compounds ..................................................................................... 27 Human tissue ............................................................................................ 28 Animals .................................................................................................... 28 Animal surgery ......................................................................................... 29 Oxycodone concentration measurement by simultaneous PET and microdialysis sampling ............................................................................. 29 

Experimental design ............................................................................ 31 Investigation of radiolabeled metabolites in vivo ................................ 31 Brain and blood profiling of [11C]carbonate and [11C]formaldehyde .. 31 Reconstruction of PET and CT images and data analysis ................... 32 Microdialysis probe recovery .............................................................. 33 

BBB transport of drugs and drug intra-brain distribution ........................ 33 Drug cassette ........................................................................................ 33 Total brain-to-plasma concentration ratio in transgenic and WT mice ..................................................................................................... 34 Equilibrium dialysis of rodent and human samples ............................. 34 

BBB integrity to large molecules during Aβ pathology and immunotherapy ......................................................................................... 35 

Experimental design ............................................................................ 35 Brain and blood analysis of fluorescent dextrans ................................ 36 Brain autoradiography of [125I]3D6 ..................................................... 36 

CAA staining in transgenic mice .............................................................. 36 Aβ measurements ..................................................................................... 37 Bioanalytical methods .............................................................................. 37 

Sample preparation .............................................................................. 37 LC-MS/MS or UPLC-MS/MS ............................................................. 38 

Statistical analysis .................................................................................... 38 

Results and discussion .................................................................................. 40 PET in the study of drug neuroPK (Paper I) ............................................ 40 

Conversion of PET total drug concentrations ...................................... 40 BBB transport investigated by PET and microdialysis ....................... 42 11C polar metabolites and their impact on brain imaging .................... 44 

BBB drug transport and integrity in health and disease (Paper II and III) ............................................................................................................ 46 

CNS pathology and BBB transport of small molecular drugs ............. 47 Aβ pathology and BBB integrity to large molecules ........................... 48 BBB integrity to large molecules after acute immunotherapy ............. 50 

Drug tissue binding in brain regions in health and disease (Paper II and Paper IV) .................................................................................................. 52 

Brain tissue binding in mice expressing Aβ pathology ....................... 52 Regional brain tissue binding in humans and rats ............................... 53 Translation of brain tissue binding from rats to humans ..................... 55 

Conclusions ................................................................................................... 58 

Populärvetenskaplig sammanfattning på svenska ......................................... 60 

Acknowledgements ....................................................................................... 63 

References ..................................................................................................... 67 

Abbreviations

Aβ Amyloid beta AβPP Amyloid beta precursor protein ABC ATP binding cassette AD Alzheimer’s disease AMT Adsorptive-mediated transcytosis ANOVA Analysis of variance AR Antonia red ARIA Amyloid-related imaging abnormalities Atot,brain_incl_blood Amount of drug per g brain ATP Adenosine triphosphate AUC Area under the concentration-time curve BBB Blood-brain barrier BG Basal ganglia CAA Cerebral amyloid angiopathy Cbrain(PET) Total drug concentration in brain from PET Cbuffer Concentration in buffer from equilibrium dialysis Cdialysate Drug concentration in dialysate Chomogenate Concentration in homogenate from equilibrium dialysisCNS Central nervous system Cplasma Concentration in plasma from equilibrium dialysis CRB Cerebellum Ctot,blood Total drug concentration in blood Cu,brain,ISF Unbound drug concentration in brain interstitial fluid Cu,brain(MD) Microdialysis unbound drug concentration in brain Cu,brain(PET) PET unbound drug concentration in brain Cu,plasma Unbound drug concentration in plasma Cu,ss Unbound steady-state concentration ELISA Enzyme-linked immunosorbent assay FITC Fluorescein isothiocyanate FrCx Frontal cortex fu,brain Fraction of unbound drug in brain fu,brain,ROI Fraction of unbound drug in brain regions of interest fu,hD,brain Buffer-to-diluted homogenate concentration ratio fu,plasma Fraction of unbound drug in plasma HIP Hippocampus ISF Interstitial fluid

i.v. Intravenous Kp,brain Total brain to total plasma concentration ratio Kp,uu,brain Unbound brain to unbound plasma concentration ratio LC-MS/MS Liquid chromatography tandem mass spectrometry mAb Monoclonal antibody MRI Magnetic resonance imaging NeuroPK Neuropharmacokinetics NVU Neurovascular unit PD Pharmacodynamics PET Positron emission tomography P-gp P-glycoprotein PK Pharmacokinetics PrCx Parietal cortex ROI Region of interest RMT Receptor-mediated transcytosis SLC Solute carrier UPLC Ultra performance liquid chromatography Vblood Volume of blood in brain Vu,brain Unbound volume of distribution in the brain WT Wild type

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Introduction

With a steadily increase in the ageing population there is today a high unmet medical need for neurodegenerative disorders like Alzheimer’s disease (AD) and Parkinson’s disease, which are both highly correlated to age. AD is the leading cause of dementia worldwide. It is characterized by brain accumula-tion of amyloid beta (Aβ) peptides, which further aggregate into insoluble ex-tracellular deposits, i.e. Aβ plaques. Another hallmark of AD is the presence of phosphorylated tau containing neurofibrillary tangles. Parkinson’s disease belongs to a different class of neurodegenerative disorders known as α-synu-cleinopathies. These diseases are characterized by the accumulation and ag-gregation of the protein α-synuclein, leading to intracellular inclusions of fi-brillary α-synuclein.

While potential disease targets seem to be fairly established, the attrition rates are high within central nervous system (CNS) drug development and the fail-ures are attributed to many factors. The accurate measurement and translation of pharmacokinetics (PK), specifically brain neuropharmacokinetics (neu-roPK), from the experimental to the clinical settings are major contributors. The translation of data from animals to humans is often confounded by species differences and the fact that human controls and patients constitute more het-erogeneous groups compared to experimental animals. The brain is also a highly protected organ with limited allowance of drug entrance and no method currently permits the direct measurement of therapeutic drug concentrations in the human brain. Neurodegenerative diseases are also suggested to influ-ence the barrier properties of the brain, which could affect a drug’s neuroPK. Altogether, this raises the question on how to adapt and optimize current avail-able methodologies for the assessment of neuroPK in humans and target pa-tient populations, with accurate back-translation to other species. Addition-ally, the resemblance in neuroPK between species, and between health and disease must be addressed, especially in the regions of interest (ROIs) and at the site of action. Increased understanding of neuroPK is required to support early drug discovery and the design of new molecular entities, while also pre-venting or lowering the high attrition rates faced within late clinical develop-ment. Enhanced knowledge would also make drug dosing in patients less ar-bitrary and more scientifically based. The use of animal models can aid in delineating the impact of specific pathological features on neuroPK. Still, fur-ther validation and confirmation must be pursued in patients.

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The blood-brain barrier and the neurovascular unit Dating back to the late 1800’s and early 1900’s, it has been shown that dyes, toxins, and molecules administered by routes other than the intrathecal, appear to be partly or fully denied entrance into the brain and the proposal of a pro-tective barrier between blood and brain was first mentioned by Stern and Gau-tier in 1918 [1]. Today this barrier is well known as the blood-brain barrier (BBB). The BBB is developed through evolution to protect the brain from toxic insults from the systemic circulation, while still providing the brain with essential nutrients. The BBB consists of the highly specialized endothelial cells forming the brain capillary walls. These cells are sealed together by tight and adherens junctions, which primarily prevent paracellular diffusion of con-stituents between blood and brain. The restrictive nature of the endothelial cells is also governed by efficient transporter systems, enzymes, and a low degree of pinocytotic activity. The BBB was for long only considered a static, impenetrable barrier. However, it is being increasingly recognized that the BBB is in fact a dynamic system capable of responding to local changes and requirements, resulting in alterations of the tight junctions and expression and activity of transporters and enzymes within the barrier.

The BBB acts in concert with other cells and structures of the brain paren-chyma and circulation to form the so called neurovascular unit (NVU, Fig. 1). The NVU holds the responsibility of maintaining the homeostasis within the brain for optimal neuronal functioning [2-4]. A gel like layer, known as the glycocalyx, is lining the endothelial cells facing the luminal side of the capil-laries. The glycocalyx is both a mechanical and negatively charged barrier, mainly consisting of proteoglycans and glycoproteins. The glycocalyx is gain-ing increased interest when it comes to the functioning of the NVU as it con-tributes to key functions of the BBB, involving BBB permeability, molecular transport, and interactions with circulatory immune cells [3, 5, 6]. Another structural component of the NVU is the basement membrane consisting of two entities, the endothelial and the parenchymal, which enfolds the endothelial cells as well as cells known as pericytes. While the basement membrane is important for the maintenance of the structure and functioning of the embed-ded cells, it also supports the multidirectional communication between these cells and interconnecting astrocytic endfeet [7]. Pericytes can be subdivided into different populations depending on their localization, morphology, and function and constitute important players of the NVU [8]. Through their com-munication with the endothelial cells, the pericytes are able to influence the permeability of the BBB, for instance by regulating transcytosis of larger mol-ecules [9]. By phagocytosis they also act as a clearance route of foreign pro-teins and tissue debris [8]. Astrocytic endfeet covers the abluminal endothelial surface and aid in the neurovascular coupling, thus responding to the needs of the neurons through communication with the vascular cells [10]. Neurons and

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brain inflammatory cells, such as microglia, also constitute important players of the NVU. In addition, peripheral inflammatory cells in the circulation also adds to the regulation of the brain microenvironment and brain response to peripheral insults [4].

Figure 1. Cells and structures of the neurovascular unit, including the blood-brain barrier, which comprises the endothelial cells of the brain capillary wall. BL, basal lamina. Figure from reference [2]. Used with permission.

Transport at the BBB Under normal physiological conditions, a variety of transporter systems act in conjunction with the paracellular tight and adherens junctions, bulk flow, the glycocalyx, as well as metabolic enzymes, to restrict constituents within the blood from entering the brain (Fig. 2). Still, other transporter systems act in the opposite direction to supply the brain with essential nutrients (Fig. 2).

Passive permeability Passive diffusion is an energy independent movement of molecules driven by concentration gradients. In most organs, paracellular transport through the ca-pillary endothelium is a given route of diffusion for many drugs from blood to tissue. The tight junctions of the BBB prevent most molecules from passing between the endothelial cells. Instead, the route of passive diffusion through cellular membranes allows for small lipophilic agents to gain entry into the brain. In this sense, the lipophilicity of small drugs has shown to correlate well with the rate of entrance into the CNS [11].

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Carrier-mediated transport Carrier-mediated transport involves the interaction between endogenous and exogenous molecules and a transport protein and can be divided into facili-tated diffusion and active transport. While carrier-mediated transport contrib-utes to the transport of lipid soluble molecules, this route also enables the transport of more hydrophilic compounds not able to partition in the lipid bi-layer of cell membranes. The movement of compounds is often unidirectional and is denoted efflux when compounds are transported out of the brain back into the blood, and influx when drugs are transported from blood to brain. The transporters can be located in either the luminal or abluminal membrane of the endothelial cells or be present in both of the domains.

The process of facilitated diffusion engages transporter proteins but is driven by a solute’s concentration gradient and does not require any energy expendi-ture in order to work. Active transport can be subdivided into secondary and primary active transport and has the potential to transport molecules against their concentration gradient. In secondary active transport, molecules can pass the BBB in the exchange (antiport) or cotransport (symport) of for instance ions. Hence, the transport is dependent on the energy in the electrochemical gradient. Primary active transport is energy dependent and usually requires the hydrolysis of adenosine triphosphate (ATP) for the transfer of molecules across the BBB.

An array of luminal and abluminal membrane proteins is responsible for me-diating solute trafficking across the BBB, allowing the passage of essential nutrients like glucose and amino acids for CNS activity, while also excreting waste products from the brain. Transporters belonging to the solute carrier (SLC) family are expressed in the BBB such as the Na+-independent L-type amino acid transporter (LAT1/SLC7A5), monocarboxylic acid transporter 1 (MCT1/SLC16A1), and facilitative glucose transporter 1 (GLUT1/SLC2A1), as well as members of the organic anion transporters (OAT), organic anion transporting polypetide (OATP) transporters, and organic cation transporters (OCT) [2, 12].

The ATP binding cassette (ABC) transporters constitute an important family of efflux transporters at the BBB. ABC transporters can hinder drugs and other molecules from direct entrance into the brain at the luminal interface or act for their active elimination within the cell membrane or brain interstitial fluid (ISF) back into the blood. This active efflux has been proposed to play a crit-ical role in CNS drug delivery and constitutes a major obstacle for CNS drug development [2]. In a study using quantitative proteomics, it was shown that the transporters P-glycoprotein (P-gp, MDR1/ABCB1), breast cancer-re-sistance protein (BCRP/ABCG2), and multidrug-resistance-associated protein

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4 (MRP4/ABCC4) are the ABC transporters mainly expressed at the human BBB [13]. This is also true for other species like rodents. However, the relative expression levels between these transporters might vary between species.

Vesicular transport The passive permeability and carrier-mediated transport mention above gen-erally involve the transport of smaller molecules. Peptides and proteins can also utilize these systems. However, these larger molecules are often targets of vesicular transport mechanisms. Vesicular transport can be divided into two primary processes at the BBB, namely receptor-mediated transcytosis (RMT) and adsorptive-mediated transcytosis (AMT). In RMT, the binding of a mac-romolecule to a receptor on the cell surface, such as the transferrin receptor, can initiate endocytosis of the molecule. The vesicle containing the receptor-ligand complex can then meet different fates within the cell. It can be recycled back to the luminal membrane where the molecule is released back into the blood stream. The vesicle can also be directed to lysosomes where the content is degraded. Alternatively, the vesicle transcytose through the cytoplasm and fuse with the abluminal membrane where the molecule is released by exocy-tosis into the brain [14]. In drug development the focus has been placed on RMT for the delivery of macromolecules to the brain, owing to the specificity in the ligand-receptor binding that can be utilized. However, due to the huge need of drug delivery to the brain, AMT is gaining increased interest as it might have a higher capacity of transporting large molecules across the BBB compared to RMT [15]. AMT is a favorable route of transport for cationic proteins and peptides where electrostatic interactions arise between the posi-tively charged moieties of the macromolecule and the negatively charged components of the glycocalyx, which triggers AMT. Whereas the anionic properties of the luminal membrane seem to be superior of that of the ablu-minal membrane, the transcytosis and externalization of the cationic com-pounds at the brain side is most probably facilitate by the anionic features of both the abluminal membrane and the interconnecting basement membrane [15].

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Figure 2. Transport routes at the blood-brain barrier. Figure from reference [2]. Used with permission.

The BBB and the NVU in neurodegenerative disease Accumulating evidence shows that the integrity and functioning of the BBB, and the whole NVU are affected in many CNS related disease conditions, both as a cause and consequence of the disease [16-21]. This has been extensively investigated in AD. The dysfunctional characteristics of the BBB vary be-tween disorders and among the most studied are changes in tight junction pro-teins, P-gp expression, and basement membrane alterations. Changes in the expression, structure, or function of tight junctions have been shown both in vitro and in vivo in association to α-synuclein or Aβ exposure [22-27].

Alterations in the efflux transporter P-gp during neurodegenerative conditions have been the target of investigation in numerous studies, with special empha-sis on AD. Clinical positron emission tomography (PET) studies in patients with AD indicate a reduced function of P-gp at the BBB, which is further sup-ported by findings of reduced P-gp expression in post-mortem tissue from AD patients [28-31]. The reduced expression translates back to BBB cell systems and animal models of AD. Aβ concentration dependent alterations in P-gp ex-pression on both an mRNA and a protein level were detected in endothelial cells, and up to 60% reduction of P-gp expression has been reported in mi-crovessels of transgenic mice displaying AD pathology [32-36]. PET studies in Parkinson’s patients indicate a reduced P-gp function or expression in pa-tients with more advanced disease, while the same was not observed in early stages of the disease [37-39].

Aβ depositions in the basement membrane of capillaries and other cerebral vessels can be observed in approximately 80% of AD cases and is referred to as cerebral amyloid angiopathy (CAA) [7]. CAA predispose BBB integrity

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loss and brain region dependent correlations have been observed between CAA pathology and brain microinfarcts [40, 41]. Furthermore, composition changes and a thickening of the basement membrane have been detected in an animal model of AD and was also described in post-mortem tissue from pa-tients with AD or Parkinson's disease [34, 42-44].

Given the changes in the BBB during AD and Parkinson’s disease pathology, many denotes the barrier as broken, disrupted or leaky during disease. This further propose that drugs targeting the CNS as well as those acting in the periphery might gain increased or possibly decreased access to the brain with enhanced or reduced effect, or side effects as an outcome. Hence, the question has aroused regarding how pathology affects the delivery of drugs to the brain and how to best investigate this.

CNS drug development and the BBB Although the complete NVU with its physical and dynamic properties is es-sential for the normal functioning of the brain, it also constitutes a major hur-dle for CNS drug development. As the NVU, and primarily the BBB, regulates and hinders the passage of endogenous compounds into the brain, it also limits the entry of drugs targeting CNS diseases.

The high failure rates in clinical CNS drug development is often attributed to a lack of effect, and hence the pharmacodynamics (PD) of the drug [45]. Though there are many reasons to the problem of demonstrating drug efficacy in human CNS disease, lack of efficacy could to some extent relate back to inadequate neuroPK. The European medical agency conducted a survey of clinical development program applications in psychiatry and neurology from 1995 to 2014 [46]. Interestingly, it was reported that 46% of the neurology programs showed missing or inadequate PK/PD, proof-of-concept, or dose-finding studies [46]. Early failures are often a proof of inefficient drug deliv-ery to the brain, and consequently inadequate therapeutic concentrations at the target site. The lack of appropriate techniques for the evaluation of neuroPK in humans can further add to the failures considered to be due to a lack in response or poor PK/PD relationship investigations. Hence, the right disease targets might have been identified, but the concentrations of drug are not suf-ficient to elicit an appropriate response in the patient population. Moreover, the question of the accuracy in using preclinical models is continuously raised and calls for extended evaluation of these models. Both PK and PD aspects must be targeted to critically judge how suitable current translation is between species or in vitro and in vivo measurements [45]. To mitigate the high attrition rates in CNS drug development and to balance the highly unmet medical needs

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of CNS related disorders, many facets of the drug development process need to be scrutinized and possibly reevaluated.

Small molecular drugs have long dominated CNS drug research. However, the brain delivery related issues of small drugs, largely relating to the efficient efflux systems of the BBB, together with the exploration of new treatment opportunities and delivery systems, have lead the industry towards the devel-opment of larger molecules for the treatment of CNS diseases. One example is the increasing interest in monoclonal antibodies (mAb) developed for pas-sive immunotherapy targeting Aβ in AD. Many of these mAbs have shown promising results in transgenic mouse models while later failing to meet clin-ical outcomes in phase II and III studies [47]. One of the earliest developed anti-Aβ antibodies was bapineuzumab (AAB-001; Pfizer Inc., New York, NY, and Janssen Pharmaceuticals Inc., Raritan, NJ), known as 3D6 in its murine form. The development of bapineuzumab was mainly terminated due to the lack of efficacy. However, the treatment with bapineuzumab during clinical development showed an increased prevalence of adverse events like brain edema and microhemorrhages, especially in the higher dose groups [48-51]. The events have been attributed to antibody interaction with Aβ pathology in the cerebral vessel walls, indicative of a subsequent integrity loss of the ves-sels and hence the BBB [52-54]. The incidence of the adverse events in the bapineuzumab trials lead to further caution and restrictions in later develop-ment of other anti-Aβ antibodies. The analysis of so called amyloid-related imaging abnormalities (ARIA), detected by magnetic resonance imaging (MRI), is now a prerequisite and has also been observed for other anti-Aβ antibodies [47]. As patients with neurodegenerative conditions in general are treated with multiple drugs and are subjected to concomitant diseases, such treatment induced alterations of the BBB is also important to consider for the brain delivery of other co-administered drugs and their effect and side effect profiles.

In vivo models of disease The use of mice and rats currently dominate early preclinical PK and PD stud-ies in drug development and CNS research. The use of these species brings about both opportunities and limitations. The establishment and development of transgenic mice, and nowadays also rats, have revolutionized the field of CNS research. A variety of transgenic models has been developed for the stud-ies of neurodegenerative diseases and could possibly be used for investigating drug transport to the brain under these pathological conditions. Still, animal models usually lack the complexity of the disease seen in humans which limits the translation between animals and patients.

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Mouse model of amyloid beta pathology While the formation of insoluble Aβ plaques is a well-known hallmark of AD, the severity and progression of the disease seem to correlate better with solu-ble aggregates of Aβ, such as oligomers and protofibrils, which show intraneu-ronal accumulation and have proven to be the more neurotoxic species of Aβ [55, 56]. In order to delineate the importance of these soluble species in AD, the tg-APPArcSwe mouse model was generated, expressing both the Swedish (KM670/671NL) and the Arctic (E693G) mutations found in inherent forms of AD [57]. Both mutations are localized within the Aβ precursor protein (AβPP), where the Swedish mutation results in an overproduction of Aβ and the Arctic mutation results in a rapid formation of soluble Aβ protofibrils [58-60]. Indeed, these transgenic mice exhibit high levels of soluble Aβ protofi-brils, shown to accumulate inside neurons, as well as early and rapid formation of insoluble plaques closely mimicking, however not fully resembling, those observed in sporadic forms of AD [57, 61, 62]. Morphological examination of the brain of these transgenes also reveals swollen and distorted dendrites and synaptic nerve endings. Inflammatory reactions in tissues were also observed with resulting microgliosis and astrocytosis [57, 63].

Mouse model of α-synuclein pathology The α-synucleinopathies are, as the name tells, characterized by the aggrega-tion of the protein α-synuclein. These aggregates are known as Lewy bodies in both patients with Parkinson’s disease or dementia with Lewy bodies, or denoted glial cytoplasmic inclusions in multiple system atrophy. A specific missense mutation in the gene SNCA, encoding α-synuclein, cause an ex-change of alanine to proline at amino acid 30 (Ala30Pro) which leads to an inherent autosomal-dominant form of Parkinson’s disease [64]. The patho-genic mechanism of this mutation is suggested to be a consequence of an ac-celerated oligomerization of α-synuclein, showing a high potential of neuro-toxicity [65, 66]. The A30P mutation has been used to generate transgenic mice overexpressing human mutant α-synuclein, (Thy 1)-h[A30P]αSYN (tg-SYNA30P), and hence resemble part of the α-synucleinopathies previously mentioned [67, 68]. The mice usually start to accumulate proteinase K re-sistant α-synuclein aggregates in their midbrain and brainstem at an age of 12 months and become cognitively impaired at about the same age, while they also develop motor symptoms at an age of 17 months [67, 69, 70]. These mice also display increased levels of neurotoxic α-synuclein oligomers and proto-fibrils, which have shown to be associated with motor symptoms within these animals [71, 72].

With regard to the pathological manifestation of the above mentioned trans-genic animals, they appear as promising models for the investigation of the

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impact that Aβ and α-synuclein pathology might have on drug delivery to the CNS, across the BBB. However, it is important to bear in mind that these are models of disease and they do not resemble the full picture of the disorders.

PK concepts Due to the sensitive nature and differing composition of the brain as well as the physical and dynamic CNS barrier properties, preventing drug molecules from entering the brain, neuroPK parameters can be challenging to study. Still, accurate neuroPK analysis is essential for correct interpretation of PD re-sponses and to help mitigate the high failure rate in CNS drug development.

Rate and extent of BBB drug transport Drug transport across the BBB can be investigated and pharmacokinetically described by the means of rate and extent of drug transport [73]. The rate of transport is important when considering treatments where a rapid effect is needed. However, when drugs are intended to be used for the treatment of chronic conditions with repeated daily dosing, the knowledge of the extent of drug transport is of higher importance. The rate of transport can be investi-gated in vitro using cell-based methods, or in vivo in animals by the use of methods like the in situ brain perfusion technique, giving a measure of the in vivo permeability surface area product across the BBB [74]. The extent of transport can be studied in animals by several methods [75]. Restrictions on what techniques that can be applied usually depend on the species used and the compound investigated.

The extent of BBB transport in vivo is a steady state measurement, dependent on the relative capacity of properties like passive permeability, and active in-flux and efflux transport to act on a drug at the BBB, resulting in a net bidi-rectional transport or a net uptake or efflux of drug at the BBB. To further describe this equilibrium across the BBB, the unbound drug concentration in brain ISF is related to the unbound concentration of drug in plasma, resulting in a ratio denoted Kp,uu,brain (Eq. 1) [73, 76].

K , ,, ,

, ,

,

, Eq. 1

AUCu describes the area under the concentration-time curve of unbound drug in either brain or plasma. Depending on the study design, the AUC parameters after a single dose can be replaced by unbound steady-state concentrations (Cu,ss) after continuous dosing. If the transport of a drug is dominated by pas-sive diffusion in both directions across the BBB, this renders a Kp,uu,brain value

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of one, hence unity. A Kp,uu,brain below unity indicates that the BBB transport is dominated by active efflux, while a ratio above unity suggests an active net uptake of drug from blood to brain across the BBB.

Unbound in vivo concentrations of small molecular drugs can be difficult to retrieve in brain and requires invasive techniques like microdialysis, described below. Instead the assessment of BBB transport and the calculation of the Kp,uu,brain value can be based on a combination of other methods and acquired parameters. Total concentration measurements in brain and plasma samples can be used to determine the total brain-to-plasma concentration ratio of a drug, Kp,brain (Eq. 2). This ratio can be used to generate the Kp,uu,brain value by compensating for the binding of drug to plasma proteins and the nonspecific binding of drug in brain tissue (Eq. 3).

K ,,

, Eq. 2

K , ,,

,K , Eq. 3

AUCtot, in Equation 2, describes the area under the concentration-time curve of total drug in either brain or plasma, and fu,brain and fu,plasma (Eq. 3) refers to the fraction of unbound drug in brain and plasma, respectively. Hence, the Kp,brain value is dependent on not only the BBB equilibrium of a drug, but also on the binding of drug to plasma and tissue constituents. It is therefore highly important to use the Kp,uu,brain value when BBB transport is to be assessed since only unbound drug can pass across the barrier [76-78]. This is especially im-portant when investigating BBB transport properties of drugs under disease conditions or when the transport is to be compared between species, as disease and species differences can be present in all three parameters. Species differ-ences in plasma protein binding are for example common. The estimation of unbound drug concentrations in brain, compared to total, is also essential for the prediction of drug effect and for correlations with PD measurements, as it is the unbound drug molecules that can interact with the drug target and hence elicit the effect [79-81].

Intra-brain distribution The nonspecific binding properties of a drug to brain tissue components (fu,brain) or plasma proteins (fu,plasma) can be determined by a technique known as equilibrium dialysis using brain homogenate or plasma, as described below [82-84]. While the binding of drug in plasma is dominated by plasma proteins, nonspecific binding in brain, later also referred to as brain tissue binding, is

20

dominated by membrane partitioning and hence drug-lipid interactions, ren-dering good correlations between drug lipophilicity and nonspecific binding in brain [83, 85, 86].

A general assumption is that nonspecific brain tissue binding of drugs is equal between species. Thus, it has been proposed that estimates of fu,brain obtained in rats can be used as surrogates for estimates in higher species, including hu-mans [87, 88]. Estimates of fu,brain are therefore used for the prediction of brain PK properties in target patient populations, such as AD patients, and whole brain estimates are used for predictions in individual regions. Still, regional differences in brain tissue binding have not been extensively investigated while studies clearly support varying lipid compositions in different brain re-gions both in rodents and humans [89, 90]. Interestingly, Loryan et al. found differences in the nonspecific binding of antipsychotics between brain regions in healthy rats [91], highlighting the need of in-depth regional investigations of such neuroPK parameters. Proposed regional lipid expression and compo-sitional changes during neurodegenerative disease like AD also call for the need of investigating the impact of neurodegenerative disease on nonspecific brain tissue binding of drugs in specific brain regions [90, 92, 93].

By determining fu,brain through the homogenate method, the parameter only accounts for the nonspecific binding of drug in the tissue and not for the in vivo intracellular distribution, as cell membranes and cell organelles are not kept intact after homogenization. Consequently, this method cannot measure possible distribution and accumulation of a drug within cells, due to active transport mechanisms, or into organelles such as lysosomes, where especially basic drugs are distributed as a result of pH partitioning. Depending on drug specific physicochemical properties, the use of fu,brain may result in an under-prediction of intra-brain distribution when intracellular partitioning is not ac-counted for, or an overprediction when intracellular binding sites are exposed to drugs which usually do not enter cells [94]. An alternative neuroPK param-eter used to compensate for these factors is the unbound volume of distribution in the brain (Vu,brain) [94, 95]. This parameter takes both the nonspecific bind-ing of drug into account while also accounting for the intracellular and sub-cellular distribution of drugs. Hence, Vu,brain better relates unbound drug con-centrations in the ISF to the total amount of drug in brain. Therefore, Vu,brain might constitute a more physiologically relevant option than fu,brain for correc-tion of total concentrations of drug in brain when aiming to estimate the un-bound concentrations in the extracellular space. Vu,brain can be determined in vivo by the measurement of total brain and blood concentrations at steady-state, while at the same time acquiring the unbound concentrations of drug in the brain ISF through microdialysis (Eq. 4).

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V ,, _ _ ,

, , Eq. 4

Atot,brain_incl_blood is the amount of drug present per gram brain, still including the blood content within the tissue. In order to assess the amount of drug pre-sent in the brain tissue itself, compensation needs to be made for the drug concentrations in the remaining blood in brain. Therefore, Vblood denotes the physiological volume of blood in brain and Ctot,blood is the total concentration of drug in blood. Cu,brain,ISF is the unbound concentration of drug in the brain ISF. Vu,brain can also be determined by the brain slice method in vitro. As men-tioned earlier and given by Equation 4, it can be used to predict unbound con-centrations of drug in brain from total drug concentration measurements [96-99].

Methods to investigate BBB drug transport in health and disease Most of the techniques used in animals to determine brain concentrations of drugs are based on terminal sampling or invasive intra-brain measurements, while concentration measurements in humans are basically restricted to in vivo imaging techniques. Whereas unbound concentrations can be measured or re-trieved through combined analysis in animals, using both in vivo and in vitro measures, methods to determine unbound concentrations in humans are highly restricted. Hence, methods determining unbound concentrations in humans must be developed and optimized for enhanced inter-species translation to support both drug development and patient treatment.

Microdialysis Microdialysis is a technique that can be used for continuous measurements of unbound drug concentrations in brain ISF and blood over time, and thus, for the direct estimation of Kp,uu,brain and hence BBB transport [100]. The unbound concentrations measured by microdialysis can be related to PD outcomes and biomarker profiles to describe PK/PD relationships. Microdialysis is advanta-geous in the sense that drug concentrations can be measured in awake and freely moving animals, without the need of anesthesia during the actual ex-periment. Cerebral microdialysis involves the insertion of a probe into the brain parenchyma. A semipermeable membrane is located at the tip of the probe and should be allocated in the region of interest. The membrane cut off will determine the size of the molecules that are allowed passage into and out of the probe. As such, the membrane can prevent large molecules and drug bound to tissue constituents to pass, only allowing unbound drug to diffuse into the probe driven by its concentration gradient. A perfusate fluid, often

22

containing a calibrator and resembling the ionic composition of the ISF, is perfused through the probe with a continuous flow. The solution leaving the probe, also called dialysate, is collected in time intervals and analyzed for drug and calibrator. This concentration should reflect the unbound concentration of drug in the surrounding ISF. However, as the exchange of drug between the ISF and probe is not carried out under equilibrium conditions, the concentra-tions in the dialysate must be compensated for in vivo probe recovery, being individual for each drug, probe and experiment. The recovery describes the ratio between the dialysate concentration of drug and the true concentration in the ISF and can be assessed through different methods [101-104].

Microdialysis is currently the only method available for the direct measure-ment of unbound drug concentrations in brain. However, the technique is not applicable to all drug molecules owing to physicochemical properties of the compounds such as its lipophilicity. Lipophilic drugs tend to stick to microdi-alysis probe components and to the connecting tubing [105]. Microdialysis is also technically and surgically challenging and could initiate tissue responses at the site of insertion. However, these responses, as well as local changes in blood flow and glucose metabolism is suggested to resolve within 24 hours after the probe insertion under healthy conditions and as long as the surgical procedure is accurately and carefully performed [105]. However, immune re-actions have been suggested to start within two to three days after probe in-sertion and limits the time window of microdialysis experiments, or at least requires consideration and caution [105]. While its application has increased in a variety of species, the invasive nature of the technique may partly limit its use in transgenic animal models already affected by disease or pathological insults. Due to the fact that probe recovery must be determined in vivo and owing to the invasiveness of the technique, the use of brain microdialysis is also highly restricted in humans and mainly used for the monitoring of brain traumatized patients only.

Positron emission tomography PET is an imaging technique showing high translational potential between species as it can be applied to both animals and humans using similar proto-cols. PET can be used for the measurements of a range of biological processes, while also allowing for the evaluation of PK properties of drugs in biodistri-bution studies. In order to be detected by PET, molecules must be labeled with a positron-emitting radionuclide, for instance 11C, 15O, or 18F. As most drugs contain carbon it makes them amendable for labeling with 11C. By the ex-change of a naturally occurring carbon isotope to 11C in the molecule, PET tracers can be generated with preserved chemical structure and PK properties comparable to the original compound. PET radionuclides contain an excess of protons in comparison to neutrons, leading to decay events where one proton

23

is converted into a neutron, while a single positron is emitted. The position will travel a short distance within the tissue till it collides with an electron. The collision results in a positron-electron annihilation where two photons are formed, which are emitted at an angle of 180°. The photons are subsequently detected simultaneously by the PET scanner and accounted for as an event. These events can later be reconstructed into tomographic images, depicting the spatial and temporal distribution of total radioactivity within a number of planes, which can be further used for the prediction of drug concentrations within the brain. A limitation of PET in PK studies is that the method gener-ates total measures of the radiolabeled tracer (including radiolabeled metabo-lites) and hence the total drug concentrations within the blood or the tissue of interest. Thus, the standard outcome measure from PET PK studies is the total brain to plasma partition coefficient. While the nomenclature differs in PET and PK literature, this parameter is usually referred to as the volume of distri-bution, VT, in PET studies, instead of Kp [88, 106, 107]. However, the total concentration measurements obtained by PET must be corrected for nonspe-cific tissue binding and possible intracellular distribution of drug in brain, if therapeutically active, unbound, drug concentrations are to be evaluated and used for the assessment of BBB transport in animals and humans.

Plasma and tissue binding of drugs In PK studies, investigating unbound drug concentrations and BBB transport, drug binding to plasma proteins and brain tissue constituents are important factors that need to be accounted for, as mentioned above. Equilibrium dialy-sis can be used to determine the fu,plasma or fu,brain in plasma or diluted brain homogenate, respectively. High-throughput equilibrium devices have been optimized and used in recent years, often utilizing a 96-well format [82, 108]. The wells of the equilibrium dialysis apparatus are separated by a semiperme-able membrane, which prevents the immediate contact between a dialysis buffer and the plasma or tissue matrix. The membrane molecular weight cut off determines the size restriction of the passive diffusion of drugs between the two compartments. The plasma or brain homogenate is supplemented with drug, usually at concentrations ranging from 1-5µM, and is denoted “donor side”. From the donor side the drug can diffuse into the buffer, or “receiver side”, based on its concentration gradient. As a general assumption, drug-tis-sue or drug-plasma protein interactions are said to be reversible and equilib-rium is therefore rapidly reached between unbound and bound drug molecules. At equilibrium the respective unbound fractions can be determined as the ratio between the concentration of drug in the receiver versus the donor side, where a subsequent correction for the dilution of the brain homogenate is also re-quired for a correct estimate of fu,brain [109]. Drugs can either interact specifi-cally with its target or nonspecifically to off-target plasma or tissue constitu-ents. In equilibrium dialysis the drug concentrations are chosen to be well

24

above the saturation limit of specific binding. Hence, it is assumed that the specific binding is negligible to the total binding detected in the donor side.

A potential drawback of the equilibrium dialysis technique is the risk of stick-ing of drug to the membrane or walls of the apparatus, which is potentially minimized by the use of Teflon covered devices [110]. Recovery and stability of the samples during dialysis is also important factors that need to be con-trolled for.

Molecules to study BBB integrity For the investigation of specific transporter systems at the BBB, small molec-ular drugs with known mechanism of transport, or at least net transport, could be used as well as antibodies and other proteins. However, drugs can be targets of many transporters and there are multiple factors affecting their delivery to the brain. Hence, if investigating general BBB integrity, it can be difficult to delineate the cause of altered integrity using such molecules. The incidence of tight junction loss, increased or decreased expression of transporters, or a thickening of the basement membrane could all result in altered passage of molecules with different routes of entrance and have all been described in neurodegenerative conditions like AD. Therefore, the use of more inert mole-cules is favorable in studies of overall BBB integrity.

Evans blue is one of the most widely used markers of BBB integrity. However, it suffers from serious limitations as thoroughly reviewed by Saunders et al. and summarized in this section [111]. In brief, the presence of free dye, not bound to albumin at commonly used concentrations of Evans blue is likely. The free dye can pass the BBB without extensive impairment of the barrier. Furthermore, Evans blue does not bind exclusively to albumin as frequently stated and shows differences in the tightness of binding to albumin depending on species. Hence, the common readout of albumin penetrance into the brain after Evans blue injections might be faulty. Adding to this notion is an early statement that Evans blue is not stable in saline formulations, if not present together with proteins. However, in many studies investigating BBB permea-bility, physiological solutions are often used as a vehicle of Evans blue. In addition, findings indicated that the dye can bind not only to plasma proteins but also to other tissue constituents. Technical and methodological concerns in the quantification of Evans blue is also apparent, while not being a matter of concern for this marker only. Given these findings, other more appropriate markers need to be considered for the use in BBB integrity studies.

Dextrans are hydrophilic polysaccharides, with beneficial characteristics like low in vivo toxicity and immunogenicity, while also advantageous in BBB permeability studies as they are suggested not to interact with transporters of

25

the BBB [111, 112]. Dextrans can be tagged with fluorescent dyes or other labels, enabling detection with a variety of different methods. They can also be obtained in different molecular sizes. This allows for BBB integrity studies using a range of sizes of the same type of molecules with similar properties at the BBB, while the size dependent PK of the dextrans and technical consider-ations still must be accounted for [113, 114].

Cassette dosing Cassette dosing is a procedure generally applied in drug discovery for high-throughput screening of PK characteristics of multiple drugs administered simultaneously to the same animal. Using this approach, the number of exper-imental animals needed is greatly reduced, while the time required for dosing, blood and tissue sampling, and processing of samples for bioanalysis is also shortened. Whereas cassette dosing carries many advantages, the risk of drug-drug interactions is high and could result in altered PK of drugs in vivo com-pared to single drug dosing. Guidelines have been set up for cassette dosing, such as limitations of the number of compounds included (not more than ten), concentration thresholds, the inclusion of a benchmark compound, and avoid-ance of potent inhibitors of drug metabolizing enzymes and so on. However, many of the assumptions in cassette dosing have shown to lack validity and result in errors of PK estimates [115]. To detect these errors, it is highly rec-ommended to compare the PK properties of drugs between the cassette dosing and single-compound, discrete, dosing. This is of course not favorable in drug discovery but could be applied if a specific cassette of drugs is to be used in multiple experiments. While not being as reliable but more useful in the in-dustry, the inclusion of the same drugs in different cassettes could be applied to investigate PK changes [115]. To minimize the impact of interactions on drug PK, the doses should be decreased to the lowest detectable levels, while the number of drugs included in the cassette should also be kept at a minimum [115].

Potential drug-drug interactions at the BBB, as a result of cassette dosing, have been investigated. In a study by Liu et al. both P-gp and BCRP substrates and inhibitors were included in a cassette of 11 compounds, dosed at 1 or 3 mg/kg and compared to single dosing of the same compounds [116]. The comparison in Kp,brain values showed a nice agreement, within two-fold, of the two dosing approaches, indicative of a lack of interactions at the BBB [116]. Interestingly, the cassette dosing approach is also being investigated for the use in microdi-alysis studies to increase the throughput in such studies [117, 118].

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Aim

The overall aim of the thesis was to explore and evaluate clinically relevant translational tools for the interpretation of CNS drug delivery in bridging pre-clinical to clinical neuroPK.

The specific aims were:

To elucidate the ability of PET imaging, in combination with PK theory, to estimate brain unbound drug concentrations and net transport across the BBB for translational purposes, through verification of data by microdi-alysis.

To elucidate how pathological hallmarks of neurodegenerative diseases influence the extent of transport of small molecular drugs across the BBB in order to further understand pathological influence on brain drug deliv-ery.

To investigate BBB integrity with respect to large molecules during AD

pathology and after acute treatment with an anti-Aβ antibody.

To provide enhanced understanding of drug brain tissue binding in various brain regions in health and neurodegenerative disease in rodents and man, as a step in translational method building.

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Materials and methods

Study compounds Oxycodone brain concentrations and BBB transport was studied in Paper I using simultaneous PET and microdialysis measurements. Oxycodone is an interesting compound as it is one of few known drugs showing a net active influx at the BBB. It was also considered a suitable drug for isotopic labeling using 11C for the application in PET.

In Paper II the extent of BBB transport of small molecular drugs was investi-gated under healthy and pathological conditions. Oxycodone was again used to determine the influence of pathology on active influx transport at the BBB. Diazepam was included as a model drug of passive diffusion at the BBB, while levofloxacin, digoxin and paliperidone were included as markers of active ef-flux, of which the latter two are confirmed substrates of P-gp.

BBB passage of large molecules during Aβ pathology and after acute immu-notherapy was investigated in Paper III using a 4 kDa dextran labeled with fluorescein isothiocyanate (FITC) and a 150 kDa dextran labeled with Antonia Red (AR). 3D6, an anti-Aβ mAb, which is the murine version of clinically tested bapineuzumab, was used in Paper III in an acute treatment regimen to study its impact on BBB integrity. 3D6 was expressed in a murine IgG2c framework using Expi293f mammalian cells and purified in accordance with a previously published protocol [119]. For the study of antibody distribution, a fraction of 3D6 was isotopically labeled with iodine-125 (125I).

In Paper IV, brain tissue binding of small molecular drugs was investigated in brain regions from human controls, AD patients, and rats using memantine, donepezil, diazepam, paliperidone, and indomethacin. The compound selec-tion in Paper IV was mainly based on differing physiochemical properties and on the relevance to the treatment of AD or concomitant diseases in the AD patient population.

All radiolabeling (Paper I and III) of molecules with 11C or 125I was performed in house.

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Human tissue In Paper IV, human frozen brain tissue was obtained from the Brain Bank at Karolinska Institutet, Sweden, which also holds the informed consent from tissue donors (ethical approval 2011/962-31/1). Samples from frontal cortex (FrCx), parietal cortex (PrCx), basal ganglia (BG), and cerebellum (CRB) were investigated from patients with confirmed AD (n=6, median age at death 83 years) and controls with no reported neurodegenerative condition (n=6, median age at death 73 years). The experimental use of human post-mortem samples was approved by the regional Ethical Review Board in Uppsala (eth-ical approval 2014/268).

Animals All handling and use of animals in the present thesis work was performed in accordance with Swedish and European legislation and directives on animal experiments. All efforts were made to reduce the number of animals used in each study and to minimize animal suffering.

Male Sprague-Dawley rats (Taconic, Lille Skensved, Denmark) 250–320 g, were used in Paper I and IV. The animals were group housed under a 12 h light/dark cycle at 20-22°C, with food and water available ad libitum. The rats were allowed to acclimatize for at least three (Paper IV) or seven days (Paper I) before the start of the experiments. The studies were approved by the local Animal Ethics Committee in Uppsala, Sweden (reference numbers C37/15 and C43/12 for Paper I, and C189/14 for Paper IV).

To investigate pathological interference of α-synuclein with drug BBB transport, 16-19 month old female and male homozygous (Thy-1)-h[A30P] α-SYN transgenic mice, overexpressing human α-synuclein with the A30P mu-tation, were used in Paper II, referred to as tg-αSYNA30P [67, 68]. The impact of Aβ pathology on BBB drug transport and integrity was studied in Paper II and III using 16 and 18-19 months old (Paper II and III, respectively) hetero-zygous female and male (Thy-1)-h[E693G;KM670/671NL] AβPP transgenic mice. The mice express AβPP with the human Arctic (E693G) and Swedish (KM670/671NL) mutations and were referred to as tg-APPArcSwe in Paper II and tg-ArcSwe in Paper III [57, 59, 60]. Non-transgenic female and male, C57BL/6, wild type (WT) mice were used for cassette validation at an age of 2-5 months in Paper II, and as controls at an age of 16 and 18-19 months in Paper II and III respectively. All mice were bred in-house and sustained on a C57BL/6 background. The animals were housed under temperature and hu-midity controlled conditions on a 12 h light/dark cycle with free access to food and water. The studies were approved by the local Animal Ethics Committee

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in Uppsala, Sweden (reference numbers C363/12, C17/14, and C92/14 for Pa-per II, and C17/14 and 13350/2017 for Paper III).

Animal surgery In Paper I, a subset of animals was subjected to microdialysis for the meas-urement of unbound oxycodone concentrations in brain ISF and the probe im-plantation was performed one day prior to the experiment. The animals were deeply anesthetized with inhalable 2.5% isoflurane (Isoflurane Baxter®, Bax-ter Medical AB, Kista, Sweden) in 50% medical oxygen and 50% air. A ste-reotaxic instrument (David Kopf Instruments, Tujunga, CA, USA) was used to position the head of the rat and a CMA/12 guide cannula was inserted into the right striatum. The guide cannula was fixed to the skull and replaced with a CMA/12 probe with a 3 mm PAES membrane with 20 kDa molecular weight cut off. After surgery, the rats were allowed to recover for about 24 h with free access to food and water, before the start of the experiment.

For all animals in Paper I, arterial and venous catheters were inserted on the day of the experiment. For subsequent drug and tracer administration, a PE-50 cannula fused with Silastic tubing was inserted into the left femoral vein. For discrete blood sampling, a PE-50 cannula fused with a PE-10 tubing was inserted into the femoral artery. The rats were kept anesthetized until sacri-ficed at the end of the experiment.

Oxycodone concentration measurement by simultaneous PET and microdialysis sampling In Paper I, combined PET and microdialysis was simultaneously performed on rats to study oxycodone concentrations with both techniques (Fig. 3). The purpose was to investigate if the total drug concentrations, acquired through PET, could be converted into unbound concentrations, resembling those at-tained by microdialysis.

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Figure 3. Experimental design of oxycodone brain concentration measurements, us-ing simultaneous small animal PET imaging and microdialysis sampling.

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Experimental design Animals with brain microdialysis probes and inserted blood catheters, as de-scribed in the section “Animal surgery”, were placed in a small animal PET-SPECT-CT system with the head of the rat in the center of field of view in the gantry of the scanner (Triumph™ Trimodality System, TriFoil Imaging, Inc., Northridge, CA, USA). The animals were kept under general anesthesia, using isoflurane, throughout the experiment. After positioning of the animal in the scanner, the microdialysis probe was continuously perfused with Ringer solu-tion at a flow rate of 1 μL/min, containing the calibrator oxycodone-D3. Dia-lysate samples were collected in 10 min intervals during a stabilization period of 60 min. After stabilization of the probe, [N-methyl-11C]oxycodone, supple-mented with a therapeutic dose of isotopically unmodified oxycodone, was administered to the animals as an intravenous (i.v.), constant rate infusion of 0.3 mg/kg/h over a 60 min time period. A continuous PET scan was acquired throughout the 60 min drug infusion period as well as 60 min after infusion stop. The PET scan was followed by a short CT examination. In parallel to imaging, brain dialysates were collected in 10 min intervals. Discrete arterial blood samples were collected pre-dose and at 5, 15, 30, 45, 55, 65, 75, 90, and 115 min after the start of drug infusion. A well counter (GE Healthcare, Upp-sala, Sweden) was used to measure the radioactivity in blood and plasma sam-ples, the latter retrieved through centrifugation.

Investigation of radiolabeled metabolites in vivo To validate if the radioactivity and thus the total brain concentrations of ox-ycodone obtained by PET in Paper I, originated from the parent compound only without interference of radiolabeled metabolites, total PET concentra-tions were compared to total brain concentrations measured in whole brain by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A separate group of animals received an i.v. infusion of 0.3 mg/kg/h isotopically unmod-ified oxycodone for 60 min, with simultaneous microdialysis and blood sam-pling as described above. The animals were sacrificed at 60 or 120 min after the start of the infusion and total oxycodone concentrations in brain and plasma were determined.

Brain and blood profiling of [11C]carbonate and [11C]formaldehyde [11C]carbonate and [11C]formaldehyde brain and blood time-activity curves were investigated in rats as potential in vivo radiolabeled metabolites of [N-methyl-11C]oxycodone. This was done by using continuous PET measure-ments and parallel blood sampling. An equal experimental setup, excluding

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microdialysis, as previously described for [N-methyl-11C]oxycodone was ap-plied, with an intravenous constant rate infusion for 60 min and a follow-up period of 60 min. Rats either received a constant infusion of [11C]carbonate or [11C]formaldehyde alone, or in combination with oxycodone at a therapeutic dose (0.3 mg/kg/h). Discrete arterial blood samples were collected at 5, 15, 30, 45, 55, 65, 75, 90, and 115 min after infusion start, and radioactivity was measured in blood and plasma. At 45 min and 120 min the percentage of [11C]carbonate was estimated in part of the blood samples from animals ad-ministered [11C]carbonate, with or without oxycodone. Furthermore, using corresponding infusion conditions as described above and blood collection at 30, 55, 75, 90, and 115 min, the percent of [11C]carbonate formation in blood from injected [N-methyl-11C]oxycodone was determined in a separate experi-ment. [11C]carbonate in whole blood was analyzed as previously described by Shields et al. by basifying one part of a blood sample to preserve all [11C]car-bonate in the sample together with radioactivity originating from the parent compound and other metabolites, while acidifying another part of the blood sample and degassing it to remove radioactivity originating from [11C]car-bonate [120]. The percentage of [11C]carbonate activity in blood was calcu-lated by subtracting the activity of the acidified and purged blood sample from the activity in the basified sample and then dividing with the total activity.

Reconstruction of PET and CT images and data analysis To improve the measurement of regional and spatial radioactivity in the brain PET images, PET and CT data were reconstructed and the CT scans were aligned to an MRI based 3D rat brain atlas [121]. The atlas contained prede-fined brain ROIs and a spherical ROI representative of the cerebrum was man-ually constructed, accounting for the whole brain. PET images were then aligned to the CT images and the radioactivity originating from [N-methyl-11C]oxycodone was quantified in the ROIs.

The total radioactive concentration measured in brain with PET was first cor-rected for interfering radioactivity in blood, assumed to be 3% of the brain volume. Second, the radioactivity concentrations were converted into oxyco-done concentrations (Cbrain(PET)) by using the known specific activity, i.e. radi-oactivity per amount of oxycodone.

Using the previous in vivo determined Vu,brain of oxycodone of 2.20 mL/g brain tissue [122], Cbrain(PET) were converted into unbound concentrations (Cu,brain(PET)) (Eq. 5).

C ,,

Eq. 5

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To investigate if the converted PET concentrations could be used for an esti-mation of BBB transport, unbound plasma concentrations (Cu,plasma) were also required and calculated by compensating total plasma concentration of oxyco-done measured by LC-MS/MS with the fraction of unbound oxycodone in rat plasma of 0.743 [122].

Microdialysis probe recovery To determine the concentrations of unbound oxycodone in the brain ISF from microdialysis sampling (Cu,brain(MD)), acquired dialysate concentrations (Cdialy-

sate) were corrected for in vivo probe recovery (Eq. 6). Microdialysis probe recovery was calculated by the retrodialysis by calibrator method [103], which assumes that the loss of the calibrator, oxycodone-D3, is equal to the gain of the parent drug, oxycodone, from the surrounding ISF to the dialysate. The average probe recovery in Paper I was 13.9 ± 3.6%.

C , Eq. 6

BBB transport of drugs and drug intra-brain distribution In Paper II, three measurements and hence parameters, Kp,brain, fu,brain, and fu,plasma, were combined to generate an estimate of the extent of BBB transport, the Kp,uu,brain (Eq. 3) in transgenic and WT mice. In Paper IV, drug brain tissue binding was investigated in different regions of human and rat brain by deter-mining the fu,brain,ROI (Eq. 8 and 9).

Drug cassette To reduce the use of aged transgenic mice in the BBB transport studies in Paper II, the drugs were administered as a cassette. However, before using the drugs in disease models, the total brain-to-plasma concentration ratio (Kp,brain) was determined in 2-5 months old WT mice for all compounds after discrete or cassette dosing in order to detect any change in drug PK properties when administered in combination. Mice were either administered a subcutaneous dose of each drug separately or as a cassette of all five drugs at a dose of 0.25 mg/kg for oxycodone and diazepam, and 1 mg/kg for digoxin and levofloxa-cin. Paliperidone was given at a dose of 1 mg/kg during discrete dosing but at a dose of 0.25 mg/kg when included in the cassette to minimize CNS effects when three CNS active compounds were given simultaneously. The animals were euthanized under deep anesthesia by blood collection through cardiocen-tesis at 0.5 h, 1 h, or 3 h after dose and plasma was acquired after centrifuga-tion. The brains were extracted following transcardial 0.01 M PBS perfusion.

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The brains were dissected on ice in order to extract areas of the brain exhibit-ing pathology in the transgenic mice later used in Paper II. Briefly, the hemi-spheres were separated and the CRB and olfactory bulb were removed from the right hemisphere, which was further divided in a caudal portion, involving pathologically affected brain regions such as hippocampus and thalamus of the tg-APPArcSwe mice, and the midbrain and brainstem, which is affected in the tg-αSYNA30P mice [69, 123]. Total drug concentrations in brain and plasma were determined to assess Kp,brain.

Total brain-to-plasma concentration ratio in transgenic and WT mice Using terminal brain and plasma sampling, the Kp,brain was determined from brain and plasma AUCs, obtained through the linear-trapezoidal method (Eq. 2) of tg-αSYNA30P mice at an age of 16-19 months, as well as 16 months old tg-APPArcSwe and WT mice. The animals were administered a subcutaneous dose of the drugs in cassette and were sacrificed at 0.5 h, 1 h, or 3 h post dose and blood and brain were sampled and processed as described in the section “Drug cassette” above. The right caudal hemisphere was used for further con-centration measurements.

Equilibrium dialysis of rodent and human samples In Paper II, fu,brain and fu,plasma were determined by equilibrium dialysis. The right caudal hemisphere and plasma from 16-19 months old tg-αSYNA30P, and from 16 months old tg-APPArcSwe and WT mice were used. To investigate re-gional differences in binding, the rostral part of the right hemisphere of the same tg-APPArcSwe and WT mice were also included in the binding assay. Due to a restricted amount of tissue and plasma from each mouse, the respective matrices (plasma, or rostral or caudal brain) were pooled within each group to avoid extensive dilution of the samples and to retrieve sufficient technical rep-licates. For equilibrium dialysis of plasma, the plasma samples were adjusted to a pH of 7.4 at 37°C. Brain tissue homogenate was prepared from the rostral and caudal part of the right hemisphere, separately, by diluting the tissue sam-ples 5-fold with 180 mM phosphate buffer, pH 7.4, followed by sonication.

In Paper IV equilibrium dialysis of drugs was performed in FrCx, PrCx, BG, and CRB from human control and AD donors, while the hippocampus (HIP) and whole brain were also included in the analysis of rat tissue. Brain tissue homogenates from rats and humans were prepared by sonication and dilution of the tissue 10-fold in 180 mM phosphate buffer, pH 7.4. In Paper II, plasma and brain homogenates were spiked with the compounds in cassette to a final concentration of 1 µM. In Paper IV, brain homogenates were spiked to the

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same final concentration with each of the five investigated drugs in separate experiments. After spiking, the samples were loaded into a 96-well equilib-rium dialysis apparatus (HTDialysis LLC, Gales Ferry, CT, USA). All sam-ples were allowed to dialysate at 37°C at 200 rpm. Dialysis was carried out across a dialysis membrane with a molecular weight cut off of 12-14 kDa (HTDialysis LLC, Gales Ferry, CT, USA) against an equal volume of 180 mM phosphate buffer, pH 7.4. After 6 h of incubation, samples were collected from both chambers and the matrix in plasma, brain homogenate, or buffer was bal-anced with the corresponding opposite fluid. Drug recovery in the samples during equilibrium dialysis was tested using before and after dialysis samples.

The fu,plasma in Paper II, could be determined directly from bioanalysis derived concentrations of buffer (Cbuffer) and plasma (Cplasma) samples (Eq. 7)

f , Eq. 7

In Paper II and IV, fu,brain was determined by first calculating the buffer-to-homogenate concentration ratio, fu,hD,brain (Eq. 8), given the concentration of drug in diluted brain homogenate sample. This estimate was further corrected for the dilution factor, D, of the brain homogenate in order to retrieve accurate values of fu,brain (Paper II) or fu,brain,ROI (Paper IV) as described by Equation 9.

f , , Eq. 8

f ,

, ,

Eq. 9

BBB integrity to large molecules during Aβ pathology and immunotherapy In Paper III, the integrity of the BBB under the impact of AD pathology and after acute anti-Aβ-antibody treatment was investigated by analyzing BBB passage of fluorescently labeled dextrans.

Experimental design Aged tg-APPArcSwe and WT mice were subjected to i.v. injections of either 3D6, supplemented with [125I]3D6, at a dose of 10 mg/kg or PBS. At 72 h after dose, the animals where anesthetized and administered i.v. injections of a 150 kDa AR dextran, followed by an injection of a 4 kDa FITC dextran 5 min

36

later. After another 5 min, animals were sacrificed by heart puncture and blood was collected and stored at 4°C until processing. Brains were extracted after transcardial perfusion using 0.9% NaCl and the olfactory bulb removed. The hemispheres were separated and the right hemisphere, including CRB, was instantly frozen. The left hemisphere was further dissected and the cerebrum (later referred to as brain in Paper III) was diluted with PBS containing com-plete protease inhibitors (Roche, Basel, Switzerland) at a 1:3 w:v ratio and further stored at 4°C until processing and analysis. The radioactivity, originat-ing from [125I]3D6, was measured in all blood and brain samples by the use of a -counter (2480 WizardTM, PerkinElmer, Waltham, USA).

Brain and blood analysis of fluorescent dextrans In order to study the brain-to-blood ratio of the injected dextrans in Paper III, dextran concentrations were analyzed in the brain and serum samples using fluorescent detection. Brain supernatants were obtained after homogenization and centrifugation of the samples and serum was collected from the blood samples after centrifugation. Standard curves, brain supernatants and 10-fold diluted serum samples were loaded onto plates and fluorescence was analyzed using a Tecan Infinite 200 pro plate reader (Tecan, Männedorf, Switzerland) All dextran analyses were carried out within 6 h after animal euthanization.

Brain autoradiography of [125I]3D6 In Paper III, the distribution of [125I]3D6, and hence 3D6, in the brain was visualized on 20 µm sagittal sections from the right hemisphere of mice treated with the antibody using autoradiography. Along with 125I standards of known radioactivity, the sections were placed in an X-ray cassette and posi-tron-sensitive phosphor screens (MS, MultiSensitive, PerkinElmer, Downers grove, USA) were placed onto the samples. After five days of exposure, the samples were scanned in a Cyclone Plus Imager system (PerkinElmer).  

CAA staining in transgenic mice In Paper III, the tg-APPArcSwe animals were further characterized for the pres-ence of Aβ vessel pathology, CAA. Congo red staining was therefore applied to sagittal sections adjacent to those used in the autoradiography described above, according to a previous protocol [124]. The staining visualizes amyloid deposits of Aβ in both brain parenchymal plaques as well as in deposits in vessels. Using a Nikon ECLIPSE 80i microscope with a Nikon DS-Ri1 cam-era (Nikon Instruments Inc., Melville NY, USA), brightfield images of cortex and hippocampus were acquired.

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Aβ measurements To confirm the genotype and pathological state of mice (Paper II and III), as well as pathology in regional brain samples from human controls and AD pa-tients (Paper IV), different species of Aβ or α-synuclein were assessed using enzyme-linked immunosorbent assay (ELISA) according to previous pub-lished protocols with modifications [71, 125]. Soluble Aβ40 and Aβ42, repre-sentative of Aβ monomers and soluble fibrils, were extracted from brain ho-mogenate in Tris buffered saline (TBS, Paper II and IV) or PBS (Paper III). Soluble Aβ oligomers and protofibrils, which are suggested to be the predom-inant toxic species of Aβ, were extracted from the same PBS or TBS fractions in Paper III and IV. The insoluble fractions of Aβ40 and Aβ42, representative of Aβ deposited in plaques, were extracted from samples processed with for-mic acid. Capture and detection antibodies, as outlined in detail in the respec-tive Papers (II-IV), were used for the concentration measurements of the sep-arate Aβ species or α-synuclein. Signals were developed and read at 450 nm using a Tecan Infinite 200 pro plate reader (Tecan, Männedorf, Switzerland).

Bioanalytical methods For all concentration measurements of small molecular drugs in Paper I, II, and IV, samples were processed for quantitative analysis using either an LC-MS/MS or ultra performance liquid chromatography (UPLC-MS/MS) system.

Sample preparation Manual sample preparation was performed in Paper I and II, while a Biomek4000 liquid handling system with Biomek Software version 3x (Beck-man Coulter, Bromma, Sweden) was used for the sample preparation in Paper IV. The microdialysis samples in Paper I were diluted with MilliQ water, spiked with internal standard before injected into the system. Plasma, diluted brain homogenate, and equilibrium dialysis samples were all precipitated with acetonitrile, with or without 0.1-0.2% formic acid and compound specific in-ternal standards. The brain and plasma samples from the Kp,brain experiments in Paper II were evaporated and further diluted in acetonitrile and mobile phase before injected into the system. For the remaining brain (Paper I), plasma (Paper I), and equilibrium dialysis (Paper II and IV) samples, acetoni-trile supernatants were diluted with the respective mobile phase before sample injection into the LC-MS/MS or UPLC-MS/MS system.

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LC-MS/MS or UPLC-MS/MS The quantitative analysis of compound concentrations was either carried out on an LC-MS/MS (Paper I, II, and IV), or a UPLC-MS/MS system (Paper IV). By optimizing the system parameters, the employed analytical methods were developed to achieve a high resolution in a reasonable analysis time. The LC system included a SIL-HTc autosampler (Schimadzu, Kyoto, Japan) and one or two LC-10ADvp pumps connected to the column. A Zorbax Eclipse XDB-CN column (4.6 x 150 mm; Agilent Technologies, Santa Clara, CA, USA), maintained at 50°C, was used for the analysis of oxycodone in Paper I and II, and a 50×4.6 mm HyPURITY 3µ C18 column (Thermo Scientific, USA), with a pre-column comprising the same material was used for diazepam, paliperi-done, digoxin and levofloxacin in Paper II, and donepezil in Paper IV. In Paper IV, the Acquity UPLC system consisted of a binary solvent manager and a sample organizer, which was connected to an ACQUITY UPLC BEH C18 1.7 µM (2.1 X 50 mm) column (Waters Corporation, Tauton, Massachusetts, USA) for the analysis of diazepam, paliperidone, memantine, and indometha-cin. A Quattro Ultima Triple Quadrupole Mass Spectrometer or a Quattro Ul-tima Pt Triple Quadrupole Mass Spectrometer (Waters, Milford, MA, USA) were used for detection. Mass spectrometry control and spectral processing for determination of compound concentrations were performed by the use of MassLynx software version 4.0 or 4.1 together with the Application Manager QuanLynx (Waters Corporation, Milford, MA, USA). Individual standard curves showed good linearity (coefficient of determination >0.990) and all samples were within the linear range.

Statistical analysis A two-tailed, paired t-test was used in Paper I to compare the unbound brain-to-plasma concentration ratios, calculated from PET or microdialysis meas-urements together with protein binding corrected plasma concentrations.

In Paper III, GraphPad Prism 6 was used for all statistical analysis. Separate two-tailed, unpaired t-tests were used for WT and transgene comparisons of 3D6 brain concentrations and brain-to-blood ratios, as well as in comparisons of Aβ pathology between 3D6 treated and non-treated transgenes. In Paper III, a two-way analysis of variance (ANOVA) followed by Tukey’s multiple com-parisons test was used for the group comparison of the brain-to-blood concen-tration ratios of each analyzed dextran in treated and non-treated transgenes and in WT animals. Pearson correlation coefficients were used to investigate correlations between parameters in Paper III.

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In Paper IV, R version 3.3.1 [126] and the lme() function included in the nlme package [127] were used for all statistical analysis. Linear mixed effects mod-els were used in within and between group comparisons of the fraction of un-bound drug in brain regions from human controls, AD patients, and rats. Lin-ear mixed effects models were also used to analyze relationships between Aβ levels and fu,brain,ROI estimates in humans.

The significance level was set to p=0.05 for all statistical comparisons and the results are presented as mean±S.D. if not stated otherwise.

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Results and discussion

PET in the study of drug neuroPK (Paper I) Until date, methods to investigate drug concentrations and BBB transport in human brain are highly limited and neuroPK data in humans is sparse. Clinical CNS drug development is commonly guided by estimates of total drug con-centrations as the therapeutically active, unbound, drug concentrations cannot be accurately estimated. Given the total concentration estimates, it is difficult to interpret and isolate net BBB transport of drugs, which depend on unbound concentrations, and to evaluate in what sense the transport might be altered under disease conditions. Hence, it is essential to make use of techniques like PET, with high translational applicability from rodent to man, for the estima-tion of unbound drug concentrations in brain.

Conversion of PET total drug concentrations In Paper I, oxycodone was successfully labeled with 11C for the use as a model compound to study brain concentrations and to evaluate BBB transport using both PET and confirmatory microdialysis. In combination with a therapeutic dose of isotopically unmodified oxycodone, [N-methyl-11C]oxycodone was administered to rats by a constant rate infusion over 60 min. The current infu-sion regimen results in oxycodone concentrations close to steady state in both brain and blood (Fig. 4). As was shown by Boström et al., and indicated in Figure 4, oxycodone has a rapid equilibration across the BBB [122]. Boström and colleagues also showed that at 60 min the Kp,uu,brain was the same after a constant rate infusion, equal to the one used in Paper I, as after an i.v. bolus injection followed by a constant rate infusion, where oxycodone concentra-tions reached steady state much faster [122].

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Figure 4. Oxycodone unbound concentration-time profiles after a constant rate infu-sion in rats from brain microdialysis samples (filled circles) and plasma samples cor-rected for plasma protein binding (open circles). The data is presented as mean±S.D. [128].

As detected by PET, [N-methyl-11C]oxycodone concentrations in brain in-creased during ongoing infusion (Fig. 5). The total oxycodone concentrations acquired through PET were converted into unbound concentrations by com-pensation for drug binding to brain constituents and intracellular distribution, using the Vu,brain, previously determined in rat of 2.20 mL/g brain for oxyco-done [122]. The transformed unbound PET concentrations, Cu,brain(PET), nicely resembled the simultaneously acquired microdialysis concentrations, Cu,brain(MD), during infusion (0-60 min). However, after the end of infusion, at time points beyond 60 min, an increasing and time dependent aberration in the concentration profiles was observed between the brain PET and microdialysis data (Fig. 5). Thus, the oxycodone half-life in brain based on PET with a mean value of 104 min, exceeded that calculated from microdialysis measures of 27 min. The later was in line with the plasma half-life of 29 min, which in turn compared to previously published data [129].

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Figure 5. Concentration-time profiles displaying oxycodone total PET concentrations (filled diamonds), unbound PET concentrations converted by Vu,brain (open diamonds), and unbound microdialysis concentrations (filled circles), with one extrapolated time point (open circle), after a constant rate infusion of [N-methyl-11C]oxycodone supple-mented with isotopically unmodified oxycodone. Data are presented as mean±S.D. [128].

BBB transport investigated by PET and microdialysis The AUC from the unbound brain and plasma concentration-time profiles ac-quired through microdialysis and discrete blood sampling, corrected by fu,plasma, were first used to calculate the Kp,uu,brain of oxycodone, resulting in a value of 2.90 ± 0.32. Thus, microdialysis data verified previously reported net uptake of oxycodone at the BBB, also observed in Figure 4 [122]. The un-bound brain-to-plasma concentration ratio calculated from the AUC of Cu,brain(PET) data together with LC-MS/MS determined blood concentrations, the same as used in the Kp,uu,brain calculations above, was 4.06 ± 0.59. While transformed PET concentrations resulted in a higher ratio, overestimating the oxycodone brain exposure, it was not significantly higher than the obtained microdialysis value (p=0.073). To investigate how the unbound brain-to-plasma ratios for the two methods compared when the unbound concentrations showed good congruence, ratios were calculated using the AUC from start of the infusion and up to 55 min. The resulting values of 2.72 ± 0.34 and 2.93 ±

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0.47 for microdialysis and PET measures, respectively, were in good agree-ment.

The divergence in the PET and microdialysis data, with a longer observed half-life for oxycodone in PET, indicated a build-up of radioactivity within the brain that progressively affected later time frames. This was likely due to the manifestation and increasing concentrations of radiolabeled metabolites. Still, despite the presence of metabolites, the resemblance of the concentra-tion-time profiles from PET and microdialysis measurements during the infu-sion phase showed that PET can be used to determine unbound concentrations in brain and to further estimate drug BBB transport. However, accurate com-pensation must be made for drug intra-brain distribution, using Vu,brain or brain tissue binding estimates available from other species, like fu,brain. Additionally, radioactive metabolites need to be evaluated and further compensated for if other techniques, like microdialysis, cannot be used to verify the brain con-centrations, which is usually only feasible in lower species. Thus, if extending a similar PET design to humans, radiolabeled metabolites and their impact on the imaging outcome must be acknowledged in an early stage of drug investi-gation. The high translational potential of PET is however optimal for these types of comparative inter-species studies. Knowledge about the metabolite profile of the parent drug can aid in the design and implementation of the ra-diolabel into the original drug structure to keep radiolabeled metabolites at a minimum. Still, labeled metabolites might penetrate the BBB and unexpect-edly constitute a problem in PET investigations as further examined in Paper I and discussed below.

As the industry is also searching for molecules with improved entrance into the brain, the experimental design applied in Paper I can offer a methodolog-ical approach for continuous in vivo evaluation of the energy-dependent, pro-ton-coupled antiporter suggested to transport oxycodone across the BBB [130]. Further knowledge concerning the transporter and drugs that bind to it could support drug design of molecular entities targeting the transporter for enhanced brain drug delivery. PET is again a useful technique as the influx transporter can be investigated in a variety of species including humans, where there is a need to verify if the same influx transport is valid between species and as efficient in humans as in rodents. Hence, adapted PET studies need to be extrapolated to humans and it needs to be confirmed whether it is possible to use rodent measures of intra-brain distribution for the conversion of human PET concentrations. Therefore, advanced investigation is required on drug distribution in whole brain as well as specific regions in rodents and humans in both health and disease, as performed in Paper IV, for better interpretation of PET neuroPK outcomes.

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11C polar metabolites and their impact on brain imaging When using techniques, like PET and gamma counter measurements, which mainly rely on radioactivity measures in a tissue, the risk of acquiring radio-labeled metabolites affecting data readouts are almost inevitable. The simul-taneous concentration monitoring of oxycodone, using PET and microdialy-sis, revealed a pronounced deviation in the data in the elimination phase when intact oxycodone was no longer provided by systemic infusion. Total oxyco-done PET concentrations were therefore also compared to total brain concen-trations analyzed by standard LC-MS/MS. At 60 min, LC-MS/MS concentra-tions of 201 and 244 ng/g brain were observed for the two animals studied, to be compared with an average PET concentration at 60 min of 280 ± 19.0 ng/mL. While the PET concentration was only slightly higher at 60 min, the divergence at the 120 min time point in the concentrations was more pro-nounced. The LC-MS/MS data showed a decline to concentrations of 38 and 37 ng/g brain for the two animals studied, while PET data showed an average value of 148 ± 10.8 ng/mL. The results further support the theory of a for-mation of radiolabeled metabolites with half-lives exceeding that of oxyco-done, which impact the brain imaging in the elimination phase.

The radiolabeling of oxycodone was performed in a strategic position of the molecule, where the radioactivity was predicted to mainly follow the metabo-lite oxymorphone (Fig. 6). While also being subjected to active influx at the BBB, oxymorphone is formed to a low extent and was not expected to influ-ence or dominate the brain PET measurements [131]. However, the main me-tabolite noroxycodone is formed through oxycodone N-demethylation, which is also the process responsible for the metabolism of noroxymorphone, and α- and β-noroxycodol. It could be speculated that polar radiolabeled metabolites such as [11C]carbonate and [11C]formaldehyde are formed as a consequence of primarily oxycodone N-demethylation to noroxycodone, and that such metab-olites penetrate the BBB and interfere with the brain imaging. To investigate this phenomenon, [11C]carbonate and [11C]formaldehyde were synthesized and their respective blood and brain time-activity profiles were studied using a similar 60 min infusion design as used for [N-methyl-11C]oxycodone in com-bination with continuous PET scanning for 120 min. A nearly one to one brain to blood relationship was observed for [11C]carbonate during the 120 min stud-ied and the brain half-life was calculated to 53±21 min. The observed brain penetration of [11C]carbonate and the estimated half-life, which exceeded ox-ycodone half-life, indicated that a formation of [11C]carbonate from [N-me-thyl-11C]oxycodone might partly contribute to the prolonged half-life ob-served in the PET measurements compared to the microdialysis data. How-ever, as the injected [11C]carbonate might be transformed or incorporated into other entities in vivo it might not be the only factor influencing the time-activ-

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ity curves. Therefore, further investigation of the percentage of intact [11C]car-bonate in blood was performed during and after a 60 min constant rate infusion of the same. Intact [11C]carbonate in blood as a percentage of the total radio-activity resulted in mean estimates of 91% and 34% at 45 min and 120 min, respectively. These results suggested that the administered [11C]carbonate is consecutively transformed or incorporated into non-volatile metabolites in vivo which rapidly equilibrate across the BBB, given the similarity in the [11C]carbonate blood and brain time-activity profiles over time.

Figure 6. Metabolic pathways of [N-methyl-11C]oxycodone [128].

 

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The percentage of [11C]carbonate was determined after an [N-methyl-11C]ox-ycodone infusion and was shown to contribute by approximately 28% to the total radioactivity in blood. Given these data, [11C]carbonate was confirmed to be formed from [N-methyl-11C]oxycodone. Still, the formation of [11C]car-bonate alone was not sufficient to explain the discrepancy in the PET and mi-crodialysis data and the brain and blood time-activity profiles of [11C]formal-dehyde was therefore also investigated. [11C]Formaldehyde and/or sequential metabolites were able to enter the brain, even though the brain radioactivity measurements were at all times lower than the radioactivity detected in blood. Interestingly, the radioactivity in blood exhibited a rapid decrease directly af-ter the stop of the infusion, possibly due to rapid formaldehyde conversion into subsequent metabolites, eliminated or distributed elsewhere in the body. The estimated half-life of [11C]formaldehyde and/or subsequent metabolites was 143±31 min in brain and its formation from [N-methyl-11C]oxycodone might contribute to the signal and prolonged half-life observed in the previous oxycodone brain PET measurements, even if this requires further detailed evaluation.

The manifestation and impact of labeled metabolites constitutes a common limitation of PET studies and the ideal radiotracer should not generate radio-labeled metabolites. However, for the wider use of PET as a method to inves-tigate drug neuroPK, this is difficult to fully circumvent. In order to mitigate metabolite impact, a study design similar or adapted from the one used in Pa-per I, with continuous infusion of compound may be preferable. While drugs cannot always be infused to steady state due to limitations in radioactivity, the most important is that an equilibrium is reached between brain and plasma concentrations. When intact radiotracer is continuously infused it dominates the PET signal which is advantageous in comparison to single bolus injections, which at least partly restricts the impact of radiolabeled metabolites on the PET signal.

BBB drug transport and integrity in health and disease (Paper II and III) Accumulating evidence suggests that the BBB is impaired during CNS related disease conditions, further posting the probability of a leakage of drugs into the brain by the route of a compromised barrier. However, to what extent dis-ease truly influence the BBB and how pathological conditions impact drug delivery can be questioned. Whereas translational techniques like PET can of-fer means of investigating BBB transport, more knowledge is required on spe-cific disease hallmarks that might affect the BBB. While being important from a drug delivery and discovery point of view, such knowledge is also essential

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for better forward and back translation between transgenic models and pa-tients. Therefore, Paper II and III aimed to investigate how Aβ (Paper II and III) and α-synuclein (Paper II) pathology affect the transport of small molec-ular drugs (Paper II) or passage of large molecules (Paper III) across the BBB.

The transgenic models, tg-APPArcSwe (Paper II and III) and tg-αSYNA30P (Paper II), expressing Aβ or α-synuclein pathology, respectively, as well as WT lit-termates at an age of 16-19 months, were used to study the BBB transport of the five model compounds digoxin, paliperidone, levofloxacin, diazepam, and oxycodone (Paper II), or the BBB passage of a 4 kDa FITC and a 150 kDa AR dextran (Paper III). The presence or absence of pathology, Aβ or α-synuclein, was confirmed in the respective transgenes and WT included in the experi-ments. In both Paper II and III, Aβ40 came out as the dominant Aβ species expressed by the tg-APPArcSwe mice. Whereas WT animals in both Paper II and III were devoid of Aβ pathology, low levels of α-synuclein was detected in the WT mice in Paper II. However, the pathology expressed by the tg-αSYNA30P mice is dominated by human α-synuclein (Paper II), and the levels observed in WT are most probably endogenous mouse α-synuclein detected by the ELISA.

CNS pathology and BBB transport of small molecular drugs In Paper II, the small molecular drugs were investigated as a cassette. Cassette dosing was applied to minimize the number of animals needed, while also providing internal validation of the in vivo processes investigated and of later ex vivo processing and analysis. Drugs with differing mechanisms of transport at the BBB were chosen, with the purpose to investigate disease impact on efflux and influx transport, as well as passive diffusion at the BBB. Combined dosing of the drugs was found to be a feasible study approach by comparing the drug Kp,brain between discrete and cassette dosing in younger WT animals, where the cassette dosing results were within two-fold of the discrete dosing for all compounds.

By combined measurements of total drug exposure in brain and plasma as well as drug binding to plasma proteins and brain tissue, the extent of BBB transport was determined as the Kp,uu,brain in Paper II. Digoxin, paliperidone, and levofloxacin all showed a net active efflux at the BBB, with Kp,uu,brain val-ues below unity. Studies in AD transgenic models as well as in humans have shown or postulated a downregulation of P-gp, of which both digoxin and pal-iperidone are documented substrates [28, 29, 33, 34]. However, no differences were observed in the extent of transport for any of the drugs targeted to efflux when comparing WT with any of the transgenes (Fig. 7). As was also shown in rats in Paper I, oxycodone displayed a net active uptake at the BBB in both WT and transgenic mice in Paper II, with a Kp,uu,brain above unity, which did

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not differ between the groups (Fig. 7). Owing to its physicochemical proper-ties, diazepam is suggested to pass the BBB trough passive diffusion, which was also confirmed by a Kp,uu,brain value at unity for diazepam, again with no difference in the extent of transport between WT and transgenes (Fig. 7). Thus, the results of Paper II suggest that the expression of Aβ or α-synuclein pathology alone does not influence the BBB to such a degree that the extent of transport of small molecular drugs is affected.

Figure 7. Relationships, depicted as the deviation from the line of identity, in the unbound brain-to-plasma concentration ratio (Kp,uu,brain) between a) tg-APPArcSwe and WT mice, and b) tg-αSYNA30P and WT mice. Dashed lines represents a two-fold de-viation from the line of identity. Oxy, oxycodone; Diaz, diazepam; Pal, paliperidone; Lev, levofloxacin; Dig, digoxin [132].

Aβ pathology and BBB integrity to large molecules In Paper III, tg-APPArcSwe mice (referred to as tg-ArcSwe in the paper) where used at an age of 18-19 months to study the general integrity of the BBB to large molecules during Aβ pathology, by investigating the passage of different molecular sized dextrans. To reduce the number of animals, the two fluores-cent dextrans were administered as consecutive injections to the same animal. The dextran brain-to-blood ratios were in general very low with mean values not exceeding 0.0015 for the 4 kDa dextran and 0.0003 for the 150 kDa dex-tran, measured at 5 and 10 min after administration, respectively. The ratios were compared between transgenes and age-matched WT, with no difference observed in the passage of dextrans at the time points investigated. Still, a slight correlation was observed both between Aβ40 and Aβ42 concentrations and the 150 kDa dextran brain-to-blood ratio (r=0.46 and p=0.031, and r=0.54 and p=0.010, respectively). While this could indicate a higher passage of the large dextran during more advanced pathology, the relationship was primarily driven by two transgenes showing among the highest Aβ levels, and such con-clusion requires further verification using animals with more progressed dis-ease.

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In Paper III, the tg-APPArcSwe mice were further characterized with regard to the presence of Aβ deposits in cerebral vessels, so called CAA. Interestingly, widespread CAA was detected in the brain vessels of transgenes (Fig. 8). CAA is in itself said to weaken the vessel integrity and predispose vessels to a higher incidence of microhemorrhages. Still, no increase in the extent of BBB transport of small molecular drugs or a general leakage of larger molecules was observed in Paper II and III. Hence, while certain properties of the NVU might be impaired during CNS disease, the BBB seems to be capable of sus-taining sufficient function for the transport of drugs, independent of the pres-ence of Aβ or α-synuclein pathology. Moreover, this observation appears to be valid regardless of the type of BBB transport, i.e. passive diffusion, active efflux or influx. The BBB also seems to be intact to large molecules in the presence of the Aβ pathology manifested by the tg-APPArcSwe mice. The find-ings in Paper II are in line with previous results in other mouse models of AD pathology, where no change in drug transport, both when considering rate and extent of small molecular drugs, have been examined [133, 134]. Still, other studies have shown alterations in the rate of transport of drugs. The rate of diazepam and memantine, a drug suggested to utilize the same transporter as oxycodone, was shown to be decreased in an AD mouse model also expressing tau pathology, possibly due to a thickening of the basement membrane rather than a change in transporter expression or function [34, 135]. This highlights the importance of investigating both the rate and extent of drug transport at the BBB and further relate the observations to the intended treatment regimen of the drug. Interestingly, in the study by Mehta et al., which showed a de-creased rate of transport of diazepam, no change in digoxin transport was de-tected, despite an observed 42.4% reduction of P-gp expression in the vessels of the transgenic animals [34]. It was suggested that this lack of change in digoxin transport was balanced as a result of lower efflux of drug and a lower permeability across the BBB compared to healthy conditions. However, con-sidering the results in Paper II it can also be speculated that the BBB is capable of sustaining sufficient transport despite the impact of disease, up to certain concentrations, possibly in the range of therapeutically used doses. Most cer-tainly, BBB integrity is also dependent on disease stage and it is important to investigate what stage in animal models that best resembles relevant human conditions.

Interestingly, the tg-APPArcSwe mice have also been used in studies evaluating enhanced delivery of antibodies and antibody fragments across the BBB [125, 136, 137]. In support of the findings of an intact BBB to large molecules in Paper III, no previous indication of an enhanced delivery across the BBB at-tributed to a leakage of molecular complexes across an impaired barrier has been proposed in the currently used AD model [136, 138]. In addition, no BBB leakage of antibodies, as a result of a compromised barrier, was shown in a study using multiple mouse models of AD pathology [139].

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BBB integrity to large molecules after acute immunotherapy BBB impairment might not be related to pathology only but can also be con-nected to treatments aiming to target the disease. Adverse events, like brain edema and microhemorrhages, have been associated to the treatment with anti-Aβ mAbs like 3D6 (bapineuzumab). In animal models of AD, the events were suggested to result from antibody interaction with CAA and further im-pairment of vessel integrity [52, 53, 140]. These side effects are usually stud-ied after long-term treatment with the antibody. However, microhemorrhages have been suggested to occur early in the treatment [141-143]. Given the in-dications of early adverse events during anti-Aβ antibody treatment, the im-pact of acute 3D6 treatment on BBB integrity was investigated in Paper III. In the study, 3D6 was supplemented with radioactive [125I]3D6 at the time of administration, enabling the detection of the antibody in the brain at termina-tion, which was 72 hours after the injection. The concentration of the antibody as well as the brain-to-blood ratio was significantly higher in transgenes com-pared to WT (p=0.014 and p=0.007, respectively). This shows that the anti-body resides in the brain of the transgenes to a higher extent than in control animals, which lack the targeted pathology. Interestingly, autoradiography pictures did not show a uniform distribution of the antibody to plaques in the brain parenchyma (Fig. 8). Instead, local deposits or accumulations were ob-served in the brains of the tg-APPArcSwe mice. As CAA pathology was con-firmed in the same tg-APPArcSwe mice, the observed radioactive accumulations are suggestive of antibody interaction with CAA in brain vessels (Fig. 8). In further support of the presence of such interaction was the finding of an asso-ciation between the brain-to-blood ratio of [125I]3D6 and Aβ40 concentrations (r=0.73 and p=0.0021). Aβ40 is the most abundantly deposited Aβ species in the tg-APPArcSwe mice, and is also the most abundant form of Aβ in human CAA, to which 3D6 has been shown to bind. As such interactions are proposed to result in microhemorrhages, an increased dextran passage across the BBB could be expected. However, using two-way ANOVAs, followed by Tukey’s multiple comparisons tests, no increase in the brain-to-blood ratio for either the 4 kDa or the 150 kDa dextran was observed when treated transgenes where compared to treated WT and non-treated transgenes and WT mice (p=0.076 and p=0.097 for the two dextrans, respectively). The antibody-CAA interac-tion is suggested to induce an immune reaction, which further influence vessel integrity [54]. However, such immune induction might take longer time than the 72-hour time lapse used in the studies of Paper III.

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Figure 8. Representative pictures of a tg-APPArcSwe and a WT mouse a) respectively, displaying or lacking [125I]3D6 depositions in brain, mainly in the frontal cortex, vis-ualized by autoradiography, and b) presence or absence, respectively, of Aβ pathology in vessels stained by Congo red. Black scale bar 250 µm.

Animal models can be used to delineate specific processes of a disease, or to exclude or confirm the impact of certain features of the disorder on drug neu-roPK. However, studies in patients are needed to fully verify to what extent complex disease conditions or treatments might affect drug delivery to the brain. The possibility exists that even small or local changes in BBB transport of certain drugs could result in altered or unexpected effects, considering the inherent properties of the drug and its interaction with its target and further potency. Yet, the studies in Paper II and III shows that the concept of a leaking, broken or dysfunctional BBB needs to be interpreted with caution. The barrier might still be functionally intact regardless of a somewhat lower or higher expression of transporters or despite impairment in tight junctional proteins. To be noted is also the importance of defining whether a change in response to a drug is due to altered PK or PD. Studies in patients and animal models of epilepsy have been conducted to address this issue, as altered drug effects are observed in these patients and as a compromised BBB, due to the upregulation of the efflux transporter P-gp, has been suggested within epilepsy. However, the reduced drug effect in these patients and animal models seems to be a result of altered PD rather than changed PK properties across the BBB [144, 145]. On the other hand, in a study by Reeves et al., increased occupancy of dopamine receptors in AD patients treated with antipsychotics were specu-lated to reside from neuroPK changes such as an impaired BBB or altered brain distribution of the drug investigated [146]. However, it was not possible to conclude whether the neuroPK related changes were due to age or AD alone [146].

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Drug tissue binding in brain regions in health and disease (Paper II and Paper IV) In Paper I, the whole brain was mainly targeted for the investigation of un-bound drug concentrations, which is often the case when using small animal PET imaging as the spatial resolution is not as good as in clinical PET. The alignment of PET and CT images with a rat based MRI atlas still provided the possibility of regional analysis of concentrations. However, as MRI atlases rarely originate from the same animals as those involved in the study, caution must be taken when interpreting the precision in such regional analysis. In-stead, there is an increasing use of integrated PET/MRI systems for both ani-mals and humans, which can enable the investigation of drug concentrations and neuroPK properties on a regional basis. However as stated earlier, if ther-apeutically active, unbound, drug concentrations and BBB transport are to be studied in humans using these techniques, further knowledge is required on drug binding properties in different regions of the brain and factors influenc-ing binding under healthy and disease conditions must be defined. Increased awareness of regional drug tissue binding is also important for preclinical drug research where combined measurements are used to estimate BBB transport and where fu,brain is used as a key component to determine Kp,uu,brain as observed in Paper II and described by Equation 3.

Brain tissue binding in mice expressing Aβ pathology Tissue and disease heterogeneity is apparent in different parts of the brain and could affect parameter estimates of Kp,brain and fu,brain, used to calculate the Kp,uu,brain, depending on the region studied. To confirm that the estimated BBB transport in Aβ transgenic mice in Paper II was representative of the whole brain, brain tissue binding of the four model compounds paliperidone, di-goxin, oxycodone, and diazepam were investigated in both the rostral and the caudal part of the right hemisphere. However, no difference in drug brain tis-sue binding was observed for any of the drugs between WT and tg-APPArcSwe or between the caudal and the rostral part of the brain (Fig. 9).

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Figure 9. Fraction of unbound drug in brain, fu,brain, in rostral and caudal brain from aged WT and tg-APPArcSwe mice of a) paliperidone, b) digoxin, c) oxycodone, and d) diazepam. Technical replicates from pooled tissue is presented together with mean values [132].

Regional brain tissue binding in humans and rats Regional differences in nonspecific drug brain tissue binding was investigated in more detail in Paper IV by determining the fu,brain,ROI of memantine, donepezil, paliperidone, diazepam, and indomethacin in specific brain regions from human controls, AD patients, and rats. The obtained regions from hu-mans include FrCx, PrCx, BG, and CRB, while HIP and whole brain were also investigated in rats. To ascertain the presence or lack of pathology in the hu-man samples, an in house analysis of the tissue was performed. The concen-trations of soluble and insoluble Aβ42 confirmed the presence of pathology in

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the AD group versus controls. The levels of Aβ42 in the regions of AD pa-tients also followed previously documented regional progression of Aβ in AD, with the highest concentrations in FrCx and BG, and with the lowest concen-trations in CRB [147, 148]. Apart from one individual in the control group who showed high levels of insoluble Aβ42 in the brain, all remaining controls were either devoid of pathology or showed concentrations of Aβ well below the levels observed in patients. Interestingly, the analysis of soluble Aβ oligo-mers and protofibrils did not show any major difference between AD patients and controls and was not able to separate the two groups based on pathology. Aβ oligomers and protofibrils are suggested to be the most toxic species of Aβ and for future studies of regional drug tissue binding in the brain of AD pa-tients it would be interesting to target patients with higher levels of this Aβ species.

No association was observed between the detected Aβ levels and drug fu,brain,ROI values for any of the compounds in Paper IV, and no major differ-ences were observed when performing a regional comparison of drug specific fu,brain,ROI values between the AD and control group. A lower brain tissue bind-ing was only observed for paliperidone in CRB, and for donepezil in FrCx in the AD group compared to controls with 1.6-fold (95% simultaneous CI: 1.1 – 2.2, p=0.002) and 1.5-fold (95% simultaneous CI: 1.0-2.2, p=0.033) higher fu,brain,ROI values in AD, respectively. Interestingly, in regional within group comparisons, a repetitive pattern of lower drug brain tissue binding in CRB compared to other investigated regions was observed for all drugs studied apart from donepezil when considering the data from both AD and controls. The fold differences were fairly modest in the within group comparison, reaching maximally 1.6-fold. Still, the same pattern was observed for diaze-pam in rats, where CRB showed a lower binding compared to all other regions as well as whole brain. However, opposite to the diazepam rat data, donepezil showed a higher binding in CRB compared to all other regions in rats. A sim-ilarly higher binding of donepezil in CRB versus FrCx was also observed in AD patients and in general, donepezil showed a deviating pattern in the re-gional comparisons from the other compounds.

In general, the results in Paper IV were in line with the results obtained in Paper II in transgenic animals. Accordingly, no major differences were ob-served in drug tissue binding when comparing controls and subjects display-ing AD pathology. However importantly, while most regions studied showed an equal brain tissue binding of the compounds investigated, a differing brain tissue binding of drugs was observed in the CRB, which was not separately analyzed in Paper II. While often excluded in many PK analyses, CRB is an important region frequently used as a reference region in PET studies investi-gating drug PK through the simplified reference tissue model [149, 150]. An

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assumption in that model is that the nonspecific binding of drugs is equal be-tween the target tissue and the reference tissue, which is not supported by the results in Paper IV [149, 150]. The importance of accounting for regional dif-ferences in nonspecific binding in PET modeling was recently addressed in a publication by Salinas et el., showing that a violation of this assumption would cause a bias in the model outcome [151]. CRB was also used as a target region to study BBB transport of small molecular drugs using fu,brain estimates in a study in pigs [152]. Again, it might be deceiving to use CRB as a reference region for the prediction of BBB transport in whole brain or other regions, when estimates are based on fu,brain, given the results of Paper IV. This is also important given the findings of transporter differences in expression and func-tioning in the CRB compared to other regions [91, 153-155].

The general assumption of nonspecific brain tissue binding in PK studies is that it is homogenous throughout the brain. However, the results of Paper IV show that further investigations are needed, including studies in more regions and on more drugs. Elaborated studies could possibly enable a stratification on drug physicochemical properties and their relation to specific binding pa-rameters, which might vary between, and even within regions. Drug binding in brain tissue is mainly dominated by the drug interaction to membrane lipids, which is suggested to surpass protein-drug interactions [83, 85, 156]. The pri-mary lipids suggested to dominate drug binding are the ones belonging to the class of phospholipids, and would need further investigation in relation to drug tissue binding in diverse brain regions [83, 85, 157, 158], especially since studies have shown differences in lipid characteristics among brain regions [89, 92, 93, 159]. In addition, a comparative study of lipid profiles in brain regions of AD patients and mouse models showed substantial lipid changes in the AD mice compared to control animals, which were both transgene and age dependent, and which were not observed in AD patients [92]. This again high-lights the importance of investigating fu,brain in humans, as animal models are not always predictive of the clinical state.

Translation of brain tissue binding from rats to humans Species independent binding of drugs in brain tissue is widely assumed given previous findings [87, 88]. The regional differences observed in rats and hu-mans in Paper IV will hopefully initiate an awareness to these assumptions and the use of binding estimates. Of importance was the observation in Paper IV of a very low variability in drug binding between and within regions in rats and human controls, while a pronounced variability was observed in the AD group. In the AD group both the inter-individual and especially the intra-indi-vidual variability was high (Fig. 10). The differences between regions in the same individual were especially pronounced for donepezil in the AD group, with up to 3.9-fold differences.

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Figure 10. Fraction of unbound drug in regions of interest (fu,brain,ROI), presented as individual mean values, in post-mortem brain from AD (blue circles) and age-matched control donors (red circles), as well as rats (green circles). FrCx, frontal cortex; PrCx, parietal cortex; BG, basal ganglia; CRB, cerebellum; HIP, hippocampus; WB, whole brain.

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When investigating the relationship between the human control and AD fu,brain,ROI values to the values in rats in Paper IV, it was shown that rat fu,brain,ROI falls within two-fold of the human control data. Nevertheless, rat fu,brain,ROI was generally lower than the human values, which renders a constant overestima-tion of the binding of the drugs investigated if extrapolating rat data to hu-mans. Given the two-fold or higher boundary usually adapted by the industry, the results in Paper IV support the use of whole brain or regional estimates of fu,brain in rat for the estimation of fu,brain,ROI in human controls. On the other hand, the rat data was not able to capture the variability observed in drug brain tissue binding in the AD patients. Furthermore, it was shown that the use of rat data as a predictor of AD fu,brain,ROI could result in a bias of the regional values in patients spanning above the two-fold deviance from unity. This could bring about repercussions for the use of rodent binding estimates in human PK stud-ies or PET paradigms like the one used in Paper I, if aiming to extrapolate the design to patients.

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Conclusions

A hampered CNS drug development and the present limitations of techniques used to study neuroPK, highlights the demand of better methods and strategies to investigate brain drug PK. Methods and neuroPK parameters must be ex-amined under both healthy and disease conditions, with translational inter-species applicability for bench to bedside verification of data. In the current thesis, PET is proposed as a technique for the estimation of unbound, pharma-cologically active, concentrations in brain and for the determination of BBB transport, given that radiolabeled metabolites are kept minimal or can be con-trolled for. For PET to be used in such neuroPK applications, accurate com-pensation of total PET concentrations using measurements of drug intra-brain distribution and binding is essential. In the current work the parameter Vu,brain, determined from in vivo measurements, was successfully implemented to cor-rect the acquired PET concentrations. Vu,brain is advantageous to fu,brain in de-scribing the true distribution of drug within brain as it takes the intracellular and subcellular distribution into account. Hence, Vu,brain is preferable to use in similar PET designs as the one used herein. Still, the acquisition of Vu,brain estimates, both in vivo and in vitro, is currently limited to species other than humans, especially measured in rodents. For that reason, fu,brain comes out as a more practical parameter for the compensation of total concentrations if aim-ing to use PET for inter-species translation of neuroPK, as fu,brain can also be determined in human tissue.

The nonspecific binding of drug to brain tissue is a key determinant of intra-brain distribution and hence of the Vu,brain and fu,brain estimates. As the nonspe-cific binding is suggested to be homogenous between species it could be spec-ulated that any of the binding estimates determined in rat could be used for the compensation of PET data in humans. However, the current findings on inter-species translation of drug brain tissue binding only support the use of rat fu,brain as a surrogate for fu,brain in humans not affected by neurodegenerative disease. Still, a general overestimation of the binding in rat was observed in comparison to human, which might need to be considered. The use of rat fu,brain

as an alternative measurement to that in AD patients is however not recom-mended given the current data. AD patients showed a more pronounced vari-ability in the brain tissue binding of the compounds investigated, which was not captured by the rat data. The use of rat fu,brain determined in whole brain for general or region-specific estimations of unbound drug concentrations in

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the brain of AD patients might give larger than the two-fold acceptance in deviation commonly acknowledged in drug industry, depending on the drug. A dissimilar brain tissue binding of drugs was also observed between regions of rats, human controls and AD patients, with repeatedly differing binding in CRB, mainly lower, compared to other regions. The study of regional differ-ences in neuroPK is, surprisingly, only in its infancy, but requires greater at-tention for more targeted and precise analysis of PK/PD relationships.

The brain tissue binding of drugs was not correlated with the Aβ pathology investigated in the current studies, neither when considering transgenic mice or humans. Neither did the findings in transgenic mice expressing Aβ or α-synuclein pathology show any difference in BBB transporter properties in-volving efflux, influx and passive diffusion, compared to age matched WT animals. The integrity to large molecules was also preserved in transgenic mice displaying Aβ pathology. All together, the findings support the presence of an intact barrier function and integrity despite the manifestation of disease specific hallmarks. Yet, other features of neurodegenerative disease, not tar-geted here, must be investigated in relation to BBB transport and drug intra-brain distribution, especially considering the complexity and diversity of CNS diseases and the variability in the patient populations. Studies are needed, in-cluding more compounds and more subjects, to bring further light into the question of a presence or absence of disease related changes in nonspecific drug binding to brain tissue. This is important given that fu,brain determined in patient tissue might be the sole alternative compensatory parameter for BBB transport estimations in AD using PET.

Animal models can help delineating the impact of different features of a dis-ease on neuroPK parameters. However, confirmatory studies in humans are required. Possibly by adapting promising new imaging techniques like com-bined PET/MRI systems to similar designs and with relevant parameter esti-mates as proposed in this thesis. PET/MRI would allow for simultaneous in-vestigation of BBB impairment and drug neuroPK. The translational potential of the methods would further enable similar studies in late preclinical investi-gations and early clinical studies. The current thesis presents different means of analyzing neuroPK parameters in rodents and models of pathology, with extrapolation to human parameters and applications. Still, many facets of the processes involved in drug discovery and development need to be scrutinized and reevaluated for a successful progress and outcome of drugs aimed for CNS disease, ultimately resulting in better treatment of patients.

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Populärvetenskaplig sammanfattning på svenska

En stadigt åldrande befolkning bidrar till en ökad förekomst av neurodegene-rativa sjukdomar såsom Alzheimers sjukdom och Parkinsons sjukdom, vilka drabbar det centrala nervsystemet (CNS). I dagsläget finns inga botemedel mot dessa sjukdomar trots ett stort behov. Möjliga sjukdomsalstrande faktorer har påvisats, såsom beta-amyloid och alfasynuklein vid respektive sjukdom. Läkemedelsindustrin har länge försökt att utveckla läkemedel mot dessa sjuk-domar men har inte lyckats påvisa tillräcklig effekt på faktiska patienter. En av orsakerna till att läkemedelsutvecklingen är problematiskt är att det kan vara mycket svårt att få in läkemedel i hjärnan, som har ett selektivt upptag av det som cirkulerar i blodet. De allra minsta blodkärlen, kapillärerna, utgör den främsta platsen för utbyte av näringsämnen och andra molekyler mellan blod och vävnad. Det är också fallet i hjärnan men här består kapillärernas väggar av specialiserade celler som formar en tät barriär, den så kallade blod-hjärn-barriären. Denna barriär har också effektiva transportproteiner som förhindrar vissa molekyler från att ta sig in i hjärnan medan andra ämnen plockas upp ur blodbanan. Blod-hjärnbarriär reglerar således in- och utpassage av både kroppsegna molekyler och läkemedel. För att kunna ge effekt måste tillräckligt höga koncentrationer av läkemedel nå in i hjärnan. Det är dock bara de fria läkemedelsmolekylerna som kan pas-sera cellmembran och alltså transporteras över blod-hjärnbarriären, inte de som är bundna till exempelvis albumin i blodbanan eller till vävnadskompo-nenter i hjärnan. Det är också bara det fria läkemedlet som faktiskt kan inte-ragera med sina specifika mål, vilket leder till en effekt i kroppen och därmed är av relevans för behandlingen av en sjukdom. När man vanligtvis mäter kon-centrationer av ett läkemedel så mäter man både den del som är ospecifikt bundet och den del som förekommer fritt. Prekliniska metoder har under den senaste 10-årsperioden utvecklats för att kunna särskilja de fria hjärnkoncent-rationerna från det som är ospecifikt bundet. I och med detta går det också att få en uppfattning om hur läkemedlet transporteras över blod-hjärnbarriären och hur mycket läkemedel som finns tillgängligt för att kunna ge effekt i hjär-nan. Däremot saknas metoder för liknande studier i människa. Djur och män-niskor skiljer sig på många sätt, speciellt vid sjukdom, och det är svårt att veta hur bra resultaten från djurstudier kan översättas till människa och till faktiska

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patienter. Eventuellt kan hjärnans skyddande barriär vara skadad eller dys-funktionell vid neurodegenerativa tillstånd, vilket skulle kunna medföra att läkemedelstransporten till och från hjärnan påverkas. Detta skulle i sin tur in-nebära att doseringen av läkemedel bör justeras beroende på sjukdomsgrad. Här kan djurmodeller som återspeglar den kliniska sjukdomen användas för att utröna vilka sjukdomsfaktorer som faktiskt har en viktig påverkan på blod-hjärnbarriärens integritet. Syftet med detta avhandlingsarbete var att utforska metoder och farmakokine-tiska parametrar, såsom ett läkemedels blod-hjärnbarriärtransport och fördel-ning i hjärnan, vilka kan användas eller studeras likvärdigt i både djur och människa. Detta för att vidare kunna mäta terapeutiskt aktiva, fria läkemedels-koncentrationer i hjärnan. Syftet var också att studera hur läkemedelstransport till och från hjärnan och läkemedels distribution i hjärnan påverkas av neuro-degenerativ sjukdom. Den medicinska avbildningstekniken positronemissionstomografi (PET) an-vänds idag inom läkemedelsstudier och vid sjukdomsdiagnostik på både män-niska och djur. Tekniken betraktas på så vis som fördelaktig för translationella läkemedelsstudier där man vill genomföra liknande analyser i olika arter. PET är beroende av att det man studerar är radioaktivt märkt. Kameran detekterar allt radioaktivt material och det går inte att skilja fria från ospecifikt bundna koncentrationer av läkemedel. I det första delarbetet i denna avhandling påvi-sades därför hur man i råtta kan beräkna de fria koncentrationerna från de to-tala PET koncentrationer av läkemedlet oxikodon. Genom att kompensera de totala koncentrationerna för ospecifik bindning av läkemedlet till hjärnvävnad och distribution av läkemedel in i celler, mätta med andra metoder, kunde fria koncentrationer erhållas. Då de fria koncentrationerna bestämts kunde vidare beräkning av läkemedlets transport över blod-hjärnbarriären göras. Slutsatsen är att en anpassad PET-design med stor sannolikhet kan användas för studier också i människa. Mycket tyder på att tillstånd som Alzheimers och Parkinsons sjukdom leder till förändringar i blod-hjärnbarriären. Trots detta kunde delarbete två och tre i denna avhandling visa att barriären inte påverkas avsevärt av sjukdomsrela-terade faktorer vad gäller transport av vanliga småmolekylära läkemedel eller, för beta-amyloid även, passage av större molekyler. Studierna genomfördes i transgena möss som enbart uttrycker delar av sjukdomen och det är viktigt att bekräfta resultaten i ytterligare modeller som uttrycker andra sjukdomsfak-torer för att fastställa vad som eventuellt bidrar till förändrad integritet hos blod-hjärnbarriären. Slutligen krävs bekräftande studier i människa för att kunna beskriva till vilken grad en potentiell funktionsnedsättning kan påverka läkemedelstransporten.

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För att ytterligare förstå översättningen mellan djurdata och data från männi-ska, undersöktes i det fjärde delarbetet hur den ospecifika bindningen av läke-medel skiljer sig i olika hjärnregioner mellan råtta och människa, samt mellan kontrollpersoner och personer med Alzheimers sjukdom. Arbetet visade att åldersmatchade kontroller och Alzheimerpatienter till stor del uppvisar lik-nande ospecifik bindning av läkemedel när samma regioner jämfördes mellan grupperna. Däremot observerades att den ospecifika läkemedelsbindningen skiljer sig åt mellan olika hjärnregioner både inom kontroll- och Alzheimer-gruppen, vilket kan vara viktigt att ta i beaktande i studier där läkemedelskon-centrationer ska bestämmas i specifika regioner. Samtidigt visade studien att data som tagits fram i råtta troligtvis kan användas för korrigering av PET koncentrationer i kontrollpersoner men inte för personer med Alzheimers sjukdom, där variationen var hög både mellan individer och mellan regioner i samma individ. Till dess att läkemedels bindningsegenskaper har undersökts vidare och karaktäriserats ytterligare i patientmaterial, kan tillämpningen av råttdata, utan ytterligare kompensation, leda till missvisande resultat vid sjuk-dom och guida potentiell läkemedelsutveckling i fel riktning. Arbetet i denna avhandling bidrar till en ökad förståelse och applicerbarhet av metodologiska och farmakokinetiska tillämpningar som kan hjälpa eller guida läkemedelsstudier och läkemedelsutveckling. Framförallt i utvecklingen av CNS-aktiva läkemedel från djur till människa. Det allmänna antagandet om en försämrad blod-hjärnbarriär vid CNS-sjukdom, vilket skulle kunna med-föra att läkemedel läcker in i hjärnan, styrks inte i detta arbete. Blod-hjärnbar-riären verkar således vara funktionellt intakt oberoende av förekomsten av sjukdomsalstrande egenskaper kopplade till hjärnans sjukdomar.

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Acknowledgements

The work presented in this thesis was carried out at the Department of Phar-maceutical Biosciences, Faculty of Pharmacy, in great collaboration with the Department of Public Health and Caring Sciences, Faculty of Medicine, and the SciLifeLab Pilot Facility for Preclinical PET-MRI, all at Uppsala Univer-sity, Sweden. I would like to acknowledge the Swedish Academy of Pharma-ceutical Sciences (Apotekarsocieteten) and Anna-Maria Lundin’s Foundation at Smålands Nation for the obtained travel grants.

I am sincerely grateful to all of you who have contributed to this thesis work and encouraged me during my time as a PhD student and a special thanks to: My main supervisor Margareta Hammarlund-Udenaes, for introducing me to a new field of research and for always allowing me to drop by your office with that one “quick question”. Thank you also for always calming me down and for your care in times when life has been challenging. My co-supervisor Stina Syvänen, for everything that you have done for me and my projects during these years. Thank you for always being calm, realistic and for giving me so much of your time and scientific input. You are a pillar of strength and I hope you know how valuable that has been to me.

My co-supervisor Irena Loryan, for the many and long talks about science and life, sharing laughter and tears. Every time we talk you always amaze me with your invaluable knowledge. Thank you also for our great stay in Australia, which you organized to perfection. Anneli Hällgren, my former manager and colleague, who is to be held respon-sible for the start of this PhD journey. I will take the opportunity to thank you for being a role model and for giving me chances to develop and grow. My co-authors and collaborators, Jonas Eriksson, Olof Eriksson, Gunnar An-toni, Veronica Lindström, Martin Ingelsson, Dag Sehlin, Tobias Gustavsson, Sahar Roshanbin, Greta Hultqvist, and Erik Lampa. Thank you all for keeping up with my short deadlines and for excellent collaborations. I have really ap-preciated your invaluable comments, discussions, personal support, and for allowing me to be part of your field of expertise and learn from you.

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Jessica, for your help in the lab. Sorry for early mornings, draughty environ-ments, a certain degree of radioactive exposure, and setting off alarms (again, sorry for that Sergio, but you said you would be there early). Also, thank you for sharing life and laughter whenever time allowed.

Britt, for your help in the lab and technical know-how. I am always impressed by your many stories and by your exceptional memory.

Annika L, for being an excellent roommate and for your indefinable patience to all of my questions and “I can’t stop my mouth from talking to you” situa-tions. Thank you for all the support you have given me and for lending me a shoulder to cry on or an opportunity for laughter.

Erik, Nebojsa, Yang, and Waqas, for the nice discussions and fun times at group meetings and conferences. There has always been an open door to any-one of you for quick chats and helpful thoughts and suggestions.

Thomas, you came to the group as an equal and I felt like home, and then you left… But never mind. Thank you for introducing me to the Rudbeck people (I don’t generalize you guys, I’ve had so much fun with you, it’s just for the ease of writing) and for always being so cheerful. Markus and Erica B, for inspiring talks and for the fun time spent together at AAPS in San Diego, with science in focus and beer tasting in the periphery.

Marina S, my one and only master student, who turns out to defend before me. Your accuracy and will to progress and learn has been great to follow. Thank you for every time that you have passed by my office to see how it goes and to help me out with committee matters during our final deadlines.

All of my roommates (not already mentioned). Oskar A and Oskar C, I am glad that I got the chance to pursue large parts of this PhD journey with you guys. You have always been supportive, cheering me up and listening to my doubts (at least you made me believe that you were listening, I know I tend to talk a lot), while also sharing moments in life. Oskar A, a big thanks for all the help with my “sorry I don’t have time to ask how you’re doing, instead I have five questions about this thesis writing” messages and emails. Chunli, it was really enjoyable and helpful to share the common PhD thoughts and feelings with you in our early days. Emilie and Anne-Gaëlle, your French aura em-braced the room and you have always contributed to good spirits. Yasunori, thank you for great talks, mostly concerning life, and for the emotional roller coasters that finally ended in happiness. Ana, for giving me a perspective on handling family, commuting, and work. It was just great having you as a

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roomie. Sebastian, sorry that I even considered taking the desk by the window. I haven’t been the most present roommate lately but it has always been nice to see you when we have both been in the office and to share the daily children talks.

All of you in who’s offices I tended to end up in for long talks. Especially Anders K, who always has an answer to everything, and Elin, it got empty when you left the department with that doctor’s title. I have missed our front row seats, listening to that music and spoken word that passionate us both.

Linda A, Shima, Elsa, Emelie, Sara and Magnus, for relieving talks during PhD after works and stairway discussions.

Dag, I’m happy to have experienced your technical expertise and knowledge and thank you for all the questions that you have answered. I hope you know how grateful I am to you and your wife when she allowed you to stay when I (nearly) cried, with post-it notes up to my nose, being left alone in the lab.

Joakim B, for sharing knowledge and anecdotes during lunch time at Rudbeck.

All of you in the Molecular Geriatrics group who have guided me in your labs and welcomed me in your discussions (sometimes, possibly involuntarily from your side as I tend to sneak in to discussions, which is really none of my business).

Sergio, Ram, Veronika, and Ola at the Preclinical PET-MRI Platform. Thanks for the guidance in your lab. Especially Ram for the runs in the culvert between the hospital and PPP to save all the valuable radioactivity that we could.

All the excellent (even if you don’t have it in your title) teachers that I have had the chance to work with. Thank you for always being there to offer a hug and a moment to breath and for letting me enjoy your teaching skills and knowledge. There are many teaching moments with you that I will never for-get, with me being the actor (let’s look at it that way for now) and you being the examiner trying to keep up with all the talk. Jörgen, thank you for being an inspiring teacher and mentor in how to teach.

Karin, Marina R, and Ulrica B, for the frequent stops at your offices, discuss-ing everything from administrative stuff to life in general. Elisabet (Bettan) and Maria (Mia), it has always been comforting to know that even if I have been away from the department for some time, there are at least always some people who know me and we can pick up from where we left. Agneta E, for your positive spirit and support when I needed it the most.

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Time is short when I write my final acknowledgements and obviously I’m not very good at keeping things short in general (note to coming thesis authors: they say start early with the acknowledgements, and they are right). Hence, to past and present colleagues at the department, thank you all for the enlighten-ing discussions and for contributing to cheerful talks and moments during these recent years. My former colleagues and friends from Karo Bio. I still enjoy our gatherings and a special thanks to Maria B and Joakim L for your support and for our nice lunch breaks in Uppsala. Erika S and Emelie A, you guys deserve to be specially mentioned just because you are you. Linda B, I definitely think it’s time for us to move closer, to again be able to share daily struggles and happiness. In other words, I miss you. I know that you are always there and I’m so grateful for having you as a friend. Annika B and Pia, for being my friends throughout the years and no matter how long we’ve been apart it feels like yesterday when I see you. Inger and Bosse, thank you for all your support and interest in my work and for being a helping hand in life. My family, Mamma, Pappa, Susanna, Hedvig, Hilda, farmor, farfar, Ulrika, Tonny, Max, Lukas, and Ann-Christin, thank you for believing in me and al-ways helping me out whenever I need you and for all the fun times we share. Jag hoppas att ni vet hur mycket ni betyder för mig. Mojje, through night and day you have been with me. Showing your love and support in your own way. Matte älskar dig!

William, you are my anchor to reality and you have given me an invaluable perspective on life. Every day you remind me of why I love science and the power of learning new things. Mamma älskar dig! Carl, I think we both know that without you in my life none of this would have been possible. You are definitely the one to lean on and I love you be-yond everything. Together we will continue to manage what comes in life, live, and enjoy. Sofia Solna (working from home), April 2018

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Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 254

Editor: The Dean of the Faculty of Pharmacy

A doctoral dissertation from the Faculty of Pharmacy, UppsalaUniversity, is usually a summary of a number of papers. A fewcopies of the complete dissertation are kept at major Swedishresearch libraries, while the summary alone is distributedinternationally through the series Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty ofPharmacy. (Prior to January, 2005, the series was publishedunder the title “Comprehensive Summaries of UppsalaDissertations from the Faculty of Pharmacy”.)

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