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Tuulia Huhtala Biodistribution Studies in Small Animal Models Using Pre‑Clinical SPECT/CT Imaging Publications of the University of Eastern Finland Dissertations in Health Sciences TUULIA HUHTALA Biodistribution studies in small animal models using pre-‐‑clinical SPECT/CT imaging To be presented by permission of the Faculty of Health Sciences, University of Eastern Finland for public examination in Auditorium L22 in Snellmania, Kuopio, on Thursday, December 5th, 2013, at 12 noon. Publications of the University of Eastern Finland Dissertations in Health Sciences Number 200 School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland Kuopio 2013 II Kopijyvä Oy Kuopio, 2013 Series Editors: Professor Veli-‐‑Matti Kosma, M.D., Ph.D. Institute of Clinical Medicine, Pathology Faculty of Health Sciences Professor Hannele Turunen, Ph.D. Department of Nursing Science Faculty of Health Sciences Professor Olli Gröhn A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences Professor Kai Kaarniranta, M.D., Ph.D. Institute of Clinical Medicine, Ophthalmology Faculty of Health Sciences Lecturer Veli-‐‑Pekka Ranta, Ph.D. (pharmacy) School of Pharmacy Faculty of Health Sciences Distributor: University of Eastern Finland Kuopio Campus Library P.O. Box 1627 FI-‐‑70211 Kuopio, Finland http://www.uef.fi/kirjasto ISBN: 978-‐‑952-‐‑61-‐‑1294-‐‑7 ISBN: 978-‐‑952-‐‑61-‐‑1295-‐‑4 (PDF) ISSNL: 1798-‐‑5706 ISSN: 1798-‐‑5706 ISSN: 1798-‐‑5714 (PDF) III Author’s address: Charles River Discovery Research Services Finland Microkatu 1 FI-‐‑70211 Kuopio Supervisors: Docent Ale Närvänen School of Pharmacy, Pharmaceutical chemistry Faculty of Health Sciences University of Eastern Finland P.O. BOX 1627, FI-‐‑70211 Kuopio Professor Jouko Vepsäläinen School of Pharmacy, Pharmaceutical chemistry Faculty of Health Sciences University of Eastern Finland P.O. BOX 1627, FI-‐‑70211 Kuopio Reviewers: Docent Anu Airaksinen Laboratory of Radiochemistry University of Helsinki P.O. Box 55, FI-‐‑00014 University of Helsinki Ph.D. Thuy Tran Lund University Bioimaging Center Lund University Faculty of Medicine, BMC D11 SE-‐‑221 84 Lund Opponent: Docent Merja-‐‑Haaparanta-‐‑Solin Turku PET Centre University of Turku, Fi-‐‑20520 Turku IV V Huhtala, Tuulia. Biodistribution studies in small animal models using pre-‐‑ clinical SPECT/CT imaging Publications of the University of Eastern Finland. Dissertations in Health Sciences. 200. 2013. 96 p. ABSTRACT Imaging can be beneficially utilized in molecular medicine. Present imaging modalities can be divided to anatomical and functional methods, which can be co-‐‑ordinated by combining different methods. Single photon emission computed tomography (SPECT) combined with CT is one example where functionality of a contrast agent can be accurately localized anatomically. For successful SPECT imaging both chemically and physically appropriate radionuclides are required. Since nuclides have different physicochemical properties, variable labelling methods are needed to combine emitting atom with its targeting part. Using of long-‐‑lived nuclides, in vivo follow up of the radioactive signal can be done for several days whereas short-‐‑ lived nuclides enable repeated administrations with brief intervals. Proper animal models are key factor in successful pharmaceutical or medicinal experiment. To reduce animal number for ethical and financial reasons, cost-‐‑efficient methods where high quantities of data are achieved fast are optimal. Biodistribution and pharmacokinetic studies of therapeutic or diagnostic agents by SPECT/CT imaging followed by post mortem analysis in disease model gives good start point for further steps toward clinical applications. In this thesis, distribution properties of different biomolecules and viruses have been studied after radiolabelling them. Targeting properties, biodistribution and pharmacokinetics of these agents have been studied in orthotropic animal models using SPECT/CT imaging and post mortem analyses. National Library of Medicine Classification: WN 206, WN 450, QY 58 Medical Subject Headings: Tomography, Emission-‐‑Computed, Single-‐‑Photon; Diagnostic Imaging; Contrast Media; Radioisotopes; Models, Animal; Animal Experimentation; VI VII Huhtala, Tuulia. SPECT/CT-‐‑kuvantaminen ja biodistribuutiokokeet pieneläinmalleissa Itä-‐‑Suomen yliopiston julkaisuja. Terveystieteiden tiedekunnan väitöskirjat. 200. 2013. 96 s. TIIVISTELMÄ voidaan Kuvantamista hyödyntää monin tavoin molekulaarisessa lääketieteessä. Nykyiset kuvantamissovellukset voidaan jakaa esimerkiksi joko anatomisiin tai toiminnallisiin menetelmiin niistä saatavan tiedon perusteella. Eri menetelmien yhdistäminen on myös tärkeää, esimerkiksi yksifotoniemissiotomografian (SPECT) ja kolmiulotteinen röntgenkuvantamisen avulla merkkiaineen toiminnallisuus voidaan tarkasti paikantaa anatomisesti. Tuloksellinen SPECT-‐‑kuvantaminen edellyttää fysikokemiallisesti sopivien radionuklidien käyttöä. Koska nuklidit yksinään eivät ole soveltuvia funktionaaliseen kuvantamiseen, erilaisia leimausmenetelmiä tarvitaan yhdistämään emittoiva ydin sekä sen kohdentava osa. Hyödynnettäessä pitkäikäisiä nuklideja, voidaan lääke-‐‑ehdokkaiden tai biologisten merkkiaineen radioaktiivista signaalia seurata useita päiviä elävässä kohteessa kun taas lyhytikäisten nuklidien käyttö mahdollistaa toistuvat annostelut lyhyin aikavälein. Tarkoituksenmukaiset eläinmallit ovat erottamaton osa onnistunutta koeasetelmaa farmaseuttisessa ja molekulaarisessa lääketieteessä. Eläinmäärän pienentämiseksi sekä eettisistä että taloudellisista syistä menetelmät, joissa saavutetaan luotettavaa ja kvantitoitavaa tietoa tehokkaasti ajallisesti ja taloudellisesti ajatellen, ovat optimaalisia. Tutkitun yhdisteen seuraaminen SPECT/CT:llä kyseisessä tautimallissa luo hyvän pohjan kohti kliinisiä kokeita. Tässä väitöskirjassa biomolekyylejä sekä viruksia tutkittiin leimaamalla niitä radioaktiivisesti. Käytettyjen yhdisteiden kohdentavia ominaisuuksia, biodistribuutiota sekä farmakokinettiikkaa selvitettiin ortotooppisissa eläinmalleissa käyttäen SPECT/CT-‐‑kuvantamista ja post mortem-‐‑analyysejä. Luokitus: WN 206, WN 450, QY 58 Yleinen suomalainen asiasanasto: kuvantaminen, lääketiede; gammakuvaus; nuklidit; merkkiaineet; markkerit; eläinkokeet VIII IX Foreword The present study was started at the University of Kuopio, Department of Chemistry and A. I. Virtanen Institute in 2007 and finished at the University of Eastern Finland, School of Pharmacy in 2013. During this period I’ve also worked part time and full time in two private companies, Imanext and Charles River Discovery Research Services Finland, which is also my current employer. At first I want to thank my principal supervisor Docent Ale Närvänen for his endless ideas and optimism towards scientific challenges. He has taught me to convert almost impossible to possible. Also, your knowledge to combine organic chemistry and biochemistry to in vitro and in vivo testing has given me a great overall understanding in the field of natural sciences from medicinal point of view. Considering the positive feedback from my present employer your demands have been worthwhile. I also want to thank my second supervisor Professor Jouko Vepsäläinen for all of his guidance to finalize studies as well support and encourage during my years in Kuopio. I want to express all my gratitude to the reviewers Docent Anu Airaksinen and Dr. Thuy Tran for their valuable and constructive comments to finalize my thsesis. I also want to thank Docent Pirjo Laakkonen for his guidance concerning angiogenesis, lymphangiogenesis and immunohistological methods. Contribution of Dr. Jani Räty, Dr. Hanna Lesch and Dr. Minna Kaikkonen to introduce me in the world of viruses and in vitro solutions has been crucial. I also want to thank M.Sc. Jussi Rytkönen for his support with nanoparticles, HPLC and iodination works. I wish to express my gratitude also for Dr. Raili Riikonen and Dr. Hanna Sallinen for their help in studied diseases. I also want to thank Docent Timo Liimatainen and Professor Jukka Jurvelin for their excellent teaching of physics in the field of medical imaging. X Even though this thesis excludes almost all my co-‐‑written and published data with stem cells, I’m also grateful for Professor Jukka Jolkkonen to our co-‐‑operation. To my knowledge we have total of four publications together which consist the use of labelled stem cells in stroke models studied with SPECT/CT imaging. I wish to thank all staff of the former Department of Chemistry. I worked and partied with you roughly ten years. Those years include the best possible atmosphere for working and the best possible crew for leisure. It has been honour to be with you and discuss about science, life, philosophy, literature and everything everyone can ever imagine. Those years include unforgettable memories. In the same chapter I want to thank all the people from “Pikkupiha”, it seems that you represent my “new Department of Chemistry”. I’m deeply thankful to my parents, Päivi and Terho, as well grandparents, Mummi (Elsi) and Pappa (Sauli), for their parenting and way of thinking. You have raised me to be passionate, independent, hard working and open-‐‑minded. I also want to thank my little sister, Karoliina, for all the moments we have spent together. Finally I want to thank my spouse Ari who have had patience to live with me all these years. I’m always grateful for your understanding to all the time, which I’ve used to work or calm my nerves by riding or with salukis. We have had great years together so let’s continue the same way. Kuopio, June 2013 Tuulia Huhtala XI Contents 1 Prologue ..................................................................................................... 1 2 From atoms to nuclear imaging .............................................................. 5 2.1 Overview of the atomic structure ........................................................ 5 2.2 Radioactive decay .................................................................................. 6 2.2.1 Basic units and equations of activity ........................................ 7 2.2.2 α emission and nuclear fission .................................................. 8 2.2.3 β emission ..................................................................................... 9 2.2.4 γ emission ................................................................................... 11 2.2.5 Isomeric transition (IT) and internal conversion (IC) .......... 11 2.3 Radiation safety .................................................................................... 12 2.3.1 ALARA principle ...................................................................... 12 2.3.2 Factors influencing to radiation dose ..................................... 12 2.4 Nuclear medicine imaging techniques ............................................. 13 2.4.1 SPECT ......................................................................................... 14 2.4.2 PET .............................................................................................. 16 2.4.3 Hybrid scanning techniques .................................................... 16 3 Radiopharmaceuticals in nuclear medicine ........................................ 21 3.1 Isotopes used in SPECT imaging ....................................................... 22 3.2 Isotopes used in PET imaging ............................................................ 24 3.3 Agents for imaging and biodistribution studies ............................. 26 3.3.1 Conventional pharmaceutical compounds ........................... 27 3.3.2 Peptides ...................................................................................... 28 3.3.3 Proteins ....................................................................................... 29 3.3.4 Viruses ........................................................................................ 31 3.3.5 Living cells ................................................................................. 33 3.3.6 Nanoparticles ............................................................................. 34 4 The aims of this study ............................................................................ 37 XII 5 Biodistribution and pharmacokinetics of IGF-‐‑1 complexed with carrier protein or nanoparticles .................................................................. 39 5.1 Background .......................................................................................... 39 5.2 Labelling and conjugation of IGF-‐‑1 .................................................. 41 5.3 Used animal model ............................................................................. 42 5.3.1 Clearance and biodistribution of native and complexed IGF-‐‑ 1 ..................................................................................................... 44 5.3.2 Conclusions ............................................................................... 47 6 Biodistribution of 111 In-‐‑labeled antibodies in SKOV-‐‑3m induced mice ................................................................................................................. 49 6.1 Ovarian carcinoma .............................................................................. 49 6.2 Experimental part ................................................................................ 51 6.3 Immunohistochemical analysis confirmed specific antibody binding in lymph nodes ............................................................................... 55 6.4 Conclusions .......................................................................................... 56 7 Novel conjugation method to study biodistribution of avidin displaying lentivirus .................................................................................... 57 7.1 Background .......................................................................................... 58 7.1.1 Gene therapy ............................................................................. 58 7.2 Lentiviral construct ............................................................................. 59 7.3 Biotinylation of DTPA-‐‑antibody conjugates ................................... 59 7.4 Conjugation of antibodies had no effects to activity ...................... 60 7.5 Radiolabelling ...................................................................................... 60 7.6 Cell line and animals ........................................................................... 61 7.7 SPECT/CT ............................................................................................. 61 7.8 Post mortem analysis of antibody targeted 111In-‐‑labeled viruses ... 63 7.9 Conclusions .......................................................................................... 66 8 Epilogue ................................................................................................... 69 9 References ................................................................................................ 73 XIII ABBREVIATIONS A activity or mass number ALARA as low as reasonable achievable ALS amyotrophic lateral sclerosis BBB blood-‐‑brain barrier BCA bifunctional chelating agent β-‐‑CIT (-‐‑)-‐‑2β-‐‑carbomethoxy-‐‑3β-‐‑(4-‐‑iodophenyl)tropane BGO bismuth germinate BNCT boron neutron capture therapy BPA boronated phenylalanine Bq becquerel CaCo-‐‑2 continuous line of heterogeneous human epithelial colorectal adenocarcinoma cells CD4 a glycoprotein expressed on the surface of T helper cells, monocytes, macrophages, and dendritic cells Ci curie CNS central nervous system CT computed tomography Da dalton DC dendritic cell DMSO dimethyl sulfoxide DOTA 1,4,7,10-‐‑tetraazacyclododecane-‐‑1,4,7,10-‐‑tetraacetic acid DTPA diethylene triamine pentaacetic acid EC electron capture EGFR epidermal growth factor receptor EIA enzyme immunoassay ELISA enzyme-‐‑linked immunosorbent assay EMEA European Medicines Agency eV electron volt FBS fetal bovine serum FDA Food and Drug Administration FDG fluorodeoxyglucose FSH follicle stimulating hormone XIV GFP green fluorescent protein HD Huntington’s disease HeLa derived cell line from cervical cancer from Henrietta Lacks HEPES 2-‐‑[4-‐‑(2-‐‑hydroxyethyl)piperazin-‐‑1-‐‑yl]ethanesulfonic acid hIgG human immunoglobulin G HIV human immunodeficiency virus HMPAO hexamethylpropyleneamine oxime hNIS human sodium iodide symporter HSV-‐‑tk thymidine kinase from Herpes Simplex virus HR1 first hepta-‐‑repeat HRP horseradish peroxidase IC internal conversion IFN interferon IGF insulin-‐‑like growth factor IGFBP IGF binding protein INCL infantile neuronal ceroid lipofuscinoses IT isomeric transition ITLC instant thin-‐‑layer chromatography i.p. intraperitoneal i.v. intravenous LDLR low-‐‑density lipoprotein receptor LLD lower-‐‑level discriminator LV lentivirus m metastable state mAb monoclonal antibody MDP methyl diphosphonate MMP matrix metalloproteinase mol mole MRI magnetic resonance imaging MRS magnetic resonance spectroscopy N neutron number or number of nuclides n quantum number NaI(Tl) sodium iodide activated with thallium XV NP nanoparticle NMR nuclear magnetic resonance PBS phosphate buffered saline PBST phosphate buffered saline tween PD Parkinson’s disease PEG polyethylene glycol PET positron emission tomography PHA pulse height analyzer PMT photomultiplier tube PPT1 palmitoyl-‐‑protein thioesterase 1 OI optical imaging RF radiofrequency RNA ribonucleic acid RT room temperature SI Le Système International siRNA small interfering RNA SKOV-‐‑3 human ovarian adenocarcinoma cell line SKOV-‐‑3m primary cell line derived from the SKOV-‐‑3 cell line SPECT single photon emission computed tomography SPPS solid phase peptide synthesis T½ half-‐‑life THCPSi thermally hydrocarbonized mesoporous nanoparticle TMB 3,3′,5,5′-‐‑tetramethylbenzidine TU transduction unit ULD upper-‐‑level discriminator USPIO ultra small super paramagnetic iron oxide VEGFR vascular endothelial growth factor receptor Z atomic number silicon 1 1 Prologue Imaging technologies can be beneficially used in molecular medicine. Imaging of potential therapeutic or diagnostic agents in animal models in the early stage of research is an important step towards pre-‐‑clinical studies and clinical trials in humans. Modern transient medicine provides totally new approaches to deliver therapeutic agents to the patient starting from conventional small molecules to virus based gene therapy. Small animal imaging provides excellent method for development of new generation diagnostic and therapeutic agents. The aim of the research was to develop more specific and efficient agents with minimum side effects. Furthermore, early diagnosis of diseases and accurate follow-‐‑up is an important part of the therapy. These requirements have lead to more complicated bioactive molecules and their carriers. Due to the instability and complex nature of bioactive macromolecules like peptides, proteins, nanoparticles, cells or viruses, novel information about safe modification to study their properties in vivo is needed. Due to the complexity of new diagnostic and therapeutic agents, their biodistribution and pharmacokinetic profiles in vivo is difficult to predict. Besides toxicity which is one of the main concerns to conventional small pharmaceutical compounds, new agents have to face defence mechanisms like mononuclear phagocyte system, immunological response and liver as well. Larger size may also affect to the bioavailability of the agent. To overcome these problems comparative biodistribution studies with potential candidates should be started in early phase of the development. Non-‐‑invasive imaging has become important part of the basic and applied research. It allows biodistribution studies within same animal in different time points and phases of the disease. This is important for accurate monitoring since variations between individuals should be minimized to achieve reliable results in used model. Using imaging applications information from several time points and usually 2 with smaller variation within a group can be achieved compared to traditional methods based on post mortem or dosing studies with behavioural assays. Due to usual small variation in groups in radionuclide assays reliable results can be obtained with fewer animals, which is cost-‐‑effective and also in accordance to 3R principle (Russell & Burch 1959). With imaging behaviour of the studied compound in vivo is immediately seen and changes e.g. for the formulation and dosing route can be done in relative short time interval compared to dosing studies which may last several months before any effects or results are obtained. In pharmacological aspect several in vivo modalities for small animal imaging exist today. Magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT), positron emission tomography (PET) and optical imaging (OI) are widely used. Choosing the most suitable imaging application for a certain study depends of the prioritization of features. Small animal SPECT/CT imaging unit was established at the end of 2005 in the former University of Kuopio, which purchased also the first rodent SPECT/CT scanner in Scandinavia. This dissertation is primarily based on the following three publications in which the author has been involved as labelling and imaging specialists during the development of imaging methods in this laboratory. 1. Huhtala T, Laakkonen P, Sallinen H, Ylä-‐‑Herttuala S, Närvänen A. In vivo SPECT/CT imaging of human orthotropic ovarian carcinoma xenografts with 111In-‐‑labeled monoclonal antibodies. Nucl Med Biol. 2010; 37(8):957-‐‑64. 2. Huhtala T. Rytkönen J, Jalanko A, Kaasalainen M, Salonen J, Riikonen R, Närvänen A. Native and complexed IGF-‐‑1: biodistribution and pharmacokinetics in Infantile Neuronal Ceroid Lipofuscinoses. J Drug Delivery 2012; 1-‐‑8. 3. Huhtala T, Kaikkonen MU, Lesch HP, Viitala S, Ylä-‐‑ Herttuala S, Närvänen A. Antitumor effect of cetuximab targeted lentivirus. Accepted to Nucl. Med. Biol.; September 2013. 3 In addition to the primary publications, the following publications including author’s labelling, imaging, stem cell and animal model expertise have been published in the imaging unit. For the most consistent content of this thesis, these publications are only cited here or in the following chapters. 1. Mitkari B, Kerkelä E, Nystedt J, Korhonen M, Mikkonen V, Huhtala T, Jolkkonen J. Intra-‐‑arterial infusion of human bone marrow-‐‑derived mesenchymal stem cells results in transient localization in the brain after cerebral ischemia in rats. Exp Neurol. 2012 S0014-‐‑4886(12)00380-‐‑9. 2. Mitkari B, Kerkelä E, Nystedt J, Korhonen M, Huhtala T, Jolkkonen J. Toward a more effective intravascular cell therapy in stroke. Advances in the Treatment of Ischemic Stroke (Ed. Balestrino, M.), Intech, 2012, pp. 141-‐‑160. 3. Ruotsalainen J, Martikainen M, Niittykoski M, Huhtala T, Aaltonen T, Heikkilä J, Bell J, Vähä-‐‑Koskela M, Hinkkanen A. Interferon-‐‑β sensitivity of tumour cells correlates with poor response to VA7 virotherapy in mouse glioma models. Mol Ther. 2012, 20(8):1529-‐‑39. 4. Sankala E., Weisell J., Huhtala T, Närvänen A., Vepsäläinen J. Synthesis of novel bisphosphonate polyamine conjugates and their affinity to hydroxyapatite. Akivoc 2012 IV: 233 – 241. 5. Huhtala T, Närvänen A. Small Animal Imaging in Development of New Generation Diagnostic and Therapeutic Agents. Pharmacotherapy 2012 ISBN 978-‐‑953-‐‑ 51-‐‑0532-‐‑9, 194 pp. 6. Kaikkonen MU, Lesch HP, Pikkarainen J, Räty JK, Vuorio T, Huhtala T, Taavitsainen M, Laitinen T, Tuunanen P, Gröhn O, Närvänen A, Airenne KJ, Ylä-‐‑Herttuala S. (Strept)avidin-‐‑ displaying Lentiviruses as Versatile Tools for Targeting and Dual-‐‑imaging of Gene Delivery. Gene Ther. 2009 Jul 16;7:894-‐‑904. 4 7. Lappalainen RS, Narkilahti S, Huhtala T, Liimatainen T, Suuronen T, Närvänen A, Suuronen R, Hovatta O, Jolkkonen J. The SPECT imaging shows the accumulation of neural progenitor cells into internal organs after systemic administration in middle cerebral artery occlusion rats. Neurosci Lett. 2008 Aug 8;440(3):246-‐‑50. 8. Helppolainen SH, Nurminen KP, Määttä JA, Halling KK, Slotte JP, Huhtala T, Liimatainen T, Ylä-‐‑Herttuala S, Airenne KJ, Närvänen A, Jänis J, Vainiotalo P, Valjakka J, Kulomaa MS, Nordlund HR. Rhizavidin from Rhizobium etli: the first natural dimer in the avidin protein family. Biochem J. 2007 Aug 1;405(3):397-‐‑405. 9. Räty JK, Liimatainen T, Huhtala T, Kaikkonen MU, Airenne KJ, Hakumäki JM, Närvänen A, Ylä-‐‑Herttuala S. SPECT/CT imaging of baculovirus biodistribution in rat. Gene Ther. 2007 Jun;14(12):930-‐‑8. 10. Mäkinen S, Kekarainen T, Nystedt J, Liimatainen T, Huhtala T, Närvänen A, Laine J, Jolkkonen J. Human umbilical cord blood cells do not improve sensorimotor or cognitive outcome following transient middle cerebral artery occlusion in rats. Brain Res. 2006 Dec 6;1123(1):207-‐‑15. 11. Räty JK, Liimatainen T, Wirth T, Airenne KJ, Ihalainen TO, Huhtala T, Hamerlynck E, Vihinen-‐‑Ranta M, Närvänen A, Ylä-‐‑Herttuala S, Hakumäki JM. Magnetic resonance imaging of viral particle biodistribution in vivo. Gene Ther. 2006 Oct;13(20):1440-‐‑6. 12. Mäntylä T, Hakumäki JM, Huhtala T, Närvänen A, Ylä-‐‑ Herttuala S. Targeted magnetic resonance imaging of Scavidin-‐‑receptor in human umbilical vein endothelial cells in vitro. Magn Reson Med. 2006 Apr;55(4):800-‐‑4. 5 2 From atoms to nuclear imaging 2.1 Overview of the atomic structure An ancient Greek philosopher Democritus coined the atomic hypothesis around 450 BCE. He defined term átomos, which means "ʺuncuttable"ʺ or "ʺthe smallest indivisible particle of matter"ʺ and this is the background also for modern description. All matter is consisted of atoms which are the smallest units to break down without losing its chemical nature. Atoms are composed of positively charged nucleus and negatively charged cloud of electrons. Niels Bohr presented 1913 a model called Bohr atom in which electrons are on stable electron orbits without loss of energy. Different orbits starting from the innermost shell are called K, L, M, N shell and so forth or expressed as quantum numbers (n) with integer values (K: n = 1, L: n = 2 etc.). The shell’s quantum number defines the maximum number of electrons at one shell to be 2n2. (Cotton 1995) If an electron is shifted from the inner shells of an atom, an electron from outer shell immediately fills the vacancy and energy is released. The released amount of energy is equal to the difference in binding energies between these two shells. The released energy may emerge as a photon of electromagnetic radiation. Since the electron binding energies between shells are exact for different elements, the photon emission energies are called characteristic x-‐‑rays. (Cherry 2003) Another way to fill the energy gap after a removed electron is process called Auger effect. In that case, an electron from outer shell again fills the vacancy of the lower shell but released energy is transferred to another orbital electron which is emitted from the atom. These ejected electrons are called Auger electrons. Since Auger electrons leave orbital vacancies, they are further filled by electrons from the outer shells resulting other characteristic x-‐‑rays or Auger electrons. (Kónya 2012) 6 Nucleus contains positively charged protons which also define atomic number (Z) and uncharged neutrons (N). Mass number (A) implicates summarized mass of protons and neutrons. A nuclide is characterized as a specific nuclear composition which has exact Z, N and A. A nuclide also has to have a measurable long existence, which is greater than 10-‐‑12 s lifetime. (Cherry 2003) Number of protons also determines the number of orbital electrons and chemical element to which the atom belongs and hence the chemical nature of the element. Nuclides which have equal number of protons but the number of neutrons varies are called isotopes. Chemical behaviour of different isotopes is the same but the mass number varies. Nuclides which have same mass number are called isobars and nuclides with same neutron number are isotones. The most stable state of the nucleus is the ground state. Extremely unstable arrangements in nucleons are called excited states. Metastable states are also unstable but they are relatively long-‐‑lived before transforming another state. These are called isomeric states. Nuclides which are metastable states are indicated by the letter m (e.g. 99m Tc). The average lifetime of metastable state can be several hours as on the other hand the distinction between excited and metastable state is only 10-‐‑12 s. There is a discrete and exact energy difference between different states. Nuclear transitions from higher to lower states release energy which is emitted as particles or photons. (Williams 1987) 2.2 Radioactive decay Radioactive decay is a consequence of unstable nucleus which results from surplus energy or it owns wrong proton-‐‑neutron relation. Unstable nucleus transforms to more stable state by emitting particles and/or photons and releasing energy at the same time. Radiation can be particulate radiation or electromagnetic radiation. Particulate radiation consist atomic or subatomic particles whereas in electromagnetic radiation energy is carried by oscillating electrical and magnetic fields travelling at the speed of light. Each nuclide has specific properties 7 such as mode of decay, type of emissions, average lifetime and the transitions energy. (Cherry 2003) Radioactive decay is a spontaneous process of the nucleus and hence the chemical structure is irrelevant to the decay. One exception to the chemical behaviour is so called isotope effect. Since there is a small difference in mass between different isotopes there may also be small chemical differences especially if the relative mass differences are large, for example 3H versus 1H. The isotope effect is important in some studies, e.g. chemical bond strength measurements, but it has no practical consequence in nuclear medicine. (Wade 1999) 2.2.1 2.2.1.1 Basic units and equations of activity Electron volt The basic energy unit in radioactive decay is electron volt (eV). It is the amount of energy acquired when an electron is accelerated through an electrical potential of 1 V. In radiation physics typical values are kilo or mega electron volt magnitude. The conversion between SI units and the electron volt is 2.2.1.2 1 eV = 1.6022 × 10-‐‑19 J (2 – 1) Activity Activity (A) is described as nuclear changes at certain time and it is proportional to the amount of nuclei (N) A = λN (2 – 2) where λ is the decay constant for the radionuclide and can defined as the average decay rate i.e. activity ΔN/Δt = -‐‑λN (2 – 3) The decay rate is characteristic for each nuclide and the SI unit of activity is becquerel (Bq). The relation between activity and decay is 1 Bq = 1 s-‐‑1 (2 – 4) The former unit for radioactive decay is curie (Ci) which was originally defined as the activity of 1 g of 226Ra. The conversions between Ci and Bq are 1 Ci = 3.7 × 1010 Bq and (2 – 5) 8 1 Bq = 2.7 × 10-‐‑11 Ci (2 – 6) Since the activity or number of radioactive atoms decreases continuously according to exponential function of time N(t) = N(0) e-‐‑ t λ (2 – 7) and A(t) = A(0) e-‐‑ t λ (2 – 8) The half-‐‑life (T½) of the radionuclide is the time where half of the nuclides have decayed. The half-‐‑life and decay constant are related T½ = ln 2/λ (2 – 9) and λ = ln 2/T½ (2– 10) Combining equations (1 – 8) and (1 – 10) a common equation of radioactive decay is achieved. 2.2.1.3 A = A(0) e-‐‑ln 2/T½t (2 – 11) Purity qualifiers Specific activity represents radioactivity per gram [Bq/g]. In literature also activity per amount of substance [Bq/mol] is commonly used. Radionuclidic purity indicates the fraction of the desired radionuclide from total activity in a sample. This is important in medical studies since impurities increase the dose for the patient and may also affect to the measurements. Radiochemical purity is the fraction of activity in desired chemical form from the total activity of the sample. Radiochemical impurities results often from competing reactions during labelling process. They are also unwanted since their distribution in body usually differs from the studied chemical form increasing background to the images. Radiochemical purity should be at least 95%. (Harapanhalli 2010) 2.2.2 α emission and nuclear fission α-‐‑particle emission or nuclear fission occurs among very heavy elements. In decay by α-‐‑particle emission, one 4He nucleus is emitted with kinetic energy between 4 and 8 MeV. Although the energy of emitted particles is quite high, the range especially in solid material is 9 very short. For example in body tissues the range is only about 0.03 mm. For this reason α-‐‑particles are difficult to detect and measure. (Cherry 2003) Since the range of the α-‐‑particles is so short they are also extremely hazardous in living systems because the local dose easily raises to very high levels starting to cause regional damage at the tissue resulting to problems at organ and finally at the whole body level. The usage of α-‐‑particles in medicine is mainly focused on therapeutic agents where compound containing α-‐‑emitter is administrated to the tumour where it is desirable to achieve tissue damage and radiation is limited to small area. (Holland et al. 2010, Lucignani 2008, Zhang et al. 2006, Zalutsky 2006, McDevitt et al. 2001, Macklis et al. 1988) One interesting field is elements which have usable isotopes for nuclear imaging and α-‐‑emitters. These nuclides could be used for both imaging and site-‐‑directed radiotherapy. Another attractive but still rare medical application of α-‐‑ emitters is so called boron neutron capture therapy (BNCT). In this method boronated phenyl alanine (BPA) is injected into patient and it accumulates to the tumour. Stable 10B isotope is used in this method. When the tumour area is irradiated with low energy neutrons, 10B splits up and forms α-‐‑particles and lithium nuclei, which are very destructive to the tumour tissue. The local high dose of radiation is lethal to the cancer cells while saving healthy tissue. (Barth 2009) 2.2.3 β emission Radioactive decay by β emission is due to wrong neutron/proton ratio in the nucleus which transfers to the lower energy state. In β decay process the charge of nucleus changes but the mass number of nucleus stays unchanged. The nucleus has different ways to transform to the lower energy states which are β-‐‑ emission, β+ emission and electron capture (EC). (Kónya 2012) 10 2.2.3.1 β -‐‑ emission In radioactive decay by β-‐‑ emission a neutron in the nucleus is transformed to a proton. An electron and neutrino are ejected from the nucleus and carry away energy. n → p+ + e-‐‑ + ν + energy (2 – 12) The ejected electron is called β -‐‑particle. Neutrino is a “particle” -‐‑ without mass or electrical charge but it carries away energy. Since the energy of emitted β-‐‑-‐‑particle varies between zero and the maximum energy of the nucleus, decay energy is shared between the β-‐‑-‐‑particle and the neutrino which result to continuous energy spectrum. Beta particles can penetrate only relatively short distance from their origin, for example the maximum range is only a few millimeters in soft tissues. Because the penetration range of β-‐‑-‐‑particles is so short, they are difficult to detect outside the body and also special detector systems are needed to find β-‐‑-‐‑particles. For these reasons pure β-‐‑ emitters have no practical use in in vivo measurements or imaging studies. Most commonly used β-‐‑ emitters are 3H and 14C which can be used e.g. in in vitro studies or preclinical biodistribution studies. In those methods 3H or 14C labelled compound is administrated in vivo, the animal is terminated at certain time point and autoradiography or liquid scintillation methods are used to detect activity from different tissues. It is worthwhile to mention that detection of pure β-‐‑-‐‑particles from tissues needs some preparations before activity measurements and can be performed only post mortem. Occasionally β-‐‑ emission results to radioactive daughter nuclide which may further decay to the ground state by γ emission. Since gamma rays are much more penetrating than β-‐‑-‐‑particles they are much more suitable to use in nuclear medicine. Most commonly used (β-‐‑, γ) nuclides in medical studies include 131I, 133Xe and 137Cs. (Cherry 2003) 2.2.3.2 β + emission Decay by β+ emission is the opposite process to β-‐‑ emission. In this case a proton is transformed to a neutron. A positive electron i.e. positron or 11 β+-‐‑particle and neutrino are ejected from the nucleus and carry away energy. p+ → n + e+ + ν + energy (2 – 13) As in β decay, also β decay emitted particles lose their kinetic energy + -‐‑ very rapidly and can penetrate only a few millimetres in body tissues. However, when a positron collides with ordinary electron in an annihilation reaction, their masses are converted to two 0.511 MeV annihilation photons which proceed at 180 degrees to each other. Imaging technique called PET is based to this phenomena making it very popular method in nuclear imaging. (Williams 1987, McQuaid 2002) 2.2.3.3 Electron capture (EC) In EC process a proton forms a neutron by capturing its own orbital electron which is usually from K or L shell. p+ + e-‐‑ → n + ν + energy (2 – 14) Since EC results to vacancy on captured electron orbital it is filled with outer shell electron and this produces characteristic x-‐‑rays or Auger electrons as previously described. The EC itself cannot be detected but resulting characteristic x-‐‑rays are measurable. Also the daughter nuclide is often on metastable or excited state emitting γ-‐‑rays-‐‑ Medically interesting EC and (EC, γ) nuclides include e.g. 67Ga, 111In, 123I and 125I. (Cherry 2003) 2.2.4 γ emission A nuclear transition from higher to lower states release energy which is emitted as a photon and called γ emission. Since excited states are exactly quantified for different isotopes, the photon emission energies are always equal. 2.2.5 Isomeric transition (IT) and internal conversion (IC) If a daughter nucleus of a radioactive parent is on relatively long-‐‑lived metastable state, it further decays by γ emission in process called isomeric transition (IT). Internal conversion (IC) is an alternative for γ emission and typical for metastable states. In this case the nucleus 12 transforms energy to the K or L shell orbital electron which is ejected. Vacancy of the shell is further filled with outer shell electron resulting to characteristic x-‐‑rays or Auger electrons. The most used radionuclide in nuclear medicine is 99mTc which decays by IT. (Cherry 2003) 2.3 Radiation safety Since ionizing radiation contains enough energy to break molecules it can cause damage to living cells, tissues and the whole organism. Therefore safety and handling issues need to be taken care of while managing radioactive material. The effects of radionuclides and dose depend also greatly whether the source is inside or outside of the body. Because ionizing radiation is potential health risk, laboratory control, education of the personnel, waste handling, general instructions for the possible accidents and monitoring of the personnel and rooms need to be taken care of as defined in law. Regular use of radionuclides for research and medical use need also licensing from the national radiation authority. (Moores 2006) 2.3.1 ALARA principle ALARA stands for as low as reasonable achievable (ALARA) and means that only that amount of radioactivity is used what is needed for validate results. If there is a possibility to choose between different radionuclides, the least harmful should be used if equal results are obtained. (Farman 2005, Harvey 2008) 2.3.2 Factors influencing to radiation dose When using radioactive material and especially open sources many issues need to be considered. The physical and chemical form of the radionuclide, emission type and the amount of emitted energy, adequate air conditioning or working at hood, amount of used activity, proper shielding, correct detectors for the type and amount of radiation to be measured and good laboratory working methods affect to the total dose. Since radiation is emitted isotropically, the flux of radiation decreases as the distance from the source increases according to 13 inverse-‐‑square law. By increasing distance to the source decreases the exposure to the radiation. Many factors affect to the absorbed dose. The part of the body exposed to the radiation affects to the possible harmful effects. It is worse if the whole body is exposed than if only part of the body. Especially sensitive parts of the body to ionizing radiation are organs in trunk, blood-‐‑forming organs, gonads and eyes. Obviously time also affects to the dose. It is more harmful if great amount of radiation is given over a short period of time than if the same amount is delivered over a long period of time. However, when handling radionuclides in laboratory, experiments should be planned so that exposure time for the nuclide is a short as possible. The age of the exposed individual affects also to the sensitivity of radiation. Embryos, foetuses and children are particularly susceptible to ionizing radiation since there is a great number of dividing cells to which mutations can be very harmful. The type of radiation also affects to injuriousness. In general less densely ionizing radiation like β-‐‑particles or γ-‐‑rays are less destructive than densely ionizing radiation such as α-‐‑particles. However, the range of densely ionizing radiation is much shorter than for example γ-‐‑rays and it is also easier to shield. Personnel who are regularly working with radionuclides, personal dosimeters are required to follow up total exposure and dose at work. Maximum annual values for dose limits have been legislated. (Holmberg et al. 2010, Engel-‐‑Hills 2006) 2.4 Nuclear medicine imaging techniques Nuclear medicine imaging techniques are based on radioactively labelled compounds, which behaviour is studied by dedicated imaging equipment to diagnose or treat patients. In principle, pharmaceutical compound is labelled with a radionuclide and administrated into the patient usually intravenously (i.v.) or orally. Biodistribution, accumulation or pharmacokinetic profile of the radiopharmaceutical is measured with external detectors. In nuclear medicine imaging 14 techniques gamma camera, SPECT or PET is used. (Koba et al. 2013, Lancelot & Zimmer 2010) Imaging can be performed two-‐‑dimensionally (2D) or three-‐‑ dimensionally (3D) depending of gathered information. If fast processes of certain organ are measured like in renography, 2D images are obtained. 3D tomographic techniques enable reconstructed slices from different planes of the area of interest allowing precise examination of the target region from different angles. SPECT and PET are used to acquire functional information of the body compared to X-‐‑ray, magnetic resonance imaging (MRI) or ultrasound, which are mainly used for anatomical imaging. Applications of nuclear medicine imaging techniques obtain information rather of an organ than specific area of the body. 2.4.1 SPECT In SPECT imaging gamma emitting radiopharmaceuticals are used. In principle, in SPECT imaging photons are emitted from the patient and filtered by collimators. Filtered gamma rays are detected by scintillation material which produces light and light is multiplied at photomultiplier tubes (PMT). PMTs also convert the light signal to an electrical pulse which can be analyzed. (Gomes et al. 2011, Pani et al. 2004, Shayduk et al. 2012) 2.4.1.1 Gamma camera After the administration of radiolabelled pharmaceutical, the patient is scanned with SPECT system consisting of one or more gamma cameras around the target. Gamma cameras consist of scintillating material, which produces light when a photon collides to it. Sodium iodide activated with thallium, NaI(Tl), crystals is often used in SPECT detectors since its dense material and effective absorber of gamma rays (50 – 300 keV), it doesn’t absorb the produced light, large crystals can be manufactured and the PMTs are sensitive for the wavelength of produced light. However, NaI(Tl) crystals are also fragile, hygroscopic and deficient with high energy photons resulted to development of other scintillation crystals. 15 The light from the crystal is detected and amplified on PMTs to produce an electrical pulse. Light photon is caught on the photocathode resulting to a photoelectron which is multiplied through series of dynodes in a vacuum. After intensification light is gathered at the anode and transformed to an electrical pulse. The height of the pulse is proportional to the radiation energy and the quantity is proportional to the activity. Since after the scintillation crystal several PMTs are positioned, X and Y spatial information can be computed. The attained electrical pulse is further evaluated with pulse height analyzer (PHA). The input voltage and hence the energy of photon can be quantified with energy sensitive detectors i.e. pulse voltage amplitude is proportional to radiation energy. With reference material a voltage range can be adjusted with lower-‐‑level discriminator (LLD) and upper-‐‑level discriminator (ULD). For further data analysis only pulses which energy is between the LLD and ULD energies are counted. (Ricard 2004, Varga 2012, Scheiber 2000); 2.4.1.2 Collimators Radiation is emitted isotropically and since only orthogonally emitted primary photons are wanted to detect on gamma cameras. Collimators enable spatial resolution of the imaging target. They are made of lead which stops effectively diagonal photons. Various collimators are available for different purposes and emitters. The higher the energy of the nuclide the thicker of the lead layer has to be. Most common collimator types are parallel hole, pin hole, divergence, convergence, high resolution and high sensitivity collimators. (Yang et al. 2008, Park et al. 2006) 2.4.1.3 Different imaging modalities Information from the imaging target can be gathered 2D or 3D. For a single 2D image a static planar image is acquired. In dynamic imaging function of an organ or kinetics of the labelled compound is wanted to measure and several planar images are scanned with appropriate intervals. 16 To measure the function of the heart and myocardial perfusion gated imaging is used. In this modality electrocardiogram guides the acquisition and results to SPECT images of contracts of the heart. For 3D imaging tomography mode is used. In that case cameras rotate around the patient gathering radiation information from different angles. From this raw data a reconstruction is calculated and activity profile can be studied from different angles and slices. 2.4.2 PET For PET imaging a positron emitting nuclide is used as a radiotracer and a ring of detectors are around the patient. PET is based on the annihilation process (see chapter 2.2.3.2) and detection of produced 511 keV photons. Since the two photons proceed in 180° angle at the speed of light, PET imaging is based on the simultaneous detection of the opposite photons and so collimators are not needed for the spatial resolution. (Cherry 2003) In PET imaging 18F labelled fluorodeoxyglucose (FDG) is the most used imaging agent. Especially tumour tissues use more glucose than health tissue which can be seen as more active metabolism and accumulation of FDG. (Wadsak & Mitterhauser 2010) In PET systems bismuth germanite (BGO) detectors are mainly used since its highest stopping power (Saha 2010). Otherwise the detection camera composition is mainly similar to gamma camera as discussed previously. One important limitation for PET resolution is that the place of positron emission is not calculated but the place of annihilation. β-‐‑range depends of the energy of the particle and also the density of tissue, for example average β-‐‑range for 18F in water is 0.064 cm (Cherry 2003). 2.4.3 Hybrid scanning techniques Since nuclear medicine imaging techniques give information mainly function of the target organ and their anatomical information is rather poor, hybrid scanning techniques are commonly used. In most of the cases SPECT or PET is combined to x-‐‑ray computed tomography (CT) unit (Figure 1). However, since CT’s soft tissue contrast is limited, 17 combination of magnetic resonance imaging (MRI), which soft tissue contrast is excellent, and PET have also been developed. 2.4.3.1 SPECT/CT and PET/CT In principle, CT unit consist of high-‐‑voltage x-‐‑ray tube and oppositely located detector. Both x-‐‑ray source and detector rotate around the patient and a 3D reconstruction of the target can be made. Contrast is based on the ratio of the radiation which is passed through and absorbed in the patient. Function of the x-‐‑ray tube is based on high energy electrons which are produced on the cathode by heating wolfram over 2000 °C in a vacuum. At this temperature part of the electrons leak and are further accelerated towards anode material which often is tungsten, molybdenum or copper. When these high energy electrons hit the anode, bremsstrahlung and characteristic x-‐‑rays are produced. (Sutton 1994) In contrast to SPECT and PET where radiation comes from the patient, in CT radiation is produced in the imaging equipment and the fraction of radiation passing through the target is measured. Since differences in linear attenuation coefficients for soft tissues are small, contrast in soft tissues is limited with x-‐‑ray CT technique (Brown et al. 2008). Also the patient’s radiation dose raise significantly in CT studies. Figure 1. Example of combined SPECT/CT small animal imaging unit. Two oppositely located gamma cameras (red arrow) are in same gantry as the CT unit (place of CT, blue arrow) enabling easy combination of functional and anatomical data. 18 2.4.3.2 PET/MRI and SPECT/MRI For good soft tissue anatomical imaging MRI is ideal method. It is based on nuclear magnetic resonance (NMR) and the nature of proton nuclei. Since approximately 63 % of human atoms are protons, NMR offers excellent tool to study proton densities and its environment in medical imaging. Protons have magnetic properties (spin) and as natural environment the spins are indiscriminately settled and the net magnetization is zero. Isotopes that contain an odd number of protons and/or neutrons and have an intrinsic magnetic moment and angular momentum, like 1H, 13C, 2H, 15N and 31P can be used for MRI. When an isotope with magnetic properties (usually a proton) is put into a strong magnetic field, the nucleus of the isotope is aligned with the magnetic field. If then a short radiofrequency (RF) pulse is applied, the nucleus will align itself with the magnetic field generated by the RF pulse. After the pulse, the nucleus will return on its natural state at certain rate emitting an RF signal which can be recorded. The RF signal is analyzed and used to produce MR image. (Brown 2010) Since the environment of the proton affects strongly to the relaxation time, contrast is achieved between tissues. Furthermore, using MRS the relative concentrations of molecules in the target tissue can be calculated (Liimatainen et al. 2006b, Liimatainen et al. 2006a). Combination PET/MRI is relatively new and rare hybrid scanning technique but very fascinating (Pichler et al. 2008, Bisdas et al. 2010, Antoch & Bockisch 2009). Especially in brain imaging combination of PET and MRI seems advantageous and promising (Heiss 2009). With combined PET and MRI imaging gives valuable information about function of the heart (PET) and also information about ventricular structure of the heart (MRI) (Nekolla et al. 2009). Combination of SPECT and MRI is available only for animal studies (Goetz et al. 2008). In MRI contrast agent is not always a necessity but ferric, gadolinium or manganese based labels can be used to enhance contrast as they shorten protons’ relaxation time. In ferric labels ultra small 19 super paramagnetic iron oxide (USPIO) particles are widely used for various applications as vascularity and macrophage content in atherosclerotic carotid plaques (Metz et al. 2011), lymph node metastasis (Lei et al. 2010), tumour vascular morphology and blood hemodynamics (Gambarota et al. 2010), diffusion in the brain disorders (Chin et al. 2009, Vellinga et al. 2009), cell number quantification (Cheung et al. 2006) and oncological studies (Gambarota et al. 2006, Baghi et al. 2005, Keller et al. 2004). For MRI studies gadolinium-‐‑based and manganese based contrast agents have also been used. They require, like technetium and indium, chelates for labelling. Gadolinium ion as a water soluble salt is also quite toxic to animals and chelating reduces significantly its toxicity. However, the sensitivity of contrast agents in MRI or MRS is significantly lower than in SPECT or PET techniques. In MRI millimolar concentrations are needed whereas pico and even nanomolar concentration of radionuclides gives reliable SPECT or PET imaging results. (Hakumaki & Liimatainen 2005, Cheng et al. 2013) 20 21 3 Radiopharmaceuticals in nuclear medicine Nuclear medicine imaging methods are ideal to study biodistribution of various types of molecules or even viruses and cells. Since even nanomolar concentration of radiolabel gives adequate signal, small amounts of label is needed for obtaining preliminary results of the biodistribution, accumulation, pharmacokinetics and metabolic routes of the studied compound (Meikle et al. 2006). Other advantages include smaller animal groups than traditional pharmacological studies since whole body results can be achieved from multiple time points without terminating the animal. To study biological behaviour of agents by using radiochemical assays, they contain at least the targeting part and a radionuclide. For the detection of labelled agent, radionuclide provides acquirable signal as the agent acts as a targeting part for the nuclide. Since different tracers have different physicochemical properties, labelling of the molecules or living particles for imaging purposes requires knowledge in biochemistry, chemistry as overall and radiochemistry. Also, since the labelled agents are used in vivo, final product needs to be physiologically safe to administrate. However, final formulation steps and proper storing before administration has to sustain the structure and radiochemical purity of labelled agent. Final formulation of labelled agent also brings challenges in pre-‐‑clinical studies since dosed volumes for rodents are very small and administration is usually done as a fast bolus. An interesting field of study is to develop targeted molecules and also the labelling methods since the labelling shouldn’t affect to the behaviour of the labelled molecule. Also nature of the labelled molecule has to be considered that the labelling won’t inactivate or degrade the molecule. The following chapters will review commonly used imaging approaches and their radiolabelling procedures for 22 nuclear imaging. (Hargreaves & Klimas 2012, Rudin 2009, Cusanno et al. 2004, Contag 2002) 3.1 Isotopes used in SPECT imaging Invention of the 99mTc generator in the late 1950s by Walter Tucker and Margret Greene enabled a new era on the field of medical imaging. Nowadays 99mTc labelled products play a waist majority of clinical SPECT imaging studies since its optimal half-‐‑live (6 h) and energy properties (Emax = 140 keV). Other commonly used isotopes for clinical SPECT studies are 67Ga, 111In and 123I. Different isotopes, their physical properties and some of their applications are listed in Table 1. (Welch & Redvanly 2002) Table 1. Selected clinical SPECT imaging isotopes and their properties. Isotope 67 Ga Half-‐‑life 3.26 d Principal photon Decay Imaging energies mode applications 93, 185, 300 keV EC Tumour localization 99m Tc 6.03 h 140 keV IT Bone metabolism Myocardial, renal or brain perfusion 111 In 2.81 d 172, 247 keV EC Infection sites 123 I 13.0 h 159 keV EC Brain receptors 201 Tl 3.05 d 135, 167 keV EC Myocardium functionality EC, electron capture; IT, isomeric transition However, metals like 99mTc and 111In have to form complexes with donor ligands or chelates prior administration. If the molecule itself lacks chemical structures, which react with the metal as a ligand like sulphur fingers, the molecule has to be chelated. Commonly used chelates for Tc are bisphosphonates (e.g. methyl diphosphonate, MDP), 23 sestamibi, tetrofosmin and hexamethylpropyleneamine oxime (HMPAO). Bisphosphonates are used to scan bones, sestamibi and tetrofosmin cardiac function and HMPAO is used for cell distribution and brain perfusion studies (Anonymous, Beller & Heede 2011, Mills et al. 2013, Chuang et al. 2013, Glaudemans et al. 2013). Even though new chelates are continuously synthesized, diethylene triamine pentaacetic acid (DTPA) and 1,4,7,10-‐‑ tetraazacyclododecane-‐‑1,4,7,10-‐‑tetraacetic acid (DOTA) remain one of the most commonly used chelates in imaging (Figure 2) (Stasiuk et al. 2012). Figure 2. The most commonly used chelating agents in nuclear medicine, DTPA (left) and DOTA (right). These chelates are also available as bifunctional chelating agents (BCA) with e.g. succinimidyl or isothiocyanate derivatives (Chakraborty & Liu 2010, Liu & Edwards 2001). BCA increases the molecular weight of the target molecule and may also change the overall charge. This may cause steric hindrances and change in total charge within small molecules. For larger molecules like peptides and proteins the use of chelates is more common. In some cases the use of the linker between chelating part and reactive part may change pharmacokinetic properties (Garrison et al. 2008, Qu et al. 2001). Iodine radiolabels have the longest history in biochemistry and cell biology. Over 30 isotopes of iodine have been reported of which around ten has been evaluated for biomedical applications (Welch & Redvanly 2002). The choice of isotope depends on the purpose of the study. 123I decays with practical energy for imaging studies (159 and 127 keV), but its relative short half-‐‑life (13 hours) limits its usage. The half life of 125I is 60 days but emission energy is only 36 keV, which 24 makes it impractical for human studies but adequate for animal experiments, especially in mice. Its long half-‐‑life enables imaging studies for several weeks with single labelling batch if the target agent can be preserved. Typically the target molecule for iodination should contain benzene ring with ortho-‐‑substitution which in the most cases is OH-‐‑ group, like tyrosine in the peptides or proteins. Oxidated iodine reacts to the positions 3 or 5 or both. Iodine is oxidated by Chloramine-‐‑T, Iodogen or lactoperoxidase in direct chemical methods (Hunter & Greenwood 1962, Fraker & Speck 1978, Marchalonis 1969). The most convenient method for iodination of biologically active molecules is commercially available Iodogen tubes, which are coated with an oxidative agent (1,3,4,6-‐‑tetrachloro-‐‑3α-‐‑6α-‐‑diphenylglycouril). Due to the high hydrophobicity, this toxic oxidative compound is insoluble to water based buffers and remains in the walls of test tubes hence enabling excellent surface to labelling e.g. sensitive biomolecules in aquatic environment without interference of the reactant to the reaction mixture. The method is optimal for sensitive molecules. Firstly, the toxic compound remains intact with labelled compound and secondly, only unreached iodine may have to be purified before in vivo studies. If the agent of interest lacks a benzene ring, indirect labelling methods for iodination can be used where the conjugation of the additional radioiodinating reagent can be used. The most common and commercially available reactants containing a benzene ring are Bolton-‐‑ Hunter reagent, succinimidyl and isothiocyanate derivatives (Bolton & Hunter 1973, Zalutsky & Narula 1987, Vaidyanathan et al. 1997, Gabel & Shapiro 1978). They react with primary amines, which are common in bioactive peptides, proteins, viruses and cells enabling iodination position to the target molecule. 3.2 Isotopes used in PET imaging Labelling of PET radiopharmaceuticals is complicated and requires on-‐‑ site cyclotron and highly educated personnel. Due to the short half-‐‑ lives of tracers the cyclotron facilities should be near the research 25 laboratory. 18F, 64Cu and 89Zr have adequate long half-‐‑life that the delivery time can be hours instead of minutes. It’s also notable that even the half-‐‑lives of PET nuclides are shorter than SPECT nuclides the dose effect may be larger with PET nuclides since their emission energies are much higher than those of conventional SPECT nuclides. However, the metabolic elimination rate of each radiolabelled agent has also high impact for the total effective dose of a patient. Both biological excretion (tb) as well physical half life of the nuclide (tp) result to the effective half-‐‑life (te) of the tracer. 1/te = 1/tp + 1/tb In PET studies 18 FDG is the most commonly used PET radiotracer. Others, such as sodium 18 fluoromisonidazole, (3-‐‑1) and 64 18 fluoride, Cu-‐‑labeled 18 fluorothymidine, diacetyl-‐‑bis N4-‐‑ methylthiosemicarbazone are in clinical use but not approved by the FDA (Vallabhajosula et al. 2011). 64Cu would also have long enough half-‐‑life for transportation. However, its availability is still moderate. Additionally 89Zr has long half-‐‑life, but its high primary gamma energy and hence requirements to obtain effective dose moderate, safe handling and shielding may have limited its usage (Zhang et al. 2011). If the PET facility has short access to cyclotron, very short-‐‑lived nuclides such as 11C, 13N, 15O can be also conjugated to different organic molecules. One advantage of PET isotopes is that the elements are found in naturally occurring molecules and hence only certain atom of the compound has to be changed during labelling with negligible effects to the compounds natural behaviour. Table 2 summarizes the most commonly used PET isotopes, their physical properties and imaging applications. (Welch & Redvanly 2002) 26 Table 2. Selected clinical PET imaging isotopes and their properties. Principal photon energy for all is 511 keV, for 89Zr principle gamma energy is 900 keV. Isotope Half-‐‑life Decay mode Imaging applications 11 C 20.3 min EC, β+ Neuroimaging 13 N 10.0 min EC, β+ Cerebral and myocardial blood flow 15 O 2.07 min EC, β+ Oxygen metabolism, blood volume and flow 18 F 109.8 min EC, β+ Brain metabolism, oncology, cardiac innervations 64 Cu 12.7 h β-‐‑, EC, β+ Blood flow, hypoxia 68 Ga 68.0 min EC, β+ Lung injury 89 Zr 78.1 h EC, β+ Oncology, antibody labelling EC, electron capture 3.3 Agents for imaging and biodistribution studies Although most of the pharmaceutical compounds are small and relatively simple structures, there is growing number of other type of molecules for therapy and diagnostics. Exactly speaking it is inaccurate to speak novel therapeutic molecules, since there are also other solutions to deliver the therapeutic agents and affect the target tissue or cells. In recent year’s nanoparticles (NP), viruses and stem cells have been used in therapy. The first aim in development of novel diagnostic and therapeutic agents is to improve specific targeting moieties to the pathological alterations in tissues or cells. Secondly, they are multifunctional containing several biological of chemical activities like targeting, drug, carrier and tracer moieties. Thirdly, side effects should be minimized. Gene technology provides totally new approach to therapeutic field by delivering genes to the host cell, which transcription and translation machinery is used as "ʺdrug factory"ʺ. 27 3.3.1 Conventional pharmaceutical compounds Most of the commercially available pharmaceutical compounds are small molecules below 500 g/mol and they typically do not have homing properties but the effect is based on specific binding as an agonist or antagonist in the target tissue or cell. It’s also worth to mention that if small organic tracer is used, the nuclide should be directly labelled to the molecule structure or the molecule has to have chelating properties if a metal is used. One of the most commonly used small organic imaging agent is FDG, a glucose derivate, which accumulation through the body is related to tissue glucose consumption. This phenomenon is utilized in several applications of brain, tumour, myocardial and lung metabolism (Berti et al. 2010, Miletich 2009, Chen & Chen 2011, Kopka et al. 2008). Today FDG is widely used especially in neurosciences including drug research and development, as well follow up in oncology. With FDG it is possible to determine activation of certain brain areas and hence applications are numerous, e.g. the sensitivity of brain areas to drugs as well as behavioural and therapeutic effects of the drug (Welch & Redvanly 2002). 99m Tc is the most commonly used isotope in nuclear medicine. When it’s conjugated with DTPA, it’s used to measure functionality of the kidneys (Eckelman & Richards 1970), as a pyrophosphate for skeletal imaging (Thrall 1976) and with HMPAO (Ceretec™) brain perfusion and cell labelling (Leonard et al. 1986a). 111In is chelated to oxine which in clinics is used to label white blood cells or platelets to study sites of acute inflammation and infection but also thrombocytopenia (Leonard et al. 1986b, Thakur et al. 1977, Thakur 1977, Rodrigues et al. 1999, Louwes et al. 1999). Bisphosphonates can also chelate metals and are also used for bone scintigraphy. They can be also conjugated e.g. with polyamines as novel imaging agents for calcium plaques in the brain (Sankala et al. 2012) One widely used imaging agent is (-‐‑)-‐‑2β-‐‑carbomethoxy-‐‑3β-‐‑(4-‐‑ iodophenyl)tropane (β-‐‑CIT or RTI-‐‑55). In SPECT and PET imaging it has been used as 123 I labelled or 18 F (FP-‐‑CIT) labelled to map 28 distribution of dopamine transporters and serotonin transporters in the brain e.g. in Parkinson’s disease and supranuclear palsy (Zubal et al. 2007, Shaya et al. 1992, Shang et al. 2007, Staffen et al. 2000, Seppi et al. 2006). 3.3.2 Peptides Peptides are short chains consisting of amino acids. As a distinction to proteins, the size (ca. 50 amino acids) has been used as a benchmark between these two. Although peptides and polypeptides have been used for therapeutic purposes already for over 80 years when insulin was taken in clinical use, only few novel peptide based drugs have been approved by FDA or EMEA. Most of the drugs are direct copies from nature like follitropin beta, which is a synthetic copy of follicle stimulating hormone (FSH) (Fares et al. 1992, Shome et al. 1988). Second generation peptide drugs are modified from the original molecule or are part of the larger proteins. Octreotide is a long-‐‑acting octapeptide with pharmacologic properties mimicking those of the natural hormone somatostatin (Bornschein et al. 2009, Anthony & Freda 2009, Stajich & Ashworth 2006). Fuzeon (Enfuvirtide) is a 36 residue synthetic peptide that inhibits HIV-‐‑1 fusion with CD4 cells. Enfuvirtide binds to the first hepta-‐‑repeat (HR1) in the gp41 subunit of viral envelope glycoprotein and prevents the conformational changes required for the fusion of viral and cellular membranes. It works by disrupting the HIV-‐‑1 molecular machinery at the final stage of fusion with the target cell, preventing uninfected cells from becoming infected. Enfuvirtide is a biomimetic peptide that was rationally designed to mimic components of the HIV-‐‑1 fusion machinery and displace them, preventing normal fusion. (Joly et al. 2010, McKinnell & Saag 2009, Makinson & Reynes 2009) The situation may change due to new screening methods for novel peptides. Epitope scanning of amino acid sequences of the proteins and phage display libraries produce novel biologically active peptides with specific binding properties to target proteins such as receptors and proteases (Nilsson et al. 2000). Some of the identified 29 receptors have been found from highly specialized tissues providing a possibility to use peptides for targeting (Laakkonen et al. 2002). These peptides serve as lead molecules for development of molecules for tumour imaging and therapy. Both natural peptides and peptides characterized by phage display are sensitive to metabolic processes like protease activity. This limits their usefulness as diagnostic and therapeutic agents. Rationale design of chemical modifications to maximize enzymatic bioavailability while preserving the potency and specificity of the peptide is needed (Adessi & Soto 2002). Typically peptides are cyclised or the amino acid side chains or bridge structures are modulated by using unnatural structures, peptidomimetics (Pakkala et al. 2007, Pakkala et al. 2010). Peptides and their modifications are typically produced by using solid phase peptide synthesis method (SPPS). Today synthesis is made with automated synthesizer and the time to produce a peptide is relatively short and several companies provide synthesis services for reasonable price. For labelling an additional reactive amino acid like tyrosine or cysteine are easy to add to the sequence for further labelling or conjugation purposes. 3.3.3 Proteins Unlike peptides proteins are large and contain secondary, tertiary and some cases even quaternary structures on which the biological activity is based. Due to their defined tertiary structure and size, they usually denature rather easily and hence sensitive labelling and purification methods have to be used. Also immunological response has to be considered when proteins are evaluated as drug candidates. On the other hand, surface of the proteins contains several different chemically active amino acid side chains or polysaccharides, which can be used for labelling purposes. Typically proteins are labelled via the ortho-‐‑hydroxy benzene ring of tyrosine or via primary amino groups of either the amino terminus or the side chain of lysine. For the imaging purposes proteins are labelled with iodine or conjugated with chelates as previously 30 described. After the conjugation proteins can be purified with conventional size-‐‑exclusion chromatography, dialysis or ultra filtration using physiological conditions. In addition, chelate conjugated proteins can be labelled with 99mTc or 111In without further purification steps. Labelling and SPECT/CT imaging of e.g. natural and modified proteins gives also an excellent tool to compare their biodistribution in vivo (Helppolainen et al. 2007). One of the most interesting families of diagnostic and therapeutic proteins are monoclonal antibodies. Already 50 products have been passed the long and very expensive way from the primary finding to the licensed drug (www.biopharma.com, 2011). The limiting factors of antibodies are large size (150 000 Da), which may interfere the penetration of the molecule to the target tissue and possible squeamishness. Large size can be bypassed with Fab1 or Fab2 fragments of the antibodies or as in human therapy using humanized monoclonal antibodies. The advantage of antibodies is high affinity compared to other protein -‐‑ ligand interactions but also relatively easiness and diversity of modification chemistry without losing binding activity. On the other hand, antibodies have slow pharmacokinetics and it differs from that of small pharmaceutical molecules and peptides in vivo (Aerts et al. 2009). Antibodies require long monitoring times and the selection of the tracer with long half-‐‑life is important. Antibodies have been successfully used as imaging agents and further in cancer therapy. Cetuximab (Erbitux) is a humanized monoclonal antibody against epidermal growth factor receptor (EGFR), which is over expressed in various cancers (Vincenzi et al. 2008, Rivera et al. 2008). It has been successfully used in the treatment of colon carcinoma in humans. Same antibody has been studied as versatile SEPCT and PET imaging agent in several cancer models, e.g. malignant mesothelioma, prostate cancer, head-‐‑and-‐‑neck squamous cell carcinoma, ovarian carcinoma, colon cancer and universally EGFR positive tumours (Nayak et al. 2011, Malmberg et al. 2011, Hoeben et al. 2011, Huhtala et al. 2010, Cho et al. 2010, Ping Li et al. 2008). 31 Monoclonal antibodies binding to the EGFR ligand-‐‑binding domain prevent signalling via the receptor and lead to inhibition of cell growth, neovascularisation and induction of apoptosis in some models. 3.3.4 Viruses This very exiting approach is based on natures own gene delivery method. After delivery, modified virus in target cell begins to use cell’s natural amplification techniques to produce therapeutic molecules. Today there are both transient (Adenoviruses) and stable (Lentiviruses) viral delivery systems (Rissanen & Yla-‐‑Herttuala 2007, Mahonen et al. 2010, Lesch et al. 2009). Biodistribution studies using non-‐‑invasive imaging is an important part of the development of virus based therapeutic agents. Viruses for the therapy are modified, they are unable to multiply and additional therapeutic and/or reporter genes are added to the viral genome. Expressed reporter genes can be imaged by using radiolabelled ligands. Using sodiumiodine symporter (hNIS) gene together with cancer-‐‑specific human telomerase promoter, human colon carcinoma xenograft has been imaged using radiolabelled iodine with SPECT/CT in animal model (Merron et al. 2007). The most extensively investigated approach in suicide gene therapy is thymidine kinase from herpes simplex virus (HSV-‐‑tk) inserted e.g. in adenovirus. Briefly, the virus including HSV-‐‑tk is administrated and infected cells start to express HSV-‐‑tk, which acts as a receptor for HSV-‐‑tk ligands like ganciclovir (GCV) or acyclovir (ACV). GCV is a nucleoside, which is phosphorylated by HSV-‐‑tk into a monophosphate form and subsequently converted by cellular kinases into toxic GCV-‐‑triphosphate. (Asklund et al. 2003, Chiocca et al. 2003, Hayashi et al. 2006) GCV triphosphates bound to the herpes DNA polymerase and inhibit the polymerase or are incorporated into DNA, terminating viral DNA chain elongation (Chiocca et al. 2003). Due to the bystander effect, not only the HSV-‐‑tk expressing cells but also many of the surrounding cells will die by apoptosis (Asklund et al. 2003, Maria & Friedman 1997). As itself, HSV-‐‑tk can be also used as a 32 reporter gene for transgene expression imaging. Fluorination or iodination of arabinofuranosyl nucleosides has enabled to image in vivo HSV-‐‑tk expression e.g. cell tracking and inflammation (Wu et al. 2013, Pullambhatla et al. 2012, Yaghoubi et al. 2001, Sun et al. 2001). Fusion proteins composed of avidin and either macrophage scavenger or low-‐‑density lipoprotein receptors (LDLR) have been constructed in order to target biotinylated molecules to cells of the desired tissues. Using adenovirus mediated gene transfer the transient expression of the fusion protein on the cell membrane was achieved. (Lehtolainen et al. 2002, Lehtolainen et al. 2003) When biotinylated molecule binds to the fusion receptor, it is internalized into the cell. Local gene transfer to target tissues could be used as a universal tool to deliver therapeutic agents at systemic low concentrations. Using biotinylated tracers like biotin-‐‑DTPA or biotin-‐‑DOTA complexes these cells can be imaged in vivo. An alternative method to study biodistribution is avidin-‐‑ expressing viruses. Their homing properties to the target tissue may be enhanced using biotinylated moieties like antibodies or peptides. For imaging purposes biotinylated radiotracer is conjugated on the virus surface and biodistribution of the labelled virus is followed by SPECT (Raty et al. 2007, Raty et al. 2006, Kaikkonen et al. 2009). Homing properties of viruses can be modified with biochemical methods using hybrid peptide with poly-‐‑lysine spacer together with cyclic peptide HWGF (His-‐‑Tyr-‐‑Gly-‐‑Phe), which binds to matrix metalloproteinase’s, MMP-‐‑2 and MMP-‐‑9 (Koivunen et al. 1999). This peptide has been conjugated with transglutaminase enzyme on the surface of Adenovirus. The use of enzyme for conjugation is gentle and do not decrease the infectivity of the virus. Conjugated receptor specific peptide enhanced the tropism of the virus in vivo in rabbits. (Turunen et al. 2002) 33 3.3.5 Living cells Nuclear medicine has been used to image leucocytes in infectious or inflammatory processes in vivo already over four decades. The techniques detect inflammatory processes to which leukocytes migrate, such as those associated with abscesses or other infection. During 1970’s a new cell membrane tropic radioactive compound was developed. 111In-‐‑oxine is a lipophilic complex and penetrates through cell membrane without interference of the membrane bound molecules like receptors. Penetration is unspecific and all cell types can be labelled (Thakur et al. 1977, Becker & Meller 2001). In clinical studies red blood cells are also labelled using 99mTc and blood pool imaging, detection of vascular malformations, red cell mass determination, detection of gastrointestinal bleeding, and hemangiomas can be studied (Srivastava & Chervu 1984). Stem cells are immature cells, which have regenerative potential in various diseases. Especially neurodegenerative disorders have been in the focus due to the poor regenerative properties on neuronal cells. The regenerative properties of the stems cells have been studied in Parkinson disease (PD), amyotrophic lateral sclerosis (ALS), Huntington'ʹs disease (HD) and stroke (Lindvall et al. 2004). For in vivo biodistribution studies stem cells have been labelled either with paramagnetic nanoparticles and followed with MRI (Raty et al. 2006, Mantyla et al. 2006, Arbab et al. 2003, Frank et al. 2003) or with 111In-‐‑ oxine for SPECT imaging (Lappalainen et al. 2008, Makinen et al. 2006, Mitkari et al. 2012, Mitkari et al. 2013). Radiolabelling of living cells is probably the most challenging labelling process since several issues has to be considered. Firstly, labelling conditions have to be effective, mild, temperate, fast and without complicated purification steps. Secondly, aseptic techniques have to be followed and last, appropriate dose for the cell batch has to be evaluated avoiding too high dose for the cells. For these reasons labelling conditions must always plan carefully for each different cell type according their usual cultivation techniques. If longer (i.e. over 24 h) biodistribution studies are measured, effect of the labelling to the 34 viability of the cells in vitro during timescale is worth to analyze. This is important since only the nuclide is seen in in vivo imaging but none information is achieved about the absolute condition of the cells viability. 3.3.6 Nanoparticles There have been invasion of basic nanoparticle research in biomedicine. Many therapeutic agents like small organic compounds, nucleic acids, peptides and proteins are unstable in vivo and novel delivery technologies should be developed to improve their pharmacokinetic properties. Development of nanoparticle based delivery could enable sustained and hence regular release of drug. If NPs are also targeted, in the ideal case they would concentrate to the desired area and allow steady release of the drug to the circulation or locally if needed. This would be beneficial for the patient as fewer drug intakes, steadier effect of the drug but maybe also economically cost-‐‑effective. The size range of nanoparticles is comparable to viruses. Conventionally nanosized materials like polymeric nanoparticles, liposomes and micelles are prepared from organic materials although they have limited chemical and mechanical stability and inadequate control over the drug release rate (Arruebo et al. 2006). Today there are NPs made of inorganic materials like silica or silicon (Haley & Frenkel 2008, Salonen et al. 2008). Inorganic material allows the production of porous or mesoporous nanoparticles with particle size in range of 50 – 300 nm and the pore diameter in the range 5 – 50 nm. The porous structure allows high loading capacity for the therapeutic therapeutic agents and/or tracers, like fluorescent, radioactive compounds or paramagnetic iron (Wiekhorst et al. 2006, Alexiou et al. 2006a, Alexiou et al. 2006b). Furthermore, the transportation and release of the molecules can be controlled. Mesoporous silicon nanoparticles have also shown to be non-‐‑toxic and stable (Salonen et al. 2008, Brigger et al. 2002, Limnell et al. 2007, Salonen et al. 2004). The surface of the nanoparticles can be derivatized with chemically active groups like primary amines or 35 carboxylic acids and conjugated with several biologically and chemically active molecules and further labelled with radionuclides (Lee et al. 2013, Zhou et al. 2010, Huang et al. 2012, Di Pasqua et al. 2013). Large surface area allows conjugation of several different molecules on the same particle. Using targeting moieties the tropism of NPs can be modulated (Kukowska-‐‑Latallo et al. 2005, Costantino et al. 2005). 36 37 4 The aims of this study The aim of this thesis was to study targeting and biodistribution behaviour of different types of biomolecules, antibodies and viruses by radiolabelling and imaging them by SPECT/CT and using ex vivo analysis. In practice, the main objectives were: I) To develop the most suitable labelling method for different types of bioactive molecules (Chapters 5, 6 and 7). II) To study their biodistribution in vivo in appropriate animal model using SPECT/CT imaging and/or ex vivo analysis (Chapters 5, 6 and 7). III) To perform accurate post mortem analysis of studied compounds (Chapters 6 and 7). In this thesis, biodistribution and pharmacokinetics of a peptide (insulin like growth factor 1, IGF-‐‑1) without conjugation and after complexation with binding protein or with nanoparticle was studied in a knockout mouse model mimicking human INCL. In addition, first-‐‑ hand information of possible dosing intervals for further treatment study was estimated. At the following parts SPECT/CT imaging was used to study differences in biodistribution and targeting properties of commercially available Cetuximab and novel monoclonal antibody mF4-‐‑31C1 in human cell line ovarian carcinoma mouse model. To achieve this, basic conjugation and labelling methods were used and the behaviour of the antibodies was tested using SPECT/CT imaging. For the avidin-‐‑displaying viruses, sensitive but effective labelling method to study their biodistribution in vivo was developed. Labelling of avidin displaying lentivirus enabled to study their targeting and transduction properties in ovarian carcinoma mouse model. This construction could be further used as site for biotin targeted drugs. All animal procedures were performed according to protocols approved by the ethical boards for animal experimentation of the 38 National Public Health Institute and University of Helsinki, as well as National Animal Experiment Board of Regional State Administrative Agencies of Southern Finland. Agreement numbers for these studies were ESLH-‐‑2008-‐‑03724, ESLH-‐‑2009-‐‑06737 and ESLH-‐‑2010-‐‑03429. All experiments were done in accordance with good practice of handling laboratory animals and of genetically modified organisms. 39 5 Biodistribution and pharmacokinetics of IGF-‐‑1 complexed with carrier protein or nanoparticles (This chapter is summarized from Huhtala T., Rytkönen J., Jalanko A., Kaasalainen M., Salonen J., Riikonen R., Närvänen A. Native and complexed IGF-‐‑1: biodistribution and pharmacokinetics in Infantile Neuronal Ceroid Lipofuscinoses. J Drug Deliv. 2012.) Insulin-‐‑like growth factor 1 (IGF-‐‑1) is important in embryonic development and is considered as a potential therapeutic agent for several disorders of peripheral and central nervous system. In circulation IGF-‐‑1 is mainly bound to its carrier protein IGFBP-‐‑3. Our aim was to compare the biodistribution of free IGF-‐‑1, IGF-‐‑1/IGFBP-‐‑3 and IGF-‐‑1/NP complex in selected organs over the time in Cln1-‐‑/-‐‑ mouse model if IGF-‐‑1 could be used as a potential drug to treat INCL. It has been suspected that IGF-‐‑1/IGFBP-‐‑3 is too large to cross the BBB, but since IGF-‐‑1 is shed, it may eventually cross the BBB by specific transport systems (Pan & Kastin 2000, Yu et al. 2006). In Cln1-‐‑/-‐‑ mice there is inflammation, and prominent alterations involved in the immune response (Jalanko et al. 2005, von Schantz et al. 2008). Consequently, in the INCL patients, there might be inflammatory changes that could open BBB and the IGFs could cross it better than expected in healthy people. 5.1 Background Infantile Neuronal Ceroid Lipofuscinoses (INCL) is a severe neurodegenerative disorder of childhood characterized by selective death of cortical neurons (Santavuori 1988). It is caused by recessive mutations in the CLN1 gene encoding palmitoyl-‐‑protein thioesterase (PPT1) (Vesa et al. 1995). Normal PPT1 activity is essential for the 40 development and survival of cortical and cerebellar neurons in human and mouse (Jalanko et al. 2005, Riikonen et al. 2000, Macauley et al. 2009). IGF-‐‑1 concentration in cerebrospinal fluid is lower in patients with INCL (Riikonen et al. 2000) suggesting that decreased levels of IGF-‐‑1 in brain may accelerate neurodegenerative disorders. To consistently study pathogenesis and treatment of INCL and other types of neuronal ceroid lipofuscinoses (NCL), different mouse models have been established (CLN1, CLN2, CLN3, CLN5) and also naturally occurring NCL mouse models exist (CLN8/mnd; CLN6/nclf) (Jalanko & Braulke 2009). The Cln1-‐‑/-‐‑ knockout mouse model is analogous to INCL in humans, with a severe phenotype and the overall neurologic features are highly similar to the clinical symptoms of INCL (Jalanko et al. 2005). Insulin-‐‑like growth factors, IGF-‐‑1 and IGF-‐‑2, are members of the insulin gene family and they play an important role in physiological development of humans and animals. IGF-‐‑1 and 2 stimulate cell proliferation and differentiation during embryonic and postnatal development. IGF-‐‑1 is the main trophic factor in the central nervous system (CNS) during early brain development (Torres-‐‑Aleman et al. 1998, Werther et al. 1998) and its relevance is greater to IGF-‐‑2. IGF-‐‑1 signals many neurodegenerative diseases (Trejo et al. 2004, Riikonen 2007) and lack of IGF-‐‑1 in the brain leads to apoptosis. The therapeutic effect of IGF-‐‑1 has been tested in several human disorders. Positive effects in adults have already been shown in body and brain growth (Laron et al. 1992, Laron 1999, Laron 2004), insulin resistance (Vestergaard et al. 1997) and head injury (Hatton et al. 1997). In children it was used in growth hormone insensitivity syndrome (Laron syndrome). In adolescents with type 1 diabetes it has been used to promote insulin sensitivity. However, undesired acute adverse reactions and the absence of suitable IGF-‐‑1 preparations for treatment have become major concerns among paediatric endocrinologists worldwide. The main problems in IGF-‐‑1 treatment include BBB permeability, side effects like hypoglycaemia and short intervals in administration of the drug. 41 In animal experiments neurotrophins have shown to have therapeutic effects on motor neuron disorder (Zhong et al. 2009), chemotherapy-‐‑induced peripheral neuropathy (Apfel et al. 1993), myelination and brain growth (Carson et al. 1993), asphyxia (Guan 2008), cerebellar ataxia (Fernandez et al. 1998), retinopathy of prematurity (Hellstrom et al. 2001), cognitive impairment (Lupien et al. 2003), and experimental autoimmune encephalitis (Lovett-‐‑Racke et al. 1998, Yao et al. 1996). It has been shown that only one week treatment with IGF-‐‑1 partially restored interneuronal number and reduced hypertrophy in the mnd/mnd mouse model of neuronal ceroid lipofuscinoses (CLN8) (Cooper et al. 1999, Ranta et al. 1999). 5.2 Labelling and conjugation of IGF-1 IGF-‐‑1 and IGFPB-‐‑3 were offered by Insmed Incorporation (Richmond, VA). IGF-‐‑1 and IGFBP-‐‑3 were radiolabelled with 125 I by Iodo-‐‑Gen method. Briefly, pre-‐‑coated iodination tubes (Pierce) were rinsed with 1 ml of phosphate buffered saline, pH 7.4, (PBS) and 125I (22 MBq, Map Medicals, Finland) was incubated at room temperature for 10 minutes. After incubation IGF-‐‑1 (200 µμg) or IGFBP-‐‑3 (200 µμg) was added and the reaction mixture was further incubated for 15 minutes at RT. The solution was purified using HiTrap Sephadex column (GE Healthcare) using PBS as a mobile phase at flow-‐‑rate 1 ml/min. Labelling efficiency was 29 – 43 % with specific activity of 0.22 MBq/nmol and 0.37 MBq/nmol for the IGF-‐‑1 and 11 – 17 % for the IGFBP-‐‑3 with specific activity of 0.29 MBq/nmol and 0.33 MBq/nmol. Thermally hydrocarbonized mesoporous silicon nanoparticles (THCPSi) were provided by Dr Jarno Salonen, University of Turku, Finland (Salonen et al. 2004). Nanoparticles (800 µμg) were mixed with of radiolabelled IGF-‐‑1 (200 µμg) in 2 ml of 10 mM HEPES pH 7.4. The suspension was mixed at RT for two hours sonicating every 30 minutes. 94% of IGF-‐‑1 was incorporated in the particles and the loading degree was 23.5% (w/w). The in vitro release was studied using fresh mouse plasma diluted 1:2 in PBS. Nanoparticles were mixed with diluted plasma and incubated at +37 °C. A sample of the particles was 42 centrifuged immediately and at 20, 60, 120 and 240 minutes time points (n = 3/time point). Radioactivity of the supernatant was measured using gamma counter (1277 Gammamaster automatic gamma counter, LKB Wallac, Finland). There was a burst immediately after mixing with the plasma releasing 20% of the incorporated IGF-‐‑1. After 20 minutes 50% of the IGF-‐‑1 was released and detachment rate was further decreased, 60% of IGF-‐‑1 was uncoupled at 240 min time point. 5.3 Used animal model A homozygous knockout mouse model Cln1-‐‑/-‐‑, showing overall neurologic features highly similar to the clinical symptoms of INCL was used in this study (Jalanko et al. 2005). The Cln1-‐‑/-‐‑ mice were backcrossed to C57BL/6 from for more than 10 generations, and the congeneity was confirmed with the Mouse Medium Density SNP Panel (Illumina). The genotypes of the mice were determined by PCR of DNA from tail biopsies. Total of 36 nine-‐‑week-‐‑old (n=3/group) female mice were used for the biodistribution studies. The mice received chow and water ad libitum. Animals were anesthetized by 1.5 – 2 % isoflurane in N2/O2 with ratios 70:30, respectively. Labelled IGF-‐‑1, IGF-‐‑1/IGFPB-‐‑3, IGF-‐‑1/NP or IGFBP-‐‑3 was injected i.v. via tail vein using 10 µμg of IGF-‐‑1; 0.2 – 0.6 MBq/animal. Also 1 ml of 5 % glucose was administrated i.p. to prevent hypoglycaemia. The doses used in our study have been the same as in experimental autoimmune encephalomyelitis mice where positive effects on inflammatory, demyelinating, and demyelinated lesions have been seen when using IGF-‐‑1 (Li et al. 1998). Animals were terminated at 20, 120 or 240 min post injection. Tissue samples were collected in tarred tubes and radioactivity was measured using automated gamma counter. Corrections were made for background radiation and physical decay during counting. The activity in all organs and tissue samples was expressed as percentage of injected dose per gram (%ID/g). All data were expressed as mean ±standard error of the mean (S.E.M) (Table 3). 43 Table 3. Biodistribution of unbound IGF-‐‑1, IGF-‐‑1/IGFBP-‐‑3, IGFBP-‐‑3 and IGF1/NP complex 20 min, 120 min and 240 min post i.v. injection in CLN1-‐‑/-‐‑ mice (n = 3 / group). The activity in all organs and tissue samples is expressed as percentage of injected dose/tissue sample weight (%ID/g). All data are expressed as mean ± standard error of the mean (S.E.M). IGF-‐‑1 complex Organ IGF-‐‑1 IGF-‐‑1/IGFBP-‐‑3 IGFBP-‐‑3 IGF-‐‑1/NP 20 min Blood 10.21 ± 0.92 15.69 ± 0.90 12.24 ± 0.49 8.66 ± 0.04 Heart 5.35 ± 0.49 7.70 ± 0.23 4.29 ± 0.55 2.45 ± 0.06 Lung 11.37 ± 1.00 7.81 ± 0.24 10.30 ± 1.95 9.00 ± 0.62 Kidney 148.3 ± 34.48 124.2 ± 9.90 34.98 ± 8.90 42.96 ± 3.83 Liver 5.52 ± 0.50 50.06 ± 2.21 61.19 ± 16.80 56.45 ± 8.44 Spleen 6.22 ± 0.39 28.37 ± 1.22 25.50 ± 13.71 47.15 ± 4.11 Ovary 12.71 ± 1.46 4.22 ± 0.57 3.92 ± 0.52 3.09 ± 0.45 Muscle 2.25 ± 0.24 0.77 ± 0.05 0.73 ± 0.06 0.85 ± 0.00 Brain 0.60 ± 0.04 0.48 ± 0.04 0.42 ± 0.00 0.30 ± 0.00 120 min Blood 8.01 ± 0.92 10.05 ± 2.66 15.82 ± 2.01 6.30 ± 0.21 Heart 2.95 ± 0.45 3.76 ± 1.22 5.50 ± 0.50 1.71 ± 0.08 Lung 6.57 ± 0.97 7.35 ± 2.42 9.49 ± 1.06 6.37 ± 0.57 Kidney 14.32 ± 2.23 10.31 ± 4.21 12.40 ± 1.19 10.27 ± 1.97 Liver 5.31 ± 0.74 7.95 ± 4.78 8.45 ± 1.28 17.75 ± 0.83 Spleen 5.30 ± 0.56 9.45 ± 3.28 9.40 ± 3.28 14.71 ± 2.49 Ovary 7.04 ± 1.66 7.78 ± 2.14 8.27 ± 0.55 2.99 ± 0.11 Muscle 1.74 ± 0.43 1.75 ± 0.48 1.67 ± 0.07 0.91 ± 0.03 Brain 0.35 ± 0.08 0.40 ± 0.16 0.51 ± 0.04 0.24 ± 0.01 44 Table 3. Continues. Organ IGF-‐‑1 240 min Blood 5.71 ± 0.41 4.68 ± 0.17 6.24 ± 0.43 4.15 ± 0.39 Heart 1.89 ± 0.15 1.46 ± 0.08 2.03 ± 0.13 1.00 ± 0.12 Lung 4.10 ± 0.46 3.23 ± 0.03 3.93 ± 0.46 3.25 ± 0.49 Kidney 4.20 ± 0.25 4.36 ± 1.14 3.87 ± 0.31 3.08 ± 0.42 Liver 2.29 ± 0.19 1.79 ± 0.17 2.74 ± 0.10 11.29 ± 0.63 Spleen 3.41 ± 0.32 3.16 ± 0.38 2.93 ± 2.93 9.05 ± 1.01 Ovary 4.33 ± 0.43 3.49 ± 0.16 2.94 ± 0.43 1.47 ± 0.03 Muscle 0.97 ± 0.06 0.90 ± 0.14 0.89 ± 0.19 0.54 ± 0.03 Brain 0.20 ± 0.01 0.13 ± 0.01 0.19 ± 0.01 0.14 ± 0.01 5.3.1 IGF-‐‑1 complex IGF-‐‑1/IGFBP-‐‑3 IGFBP-‐‑3 IGF-‐‑1/NP Clearance and biodistribution of native and complexed IGF-‐‑1 Earlier studies in mice have shown that IGF-‐‑1 injected as such is bound to the plasma proteins immediately after the injection forming different size of complexes and is cleared via the kidneys (Sun et al. 2000, Sun et al. 1997). In our biodistribution and pharmacokinetic studies iodinated IGF-‐‑1 was complexed to IGFBP-‐‑3 or nanoparticles in vitro before the injection. As expected, most of the unbound IGF-‐‑1 was cleared through the kidneys within 120 minutes (Figure 3A). The clearance of IGF-‐‑ 1/IGFBP-‐‑3 through the kidneys was also high indicating fast dissociation since the labelled IGFBP3 was mainly excreted trough the hepatic route. However, substantial part (50.1 % ID/g) of IGF-‐‑1/IGFBP-‐‑ 3 was also eliminated through the liver after 20 min. Excretion of IGF-‐‑ 1/NP via the kidneys was significantly inferior to unbound IGF-‐‑1 or IGF-‐‑1/IGFBP-‐‑3 (42.3 %ID/g; 148.3 and 124.2 %ID/g) indicating sustained release of IGF-‐‑1 from the nanoparticles. The level of IGF-‐‑1/IGFBP-‐‑3 in blood decreased faster in both time frames 20 – 120 min (36%) and 120 – 240 min (53 %) than IGF-‐‑1/NP (Table 3). Concentration of IGF-‐‑1/NP in blood stayed steadier between both time frames (20 – 120 min, 27 %; 120 – 240 min, 34 %) like unbound IGF-‐‑1 (Figure 3B). This suggests that IGF-‐‑1/NP is 45 concentrated in liver and rather dissociated to circulation enabling longer bioavailability to other tissues than excreted immediately. IGF-‐‑1 accumulated strongly in the liver only as a protein or nanoparticle complex. However, the protein complexed IGF-‐‑1 was removed by active excretion whereas IFG-‐‑1/NP complex is concentrated into the liver and rather dissociated to the circulation than excreted immediately. It has been shown that intravenously administered mesoporous silicon microparticles loaded with siRNA encapsulated into nanoliposomes accumulate into the liver and spleen, but remains in the sinusoidal space, enabling sustained release of siRNA loaded nanoliposomes (Tanaka et al. 2010). In our other studies we have analyzed behaviour of 125I labelled undecylenic acid functionalized thermally hydrocarbonized mesoporous silicon nanoparticles in liver using combined data of autoradiography and electron microscopy (Rytkönen et al. 2012). Similar nanoparticles as used in this study were seen in hepatic veins and sinusoids but not internalized into macrophages or hepatocytes. In addition Bimbo et al. reported that THCPSi nanoparticles are not phagocyted in extent by CaCo-‐‑2 or RAW 264.7 macrophages in vitro. Instead they showed a strong cellular association, as majority of the nanoparticles remained attached to cell membranes. (Bimbo et al. 2010) Relatively low levels of IGF-‐‑1 with or without IGFBP-‐‑3 or nanoparticles accumulated to the brains in all studied time points (Figure 3B). The amount crossing the BBB might, however, be sufficient to affect the physiological functions and modulate neuroendocrine and behavioural responses. The highest accumulation of IGF-‐‑1 was achieved at 20 min time point (0.60% ID/g), which was 25% more than IGF-‐‑1/IGFBP-‐‑3 (0.48 %ID/g) and twice as much as IGF-‐‑1/NP (0.30% ID/g). However, accumulation of IGF-‐‑1 drops dramatically between 20 – 120 min (42 %) whereas levels of IGF-‐‑1/IFGBP-‐‑3 or IGF-‐‑1/NP decreased only 17 % and 18%, respectively indicating more constant delivery of the IGF-‐‑1 in the brain. 46 Figure 3. A) Clearance of unbound and complexed IGF-‐‑1 in vivo. Pharmacokinetics of free IGF-‐‑1, IGF-‐‑1/IGFBP-‐‑3 complex, free IGFBP-‐‑3 and IGF-‐‑1/NP complex in kidney, liver and spleen. Pharmacokinetics of free and complexed IGF-‐‑1 in selected organs. B) 47 This data shows that the complexed forms of IGF-‐‑1 doesn’t enhance delivery of IGF-‐‑1 in the brain but gives more stable concentrations. After 240 min IGF-‐‑1 accumulation in the brain was the same with or without complex. The sustained release to blood and low tissue concentrations of IFG-‐‑1 delivered with nanoparticles may decrease the side effects like hypoglycaemia without losing the therapeutic effect. Low blood and tissue concentrations together with constant and sustained release may be beneficial for the continuous IGF-‐‑1 therapy for INCL. 5.3.2 In Conclusions summary, we have studied the biodistribution and pharmacokinetics of human IGF-‐‑1 administrated free or complexed to its natural binding protein IGFBP-‐‑3 or nanoparticles in Infantile Neuronal Ceroid Lipofuscinoses (INCL) mouse model. IGF-‐‑1 conjugated to nanoparticles accumulated and also remained in liver probably in the hepatic veins and sinusoids at high concentration in contrast to IGF-‐‑1/IGFPB-‐‑3 complex, which dissociated and was actively excreted via kidneys and liver during studied time points. Since IGF-‐‑ 1/NP level also in blood decreased moderately compared to IGF-‐‑ 1/IGFBP-‐‑3 this data demonstrates steadier release of IGF-‐‑1 in to the circulation and longer bioavailability of IGF-‐‑1. Also IGF-‐‑1/NP concentration in all other studied tissues except spleen was lower than with protein complex or unbound IGF-‐‑1, which may result in milder side effects and is beneficial for the long term hormone therapy. Our biodistribution and pharmacokinetic results of IGF-‐‑1 offer relevant preliminary data for further therapy studies in INCL model. 48 49 6 Biodistribution of 111In-‐‑labeled antibodies in SKOV-‐‑3m induced mice (This chapter is summarized from original article of Huhtala T., Laakkonen P., Sallinen H., Ylä-‐‑Herttuala S., Närvänen A. In vivo SPECT/CT imaging of human orthotropic ovarian carcinoma xenografts with 111In-‐‑labeled monoclonal antibodies. Nucl.Med.Biol., 2010, 37, 8, 957-‐‑964.) In this study we investigated potential of two indium-‐‑labelled antibodies, Cetuximab and mF4-‐‑31C1, in SPECT/CT imaging of human ovarian carcinoma in orthotropic murine model in vivo. As an ovarian cancer model we used SKOV-‐‑3m cell line, which proceeds aggressively with several clinical, histological and pathological similarities with human cancer (Sallinen et al. 2006). 6.1 Ovarian carcinoma Ovarian cancer is the sixth most common cancer worldwide and at the time of diagnosis cancer has progressed to stage III or IV in most of the cases. Diagnosis of ovarian cancer at stage III or IV means that it has already metastasized to the peritoneum or distant sites and the overall 5-‐‑year survival rate is only 35 – 45 % (Auersperg et al. 2001, Friedlander 1998, Singh et al. 2008, Jemal et al. 2006). Therefore, further studies are needed for more efficient early diagnosis and treatment. Also the treatment is mainly focused to surgical methods which are challenging in practice (Singh et al. 2008). An effective therapy requires other approaches, e.g. targeted delivery of therapeutic agents for more accurate and effective treatment of ovarian carcinoma. Endothelial growth factor receptor (EGFR) belongs to the receptor tyrosine kinase family (ErbB-‐‑family), which plays an important role in cell proliferation, differentiation and survival (Yarden & Sliwkowski 2001). In multiple malignancies EGFR is over-‐‑expressed 50 which has also shown to correlate with poor prognosis in advanced stages of ovarian cancers (Psyrri et al. 2005). EGFR also affects tumour cell proliferation, angiogenesis and metastasis by induction of prometastatic matrix metalloproteinases (Cowden Dahl et al. 2007). Increased EGFR expression is observed in ~70 % of ovarian carcinomas (Psyrri et al. 2005, Kohler et al. 1989). Monoclonal antibodies binding to EGFR ligand-‐‑binding domain prevent signalling via the receptor and lead to inhibition of cell growth, neovascularisation and induction of apoptosis in some models. Anti-‐‑ EGRF antibodies have been used to treat several different types of cancer including head-‐‑and-‐‑neck, lung and breast cancers. One of the most widely studied therapeutic antibody is a-‐‑EGFR Cetuximab. It was approved by the FDA (Erbitux®) for the treatment of patients with metastatic colorectal cancer and it is currently in advanced clinical trials (phase II) in several other indications such as ovarian cancer (Secord et al. 2008). Role of lymphangiogenesis has been widely studied in various metastatic cancers. Spread of tumour cells via lymphatic vessels to regional lymph nodes is an important indicator of tumour aggressiveness (Skobe et al. 2001, Alitalo et al. 2005). Vascular endothelial growth factor receptor 3 (VEGFR-‐‑3) and its ligands VEGF-‐‑C and D are essential for remodelling and maturation of embryonic blood capillaries, and in the absence of the VEGFR-‐‑3 embryos die already at embryonic day 10 due to cardiovascular failure. After blood vessel maturation expression of VEGFR-‐‑3 becomes restricted to lymphatic endothelial cells. Expression of VEGF-‐‑C/D occurs in variety of human tumours and increased expression of VEGFR-‐‑3 has been detected in lymphatic endothelia adjacent to cancer cells and in lymph nodes containing metastatic tumour cells. VEGF-‐‑C has been shown to facilitate lymphatic metastasis by inducing dilation of peritumoral lymphatic capillaries via the VEGFR-‐‑3. Only a few tumour cells express VEGFR-‐‑3 and its expression is restricted to tumour-‐‑associated lymphatic vessels and angiogenic blood vessels (Petrova et al. 2008). Interestingly, VEGR-‐‑3 51 expression is not only up regulated during angiogenesis but it is functional in angiogenic blood vessels during development and in pathological conditions (Petrova et al. 2008, Tammela et al. 2008). Since growth factor receptors are key factors in neovascularisation and thus tumour growth and metastasis, inhibition of the receptor function might provide means for development of cancer therapies. Recently, blocking antibodies against growth factors or their receptors have been described as potential tool to inhibit signalling via these receptors. A mouse monoclonal antibody (mF4-‐‑ 31C1) against VEGFR-‐‑3 has been shown to inhibit lymphatic regeneration in athymic mice implanted with human breast cancer cells (Pytowski et al. 2005), and growth of xenografted human large-‐‑cell lung carcinoma in immunodeficient mice via inhibition of tumour angiogenesis (Laakkonen et al. 2007). 6.2 Experimental part To study biodistribution of monoclonal antibodies against EGFR (Cetuximab) and VEGFR-‐‑3 (mF4-‐‑31C1) in a human ovarian carcinoma model, SKOV-‐‑3m cells were injected intraperitoneally in nude mice. Healthy mice were used as control animals. All SKOV-‐‑3m induced animals developed tumours and carcinosis in peritoneal cavity as previously described (Sallinen et al. 2006). Tumour-‐‑bearing and healthy control mice were injected i.v. via tail vein with 111In-‐‑labeled Cetuximab or mF4-‐‑31C1 (25 µμg, 5 MBq), and imaged three times during the following 48 hours using SPECT/CT (Huhtala et al. 2010). 2D dynamic images were taken immediately after the injection. Accumulation of the antibodies was detected in lung and liver area as well as in bladder in all animals. 3D static images were taken 24 and 48 hours after the injection. In healthy control animals lung and liver accumulation was seen at 24 and 48 h time points whereas the signal from bladder had waned (Figure 4A). In tumour-‐‑bearing animals a clear accumulation of Cetuximab was seen in tumour area, liver and lungs already 24 hours after injection (Figure 4B). Radioactive signal 52 had decreased in the lungs and liver while the tumour still showed a very distinct signal after 48 hours. Tumour-‐‑bearing animals injected with 111In-‐‑labeled mF4-‐‑31C1 showed accumulation of the antibody mostly in liver 24 hours after injection and a weak signal in the tumour area was observed. However, a substantial accumulation of mF4-‐‑31C1 in tumour area was detected in SPECT/CT images in all tumour-‐‑bearing animals (5/5) at 48 hrs post injection. Interestingly, 2/5 animals showed a strong signal in left axillary area 48 hours after administration of the antibody suggesting that the antibody had accumulated in left axillary lymph node (Figure 4B). This was specific for the a-‐‑VEGFR-‐‑3 antibody since no accumulation of Cetuximab in any lymph node area was observed. 53 Figure 4. Combined 3D SPECT/CT images of healthy control and tumour bearing mice. After a single i.v. injection of 111In-‐‑Cetuximab or 111In-‐‑mF4-‐‑31C1, animals were imaged at 24 h and 48 h post administration. In healthy animals both antibodies were seen only in the liver and lung areas (A). A clear accumulation of Cetuximab is visualized on the left side of peritoneal cavity at the tumour swelling area (white arrows) on both time points. Using mF4-‐‑31C1, a weak accumulation in the tumour area can be seen 24 h after the injection and a strong signal at the tumour area (white arrows) in all five animals as well as in the left axillary area in 2 out of 5 animals after 48 h post-‐‑injection (B). After the last imaging animals were terminated and radioactivity of the organs and tissues was measured using a gamma counter. Activities in tumour were 8.78 ± 0.74 %IDg-‐‑1 for the Cetuximab and 5.77 ± 0.62 %IDg-‐‑1 for the mF4-‐‑31C1, respectively (Table 4). 54 Table 4. Radioactivity in the organs derived from the SKOV-‐‑3m tumour-‐‑bearing and healthy mice at 48 h post-‐‑injection of the 111In-‐‑ labeled Cetuximab and mF4-‐‑31C1 (n=5 in each group). Results are shown as mean percentage of the injected dose per g tissue (% ID g-‐‑1) ±SEM. SKOV-‐‑3m tumour-‐‑bearing mice Healthy controls Organ Cetuximab mF4-‐‑31C1 Cetuximab mF4-‐‑31C1 Tumour 8.78 ± 0.74 5.77 ± 0.62 -‐‑ -‐‑ Blood 3.71 ± 0.84 3.17 ± 0.67 10.54 ± 0.24 12.40 ± 1.61 Muscle 0.83 ± 0.06 1.10 ± 0.19 1.07 ± 0.17 1.57 ± 0.25 Ovary 3.28 ± 0.45 6.03 ± 0.43 5.23 ± 0.54 7.33 ± 0.79 Liver 4.05 ± 0.19 13.23 ± 0.22 4.59 ± 0.22 10.25 ± 0.40 Spleen 3.01 ± 0.25 10.58 ± 0.31 3.82 ± 0.31 8.31 ± 0.47 Kidney 1.90 ± 0.12 3.58 ± 0.19 3.81 ± 0.05 6.31 ± 0.79 Heart 1.40 ± 0.22 1.87 ± 0.12 3.33 ± 0.13 4.27 ± 0.30 Lung 2.80 ± 0.21 7.05 ± 0.36 6.79 ± 0.25 11.07 ± 0.55 Brain 0.14 ± 0.01 0.27 ± 0.02 0.26 ± 0.02 0.38 ± 0.02 Furthermore, tumour-‐‑to-‐‑blood and tumour-‐‑to-‐‑muscle ratios with both antibodies exceeded value of one indicating specific binding to their respective receptors in tumour which enabled specific tumour imaging separately from circulating blood and surrounding tissues (Huhtala et al. 2010). Cetuximab showed greater tumour-‐‑to-‐‑tissue ratio than mF4-‐‑ 31C1. Especially in the liver, spleen and lung the uptake of mF4-‐‑31C1 was higher than that of Cetuximab. It has been previously reported that conjugation of monoclonal a-‐‑EGFR-‐‑antibody C255 (Cetuximab) to polyethylene glycol (PEG) decreases the liver uptake and greatly improves the imaging properties of the EGFR-‐‑positive human squamous carcinoma (A431) xenografts in mice (Wen et al. 2001). In average, healthy animals showed higher organ accumulations than tumour-‐‑bearing animals with both antibodies. 55 In an earlier study imaging properties of Cetuximab have been studied in four different subcutaneous tumour models using PET. Uptake of 89Zr-‐‑Cetuximab varied between < 1 and 9 %IDg-‐‑1 in tumour tissue. Uptake of Cetuximab in those tumour models rose significantly up to 24 and 48 hours. In PET imaging the best resolution in colon carcinoma-‐‑bearing mice was reached at 72 hour post injection (Aerts et al. 2009). This is in concordance with our results. 6.3 Immunohistochemical analysis confirmed specific antibody binding in lymph nodes Cancer progression involves several functional changes both in tumour and adjacent tissues. Especially neoangiogenesis plays important role in tumour growth and dissemination. Growth factors EGF and VEGF-‐‑C are key factors in these processes. Previously, a report by Burton and colleagues verified that over expression of VEGF-‐‑C resulted in an increase in disseminated tumour cells to brachial and axillary lymph nodes in xenografted prostate cancer model in mice (Burton et al. 2008). Interestingly, in SPECT/CT images 2/5 of tumour-‐‑bearing animals showed a robust accumulation of mF4-‐‑31C1 in left axillary lymph nodes. To verify strongly accumulation of mF4-‐‑31C1 in the SPECT/CT images in left axillary area we studied axillary and inguinal lymph nodes of the tumour-‐‑bearing and healthy mice using immunohistochemistry. Tissue section stainings from SKOV-‐‑3m induced mice revealed that systemically administered a-‐‑VEGFR-‐‑3 antibody accumulated in vessel-‐‑like structures in lymph nodes (Huhtala et al. 2010). Left axillary node contained more accumulated antibody than the other nodes studied. Accretion of mF4-‐‑31C1 was not detected in healthy control mice. We were not able to observe any bounded Cetuximab in lymph node sections from tumour-‐‑bearing or control mice. Although the strongest staining was seen in the left axillary node, faint staining was also seen in other lymph nodes. The accumulation of 111In labelled mF4-‐‑31C1 was seen in SPECT only in left axillary lymph node due to the higher receptor density. Accumulation 56 to the lymph node was specific for the mF4-‐‑31C1 since we detected no Cetuximab-‐‑derived signal in the lymph nodes in the SPECT/CT images or in immunohistological stainings. Cetuximab accumulated only in the tumour area suggesting that at the time of the study there are either no metastatic tumour cells in the lymph nodes but a remote activation of lymph node angiogenesis and lymphangiogenesis by the primary tumour by VEGF-‐‑C (Skobe et al. 2001). This may reflect the situation in early phase metastatic progress of the ovarian cancer in humans. 6.4 Conclusions SPECT has shown to be efficient tool for the biodistribution studies of different antibodies in animal models. Since the pharmacokinetics of the large molecules like antibodies is slow the total monitoring time should be rather days than hours. We used 111indium in our studies which half-‐‑life is 67 hours making it ideal for long term SPECT imaging. This study clearly demonstrates the possibility of monoclonal antibodies, Cetuximab and mF4-‐‑31C1, to detect ovarian carcinoma using SPECT/CT imaging in vivo. Our very promising results showed that intravenously administered mF4-‐‑31C1 was able to target the lymph nodes with increased VEGFR-‐‑3 expression and warrants further evaluation of VEGFR-‐‑3 in the development of diagnostic and screening methods for ovarian carcinoma and its metastatic spread. Accumulation of mF4-‐‑31C1 antibody to the lymph nodes suggests the remote activation of VEGFR-‐‑3 by the primary tumour. 57 7 Novel conjugation method to study biodistribution of avidin displaying lentivirus (This chapter is modified from Huhtala T., Kaikkonen M., Lesch H., Viitala S., Ylä-‐‑Herttuala S., Närvänen A. Antitumor effect of cetuximab targeted lentivirus. Accepted Nucl. Med. Biol., September 2013. Also previous publications from Räty J., Liimatainen T., Huhtala T., Kaikkonen M., Airenne K., Laitinen O., Hakumäki J., Närvänen A., Ylä-‐‑Herttuala S. SPECT/CT imaging of Baculovirus biodistribution in rat. Gen. Ther., 2007, 14, 12, 930 – 938. and Kaikkonen M., Lesch H., Pikkarainen J., Räty J., Vuorio T., Huhtala T., Taavitsainen M., Laitinen T., Tuunanen P., Gröhn O., Närvänen A., Airenne K., Ylä-‐‑Herttuala S. (Strept)Avidin-‐‑displaying Lentiviruses as Versatile Tools for Targeting and Dual-‐‑imaging of gene delivery. Gene Ther.,2009, 16,7, 894 – 904. have been as a background for this experiment. In this study we have developed a novel method to target avidin-‐‑ displaying lentivirus with biotinylated Cetuximab in SKOV-‐‑3m induced ovarian carcinoma mouse model. Biotinylated human polyclonal IgG was used as a control targeting antibody with same virus construction. Biodistribution of radiolabelled antibody-‐‑virus conjugates was followed for six days after single injection of the virus. 58 7.1 Background Targeted drug delivery has been considered one of the major challenges in the area of pharmaceutical development (Gonzalez-‐‑ Angulo et al. 2010, Phillips & Giannoukakis 2010). One of the main problems in modern cancer therapy medication is the low targetability and hence extensive toxic effects also to the healthy cells. By targeting the dose can be reduced since better drug accumulation at the desired tissue can be achieved which also results to milder side effects. In direct targeting antibodies are most commonly used because of their selective binding towards antigen on the target cell surface. One of the widest studied targeted antibody is Cetuximab™ which was approved by the FDA (Erbitux®) for the treatment of patients with squamous cell carcinoma of head and neck and metastatic colorectal cancer (Chen et al. 2009). However, quite often antibody-‐‑based treatment alone is not efficient enough in cancer treatment and combination with surgery, radiotherapy, chemotherapy and immunotherapy is needed (Goldenberg et al. 2006). Based on epidemiological studies, it seems to be that family history is the strongest risk factor for ovarian cancer (Auersperg et al. 2001). Mutations in human tumour suppressor genes BRCA1 and BRCA2 are considered to be particularly important (Lynch et al. 1998). Viral-‐‑based gene therapy has shown to be a promising approach to different types of cancers, including ovarian cancers (Kimball et al. 2006, Rocconi et al. 2005, Lo et al. 2005). Lentiviruses can mediate robust gene transfer and stable long-‐‑term transgene expression into human cells in vivo and in vitro without compromising normal cellular functions. 7.1.1 Gene therapy Gene therapy represents a promising methodology to treat various diseases like cancers, cardiovascular, monogenic, infectious, neurological and ocular diseases (Aiuti & Roncarolo 2009, Aiuti & Roncarolo 2009, Nanou & Azzouz 2009, Gray & Samulski 2008, Ferrer 59 2006, Lundstrom 2004). However, only a limited number of gene therapy clinical trials have been performed. Most concerning safety issues in gene therapy area are the lack of targetability, immune responses against the vector and the possibility of cancer by insertion mutagenesis. Lentivirus is a good tool for targeted gene therapy since it can transduce both dividing and non-‐‑dividing cells by integrating to host cell genome enabling long term transgene expression in vivo and in vitro (Naldini et al. 1996, Kafri et al. 1997). In viral vector targeting the affinity is based on the recognition between viral surface protein and the receptor of the cell surface. By modifying surface of the virus it can be targeted to different cells. After viral attachment to the cell virus internalizes, infects the cell and starts to produce its own proteins. By inserting avidin displaying gene into viral genome gives excellent tool for indirect targeting. Various molecules can be conjugated with biotin, which is then targeted to the avidin displaying cells infected by the virus. 7.2 Lentiviral construct Streptavidin displaying lentiviruses expressing GFP were generated by five plasmid transfection of 293T cells and concentrated by ultracentrifugation as described earlier (Kaikkonen et al. 2009). Lentivirus titers (Transforming units (TU)/ml)) were determined by analyzing the number of virus particles able to transduce HeLa cells (Lesch et al. 2008). The amount of lentiviral capsid protein p24 (pg/ml) was determined by p24 ELISA kit (PerkinElmer, Waltham, MA, USA). 7.3 Biotinylation of DTPA-antibody conjugates Cetuximab (Erbitux®, IMC-‐‑C225 Merck) and polyclonal human IgG were conjugated with p-‐‑SCN-‐‑Bn-‐‑DTPA (Macrocyclics, Dallas, USA). After buffering the antibodies (1 mL, 2 mg/mL in PBS) with equal volume of 0,1 M NaHCO3 pH 9,5 DTPA (40 µμL, 5 mg/mL in DMSO) was added to the solution and mixed at room temperature for 2 hours. Unbound DTPA was removed by ultra filtration (VivaSpin filter 30 60 kDa, Millipore, twice 10 min at 3000 g). After filtration PBS was added to the concentration of 2 mg/mL. For the biotinylation NHS-‐‑biotin (Pierce) was mixed with DTPA-‐‑conjugated antibodies. Briefly, biotin (400 µμL, 1 mg/mL in DMSO) was added to the DTPA-‐‑conjugated antibodies and mixed for 2 hours at 37 °C. After mixing the unbound biotin was removed by ultra filtration as described above. The concentration of the antibodies was adjusted to 2 mg/mL. 7.4 Conjugation of antibodies had no effects to activity Indirect enzyme-‐‑linked immunosorbent assay (EIA) was used to compare the activity of conjugated and natural antibodies. A serial dilution of conjugated and natural antibodies was incubated on 96-‐‑well plate overnight. After incubation wells were washed with PBST and secondary incubated with 1 % BSA in PBST for 1 h. After washing goat anti-‐‑human IgG conjugated horseradish peroxidase (HRP) or avidin conjugated HRP were incubated for 30 min. For the detection TMB substrate (10 min at dark) was used. Absorbance of the different wells was measured at 450 nm (OD450) using an ELISA reader (Labsystems Multiscan RC, Thermo Labsystems Oy, Finland). Similar attachment of goat anti-‐‑human IgG-‐‑HRP to the conjugated and natural antibodies demonstrated that DTPA and biotin conjugation of antibodies didn’t decrease affinity of the antibodies. Also complexation of avidin conjugated HRP with TMB substrate was used to detect biotin on Cetuximab and hIgG surfaces, which demonstrated biotinylation of the antibodies. All EIA experiments were performed as duplicates. 7.5 Radiolabelling 111 In-‐‑labelling of the Biot-‐‑DTPA-‐‑antibody was performed by mixing the antibody conjugate (125 µμg) with 10 mM sodium acetate solution pH 5 after which 111 InCl3 (38 MBq, 370 MBq/ml on the reference day, Mallinckrodt Medical BV, Petten, Netherlands) was added so that the 61 total volume of the solution was adjusted to 300 µμl. The solution was incubated at room temperature for 1 hour. After incubation the radiochemical purity was tested by using instant thin-‐‑layer chromatography (ITLC, SG, Pall Corporation, USA) on a 13 × 1.5 cm strip using 0.3 M sodium citrate pH 5 as a mobile phase. Radiochemical purity of the Biot-‐‑DTPA-‐‑Cetuximab was 93 % and for Biot-‐‑DTPA-‐‑hIgG 77 %. After 111 In-‐‑labeling, the antibodies were mixed with equal volume of biotin-‐‑free Optimem to achieve neutral pH and the avidin displaying lentivirus (100 µμL; 7,42 × 105 TU) was added to the solution. Virus was incubated with the antibodies for 30 min on ice. After incubation the unbound antibody and 111 InCl3 were removed by filtration (Nanosep 300K Omega, Pall Corporation). After filtering the total volume of the labelled virus stocks were adjusted to 600 µμL with PBS. Total labelling efficiency of the virus was 72 % with Biot-‐‑DTPA-‐‑ Cetuximab (Cet-‐‑LV) and 79 % with Biot-‐‑DTPA-‐‑hIgG (IgG-‐‑LV). After ultra filtration the labelled viruses were titrated in order to verify the viability. 7.6 Cell line and animals The human ovarian carcinoma model, SKOV-‐‑3 (Sallinen et al. 2006) known to express EGF receptor in vivo (Sheng & Liu 2011) was used in biodistribution studies. SKOV-‐‑3m cells were cultured in McCoy’s 5A medium (Sigma) containing and supplemented with 10% FBS, 1% Glutamax, 100 µμg/mL penicillin and 100 µμg/mL streptomycin. For each animal, 107 cells (200 µμl) suspended in Optimem, were injected into the left peritoneal cavity of the six to seven-‐‑week-‐‑old (n=10) Balb/c nu-‐‑nu mice and the virus experiment was started 12 days after the implantation. The mice received chow and water ad libitum. 7.7 SPECT/CT The animals were anesthetized by 1.5 – 2 % isoflurane in N2/O2 with ratios 70:30, respectively. Indium labelled virus-‐‑antibody particle (~5 MBq in 100 µμl), either Cet-‐‑LV (n=4) or IgG-‐‑LV (n=3, one mouse was 62 died from fighting), was administrated into the mouse intravenously via tail vein and 3D SPECT imaging was performed on days 4, 5 and 6 after injection using combined SPECT/CT device with equal imaging parameters as described previously (see chapter 5.2.2). These time points were chosen based on our previous experience in antibody distribution profile as well the time requested a virus to internalize and start the gene expression. At the end of the study animals were terminated with CO2 immediately after the sixth day imaging and tissue samples were collected in tarred tubes. Major organs and tissues were weighted and radioactivity in each organ or tissue sample was measured in an automated gamma counter (Clinigamma, Wallac, Finland). Corrections were made for background radiation and physical decay during counting. The activity in all organs and tissue samples was expressed as percentage of injected dose per gram (% ID / g). Clear accumulation was seen in the liver area of all animals. In one mouse, which received Cet-‐‑LV a weak accumulation was seen in the tumour area on the sixth day after a single injection (Figure 5A). Since the signal came near the bladder, the localization was verified to be from the tumour and not from the bladder with axial CT sections combined to SPECT images (Figure 5B). Tumour area was not seen in the earlier time points suggesting that the tumour to blood ratio was not high enough for a visible effect. In the other mouse which received Cet-‐‑LV tumour mass was mainly developed in the diaphragm and liver area making tumour and liver signal uniform and inseparable. Two animals, which received Cet-‐‑LV had not any tumour mass so only liver areas could be seen in the SPECT images. However, in previous studies it has been shown that in vivo administration of lentiviral vectors triggers a type I interferon response that restricts hepatocyte gene transfer and promotes vector clearance (Brown et al. 2007). A high incidence of transgenic specific immunity has also been observed in studies using LVs for in vivo gene delivery. This has resulted in elimination of transgenic cells and eradicated the advantages of gene transfer. Brown et al showed that interferonαβ 63 (IFN) response is activated within 4 hours after single LV injection in mice. Kim et al have studied different ovarian carcinoma cell lines in vitro and found out SKOV-‐‑3 to be IFN-‐‑γ resistant (Kim et al. 2002). Banerjee et al on the other hand have shown glioma cells coated with anti-‐‑EGFR antibody, Cetuximab, led to enhance tumour-‐‑specific interferon-‐‑γ producing CD8+ T cells by dendritic cells (DC) (Banerjee et al. 2008). Studies with wild-‐‑type HIV, which is the parent-‐‑virus of many LVs, activated human DCs, which in response produce high levels of IFNαβ and can further activate the adaptive immune system (Brown et al. 2007). Furthermore, there was clear difference in the whole body CT images between Cet-‐‑LV and IgG-‐‑LV administrated mice. Abdominal cavity of the control animals is seen very uniform and internal organs cannot be distinguished probably due to the ascites, whereas regular body shape was seen in animals treated with Cet-‐‑LV (Figure 6). 7.8 Post mortem analysis of antibody targeted 111 In-labeled viruses Followed by the imaging at sixth day post-‐‑injection the animals were terminated and selected organs were removed for further analyses. In our previous studies (Huhtala et al. 2010) and unpublished data all the SKOV-‐‑3m introduced animals developed massive tumours and carcinosis in the peritoneal cavity and tumour mass was seen in SPECT/CT images two days after single 111 In-‐‑labeled Cetuximab injection. In this study all animals were terminated 6 days after virus injection. Mice injected with IgG-‐‑LV had massive tumour mass. However, two out of four mice, which received Cet-‐‑LV had no tumours. The tumour mass in the rest two Cet-‐‑LV animals was notably smaller (11.1 mg and 291.0 mg, average 151.1 mg) than in control group where the average tumour mass was 374 mg ± 61 mg. Radioactivity of the tissue samples was measured using a gamma counter and expressed as %IDg-‐‑1 of tissue weight. As it was seen in 3D SPECT images most of the radioactivity was found in the liver (Table 5). The main differences in accumulation between Cet-‐‑LV 64 and IgG-‐‑LV are seen in tumour, spleen and kidney. The activities in the tumour were 1.13 %IDg-‐‑1 for the Cet-‐‑LV and 0.76 %IDg-‐‑1 for the IgG-‐‑ LV. Furthermore, the average of tumour-‐‑to-‐‑blood ratio of Cet-‐‑LV was 8.26 as the average ratio of IgG-‐‑LV was only 4.56. Figure 5. (A) SPECT/CT image of Cet-‐‑LV injected animal expressing tumour in peritoneal cavity on D6 after single i.v. administration. (B) Since the SPECT signal was seen in the bladder area, signal was further confirmed to originate from the tumour and not the bladder, which is clearly seen (red arrow) in the upper image without any SPECT signal. 65 Figure 6. Representative sagittal and horizontal (anterior-‐‑posterior) CT images of mouse administrated with Cet-‐‑LV (left) or IgG-‐‑LV (right). Abdominal cavity of Cet-‐‑LV dosed mouse was typical compared to IgG dosed animal which peritoneal cavity was full of tumour mass and ascites resulted in forwarded diaphragm, twisted alignment of heart and rounded shape of flanks. 66 Table 5. Biodistribution of Cetuximab-‐‑targeted lentivirus and hIgG-‐‑ targeted lentivirus in SKOV-‐‑3m mouse model. Values are expressed as averages, for Cetuximab-‐‑targeted virus n = 4 and for hIgG-‐‑targeted virus n = 3, one control animal was killed in fighting at the following night after injection. *For the tumour activities n = 2 since only two mice which received Cetuximab-‐‑targeted lentivirus had visible tumour mass. Cetuximab-‐‑virus hIgG-‐‑virus Blood 0.18 ± 0.05 0.17 ± 0.01 Heart 0.65 ± 0.07 0.56 ± 0.04 Lung 1.28 ± 0.20 1.28 ± 0.03 Liver 44.69 ± 0.98 43.30 ± 3.35 Spleen 10.56 ±1.67 8.06 ± 1.86 Kidney 1.98 ±0.07 2.61 ± 0.09 Tumour 1.13* ± 0.81 0.76 ± 0.25 Ovary 0.96 ± 0.18 0.91 ± 0.18 Muscle 0.08 ± 0.01 0.08 ± 0.03 Brain 0.02 ± 0.00 0.02 ± 0.00 Sample 7.9 Conclusions Viral vectors are one of the key methods in the field of gene therapy. However, for a successful and safe delivery of the transferred gene, only limited studies have been made for specific targeting the viral particles to the desired tissue. In this study we have used avidin fusioprotein expressing lentivirus together with biotinylated anti-‐‑EGFR antibodies (Cetuximab) for targeting studies in ovarian cancer. Biodistribution studies showed that most of the viruses conjugated with Cetuximab or control human IgG accumulated to liver and spleen. In SPECT/CT images an accumulation to the tumour could be detected with Cetuximab targeted virus. hIgG targeted virus could be seen only in the liver and spleen area. In addition, only two out of 67 four mice receiving Cetuximab targeted virus developed tumour mass compared to control group of hIgG targeted virus which all had massive tumours and carcinosis in peritoneal cavity. These results demonstrate a novel method for combined targeting and labelling of lentivirus. This construction also may have triggered an immunological defence activation in vivo, which resulted to prevention of tumour progression. 68 69 8 Epilogue Several imaging modalities for small animal pre-‐‑clinical studies have been developed. Different modalities provide different information about biodistribution, pharmacokinetics and effect of potential therapeutic agents. Using SPECT or PET, biodistribution of the labelled agents can be easily followed over the time in animals with high sensitivity. Due to the high spatial resolution, chances in fine structure and furthermore chemical chances of the target tissue can be studied using MRI and MRS. Contrast of CT is not optimal for soft tissue studies in small animals in vivo, but using combined images with SPECT and PET it facilitates the localization of the labelled bioactive agents. Today several new therapeutic and diagnostic agents are large and/or complex structures like viruses, stem cells or nanoparticles. Due to high variety of the structures in new agents, requirement of special skills is growing starting from basic organic chemistry to virology and cell biology. To achieve reliable and possibly translational methods and results, understanding of anatomy, physiology and imaging physics is as well crucial. More information is needed quickly and reliably about the biodistribution and pharmacokinetics before clinical trials. Using different imaging modalities and combining the information excessive preliminary knowledge of the behaviour of the studied complex in vivo can be achieved cost-‐‑efficiently. In this thesis different disease models were studied using SPECT/CT imaging as well optimization of labelling method in each case. In chapter 5 biodistribution and pharmacokinetics of human IGF-‐‑ 1 administrated free or complexed to its natural binding protein IGFBP-‐‑ 3 or nanoparticles in INCL mouse model was studied. Different pharmacokinetic profiles were seen between varying complexation structures offering relevant preliminary data for further therapy studies in INCL model. 70 In chapter 6 biodistribution of novel monoclonal antibody mF4-‐‑ 3ICI to VEGFR-‐‑3 was compared to Cetuximab in SKOV-‐‑3m ovarian carcinoma mouse model. This study clearly demonstrated the possibility of monoclonal antibodies, Cetuximab and mF4-‐‑31C1, to detect ovarian carcinoma using SPECT/CT imaging in vivo. Interestingly, intravenously administered mF4-‐‑31C1 was able to target the lymph nodes with increased VEGFR-‐‑3 expression and warrants further evaluation of VEGFR-‐‑3 in the development of diagnostic and screening methods for ovarian carcinoma and its metastatic spread. Accumulation of mF4-‐‑31C1 antibody to the lymph nodes suggests the remote activation of VEGFR-‐‑3 by the primary tumour. As we showed in chapter 6, Cetuximab could be safely labelled with 111 In and further detect and target ovarian carcinoma in mouse model. Hence Cetuximab was chosen as a targeting part for a virus in the last experimental phase described in this thesis (chapter 7). The main goal in that study was to develop a methodology to construct labelled and targeted virus complex and further study how it would behave in vivo. Accumulation of labelled Cetuximab alone or conjugated with virus to tumour showed ca. 8 times difference. Significantly lower Cetuximab-‐‑virus tumour ratio compared to Cetuximab alone may be due to steric hindrance, but also due to different end points. Also, virus containing Cetuximab accumulated ca. 10 times higher amount in liver, which is the natural route of viral elimination. SPECT/CT images showed, that the tumour could be detected with Cetuximab targeted virus compared to control hIgG targeted virus, which could be seen only in the liver and spleen area. In addition, this construction also may have triggered an immunological defence activation in vivo, which resulted to prevention of tumour progression. As a summary, different methods to tag various types of molecules or even living viruses using radioactive tracers have been developed. In addition to labelling, pharmacokinetic behaviour of labelled targeting molecules was studied in representative animal model in vivo using SPECT/CT imaging offering novel tools and 71 information to further evaluate therapeutic and pathological pathways in these diseases. As a final chapter, information gathered during these years provided tools to safely label various types and sizes of bioactive molecules, particles or even living cells. Complete distribution profile of labelled compounds has been further analyzed using SPECT/CT and gamma counter analysis. As important to labelling part, use of proper and representative animal model is also in crucial part to achieve reliable results. 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