Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 73

Synthesis, Characterisation and Application of 68Ga-labelledMacromolecules

IRINA VELIKYAN

ISSN 1651-6214ISBN 91-554-6295-2urn:nbn:se:uu:diva-5876

ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2005

“Results! Why man, I have gotten a lot of results. I know several thousand things that won't work.”

– Thomas Alva Edison American inventor & industrialist

To my beloved Misha and Sona

Papers included in the thesis This thesis is based on the following papers and appendices, which are re-ferred to in the text by their roman numerals.

I Velikyan I, Beyer GJ, Långström B. Microwave-supported prepara-tion of 68Ga-bioconjugates with high specific radioactivity, Biocon-jugate Chemistry 2004, 15, 554-560.

II Velikyan I, Lendvai G, Välilä M, Roivainen A, Yngve U, Bergström M, Långström B. Microwave accelerated 68Ga-labelling of oligonu-cleotides, Journal of Labelled Compounds & Radiopharmaceuticals2004, 47, 79-89.

III Velikyan I, Liljegren Sundberg Å, Lindhe Ö, Höglund AU, Eriksson O, Werner E, Carlsson J, Bergström M, Långström B, Tolmachev V. Preparation and evaluation of 68Ga-DOTA-hEGF for visualisation of EGFR expression in malignant tumours, Journal of Nuclear Medi-cine 2005, accepted.

IV Velikyan I, Bergström M, Beyer GJ, Bergström-Pettermann E, Frändberg P, Långström B. On the high specific radioactivity of 68Ga-bioconjugates exemplified with 68Ga-DOTATOC, 2005, pre-liminary manuscript.

V Lendvai G, Velikyan I, Bergström M, Laryea D, Välilä M, Salomäki S, Långström B, Roivainen A. Biodistribution of 68Ga -labelled phosphodiester, phosphorohtioate, and 2'-O-methyl phosphodiester oligonucleotides in normal rats, European Journal of Pharmaceuti-cal Sciences 2005, 26, 26-38.

VI Lavén M, Velikyan I, Djodjic M, Ljung J, Berglund O, Markides KE, Långström B, Wallenborg S. Imaging of peptide adsorption to microfluidic channels in a plastic disc using a positron emitting ra-dionuclide, Lab on a chip 2005, 5, 756-763.

VII Appendix I: Velikyan I, Långström B. 68Ga-labelling of an Affibody® ligand

VIII Appendix II: Velikyan I, Långström B. Direct 68Ga-labelling of Lactoferrin

Reprints were made with kind permission from the publishers: The Ameri-can Chemical Society (I), John Wiley & Sons Ltd. (II), Elsevier Science (V), The Royal Society of Chemistry (VI).

Contribution report The author was responsible for planning, carrying out and developing syn-thetic and labelling chemistry and the consequent chemical characterisation of the products in papers I-VIII. In paper V the author was also responsible for the planning and developing of a method for metabolite analysis. The study of adsorption on CDs was carried out together with Martin Lavén and Susanne Wallenborg (paper VI). The author wrote papers I-IV and VII-VIII, paper I together with Gerd Beyer, and paper III together with Vladimir Tol-machev and Åsa Liljegren Sundberg. The author also shared the writing of paper V with Gabor Lendvai and paper VI with Martin Lavén and Susanne Wallenborg.

Related papers and patent applications

Velikyan I, Liljegren Sundberg Å, Långström B, Tolmachev V. [68Ga] DOTA hEGF for non-invasive measurement of EGFR expression in tumours using PET, Filed patent application, PH0505, 2005

Velikyan I, Långström B, Beyer G, Method of obtaining gallium-68 and use thereof and device for carrying out said method, WIPO (World Intel-lectual Property Organization), WO 2004/089517, 2004

Velikyan I, Långström B, Microwave method for preparing radiolabelled gallium complexes, WIPO (World Intellectual Property Organiza-tion), WO 2004/089425, 2004

Lavén M, Wallenborg S, Velikyan I, Bergström S, Djodjic M, Ljung J, Ber-glund O, Edenwall N, Markides KE, Långström B. Radionuclide imaging of miniaturized chemical analysis systems, AnalyticalChemistry 2004, 76, 7102-7108.

Roivainen A, Tolvanen T, Salomäki S, Lendvai G, Velikyan I, Numminen P, Välilä M, Sipila H, Bergström M, Harkonen P, Lönnberg H, Lång-ström B. 68Ga-labelled oligonucleotides for in vivo imaging with PET, Journal of Nuclear Medicine 2004, 45, 347-355.

Bergström SK, Edenwall N, Lavén M, Velikyan I, Långström B, Markides KE. Polyamine Deactivation of Integrated Poly(dimethylsiloxane) Structures Investigated by Radionuclide Imaging and Capillary Elec-trophoresis Experiments, Anal Chem 2005, 77, 938-942.

Front cover picture (A) represents a PET examination image of a patient with mid-gut carcinoid tumour originating from the intestine, with multiple liver metastases. The tracer, 68Ga-DOTATOC, was synthesised employing microwave heating.

Contents

1 INTRODUCTION .............................................................................111.1 Aims of the study ...........................................................................12

2 BACKGROUND ................................................................................132.1 Imaging techniques: Positron Emission Tomography ...................132.2 The generator produced 68Ga radiometal .......................................152.3 Radiometallation ............................................................................16

2.3.1 Microwave heating in radiolabelling chemistry ....................192.4 Macromolecular tracers..................................................................20

2.4.1 Peptides/proteins/antibodies ..................................................202.4.2 Oligonucleotides ....................................................................22

2.5 Specific radioactivity .....................................................................242.6 68Ga-labelled peptides as a tool to study analytical devices...........24

3 RESULTS AND DISCUSSION ........................................................263.1 The preparation of 68Ga(III) ...........................................................26

3.1.1 68Ge/68Ga generator characterisation......................................263.1.2 Preconcentration/purification of 68Ge/68Ga generator eluate .27

3.2 Conjugation of DOTA with macromolecules ................................293.3 Chelator mediated 68Ga-labelling of the bioconjugates .................32

3.3.1 The influence of microwave heating......................................373.4 Specific radioactivity .....................................................................383.5 68Ga-labelling of DOTA-Trastuzumab...........................................403.6 Direct complexation of 68Ga with Lactoferrin ...............................413.7 Characterisation of the macromolecular conjugates and their

68Ga-labelled counterparts..............................................................423.7.1 Chemical characterisation......................................................423.7.2 Preliminary chemical and biological examination of the

68Ga-labelled oligonucleotide conjugates..............................433.7.3 Biological characterisation of the 68Ga-labelled peptide

conjugates..............................................................................473.8 68Ga-labelled peptides for adsorption studies.................................49

4 CONCLUSIONS ................................................................................50

5 OUTLOOKS ......................................................................................51

6 ACKNOWLEDGEMENTS ..............................................................52

7 SUMMARY IN SWEDISH ...............................................................55

8 REFERENCES ..................................................................................57

Abbreviations

+ Positron Bmax Total receptor number (concentration) DNA Deoxyribonucleic acid DOTA 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic

acidDOTATOC 4,7,10-Tricarboxymethyl-1,4,7,10-

tetraazacyclododecan-1-yl-acetyl-D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-L-threoninol (DOTA-D-Phe1-Tyr3-Octreotide)

DOTA-RGD Cys2-6; c[CH2CO-Lys(DOTA)-Cys-Arg-Gly-Asp-Cys-Phe-Cys]-CCX6-NH2)

EC Electron capture EDC 1-Ethyl-3(3-dimethylaminopropyl)carbodiimide EGF Epidermal growth factor ESI Electrospray ionisation HEPES N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid HER2 Human epidermal growth factor receptor 2 HPLC High-performance liquid chromatography ICP-AES Inductively Coupled Plasma Atomic Emission Spectros-

copy LC Liquid chromatography Lf Lactoferrin mAb Monoclonal antibody mRNA Messenger-RNA MS Mass spectrometry NOTA 1,4,7-triazacyclononane-1,4,7-triacetic acid NMR Nuclear Magnetic Resonance NODAGATATE 1,4,7-Tricarboxymethyl-1,4,7-triazacyclononan-1-yl-

acetyl-D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-L-Thr (NO-DAGA - Tyr3 - Octreotate)

PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffered saline PET Positron emission tomography pI Isoelectric point RAI Radioactivity incorporation RNA Ribonucleic acid

RP Reverse phase RSD Relative standard deviation SE Size exclusion SPE Solid phase extraction SPECT Single photon emission computed tomography SRA Specific radioactivity SST Somatostatin SSTR Somatostatin receptor SSTR2 Somatostatin receptor subgroup 2 Sulfo-NHS 1-Hydroxy-2,5-dioxo-3-pyrrolidinesulfonic acid SUV Standardized uptake value TATE octreotate TEAA Triethylammonium acetate Tf Transferrin TFA Trifluoroacetic acid TLC Thin layer chromatography Tyr Tyrosine UV UltravioletVIP Vasoactive intestinal peptide

11

1 INTRODUCTION

Positron emission tomography (PET) is a non-invasive imaging technique of high resolution, sensitivity and accurate quantification. It provides informa-tion about the physiological function of the body since it employs biologi-cally active tracer molecules. The latter must comprise a positron emitting radionuclide in order to be detected. Those radionuclides are short-lived and thus require fast labelling syntheses. Diverse biologically active molecules have been labelled with biogenic elements (11C, 13N, 15O), radiohalogens (e.g. 18F, 76Br, 124I) and radiometals (e.g. 64Cu, 66Ga, 68Ga). The choice of a radionuclide depends on its physical half-life, decay mode, chemistry for the tracer preparation, in vivo kinetics and availability/cost. Large biological macromolecules such as peptides/proteins/antibodies and oligonucleotides are mostly labelled with either radiohalogens or radiometals. A straightfor-ward preparation of a tracer using radiometallation with generator produced radionuclides may result in kit type production of PET radiopharmaceuticals and make PET examinations possible at centers lacking accelerators. The 68Ge/68Ga generator produced positron emitting 68Ga is a suitable radiometal. However, such obstacles as the chemical form of 68Ga after generator elu-tion, the large elution volume and the contamination of other cations origi-nating from the column material and 68Ge breakthrough may have limited its use. Methods to surmount these drawbacks would promote the wider use of 68Ga-based radiopharmaceuticals.

The labelling of macromolecules with radiometals can be direct or chela-tor mediated. The direct labelling utilizes the chelating ability of the macro-molecule itself as, for example, Lactoferrin. Macromolecules lacking com-plexing ability and requiring site-specific labelling might be conjugated to a bifunctional chelator prior to the labelling. Macrocyclic bifunctional chela-tors can form stable complexes with radiometal cations and covalently bind to macromolecules. The fact that the same chelator can complex different cations makes it possible to use the same biologically active molecule for diagnosis and therapy, employing corresponding radiometals. 68Ga may have the potential for diagnosis, dosimetry, dose planning for radiotherapy and follow up of response to chemo- and radiotherapy. This might require accu-rate quantification, which for some applications is dependent on the specific radioactivity (SRA) of a tracer. This is especially important for the charac-terisation of high affinity binding sites, such as many peptide receptors. An-other factor that necessitates the high SRA is the labelling of highly potent

12

receptor agonists which can induce side effects. It was thus essential to de-velop a fast and relilable method for 68Ga-labelling of various macromole-cules with high SRA.

The identity, purity and amount of a radiolabelled compound should be confirmed prior to its use. Moreover, modifications introduced into biologi-cally active macromolecules during the preparation of tracers might affect the biological activity. Thus, the radiolabelled bioconjugates should be char-acterised both chemically and biologically.

Tracers comprising positron emitting radionuclides can be used to study not only biological but also mechanical systems. Another imaging technique such as autoradiography can be employed for detection and quantification. A method that would allow imaging and quantitative investigation of peptide adsorption to different surfaces within analysis systems might find a wide use in the development and evaluation of miniaturised chemical analysis devices.

1.1 Aims of the study The aim of the thesis was exploration of scope and limitations of meth-ods suitable for 68Ga-labelling of macromolecules, such as peptides, pro-teins, antibodies and oligonucleotides, as well as characterisation and application of the resulting tracer candidates. In particular, the following tasks were addressed:

To characterise the performance of a commercially available 68Ge/68Ga generator in order to determine the potential for its long term efficient utilization for 68Ga radiopharmaceutical preparation. To develop a method for preconcentration and purification of 68Ge/68Ga generator eluate. To conjugate biological macromolecules to a bifunctional chelator. To develop a fast method for 68Ga-labelling of macromolecules with high specific radioactivity. To perform chemical characterisation and preliminary biological ex-amination of the bioconjugates and their corresponding 68Ga-labelled counterparts. To develop 68Ga-labelled peptide tracers for imaging peptide adsorp-tion in chemical analysis systems.

13

2 BACKGROUND

2.1 Imaging techniques: Positron Emission Tomography

The radioisotope based imaging techniques utilize the tracer concept which was first described by George de Hevesy in 1923.1 A tracer is defined as a substance which is introduced into a biological or mechanical system and can be followed through the course of a process. The tracer is used in low amounts in order to provide information on the pattern of events in a process without disturbing the studied system. Tracers containing radioactive nu-clides that give detectable radiation, are used in medicine and in biomedical research for studies of physiological processes, in vivo pharmacokinetics, in drug development as well as for diagnosis and radiotherapy.2-9 Analytical chemistry is another field where tracers can be used in method development and studies of analytical systems.10,11

Positron emission tomography (PET) is a highly sensitive non-invasive mo-lecular imaging technology introduced by Ter-Pogossian et al. in early 1970s.12,13 PET requires a tracer, e.g. a radiopharmaceutical labelled with a positron-emitting radionuclide ( +), and a PET camera for imaging the pa-tient (Figure 1).14,15

Positron emitting isotope 511keV Annihilation photon

Positron

Electron

511keV Annihilation photon

Annihilation photons

A

Figure 1. (A) A positron and an electron annihilate producing two 511 keV annihila-tion photons travelling in opposite directions and (B) registered externally by radio-detectors in the PET camera.

14

PET may provide information on cellular function of the body when employ-ing biologically active tracer molecules. Positron scan registration is based on the 180° correlation of the 511 keV photons arising from the annihilation of positrons with electrons and detection by means of two opposing counters recording only coincident events (Figure 1).12,15 The registered events are reconstructed into images representing the spatial distribution of the radioac-tive source in the body. PET is a quantitative imaging method allowing the measurement of regional concentration of the tracer. Positron emission is exclusively a property of the neutron deficient nuclides. Some positron emit-ting nuclides are presented in Table 1.

Table 1. Some positron emitting radionuclides used in PET, their decay properties and production mode.

Radionuclide Half-life Emax( +),keV

+ decay, % Production 11C 20.3 min 961 100 Cyclotron 13N 9.97 min 1190 100 Cyclotron 15O 2.1 min 1732 100 Cyclotron 18F 110 min 634 97 Cyclotron

52Fe 8.2 h 800 57 Cyclotron 62Cu 9.8 min 2910 98 Generator 64Cu 12.8 h 656 19 Cyclotron 66Ga 9.5 h 4153 56 Cyclotron 68Ga 67.6 min 1899 89 Generator 76Br 16.2 h 1310 54 Cyclotron 82Rb 76 sec 3150 95 Generator 86Y 14.7 h 3150 34 Cyclotron 124I 4.17 d 2100 23 Cyclotron

Most positron-emitting nuclides are produced in accelerators. A generator system consisting of a long-lived parent radionuclide, which decays to a shorter-lived daughter radionuclide is an alternative production method.16

The daughter radionuclide is radiochemically separated from the decaying parent so that the former is obtained in a pure radionuclide and radiochemi-cal form. Key features of radionuclide generators include relatively low cost and the convenience of obtaining the desired daughter radionuclide on de-mand.16 Thus, about 80% of single photon emission computed tomography (SPECT) medical examinations using metal radionuclides are performed with 99mTc partly due to the fact that it is readily available from the 99Mo/99mTc generator, even though the latter has only one week shelf-life.17,18

The choice of a radionuclide depends on: i) the physical half-life that should allow the production and application of the radioactive tracer and should match the biological half-life of a corresponding tracer in order to

15

avoid unnecessary irradiation; ii) the decay mode; iii) chemistry for the tracer preparation; iv) the availability and cost. Generally, a tracer is chosen that will be selectively taken up by a certain type of tissue, e.g. cancer cells. The most common applications of PET are in the fields of oncology, cardi-ology, and neurology. For example, PET has been employed to follow re-sponse to therapy19, image tumours20 as well as to study neurotransmission21

and measure blood flow7,12. A number of 68Ga-based imaging agents to study pulmonary, myocardial and cerebral perfusion as well as renal and hepatobil-iary function, to detect blood-brain barrier defect, to image tumour, brain, and bone has been investigated.22-24 PET tracers covering a broad range from small molecules to large biomolecules have been developed, promoting the growth of the PET field.25

2.2 The generator produced 68Ga radiometal Naturally occurring gallium consists of two isotopes 69Ga (60.1% natural abundance) and 71Ga (39.9% natural abundance). Three radioisotopes can be produced for labelling of radiopharmaceuticals. Two of these, 66Ga (T1/2 = 9.5 h) and 68Ga (T1/2 = 68 min), decay by +-emission and are therefore suit-able for PET imaging, 67Ga (T1/2 = 78 h) decays by -emission and is used for SPECT imaging. 68Ga is a generator26 produced nuclide and does not require a cyclotron on site. The parent 68Ge is accelerator-produced by the 69Ga(p,2n) reaction.27 68Ge decays with a half-life of 270.8 days solely by orbital electron capture to 68Ga. The latter disintegrates by the emission of positrons of 1.9 MeV max energy and 11% by electron capture to stable 68Zn. The half-life of 68Ga permits production and utilization of 68Ga–based radiopharmaceuticals.

The only stable chemical form in aqueous solution is the Ga(III) cation, which can hydrolise and precipitate at pH 3-7 in the form of insoluble trihy-droxide if the concentration exceeds nanomolar level. However, in presence of stabilising agents precipitation can be avoided.23,28 At physiological pH 7.4 the total solubility of Ga is high because of the almost exclusive forma-tion of [Ga(OH)4]¯ ions.29 The Ga(III) cation is classified as a hard acid metal. It forms stable complexes with many ligands containing oxygen and nitrogen as well as sulfur as donor atoms.23,30,31 Thus, Ga(III) is suitable for complexation with chelators, naked or conjugated with peptides or other macromolecules. The coordination chemistry of Ga(III) is defined by coor-dination number six and the octahedral coordination sphere.

16

2.3 RadiometallationAs already mentioned Ga(III) can be coordinated by bifunctional complex-ing agents covalently linked to a targeting vector, which in its turn can bind to a target. The advantage of such complexes is that the bifunctional chelat-ing agent can be labelled with various radiometals for certain diagnostic or therapeutic applications. Acyclic bifunctional chelators, such as ethyl-enediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), desferrioxamine (DFO), N,N'di(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED) and their derivatives have been used for labelling of macromolecules with 111In, 67Ga, 68Ga, 90Y for tumour imaging and ther-apy (Figure 2).32-39 Most of these complexes showed low stability in vivo andin vitro, which has been related to the tendency of such anionic complexes to undergo acid- or cation-promoted dissociation in vivo.32,40-43

HOOC N

HOOC

N

COOH

COOH HOOC

N

HOOC N

COOH

N

COOH

COOH

NH2NNHN

NHNCH3

OH

O

O

OH

O

O

OH

O

EDTADTPA

DFO

OH

NH+NH+

O-

O

O-

O

OH

HBED

Figure 2. Some open-chain metal chelators used for radiopharmaceuticals.

17

The released radioisotope may then be bound by serum proteins, such as transferring, or may build up in radiosensitive organs, such as bone/bone marrow or in the gastrointestinal mucosa.29,41 The introduction of macrocyc-lic bifunctional complexing agents has led to more stable complexes.44-46

Chelators such as 1,4,7-triazacyclononanetriacetic acid (NOTA), 1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) and their deriva-tives have been used for complexation with 64/67Cu,, 66/67/68Ga, 86,90Y, 111In,177Lu and 225Ac (Figure 3).3,17,39,41,45,47-54 DOTA and NOTA chelators can be attached to a carrier molecule via the carboxylate group or the functionalised skeleton carbon. The former method allows a one step coupling reaction to amines using carbodiimide chemistry.55 Modifications on the macrocyclic bifunctional chelators allow variation of the overall charge and hydrophilic-ity of the metal-chelate moiety, thus regulating the biodistribution pattern, excretion ways and resistance to acid/cation-promoted dissociation over a wide pH range.

O

N N

NN

O

O

O

OH

OH

OH

OH

O

N

NN

O

OOH

OH

OH

N N

NN

OO

O

OH

OH

OH

OOHNOTA

DOTA

TETA

Figure 3. Examples of macrocyclic chelators used for radiolabelling.

The high stability of tetraazamacrocycles is provided by the extremely slow dissociation reactions. Typical dissociation constant values are 105 to 107

times lower than those of open-chain analogues. The hole-size effects influ-

18

ence both thermodynamics and kinetics of macrocycle complexes. This is because a chelator in its minimum energy metal-binding conformation will be optimized for a particular size of a metal ion, and when other metal ions are bound, the chelator conformational energy will rise with a resultant de-crease in stability of the complex. Thus, macrocycles are selective for metal ions.56-58 For a given ionic size the stability of the complex increases with increasing charge. Another factor which influences the complexation rate and the stability is the presence of pendant arms, which help to achieve the full coordination number of the metal ion.59-61 In addition, a pendant arm can be used for the conjugation to a macromolecule.62

A bifunctional chelator should meet the following criteria: i) A chelator conjugated to a macromolecule should bind the radioisotope rapidly and sufficiently; ii) The formed complex should be kinetically stable to cation release over a pH range of 2-8 and in the presence of cations like Ca2+, Zn2+

and Mg2+ which can be found in serum.41 Other factors to consider include redox properties, charge and lipophilicity.63,64 Neutral complexes are less sensitive to acid/cation-promoted dissociation compared with anionic com-plexes. For example, the neutral Ga(III) complex of NOTA has been shown to undergo acid-catalysed dissociation only at very low pH which is unlikely to happen under physiological conditions. The log stability constant (logK) of the Ga(III)-NOTA has been determined to 30.9865, while the log stability constants of the Ga(III) complex of DOTA has been determined to 21.3366.The X-ray crystallography study of NOTA complex with Ga(III) revealed the octahedral coordination of Ga(III).41 The cavity of NOTA is defined by the facial triaza plane and an opposite facial plane consisting of three car-boxylate oxygen atoms (Figure 4). The compactness of the triazanonane ring and the steric efficiency of the pendant acetate groups lead to the formation of complexes of unusually high stability and selectivity for the Ga(III).65

O

N

NN

O

O

O

O

O

Ga

O

O

N N

NN

O

O

O

Phe-HN

OH

GaO

Figure 4. Molecular structure of the gallium complex with NOTA (left) and DOTA-D-Phe-NH2 (right).

19

DOTA has a larger cavity than NOTA, which results in lower stability of the Ga(III) complex. In the Ga(III) complex of DOTA-D-Phe-NH2, DOTA adopts a cis-pseudooctahedral geometry with a folded macrocyclic unit.51,67

Gallium radiopharmaceuticals must be stable enough to avoid trans-chelation of Ga(III) to various iron binding proteins, particularly transferrin. Transferrin has two binding sites, for which the gallium binding constants are 20.3 and 19.3.29 Therefore, a polydentate chelator, forming thermody-namically stable and kinetically inert complexes with six-coordinate Ga(III), is required. The kinetic stability of 67Ga-DOTA-D-Phe1-Tyr3–Octreotide (67Ga-DOTATOC) under physiological conditions has been studied by measurements of the rate of radiometal exchange in blood serum, and the half-life of the exchange has been estimated to 1250h.67 Moreover, the Ga-labelling of DOTA-peptides assures site-specific labelling and consequently, higher in vivo stability as, for example, compared to 99mTc which can non-specifically bind to peptides comprising cysteine residues.52,68

2.3.1 Microwave heating in radiolabelling chemistry The radiolabelling reaction time has to be minimised due to the short half-life of the radionuclide. Usually, the total time including the synthesis, puri-fication and utilization of a tracer should not exceed three half-lives of the radionuclide. The labelling synthesis is considered complete when the maxi-mum amount of radioactivity is incorporated into the tracer molecule. This corresponds to the highest possible radiochemical yield, which is a compro-mise between the chemical yield and the radionuclide decay.69

Microwave heating, providing acceleration of reactions70, is an attractive tool for radiolabelling chemistry of short-lived radionuclides. Moreover, during the conventional heating using an oil bath or oven, the walls of the vessel get heated up first, causing a temperature gradient in the solution. Under microwave irradiation the sample is heated from inside more uni-formly at each point resulting in very fast heating.70 Microwave heating is especially useful for microscale organic chemistry, such as radiolabelling where the sample size is comparable to the penetration depth of the micro-wave field.71,72 The technique has been used for labelling of different organic molecules with 131I, 11C, 15O, 18F and 13N.71

The slow radiolabelling kinetics of DOTA-based bifunctional chelators requires elevated temperatures and time.18 The extensive heating is undesir-able because of the potential damage to macromolecules and the relatively short half-life of 68Ga. Microwave heating is an attractive tool that can pro-vide acceleration of the labelling.

20

2.4 Macromolecular tracers 2.4.1 Peptides/proteins/antibodiesPeptides, proteins and antibodies are polyamides consisting of monomeric units of different -amino acid residues. The side chains of the residues bear functional groups which can be modified.73 The functionalities commonly used for the conjugation to bifunctional chelators are terminal and side-chain amino and carboxy groups as well as sulfhydryl group of cysteine.18,39

Peptides are important regulators of growth and cellular functions in nor-mal tissue and tumours. Radiolabelled regulatory peptide analogues might be used for in vivo localization and therapy of tumours (Figure 5).2,17,46,51,74-80 Iflabelled with a positron-emitting radionuclide, they might be used to make an accurate tumour diagnosis, to quantify the radiation dose to tumours and critical organs, thus allowing dose planning and dose monitoring for success-ful radiotherapy, and to follow tumour response to chemo- and radiother-apy.81-83 The failure to estimate patient dosimetry might be one of the rea-sons why radioimmunotherapy has advanced slowly during the last 20 years.84 The new approaches in radionuclide therapy based on radiolabelled peptides call for individualised imaging protocols.2,5,22,85-89

Figure 5. Schematic representation of a vector macromolecule labelled with a radi-ometal nuclide (M) via a covalentely attached chelator.

Peptides have been labelled with radiohalogens, 11C and radiometals.43,52,81,90-

95 Design of metal-based peptide radiopharmaceuticals requires knowledge of the cation coordination geometry, chelator selectivity, metal-chelator complex structure, lipophilicity and thermodynamic/kinetic stability. Some of these factors have been studied, for example, for somatostatin (SST) (Fig-ure 6) analogues such as octreotide, [Tyr3]octreotide and [Tyr3]octreotate (Figure 7).17,43,96-98 These are cyclic peptides with one cysteine disulfide

Radionuclide

Vector

Linker

ChelatorReceptor

M

21

bridge which restricts the conformational flexibility of the peptides. They have been conjugated with bifunctional chelators such as DTPA, DFO99,100,EDDA/HYNIC101,102, DOTA97,103 and NOTA51 (Figure 8 and Figures 2 and 3 for the chelates). The DOTA-D-Phe1-Tyr3–Octreotide (DOTATOC) (Figure 8) has been labelled with a variety of metals in the +3 oxidation state, such as Ga22,103-106, In107, Y79,80 and Lu78.

The short half-life of 68Ga in combination with fast target localization and blood clearance of DOTATOC is of special interest for clinical use.22

Figure 7. Structural formulae of eight amino acid-containing analogues: (A) octreo-tide, (B) [Tyr3]octreotide, (C) [Tyr3]octreotate.

The receptor binding affinity, internalisation and biodistribution of different SST ligands have been shown to be dependent on the peptide chemical struc-ture, chelator type (DOTA, DTPA and their derivatives) and metal cations (In, Y, Ga, Tc, Lu).43,97,107-114 Thus, for example, replacement of In or Y in DOTATOC by Ga improved SST2 binding affinity, in vivo tumour imaging and decreased kidney uptake.67,103,113,115 Structural nuclear magnetic reso-nance (NMR) studies of Ga(III)- and Y(III)-DOTATOC atributed the bioac-tivity variations to the differences in coordination sphere of the metal cations.98 In contrast to Ga(III), Y(III) interacts with carbonyl oxigen of the amide bond of D-Phe1 resulting into a cis-trans isomerization across the DOTA-peptide linker. Thus, D-Phe1 has been found essential for the binding

(A) D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr(OH)

(B) D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr(OH)

(C) D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr

(A) D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr(OH)(A) D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr(OH)

(B) D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr(OH)(B) D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr(OH)

(C) D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr

Ala—Gly—Cys—Lys—Asn—Phe—Phe—Trp

Cys—Ser—Thr—Phe—Thr—Lys

Ala—Gly—Cys—Lys—Asn—Phe—Phe—Trp

Cys—Ser—Thr—Phe—Thr—Lys

Figure 6. Structural formula of somatostatin (SST-14), a cyclic disulphide-containing peptide hormone of 14 amino acids.

22

activity of DOTATOC to SSTR.98 Comparative studies of imaging the soma-tostatin receptor in neuroendocrine tumours using 111In-octreotide (SPECT detection) versus 68Ga-DOTATOC82,104 and 68Ga-DOTA-1-Nal3-octreotide116

(PET detection) have also been conducted revealing a better performance of the PET-tracers.

O

HO

N N

NN

OH

O

OOH

O

NN

NHO

HOO

O

O

S

NN

NN

O

O

ON

OS

NH2

HO

NH

OH

H

H

H

H

HH

H

H

Figure 8. Primary structure of DOTATOC.

2.4.2 Oligonucleotides

An antisense oligonucleotide (Figure 9) is a short, synthetic nucleic acid that manifests its inhibiting effect on the gene expression by selectively hybridis-ing with its complementary “sense” sequences in mRNA through Watson-Crick base-pairing117 (Figure 10). Antisense oligonucleotides are of consid-erable interest for biological studies and particularly molecule-targeted therapies of cancer.117 Radionuclide labelled antisense oligonucleotides may be used for in vivo imaging of gene expression.118 A number of radiolabel-ling studies has been conducted with gamma emitters, such as 99mTc, 111Inand 125I for in vitro studies and for imaging with gamma cameras and SPECT.118-123 Methods for the labelling of oligonucleotides with positron emitting radionuclides, such as 11C, 18F and 76Br, have also been pre-sented.119,124-129

23

DNA

3´-end

5´-end

Guanine

Cytosine

Adenine

Thymine

O

OPOO

O

N

N

NH

N

O

NH2

ON

N

O

OPOO-

O

NH2

ON

NH

O

O

H3C

OPOO-

O

OPOO-

O

O

OPOO-

O

N

N

N

N

NH2

Figure 9. An oligonucleotide is a linear polymer built up of monomeric units, the nucleotides. A nucleotide consists of three molecular fragments: sugar, heterocycle, and phosphate. The oligonucleotide biological function like replication of DNA, messenger RNA synthesis and protein synthesis is influenced by its three-dimensional structure and its ability to form hydrogen bonding.

Figure 10. (A)The diagram depicts the flow of genetic information from DNA to the protein. The information contained in genes (DNA) is eventually expressed as the phenotype (protein). (B) An antisense oligonucleotide hybridises to the complemen-tary target mRNA and causes blocking of protein translation.

A B

24

A prime requirement of a modified oligonucleotide is that it should maintain the capacity to hybridise and be chemically and metabolically sta-ble.117,119,130-134 A number of biological/pharmacological aspects play impor-tant roles, such as penetration of cell membranes, non-specific accumulation in organs and non-specific binding to proteins and other structures as well as toxicity.118-120 In order to improve these characteristics, phosphorothioate, methylphosphonate, phosphoramidate, phosphoramidate morpholino, 2 -O-methyl oligonucleotides as well as peptide nucleic acids have been designed and studied.119,120,130-133,135

2.5 Specific radioactivity SRA is defined as the concentration of a radioactive material in a sample and in this thesis is expressed as radioactivity per macromolecule amount (Bq/mol). The requirement of high SRA is dictated by biological factors such as limited amount of receptors, tracer affinity and possible pharmacol-ogical side effects of the tracer.

In particular, due to competition with the labelled macromolecules for the same target, the presence of unlabelled vectors may decrease the uptake of the radioactive tracer. Moreover, high SRA might be critical for providing a sufficient contrast of a PET image and it assures the possibility for an opti-misation of the tracer SRA. It might also be of interest to explore if an in-creased SRA would allow more refined evaluations of receptor binding pa-rameters, especially if it would be possible to determine in vivo the total receptor number (Bmax) in e.g. tumours.

Peptides with potent agonistic properties may provoke side effects.17

Minimization of the administered amount of such peptides is critical. This might be achieved by labelling procedures which provide high SRA of a peptide tracer.

2.6 68Ga-labelled peptides as a tool to study analytical devices

Analytical devices in chemistry and biochemistry are developing towards miniaturisation and the possibility of performing parallel separations on a single microchip device.136-139 It is necessitated by the minute amounts of analytes as well as economical and environmental concern. But the reduced dimensions and increased surface-to-volume ratios in microstructures require careful control of liquid flow rate, surface chemistry and a reliable and re-producible manufacturing process.140,141 Quantitative radionuclide imaging might be of significant value in the evaluation and optimisation of such

25

miniaturized analytical systems. The relatively energetic positrons can pene-trate high optical density materials and be registered by PhosphorImager plates to enable imaging. Moreover, tracers labelled with high SRA may allow highly mass sensitive analysis and thus imaging of small amounts of analytes.

26

3 RESULTS AND DISCUSSION

An improved SRA of the 68Ga-bioconjugates was achieved due to precon-centration/purification of the 68Ga preparations and microwave heating. The influence of the SRA on the performance of a tracer was preliminarily tested and the importance of SRA levels is discussed. Factors that might influence the 68Ga-labelling efficiency and the SRA were studied and the results of these pilot experiments are discussed. The possibility of a direct 68Ga-labelling of a chelating protein as well as the labelling of a temperature sen-sitive antibody is described. Chemical and biological characterisations of 68Ga-labelled bioconjugates are presented. Finally, the application of 68Ga-labelled peptides to study peptide adsorption is described.

3.1 The preparation of 68Ga(III)3.1.1 68Ge/68Ga generator characterisation Three units of a commercial 68Ge/68Ga generator (Cyclotron Co., Ltd, Obninsk, Russia) were evaluated over a period of up to 2.5 years (paper I).

Figure 11. (left) 68Ga elution efficiency for generator-1 during 29 months, generator-2 during 14 months and generator-3 during 3 months . (right) Elution profile of the 68Ge/68Ga generator, one fraction was 1 ml, (fraction 1 = 0.3 ml, fraction 7 = 0.7 ml) giving a total eluted volume of 6 mL. The profiles for the 68Ga and the 68Ge break-through are similar, the 68Ge breakthrough is ~10-3 % of total radioactivity. Fraction 3 (1 mL) contains over 60 % of the available 68Ga radioactivity.

0

20

40

60

80

100

0 200 400 600 800 1000

Time, [day]

68G

a yi

eld,

[%]

Generator-1 Generator-2 Generator-3

0

200

400

600

800

1000

1 2 3 4 5 6 7

Fraction

Elu

ted

activ

ity p

er fr

actio

n 68Ga activity given in [MBq]

68Ge breakthrough given in [Bq]

volume 6 mL

27

During the first month the elution efficiency was 78±5% of the theoretical amount and then decreased slowly and continuously down to 41±3% after 29 months use. The generator elution efficiency over time for the three units was found reproducible (RSD<10%, n=3) (Figure 11 left). The 68Ga elution profile and the 68Ge breakthrough are presented in Figure 11 right. By frac-tionating the 68Ga eluate, approximately 65% of the available radioactivity could be obtained in 1 mL (the third fraction, Figure 11 right). The 68Gebreakthrough with respect to the eluted 68Ga radioactivity was found to be 0.001 - 0.007% and did not change during the investigation period. It has previously been shown that 68Ge breakthrough losses of 0.001% per elution are insignificant compared with 68Ge decay losses, assuming two elutions per day.142

Since the major hindrance in complexation chemistry of 68Ga is the pres-ence of competing metal ions in the eluate, special attention was paid to metal ion analysis and optimisation of the elution of 68Ga. The results sug-gested a daily elution of the generator in order to keep the concentration of the contaminant metal ions as low as possible. A preventive elution 3-4 hours prior to the radiosynthesis is recommended, since the interfering metal ion concentration thus can be kept at its minimum value. The non-toxic gen-erator matrix material (TiO2) may cause relatively high Ti concentrations (close to 1000 ppm) in the eluate if the generator is not eluted regularly. Breeman et al.143 have recetly characterised the same type of generator and obtained similar results, thus supporting our approach.

3.1.2 Preconcentration/purification of 68Ge/68Ga generator eluate

The obstacle for a wider use of 68Ga has been its chemical form upon elution from the generator, the low 68Ga concentration and the presence of other metal ions and parent 68Ge in the generator eluate.16,144-150

To overcome these disadvantages, a method based on anion exchange to purify and to reduce the volume (preconcentrate) of the generator eluate has been developed (paper I). The adsorption behaviour of metal ions from HCl-solutions on the anion exchanger is well known.151,152 In HCl solution gal-lium forms strong anionic complexes with Cl¯. The corresponding [GaCl6]3¯and [GaCl4]¯ complexes are strongly adsorbed at the mentioned anion exchange resin from HCl concentrations > 3 M, while germanium is practically not adsorbed from < 5 M HCl solution.

A number of strong anion exchange resins (AG 1, Bio-Rad, USA) with different mesh size and crosslinking percent parameters, as well as commer-cially available cartridges with a quarternary ammonium functional group were investigated (paper I). The resin type, cartridge size, sample pretreat-ment, column conditioning, sample addition, column wash, column drying

28

and analyte elution procedures were optimized. The AG 1-X8 (200-400 mesh) resin and Chromafix SAX SPEC cartridge showed comparable results with respect to retention, recovery and elution profile. However, the Chro-mafix SAX SPEC cartridge is, as a standardized commercial product, much more suited for further standardization and automation. The adsorption of 68Ga from the HCl solution at the cartridge increased rapidly from 0.8% at 0.1M HCl to almost 100 % at 3.8 M HCl in agreement with literature data on distribution coefficients151 (D > 105) (Figure 12). The distribution coeffi-cients D of 68Ga between the stationary and mobile phase were determined by the column method as weighted distribution coefficients, D.

The steeply rising portion of the distribution function is due to the forma-tion of the negatively charged complexes. Since the parent nuclide 68Ge is not retained at the resin, the concentrating step is also purification of the 68Ga from 68Ge. The eluate was purified from Al(III) and In(III) as well, since the adsorbability of these ions decreased rapidly with increasing con-centration of HCl and became negligible above 3 and 1 M, respectively, in agreement with literature data153. In the same way the original generator eluate was purified from Ti by 90% as shown by the ICP-AES analysis data.

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

1 2 3 4 5 6

HCL in Mol/L

Dis

trib

utio

n co

efic

ient

D

The elution with deionised water finally resulted in a 68Ga recovery of 93±2% in a total volume of 200±20 L only. In total the preconcentra-tion/purification procedure required 4-6 min. The technique could be applied for the preconcentration of the eluates of at least two generators (2 x 6mL).

Figure 12. Distribution coefficient D for the adsorption of 68Ga on the anion-exchange resin of the com-mercial Cartridge: SEX SPEC, Chromafix, that contains 45 mg of resin.

res

liq

liq

res

mm

AAD

where, Ares = 68Ga radioactivity in [Bq] of the dry resin, Aliq = 68Ga radioactivity of the solution passed through the column in [Bq], mres = amount of dry resin in [g], mliq = amount of the solution in [g].

29

3.2 Conjugation of DOTA with macromolecules

The carboxylic functionality of the DOTA (5) chelate can be used to form an amide bond with an amine functionality of a macromolecule (papers II,III,V,VI). Peptides, proteins and antibodies have primary amine groups at the N-terminal and at lysine amino acid residues (Figure 13). The oligonu-cleotides used in these studies were modified at 5 - or 3 -end with an amino-hexyl linker in order to introduce the amine functionality (Figure 14). The major work on oligonucleotides presented in the thesis was conducted with four modified antisense oligonucleotides (1 - 4) specific for the activated human K-ras oncogene117: 17-mer phosphodiester oligonucleotide with a hexylaminolinker at the 5 end (PD, 1); 17-mer phosphodiester oligonucleo-tide with a hexylaminolinker at the 3 end (2); 17-mer phosphorothioate oli-gonucleotide with a hexylaminolinker at the 5 and (PS, 3); and 2 -O-methyl phosphodiester with a hexylaminolinker at the 5 end (OMe, 4).

The macromolecules (peptides/antibody and oligonucleotides, 6) bearing amine functionality were reacted with the N-hydroxy-sulfosuccinimide ester of DOTA (7), generated in situ using the water-soluble 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC, 8) as coupling reagent to give DOTA-macromolecule bioconjugate (9) (Figure 15). The advantage of add-ing N-hydroxy-sulfosuccinimide (sulfo-NHS , 10) to the EDC (8) reaction is that it increases the stability of the active intermediate.55,73 EDC reacts with the carboxylate group to form an active ester (O-acylisourea, 11) leaving group. Subsequent formation of the sulfo-NHS ester intermediate extends the half-life of the activated carboxylate which reacts with an amine to give a stable amide linkage. Alternatively, the commercial N-hydroxy-sulfosuccinimide ester of DOTA was used. It resulted in a decreased re-quired amount of the reagents, compared with the in situ generated ester, and, consequently, more efficient purification of the product.

Lysine NH2

Terminal NH2

NH 3+

H O

O -

N H 3+

Figure 13. Lysine amino acid residue.

30

1 X= O-, X' = H

3 X= S-, X' = H

4 X= O-, X' = O - CH3

O

O

O Base

PX O

O

P

O

X

X'

OH2N

P

O

O-O

O

O

O Base

P-O O

OH2N

OH

X'

2 X' = H

Figure 14. 1 - phosphodiester oligonucleotide with a 5'- Hexylamine modification (PD); 2 - phosphodiester oligonucleotide with a 3' - Amino-C7 modification; 3 - phosphorothioate oligonucleotide with a 5'- Hexylamine modification (PS); 4 - 2'-O-methyl phosphodiester oligonucleotide with a 5'- Hexylamine modification (OMe). The antisense and sense sequences used were, respectively, 5'-CTA CGC CAC TAG CTC CA and 5 -TGG AGC TAG TGG CGT AG.

R1 O

OH

CH3 N

N NH +

CH3

CH3

Cl-

CH3 NH N N+

CH3CH3

O

R1

OH

Cl-

R1 NHR

ON R

H

H

NOH

O

O

SO

O

ONa

5

6

7

8

9

10

11

O

OH

N N

NN

OOH

O OH

R1 =

NO

O

O

SO

O

ONa

R1

O

Figure 15. Reaction scheme of the amide bond formation via sulfo-NHS ester inter-mediate. R stands for an oligonucleotide modified with a hexylamine linker or a peptide/antibody.

The bifunctional chelator, DOTA, has been conjugated in solution to pep-tides, an antibody and oligonucleotides (Tables 2 and 3). The peptides had varied pI values, constitution and length ranging from 8 to 53 amino acid

31

residues. The oligonucleotides were of various sequences and length with modifications in backbone, sugar moiety and both 3' and 5' ends with a mo-lecular weight up to 9.8 kDa. The number of DOTA molecules coupled to a macromolecule depended on the number of primary amino groups available. For example, a 28 amino acid residue peptide, vasoactive intestinal peptide (VIP), contains one terminal and three lysine amino groups, whereas angio-tensin II has only a terminal amino group. Consequently, the conjugation reaction of angiotensin II resulted in the formation of a conjugate bearing one DOTA molecule, as determined by LC-MS analysis.

Table 2. Peptides and a monoclonal antibody (mAb) conjugated to DOTA bifunc-tional chelator.

Peptides/mAb Residuenumber

NH2groups pI MW,

Da

Angiotensin II 8 1 6.74 1046.20 Bombesin 14 1 6.85 1619.00

Pancreastatin Fragment 37-52, human 16 1 9.75 1819.00 Neuropeptide Y Fragment 18-36 19 1 10.42 2456.80

Secretin human 27 1 9.45 3039.00 VIP 28 4 9.82 3325.80 EGF 53 3 4.78 6243.70

Trastuzumab (mAb) - - - 155000

Table 3. Oligonucleotides modified with aminohexyl linker either at 3' or 5' end conjugated to DOTA bifunctional chelator.

Oligonucleotide Residuenumber

NH2groups

MW, Da

Phosphodiester (3'- modified) 17 1 5249.80 Phosphodiester (5'- modified) 17 1 5249.80

Phosphorothioate (5'- modified) 17 1 5522.13 2 -O –methyl Phosphodiester (5'- modified) 17 1 5724.00

Phosphodiester (5'- modified) 30 1 9172.00 Phosphorothioate (5'- modified) 30 1 9776.70 Phosphodiester (5'- modified) 18 1 5360.00

Phosphorothioate (5'- modified) 18 1 5748.00 Phosphodiester (5'- modified) 15 1 4837.10

Phosphorothioate (5'- modified) 15 1 5061.90

The VIP conjugate consisted of a mixture of molecules with two, three and four DOTA fragments with the corresponding abundance of 24%, 61% and 15% (data from MS analysis).

32

3.3 Chelator mediated 68Ga-labelling of the bioconjugates

The 68Ga-labelling of the chelator conjugated macromolecules was con-ducted with varied temperature, heating methods, pH, time, buffering sys-tems, bioconjugate concentration as well as 68Ga preparation method (Figure 16) (papers I-VII).

O

O

N N

NN

O

O

OO

-

GaO

NH

R

O

O-

N N

NN

O

O

OO

-

O-

NHR

Buffer

68Ga3+ in HCl

Figure 16. Reaction scheme for the complexation of 68Ga (III) with a DOTA-conjugated macromolecule, where R is a peptide/antibody or — (CH2)6—oligonucleotide.

Labelling at room temperature required relatively long incubation time and resulted in low RAI. The temperature was elevated using conventional heat-ing and microwave heating. The latter showed advantages in terms of time shortening and RAI. The kinetics studies were carried out at room tempera-ture and conventional heating for 30-40 minutes. The microwave heating was applied for 30-120 seconds. The microwave heating was applicable without observed degradation of the peptides and oligonucleotides with re-spective molecular weights of at least up to 7.1 and 9.8 kDa. However, larger biomolecules like proteins and mAb do not tolerate high temperatures and were therefore labelled at room temperature or 35-45 °C provided by con-ventional heating. The pH of the reaction media was adjusted by sodium acetate or N-2-Hydroxyethylpiperazine-N´-2-ethanesulfonic acid buffer (HEPES) as well as sodium hydroxide. The pH range of 1.0-8.0 was investi-gated. The concentration of the bioconjugates was decreased in order to pos-sibly increase the SRA of the radiolabelled product. The labelling was con-ducted using both original and preconcentrated 68Ge/68Ga generator eluate, resulting in higher RAI when using the latter. The oligonucleotide counter-

33

parts were labelled with decay corrected radiochemical yields in the range of 30 to 52%. The RAI for peptide conjugates was usually higher than 70%.

In order to investigate the influence of the 68Ga preparation method on the labelling efficiency, labelling of a test molecule, DOTATOC, was performed using three different preparations: i) 6 mL of the non-treated generator elu-ate; ii) 1 mL peak fraction of the non-treated generator eluate; and iii) preconcentrated/purified generator eluate.

i) The complexation reaction of 68Ga with the macrocyclic chelator con-jugated to the peptide started at room temperature, but the RAI did not ex-ceed 24% and 30% for sodium acetate and HEPES buffers, respectively (Figure 17A). Conventional heating improved the RAI. In the case of HEPES buffer, the 68Ga incorporation time was shorter. Microwave heating of the reaction mixture shortened the reaction time considerably. Neverthe-less, a final purification of the labelled peptide conjugate was still required.

Figure 17. Time course of the 68Ga complexation reaction conducted using the full original 68Ga eluate (6mL) (A) and using only the 1 mL peak fraction of the genera-tor eluate (B) at room temperature (dashed line), conventional heating in a heating block at 95 °C (solid line) and with microwave heating for 1 min at 90±5 °C (cir-cled) for two different buffer systems: sodium acetate buffer, pH = 4.6, 20 nano-mols of DOTATOC; HEPES buffer, pH = 4.2, 20 nanomols of DOTATOC (A) and 5 nanomols of DOTATOC (B)

ii) An increase of 68Ga concentration was achieved by fractionation of the generator eluate. The third fraction of the eluate (1 mL) contained > 60 % of the total available 68Ga radioactivity (Figure 11 right). The RAI of the reac-tion at room temperature, with conventional heating or microwave heating was improved using this fraction (Figure 17B). With microwave heating the labelling reaction was complete within 1 min, and the incorporation of the radioactivity was quantitative (>95%). No further purification of the labelled peptide conjugate was required. The amount of peptide needed for a quanti-tative 68Ga incorporation was 15 nanomols, at least when using sodium ace-tate buffer, and 5 nanomols when using HEPES (Figure 18). It is worth men-

0

20

40

60

80

100

0 5 10 15 20

Time, [min]

RA

I, %

B

0

20

40

60

80

0 5 10 15 20Time, [min]

RA

I, %

A

34

tioning that the influence of the buffer is more pronounced at lower peptide quantities. The reason might be sensitivity of the reaction to potential metal impurities present in the buffers, as well as complexing ability of the sodium acetate itself, competing with DOTA when used at high concentration (>1M).

0

20

40

60

80

100

120

0 5 10 15 20

DOTATOC, [nanomol]

RA

I, %

68Ga-DOTATOC of the described preparations using microwave heating was used in 16 patient examinations (unpublished data, see for midgut carcinoid tumour image the thesis title page picture A).

iii) Although the 68Ga incorporation was quantitative using only 5 nano-mols of DOTATOC, there were still drawbacks in the approach when using a 1 mL peak fraction of the generator eluate. Firstly, about 40% of the 68Garadioactivity was wasted and secondly, even smaller peptide amounts might be requested. Thus, a technique for rapid purification and concentration of 68Ge/68Ga generator eluate was introduced (Section 3.1.2).

The influence on the RAI of the concentration/purification step of the 68Ga eluate in combination with microwave heating is demonstrated in Fig-ure 19 and Table 4. Figure 19 illustrates the comparison of conventional and microwave heating.

0

20

40

60

80

100

120

0.001 0.01 0.1 1 10

DOTATOC, [nanomol]

RA

I, %

Figure 18. Influence of the buffer-ing system ( sodium acetate, HEPES) on the 68Ga RAI for dif-ferent DOTATOC quantities under 1 min microwave heating at 90±5°C. The reaction was con-ducted using the 1 mL peak frac-tion of the original generator eluate (see Figure 11 right). Data are presented as mean ±SD (n=4).

Figure 19. Influence of the DOTATOC amount on the RAI for the 68Ga com-plexation reaction in HEPES buffer using the total 68Ga radioactivity in 200 µL volume after the preconcentration and purification. Solid line : 1 min micro-wave heating at 90±5 °C; dashed line: 5 min conventional heating at 95 °C. Data are presented as mean ±SD (n=3-5).

35

The preconcentrated/purified 68Ga(III) could quantitatively be incorpo-rated into less than 1 nanomol of DOTATOC when microwave heating was used. Whereas labelling under the conventional heating reqired 5-10 nano-mols of DOTATOC. Table 4 presents details concerning age of the genera-tor, variation of the bioconjugate and its amount.

Table 4. Labelling conditions and practical quality parameters of the 68Ga-labelledpeptide conjugates obtained from preconcentrated/purified 68Ga preparations (200-220 µL) under microwave heating. Due to quantitative radionuclide incorporation no further purification of the labelled product was needed.

Generatorage,

[month]

Peptide conjugate [nanomol]

RAI[ % ]

68Ga from generator[MBq ]

SRA after the synthesis [MBq/nmol ]

*SRA

1 DOTATOC 1 >99 1289 997 0.77

1 DOTATOC 0.5 >99 1286 2003 0.78

1 DOTATOC 0.3 >98 1251 3307 0.79

14 DOTATOC 1 >99 357 308 0.86

14 DOTATOC 0.5 >99 337 537 0.80

14 DOTATOC 0.3 >99 329 843 0.77

29 DOTATOC 1 >99 78 61 0.78

1 NODAGATATE 1 >99 1275 1011 0.79

1 NODAGATATE 0.5 >99 1110 1758 0.79

14 NODAGATATE 1 >98 357 281 0.79

14 NODAGATATE 0.5 >96 323 493 0.76

29 NODAGATATE 2 >98 77 30 0.78

1 DOTA-RGD 1 >99 1253 976 0.78

1 DOTA-RGD 0.5 >99 1213 1869 0.77

1 DOTA-RGD 0.25 >96 1256 3756 0.75

14 DOTA-RGD 0.5 >99 362 570 0.79

14 DOTA-RGD 0.25 >96 369 953 0.65

29 DOTA-RGD 1 >98 72 56 0.78

*SRA is the SRA after synthesis devided by corresponding 68Ga radioactivity available from the generator and multiplied by the amount of peptide conjugate used.

Independent on age of the generator, overviewing a period of 2.5 years, a stable and repeatable (RSD = 2.0%, n=54) 68Ga incorporation of >95 % into 0.3 – 1 nmols of the peptide conjugate was obtained. It is worth noting that even with the lowered 68Ga amount with time (upto 2.5 years), it was possi-ble to get a quantitative incorporation with one nanomol of the bioconjugate. This means that the SRA is basically dependent only on the 68Ga radioactiv-ity amount available from the generator. Consequently, the SRA can be

36

maintained constant throughout the generator life time by adjusting the amount of peptide conjugates used for the labelling. This should provide a possibility for carrying out experiments under comparable conditions during long time course. The last right column (*SRA) presents SRA normalized to the 68Ga radioactivity available from the generator and the amount of bio-conjugate, with RSD = 3-5%, n=18.

Another DOTA-conjugated eight amino-acid residue peptide (DOTA-GGGGKGGGG) has also been quantitatively labelled using only one nano-mol of the bioconjugate (unpublished data), thus proving the concept further. Moreover, this labelling method was applicable to a larger DOTA compris-ing peptide, DOTA-Z00342-2, belonging to Affibody® ligand family,154,155

with a molecular weight of 7.1 kDa and constituted of 58 amino acid resi-dues (paper VII). The peptide conjugated with DOTA at the terminal amino group was quantitatively labeled, using 2 nanomols of the bioconjugate. This indicates that the described method can be applied to label larger peptides, at least up to 7 kDa.

The further investigation (paper IV) of the influence of the purifica-tion/preconcentration on the RAI, using labelling with commercial 67Ga,preliminarily suggested that the gain in RAI was achieved mainly by precon-centration of the generator eluate. However, the purification from Ge, Ti, In and Al improved the labelling efficiency and the tracer preparation quality. Even though, it did not eliminate Fe(III) which may compete with Ga(III) in the complexation reaction. The latter is adsorbed with a logD of 4 in the same range of HCl molarity as Ga(III). Thus it is difficult to purify the Ga(III) solution from Fe(III) with the presented technique. Therefore, alter-native approaches should be employed to purify the 67Ga and 68Ga prepara-tions from Fe(III).

To summarise, the developed method comprising preconcentra-tion/purification of the generator eluate and the microwave heating has been shown to be robust and fast (~15 min). The small reaction solution volumes (200 µL) allowed use of small amounts of bioconjugates. The labelled prod-uct purification could be omitted, since incorporation of radioactivity was quantitative (>95%) and the preparation buffer (HEPES) is eligible for hu-man use. The SRA was considerably increased compared to the previously used methods. The method was amenable to automation and might result in a device for peptide radiopharmaceutical kit production.An automated system is currentely under evaluation demonstrating quantitative incorporation of 68Ga(III). A high labelling efficiency and the elimination of purification step minimizes the amount of radioactivity needed from the generator system. Thus, a commercial 1850 MBq generator might for more than two years be used for the production of 68Ga-based peptide tracers for patient studies. Preconcentration of at least two generator eluates can be performed and might prolong further the shelf life of the generator.

37

The interest towards 68Ga-based peptide tracers is increasing, thus, re-cently, Meyer et al.156 have reported on an automated system which provides 68Ga-labelling yields of 50% using 10-24 nanomols of small peptide biocon-jugates.

3.3.1 The influence of microwave heating The macromolecules, their conjugates and 69,71Ga-comprising counterparts were exposed to microwaves and then analyzed by radio-UV-HPLC and/or LC-ESI-MS to confirm their stability. Compared to synthesis with conven-tional heating, the application of microwave heating shortened the synthesis time considerably (papers I-VII). It should be mentioned that the radio-chemical yield of a tracer comprising 68Ga radiometal decreases by ~10% with additional 10 min due to 68Ga decay.

Figure 20. HPLC-radiochromatogram of a 68Ga-DOTATOC: (A) 5 nanomols of DOTATOC, 1 min microwave heating at 90±5 °C; (B) 5 nanomols of DOTATOC, 5 min conventional heating at 95 °C.

But the microwave heating not only reduced the chemical reaction time, it also eliminated side reactions, increased the RAI, and improved the repro-ducibility (Figure 20). The 68Ga-labelling under microwave heating was performed with small peptides with a molecular weights varying between 1.4-3.3 kDa (paper I, IV, VI) as well as larger peptides with a molecular weight of 6.2 and 7.1 kDa (paper III, VII). A cell binding assay was usually

0 5 10 Minutes

A

B Impurities

68Ga-DOTATOC

68Ga-DOTATOC

0.6

0.0

0.0

0.4

0.3

0.2

38

performed in order to assess the maintenance of receptor binding capability of the tracers. Oligonucleotides exposed to the microwaves were subjected to hybridisation tests for the preliminary characterization (paper II, V).

In order to study the influence of the heating mode on the complexation reaction selectivity, the complexation reaction of DOTATOC was carried out with equimolar concentrations of the stable isotopes of Fe (III), In(III), Ga(III) and DOTATOC both under conventional and microwave heating (paper IV). The results of these pilot experiments with Fe(III) did not reveal any advantage of microwave heating over conventional heating in terms of the reaction selectivity. On the other hand, the complexation of Ga(III) seems to be more favorable compared to In(III) when using microwave heat-ing. The preliminary conclusion was that the microwave heating might influ-ence the selectivity of the complexation reaction. The developed method was found appropriate for the study of the complexation reaction. However, more thorough investigation involving other competing trivalent cations is re-quired in order to answer the question if the microwave heating influences the selectivity of the DOTA complexation reaction.

To elucidate if the microwave acceleration of the complexation reaction of 68Ga with DOTATOC occurred due to the increased temperature or elec-tromagnetic interaction, the 68Ga-labelling of DOTATOC was performed under microwave irradiation with simultaneous cooling of the reaction ves-sel. The results (paper IV) suggested that the acceleration of the complexa-tion reaction takes place due to thermal rather than electromagnetic effect of microwaves.

3.4 Specific radioactivity

The theoretical SRA of 68Ga is 100 GBq/nmol. The developed labelling tech-nique (paper I) allowed high SRA values up to 3.3 GBq/nmol considering the generator eluted 68Ga radioactivity of 1.25 GBq. This allows a broad range of SRA values and possibility for optimisation of an applied tracer amount in terms of the required mass transport, receptor saturation and im-age contrast.

The importance of the tracer SRA for the investigation of receptor bind-ing properties was studied by binding saturation of 68Ga-DOTATOC to Rhesus monkey brain targeting cortex (section 3.7.3.1) (paper IV). The data presented in Figure 21 was derived from binding saturation experiments using 68Ga-DOTATOC with a SRA value of 390 MBq/nmol. The graph pre-sents the ratio of the amount of the receptor bound tracer to a free tracer (Bound/Free) as a function of SRA. The ratios of the local concentrations of 68Ga-DOTATOC bound specifically to the receptors, and the free 68Ga-

39

DOTATOC reflect the contrast of an image which is critically dependent upon SRA if the amount of radioactivity is kept constant.

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where B the is the concentration of the ligand-receptor complex; F is the concentration of free ligand; B* is the concentration of radioactive ligand-receptor complex; F* is the concentration of radioactivity; Bmax is the maxi-mum concentration of ligand-receptor complex; KD is the equilibrium disso-ciation constant; and SRA is specific radioactivity which equals F*/F. This equation notifies that the bound to free ligand ratio approaches zero when the SRA approaches zero, thus resulting in decreased image contrast. The expression approaches Bmax/KD when SRA reaches infinity. At certain level of SRA, B/F reaches the plateau and does not change with increasing SRA. The dependence of the signal-to-background ratio on the SRA is critical around the inflection point. Small decrease in SRA values might bring con-siderable deterioration of image contrast, and consequently cause irrepro-ducible results. This could be the case when a certain amount of radioactivity is needed to obtain a sufficient signal in vitro, or when the radiactivity dose is the limiting factor as in vivo. In such cases the value of the SRA should be high enough for B/F to lie on the plateau where the signal-to-background ratio becomes independent on variations in SRA. This would provide high reproducibility and robustness of in vivo and in vitro studies, since variations in SRA from one experiment to another would not influence the quantifica-tion.

Figure 21. The ratio of the receptor bound and the free ligand as a function of the SRA, assuming constant radioac-tivity concentration. The data were fitted to a sigmoid two-parametric emax-model. The optimisation was made in MATLAB 7.0 using a least square method (LSQNONLIN).

SRAKF

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40

The pilot experiment results (paper IV) have shown that a sufficiently high SRA might be necessary for receptor quantification, evaluation and sufficient contrast of images. Furthermore, high SRA, achieved by a combi-nation of the preconcentration/purification of 68Ga and microwave heating, enables investigation of radioactivity uptake as a function of SRA157,158 for optimisation. Moreover, an optimisation of the SRA might be performed for patient studies when planning the dose for radiotherapy. Further improve-ments of the SRA might also open for a possible determination of Bmax in tumours in vivo.

In paper I the SRA was increased by a factor of 100-150 compared to the previously applied technique using the non-treated generator eluate and to literature data.82,103,104,156 Labelling tests (paper I) with equimolar quantities (68Ga : bioconjugate = 1 : 1) using 6 pmols of DOTATOC resulted in an RAI of 11 % and a SRA of the labelled bioconjugate a factor of only 10 less than the theoretical. Recently, another report143 has claimed the obtaining of as high SRA as 1GBq/nmol, using the top fraction of the generator eluate as decribed in paper I.

3.5 68Ga-labelling of DOTA-Trastuzumab

Trastuzumab, also known as Herceptin or rhuMAb HER2, is the most exten-sively studied monoclonal antibody (mAb).159 It binds with high affinity to the human epidermal growth factor receptor 2 (HER2) and is used against cancer cells overexpressing HER2.160 Trastuzumab has previousvly been labelled with radiometals and radiohalogens.161-168 As a large biomolecule with a molecular weight of 155 kDa, Trastuzumab demonstrates relatively slow tumour penetration and clearance. Thus, the short half-life of 68Ga might in this case not be sufficient for biological or clinical studies. How-ever, the availability of 68Ga makes it useful for the development of the la-belling chemistry of Herceptin, after which it can be replaced with the more long-lived positron emitting 66Ga (T1/2=9.5 h).

Large fragile biological macromolecules like mAb are very sensitive, in terms of tertial structure, to organic solvents and elevated temperatures, thus requiring milder labelling conditions. Trastuzumab was conjugated to DOTA using 50 fold excess of N-hydroxy-sulfosuccinimide ester of DOTA.169

Thereafter DOTA-Trastuzumab was labelled with preconcentrated/purified 68Ga at RT (30 min) or 30 °C (10 min) with a resulting RAI of ~70%. 68Ga-DOTA-Trastuzumab proved to be stable in the reaction mixture and PBS buffer during the three hour stability assay with no additional radio-HPLC signals. DOTA-mediated complexation of 68Ga to Trastuzumab was con-firmed by performing the labelling reaction with both conjugated and non-conjugated macromolecules. No product was detected during 300 minutes in

41

the reaction with nonconjugated macromolecules, indicating that the label was attached to the DOTA chelator.

The binding of 68Ga-DOTA-Trastuzumab to HER2-expressing cell line, SKOV-3, could be partially prevented by receptor saturation with non-labelled Trastuzumab. One and two hour incubations resulted, respectively, in 47% and 64% blockage. This suggests that the binding of the labelled conjugate might be receptor specific. Further investigation should be con-ducted in order to find out the reason for incomplete blocking and verify the binding specificity.

3.6 Direct complexation of 68Ga with Lactoferrin Lactoferrin (Lf), like transferrin (Tf), is an iron binding glycoprotein that is present in several mucosal secretions. It consists of 692 amino acids and the molecular weight is 80 kDa.170,171 The polypeptide chain of the protein is folded into two globular lobes that can bind one metal cation each.172 It has been shown that Lf facilitates iron absorption when there is a shortage of iron stores in the body.173 Tumour cells have a higher iron need than normal cells.174 This property could be used to develop a tumour seeking tracer based on Lf. Previously, Lf has been labelled with 125I and 59Fe.173,175,176 Lac-toferrin is known to bind two equivalents of ferric ion. The coordination sites are occupied by two tyrosines, one histidine, one aspartic acid and two bi-dentate carbonate ligands as a synergestic anion.177,178 Equilibrium constants for successive binding of Ga(III) to Lf have previously been measured (logK1=21.43±0.18 and logK2=20.57±0.16) and revealed stronger binding as compared to Tf.179

The tertial structure of Lf is crucial for the chelating ability of the protein. Elevated temperatures or low salt concentrations might damage the tertial structure. Thus, Lf was labelled with 68Ga at room temperature (paper VIII). The direct complexation was performed in the presence of sodium bicarbon-ate and using the original 68Ge/68Ga generator eluate. The pH of the reaction solution was adjusted with HEPES and sodium hydroxide. In order to im-prove the SRA, the labelling reaction was conducted with varying amounts of Lf (0.01-6.25 nanomoles). Quantitative 68Ga-labelling of Lf was achieved using 1.25 nanomoles of the protein. The use of 0.01 nanomoles of Lf re-sulted in ~30% RAI. The 68Ga-Lf was stable in the reaction mixture at room temperature for at least 4 h. Further optimisation will be aimed at involving higher radioactivity amounts and getting the highest possible SRA. It might be accomplished by using preconcentrated/purified preparations of 68Ga.

42

3.7 Characterisation of the macromolecular conjugates and their 68Ga-labelled counterparts

3.7.1 Chemical characterisationChemical characterisation and analysis prior to the further application of a radiolabelled compound are necessary to ensure its identity, purity and amount. The analysis should be performed within short time to minimize the loss of radioactivity. For macromolecular bioconjugates, appropriate means of characterisation generally include HPLC and mass spectrometry (MS). The most commonly used method is the addition of the authentic reference substance to the tracer and coelution on an HPLC column connected in se-ries with a radioactivity and a UV detector. This method is convenient and can easily be performed for each synthesis. The HPLC analysis developed in this study is accomplished within 10 min allowing fast quality control (QC) of the peptide-based radiopharmaceutical prior to clinical application (Figure 22) (paper I, III, IV, VI, VII). The authentic reference substance was synthe-sized under the same conditions as its radioactive counterpart, but using a mixture of 68Ga and 69,71Ga cations. The aim of the use of a mixture of radio-active and stable gallium isotopes was twofold: 1. to create a reaction condi-tion identical to the labelling procedure; 2. to make it possible to follow the reaction. The identity of the compounds was confirmed by LC-ESI-MS. Theposition of the 68Ga-label was assessed by performing the labelling reaction with both conjugated and nonconjugated macromolecules.

The stability of the radiolabelled bioconjugates both in preparation and application buffers was usually monitored by radio-HPLC with analysis of aliquots taken from the labelling reaction mixture during 3-4 hours to control possible appearance of additional radio-HPLC signals. The samples incu-bated for 12-24 hours were analyzed by UV-HPLC or LC-ESI-MS. The ra-diochemical purity of the 68Ga-bioconjugates used in the applied studies was >95% for at least four hours. This time corresponds to 3-4 physical half-lives of 68Ga and is the time required for the applied experiments. In addition, the macromolecules, bioconjugates and the bioconjugate complexes with the stable gallium isotope were analyzed regarding stability by UV-HPLC or LC-ESI-MS.

To assess the reliability of the designed HPLC system, the quantity of 68Ga–labelled bioconjugate and radio-impurities retained on the column was determined by measuring the radioactivity of the sample injected on the col-umn and the fractions collected from the outlet with a crystal scintillation counter. The overall loss on the system was then estimated and depending on separation methods was 10-15%.

Purification of radiolabelled compounds is usually performed using semi-preparative liquid chromatography (LC) or solid phase extraction (SPE). The

43

purification step causes loss of radioactivity due to the physical decay as well as loss of the labeled compound on the purification systems. To speed up this step SPE cartridges are usually used. However, in case of the 68Ga-labelled macromolecules, the radioactivity loss on the cartridge was 20-50%, since the preferred strong organic solvents cannot be used. Another compli-cation is the product elution, since most of the organic eluents are not com-patible with biological systems. Therefore either evaporation of the product solvent or change of the solvent using SPE cartridges is needed. This addi-tional step decreases the radiochemical yield further. In the present study (paper I, IV, VII), the omission of the purification step was possible because of the developed method for quantitative RAI (Figure 22). Moreover, the labelling product was obtained in HEPES buffer which is compatible with biological systems and eligible for human use.

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It should be mentioned that the labelling efficiency is strongly dependent on the purity of a bioconjugate. Even trace amounts of a free bifunctional chela-tor decrease the RAI drastically. Thus, HPLC and/or filter ultracentrifugation purification is preferable in the bioconjugate preparation.

3.7.2 Preliminary chemical and biological examination of the 68Ga-labelled oligonucleotide conjugates

The impact of the modifications and labelling on the oligonucleotide probe performance was investigated conducting: i) specific hybridisation of 68Ga-labelled antisense oligonucleotide to a complementary 17-mer phosphodi-ester sense oligonucleotide in solution (paper II, V); ii) radioactivity organ distribution in rats (paper V).

i) The hybridisation products were analysed using polyacrylamide gel electrophoresis (PAGE), and the results were visualised by ethidium bro-mide staining and autoradiography. The results of the concentration depend-

Figure 22. HPLC-radiochromatogram of a 68Ga-DOTATOC preparation (0.5 nanomols of DOTATOC, 1 min microwave heating at 90±5 °C, total reaction vol-ume 220 µL). The 68Ga incor-poration was >99% and the SRA of the labelled peptide conjugate was 2 GBq/nmol.

44

ent hybridisation of the phosphorothioate counterpart are shown in Figure 23.

Figure 23. Concentration dependent hybridisation of 68Ga-labelled 17-mer antisense phosphorothioate oligonucleotide (20 pmol in 1 L) to the complementary 17-mer sense phosphodiester oligonucleotide in solution. The antisense:sense concentration ratios in the lanes are as follows: 1) 1:1/60, 2) 1:1/30, 3) 1:1/15, 4) 1:1/5, 5) 1:1/2, 6) 1:1, 7) 1:2, 8) 1:3, 9) 1:4 and the references are sense oligonucleotide (lane 10), 68Ga-labelled antisense oligonucleotide (lane 11) and molecular weight marker (lane 12). A) PAGE picture of the concentration dependent hybridisation study; B) Autoradiography of the polyacrylamide gel (A); C) The scaled up region of interest.

Nine samples with a constant concentration of the radiolabelled antisense oligonucleotide and a gradually increasing concentration of the sense oli-gonucleotide were analysed (Figure 23, lanes 1-9). As a reference the sense oligonucleotide (lane 10), the antisense oligonucleotide (lane 11) and mo-lecular weight marker (lane 12) samples were loaded onto the polyacryla-mide gel. The gradual increase of the intensity of the hybrid bands, the ab-sence of the free radioactive bands of antisense at the higher concentrations of the sense oligonucleotide and the absence of free sense bands at the lower concentration of the sense oligonucleotide serve as an indication of the con-centration dependent hybridisation. All four 17-mer oligonucleotide coun-terparts were able to hybridise to the complementary sense oligonucleotide.

ii) Since the studied oligonucleotides do not have any biological target in rats, their tissue distribution reflects their non-specific interactions and elimination. The measurement of the organ radioactivity at 20, 60 and 120 min time points after i.v. administration of labelled phosphodiester showed the highest values in the liver followed by the urinary bladder, bone marrow and spleen.

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Figure 24. The five organs of highest radioactivity uptake after injection (20, 60 and 120 min time points) of 68Ga-labelled phosphodiester (PD), phosphorothioate (PS) and 2'-O-methyl phosphodiester (OMe) oligonucleotides. Data are presented as mean ±SD (n=3-5).

Uptake in the kidney was predominant in the phosphorothioate and 2'-O-methyl phosphodiester distribution patterns. Bone marrow, kidney, liver, spleen and urinary bladder were among the five organs with the highest standardized uptake values (SUV) in each oligonucleotide distribution pat-tern (Figure 24). The distribution pattern in almost all tissues seemed to vary with the nature of the oligonucleotide backbone.

3.7.2.1 Analysis of 68Ga-labelled 17-mer oligonucleotide metabolites in rat plasma

The metabolite analysis was intended at detection of intact 68Ga-labelledantisense oligonucleotides in plasma (paper V). A method was developed employing complementary PAGE, UV-radio-HPLC and TLC techniques. The most robust information was obtained from PAGE results. The advan-tage of PAGE and TLC was that references such as 68Ga-labelled oligonu-cleotides, 68Ga-DOTA, 68GaCl3, original oligonucleotides and DOTA-oligonucleotides could be used next to the samples for unambiguous assign-ment. Furthermore, PAGE also allowed better separation and higher resolu-tion compared to size exclusion-UV-radio-HPLC (SE-UV-radio-HPLC). The data obtained from the PAGE were complemented and supported by TLC

46

and UV-radio-HPLC results, the former to identify low molecular weight metabolites, the latter to assign intactness and free ionic 68Ga. The combina-tion of the three analytical techniques allowed differentiation between high and low molecular weight radiometabolites. The degradation extent of the oligonucleotides was in the following order: PS<OMe<PD. Radiometabo-lites were observed already 20 min after injection (Figure 25). The intact PS and OMe were found at both the 20 and 60 min time points, whereas only a small amount of PD was found at the 20 min time point. These results corre-late well with the literature data on oligonucleotide stability.129,180-184

Figure 25. PAGE radiochromatograms of the injected 68Ga-labelled oligonucleotides and extracted plasma samples 20 and 60 minutes after injection of 68Ga-phosphodiester oligodeoxynucleotide, 68Ga-phosphorothioate oligodeoxynucleotide, 68Ga-2'-O-methyl phosphodiester oligoribonucleotide.

To summarize, the modifications of the oligonucleotides, such as the intro-duction of a hexylaminolinker either at the 3 - or 5 - end, the substitution of non-bridging oxygens by sulphur or introduction of an O-methyl group at the sugar 2 position, did not influence the conjugation or radiolabelling or the hybridisation capacity of the oligonucleotides (paper II, V). The 68Ga-oligonucleotide counterparts showed different radioactivity organ distribu-tion reflecting varying metabolism and non-specific binding in rats. In plasma metabolite studies, the intact PD, PS and OMe were detected with stability order PD<OMe<PS (paper V).

The oligonucleotide conjugation and labelling methods (paper II, V) were shown to be easily transferable from one laboratory to another and were implemented at the Turku PET centre, Finland.184

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47

3.7.3 Biological characterisation of the 68Ga-labelled peptide conjugates

The maintenance of the biological activity and the targeting properties of the synthesized tracers has been tested by: i) in vitro receptor binding experi-ments performed on frozen sections of tissues and cell lines expressing cor-responding receptors (paper III, IV); ii) ex vivo studies of biodistribution of the tracers in tumour-bearing mice (paper III); iii) in vivo localization of tracers in tumour-bearing mice (paper III).

3.7.3.1 Saturation binding of 68Ga-DOTATOC A pilot study (paper IV) on saturation binding of 68Ga-DOTATOC to Rhesus monkey brain targeting cortex was performed using a frozen section autora-diography method.

Figure 26. Saturation of 68Ga-DOTATOC binding to SSTR of Rhesus monkey thalamus (left) and cortex (right). The brain sections were incubated with different concentrations of 68Ga-DOTATOC (0.01-10 nM) for 30 min at room temperature in presence and absence of 1 M DOTATOC to get non-specific and total binding of 68Ga-DOTATOC, respectively.

Previously, saturation of the tracer binding could not be observed, most probably, because of low SRA of the tracer (5 MBq/nmol) and consequently, saturation of the receptors with non-labelled DOTATOC and insufficient image contrast between the background and receptor expressing tissues. The method described in paper I for 68Ga-labelling of peptide bioconjugates al-lowed for SRA of ~390 MBq/nmol and studies of the binding saturation became feasible (Figure 26). The dissociation constant values (Kd) corre-lated well with literature data for 67Ga-DOTA-[Tyr3]-octreotide saturation binding using homogenate of rat brain cortex membranes.67,113

3.7.3.2 Biological evaluation of 68Ga-DOTA-hEGF The epidermal growth factor (EGF) is a ligand binding to EGF receptors (EGFR) overexpressed in human malignancies.185-189 It is a peptide of 6.2

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kDa, consisting of 53 amino acid residues in a single polypeptide chain. The structure of the peptide is stable, mainly due to many -structures and three disulfide bonds that are also essential for the biological activity.190,191 Detec-tion of the overexpressed EGFR in clinical practice might influence patient management, including questions of relevance of the use of EGFR-targeted drugs.95,192 EGF can be used as a targeting vector to deliver radionuclides to EGFR expressing tumour cells. EGF has earlier been labelled with 111In and 131I.95,193-196

Figure 27. left) A PET image showing a summation of frames 20-24 (20-30 min after injection). The tumours can clearly be seen at both sides of the head. right) A photograph of the positioning of the mouse.

The biological activity maintenance of the 68Ga-DOTA-hEGF tracer (paper III) has been thoroughly studied performing cell binding assays, biodistribu-tion studies and microPET imaging in tumour-bearing mice. 68Ga-DOTA-hEGF retained its capacity to specifically bind to the EGFR expressing cell lines A431 and U343 (KD=2 nM). The affinity in the low nanomolar range was compatible with the application of 68Ga-DOTA-hEGF as a tracer for in vivo imaging. The EGFR expressing xenografts were visualized in mice (Figure 27).

49

3.8 68Ga-labelled peptides for adsorption studies Two peptides (Angiotensin II and VIP) of different lengths, constitution and pI values were chosen for studies of their adsorption in a micro-device con-sisting of channels moulded in a plastic compact disc (Figure 28) (paper VI). Angiotensin II represents a low molecular weight peptide, composed of 8 amino acids, with a pI value of 6.7. VIP, of 28 amino acids, constitutes a peptide of greater length and a pI value of 9.8.

The major factors that influence the adsorption of a peptide are its charge, hydrophobicity and solubility.197 The influence on the hydrophobicity of the peptides caused by the introduction of DOTA bifunctional chelator probably was insignificant, as observed by RP-HPLC analysis. The tracers were as-sumed to be representative of the native peptide bulk in terms of adsorption properties. 68Ga-labelled Angiotensin II and VIP were employed to develop a method for evaluation and optimisation of miniaturized analysis systems. Quantitative images of the tracer distribution within the microfluidic chan-nels were obtained using a PhosphorImager system. Variation of different parameters, such as ionic strength of the buffer used, peptide concentration, pH and surface modifications, showed a great change in adsorption, thus indicating that the developed method is a useful means for the study of pep-tide non-specific adsorption to microfluidic channels in a plastic compact disc. The generator produced 68Ga radionuclide is readily available and has a relatively low price. The production of peptide tracers is straightforward. Moreover, the tracers might be prepared with high SRA that may provide mass sensitive detection, which is very important when studying miniatur-ized chemical analysis systems. In addition, the developed method can be used in laboratories that are not specialized in radiotracer chemistry.

Peptide adsorption to microstructures such as a poly(dimethylsiloxane) (PDMS) material used in integrated analytical systems has also been stud-ied.198,199

Figure 28. Drawing of the mi-crofluidic channel system (left) and image of 68Ga-DOTA-Angiotensin II tracer distribu-tion (right).

50

4 CONCLUSIONS

A 68Ge/68Ga generator has been characterised and a method for preconcentration and purification of the 68Ge/68Ga generator eluate was developed. In combination with the microwave heating the method might make it possible to use a commercial 1850 MBq gen-erator over a period of more than two years for patient studies. The technique could be applied for the preconcentration of the eluates of at least two generators, thus allowing larger radioactivity amounts and prolonging the shelf life of a generator further. Microwave heating was found to be efficient to accelerate and im-prove the complexation reaction of 68Ga with bifunctional chelators, DOTA and NOTA, conjugated to the peptides and oligonucleotides. The introduction of a 68Ga purification and concentration step in combination with the microwave heating allowed quantitative incor-poration of 68Ga and omission of the purification of the resulting 68Ga-labelled peptide conjugates. Consequentely, the specific radio-activity of the radioactive products was considerably increased. The proposed method is suited for automation and a device for peptide tracer production is under evaluation, demonstrating quantitative in-corporation of 68Ga(III).Specific radioactivity was shown to be an important parameter influ-encing the feasibility of accurate imaging data quantification. The identity of the 68Ga-labelled bioconjugates was verified. The tracers were found to be stable and their biological activity main-tained.Quantitative direct 68Ga-labelling of Lactoferrin was revealed to be feasible.68Ga-labelled peptide imaging was shown to be a suitable tool for the investigation of peptide adsorption in analytical devices.

51

5 OUTLOOKS

The investigation of the measurement viability of Bmax in tumours invivo might require even higher SRA than has been achieved. Further improvement of the SRA requires thorough investigation of the pre-requisites for efficient labelling. The chemistry of Fe(III) and Ga(III) is very similar. Iron is an abun-dant cation and can be found in glassware, SPE cartridges and chemicals. The removal of Fe(III) may improve the SRA and also omit any uncertainty over its role in the 68Ga-labelling process. However, the purification method presented in the thesis does not eliminate Fe(III). The method might be further improved by, for ex-ample, introduction of Fe(III) reduction to Fe(II). A thorough inves-tigation and search for a suitable method for purification of the 68Ge/68Ga generator eluate from Fe(III) is the next step of our study on 68Ga-labelling of bioconjugates with high SRA. Another means to improve the SRA is to design a bifunctional chela-tor with high selectivity towards Ga(III). The achieved high SRA has made it possible to investigate the influ-ence of SRA on biodistribution. Further studies on the optimisation of SRA for a certain tracer, for example 68Ga-DOTA-hEGF, might be of practical interest. 68Ga-labelling with high SRA of highly potent larger peptides and biological characterisation of the resulting tracers might be of practi-cal interest as well.It is important to develop bifunctional chelators for the production of 68Ga-labelled bioconjugates of desired biodistribution characteris-tics, in particular, low kidney uptake. This might be achieved by modification of a chelator with substituents of different charges and lipophilicity. The evaluation of the tracers used in the adsorption imaging should be performed regarding the effect of the modifications introduced to the native peptides.

52

6 ACKNOWLEDGEMENTS

I would like to thank all people who have helped me and contributed to this thesis.

First of all I express my sincere gratitude to my supervisor Professor Bengt Långström for accepting me as a PhD student, for constant support, guid-ance, and enthusiasm, for providing excellent working facilities and condi-tions.

To my co-supervisor, Professor Mats Bergström, for elegant ideas, for being always ready to give thorough and educative answers to practical and theo-retical questions on any PET topic.

To Professor Gerd Beyer, Cyclotron Unit, Geneva University Hospital, Ge-neva, Switzerland, for constructive, instructive discussions and fruitful col-laboration on the studies of preconcentration and purification of 68Ga eluate. For constant encouragement and support that kept me going on.

Gabor Lendvai for enjoyable and productive collaboration on the oligonu-cleotide projects, for your patience and gentleness. Dr. Sergio Estrada and Daniel Laryea for biological studies. Maria Välilä, Satu Salomäki and Dr. Anne Roivainen, Turku PET centre, Finland, for provision of OMe and be-ing such a good learners.

Assoc. Professor Vladimir Tolmachev and Dr. Åsa Liljegren Sundberg for the 68Ga-DOTA-hEGF project, excellent biological experimental work and exciting, encouraging collaboration. Eva Werner, Olof Eriksson, Dr. Urban Höglund and Dr. Örjan Lindhe for the PET experiments and for long work-ing hours.

Elisabeth Bergström-Pettermann for conducting excellent frozen section autoradiography experiments. Professor Gerd Beyer for continuous collabo-ration and so much needed feedback. Dr. Pernilla Frändberg for the constant help and advices with the mass-spectrometry.

53

Dr. Martin Lavén for the constant help with the mass-spectrometry, reward-ing, enjoyable, and fruitful collaboration on the peptide adsorption studies. I always remember your comment that I work like crazy. I feel so much ap-preciated. Professor Karin Markides and Dr. Susanne Wallenborg for con-structive disscutions and invaluable support when it was really needed. Jenny Ljung, Oskar Berglund and Majda Djodjic for your important experi-mental contribution.

Dr. Joachim Feldwisch, Dr. Lars Abrahmsén, Dr. Anders Wennborg and Dr. Anna Orlova (Affibody AB, Bromma, Sweden) for the Affibody project, kind permission to publish the experimental data and for constructive com-ments on the appendix.

Professor Paul H Walton, James Spearman and Dr. Phil Palmer (University of York, Heslington) are acknowledged for the kind permission to publish the experimental data on the Lactoferrin project.

Many thanks to Dr. Johanna Höglund, Dr. Martin Lavén, Assoc. Professor Tor Kihlberg and Assoc. Professor Vladimir Tolmachev for thorough dis-cussions on the thesis and constructive, instructive comments. Kristina Lundqvist and Dr. Urban Höglund for the linguistic review.

Everybody at the preclinical laboratory for creating a friendly and stimulat-ing atmosphere. In particular, Elisabeth Bergström-Pettermann for constant support and provision with laboratory stuff. Pascha Razifar, Stina Syvänen, Kayo Takahashi, Dr. Sergio Estrada, Gabor Lendvai, Azita Monazzam, Olof Eriksson, Gudrun Nylén for constant help, support and comforting atmos-phere.

Past and present members of the group BLå. In particular, Dr. Ulrika Yngve for the first introduction to the lab equipment and 68Ga-labelling in spite of being stressed out by writing your thesis. Dr. Koichi Kato, Dr. Hisashi Doi and Dr. Bert Windhorst for educative chemistry talks and instructive remarks on the “oligo” manuscript. Jonas Eriksson, Olexiy Itsenko, Julien Barletta and Dr. Obaidur Rahman for always being ready to help throughout these years. Linda Samuelsson for encouraging me to speak Swedish.

Everybody at Uppsala Imanet AB and the Department of Organic Chemistry for the support, help and for making me feel always welcome. In particular, Assoc. Professor Gunnar Antoni, Eva-Lotta Vesström, Assoc. Professor Alf Thibblin, Madeleine Svennebäck, Margareta Sprycha and Sven-Åke Gus-tavsson for the help, support and encouragement. Mimmi Lidholm for the collaboration, for an always positive and optimistic attitude and sharing the passion for Flamenco art. Dr. Yvonne Andersson, Göran Bejer and Ylva

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Ponsiluoma for your support and appreciation. Jonas Eriksson, Dr. Johan Ulin, Tommy Ferm, Daniel Johansen and Helena Wilking for the work on the “Gallea” automated system. Dr. Johanna Höglund for constructive and instructive discussions, constant help and encouragement. Eva Pylvänen, Paula Delking, Maggie Hynd, Joachim Schultz, Tomas Nyberg, Anders Nilsson, Peter Hjelm, Viktor Leander and Tommy Ferm for invaluable sec-retarial and technical assistance. Professor Lars Engman for concern and support. Eva Werner, Martin Lavén, Helena Wilking, Stina Syvänen and Susanna McMillar for regular help correcting my Swedish essays. Anna Norgren and Susanna McMillar for connecting me to the department. Joachim Schultz, Mimmi Lidholm, Lars Lindsjö, Anders Wall, Johan Mäl-man, Daniel Johansen, Johanna Höglund, Peter Hjelm, Viktor Leander, Jonas Eriksson, Tomas Nyberg, Andreas Wallberg for your enthusiasm and consent to help and participate in the disputation party entertainment pro-gram.

Group 2 pedagoger, Daniel, Eva, Hans-Christian, Henrik, Karin E., Karin R., Niklas, Veronica for the fun, jokes, chat and great time we have had, and, sure, for your professional comments on my popular scientific writing.

Dr. Hege Karlsen, Amersham Health, Olso, Norway for the provision of DOTA-RGD.

European Union COST Action B12 is acknowledged for its support. The Swedish Research Council is acknowledged for its support by grant K3464.

My old faithful friends and relatives who have always been close, suppor-tive, encouraging and understanding in spite of distance and time. I wish I had more time to communicate. My Armenian, Assyrian, Danish, English, Finnish, Hungarian, Indian, Russian, Sudanese, Swedish and Ukrainian friends for the help and fun outside the PET world during these years. Your diverse backgrounds and histories have made me rich. The common thing you all have is a big kind heart. I am a lucky person to have you in my life.

My beloved husband Misha and my beloved daughter Sona, without your love, endless patience, understanding and constant support it would not have been possible to accomplish this thesis.

Uppsala, August 2005

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7 SUMMARY IN SWEDISH

Unveckling av substanser för PET-undersökning av tumörer

En PET-undersökning kan synliggöra tumörer vilka är mycket svåra eller omöjliga att upptäcka med någon annan metod. PET-undersökningar görs med hjälp av radioaktiva kemiska substanser, så kallade spårmolekyler. I avhandlingen visas att stora biologiska molekyler tillsammans med radioaktivt gallium-68 kan användas för effektiv och enkel framställning av sådana spårmolekyler.

PET står för Positron Emissions Tomografi. Vid en PET-undersökning kan man upptäcka mycket små tumörer redan innan sjukdomen har utvecklats, vilket innebär att behandling kan påbörjas tidigt. För att göra en PET-undersökning används en PET-kamera. Det speciella med PET-kameran är att den är konstruerad så att den ”ser” radioaktiva spårmolekyler och ger värdefull information om hur de tas upp och fördelas i kroppen. Med PET-metoden kan man alltså undersöka kroppens funktioner med hjälp av olika så kallade spårmolekyler som injiceras i en patient och som kan hitta skadliga förändringar i kroppen. På så sätt kan en diagnos ställas på till exempel hjärnskador, tumörsjukdomar, demenssjukdomar och depressioner. En spår-molekyl kan vara ett kroppseget ämne, t ex glukos (druvsocker), aminosyror eller t o m vanligt vatten som innehåller en liten mängd radioaktivitet, vilket är nödvändigt för att PET-kameran skall kunna se spårmolekylernas fördel-ning och omsättning i kroppen. Spårmolekylerna som används tillverkas i specialbyggda kemilaboratorier.

Utveckling av spårmolekyler I detta arbete utvecklades metoder att producera spårmolekyler som kan användas för att diagnostisera t.ex. tumörer. Stora biologiska molekyler

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(makromolekyler), bl a peptider, proteiner och antikroppar användes, efter-som de spelar en viktig roll i många sjukdomsförlopp. Oligonukleotider, syntetiska bitar av DNA, är andra intressanta ämnen som bedöms vara an-vändbara för diagnostik av cancer och virussjukdomar. Eftersom tumörer har ett högre upptag av vissa spårmolekyler jämfört med övrig vävnad kan de lokaliseras genom att en väldesignad radioaktiv spårmolekyl injiceras i en människa eller ett djur. Vi har utvecklat metoder för att märka peptider, oli-gonukleotider och olika andra makromolekyler med den relativt kortlivade radioaktiva isotopen gallium-68. Först binds makromolekylen till en mindre molekyl, en s. k. kelator. Därefter binds gallium-68 hårt till kelatorn. Den radioaktivt märkta slutprodukten renas och karakteriseras kemiskt. Sedan undersöks molekylens biologiska egenskaper i samarbete med ett prekliniskt laboratorium för att se om de utvecklade spårmolekylerna uppför sig som den ursprungliga substansen.

Vad händer med spårmolekylerna i kroppen? Spårmolekyler binder till vissa ställen i kroppen, till exempel tumörer, bero-ende på sin funktion. Medan spårmolekylerna sitter fast på en tumör strålar gallium-68 ut så kallade positroner. En positron attraherar en elektron i ma-teria och omvandlas till två fotoner, som sänds ut i två rakt motsatta rikt-ningar. Detektorer i PET-kamera kan registrera fotonerna. Denna informa-tion överförs till en bild som visar var i kroppen tumören sitter. En peptid som är en analog till somatostatin har till exempel märkts med gallium-68 och använts för PET-undersökning av en tumör. Sammanfattningvis, har Irina Velikyan visat att gallium-68 är användbart för att framställa radioak-tivt märkta makromolekyler till PET-undersökningar.

Analytisk applikation I avhandlingen visas också att peptider märkta med gallium-68 kan använ-das till att spåra molekyler i miniatyriserade ledningssystem. Detta kan fun-gera som ett värdefullt verktyg för att undersöka och utveckla peptid- och proteinanalyssystem i mikroskala för analys av små provmängder.

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