Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Pharmacy 191
Bombesin Antagonists for
Targeting Gastrin-Releasing Peptide
Receptor-Positive Tumors
Design, Synthesis, Preclinical Evaluation and
Optimization of Imaging Agents
ZOHREH VARASTEH
ACTA
UNIVERSITATIS
UPSALIENSIS
UPPSALA
2014
ISSN 1651-6192
ISBN 978-91-554-9039-3
urn:nbn:se:uu:diva-232123
Dissertation presented at Uppsala University to be publicly examined in Rudbecksalen, Dag
Hammarskjölds väg 20, Uppsala, Friday, 31 October 2014 at 09:00 for the degree of Doctor
of Philosophy (Faculty of Pharmacy). The examination will be conducted in English. Faculty
examiner: Prof. Dr. Roger Schibli (Institute of Pharmaceutical Sciences - ETHZ).
Abstract
Varasteh, Z. 2014. Bombesin Antagonists for Targeting Gastrin-Releasing Peptide ReceptorPositive Tumors. Design, Synthesis, Preclinical Evaluation and Optimization of Imaging
Agents . Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of
Pharmacy 191. 66 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9039-3.
This thesis is focused on the development, preclinical evaluation, and optimization of
radiotracers for the detection of gastrin-releasing peptide receptor (GRPR)-expressing tumors.
The work is divided into three distinct parts: (1) the development of bombesin (BN)
antagonist (RM26)-based imaging radiotracers for the detection of GRPR-expressing tumors
using different positron emission tomography (PET) and single photon emission computed
tomography (SPECT) radionuclides (68Ga, 18F and 111In), (2) the establishment of a method to
monitor the ligand-G protein-coupled receptor (GPCR) interaction in real time without requiring
purification and stabilization of the receptors, and (3) the evaluation of radiopeptide structurerelated factors (length of mini-PEG linker and composition of chelator for metal labeling)
affecting the in vitro and in vivo characteristics of RM26-based tracers.
We demonstrated the possibility of high-contrast in vivo imaging of GRPR-expressing
xenografts despite the physiological expression of GRPR in abdominal organs. Fast
radioactivity clearance from the blood and healthy organs, including receptor-positive organs,
and long retention in the tumors resulted in high tumor-to-background ratios. A novel real-time
assay for measuring the kinetics of the radiotracers targeting GPCR was evaluated. Living cells
were used instead of purified receptors in this technology, bringing the developmental work
one step closer to the true target environment (imaging in living systems). The comparative
study of 68Ga-labeled NOTA-PEGn-RM26 with di-, tri-, tetra- and hexaethylene glycol chains
demonstrated that the addition of only a few units of ethylene glycol to the spacer is insufficient
to appreciably affect the biodistribution of the radiopeptide. Finally, a comparative study
of 68Ga-labeled PEG2-RM26 analogs N-terminally conjugated to NOTA, NODAGA, DOTA
or DOTAGA highlighted the influence of the chelator on the targeting properties of the
radiopeptide.
The main conclusion that can be drawn from this thesis is that 68Ga-NOTA-PEG2-RM26
has favorable biodistribution properties, such as rapid clearance from blood and tissues with
physiological GRPR expression levels and long retention in GRPR-expressing tumors, and that
this radiopeptide is potentially suitable for initial clinical investigation.
Keywords: Bombesin, Gastrin-releasing peptide receptor (GRPR), Antagonist, Radionuclide
molecular imaging
Zohreh Varasteh, Department of Medicinal Chemistry, Preclinical PET Platform, Dag
Hammarskjöldsv 14C, Uppsala University, SE-751 83 Uppsala, Sweden.
© Zohreh Varasteh 2014
ISSN 1651-6192
ISBN 978-91-554-9039-3
urn:nbn:se:uu:diva-232123 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-232123)
To my beloved parents
and my soulmate
for supporting me all the way!
List of Papers
This thesis is based on the following papers, which are referred to in the
text by their Roman numerals.
I
II
III
IV
V
Varasteh, Z., Velikyan, I., Lindeberg, G., Sörensen, J., Larhed, M., Sandström, M., Selvaraju, RK., Malmberg, J.,
Tolmachev, V., Orlova, A. (2013) Synthesis and characterization of a high affinity NOTA-conjugated bombesin antagonist for GRPR-targeted tumor imaging. Bioconjug
Chem, 24(7):1144–1153
Varasteh, Z.*, Åberg, O.*, Velikyan, I., Lindeberg, G.,
Sörensen, J., Larhed, M., Antoni, G., Sandström, M., Tolmachev, V., Orlova, A. (2013) In vitro and in vivo evaluation of a 18F-labeled high affinity NOTA conjugated
bombesin antagonist as a PET ligand for GRPR-targeted
tumor imaging: A murine model. PLOS ONE,
8(12):e81932
Xu, B.*, Varasteh, Z.*, Orlova, A., Andersson, K., Larhammar, D., Björkelund, H. (2013) Detecting interactions
with GPCR in real-time on living cells to understand receptor dynamics. Biochem Biophys Res Comm, 441(4):820–4.
Varasteh, Z., Rosenström, U., Velikyan, I., Mitran, B., Altai, M., Honarvar, H., Rosestedt, M., Lindeberg, G., Sörensen, J., Larhed, M., Tolmachev, V., Orlova, A. (2014) The
Effect of Mini-PEG-Based Spacer Length on Binding and
Pharmacokinetic Properties of a 68Ga-Labeled NOTAConjugated Antagonistic Analog of Bombesin. Molecules.
19(7):10455–72.
Varasteh, Z., Mitran, B., Rosenström, U., Velikyan, I.,
Rosestedt, M., Lindeberg, G., Sörensen, J., Larhed, M.,
Tolmachev, V., Orlova, A. The effect of macrocyclic chelators on targeting properties of 68Ga-labeled gastrinreleasing peptide receptor antagonist RM26. Manuscript.
Reprints were made with permission from the respective publishers.
List of papers not included in this thesis
I.
Altai, M*., Varasteh, Z*., Andersson, K., Eek, A., Boerman, O., Orlova, A. (2013) In vivo and in vitro studies
on renal uptake of radiolabeled affibody molecules for
imaging of HER2 expression in tumors. Cancer Biother
Radiopharm, 28(3):187-195.
II.
Tolmachev, V., Varasteh, Z., Honarvar, H., Hosseinimehr, SJ., Eriksson, O., Jonasson, P., Frejd, FY.,
Abrahmsen, L., Orlova, A. (2014) Imaging of plateletderived growth factor receptor β expression in glioblastoma xenografts using affibody molecule 111In-DOTAZ09591. J Nucl Med, 55(2):294-300.
III.
Malmberg, J., Perols, A., Varasteh, Z., Altai, M., Braun,
A., Sandström, M., Garske, U., Tolmachev, V., Orlova,
A., Karlström, AE. (2012) Comparative evaluation of
synthetic anti-HER2 Affibody molecules site-specifically
labelled with 111In using N-terminal DOTA, NOTA and
NODAGA chelators in mice bearing prostate cancer
xenografts. Eur J Nucl Med Mol Imaging, 39(3):481-492.
IV.
Orlova, A., Malm, M., Rosestedt, M., Varasteh, Z., Andersson, K., Selvaraju, RK., Altai, M., Honarvar, H.,
Strand, J., Ståhl, S., Tolmachev, V., Löfblom, J. (2014)
Imaging of HER3-expressing xenografts in mice using a
(99m)Tc(CO) 3-HEHEHE-Z HER3:08699 affibody molecule. Eur J Nucl Med Mol Imaging, 41(7):1450-1459.
V.
Orlova, A., Hofström, C., Strand, J., Varasteh, Z., Sandstrom, M., Andersson, K., Tolmachev, V., Gräslund, T.
(2013) [99mTc(CO)3]+-(HE)3-ZIGF1R:4551, a new Affibody conjugate for visualization of insulin-like growth
factor-1 receptor expression in malignant tumours. Eur J
Nucl Med Mol Imaging, 40(3):439-449.
VI.
Rosik, D., Orlova, A., Malmberg, J., Altai, M., Varasteh, Z., Sandström, M., Karlström, AE., Tolmachev,
V. (2012) Direct comparison of 111In-labelled two-helix
and three-helix Affibody molecules for in vivo molecular
imaging. Eur J Nucl Med Mol Imaging, 39(4):693-702.
VII.
Malm, M., Kronqvist, N., Lindberg, H., Gudmundsdotter, L., Bass, T., Frejd, FY., HöidénGuthenberg, I., Varasteh, Z., Orlova, A., Tolmachev, V.,
Ståhl, S., Löfblom, J. (2013) Inhibiting HER3-mediated
tumor cell growth with affibody molecules engineered to
low picomolar affinity by position-directed error-prone
PCR-like diversification. PLoS One, 8(5):e62791.
VIII.
Altai, M., Wållbreg, H., Honarvar, H., Strand, J., Orlova,
A., Varasteh, Z., Sandsröm, M., Löfblom, J., Larsson,
E., Strand, SE., Lubberink, M., Ståhl, S., Tolmachev, V.
188
Re-ZHER2:V2, a promising affibody-based targeting
agent against HER2-expressing tumors: preclinical assessment. J Nucl Med. [In press]
IX.
Strand, J., Varasteh, Z., Eriksson, O., Abrahmsen, L.,
Orlova, A., Tolmachev, V. (2014) Gallium-68-Labeled
Affibody Molecule for PET Imaging of PDGFRβ Expression in Vivo. Mol Pharm. [Epub ahead of print]
X.
Andersson, G.K., Rosestedt, M., Varasteh, Z., Malm1,
M., Sandström, M., Tolmachev, V., Löfblom, J., Ståhl1,
S., Orlova, A. Comparative Evaluation of 111In-Labeled
NOTA-Conjugated Affibody Molecules for Visualization
of HER3 Expression in Malignant Tumors. Conditionally
accepted to J Nucl Med.
XI.
Altai, M., Honarvar, H., Wållbreg, H., Strand, J., Varasteh, Z., Rosestedt, M., Orlova, A., Dunås, F.,
Sandsröm, M., Löfblom, J., Tolmachev, V., Ståhl, S. Selection of an optimal cysteine-containing peptide-based
chelator for labeling of affibody molecules with 188Re.
Eur J Med Chemistry. [In press]
XII.
Varasteh, Z., Orlova, A. Comparing the measured affinity of 111In-labeled ligands for cellular receptors by
monitoring gamma beta or x-ray radiation with three different LigandTracer® devices: A technical note on affinity measurement, submitted to PLOS ONE.
*Equal contribution to this work
Contents
Introduction ............................................................................................. 13
Prostate cancer .................................................................................... 13
Screening of prostate cancer: Potential and limitations ...................... 13
Staging of prostate cancer: Potential and limitations.......................... 14
Treatment of prostate cancer .............................................................. 16
Single photon emission computed tomography (SPECT) and
positron emission tomography (PET) ................................................. 16
Peptide ligand/receptor systems used for tumor imaging and
treatment ............................................................................................. 18
Bombesin, bombesin-like peptide ligands and their corresponding
receptors.............................................................................................. 19
BN receptor agonists vs. antagonists .................................................. 20
Molecular basis for selectivity of RM26 for GRPR ........................... 22
The influence of the structure of radiolabeled peptides on targeting
properties and biodistribution ............................................................. 23
The influence of the spacer between the receptor binding sequence
and the chelating moiety on the biological characteristics of BN
analogs ................................................................................................ 24
The influence of the chelator on the biological characteristics of
peptide-based targeting agents ............................................................ 25
Detecting ligand interactions with G protein-coupled receptors on
living cells in real time to measure the binding affinity ..................... 27
Bombesin analogs in the clinic ........................................................... 29
Aims of this thesis ................................................................................... 30
The present investigation......................................................................... 31
Development of imaging radiotracers based on the BN antagonist
RM26 for the detection of GRPR-expressing tumors
(Papers I and II) .................................................................................. 31
Paper I. Synthesis and characterization of a high-affinity
NOTA-conjugated bombesin antagonist for GRPR-targeted tumor
imaging. .............................................................................................. 31
9
Rationale and aims ........................................................................ 31
Methods ......................................................................................... 32
Results and discussion .................................................................. 32
Conclusions ................................................................................... 34
Paper II. In vitro and in vivo evaluation of a 18F-labeled high
affinity NOTA-conjugated bombesin antagonist as a PET ligand
for GRPR-targeted tumor imaging. .................................................... 38
Rationale and aims ........................................................................ 38
Methods ......................................................................................... 39
Results and discussion .................................................................. 39
Conclusions ................................................................................... 41
Paper III. Detecting ligand interactions with G protein-coupled
receptors on living cells in real time. .................................................. 43
Rationale and aims ........................................................................ 43
Methods ......................................................................................... 43
Results and discussion .................................................................. 43
Conclusions ................................................................................... 45
Evaluation of tracer structure-related factors affecting binding
specificity and sensitivity of RM26 (Paper IV and V) ....................... 46
Paper IV. The effect of mini-PEG-based spacer length on binding
and pharmacokinetic properties of a 68Ga-labeled NOTAconjugated antagonistic analog of bombesin. ..................................... 46
Rationale and aims ........................................................................ 46
Methods ......................................................................................... 46
Results and discussion .................................................................. 47
Conclusions ................................................................................... 48
Paper V. The effect of macrocyclic chelators on targeting
properties of 68Ga-labeled gastrin-releasing peptide receptor
antagonist RM26................................................................................. 50
Rationale and aim ......................................................................... 50
Methods ......................................................................................... 50
Results and discussion .................................................................. 50
Conclusions ................................................................................... 54
Conclusions and perspectives .................................................................. 55
Acknowledgements ................................................................................. 58
References ............................................................................................... 61
10
Abbreviations
ADT
AR
BN
BPH
DOTA
DOTAGA
Androgen deprivation therapy
Androgen receptor
Bombesin
Benign prostatic hyperplasia
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid
1,4,7,10-tetraazacyclododecane-1-glutaric acid-4,7,10triacetic acid
EC
Extracellular
EGFR
Epidermal growth factor receptor
EPR
Enhanced permeability and retention
FDG
Fluorodeoxyglucose
FDHT
Fluorodihydrotestosterone
GRPR
Gastrin releasing peptide receptor
GPCR
G protein-coupled receptor
HRPC
Hormone-refractory prostate cancer
MET
Methionine
MDP
Methylene diphosphonate
NMBR
Neuromedins B receptor
NOTA
1,4,7-triazacyclononane-1,4,7-triacetic acid
NODAGA 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid
PC
Prostate cancer
PSA
Prostate specific antigen
PET
Positron emission tomography
PEG
Polyethylene glycol
RAID
Radioimmunodetection
SPR
Surface plasmon resonance
SPECT
Single photon emission computed tomography
TRUS
Transrectal ultrasonography
11
Introduction
Prostate cancer
Prostate cancer (PC) is the most frequently diagnosed non-cutaneous
cancer and the second leading cause of cancer-related mortality in men
after lung and bronchus cancers [1]. Although PC can grow and spread
rapidly and develop into fatal and painful disease, in most cases, it grows
slowly, and symptoms do not affect men during their lifetime. PC has a
heterogeneous nature, and the characteristics of the disease diverge during its progression. The progression of PC leads to a transition from tumors with androgen-responsive growth toward hormone-refractory prostate cancer (HRPC).
Screening of prostate cancer: Potential and limitations
PC can be indicated by the prostate-specific antigen (PSA) blood test,
which measures the level of this well-characterized prostate-specific glycoprotein. Digital rectal examination (DRE), the physical examination of
the patient’s prostate from the rectum to assess prostate size abnormalities, is the other standard screening test. In cases with an elevated PSA
level and an enlarged prostate gland, the screening procedure will be
followed by the histopathological examination of multiple transrectal
ultrasonography (TRUS)-guided biopsy samples to confirm PC. However, none of the above-mentioned screening modalities are sufficiently
reliable to rule out the presence of cancer. An enlarged prostate and an
elevated PSA level can result from factors other than PC, e.g., prostatitis
(prostatic inflammation) and benign prostatic hyperplasia (BPH) (enlargement of the prostate) [2, 3]. In addition, prostate growth is agedependent [4], and the serum PSA level is related to prostate size [5].
Therefore, an increase in the PSA level due to prostate enlargement is
common in advanced age and can lead to overdiagnosis. Due to the heterogeneous expression of genetic markers associated with PC even in the
primary tumor, not all the biopsy samples are representative, which limits
the utility of biopsy data [6].
13
Staging of prostate cancer: Potential and limitations
After confirming the presence of cancer, it is necessary to determine the
extent of the disease. Staging, the detection of bone and soft tissue involvement, is essential for guiding optimal therapy for cancer patients.
The stage of disease describes how far the cancer has spread. The staging
of PC may also consider biopsy results (Gleason score), the PSA level, or
certain imaging tests. Among imaging modalities, nuclear medicine procedures are widely used.
Bone is one of the strongly preferred sites for spreading of the PC.
Bone scintigraphy with [99mTc]methylene diphosphonate ([99mTc]MDP)
using a gamma camera is the initial, sensitive, and cost- and timeefficient imaging modality for the identification of altered skeletal metabolism and metastatic spread to the skeletal system [7]. Bone scanning
with [18F]fluoride using PET is the other imaging test used for focal and
generalized bone diseases [8].
Likewise, most of the tracers used in nuclear medicine for imaging
soft
tissue
metastases
visualize
tumor
cell
metabolism.
[18F]Fluorodeoxyglucose (FDG)-PET, a metabolic imaging technique,
has shown great success for the detection of many types of hypermetabolic cancer. However, this method very poorly visualizes tumors with low
metabolism or low FDG-avidity. Because PC in most cases is a slowgrowing disease, it has a low glycolytic rate that results in low FDG uptake [9].
[11C]Acetate, another metabolic radiopharmaceutical, has been evaluated clinically for PC imaging. [11C]Acetate accumulation in tumor cells
is the consequence of enhanced cell membrane and thus lipid (fatty acid)
synthesis due to cell growth [10]. However, this tracer has shown low
specificity for prostate tumors. Data showing high [11C]acetate uptake in
normal prostate and hyperplastic tissues have been reported [11, 12].
[11C]/[18F]Choline uptake in PC tumors has been extensively studied.
The cellular uptake of [11C]/[18F]choline is primarily due to increased
phospholipid synthesis [13] and the upregulation of choline kinase induced by malignancy [9]. Depending on the lesion localization, the sensitivity of choline-based tracers in the detection of PC is as low as 4565% [14].
[11C]Methionine (MET) accumulation in tumor cells is associated with
increased amino acid active transport and metabolism, which corresponds
with the number of tumor cells [15, 16]. [11C]MET-PET has shown higher sensitivity in PC imaging by identifying significantly more lesions than
[18F]FDG-PET [17]. However, [11C]MET demonstrated low specificity
14
for the tumor cells. Methionine uptake increases by increasing the density
of inflammatory cells [18].
To overcome these limitations regarding tumor metabolic imaging
agents, a new approach based on receptor targeting was developed. This
technique provides information concerning the extent and behavior of
tumors by directing radiolabeled peptides and proteins to the pathological
or modified antigens and over-expressed target receptors. Receptormediated tumor targeting concept showed higher specificity to the site of
action in PC. Following this concept, [18F]fluorodihydrotestosterone
(FDHT)-based imaging was evaluated in prostatic carcinoma with increased androgen receptor (AR) expression. This imaging method was
used to assess the receptor status and to quantify changes in the receptor
density. However, AR mutations that occur during PC progression may
affect the sensitivity of the ligand [19].
Radiolabeled monoclonal antibodies were considered initially as target-specific imaging agents. The 111In-labeled monoclonal antibody capromab pendetide (ProstaScint) is an FDA-approved imaging radiopharmaceutical that has been introduced for staging PC. This antibody binds
to prostate specific membrane antigen (PSMA), which is overexpressed
in PC. Capromab pendetide is directed against the cytoplasmic domain of
PSMA and is thought to bind primarily to the necrotic cells in prostate
tumors. As is typical for other intact antibody-based tracers, the sensitivity and specificity of [111In]capromab pendetide is far from optimal [20,
21, 22]. The large size of the antibodies does not allow rapid kinetics, and
the slow clearance of unbound molecules from blood and non-target
compartments results in enhanced background signals and reduced targetto-background ratios. In addition, the enhanced permeability and retention (EPR) effect, which results in the nonspecific tumor uptake and retention of macromolecules (MW >40 kDa) due to defective vascular architecture in tumors, makes it difficult to evaluate the specific uptake of
antibody-based radiotracers [23].
To overcome the limitations of radioimmunodetection (RAID) of tumors, a number of more efficient approaches have been investigated. The
targeting of overexpressed receptors in tumors using radiolabeled peptide
ligands for non-invasive cancer imaging has received an appreciable attention.
15
Treatment of prostate cancer
After PC diagnosis and staging, a treatment option, which differs for each
individual, will be chosen. Selecting the treatment for PC patients depends heavily on the stage of the cancer. Radical prostatectomy (the surgical removal of the prostate gland and the seminal vesicles) and/or external radiation therapy are treatment modalities for early diagnosed locally confined prostate tumors, whereas androgen deprivation therapy
(ADT) is the first-line treatment for the palliation of symptomatic latestage PC that has already metastasized [24, 25]. Testosterone suppression
can be achieved surgically (removal of testicles to suppress testosterone
productin) or chemically (treatment with female sex hormones).
Single photon emission computed tomography
(SPECT) and positron emission tomography (PET)
Single photon emission computed tomography (SPECT) and positron
emission tomography (PET) are two major modalities used in nuclear
medicine for imaging. SPECT uses radioactive tracers labeled with gamma-emitting radionuclides and a gamma camera to obtain a projection
image of the radioactivity distribution. The projections can be reconstructed into three-dimensional images by a computer (Figure 1A). Similar to SPECT, a PET scan creates computerized in vivo image of distribution of the tracers labeled with positron-emitting radionuclides. The detectors detect pairs of annihilation photons, which are produced when the
emitted positrons interact with nearby electrons (Figure 1B). Due to the
coincidence detection in the PET camera, it is the most sensitive method
for the quantitative measurement of physiological processes in vivo. In
other words, PET provides better spatial resolution, better sensitivity, and
more accurate quantification of the radioactivity concentration in vivo.
For this reason, PET imaging typically provides better sensitivity and
improved diagnostic specificity.
The most widely used short-lived positron-emitting nuclides, 15O, 13N,
11
C and 18F, are produced by a cyclotron. Thus, the PET scanner must be
located near a cyclotron and a radiopharmaceutical laboratory that synthesizes tracers, which substantially increases the cost of PET diagnostics.
In contrast to cyclotron-produced positron emitters, 68Ga is a generator-produced radionuclide that can be eluted from a 68Ge/68Ga generator
on-site and does not require a cyclotron in the vicinity of the PET facility.
In addition, the cyclotron-PET satellite concept enables a single cyclotron
16
production facility to regularly supply 18F to several clinical PET centers
and significantly increase the number of PET scans. The list of the most
commonly used radionuclides for imaging is presented in Table 1.
Figure 1. A schematic overview of (A) gamma camera and (B) PET camera
acquisition of images.
Table 1. A tabular overview of the most commonly used radionuclides for imaging [26, 27, 28].
Radionuclide
Usage
Half-life
11
C
PET
13
N
15
maximum
positron
energy (MeV)
Mean range
of particle in
tissue (mm)
20.3 min
0.97
1.1
PET
10 min
1.2
1.5
O
PET
2 min
1.7
2.5
18
F
PET
109.7 min
0.63
0.6
68
Ga
PET
68 min
1.9
2.9
89
Zr
PET
3.2 d
0.89
1
PET
4.2 d
2.1
3.4
Tc
SPECT
6.0 h
0.141
-
-
111
In
SPECT
2.8 d
0.172 and 0.245
-
-
123
I
SPECT
13.2 h
0.159
-
-
131
I
SPECT
8d
0.364
-
-
124
I
99m
Gamma-ray
energy (MeV)
17
Peptide ligand/receptor systems used for tumor
imaging and treatment
Molecular imaging is a technique that enables the visualization of cellular
and molecular disease mechanisms and normal biological processes. It is
a rapidly growing discipline aiming toward developing new probes for
imaging specific molecular pathways involved in disease processes and
utilizing them for improved diagnostics.
The observation that some peptide receptors are overexpressed in certain tumors compared to endogenous levels in normal organs has encouraged the use of these receptors as targets and the design of peptide-based
radiotracers for targeted molecular imaging. Peptides are molecules consisting of 3 to 100 amino acid residues with a molecular mass of less than
10 kDa. Peptides are designed by nature for inhibiting, stimulating, and
regulating important fundamental biological processes through their corresponding receptors, which are located on the cell membrane. Due to
their small size, peptides usually have rapid extravasation, tissue penetration, and clearance from blood and non-target tissues. These attributes
result in high tumor-to-organ ratios—the primary requirement for diagnostic and therapeutic agents. Furthermore, the synthesis, modification,
and stabilization of small peptides to obtain optimized pharmacokinetic
parameters can be cheaper and faster than the modification of bulky antibodies. High binding affinity and low immunogenicity are other considerable advantages of the peptides.
There has been an exponential growth in the development of radiolabeled peptides providing in vivo histopathological information for diagnostic and therapeutic purposes [29]. Among these tailored radiopharmacuticals, some G protein-coupled receptor (GPCR) radioligands have
already made or are likely to make a contribution to the diagnosis and
therapy of oncological disorders. Indium-111-labeled octreotide (111InOctreoScan) became the first FDA-approved peptide-based tracer. It is
used primarily for the visualization of neuroendocrine tumors but also for
many other tumors [30]. In addition, lutetium-177-labeled octreotate has
been clinically used for many years to treat patients with somatostatin
receptor-positive tumors [31]. This example of success in developing
radiolabeled peptides for targeting receptor-expressing tumors has paved
the way for the exploration of other receptor/peptide systems.
Bombesin (BN) analogs, other well-studied GPCR-targeting peptides, are
the main focus of this thesis.
18
Bombesin, bombesin-like peptide ligands and their
corresponding receptors
BN is a natural linear amidated tetradecapeptide initially isolated from
skin extracts of the European discoglossid frog Bombina bombina [32,
33]. Subsequently, a search for BN counterparts in mammalian tissues
was initiated. The neuromedins B (NMB) from the central nervous system and the gastrointestinal bombesin (so-called gastrin-releasing peptide, GRP) are the corresponding mammalian peptides. BN-like regulatory peptides exert a spectrum of physiological activities in the nervous
system and the gut. These physiological activities are elicited after BNlike peptides bind to a group of three GPCRs in mammals and one GPCR
in amphibians. The neuromedin B receptor (BB1R/NMBR) with high
affinity for NMB, the gastrin-releasing peptide receptor (BB2R/GRPR)
with high affinity for GRP, and the orphan BN receptor subtype 3
(BB3R/BRS3) are expressed in mammalian tissues. The BN receptor
subtype 4 (BB4R/BRS4) is the amphibian equivalent [34, 35, 36, 37, 38,
39]. The receptor subtypes and the structures of their native peptide ligands are summarized in Table 2.
Table 2. The sequences for the native peptide ligands binding to different receptor subtypes (BB1R, BB2R and BB4R) are presented. Two distinct naturally
occurring BN agonists have been identified for the BB4R differing in the second
residue from C-terminal (either by Leu13 or Phe13). The potency of [Phe13]BN for
BB4R is higher than [Leu13]BN [40]. The native peptide for BB3R has not been
identified.
Receptor
subtype
Native peptide
ligand
Structure of the corresponding
ligand
BB1R/NMBR
NMB
Gly-Asn-Leu-Trp-Ala-Thr-GlyHis-Phe-Met-NH2
Mammalian
GRP
Val-Pro-Leu-Pro-Ala-Gly-GlyGly-Thr-Val-Leu-Thr-Lys-MetTyr-Pro-Arg-Gly-Asn-His-TrpAla-Val-Gly-His-Leu-Met-NH2
Mammalian
Not identified
Mammalian
Pyr-Gln-Arg-Leu-Gly-Asn-GlnTrp-Ala-Val-Gly-His-Leu-MetNH2
Pyr-Gln-Arg-Leu-Gly-Asn-GlnTrp-Ala-Val-Gly-His-Phe-MetNH2
Amphibian
BB2R/GRPR
BB3R/BRS3
[Leu13]BN
BB4R/BRS4
[Phe13]BN
Origin
19
It is known that the normal human pancreas (most abundantly), stomach,
adrenal cortex, and brain (weakly) express GRPR [41]. The abnormal
overexpression of GRPR was reported in the vast majority of cancer cells
from the breast, prostate, uterus, ovaries, colon, pancreas, stomach, lung
(small and non-small cell), head and neck squamous cell cancer and in
various CNS/neural tumors. These data suggest that GRPR could be a
potential candidate as a clinically relevant molecular target for the visualization and therapy of several tumors [34].
GRP, the endogenous ligand for GRPR, and [Leu13]BN share an amidated C-terminal seven amino acid sequence homology (highlighted in
Table 2) that governs the agonist activity of the peptides. Previously investigated BN analogs can be categorized in two different groups based
on their structures. Type A analogs are truncated with a portion of BN
retained, and type B analogs are full-length tetradecapeptides in which
one or more amino acid residues were replaced [42]. The C-terminal of
BN is required for receptor recognition and binding affinity [43]. It has
been shown that more than seven but no more than nine C-terminal amino acids from the BN sequence are required for high affinity receptor
binding [44]. Thus, BN[7-14] is the sequence most widely used for tracer
development. Previous structure-activity studies have demonstrated that
modification in the C-terminal region affects the binding affinity, and
changing L- to D-amino acids at the 8, 9, 13 or 14 positions reduced the
receptor binding affinity dramatically [43]. Therefore, the N-terminal of
these analogs was used for coupling to the chelators for loading with
radiometals or modified for labeling with other radionuclides [45, 46].
BN receptor agonists vs. antagonists
In the past few decades, peptide receptor agonists have been the ligands
of choice for tracer development and utilization in nuclear medicine. The
paradigm regarding the use of agonist-based constructs was primarily due
to receptor-radioligand complex internalization and the high accumulation of radioactivity inside the target cells. In the case of radiometallabelled peptides, the efficient receptor-mediated endocytosis in response
to agonist stimulation provides high in vivo radioactivity uptake in the
targeted tissue, which is a prerequisite for the optimal imaging and treatment of malignancies. In contrast, antagonists, which do not trigger internalization, have not been considered for tumor targeting. Subscribing to
this paradigm, a majority of radiolabeled BN derivatives for the molecular imaging of GRPR overexpression in human tumors were agonists.
20
However, a paradigm shift occurred when somatostatin receptor-selective
peptide antagonists showed preferable biodistribution, including considerably higher in vivo tumor uptake, compared with highly potent agonists
[47]. Similar results were observed for the BN antagonist Demobesin1
[48]. These results suggest that the same preference change from agonists
to antagonists could be justified for other GPCRs such as BN-based radiopharmaceuticals. The tumor-to-kidney ratio for Demobesin 1 was more
than 7-fold higher than the ratio for the corresponding BN agonist Demobesin 4 [48]. Furthermore, it has been hypothesized that side effects
observed during a clinical phase I dose escalation study of BN[7-14] agonist, including abdominal cramps, nausea and diarrhea, might be avoided
by using antagonists [49]. In addition, antagonistic GRPR-based peptides
may not induce the endocrinological side effects, such as the stimulation
of tumor-growth, that are known to occur with agonists [50]. The BNinduced activation of MAP kinase pathway by transactivating epidermal
growth factor receptor (EGFR) was reported by Xiao et al [50]. This side
effect could be even more crucial when a therapeutic dose is administrated. In contrast, BN receptor antagonists may be able to suppress tumor
growth [51]. A considerable decrease in hormone-dependent human prostate tumor cell (PC-82) proliferation was reported upon treatment with a
BN receptor antagonist [D-Tpi6, Leu13 ψ (CH2NH) Leu14]BN[6-14]
(RC-3095) alone or in combination with an agonist of luteinizing hormone-releasing hormone [D-Trp6]-LH-RH and the somatostatin analog
D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2 (RC-160) [52]. Therefore,
there has been considerable interest in the design and development of BN
analogs with antagonistic activity based on the tetradecapeptide BN agonist.
There are additional strategies for BN receptor antagonist design beyond the classical multiple side chain modification approach. Peptide
backbone modification through carbonyl reduction was found to be the
design approach of choice for BN antagonist discovery. It has been reported that the C-terminal region of [Leu14]BN (peptide bond between
residues 13 and 14) is directly responsible for triggering the receptor response, and the replacement of this bond (CO-NH group) by its reduced
form (CH2-NH group) using rapid solid-phase methods resulted in the
first sufficiently potent (IC50 = 35±7 nM) BN antagonist [Leu13ψ(CH2NH)Leu14]BN [53]. In this work, the readily oxidized Met14 residue was also replaced by a Leu14 residue.
BN receptor antagonists can also be obtained by manipulating Cterminal amino acid residues. Broccardo et al. demonstrated that the Trp8
and His12 residues play essential roles in the biological activity of the
21
peptide [54], and substitution with D-Phe in these positions (especially
His12) resulted in a BN receptor antagonist [D-Phe12, Leu14]BN
(IC50=2±0.2 µM) [55]. Additional amino acid replacements improved the
binding affinity of the antagonistic analog [D-Phe6, D-Phe12, Leu14]BN
(IC50 = 0.3±0.05 µM) [55]. The lipophilicity of position 12 was also reported to be important because substitution with a more hydrophilic residue in this position resulted in more than 40-fold reduced binding affinity
[55].
It was observed that the peptide bond between the last two amino acids
can be hydrolyzed by an activating enzyme system, and H-Leu14-NH2 was
released leading to the hypothesis that these two residues are important as
an active moiety responsible for the biological activity of BN. Therefore,
replacing this bond with a non-hydrolysable bond would result in BN
receptor antagonists. Several partial BN antagonist derivatives based on
[D-Phe6-Leu14]BN[6-14] have been reported in which Leu14 was replaced
by non-natural amino acid residues or the peptide bond between Leu13
and Leu14 was modified [56]. However, the replacement of Leu13 with a
statyl residue in [D-Phe6-Leu14]BN[6-14] ([D-Phe6, Sta13, Leu14]BN[614], IC50=2.9±0.6 nM) or introducing a hydroxyl group in place of the
amide bond between Leu13-Leu14 residues resulted in potent BN antagonistic analogs [57]. The spatial orientation of the hydroxyl group is not
important for biological activity [57].
It has also been shown that the C-terminal amide group is important
for the agonist activity of BN, and by deleting this amide function, several potent BN receptor antagonists with preserved high affinity for the BN
receptor have been obtained [57].
This thesis is focused on the development of high-affinity BN receptor
antagonist-based imaging agents using D-Phe-Gln-Trp-Ala-Val-Gly-HisSta-Leu-NH2 ([D-Phe6, Sta13, Leu14]BN[6-14]NH2), which is also known
as JMV594 or RM26 [46, 58, 59].
Molecular basis for selectivity of RM26 for GRPR
The BN receptor antagonist RM26 has more than 5000 times higher selectivity for GRPR compared to NMBR, despite the fact that these receptors share 56% overall amino acid sequence identity [60, 61]. It has been
shown that the fourth extracellular (EC) domain of GRPR is principally
involved in selectively discriminating GRPR from NMBR and responsi22
ble for high affinity binding. Substituting the fourth EC domain of GRPR
into NMBR ([e4-NMBR]GRPR) significantly decreased the binding affinity for RM26 [61]. In the reverse study, substituting the fourth EC
domain of NMBR into GRPR ([e4-GRPR]NMBR) resulted in a 300-fold
improvement in binding affinity for the same peptide analog [61]. In the
same study, site-directed mutagenesis investigations showed that among
11 divergent amino acids between GRPR and NMBR in the fourth EC
domain, Thr297, Phe302 and Ser305 are critical residues for determining the
GRPR selectivity of RM26. It is also important to mention that both human and mouse GRPRs are identical in the fourth EC domain [62, 63].
The influence of the structure of radiolabeled peptides
on targeting properties and biodistribution
Radiolabeling receptor-specific peptides and proteins provides a method
for the selective delivery of radionuclides to the tissues (over-) expressing
the target receptors. This method may also be used for imaging tumors
overexpressing certain receptors. It may also be used for the delivery of
nuclides with cytotoxic particulate emission for therapeutic purposes.
Sensitivity and specificity to the target are essential features for a successful diagnostic and therapeutic application of radiopeptides. In other
words, the success of the detection and treatment of malignancies using
radiolabeled peptides and proteins depends upon the delivery of the radionuclide specifically to the cancer cells while sparing healthy tissue. The
degree of discrimination between target and normal tissue highly depends
on the structure of the radioconjugate.
Figure 2. Radiotracer structure.
A radiopeptide is composed most importantly of the target recognition
moiety, which is conjugated with a radionuclide using different labeling
23
strategies. The labeling technique used depends primarily on the radionuclide used. Radiohalogens, e.g., iodine, can be directly integrated into a
peptide by the electrophilic substitution of a tyrosine or indirectly via a
prosthetic group. Radioactive metals, in contrast, are incorporated into a
protein via complexation by a chelating agent. In addition, a spacer could
be introduced to the structure to separate the chelating moiety from the
target-binding fragment (Figure 2).
The pharmacokinetics of radiopeptides can be tuned by altering the tracer
structure using different spacer moieties and/or different chelators with
variable chemical natures [64, 65].
The influence of the spacer between the receptor
binding sequence and the chelating moiety on the
biological characteristics of BN analogs
There are many structure-related parameters that control the behavior of
peptide-based radiopharmaceuticals in the biological system. One of
these parameters is the structure and physicochemical properties of a
spacer moiety. A chelator or a prosthetic group might be attached to a
peptide using a covalent bond (e.g., amide), but a spacer could also be
introduced to modify the pharmacological properties of the radiotracer.
It is known that the direct coupling of the radiometal-chelator complex
to the N-terminus of the BN analogs decreases the receptor binding affinity [66], whereas the introduction of a linking group prevents the interference of the chelator with the binding site of the targeting vector to the
GRPR. To maintain the radiolabeling site distant from the receptor
recognition and binding site, different spacers have been used. The analog containing no spacer (X=0) for 111In-DOTA-X-BN[7-14]NH2 has
100-fold reduced binding affinity to GRPR compared to its counterpart
containing an 8-carbon aliphatic spacer [67]. A Lys(sha)-βAla-βAla linker for coupling retro[Nα-carboxymethyl-histidine] to a BN analog for
99m
Tc(CO)3 labeling improved the affinity of the conjugate by a factor of
23 compared to the analog without the linker, [Leu13, Nle14]BN[7-14]
[68].
Subsequently, it was demonstrated that an excessive increase of the
lipophilicity of the linker group impairs the binding affinity [67]. Significantly high liver uptake for hydrophobic linker analogs is an additional
24
problem beyond the suboptimal pharmacokinetics of these tracers [67].
To improve the pharmacokinetic profiles for BN analogs, more hydrophilic spacer moieties were introduced. Schweinsbery et al. have shown
that a hydrophilic carbohydrate introduced into the linker sequence of
99m
Tc-labeled BN analogs decreased liver uptake significantly [69]. Increasing the polarity by inserting a negatively charged β3hGlu to the linker provided favorable biodistribution compared to the reference analog
(NαHis)Ac-βAla-βAla-[Leu13, Nle14]BN[7-14] labeled using 99mTctricarbonyl [70]. Consequently, modifying the spacer length and composition may influence the binding affinity of BN to GRPRs and the in vivo
kinetics.
This thesis is focused on the development of RM26-based imaging agents
using linear polyethylene glycol (PEGn)-spacers of different molecular
weights. The hypothesis was that the incorporation of the PEG-spacer
would increase tracer hydrophilicity and decrease unspecific off-target
uptake in healthy organs, particularly in the liver.
The influence of the chelator on the biological
characteristics of peptide-based targeting agents
The pharmacokinetics of radiotracers can also be tuned by choosing the
proper chelation system. The chemical structure of chelators is different,
and this can influence the targeting properties of tracers substantially.
Different chelators are reported to influence the receptor binding affinity,
internalization rate, and even the functional (agonistic/antagonistic) profile of targeting peptides. The effect of chelator modification on the biodistribution of peptide-based imaging agents has been widely investigated
for somatostatin analogs. For example, it has been reported that increasing the number of carboxylates on a triazacyclononane-based chelator
seems to decrease the overall receptor binding affinity and internalization
of 68Ga-labeled [Tyr3]octreotide conjugates, which results in unfavorable
biodistribution profiles [71]. Tumor-to-normal tissue (blood, liver, kidney, and muscle) ratios were significantly higher for the 64Cu-NODAGAconjugated than for the 64Cu-CB-TE2A-conjugated somatostatin-based
antagonist LM3 [72].
The antagonistic somatostatin analog Cpa-DCys-Asn-Phe-Phe-DTrpLys-Thr-Phe-Thr-Cys-2Nal-NH2 (406-040-15) alternated to a full agonist
when coupled to DOTA (406-051-20), as determined in the sst(3) recep25
tor internalization assay [73]. This finding demonstrates that it is not possible to predict whether the modification of a targeting peptide by the
addition of a chelator will activate or antagonize the internalization process.
The chelator has been shown to have little impact on the integrin ανβ3targeting capacity of multimeric cyclic RGD peptides. However, DOTAconjugated 3P-RGD2 showed favorable tumor-targeting properties compared to DTPA- and DTPA-Bn-coupled counterparts [74]. Both 111InDTPA-3P-RGD2 and 111In-DTPA-Bn-3P-RGD2 showed a much faster
tumor washout and lower tumor-to-background ratios than 111In-DOTA3P-RGD2. However, a rapid increase of tumor-to-organ ratios over time
for 64Cu-NODAGA-c(RGDfK) was not observed for 64Cu-DOTA-linked
RGD peptide [75]. Long retention in the blood is most likely a consequence of the limited in vivo stability of the 64Cu-DOTA complex and the
transchelation of 64Cu to blood proteins.
The effect of varying the chelator on the stability and radiochemical
properties of the 68Ga-labeled BN agonist AMBA has been reported [76].
Although NODAGA showed remarkable advantages over DOTA- and
NOTA-coupled AMBA (from a radiochemical point of view), further in
vivo comparison is required to confirm the superiority of NODAGA over
other chelators.
Figure 3. Schematic overview illustrating the structural differences of the four
chelators used in this study, NOTA, NODAGA, DOTA and DOTAGA. The chelators are shown as conjugated to a peptide via one of the carboxylic acid groups.
The profound influence of the labeling chemistry on the tumortargeting properties observed for small targeting agents was less expected
for larger molecules. However, this effect was also reported for larger
targeting agents such as affibody molecules [77]. The 111In-labeled antiHER2 affibody molecule NODAGA-ZHER2:2395 demonstrated higher tumor-to-organ ratios compared to 111In-DOTA-ZHER2:2395 despite the lower
uptake in HER2-expressing tumors. Due to rapid blood clearance and
26
lower uptake in normal organs, the NODAGA-conjugated affibody molecule improved biodistribution compared to the DOTA-coupled counterpart [78]. Similar results were obtained when the radionuclide was
changed to 68Ga. The ZHER2:2395 affibody molecule, which was labeled
with 68Ga using a maleimido derivative of NODAGA, showed a higher
tumor-to-liver ratio compared to the 68Ga-DOTA-conjugated analog [79].
In this thesis, we investigated the influence of chelators on the in vitro
and in vivo performance of PEG2-RM26. As chelators, we have chosen
four homologous macrocyclic chelators: NOTA, NODAGA, DOTA and
DOTAGA (Figure 3).
Detecting ligand interactions with G protein-coupled
receptors on living cells in real time to measure the
binding affinity
The development of radiotracers for the visualization of cancerassociated molecular abnormalities or the treatment of target-expressing
cancers requires careful characterization of the interaction of a potential
targeting probe with its target. In the development of novel radiolabeled
ligands, the measurement of their affinity to the target is critical.
There are some end-point assays in which either direct ligand-receptor
binding is measured, as in saturation assays, or binding is measured indirectly by signaling assays that measure the activity of the downstream
signaling pathway. Both saturation and activity assays for affinity measurement typically rely on equilibrium being reached at the time of treatment. This requirement has been identified as a potential source of error
for binders with subnanomolar KD values. In saturation binding studies,
the time to equilibrium (T) depends on the association rate (ka), the dissociation rate (kd), and the radioligand concentration (C). According to the
approximate expression, T ≈ 3.5/( ka×C + kd). For the strong interactions
(KD << 1 nM), the time to equilibrium can sometimes be many hours or
even days, leading to practical difficulties using equilibrium-based endpoint assays [80, 81].
Monitoring the ligand-receptor binding in real time is an alternative
method to end-point assays that does not require the interaction to reach
equilibrium to estimate the affinity. In addition, the time-resolved assays
27
provide information about not only the affinity but also the binding kinetics.
The heterogeneous nature of living cells is another important issue that
should be considered. The information on the kinetics provided by surface plasmon resonance (SPR) instruments is based on purified proteins,
which may be a poor representation of the real conditions in living cells.
Therefore, it is also more accurate to determine the kinetics from ligandreceptor binding when receptors are located on intact living cells [82].
Furthermore, it is known that radiolabeling can change receptor-ligand
binding affinity, and these changes can be more pronounced for small
peptides than for larger proteins [83, 84]. Hence it is important to measure the affinity of the ligands after being labeled with radionuclides.
Figure 4. Schematic principle of real-time kinetic assay for radioligands. Target-bound signals are collected with detectors mounted above the highest position of a sloping and rotating cell dish in the LigandTracer devices. The detectors are collimated to read a certain liquid-depleted area of the cell dish. Cells
are seeded in a local well-defined part of 10-cm cell dishes and placed inside the
instruments on a tilted support. The opposite side of the cell dish is used as reference (background radioactivity). Finally amount of bound protein to the cells
are registered after repeated differential measurements over time.
28
Thus far, most of the real-time interaction measurements have been
made in cell-free systems using label-free technologies. These measurements have proved challenging for GPCRs. The difficulty of functionally
expressing, purifying, and stabilizing GPCRs in sufficient quantities creates an obstacle to research using SPR technology for the detailed analysis of ligand-receptor interactions [85, 86, 87].
In this thesis, a novel simple and accurate technology (LigandTracer) was
used to demonstrate how radioligand binding to GPCRs can be followed
in real-time on living cells (Figure 4).
Bombesin analogs in the clinic
A limited number of radiolabeled BN analogs have been studied in humans. Most of these, including [Leu13]BN [88], [Lys3]BN [89], RP527
[90], and BZH3 [91] are BN agonists. It was reported that agonists were
capable of eliciting physiological responses. In addition, high accumulation in the liver and/or hepatobiliary clearance have been reported for
most of the studied tracers [92]. These biodistribution characteristics
make imaging in the suprabdominal and/or abdominal region problematic.
BAY 86-7548, also known as RM2, was the first BN antagonist that
was first studied in healthy men [93] and later in prostate cancer patients
[94]. The pilot clinical study confirmed the favorable biodistribution of
the 64Cu-labeled BN antagonist RM26 (64Cu-CB-TE2A-AR06) with rapid
clearance from organs with physiological GRPR expression and significantly longer retention in human PC, which resulted in steadily increasing
tumor-to-normal tissue ratios over time [95]. Tumors were visualized
with high contrast (tumor-to-prostate ratio > 4) at 4 h p.i. While these
studies showed interesting results, more systematic studies involving
more patients are needed to determine whether a BN agonist or antagonist
is better for GRPR targeting.
29
Aims of this thesis
The aims of this thesis project were as follows:
• To develop BN antagonist-based imaging radiotracers for the detection of GRPR-expressing tumors using different PET and
SPECT radionuclides. (Papers I and II)
• To establish a method to monitor the ligand-GPCR interaction in
real time on cell-associated receptors without requiring the purification and stabilization of the receptors. (Paper III)
• To evaluate radiopeptide structure-related factors affecting the in
vitro and in vivo characteristics of RM26; to study the influence
of the length of the spacer moiety and the effect of different chelators on the targeting properties and biodistribution of RM26.
(Papers IV and V)
Radionuclide
Project 2
Chelator
Spacer
Peptide
[18F]AlF
DOTA
NODAGA
Project 5
68Ga
DOTAGA
NOTA
RM26
PEG2
Project 1
Project 4
Project 3
111In
PEG3
PEG4
PEG6
Figure 5. Schematic representation of the projects regarding papers 1, 2, 3, 4
and 5.
30
The present investigation
Development of imaging radiotracers based on the
BN antagonist RM26 for the detection of GRPRexpressing tumors (Papers I and II)
Paper I. Synthesis and characterization of a highaffinity NOTA-conjugated bombesin antagonist for
GRPR-targeted tumor imaging.
Rationale and aims
As was mentioned in the Introduction, there was a general consensus
until recently that agonistic peptide ligands are preferable targeting moieties for the development of imaging agents. However, BN agonist-based
radiotracers cause undesirable side effects when administered intravenously to patients, even in trace amounts. It was hypothesized that these
issues could be avoided with radiopeptides based on BN antagonists.
The aim of this first study was to develop BN antagonist-based PET
and SPECT tracers labeled with gallium-68 and indium-111 by the sitespecific coupling of the macrocyclic chelator NOTA via a PEG2 linker.
We also aimed to establish a preclinical model for the in vitro and in vivo
evaluation of GRPR-imaging agents.
Our reasoning in the selection of this tracer design was based on the
following. NOTA is a versatile chelator, which permits labeling with 68Ga
and 111In, and it is also capable of forming stable chelates with Al18F and
64
Cu. NOTA was coupled to the N-terminal of the peptide via PEG2. This
linker is long enough to provide distance between the chelator and the
binding site of the RM26 antagonist. It was also used with the aim to
decrease the lipophilicity of the conjugate and to modulate its pharmacokinetics.
31
Methods
The RM26 conjugated with NOTA via PEG2 (NOTA-PEG2-RM26, Figure 6) was synthesized and labeled with 68Ga and 111In. The labeling stability, binding specificity to GRPR-expressing cells, inhibition efficiency
(IC50), and retention and internalization by PC-3 cells of both compounds
were investigated. The dissociation constant (KD) of 111In-NOTA-PEG2RM26 was measured using LigandTracer technology. The biodistribution
of the dual isotope 111In/68Ga-NOTA-PEG2-RM26 in both normal NMRI
mice and PC-3-xenografted BALB/c nu/nu mice was studied.
Figure 6. Structural formula of NOTA-PEG2-[D-Phe6, Sta13, Leu14]bombesin [614] (NOTA-PEG2-RM26).
Results and discussion
This study was the first step toward developing an imaging radiotracer
based on the BN-antagonist RM26.
NOTA-PEG2-RM26 was labeled with 111In and 68Ga in greater than
98% yield. The labels were stable under challenge with a 500-fold excess
of EDTA. Both 111In- and 68Ga-labeled analogs demonstrated saturable
uptake in GRPR-expressing PC-3 cells (Figure 1 in paper I). The antagonistic activity of the radiotracers was confirmed by an in vitro internalization assay (Figure 2 in paper I). More than 6-fold lower radioactivity
internalization was detected for 68Ga- and 111In-labeled NOTA-PEG2RM26 compared to the agonistic analog 125I-Tyr4-BBN after 30 min incubation with a 1 nM concentration. More than twice as many binding
sites were found for the BN-based antagonist compared to the universal
agonist 125I-Tyr4-BBN. These data are in agreement with earlier findings
for a somatostatin-based antagonist [47] and a BN-based antagonist [58].
Almost 4-fold lower IC50 values were measured for metal-loaded analogs compared to their non-labeled counterpart after 5 h incubation at 4°C
(Figure 7). The positive charges at the N-terminal of BN analogs was
reported to be beneficial for GRPR targeting by increasing the affinity,
whereas negative charges decrease this affinity [46]. The overall charge
of the gallium- or indium-NOTA compounds is +1, which could explain
32
Normalized,
% of added radioactivity
the higher affinity for the metal-loaded analogs compared to non-labeled
NOTA-PEG2-RM26. A KD of less than 36 pM was determined for the
111
In-labeled compound.
Figure 7. Inhibition of 125I-Tyr4BBN binding to PC-3 cells with
nat
nat
Ga-NOTA-PEG2-RM26,
InNOTA-PEG2-RM26, and nonlabeled NOTA-PEG2-RM26.
100
50
0
-2
0
2
Concentration (log[nM])
Variant
IC50 (nM)
Ga-NOTA-PEG2-RM26
nat
In-NOTA-PEG2-RM26
NOTA-PEG2-RM26
0.9 ± 0.2
1.2 ± 0.3
5±1
nat
The biodistribution studies in NMRI mice (Table 3, Figure 4 in paper
I) demonstrated fast blood and whole body clearance for both tracers via
renal excretion. Kidney reabsorption was low for both counterparts.
Moreover, the uptake of both 68Ga- and 111In-labeled peptides was specific in the abdominal area organs (pancreas, stomach and small intestine),
which express GRPR.
The influence of the administered peptide dose on the biodistribution
at 2 h p.i. of 111In-NOTA-PEG2-RM26 was minor. The tumor uptake after
the injection of 15 pmol was significantly higher than uptake after the
injection of lower or higher peptide doses. However, differences in tumor-to-organ ratios were not significant for doses between 3.6 and 45
pmol per mouse (Table 4, Figure 5 in paper I).
The biodistribution of both111In-NOTA-PEG2-RM26 and 68Ga-NOTAPEG2-RM26 in tumor-bearing mice was similar to the biodistribution in
NMRI mice (Table 5, Figure 6 in paper I). The rapid blood and whole
body clearance along with specific uptake in receptor-expressing organs
and tumors was shown for both counterparts. However, 111In-NOTAPEG2-RM26 showed more rapid blood clearance, and higher tumor uptake was measured for the 68Ga-labeled analog, which resulted in higher
tumor-to-organ ratios. We speculate that the difference in blood clearance
resulting in different tumor uptake could be due to the difference in the
coordination geometries of 68Ga- and 111In-NOTA complexes. These co-
33
ordination differences might result in different strengths of interaction
with blood proteins.
The high potential of 68Ga/111In-NOTA-PEG2-RM26 for the in vivo
imaging of GRPR was confirmed by microPET/CT and gamma camera
images (Figure 8). Based on biodistribution data, 2-3 h p.i. was a suitable
time point for image acquisition, when higher tumor-to-organ ratios were
obtained. These data demonstrate that 68Ga is a suitable label for NOTAPEG2-RM26, despite its short half-life.
Figure 8. Imaging of GRPR expression in PC-3-xenografted BALB/c nu/nu mice.
The gamma camera image (A) was acquired at 3 h p.i. from an animal injected
with 45 pmol of 111In-NOTA-PEG2-RM26 (260 kBq). MicroPET/CT images were
acquired at 1 h (B) and 2 h (C) p.i. from animal injected with 68Ga-NOTA-PEG2RM26 (45 pmol, 300 kBq).
Conclusions
This study demonstrated the potential of using NOTA-PEG2-RM26, a BN
antagonist-based imaging probe, for the visualization of GRPRexpressing tumors with SPECT and PET. It was shown that the biodistribution and kinetics of a peptide can be altered by small modifications,
e.g., exchanging the radionuclide.
34
0.56±0.06ab
0.48±0.08ab
1.0±0.2
0.8±0.2ab
5±1ab
0.98±0.08ab
1.4±0.5ab
5±2
0.15±0.04b
0.37±0.07b
1h
0.38±0.09
0.26±0.10
0.92±0.02
0.4±0.1
0.9±0.2
0.25±0.10
0.40±0.05
5±2
0.08±0.02
0.2±0.1
1h
(blocked)
Ga-NOTA-PEG2-RM26
0.24±0.04
0.15±0.02
0.89±0.10
0.3±0.1
1.1±0.3
0.39±0.03
0.34±0.03
3±1
0.05±0.02
0.15±0.02
2h
0.3±0.1
0.35±0.08
0.75±0.10
0.3±0.1
1.1±0.1
0.26±0.08
0.39±0.09
4±2
0.10±0.03
0.26±0.09
1h
(blocked)
0.15±0.04
0.23±0.03
0.8±0.1
0.6±0.3
2.6±0.3
0.43±0.05
0.53±0.04
3±1
0.07±0.01
0.26±0.06
2h
In-NOTA-PEG2-RM26
0.42±0.08d
0.52±0.04cd
1.1±0.2c
0.6±0.3c
9.1±0.8cd
0.85±0.09cd
1.7±0.4cd
5±2
0.17±0.03cd
0.42±0.10cd
1h
111
0.01±0.01
0.12±0.02
0.4±0.1
0.20±0.03
0.2±0.2
0.1±0.1
0.11±0.05
0.33±0.06
0.03±0.02
0.13±0.11
24 h
Significant difference (p<0.05) between 1 h and 1 h blocked for 68Ga-NOTA-PEG2-RM26. bSignificant difference between 1 h and 2 h
for 68Ga-NOTA-PEG2-RM26. cSignificant difference between 1 h and 1 h blocked for 111In-NOTA-PEG2-RM26. dSignificant difference
between 1 h and 2 h for 111In-NOTA-PEG2-RM26.
a
Blood
Lung
Liver
Spleen
Pancreas
Stomach
Small intestine
Kidney
Muscle
Bone
Organ and tissue
68
Table 3. Biodistribution of 68Ga-NOTA-PEG2-RM26 and 111In-NOTA-PEG2-RM26 (total injected mass 23 pmol) in male NMRI mice.
One group of animals was pre-injected (i.v.) with 20 nmol of non-labeled NOTA-PEG2-RM26 (designated as blocked). Data are presented as mean percentage of injected dose per gram of tissue (%ID/g ± SD, n=4).
Table 4. Dose escalation study in BALB/c nu/nu male mice bearing PC-3 xenografts at 2 h p.i. of 111In-NOTA-PEG2-RM26. Uptake values are expressed as an
average percentage of injected dose per gram of tissue and standard deviation
based on data from four mice (% ID/g ± SD).
111
In-NOTA-PEG2-RM26
3.6 pmol
15 pmol
25 pmol
45 pmol
0.26±0.07
0.52±0.08
2.0±0.3
0.60±0.28
14±3
1.8±0.6
3±2
3.8±0.6
7±2
0.12±0.03
0.26±0.10
0.5±0.1ab
0.6±0.4
2.4±0.4
0.6±0.1
17±3c
2.4±0.7
4±2
5±1
11±1ab
0.15±0.05
0.30±0.08
0.29±0.04
0.8±0.4
2.4±0.3
0.50±0.07
15±2
3±1
3±2
3.9±0.3
9±1
0.14±0.04
0.4±0.2
0.3±0.2
0.6±0.1
2.1±0.4
0.6±0.2
11±4
1.9±0.3
2.5±0.6
5±1
9±1
0.13±0.04
0.27±0.06
29±9
14±3
3.7±0.8
13±4
0.5±0.1
4.2±0.9
3±1
1.9±0.5
64±21
31±14
25±5
30±25
4.7±0.5
18±3
0.68±0.09
5±1
3±2
2.2±0.4
79±18
39±8
31±4
12±4
3.8±0.3
18±1
0.61±0.08
4±1
3±2
2.3±0.1
66±16
26±7
38±14
16±3
4.4±0.7
17±5
0.9±0.2
4.9±0.8
3.8±0.7
2.1±0.5
80±50
34±5
Organ & tissue
Blood
Lung
Liver
Spleen
Pancreas
Stomach
small intestine
Kidney
Tumor
Muscle
Bone
Tumor to organ
Blood
Lung
Liver
Spleen
Pancreas
Stomach
small intestine
Kidney
Muscle
Bone
a
Significant difference (p<0.05) between 3.6 and 15 pmol. bSignificant difference
between 15 and 25 pmol. cSignificant difference between 15 and 45 pmol.
36
0.68±0.04b
0.78±0.07
1.83±0.07
1.04±0.06b
5.7±0.8
1.89±1.04
1.4±0.5
2.7±0.4
8.1±0.4b
0.09±0.01
0.28±0.05
1h
0.3±0.1a
0.25±0.02a
0.13±0.04a
1h
(blocked)
3h
0.32±0.05c
0.7±0.2c
1.7±0.1
0.8±0.1c
0.6±0.2
0.5±0.1
0.27±0.05
1.4±0.1
7.4±1.4c
0.03±0.01
0.25±0.07
Ga-NOTA-PEG2-RM26
0.37±0.05a
0.26±0.02a
0.13±0.04a
1h
(blocked)
3h
0.10 0.01
0.28±0.05
1.5±0.2
0.41±0.07
3.38±1.08c
0.89±0.08c
0.9±0.2c
2.9±0.4c
5.8±1.3
0.063±0.005c
0.17±0.07
In-NOTA-PEG2-RM26
0.55±0.05
0.7±0.1
2.1±0.2b
0.68±0.08
12±1b
1.7±0.9
2.57±1.04
4.6±0.6b
5.7±0.2
0.163±0.005b
0.38±0.08b
1h
111
0.022±0.005
0.2±0.2
1.4±0.4
0.3±0.1
0.11±0.03
0.12±0.03
0.17±0.04
1.3±0.5
4.5±0.9
0.039±0.008
0.12±0.04
24 h
Significant difference (p<0.05) between 1 h and 1 h blocked for each of the compounds. bSignificantly (p<0.05) higher uptake in
comparison with counterpart for 1 h p.i. cSignificantly (p<0.05) higher uptake in comparison with counterpart for 3 h p.i.
a
Blood
Lung
Liver
Spleen
Pancreas
Stomach
Small intestine
Kidney
Tumor
Muscle
Bone
Organ & tissue
68
Table 7. Biodistribution of 68Ga-NOTA-PEG2-RM26 and 111In-NOTA-PEG2-RM26 (total injected mass 45 pmol) after injection in male
BALB/c nu/nu male mice bearing PC-3 xenografts. One group of animals was co-injected (i.v.) with 20 nmol of non-labeled NOTAPEG2-RM26 (designated as blocked). Data are presented as mean percentage of injected dose per gram of tissue (%ID/g ± SD, n=4).
Paper II. In vitro and in vivo evaluation of a 18Flabeled high affinity NOTA-conjugated bombesin
antagonist as a PET ligand for GRPR-targeted tumor
imaging.
Rationale and aims
The potential of NOTA-PEG2-RM26 as an imaging agent when labeled
with gallium-68 and indium-111 was demonstrated in Paper I. The second study was designed to test the hypothesis that using the more longlived positron-emitting radionuclide fluorine-18 as a label would further
improve the sensitivity of imaging using NOTA-PEG2-RM26.
The sensitivity of imaging depends directly on the contrast. The image
contrast is determined by the ratio of accumulated radioactivity in the
tumor to that in non-target normal tissue. Fast clearance of the 111In/68GaNOTA-PEG2-RM26 from blood and the receptor-expressing organs together with high uptake and long retention of the radioactivity in tumors
resulted in increasing tumor-to-normal tissue ratios over time. The use of
18
F, with its longer half-life of 109.7 min compared to 67.6 min for 68Ga,
may provide an opportunity for imaging at later time points when the
radiotracer would clear better from normal organs and higher image contrast could be obtained. In addition, the low positron energy of 18F, which
limits tissue penetration (2.3 mm in water) compared to 68Ga (8.9 mm in
water) is well suitable for high-resolution PET images.
The availability of 18F is another important attribute. 18F is the most
widely used radioisotope for PET scans, which is produced in large quantities for the synthesis of 18F-FDG (the main radiopharmaceutical for PET
scans). In addition, radiolabeling peptides with 18F using chelate-based
AlF-chemistry provides a relatively high yield of fluorinated peptides.
This one-step method utilizes the strength of the Al-F bond and the ability of NOTA to chelate aluminum [96].
The aim of this study was to test the hypothesis that using 18F would
enable imaging at later time points compared to 68Ga, which would improve imaging contrast due to better clearance of non-bound tracer.
38
Methods
NOTA-PEG2-RM26 was labeled with 18F using aluminum-fluoride chelation. In vitro binding specificity and cellular processing tests were performed. The inhibition efficiency (IC50) of the AlnatF-NOTA-PEG2-RM26
was compared to previously tested natGa-loaded peptide using 125I-Tyr4BBN as the radioligand displacer. The biodistribution in NMRI mice and
the targeting of GRPR-overexpressing PC-3 xenografts in nude mice
were studied.
Results and discussion
Normalized,
% of added radioactivity
The peptide was labeled with 18F within 1 h with a decay-corrected yield
of 60-65%. The radiochemical purity was greater than 98%. Receptor
binding capacity of NOTA-PEG2-RM26 was preserved after radiolabeling (Figure 3A in Paper II).
The low internalization of radioactivity into the cells was in good
agreement with 68Ga- and 111In-labeled analogs (3.2 ± 1.4% of cell-bound
radioactivity at 30 min of incubation). The IC50 value measured for Alnat
F-NOTA-PEG2-RM26 was in the same low nanomolar range as natGaNOTA-PEG2-RM26 after 3 h incubation at 4°C (Figure 9).
Figure 9. Inhibition of 125I-Tyr4BBN binding to PC-3 cells with
nat
Ga-NOTA-PEG2-RM26 or Alnat
F-NOTA-P2-RM26. Data are
mean values ± SD of 3 culture
dishes.
100
50
0
-2
-1
0
1
2
3
Concentration (Log[nM])
Variant
IC50 (nM)
Ga-NOTA-PEG2-RM26
AlnatF-NOTA-PEG2-RM26
3.5±0.5
4.4±0.8
nat
Al18F-NOTA-PEG2-RM26 demonstrated fast blood clearance via renal
excretion, low kidney retention and liver uptake, and high and specific
uptake in the tumors and receptor-positive organs similar to 111In/68GaNOTA-PEG2-RM26 (Table 6, Figure 4 in paper II). Low uptake in bones
was an indication of the high in vivo stability of the Al18F-NOTA chelation in Al18F-NOTA-PEG2-RM26. However, the washout of Al18F39
NOTA-PEG2-RM26 from PC-3 xenografts was faster than 111In-NOTAPEG2-RM26 (the biodistribution data obtained in previous project) (Table
7, Figure 6A in paper II). This result contradicts the results obtained for
affibody molecules [97]; Al18F-NOTA-ZHER2:2395 showed tumor retention
comparable with 111In-NOTA-ZHER2:2395 in SKOV-3 xenografts.
The biodistribution data suggest that 3 h p.i. would be the optimal time
point for in vivo imaging when the tumor-to-organ ratio was the highest
(Table 7, Figure 6B in paper II). The biodistribution data were confirmed
by in vivo imaging using a clinical PET scanner (Figure 10).
Among compounds tested in this part of the project (111In, 68Ga and
18
Al F-labeled NOTA-PEG2-RM26), 68Ga-NOTA-PEG2-RM26 showed
slightly higher decay-corrected tumor uptake (Table 1 in paper II). However, a non-decay-corrected tumor uptake of Al18F-NOTA-PEG2-RM26
at 3 h p.i. would not be lower due to the longer half-life of 18F. Al18FNOTA-PEG2-RM26 provided higher tumor-to-blood, tumor-to-lung,
tumor-to-liver, and tumor-to-spleen ratios compared with 68Ga-NOTAPEG2-RM26 at 3 h p.i (Figure 11). There was no difference in tumor-tomuscle and tumor-to-bone ratios for these two PET imaging agents. In
addition, the short positron range of 18F compared to 68Ga can improve
image quality significantly.
Figure 10. Imaging of GRPR-expression in
PC-3-xenografted BALB/c nu/nu male mice
3 h p.i. The animal was injected with 45
pmol of Al18F-NOTA-PEG2-RM26 (~2 MBq).
40
Table 6. Biodistribution of Al18F-NOTA-PEG2-RM26 in male NMRI mice 1 h p.i.
1h
1h
blocked
0.3±0.1
0.3±0.2
1.2±0.3
0.6±0.3
14±6
2±1
4.05±1.53
2.8±0.5
0.09±0.03
0.3±0.2
0.3±0.1
0.34±0.13
1.4±0.4
0.16±0.08*
0.4±0.1*
0.3±0.1*
1.7±0.7*
3.4±0.8
0.08±0.03
0.20±0.07
Organ & tissue
Blood
Lung
Liver
Spleen
Pancreas
Stomach
Small intestine
Kidney
Muscle
Bone
Total injected mass of radiolabeled
conjugate was 45 pmol and animals
in blocked group were co-injected
with 20 nmol of non-labeled peptide.
Data are presented as the mean percentage of the injected dose per gram
of tissue (%ID/g ± SD, n=4). The
asterisks denote significant differences between the blocked and nonblocked animals 1 h p.i (p<0.05).
Tumor-to-organ ratio
600
300
200
68
150
Ga-NOTA-PEG2-RM26
Al18F-NOTA-PEG2-RM26
100
50
Figure 11. Tumor-toorgan ratios for 68GaNOTA-PEG2-RM26 (data
from paper I) and Al18FNOTA-PEG2-RM26 (data
from paper II) in male
BALB/c nu/nu mice with
PC-3 xenografts, 3 h p.i.
bl
oo
d
lu
ng
liv
e
sp r
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pa en
nc
re
s t as
om
ac
h
sm
in
ki t
dn
e
m y
us
cl
e
bo
ne
0
Conclusions
Because of the wide availability and superior imaging characteristics of
18
F together with the simplicity of the facile Al18F-NOTA chelation labeling chemistry compared to classical multi-step labeling methods, Al18FNOTA-PEG2-RM26 may be a good alternative or supplement to 68GaNOTA-PEG2-RM26 for clinical PET imaging.
41
Table 7. Biodistribution of Al18F-NOTA-PEG2-RM26 in male BALB/c nu/nu mice
bearing PC-3 xenografts.
1h
Organ & tissue
Blood
Lung
Liver
Spleen
Pancreas
Stomach
Small intestine
Kidney
Tumor
Muscle
Bone
Tumor to organ
Blood
Lung
Liver
Spleen
Pancreas
Stomach
Small intestine
Kidney
Muscle
Bone
0.48±0.08
0.36±0.06
1.9±0.4
1.0±0.2
13.1±0.2
2.4±0.4
4.06±1.76
3.61±0.26
6.32±0.86
0.10±0.03
0.30±0.08
13.27±1.24
17.4±0.8
3.4±0.2
6.5±1.2
0.49±0.04
2.7±0.1
1.7±0.6
1.74±0.16
66.9±12.8
21.7±2.8
1h
(blocked)
0.40±0.15
0.44±0.25
1.6±0.3
0.67±0.23
0.29±0.15*
0.38±0.14*
2.6±0.9
3.9±0.8
0.75±0.15*
0.09±0.04
0.2±0.2
3h
6h
0.08±0.03
0.21±0.17
0.4±0.2
0.10±0.03
2.7±0.7
1.4±0.3
0.9±0.4
1.7±0.2
5.53±0.75
0.03±0.01
0.17±0.08
0.02±0.00
0.11±0.08
0.10±0.02
0.15±0.08
0.15±0.05
0.33±0.08
0.13±0.06
0.27±0.06
2.25±0.65
0.02±0.01
0.22±0.08
87±42
36±20
14±8
61±19
2.2±0.6
4.25±1.16
7.4±3.7
3.2±0.6
159±47
38±16
89±25
32.5±28.8
23±3
15.5±8.3
14.7±2.3
6.4±0.6
15.5±6.5
8.4±0.8
138±108
10.8±1.7
Total injected mass of radiolabeled conjugate was 45 pmol and animals in
blocked group were co-injected with 20 nmol of non-labeled peptide. Data are
presented as the mean percentage of the injected dose per gram of tissue (%ID/g
± SD, n=4). The asterisks denote significant differences between the blocked and
non-blocked animals 1 h p.i (p<0.05).
42
Paper III. Detecting ligand interactions with G
protein-coupled receptors on living cells in real time.
Rationale and aims
The careful characterization of the interaction of a potential targeting
probe with its target, such as measuring its affinity to the corresponding
target, is essential for the development of novel radiotracers. The methods typically used to measure ligand-receptor affinity are end-point assays, which rely on equilibrium being reached during incubation. This
requirement is a potential source of error for high-affinity binders, leading to underestimation of the affinity. In contrast, time-resolved interaction measurements using intact cells would provide information about
binding kinetics and overcome the difficulties of the expression, purification and stabilization of GPCRs in sufficient quantities.
The aim of the present study was to establish a method for real-time
measurements of radioligand-GPCR (RM26-GRPR) interactions in living
cells.
Methods
111
In-labeled BN-antagonist (111In-NOTA-PEG6-RM26) binding to the
naturally expressed GRPR system was used to evaluate the time-resolved
ligand-GPCR binding assays in their true environment. The affinity and
kinetic properties, such as association and dissociation rates were measured by following the interactions over time using LigandTracer technology.
Results and discussion
The interaction of 111In-NOTA-PEG6-RM26 with GRPR was monitored
on PC-3 cells (Figure 12A). The data fit to the one-to-one kinetic model
was poor, suggesting that the interaction is not homogeneous and more
than one type of interaction exists simultaneously. Later, when the realtime interaction data were analyzed with the Interaction Map method, two
peaks were visible, indicating two parallel interaction types (Figure 12B).
43
Figure 12. (A) Binding of 111In-NOTA-PEG6-RM26 to GRPR on PC-3 cells. Data
is presented as duplicates, measured in separate dishes (grey and black). (B)
Interaction Map, calculated from the black curve of (A). The map shows the
presence of two parallel interactions, i and ii, with different kinetic constants (ka
and kd). The darker peak (peak i), represents the stronger contribution which has
higher recognition (ka) and stability (kd), thus higher affinity.
Kinetic parameters are presented in Table 8. The two different interactions for the BN analog on PC-3 cells can be explained either by different
conformations of the receptors or post-translational modification.
Table 8. The summary of kinetic parameters for 111In-NOTA-PEG6-RM26 ̶ GRPR
interaction.
Radiotracer
Interaction
ka (M-1s-1)
kd (s-1)
KD (M)
Weight (%)
111
In-NOTA-PEG6-RM26
i
(5.9 ± 0.9) × 105
(1.4 ± 0.2) × 10-5
(24.2 ± 0.2) × 10-12
73.4 ± 4.0
ii
(2.4 ± 1.8) × 105
(2.1 ± 0.7) × 10-3
(15.6 ± 13.4) × 10-9
25.2 ± 3.7
The IC50 value is another parameter that reflects the affinity of the conjugate for its receptor. However, the accuracy of IC50 measurements relies
on equilibrium being reached at the time of the treatment (end-point).
This requirement may lead to the underestimation of binding affinity
because the time needed to reach equilibrium could be several hours for
high-affinity binders. In contrast, KD values (obtained with the LigandTracer instrument) were measured by monitoring the ligand-receptor
binding in real time and did not depend on equilibrium being achieved.
44
For instance, in our first study, the measured association (ka) and dissociation (kd) rates for 111In-NOTA-PEG2-RM26 were approximately
3.3×10+5 s-1M-1 and 1.2×10-5 s-1, respectively, which would indicate that
more than 8 h of incubation is necessary to achieve equilibrium (T ≈ 3.5/(
ka×C + kd)) for a 0.3 nM concentration of the ligand. This duration necessary to reach equilibrium could explain the more than 25-fold lower
IC50 value (where cells were incubated for 5 h) for natIn-NOTA-PEG2RM26 compared with the KD value measured in a real-time interaction
assay for 111In-labeled NOTA-PEG2-RM26. However, IC50 measurement
is a relatively appropriate method to compare the affinity of different
conjugates and exclude binders with lousy affinities.
Conclusions
This project presents a method for providing information about binding
kinetics by measuring real-time interactions with GPCRs in a living system without needing to purify and stabilize the receptors. With the use of
advanced tools, e.g., the Interaction Map, the interaction heterogeneity
related to the complexity of living cells can be further evaluated. The
application of this method might improve the understanding of ligandGPCR interactions.
45
Evaluation of tracer structure-related factors affecting
binding specificity and sensitivity of RM26 (Paper IV
and V)
Paper IV. The effect of mini-PEG-based spacer length
on binding and pharmacokinetic properties of a 68Galabeled NOTA-conjugated antagonistic analog of
bombesin.
Rationale and aims
As was indicated in the Introduction, the spacer between the receptor
binding site and the chelator can be used not only to prevent the interference of the radiometal-chelator complex with the binding site of the peptide but also to improve the pharmacokinetic properties of the radiopeptides. The introduction of hydrophilic spacer moieties may improve the
suboptimal biodistribution of BN analogs.
In this project, we aimed to investigate whether increasing the miniPEG-spacer length (reducing the lipophilicity of the tracer) would further
suppress the uptake of radioactivity in the liver, which would improve the
visualization of liver metastases of GRPR-expressing breast cancer (BC).
Because the liver is one of the major metastatic sites for BC, less radioactivity accumulation in the liver is desirable for tracer sensitivity. We have
therefore studied the effect of varying the length of the mini-PEG spacer
moiety on in vitro binding affinity as well as biodistribution, particularly
the targeting properties and excretion pathway of the peptide.
Due to the flexibility of the PEG chain, which can mask the biologically active moiety of the peptide and impair the binding affinity, no more
than 6 units of ethylene glycol were used.
Methods
Four NOTA-conjugated RM26 analogs with different mini-PEG-lengths
(di-, tri-, tetra- and hexaethylene glycol) were synthesized, radiolabeled
with 68Ga, and evaluated in in vitro and in vivo comparative studies. 111In
was used in some in vitro studies, in which the short half-life of 68Ga was
insufficient.
46
Results and discussion
Normalised,
% of added radioactivity
All variants were labeled with high yield with 68Ga and 111In, and the
labels were stable under EDTA challenge (Table 2 in paper IV). The log
D values were -2.27±0.07, -2.47±0.06, -2.49±0.10 and -2.50±0.09 for
PEG2-, PEG3-, PEG4- and PEG6-containing 68Ga-labeled variants, respectively. Small but significant increases in the overall hydrophilicity of the
conjugates were observed by increasing the spacer length from two to
three ethylene glycol units. A further decrease of lipophilicity was not
significant.
Because the short half-life of 68Ga is not enough to measure the slow
dissociation rate of the high-affinity conjugates, the KD values were
measured for 111In-labeled analogs using LigandTracer technology. The
calculated equilibrium dissociation constant values were in the low
picomolar range due to rapid on-rates and slow off-rates (KD values were
23±13, 5±3, 8±5 and 18±8 pmol for PEG2, PEG3, PEG4 and PEG6 variants, respectively). The IC50 values of natGa-loaded conjugates slightly
increased as the number of PEG units increased (Figure 13).
Figure 13. Inhibition of 125I-Tyr4-BBN
binding to PC-3 cells with natGa-NOTAPEGn-RM26 (n = 2, 3, 4 and 6). Cell monolayers were incubated 3 h at 4°C.
100
50
0
-11
Variant
nat
-10
-9
-8
Ga-NOTA-PEG2-RM26
Ga-NOTA-PEG3-RM26
nat
Ga-NOTA-PEG4-RM26
nat
Ga-NOTA-PEG6-RM26
nat
-7
-6
Concentration (log[nM])
IC50 (nM)
3.2±0.2
4.1±0.4
5.6±0.5
6.0±0.5
The biodistribution profiles of the 68Ga-labeled analogs in NMRI mice
were similar. No significantly different uptake was observed in the majority of the organs, neither in non-target organs nor in receptor-positive
organs (Figure 14, Table 5 in paper IV). However, the accumulation of
radioactivity for the PEG3 analog in the liver was slightly but significantly lower (p<0.05) than the uptake of PEG4 and PEG6 analogs.
On the basis of the measured affinity and biodistribution data in NMRI
mice, 68Ga-NOTA-PEG3-RM26 was selected as the most promising conjugate for biodistribution, in vivo binding specificity, and imaging studies
47
in tumor-bearing mice. Specific uptake was found in receptor-positive
organs and the tumor xenografts formed by PC (PC-3) and BC (BT-474)
cells (Figure 15, Table 6 in paper IV). Both tumor xenografts were clearly visualized with a microPET/CT camera at 2 h p.i (Figure 16). We
speculate that implanted estradiol pellets may affect experimental outcomes because the radioactivity accumulation in the liver and pancreas
was significantly lower in mice implanted with BT-474 cells both in ex
vivo measurements and in imaging studies.
A
6
3
2
1
1
0
0
PEG2
PEG3
PEG4
PEG6
bl
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liv
sp er
pa lee
nc n
r
st ea
sm om s
in ach
te
st
in
ki e
dn
m ey
us
cl
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bo
ne
2
bl
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liv
sp er
pa lee
nc n
r
st ea
sm om s
in ach
te
st
in
ki e
dn
m ey
us
cl
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bo
ne
Uptake, % ID/g
3
B
PEG2
PEG3
PEG4
PEG6
Figure 14. Biodistribution of 68Ga-NOTA-PEGn-RM26 (n = 2, 3, 4 and 6) in
female NMRI mice at (A) 1 h and (B) 2 h p.i. Data are presented as the mean
percentage of the injected dose per gram of tissue (%ID/g ± SD, n=3). Uptakes
in GI tract and carcass are presented as %ID per whole sample.
Conclusions
This study demonstrates that the addition of only a few PEGs to the spacer sequence is not sufficient to change appreciably the biodistribution of
NOTA-PEGn-RM26. The hepatic uptake was low for all the conjugates,
and the difference in the radioactivity accumulated in the liver was marginal when di-, tri-, tetra- and hexaethylene glycol were compared. However, the modification of the spacer moiety may be suitable for finetuning the targeting properties of tracers.
48
B
3
3
2
2
1
1
0
0
bl
oo
d
4
bl
o
Non-blocked
Blocked
5
4
od
liv
e
sp r
le
p
e
sm an n
al cr
e
li
nt as
es
tin
k e
tu idn
m
or ey
PC
m -3
us
cl
e
bo
ne
Uptake, % ID/g
5
Non-blocked
Blocked
liv
e
sp r
le
s m p a n en
al c r
e
li
n t as
es
tin
tu
k e
m idn
or
e
B y
T47
m 4
us
cl
e
bo
ne
A
Figure 15. In vivo binding specificity test and biodistribution of 68Ga-NOTAPEG3-RM26 in female BALB/c nu/nu mice bearing (A) PC-3 or (B) BT-474 xenografts.
Figure 16. Imaging of GRPRexpression 2 h p.i. in (A) PC-3
and (B) BT-474 xenografts in
BALB/c nu/nu female mice. The
mice were injected with 45 pmol
of 68Ga-NOTA-PEG3-RM26.
49
Paper V. The effect of macrocyclic chelators on
targeting properties of 68Ga-labeled gastrin-releasing
peptide receptor antagonist RM26
Rationale and aim
The chelators are used to coordinate metal radionuclides and attach them
to the targeting peptides. However, they may also modify the targeting
properties of a radiopeptide. Different chelator-metal complexes have
different charge and structures, which might affect their interaction with
adjacent amino acids in the targeting peptides and determine their preferred conformation in solution, thus influencing their interaction with a
target receptor. Furthermore, the variation of charge, conformation, and
distribution of lipophilic patches along a radiopeptide may influence its
off-target interactions, e.g., binding to the blood proteins and the scavenger receptors in excretory organs.
The aim of this study was to select an optimal chelator for labeling
PEG2-RM26 with 68Ga. The commercially available macrocyclic chelators NOTA, NODAGA, DOTA and DOTAGA, which provide thermodynamically stable and kinetically inert chelates with gallium, were selected
for this study. The highest tumor-to-organ ratios were selected as an optimization criterion.
Methods
In this investigation, four homologous macrocyclic chelators, NOTA,
DOTA, NODAGA and DOTAGA, were coupled to PEG2-RM26. The
properties of the 68Ga-labeled analogs, including labeling efficiency, stability, in vitro binding specificity, and affinity, were compared among the
four radioligands. Their biodistributions were compared in normal and
tumor-bearing mice.
Results and discussion
All four conjugates were labeled with 68Ga with high yield and demonstrated high stability in EDTA challenge (Table 1 in paper V). The compounds demonstrated specific uptake and slow internalization after binding to the receptors (Figure 1 and 3 in paper V). However, the DOTAconjugated analog showed two-fold greater internalization compared with
NOTA-, NODAGA- and DOTAGA-conjugated peptides. This effect is
50
Normalized,
% of added radioactivity
similar to that of the same chelator on the full antagonist somatostatin
analog 406-040-15, which converted to a full agonist when coupled with
DOTA (406-051-20) [73].
This study demonstrates that the tracer affinity depends on the chelator. Although all natGa-loaded variants showed subnanomolar IC50 values,
nat
Ga-NOTA-PEG2-RM26 demonstrated the highest affinity (Figure 17).
It is already known that the positive charge at the N-terminus of BN analogs increases the affinity whereas negative charge decreases their affinity [46, 98]. The overall charges of the NOTA, NODAGA, DOTA and
DOTAGA complexes with gallium are +1, 0, 0, and ̵1, respectively
(Chart 2 in paper V), which can explain almost 4-fold lower IC50 value of
nat
Ga-NOTA-PEG2-RM26 compared to natGa-DOTAGA-PEG2-RM26.
Figure 17. Inhibition of 125I-Tyr4BBN binding to PC-3 cells with
nat
Ga-X-PEG2-RM26 (X = NOTA,
NODAGA, DOTA and DOTAGA).
100
50
0
-2
0
2
Concentration (log[nM])
Variant
IC50 (nM)
nat
Ga-NOTA-PEG2-RM26
2.3±0.2
nat
Ga-NODAGA-PEG2-RM26
3.0±0.3
nat
Ga-DOTA-PEG2-RM26
2.9±0.3
nat
Ga-DOTAGA-PEG2-RM26 10.0±0.6
Biodistribution studies in NMRI mice demonstrated rapid blood clearance via kidney ultrafiltration for all 68Ga-labeled conjugates (Figure 18,
Table 2 in paper V). However, NOTA- and NODAGA-conjugated analogs showed lower normal organ (lung, muscle and bone) uptake compared to DOTA- and DOTAGA-coupled counterparts. The radioactivity
in blood was twice as high for 68Ga-DOTAGA-PEG2-RM26 compared to
the NOTA-conjugated counterpart 2 h p.i.
In good agreement with the biodistribution in NMRI mice, NOTAand NODAGA-conjugated analogs demonstrated lower uptake in normal
organs in tumor-bearing mice biodistribution studies (Figure 19, Table 3
in paper V). Better blood clearance was also observed for these analogs
compared to DOTA- and DOTAGA-coupled compartments. The higher
affinity of NOTA-PEG2-RM26 was reflected in a higher tumor uptake
and resulted in higher tumor-to-non-tumor ratios (Figure 20, Table 4 in
paper V).
51
Uptake, % ID/g
A
8
4
3
NOTA
NODAGA
DOTA
DOTAGA
2
1
B
4
Uptake, % ID/g
v
Sp er
P a le e
nc n
Sm St rea
al om s
l i ac
nt
es h
tin
ki e
dn
M ey
us
cl
e
B
on
e
ng
Li
Lu
B
lo
od
0
NOTA
NODAGA
DOTA
DOTAGA
3
2
1
52
v
Sp e r
Pa lee
nc n
Sm St rea
al om s
l i ac
nt
es h
t in
ki e
dn
M ey
us
cl
e
B
on
e
ng
Li
Lu
B
lo
od
0
Figure 18. The biodistribution results of 68Ga-X-PEG2RM26
(X
=
NOTA,
NODAGA, DOTA and DOTAGA) in NMRI mice at (A) 1
h and (B) 2 h p.i.
2
6
non-blocked
blocked
4
2
0
Uptake, % ID/g
4
68
68
d
lu
ng
liv
sp er
pa lee
nc n
s re
sm tom as
in ac
te h
st
i
ki ne
dn
e
tu y
m
m o
us r
cl
e
bo
ne
bl
oo
Uptake, % ID/g
non-blocked
Uptake, % ID/g
d
lu
ng
liv
sp er
pa lee
nc n
s re
sm tom as
i n ac
te h
st
i
ki ne
dn
e
tu y
m
m o
us r
cl
e
bo
ne
bl
oo
Ga-NOTA-PEG 2-RM26
0
Ga-DOTA-PEG 2-RM26
d
lu
ng
liv
sp er
pa lee
nc n
s re
sm tom as
in ac
te h
st
i
ki ne
dn
e
tu y
m
m o
us r
cl
e
bo
ne
Uptake, % ID/g
6
bl
oo
d
lu
ng
liv
sp er
pa lee
nc n
s re
sm tom as
in ac
te h
st
i
ki ne
dn
e
tu y
m
m o
us r
cl
e
bo
ne
bl
oo
68
68
Ga-NODAGA-PEG 2-RM26
6
non-blocked
blocked
4
2
0
Ga-DOTAGA-PEG 2-RM26
6
non-blocked
blocked
4
2
0
68
Figure 19. The biodistribution and in vivo binding specificity test results of
Ga-X-PEG2-RM26 (X = NOTA, NODAGA, DOTA and DOTAGA) in PC-3
xenografted BALB/c nu/nu mice at 2 h p.i.
53
Tumor-to-organ ratio
150
100
NOTA
NODAGA
DOTA
DOTAGA
40
Figure 20. Tumor-toorgan ratios of 68GaX-PEG2-RM26 (X =
NOTA,
NODAGA,
DOTA
and
DOTAGA) in PC-3
xenografted BALB/c
nu/nu mice at 2 h p.i.
20
0
blo
od lung liver leen reas ach tine ney cle one
b
sp anc stom intes kid mus
p
m
s
Conclusions
This study suggests that the modification of the chelating moiety can
appreciably alter the targeting properties of a peptide. In this study, the
triaza chelators NOTA and NODAGA provided better imaging properties
(higher tumor-to-organ ratios) compared to the tetraaza chelators DOTA
and DOTAGA for 68Ga-labeled PEG2-RM26. 68Ga-NOTA-PEG2-RM26
was the best conjugate, providing significantly higher tumor-to-lung,
tumor-to-kidney and tumor-to-muscle ratios compared with 68GaNODAGA-PEG2-RM26.
54
Conclusions and perspectives
Accurately defining the location and extent of cancer is important factor in
treatment selection. Thus, the ability to visualize disease throughout the
body is increasingly important for treatment planning, and many radiotracers
have been developed for this purpose.
This thesis describes an in vitro and in vivo evaluation of radiolabeled antagonistic BN-analogs for PET and SPECT imaging of GRPR. The main
aims were to develop radiotracers for the visualization of GRPR-expressing
tumors and to optimize their targeting properties using different lengths of
mini-PEG spacers and chelator moieties. It has been shown that BN receptor-based antagonists can perform as well as or even better in terms of high
in vivo tumor uptake and long tumor retention than the corresponding agonists [47, 48]. Despite poor internalization into tumor cells, optimal pharmacokinetics of the antagonists suggests that the consensus of tracer development may be changed for GPCR targeting.
In this thesis, a high affinity, hydrophilic, antagonistic, BN-based imaging
agent, RM26, was used. NOTA, allowing stable labeling with several radionuclides, e.g., 68Ga, 111In and 18F(AlF), was coupled to RM26 via a PEG2
spacer. It was hypothesized that the incorporation of the mini-PEG spacer
would decrease the lipophilicity and thus decrease the non-specific off-target
uptake in healthy organs, particularly in the liver. NOTA-PEG2-RM26 was
labeled with 68Ga for PET and 111In for SPECT imaging, and the targeting
properties of these peptides were directly compared in pre-clinical models.
The significant conclusion from this part of the study was that the radiometal-labeled NOTA-PEG2-RM26 was rapidly cleared from the blood and
whole body (even from receptor positive organs), but tumor uptake was high
and stable, thus resulting in high tumor-to-organ ratios shortly after administration. The biodistribution profile of radiometal-labeled NOTA-PEG2RM26 demonstrated that short-lived positron-emitting isotopes (such as 68Ga
and 18F) could be used for imaging of GRPR-expressing tumors using NOTA-PEG2-RM26. However, tumor-to-organ ratios increased over time,
which indicates that more long-lived positron-emitting radionuclides than
68
Ga might be preferable radioisotopes for PET.
55
To test the hypothesis that a more long-lived label would provide better
contrast and therefore better sensitivity, short-lived 68Ga (T1/2 ~ 68 min) was
exchanged with the longer half-life radionuclide 18F (T1/2 ~ 110 min) for PET
studies. The Al18F-NOTA concept developed by McBride et al [93] was used
for radiolabeling NOTA-PEG2-RM26.
The main observation with regard to the biodistribution of Al18F-NOTAPEG2-RM26 in PC-3 xenografted mice was that the radioactivity retention in
the tumors was not as high as it was for the 111In-labeled counterpart. Therefore, an optimal imaging time for Al18F-NOTA-PEG2-RM26 should be approximately 3 h p.i. Al18F-NOTA-PEG2-RM26 provided higher tumor-toblood, tumor-to-lung, tumor-to-liver, and tumor-to-spleen ratios compared
with 68Ga-NOTA-PEG2-RM26 at 3 h p.i. There was no difference in tumorto-muscle and tumor-to-bone ratios for these two PET imaging agents. Wide
availability and a short positron range in tissue make 18F an attractive label
for NOTA-PEG2-RM26. Al18F-NOTA-PEG2-RM26 might be alternative for
68
Ga-NOTA-PEG2-RM26 if a 68Ga generator is not available.
The literature suggests that the in vivo targeting properties of a tracer are
not determined solely by the receptor targeting moiety of the tracer. These
properties can be radically modified by a spacer or chelator. The influence of
a mini-PEG spacer length and a macrocyclic chelator structure on the targeting properties of 68Ga-labeled RM26 were studied to further optimize its
imaging properties.
The study comparing the biodistribution of 68Ga-labeled RM26 conjugated
with NOTA via a mini-PEG polymer of different lengths showed that the
length of the PEG-spacer had minor influence on the biological outcome
when two, three, four and six ethylene glycol units were compared.
Comparing NOTA, NODAGA, DOTA and DOTAGA as chelators for labeling PEG2-RM26 with 68Ga demonstrated the strong influence of the chelator
on the targeting properties of this BN analog. In this study, triaza chelators
provided appreciably better targeting properties than tetraaza counterparts.
For imaging the local metastases of PC, high ratios of radioactivity in tumors
to radioactivity in blood, muscle and bone are critical. The best variant,
68
Ga-NOTA-PEG2-RM26, provided almost 4-fold higher tumor-to-bone and
tumor-to-blood and more than 7-fold higher tumor-to-muscle ratios compared to the poorer variant, 68Ga-DOTAGA-PEG2-RM26.
The favorable biodistribution of 68Ga-NOTA-PEG2-RM26, including rapid clearance from blood and tissues with physiological GRPR expression and
long retention in GRPR-expressing prostate tumors, observed in the preclini56
cal studies, make it the best candidate for imaging PC among tested variants.
Clinical studies using 68Ga-NOTA-PEG2-RM26 to image PC are currently
planned in Uppsala University Hospital.
SPECT remains a more available clinical imaging modality than PET.
111
In is a commonly used radionuclide for SPECT. We need to take into consideration that the choice of radionuclide is equally important as the choice
of chelator. The substitution of 68Ga with 111In in complex with structurally
different chelates might influence the biodistribution of targeting agents by
interfering in different ways with both on-target and off-target interactions.
Future studies should evaluate the possible effect of different radionuclides
on the pharmacokinetics of the analogs with different chelators.
NOTA is also a suitable chelator for 61Cu and 64Cu, as long-lived PET radionuclides. Imaging with 61Cu- or 64Cu-labeled RM26 can be performed
over several hours or next day because of the relatively long half-life of 61Cu
(3.33 h) and 64Cu (12.7 h). This long half-life would allow for better radioactivity clearance from receptor-expressing organs and provide images with
better contrast. 99mTc (a cheap and widely used radionuclide in nuclear medicine) and 55Co (relatively long-lived PET radioisotope that forms stable
complex with DOTA) should also be considered.
Another important study that needs to be considered is using RM26 in
combination with PSMA inhibitors as dual targeting radiopharmaceuticals
for the diagnosis, staging, and treatment of PC. PSMA inhibitors based on
glutamate-urea-lysine analogs are promising targeting agents that were recently developed [99, 100]. The phase 1 study using two derivatives, 123IMIP-1072 and 123I-MIP-1095, demonstrated the capability of PSMA inhibitors to detect lesions in soft tissue, bone and the prostate gland as early as 14 h after injection [101]. PSMA is expressed by nearly all prostate cancers,
and its expression is further increased in late-stage, poorly differentiated,
metastatic, and hormone-refractory carcinomas [102, 103]. In contrast,
GRPR, with almost 100% expression in primary prostate tumors and over
50% in metastases, is highly expressed in early-stage PC, and its expression
decreases during disease progression [103]. Therefore, a bi-specific heterodimeric molecule addressing both targets (PSMA and GRPR) simultaneously may significantly improve PC imaging and therapy. Targeting vectors
covering two receptor entities, which could be used for the whole period of
PC progression from onset to recurrent PC, might lead to an improved diagnostic sensitivity and therapeutic efficiency.
57
Acknowledgements
Over these past years I have learnt so much from so many and I would like
to thank all those who supported me in my personal journey as well as a
scientific or educational one.
I would like to express my deepest sense of gratitude to my supervisor Anna
Orlova, for her support and her guidance during my work in the cell lab and
animal experiments. Thanks a bunch for the everyday patience and inspiration.
My very special appreciation goes to my co-supervisor Vladimir Tolmachev for his bottomless scientific knowledge, his support and always helpful
advice. I would also like to thank you and Anna for your hospitality and
interesting discussions in and out of research at your home.
Special thanks go to my co-supervisors Bo Stenerlöw, for all the interesting
radiobiology lectures and Irina Velikyan, for sharing her precious
knowledge of 68Ga-labeling, purification and analysis methods.
Many thanks to Ola Åberg for the long hours you spent teaching me Al18FNOTA labeling.
I would like to thank Gunnar Lindeberg and Ulrika Rosenström for our
fruitful collaboration and for providing us with the peptides we investigated
in this thesis.
Thanks to Karl Andersson, Hanna Björkelund and Jonas Stenberg for
their precious help and knowledge in experiments regarding LigandTracer
technology.
Many thanks to all my group-mates at PPP and BMS, especially Bogdan
Mitran who made all (that I don’t remember how many) inhibition experiments joyful, Mohamed Altai who always has simple and understandable
explanations for complicated chemistry problems, Ram Kumar Selvaraju
for all your helps in the imaging studies and of course disturbing me and my
computer every now and then, Jennie Andersson, Maria Rosestedt, Hadis
58
Honarvar, Joanna Strand, Javad Garousi for all your helps in animal
experiments.
Special thanks to my co-workers at PPP for the help and support they offered.
I want to express my love to each and every member of the women’s exclusive group, Tonge, Fernanda, Vicky, Maryam and Ruta. I am feeling
home among you.
Nazila! Sanırım artık seninle Türkçe irtibat kura bilirim. Hastalıkta ve
sağlıkta, İsveç'in karanlık ve soğuk günlerinde bir vefalı koca gibi 
yanımda olduğun için memnunum. Seni her zaman özleyeceğim!
Gamzecik! Senin yerin benim için apayri, Hep ‘’Mesut(la)’’  ol sen!
Many thanks to Maria, who made me meet the sweetest γιαγιά in the world.
We should have asked her to make τραχανάς for us!
Mari and Ankur! Thanks for the cozy evenings with Italian food we had
together. I still remember the sky trip ! Hope to have a chance to repeat it.
Erika, Arash and Anahita, I am really glad to know you. Arash! I promise
one day I will start taking proteins with carbs, but I cannot promise about
Creatine !
Mom. and Dad. What can I say other than thank you! You have given me
the greatest gifts of all, the chance at education and the freedom of choice. I
would not be who and where I am today without your support.
Eshgham! My truly better other half, thanks for your unwavering support,
patience and humor. I can boldly say that ‘’Hamsahoo loves you!’’
59
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Acta Universitatis Upsaliensis
Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Pharmacy 191
Editor: The Dean of the Faculty of Pharmacy
A doctoral dissertation from the Faculty of Pharmacy, Uppsala
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