1. health and fitness
2. disease
3. diabetes

Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Pharmacy 202
[68Ga]Exendin-4: Bench-to-Bedside
PET molecular imaging of the GLP-1 receptor for
diabetes and cancer
RAM KUMAR SELVARAJU
ACTA
UNIVERSITATIS
UPSALIENSIS
UPPSALA
2015
ISSN 1651-6192
ISBN 978-91-554-9323-3
urn:nbn:se:uu:diva-261629
Dissertation presented at Uppsala University to be publicly examined in Fåhraeussalen,
Rudbecklaboratoriet (hus C5), Dag Hammarskjölds väg 20, 751 85, Uppsala, Friday,
23 October 2015 at 09:15 for the degree of Doctor of Philosophy (Faculty of Pharmacy).
The examination will be conducted in English. Faculty examiner: Professor Martin Gotthardt
(Clinical Head of Nuclear Medicine, Radboud University, Nijmegen, Netherlands).
Abstract
Selvaraju, R. k. 2015. [68Ga]Exendin-4: Bench-to-Bedside. PET molecular imaging of the
GLP-1 receptor for diabetes and cancer. Digital Comprehensive Summaries of Uppsala
Dissertations from the Faculty of Pharmacy 202. 72 pp. Uppsala: Acta Universitatis
Upsaliensis. ISBN 978-91-554-9323-3.
Diabetes epidemic is underway. Beta cell dysfunction (BCF) and loss of beta cell mass (BCM)
are known to be key events in its progression. Currently, there are no reliable techniques to
estimate or follow the loss of BCM, in vivo. Non-invasive imaging and quantification of the
whole BCM in the pancreas, therefore, has a great potential for understanding the progression
of diabetes and the scope for early diagnosis for Type 2 diabetes.
Glucagon-like peptide-1 receptor (GLP-1R) is known to be selectively expressed on the
pancreatic beta cells and overexpressed on the insulinoma, a pancreatic neuroendocrine tumor
(PNET). Therefore, this receptor is considered to be a selective imaging biomarker for the
beta cells and the insulinoma. Exendin-4 is a naturally occurring analog of GLP-1 peptide. It
binds and activates GLP-1R with same the potency and engages in the insulin synthesis, with
a longer biological half-life. In this thesis, Exendin-4 precursor, DO3A-VS-Cys40-Exendin-4
labeled with [68Ga], [68Ga]Ga-DO3A-VS-Cys40-Exendin-4 ([68Ga]Exendin-4), was evaluated in
different species models, namely, immune deficient nude mice, rats, pigs, non-human primate
(NHP), and clinically in one insulinoma patient by positron emission tomography (PET), for its
potential in beta cell imaging and its quantification as well as for visualizing the insulinoma.
From internal dosimetry, the possible number of repetitive [68Ga]Exendin-4-PET/CT scans was
estimated.
Pancreatic uptake and insulinoma tumor uptake of [68Ga]Exendin-4 were confirmed to be
mediated by the specific binding to the GLP-1R. Pancreatic GLP-1R could be visualized and
semi-quantified, for diabetic studies, except in rats. Nonetheless, we found conflicting results
regarding the GLP-1R being a selective imaging biomarker for the beta cells. PET/CT scan of the
patient with [68Ga]Exendin-4 has proven to be more sensitive than the clinical neuroendocrine
tracer, [11C]5-HTP, as it could reveal small metastatic tumors in liver. The kidney was the doselimiting organ in the entire species model, from absorbed dose estimation. Before reaching
a yearly kidney limiting dose of 150 mGy and a whole body effective dose of 10 mSv, 2–4
[68Ga]Exendin-4 PET/CT scans be performed in an adult human, which enables longitudinal
clinical PET imaging studies of the GLP-1R in the pancreas, transplanted islets, or insulinoma,
as well as in healthy volunteers enrolled in the early phase of anti-diabetic drug development
studies.
Keywords: PET, [ 68Ga]Exendin-4 ,beta cell imaging ,insulinoma ,dosimetry
Ram kumar Selvaraju, Department of Medicinal Chemistry, Preclinical PET Platform, Dag
Hammarskjöldsv 14C, Uppsala University, SE-751 83 Uppsala, Sweden.
© Ram kumar Selvaraju 2015
ISSN 1651-6192
ISBN 978-91-554-9323-3
urn:nbn:se:uu:diva-261629 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-261629)
It’s Not Who I Am Underneath,
h,
But What I DO,
That Defines Me.
To my family
(Past, present, and future)
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.
Selvaraju RK, Velikyan I, Johansson L, Wu Z, Todorov I, Shively J,
Kandeel F, Korsgren O, Eriksson O. In vivo imaging of the glucagon
like peptide 1 receptor in the pancreas with 68Ga-Labeled DO3AExendin-4. J Nucl Med, 2013; 54(8):1458-63.
Nalin L*, Selvaraju RK*, Velikyan I, Berglund M, Andréasson S,
Wikstrand A, Rydén A, Lubberink M, Kandeel F, Nyman G,
Korsgren O, Eriksson O, Jensen-Waern M. Positron Emission Tomography imaging of the glucagon like peptide-1 receptor in healthy
and streptozotocin-induced diabetic pigs. Eur J Nucl Med Mol Imaging, 2014; 41(9):1800-10. *Equal Contribution
Selvaraju RK, Velikyan I, Asplund V, Johansson L, Wu Z, Todorov
I, Shively J, Kandeel F, Eriksson B, Korsgren O, Eriksson O. Preclinical evaluation of [68Ga]Ga-DO3A-VS-Cys40-Exendin-4 for imaging of insulinoma. Nucl Med Biol, 2014; 41(6):471-6.
Eriksson O*, Velikyan I*, Selvaraju RK, Kandeel F, Johansson L,
Antoni G, Eriksson B, Sörensen J, Korsgren O. Detection of metastatic insulinoma by positron emission tomography with
[68Ga]Exendin-4 - a case report. J Clin Endocrinol Metab, 2014;
99(5):1519-24. *Equal contributions.
Selvaraju RK*, Bulenga TN*, Espes D, Lubberink M, Sörensen J,
Eriksson B, Estrada S, Velikyan I, Eriksson O. Dosimetry of
[68Ga]Ga-DO3A-VS-Cys40-Exendin-4 in rodents, pigs, non-human
primates and human – repeated scanning in human is possible. Am J
Nucl Med Mol Imaging, 2015; 5(3): 259–269.*Equal contributions.
Reprints were made with permission from the respective publishers.
Additional contributions
1. Velikyan I, Bulenga TN, Selvaraju RK, Lubberink M, Espes D,
Rosenström U, Eriksson O. Dosimetry of [177Lu]-DO3A-VSCys40-Exendin-4 – impact on the feasibility of insulinoma internal radiotherapy. Am J Nucl Med Mol Imaging, 2015; 5(2):10926.
2. Eriksson O, Selvaraju RK, Johansson L, Eriksson JW, Sundin A,
Antoni G, Sörensen J, Eriksson B, Korsgren O. Quantitative Imaging of Serotonergic Biosynthesis and Degradation in
the Endocrine Pancreas. J Nucl Med, 2014; 55(3):460-5.
3. Eriksson O*, Espes D*, Selvaraju RK, Jansson E, Antoni G,
Sörensen J, Lubberink M, Biglarnia AR, Eriksson JW, Sundin A,
Ahlström H, Eriksson B, Johansson L, Carlsson PO, Korsgren O.
The positron emission tomography ligand [11C]5-Hydroxytryptophan can be used as a surrogate marker for the human endocrine pancreas. Diabetes, 2014; 63(10):3428-37. *Equal contributions.
4. Eriksson J, Åberg O, Selvaraju RK, Antoni G, Johansson L and
Eriksson O. Strategy to develop a MAO-A resistant 5-hydroxy-L[ș-11C]tryptophanisotopologue based on deuterium kinetic isotope effects. EJNMMI Res, 2014; 4:62.
5. Eriksson O, Selvaraju R, Borg B, Asplund V, Estrada S, Antoni
G. 5-Fluoro->ȕ-¹¹C]-L-tryptophan is a functional analogue of 5hydroxy->ȕ-¹¹C]-L-tryptophan in vitro but not in vivo. Nucl Med
Biol, 2013; 40(4):567-75.
List of other Publications
In addition to the enclosed publications related to the thesis, the author has
also collaborated with other research groups
1. Tugues S, Roche F, Noguer O, Orlova A, Bhoi S1, Padhan N,
Akerud P, Honjo S, Selvaraju RK, Mazzone M, Tolmachev V,
Claesson-Welsh L. Histidine-Rich Glycoprotein uptake and turnover is mediated by mononuclear phagocytes. PLoS One, 2014;
9(9):e107483.
2. Honarvar H, Strand J, Perols A, OrlovaA, Selvaraju RK,
Karlström AE, Tolmachev V. Position for site-specific attachment of a DOTA chelator to synthetic affibody molecules has a
different influence on the targeting properties of 68Ga-, compared
to 111In-labeled conjugates. Mol Imaging, 2014; 13(0):1-12.
3. Rosik D, Thibblin A, Antoni G, Honarvar H, Strand J, Selvaraju
RK, Altai M, Orlova A, Eriksson Karlström A, Tolmachev. Incorporation of a triglutamyl spacer improves the biodistribution
of synthetic Affibody molecules radiofluorinated at the Nterminus via oxime formation with 18F-fluorobenzaldehyde. Bioconjugate Chem, 2014; 25 (1): 82–92.
4. Orlova A, Malm M, Rosestedt M, Varasteh Z, Andersson K, Selvaraju RK, Altai M, Honarvar H, Strand J, Ståhl S, Tolmachev
V, Löfblom J. Imaging of HER3-expressing xenografts in mice
using a 99mTc(CO)3-HEHEHE-Z08699 affibody molecule. Eur J
Nucl Med Mol Imaging, 2014; 41(7):1450-9.
5. Spiegelberg, D, Kuku, G, Selvaraju R, Nestor, M. Characterization of CD44 variant expression in head and neck squamous cell
carcinomas. Tumor Biol, 2014; 35(3): 2053–2062.
6. Altai M, Strand J, Rosik D, Selvaraju RK, Eriksson Karlström A,
Orlova A, Tolmachev V. Influence of Nuclides and Chelators on
Imaging Using Affibody Molecules: Comparative Evaluation of
Recombinant Affibody Molecules Site- Specifically Labeled with
68
Ga and 111In via Maleimido derivatives of DOTA and
NODAGA. Bioconjug Chem, 2013; 24 (6):1102–1109.
7. Strand J, Honarvar H, Perols A, Orlova A, Selvaraju RK, Eriksson Karlström A, TolmachevV. Influence of macrocyclic chelators on the targeting properties of 68Ga-labeled synthetic Affibody molecules: comparison with 111In-labeled counterparts.
PLoS One, 2013; 8(8):e70028.
8. Varasteh Z, Velikyan I, Lindeger G, Sörensen J, Larhed M,
Sandström M, Selvaraju RK, Tolmachev V, Orlova A. Synthesis
and characterization of a high-affinity NOTA-conjugated
bombesin antagonist for GRPR-targeted tumor imaging. Bioconjug Chem, 2013; 24(7):1144-53.
9. Perols A, Honarvar H, Strand J, Selvaraju R, Orlova A,
Karlström AE, Tolmachev V. Influence of DOTA chelator position on biodistribution and targeting properties of (111)In-labeled
synthetic anti-HER2 affibody molecules. Bioconjug Chem, 2012;
23(8):1661-70.
Front cover: [68Ga]Exendin-4’s in vitro evaluation in INS-1 tumor autoradiography (ARG), and by in vivo PET/CT imaging in mice, rat, pig, nonhuman primate(NHP) and in human.
Supervisors, faculty opponent, and members
of the committee
Supervisors
Olof Eriksson, Ph.D., M.Sc.
Assoc. Prof., Preclinical PET Platform
Department of Medicinal Chemistry
Uppsala University, Uppsala, Sweden
Olle Korsgren, M.D., Ph.D.
Professor of Cell Transplantation
Department of Immunology, Genetics and Pathology
Uppsala University Hospital, Uppsala, Sweden
Lars Johansson, Ph.D.
Senior lecturer, Department of Surgical Sciences, Radiology
Uppsala University Hospital, Uppsala, Sweden
Faculty Opponent
Martin Gotthardt, M.D., Ph.D.
Professor, Clinical Head of Nuclear Medicine
Radboud University Nijmegen, Netherlands
Members of the committee
Ulf Ahlgren, Ph.D.
Professor, Umeå Centre for Molecular Medicine (UCMM)
Umeå University, Umeå, Sweden
Sven-Erik Strand, M.D., Ph.D.
Professor, Medical Radiation Physics
Lund University, Lund, Sweden
Andrea Varrone, M.D., Ph.D.
Senior lecturer, Department of Clinical Neuroscience (CNS)
Karolinska Universitetssjukhuset, Stockholm, Sweden
Contents
Overview of the Thesis ................................................................................. 13
Overview of the included publications ..................................................... 13
Paper I .................................................................................................. 13
Paper II ................................................................................................ 13
Paper III ............................................................................................... 13
Paper IV ............................................................................................... 13
Paper V ................................................................................................ 14
Introduction ................................................................................................... 15
Islets of Langerhans ................................................................................. 15
Diabetes ............................................................................................... 16
BCM and onset of diabetes .................................................................. 17
Beta cell imaging ................................................................................. 18
Insulinoma ........................................................................................... 19
GLP-1R .................................................................................................... 20
Exendin-4 ................................................................................................. 21
PET ........................................................................................................... 21
PET image acquisition ......................................................................... 22
PET in vivo tracer quantification ......................................................... 23
Internal dosimetry .................................................................................... 25
Material and Methods ................................................................................... 27
[68Ga]Ga-DO3A-VS-Cys40-Exendin-4 Synthesis (I–V) ........................... 27
Animal Studies (I–V) ............................................................................... 27
Clinical Study (IV) ................................................................................... 28
Cell Culture (III) .................................................................................. 28
Animal Models (I–III) ......................................................................... 28
In vitro autoradiography (III)............................................................... 29
Ex vivo autoradiography (V)................................................................ 29
Immunofluorescence (IF) insulin staining (V) .................................... 30
Insulin staining (II) .............................................................................. 30
Ex vivo organ distribution (I, III, and V) ............................................. 31
PET/CT Imaging (I–IV) ...................................................................... 31
PET/CT Image analysis (I–V) .................................................................. 32
Dosimetry (V) ...................................................................................... 33
Statistical Analysis ................................................................................... 34
Results........................................................................................................... 35
[68Ga]Ga-DO3A-VS-Cys40-Exendin-4 Synthesis..................................... 35
Beta cell Imaging and quantification........................................................ 36
Rat Model (I and V)............................................................................. 36
NHP model (I) ..................................................................................... 37
Pig model (II)....................................................................................... 39
Insulinoma Imaging.................................................................................. 41
Murine model (III) ............................................................................... 41
Clinical Study (IV) .............................................................................. 43
Dosimetry (V) .......................................................................................... 45
[68Ga]Exendin-4 in different animal models........................................ 45
Residence time and estimated absorbed dose ...................................... 47
Discussion ..................................................................................................... 49
[68Ga]Exendin-4 for diabetes.................................................................... 49
Importance of specific radioactivity .................................................... 49
Choice of animal model ....................................................................... 50
Uptake of [68Ga]Exendin-4 in the pancreas of different species ......... 51
Is GLP-1R a BCM biomarker? ............................................................ 53
[68Ga]Exendin-4 for islet transplantation. ............................................ 54
68
[ Ga]Exendin-4 for Cancer ..................................................................... 55
[68Ga]Exendin-4 for insulinoma preoperative localization .................. 55
Potential use of [177Lu]-Exendin-4 for radiotherapy ............................ 56
68
[ Ga]Exendin-4 dosimetry ...................................................................... 58
[68Ga]Exendin-4 versus other Exendin-4 derivatives .......................... 58
Pancreatic model for internal dosimetry .............................................. 58
Stability of [68Ga]Exendin-4..................................................................... 60
Conclusions ................................................................................................... 62
Acknowledgements ....................................................................................... 63
References ..................................................................................................... 67
Abbreviations
1TCM
2 TCM
3-D
[11C]
[11C]DTBZ
[18F]
[64Cu]
[68Ga]
[68Ga]Exendin-4
[99mTc]
[111In]
[177Lu]
BCF
BCI
BCM
Bq
CT
Cp
Ct
cps
DOPA
DPPIV
EC
FOV
FDG
GLP-1
GLP1-R
HTP
IV
K1
k2
k3
k4
LOR
Molwt
MRI
1-Tissue compartment modeling
2-Tissue compartment modeling
3-dimensional
Carbon-11
[11C]-dihydrotetrabenazine
Flourine-18
Copper-64
Gallium-68
[68Ga]Ga-DO3A-VS-Cys40-Exendin-4
Technetium-99m
Indium-111
Lutetium-177
Beta cell function
Beta cell imaging
Beta cell mass
Becquerel
Computed tomography
Plasma concentration
Tissue concentration
Counts per Second
Dihydroxyphenylalanine
Dipeptidyl peptidase-4 protease
Electron capture
Field-Of-View
Fluorodeoxyglucose
Glucagon like peptide-1
Glucagon like peptide-1 receptor
Hydroxytryptophan
Intravenous
Influx rate constant (plasma to tissue)
Efflux rate constant (tissue to plasma)
Rate constant (kon)
Rate constant (koff)
Line of response
Molecular weight
Magnetic resonance imaging
NHP
PBS
p.i
PET
PNETs
PVE
ROI
SPECT
SRA
STZ
SUV
T1D
T2D
TAC
Tris
VOI
vB
vT
WB
Non-human primate
Phosphate-buffered saline
Post injection
Positron emission tomography
Pancreatic neuroendocrine tumors
Partial volume effect
Region of interest
Single photon emission computed tomography
Specific radioactivity
Streptozotocin
Standardized uptake value
Type1 diabetes
Type 2 diabetes
Time activity curve
Tris(hydroxymethyl)aminomethane
Volume of interest
Fractional volume
Distribution volume
Whole body
Overview of the Thesis
To evaluate the potential of a new PET tracer [68Ga]Exendin-4 for its efficacy in imaging and quantification of the pancreatic BCM and for visualizing
insulinoma, in vivo.
Overview of the included publications
Paper I
To image and quantify the GLP-1R in the pancreatic beta cells, using
[68Ga]Exendin-4. The in vivo specificity of the tracer to the beta cells was
first evaluated by dose escalation studies in normal rats and by comparing
the tracer uptake in normal rats and in Streptozotocin (STZ)-treated diabetic
rats (organ distribution) at baseline dose. The process of in vivo visualization
and quantification of GLP-1R in the pancreas was assessed in non-diabetic,
NHP animal model by dose escalation studies (PET/CT).
Paper II
To assess the feasibility of non-invasive visualization and quantification of
the pancreatic GLP-1R in non-diabetic pig and STZ-induced diabetic pig
with [68Ga]Exendin-4. Specifically, perfusion in the pancreas, specific BCM
uptake of the tracer, imaging and kinetics of the tracer were investigated in a
clinically relevant diabetic animal model, pig.
Paper III
To investigate the potential of [68Ga]Exendin-4 for visualizing the insulinoma at preclinical setting. The tracer was studied in immunodeficient nude
mice (nu/nu balb C), INS-1 xenograft, and PANC-1 xenograft (nu/nu balb
C), by ex vivo biodistribution. Insulinoma imaging with [68Ga]Exendin-4
was compared with clinical neurendocrine marker [11C]5-HTP in small animal PET/CT scan.
Paper IV
To visualize the insulinoma metastasis using [68Ga]Exendin-4 in a patient
diagnosed with insulinoma. Ultrasound, CT, and [11C]5-HTP PET/CT failed
to conclusively detect the extent of the small metastatic lesions. This was a
compassionate care case study performed in only one female patient.
13
Paper V
To estimate the possible number of repetitive PET/CT scans in an adult human with [68Ga]Exendin-4 using dosimetry, before reaching a yearly limiting dose in a critical organ and whole body effective dose. Retrospective
kinetic data of the tracer from rats, pigs, NHPs, and patient at baseline peptide dose were used for computation.
14
Introduction
Islets of Langerhans
The islets of Langerhans, named after its discoverer Paul Langerhans, are the
endocrine components of the pancreas. They are responsible for the maintenance of glucose homeostasis, primarily by secreting the hormones insulin
(by beta cells) and glucagon (by alpha cells) into the bloodstream. The size
and components of an individual pancreatic islet are illustrated in figure 1. In
all species, the beta cells are the most abundant (50–80%), followed by the
alpha cells (20–30%). The rest of the cell types such as delta cell, polypeptide (PP) cells, etc. constitute the remaining [1].
Islets of Langerhans constitute approximately 2% of the pancreas, and
most of the islets are small in diameter (~50–500 μm). In a healthy pancreas
(~100g), approximately one million islets are distributed heterogeneously,
and there has been reports showing that the concentration of islets tend to be
higher in the tail (cauda) than in the head (caput) or body (corpus) [2-3].
The collective beta cell numbers in the islets are referred to as beta cell
mass (BCM), and the proper release of the insulin in response to the glucose
levels in the blood is referred to as beta cell function (BCF). By these definitions, this regulation declines with the onset of diabetes. In both type 1 diabetes (T1D) and type 2 diabetes (T2D), there is an impaired glucose mediated insulin release, insulin resistance, and decrease in the BCM [4-6].
Exocrine
Ductal
System
~98%
Islets of Langerhans (~2%)
Liver
Alpha cells
(glucagon)
Stomach
ch
Beta cells
(insulin)
Pancreas
Large
Intestine
Delta cells
(somatostatin)
Small
Intestine
50-500 μm
PP cells
(pancreatic polypeptide)
Capillaries
Figure 1.Schematic representation of human islets of Langerhans and its composition.
15
Diabetes
Diabetes mellitus, commonly referred to as diabetes, is a disease characterized by unusually high glucose levels in the blood and of several metabolic disorders noted by excessive urination, persistent thirst, and increased
hunger. It is a long-standing disease affecting mankind. In its earliest record
in ancient Hindu writings, are dated back to 1500 BC, Indian physicians
called it madhumeha (‘honey urine’), as ants were attracted to the urine of
people with a mysterious emaciating disease [7-8].
Diabetes can be broadly classified into three categories: type 1 (T1D),
type 2 (T2D), and gestational diabetes. These categories are compared in
table 1.
Table 1.Comparison between the different categories of Diabetes.
Features
T1D[9]
T2D[9]
Gestational[9-11]
Pattern of onset
Acute
Gradual
During pregnancy
Pathogenesis
Autoantibodies
Autoimmunity?
Present
Insulin resistance
Rare
Insulin resistance
Rare
Age of onset
Mostly Young
Mostly in adult
First detected in
pregnancy
Body Habitus
Normal/thin
Generally obese
Often obese
Ketoacidosis
Common
Rare
Rare
Endogenous insulin
Absolute insulin
deficiency
Decreased
(Insulin resistant)
Commonly insulin
resistant-placental
hormones
Beta cell mass
Decreasing
Increasing/mild beta
cell depletion
Similar to T2D
Prevalence
Less
More prevalent
Becoming more
prevalent
Family history
Indefinite
Strong
Strong with T2D
Gene link
HLA linked
No HLA association
No HLA association
Treatment
Insulin administration
required for survival;
islet transplantation
Healthy diet and
lifestyle; may require
insulin injection or
oral hypoglycemic
tablets
Healthy diet and
lifestyle; medication
based on limited
affect to the growing
baby
In 2014, 400 million people were reported to have diabetes worldwide and it
is expected to reach 600 million by 2035(http://www.idf.org/diabetesatlas).
16
BCM and onset of diabetes
Beta cell mass (BCM)
Diabetes is a rising epidemic throughout the world. Beta cell dysfunction
and a decrease in its mass are the key events in the prognosis of diabetes. In
both T1D and T2D, there is a loss of BCM by ~80–95% and ~60% in time
(figure 2), along with dysfunctional glucose mediated insulin secretion and
insulin resistance [12-16].
In humans, BCM grows rapidly during childhood and its differentiation
into insulin secreting BCM is complete within 5 years of age [17].In T1D,
the BCF and its mass proportionally decline over many years with the onset
of hyperglycemia [4, 9]. Although the autoimmune mediated onset of T1D is
still debatable, the reasons or factors for “good guys” turning “bad” are still
under investigation. In T2D, the BCF progressively declines over many
years before alterations in its BCM. Currently, there is no methodology to
understand the extent to which this loss of BCF is associated with BCM at
its onset, in vivo. The onset of T2D is poorly defined and it has been estimated that most patients with T2D are not diagnosed for several years after
its onset [9, 18].
NDB
T2D
T1D
Juvenile
Adult
Time
Figure 2. Author’s schematic presentation of the change in the pancreatic BCM at onset
ofT1D and T2D with time, compared to a non-diabetic (NDB) pancreas.
Although measurements of circulating insulin and C-peptide may provide
indirect assessment of the BCM, it lacks sensitivity regarding subtle changes
in the BCM on onset of disease, at least in T2D. At present, the BCM in the
pancreas can only be determined by insulin staining for beta cells in the pan-
17
creatic biopsies. Pancreatic dissection is complicated, dangerous, and further
autopsy is usually limited to selected parts of the pancreas. Insulin staining
of these sections may not represent the total pancreatic BCM. Also, the pancreas is among the organs that undergo autolysis after death. This may affect
the BCM estimation in postmortem analysis. Hence, there are no practical or
reliable ways to assess the complete BCM in order to understand the subtle
changes in the onset of diabetes. Thus, a method for non-invasive imaging of
the entire BCM is urgently needed in order to study the diabetes progression.
Beta cell imaging
The non-invasive imaging of the native beta cells in the pancreas is analogous to the phrase, “looking for a needle in a haystack.” As described earlier
in the section “Islets of Langerhans,” a healthy human pancreas in adults
weighs around 100g and the islet of Langerhans comprise merely 2% of the
whole pancreas. Islets, between 50–500 μm in diameter, are composed of
~60% beta cells. Thus, the target is less than 1% of the whole pancreas, heterogeneously distributed in the organ and is expected to alter with the onset
of diabetes.
Its anatomical limitation (size and level of target expression) leads to further challenges in the development of ligand (specific activity and stability)
and drawbacks in currently available non-invasive imaging modalities such
as magnetic resonance imaging (MRI), PET, single photon emission tomography (SPECT), and CT (spatial resolution)[19].
However, in the past few years, researchers have been developing noninvasive imaging methods for visualizing the native pancreatic beta cells and
the transplanted islets. Some of the tracers/targets investigated in the clinical
settings and large animal models are summarized in table2.
Table 2.Tracers and targets evaluated in clinical settings.
Imaging Tracer
Modality
Target
Target function
SPECT
[111In]-DTPA-Exendin-3[20]
GLP-1R
Insulin release
PET
[11C]5-HTP[21]
Serotonin Precursor
Serotonin pathway
VMAT2
Monoamine transporter
Glucose metabolism
Glucose metabolism
VMAT2
Monoamine transporter
11
[22]
PET
[ C]DTBZ
PET
[18F]F-FDG[23]
PET
18
[ F]FP-DTBZ
[24]
Other promising targets for BCI that were studied at preclinical settings are:
Transmembrane protein 27 (TMEM27) [25, 26]; Polysialylated Neural Cell
Adhesion Molecule (PSA-NCAM) [27, 28]; Zinc (Zn2+) agents [29]; Sulfonylurea receptors [30-32]; Manganese (Mn2+) [33, 34]; and sphingomyelin
(SM) [35].
18
The ideal target and tracers for BCI are still under investigation. Such
non-invasive imaging and possible quantification of the BCM will be a major breakthrough and a key tool for understanding the etiology of diabetes.
This would allow for early diagnosis of T2D and give a scope for therapies
or allow one to change their lifestyle in order to recover, as these individuals
have a larger functioning BCM, thus, ultimately specifying personalized
treatments. Understanding the disease would allow clinicians to assess the
correlation between BCF and BCM, in vivo, as well as evaluate the success
of islet transplantation; moreover, in cancer research, such knowledge would
make it possible to diagnose the beta cell derived tumors such as insulinoma.
Insulinoma
Insulinoma is the most common form of pancreatic neuroendocrine tumors
(PNETs) of beta-cell origin. Although rare in the overall population, insulinomas are the most common cause of hyperinsulinemic hypoglycemia in
the adult population [36- 38]. The incidence has been reported as higher in
autopsy studies (0.8% to 10%), suggesting that these tumors frequently remain undiagnosed [39, 40].A major proportion of tumors are benign (>90%)
in the pancreas, and a small percentage (<10%) is metastasized. The most
common sites of metastasis are the liver and the lymph nodes. The prognosis
of survival for patients with liver metastasis has been estimated to be less
than 2 years [41].
The diagnosis of insulinoma includes inappropriately high insulin/proinsulin (>35 mEquivalent/L; normal <11), C-peptide (>1.5 nmol/L;
normal, <1.5), and low glucose (<2.5 mmol/L; normal, >4) during prolonged
fasting (up to 72 h) [42]. Such severe hypoglycemia can result in seizures or
unconsciousness. Once the diagnosis of an insulinoma is positive, localization procedures are the next step. Curative surgery is the golden standard
treatment, if these lesions can be localized. Hence, preoperative imaging is
crucial for surgery.
Due to the small size of the tumors (<1–2 cm), they are often difficult to
detect by ultrasound (US), CT, and MRI. Functional imaging (SPECT/PET)
with octreotide is incomprehensible in 50% of the cases since benign tumors
have low somatostatin receptor expression (subtype 2 and 5) [43]. [11C]5HTP-PET/CT imaging, has been shown to be a promising and sensitive tracer for neuroendocrine tumors that surpass the octreotide scintigraphy and
SPECT/CT [44].Nonetheless, in a recent study, [11C]5-HTP failed to detect
two out of six insulinoma, although it had higher sensitivity than the other
imaging methods [45].
Glucagon-like peptide-1 receptor (GLP-1R) is known to be expressed in
human beta-cells and highly overexpressed in insulinomas [46, 47]. Hence,
GLP-1R is considered to be an imaging biomarker that could aid in the de-
19
velopment of a tracer for beta cell imaging and for visualizing the occult
insulinoma.
GLP-1R
GLP-1Rs are G protein-coupled receptors of the glucagon receptor family.
GLP-1 receptors are expressed both in the central (brainstem and hypothalamus) and the peripheral (pancreatic islet cells and heart) organs, figure3
[48]. This thesis work focuses on their peripheral function. GLP-1R is
known to be expressed in the pancreatic beta cells. They mediate the functional connection between the intestine and the islets of Langerhans in the
pancreas.
On ingestion of food, the glucagon-like peptide-1 (GLP-1) released by the
intestinal L-cell activates the GLP-1R on the islet beta cells. The activation
of the GLP-1/GLP-1R stimulates the adenylyl cyclase pathway, which results in increased insulin synthesis and release of insulin, in a glucosedependent manner [49-51].
Figure 3.GLP-1/GLP-1R roles in the central and peripheral tissues.
Therefore, the GLP-1R has been suggested as a potential target for the
treatment of diabetes. GLP-1Rs are also highly expressed on the insulinoma,
the functional tumors of the pancreatic beta cell origin. This facilitates the
precise localization of the lesions and staging for adequate patient management.
20
Exendin-4
GLP-1 has a short biological half-OLIH”PLQGXHWRFOHDYDJHE\WKHFLUFulating protease DPPIV [52]; therefore, biologically stable GLP-1 analogues
have been proposed as a potential imaging agent. Exendin-4 is a peptide of
39 amino acids, which have been isolated from the venom of the lizard Heloderma Suspectum (Gila monster) [53].
It is a naturally occurring analog of the GLP-1, which binds and activates
the GLP-1 receptor with the same potency as the GLP-1. Unlike GLP-1,
Exendin-4 is resistant to cleavage by protease dipeptidyl peptidase 4 (figure
4) and has a markedly increased biological half-life in vivo [54, 55].It is
currently approved for the treatment of T2D [56].
Figure 4. GLP-1 (7-37)and Exendin-4 amino acid sequence.
For the past few decades, several derivatives of GLP-1 analogues for MR
and nuclear imaging of native beta cells, transplanted islets, and beta cell
derived tumor insulinoma have been investigated [57-64].
In the present investigation, Exendin-4 precursor, DO3A-VS-Cys40Exendin-4 labeled with 68Ga, [68Ga]Ga-DO3A-VS-Cys40-Exendin-4
([68Ga]Exendin-4) was evaluated in different species models, namely, immune deficient nude mice, rats, pigs, NHP and clinically, in one insulinoma
patient by PET/CT, for its application in the BCI and its quantification as
well as for visualizing the insulinoma.
The development of an imaging agent based on the ligand to GLP-1R
such as Exendin-4 and positron emitting radionuclide would provide means
for specific, sensitive, quantitative, and non-invasive diagnosis using PET.
The use of a generator produced positron emitting 68Ga [physical half-life
(t1/2) = 68.3 min, 89% pRVLWURQ (PLVVLRQ ȕ+), and electron capture (EC) =
11%] radionuclide would afford worldwide accessibility to the agent.
PET
PET has in-built design and instrumentation to acquire tomographic data,
attenuation correction (assisted with computed tomography), and to register
dynamic studies. It uses positron emitting radio-isotopes such as carbon-11,
nitrogen-13, oxygen-15, and fluorine-18 of naturally occurring elements
21
such as carbon-12, nitrogen-14, oxygen-16, and fluorine-19. These can be
labeled without compromising the biocompatibility of the ligand.
Electronic collimation increases its sensitivity, compared to gamma cameras. The convergence of PETs higher sensitivity and the high specific activity of PET radionuclide achievable by labeling chemistry enable the administration of doses less than a few micrograms into the subjects for qualitative
and quantitative imaging. This approach known as “micro-dosing” triggers
merely a cellular response, without causing any pharmacological effects
[65]. This technique can aid in leading drug selection and its rapid translation from bench to bedside.
However, the disadvantage of PET is its cost. Most commonly used PET
isotopes such carbon-11 and fluorine-18 require an on-site cyclotron, and
detectors of PET are costlier than that of SPECT. Nevertheless, PET is widely used in preclinical research studies and in clinical settings. Generator produced PET isotopes such as 68Ga is supplementing the PET’s popularity in
the developing world.
PET image acquisition
PET is a functional imaging modality. It uses the unique decay characteristics of
radionuclides by positron emission. These radionuclides are produced in a cyclotron or a generator and are then labeled to a ligand of biological interest. The
labeled compound is introduced into the body, usually by intravenous injection,
and is distributed in the tissues depending on its biochemical properties. Data
acquisition begins only a few seconds to minutes after the tracer administration,
depending on the study design. When an unstable atom on a particular molecule
decays, a positron is ejected from the nucleus, which annihilates with an electron, ultimately leading to the emission of high-energy photons (511 keV) that
have a good probability of escaping from the body.
A typical clinical PET scanner’s open gantry consists of a set of detectors,
arranged in a circle at a distance of 80 cm in diameter from the center and
approximately 15 cm in length. The subject is positioned in the center of the
gantry covering the organs of interest in the field of view (figure 5).
The detectors are designed to utilize the concept that two photons sensed
by two opposed detectors in the ring are likely to have originated from a
single annihilation event in the body, somewhere along a line of response
between the two detectors, within a timeframe of 8–12 nanoseconds. Such a
simultaneous detection is termed a “coincidence.” The principle of coincidence detection provides a so-called “electronic collimation.” This electronic
collimation makes PET scanners much more efficient than gamma cameras,
with markedly better signal to noise ratios leading to better spatial resolution. Photo multiplier tubes or photodiodes connected to the detectors aid in
converting these coincidence photons into an electrical signal that can be fed
to subsequent electronics.
22
PET Detectors
Tracer
acer
Coincidence
Processing Units
Radionuclide
Radio
Peptide
Sinogram or
List-mode Data
A
A
Z
Z-1
0
;ĺ<H
+1
0
0
HHĺȖ
+1
-1
Image Reconstruction & Analysis
Figure 5. Schematic representation of the PET imaging protocol.
These events are corrected for a number of factors and then reconstructed
using mathematical algorithms.
The output of the reconstruction process is a three-dimensional (3-D) image, where the signal intensity in any particular image voxel is proportional
to the amount of the radionuclide (hence, the amount of the labeled molecule
to which it is attached) in that voxel, upon appropriate calibration. By taking
a time sequence of images, the tissue concentration of the radiolabeled molecules as a function of time is measured, and with appropriate mathematical
modeling, the rate of specific biological processes can be determined.
PET in vivo tracer quantification
The prospect of tracer quantification in vivo by PET examination is its major
highlight. The quantification of tracer uptake can aid in the diagnosis and
has the potential to stratify responding and non-responding patients in therapy studies. When a PET camera is properly calibrated, in a reconstructed
PET image, the signal intensity in any particular image voxel is linearly
proportional to the amount of the radionuclide in term of becquerals (Bq)
and hence the tracer concentration in that voxel. Thus, the 3-D images can
estimate the concentration of the tracer in the tissue or the organ as Bq/g.
Commonly used ‘semi-quantitative’ methods for analyzing the tracer activity are presented in equation 1 and 2.
23
1. Percentage injection dose per gram (%ID/g)
(1)
%ID/g = ‫ܦ‬t/Wt ‫ כ‬1/Dinj ‫ כ‬100 (%/g)
Where, Dt =Tissue uptake (Bq); Wt= Tissue weight (g); Dinj= Dose injected
(Bq)
2. Standardized Uptake Value (SUV)
SUV = (‫ܦ‬t/Wt)/(Dinj/Wp)
(2)
Where, Dt=Tracer uptake in tissue (Bq); Wt= Tissue weight (g); Dinj= Dose injected
(Bq); and Wp= weight of the patient.
%ID/g is commonly used for small animal studies. Both parameters are referred to as semi-quantitative because they represent the tracer uptake in a
selected tissue or organ at specific time points, rather than measuring the rate
of tracer uptake in tissue or organ of interest. Additionally, they do not consider metabolites or other functional data such as blood flow or perfusion
rate in the organs that might affect the tracer uptake in the tissue. For example, the glucose utilization rate and the SUV estimation for the [18F]FDG
scan in patients undergoing chemotherapy [66].
Kinetic modeling has the potential to extract a physiological process and
respond to the above limitations [67]. It uses mathematical compartments to
represent the delivery and the tracer behavior. Metabolic (parent or metabolite), biochemical (bound or unbound), and physical space (blood,
intra, or extra-cellular) are used as compartCapillary membrane
ments, and along
with rate conTissue
stants, to estik1
k3
mate the strength
[18F]FDG
18
[18F]FDG
[ F]FDG-6-P
in tissue
in Plasma
of the tracerin tissue
k2
k4
receptor
interaction or the
Figure 6. Two-tissue compartment modeling of the [18F]FDG.
rate of the tracer
The arterial plasma time course of the [18F]FDG activity and dytransport across
namically acquired tracer uptake in the organs (VOI) from the PET the cell mem
data are used as input functions in the compartmental modeling. The -brane, etc.
rate constants (K1& k2) represent the diffusion or glucose transport
figure 6.
across the membrane and (k3& k4) represent the rate of phosphorylation and dephosphorylation of the intracellular FDG.
24
Internal dosimetry
In any use of ionizing radiation, one must minimize the risks from radiation
while allowing its beneficial applications. The quantity-absorbed dose, defined as the energy deposited per unit mass of organ, unit J/kg or Gray (Gy),
is used in the prediction of such harmful biological effects. Dosimetry is the
measurement of radiation dose to organs from a radioactive source.
To estimate the organ dose in internal dosimetry, the quantification of
tracer uptake is of crucial importance and the possibility of such by PET
scans adds to its advantage. Nevertheless, data collection is limited to a few
time points from PET scans, which may increase the uncertainty in the dose
calculations.
Other factors affecting the dose estimation are half-life of the radionuclide, type of decay, energy of the gamma radiation, and crossfire from the
nearby organs.
The estimation of the absorbed dose by dosimetry in/to human has the following steps:
1. Decay corrected values to “decay-uncorrected” values
Generally, tracer uptake in the organ is decay-corrected to a reference point, most often to the time of injection. These values are decay “uncorrected” for true uptake in an organ at a given time point.
2. Extrapolation of animal data
The tracer kinetics data gathered from the animal studies can be
used to predict the tracer uptake in the human. This is commonly
performed, according to the kg/g method equation 3 [68].
൤
g ୭୰୥ୟ୬
%
= SUV୅ x ቆ
൨
ቇ
organ ୦୳୫ୟ୬
kg ୵ୣ୧୥୦୲
(3)
୦୳୫ୟ୬
Where, SUVA is SUVs in the animal organs; g organ and kg weight
are, respectively, standard organ weight and standard total body
weight of a human phantom.
3. Residence time (ȫ) calculation
Residence time is defined as the ratio of cumulative activity (Ã) in the
source organ over time, normalized to the injected dose (A0). Ã is calculated as the area under the TAC curve from the above step by trapezoidal approximation [69], followed by the extrapolation of the remaining points from the last time point to infinity by a single monoexponential fit. While using SUV as unit of tracer activity, each point is
normalized for injected dose value (eq.2). So, residence time can be
calculated as the area under the TAC curve as shown in figure 7.
25
Tracer Activity (SUV)
Area under curve (Ã)
= (T1-T0)* [(h0 + h1)/2] + (T2-T1)* [(h1 + h2)/2]
+ (T3-T2)* [(h2 + h3)/2] + (T4-T3)* [(h3 + h4)/2]
+ (T5-T4)* [(h4 + h5)/2 + h5* (t1/2 /ln 2)
h2
h1
h3
h4
h5
T1
T2
T3
T4
T5
Time (h)
Figure 7. Schematic representation of trapezoidal method to estimate the residence time. For
time points >T5, it is assumed for the tracer to decay by physical half-life; thus the integrated
cumulative activity from the last time point to infinity is estimated by a single monoexponential fit.
4. Dose Calculation
The mean absorbed dose to target organ from the source organ can
be expressed with the following equation:
Dr(rt ĸUs) = IJ S(rt ĸUs)
(4)
Where, Dr(rtЋrs): mean absorbed dose to target organ (rt) from the
cumulated activity in the source organ (rs); ȫ: residence time; and
S(rtЋrs): dose conversion factor or S-factor deals with the radionuclide and accounts for the energy released from each decay per mass
of source and target organ.
The estimation of the absorbed dose from the residence time is
handled by Organ Level Internal Dose Assessment with Exponential
Modeling (OLINDA/EXM 1.1) [70], where the calculations are
based on in-built phantom models and S-factor values to obtain the
intended absorbed dose estimate in humans (ICRP60).
26
Material and Methods
Various protocols were used in the present investigation. The procedures are
herein referred to, with their respective publications (I-V). (See p.13).
[68Ga]Ga-DO3A-VS-Cys40-Exendin-4 Synthesis (I–V)
Throughout the experiments,68Ga was eluted from two 68Ge/68Ga generator
systems (1,850 MBq; Eckert & Ziegler Eurotope GmbH and 1,850 MBq;
IDB Holland BV) in order to increase the initially available radioactivity.
The first fraction of 1.5 ml from the generators was discarded and the next
1.5 ml, containing over 90% of the total radioactivity, was collected and
buffered with 200 μl of acetate buffer and 60 μl of sodium hydroxide to
provide a pH of 4.6 ± 0.4. Then, 10 nmol of DO3A-VS-Cys40-Exendin-4
(CS Bio Company, Inc, CA, USA) was added, and the reaction mixture was
incubated at 750C for 15min.The product, [68Ga]Ga-DO3A-VS-Cys40Exendin-4, was formulated in PBS, and its stability was monitored for 4 hr.
Animal Studies (I–V)
All animals (table 3) were housed under the proper lab conditions (mice and
rats) /breeder [pigs and NHP]. All procedures were approved by the local
ethical committee for animal experimentation in Uppsala, Sweden
Table 3. Animal models used in this research work. (NDB: non-diabetic; DB: diabetic)
Species
Breed
n (total)
NDB/DB
Study
Ethical Permit
Mice
nu/nu balb/c
31
31/-
III
C52/10
Rat
Lewis
42
35/7
I
C106/11
4
4/-
V
C242/11
Rat
Sprague Dawley
Pig
(Yorkshire Swedish
Landrace x Hampshire)
7
4/3
II
C404/12
NHP
Cynomolgus monkey
3
3/-
I
C160/11
27
Clinical Study (IV)
Use of [68Ga]Exendin-4 in the patient was approved as compassion care after
consultation with the Swedish Medical Agency. The patient gave written
consent for publication of the case report.
In 2012, a patient (female, 35y.o,) was referred to the Uppsala University
Hospital and examined for suspected pancreatic insulinoma. The contrastenhanced CT and the US were negative, but the endoscopic ultrasonography
indicated a small lesion in the pancreas, and an exploratory laparotomy was
urgently performed. In surgery, 2-cm tumor in the tail of the pancreas and an
adjacent lymph node metastasis were found. A distal pancreatic resection
plus splenectomy was performed.
Histopathology showed a World Health Organization classification grade
II insulinoma. Postoperatively, hypoglycemia persisted. It was then evident
that the insulinoma had metastasized. The common site of metastasis is the
liver, and they are generally very small in size (<2 cm). Such small lesions
were undetectable by morphological techniques such as CT or US, or with
molecular imaging techniques such as PET/CT scans with [11C]5-HTP (clinical neuroendocrine marker) or [18F]FDG-PET/CT. Due to the deteriorating
health condition of the patient,[68Ga]Exendin-4-PET/CT scan was performed.
Cell Culture (III)
The INS-1 cell line (GLP-1R positive), derived from the rat insulinoma was
cultured in RPMI-1640 medium, supplemented with 10% (v/v) heatinactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/ml peniFLOOLQVWUHSWRP\FLQ8POP0VRGLXPS\UXYDWHȝ0ȕ- mercaptoethanol, and 10 mM HEPES.
The PANC1 cell line (GLP-1R negative), derived from the human pancreatic ductal cells, was cultured in the DMEM medium, supplemented with
10% (v/v) heat-inactivated FBS and 100 units/ml penicillin/streptomycin
(10.000U/ml). All media and supplements were bought from Biochrom AG.
Cells were incubated at 37°C, 90% humidity, and 5% CO2.
Animal Models (I–III)
Subcutaneous INS-1 and PANC-1 xenografted mice (III)
INS-1 cells (10–15 × 106; n=16) mixed with RPMI-1640 with supplements
or PANC1 cells (25–30 × 106; n=5) mixed with DMEM with supplements
were injected subcutaneously into the right front leg of the nu/nu balb/c
mice.
Drinking water was supplemented with 50 mg/ml of glucose for the mice
bearing INS-1 xenografts, as an inappropriate amount of insulin secreted
28
into blood from the tumor may cause hypoglycemia in the animal. Twentyfour hours before any experiment, the glucose-supplemented water bottles
were replaced with normal water.
STZ-induced T1D rats (I)
Lewis rats (n=7) were treated with STZ (Sigma S0130, Stockholm, Sweden)
at 55 mg/kg to induce diabetes by beta-cell destruction. A Bayer COUNTER
monitoring unit (Bayer AG) was used to measure the glucose in the blood
from the tail vein, and diabetes was defined as sequential measurements of
>20mmol/L glucose.
STZ-induced T1D Pigs (II)
Diabetes was induced in pigs (n=3) by an IV administration of STZ (150
mg/kg; Sigma S0130, Stockholm, Sweden) following a previously developed protocol [71].
An experienced veterinarian carefully monitored these pigs. Subcutaneous (SC) insulin treatment with intermediate acting porcine insulin (Caninsulin® vet. 40 IU/mL, Intervet AB, Sweden) was initiated 7 days post STZ.
In vitro autoradiography (III)
Biopsies from the INS-1 xenograft mice were frozen to -80°C and processed
LQWRȝPVHFWLRQV7RVWXG\the tracer binding properties of the tissue, the
sections were incubated in several concentrations (0.3–30 nM) of
[68Ga]Exendin-4 in 200 mM TRIS + 1% BSA for 60 min at room temperature (RT). For blocking, 200 nM of unlabeled Exendin-4 (native peptide) or
DO3A-VS-Cys40-Exendin-4 (tracer precursor) was added to the incubation
buffer 10 min before the tracer administration. Tissue slices were then
washed 3 times for 4 min in 150 ml 200 mM TRIS at RT to remove the excess tracer and then dried at 37°C for 10 min.
The sections were then exposed against a phosphor-imager screen for 2
hours, digitalized using a Phosphorimager SI (Molecular Dynamics,
Sunnyvale, CA, U.S.A.) and analyzed using Image Quant (Molecular Dynamics, Sunnyvale, CA, U.S.A.).
The affinity (expressed as the dissociation constant Kd) and the GLP-1R
density (Bmax) were determined by non-linear regression of total and nonspecific binding using GraphPad Prism 5 (San Diego, CA, U.S.A.).
Ex vivo autoradiography (V)
The Sprague-Dawley rats (n=4) were administered a peptide mass of 0.1
μg/kg. The animals were euthanized by CO2, 60 min post injection (p.i). The
pancreas from the animals were removed post mortem, immediately frozen,
and sectioned by a microtome (Microm 560 cryostat, CellabNordiaAB,
29
Sweden) into 20 μm sections and placed upon object glasses. The object
glasses from each animal were exposed to Super Resolution (SR) storage
phosphor screens (PerkinElmer, Downers Grove, IL, U.S.A.), for 2 hr. The
plates were scanned using a Cyclone plus phosphor imager (PerkinElmer,
Model no. C4-31200, Downers Grove, IL, U.S.A.).The pancreatic sections
were immediately re-frozen and later thawed and stained for insulin by the
immune florescence method (IF).
Immunofluorescence (IF) insulin staining (V)
The sections were fixed in ice-cold acetone for 10 minutes and then washed
in PBS for 3 minutes, followed by incubation with DAKO Serum free protein block (Agilent Technologies, Glostrup, Denmark) for 30 minutes. The
sections were then incubated overnight at 4°C in the insulin primary antibody (Insulin A, Santa Cruz, SC-7839, goat-polyclonal (1:100) followed by
washing in PBS (3 × 3 minutes). Slides were then incubated with secondary
antibody Alexafluor 555 (Invitrogen, Carlsbad, CA, U.S.A.; donkey antigoat; dilution 1:1000) for 60 min in a humidified dark chamber, again followed by washing with PBS (3 × 3 minutes). ProLong ® Gold Antifade
reagent (Life Technologies, Rockville, MD, U.S.A.) was used for mounting
the slides. Tile scan images were acquired with a Zeiss LSM780 confocal
microscope at 10 × magnification.
The autoradiograms and the IF stained images were co-registered and analyzed using Image J 1.48 v (National Institutes of Health, Bethesda, MD,
U.S.A.).Manual regions of interests (ROIs) were drawn on the regions with a
high radioactive signal density (hotspot-assumed to correspond to islets of
Langerhans) and within the regions with weak radioactivity signal (exocrine
tissue). The overall phosphor image plate background was also assessed and
subtracted from all the data samples. The hotspot-to-exocrine uptake was
expressed as a ratio for each analyzed section. Several sections (more than 3)
from each animal were analyzed in this fashion.
Insulin staining (II)
&RQVHFXWLYHȝPVHFWLRQVIURPHDFKELRSV\ZHUHVWDLQHGIRULQVXOLQXVLQJ
A0564 from DAKO (Denmark) as the primary antibody. The stains were
developed by the Envision DAB system K4010 (DAKO, Denmark) using an
anti-rabbit secondary antibody. All sections were counterstained using Mayer’s Hematoxylin. The slides were captured digitally at 10 x magnification
by using an Axio Imager 2 microscope (Carl Zeiss Microscopy GmbH,
Germany) mounted with an Axiocam MRm camera. Images were analyzed
in AxioVision (Version 4.8.2.0).
30
Ex vivo organ distribution (I, III, and V)
Beta cell imaging (I, V)
In vivo specificity of the tracer to the beta cells was evaluated in rodent animal model (Male Lewis rats; n=35) by dose escalation studies (ex vivo biodistribution) with [68Ga]Exendin-4,co-injected in escalating doses of unlabeled DO3A-VS-Cys40Exendin-4 peptide (0.1, 1, 2, and 100 μg/kg).
Dose escalation studies were performed at three different time points (p.i)
30, 60 and 80 min, after euthanasia by CO2.
Specificity of the tracer to the beta cells was investigated between the
pancreas of healthy rats and the STZ-treated diabetic rats (n=7), at baseline
dose of 0.1 μg/kg. The tracer was injected into the rodents via the tail vein,
in sedated condition, anesthesia - 3.5% isoflurane in 50%/50% medical oxygen: air at 450mL/min. The tracer uptake in the organs is presented as tissueto-blood ratio.
Insulinoma Imaging (III)
In vivo specificity of the tracer to the GLP-1R in the insulinoma was evaluated in the murine models. The biodistribution of [68Ga]Exendin-4 was analyzed at WZRGLIIHUHQWGRVHVRISHSWLGHȝJNJEDVHOLQHLQQRUPDOQ INS-1 tumor bearing (n=5); PANC1 tumor bearing nu/nu Balb/C mice (n=5)
DQG ȝJNJ EORFN in normal (n=4) and INS-1 tumor bearing (n=5).The
tracer was injected intravenously through the tail vein under general anesthesia (isoflurane 3.0%). The animals were allowed to wake up after the
tracer administration and organs were resected 80 min later, after euthanasia
by CO2. Organs were excised and weighed, and the radioactivity uptake was
measured in a well-counter. The results are presented as tissue-to-blood ratio.
PET/CT Imaging (I–IV)
Small animal imaging (I and III)
All small animal PET scans were performed in an animal PET/CT scanner
(Triumph™ Trimodality System, TriFoil Imaging, Inc. Northridge, CA,
U.S.A.).
In study I, the sedated rats (isoflurane 1.0%–2.5% in 50%/50% medical
oxygen: air at 450 mL/min) were positioned in a PET gantry with the abdomen as the center of the field of view (FOV) and examined for 60 min as
dynamic scan, after a tracer injection (0.1 μg/kg) as bolus through a catheter
in the tail vein.
In study III, xenografts with pre-injected [68Ga]Exendin-4 (80 min p.i) or
11
[ C]5-HTP (30 min p.i.) were euthanized, by CO2, and examined with a
31
whole body PET for 60 min. A sequential CT scan (3 min) was performed
after all the PET studies.
The PET data were reconstructed into dynamic (I)/ static images (III) using a MLEM 2D algorithm (10 iterations). The CT raw files were reconstructed using a Filter Back Projection (FBP). PET and CT dicom files were
fused and analyzed using PMOD v3.508 (PMOD Technologies Ltd., Zurich,
Switzerland).
PET/CT in NHPs, pigs and human (I, II, and IV)
The subject handling, anesthesia (I–II) and camera PET/CT procedures were
under the control of veterinarians and nurses.
The NHPs and pigs were positioned in the Discovery ST PET/CT (GE
Healthcare, MI, U.S.A.) to include the pancreas in the center of FOV, and in
human, the liver, assisted by a low-dose CT scout view for dynamic PET
scans. The CT scans were acquired for attenuation correction. A whole body
scan was acquired by multi-bed scans (five partly overlapping bed positions), following the dynamic baseline to investigate the whole body biodistribution. The PET data were acquired for 4 min at each bed position. Attenuation correction and morphological CT were obtained in the same manner
as for the dynamic scan.
In study II, [15O]WAT scans were performed before the [68Ga]Exendin 4
scans to analyze the tissue perfusion.
In studies 1 and II, venous blood samples (0.2–0.5 mL) were collected at
different time points after each injection to measure the radioactivity concentrations in the whole blood and plasma.
PET/CT Image analysis (I–V)
PET/CT data were analyzed using PMOD v3.13 (I, PMOD Technologies
Ltd., Zurich, Switzerland), PMOD v3.508 (III-IV), VOIager 4.0.7 software
(II, GE Healthcare, Sweden) and in xeleris (IV, GE Healthcare, Sweden).
ROI were delineated on the trans-axial CT slices. Entire organs were delineated on the sequential slices and combined into volume of interest (VOI).
Kinetic Modeling (I and II)
Perfusion was assessed by a one-tissue compartment model from the dynamic pancreatic [15O]WAT-PET data, using an aortic VOI as the input function
(4–5 pixels in 13 consecutive slices).
The dynamic [68Ga]Exendin-4 PET data from the pancreas was fitted
to a single tissue compartment model, 1--TCM (figure 8) using the PMOD
v3.508 PKIN kinetic modeling module (PMOD Technologies Ltd., Zurich,
Switzerland). The output parameter total volume of distribution (vT), defined as the ratio K1/k2, was used to assess the changes in the tracer uptake,
32
receptor occupancy, and availability in dose escalation studies in NHPs (I),
as well as differences in the tracer uptake between the non-diabetic and diabetic pigs (II).
Figure 8. Schematic representation of 1TCM.
In this setting, K1 can be interpreted as rate of tracer internalization intracellularly by interaction with GLP-1R, and k2 represents the washout of the
unbound tracer from the tissue. The GLP-1R occupancy was estimated using
the equation 5:
Occupancy(%) 100 QT ( Pg / kg )
*100
QTbaseline
(5)
Where, vTbaseline represents zero occupancy and vT after co-injection of increasing doses of unlabeled Exendin-4.
Dosimetry (V)
[68Ga]Exendin-4 kinetics from the non-diabetic animal models (table 4) at
their baseline doses were used for the absorbed dose estimation.
Table 4.Summary of the species models and exendin-4 peptide dose used in the studies
Animal Model
n
Peptide dose (μg/kg)
Time points (min)
Rat
Pig
NHP
Human
12
4
3
1
0.1
0.021±0.003
0.037±0.023
0.17
30,60, 80
0–60
0–90
0–40, 100, 120
Tracer uptake in the organs of different animal models was decayuncorrected and extrapolated to adult human (73.7 kg) and adult female
(58kg) phantoms from OLINDA/EXM 1.1 database.
33
Residence time calculations
Residence times (MBq-h/MBq) were assessed by trapezoidal approximation
of the collected kinetic data, followed by the extrapolation of the remaining
points from the last time point to infinity by a single mono-exponential fit.
The remainder of the body residence times for [68Ga]Exendin-4 was obtained by taking the theoretical residence time of [68Ga] minus the total residence times in the source organs, and the difference was assumed to be homogeneously distributed in the body.
Absorbed Dose Calculations
The estimation of the absorbed dose was performed by OLINDA/EXM 1.1
software where the calculations were based on the adult reference male or
female phantom to obtain the intended absorbed dose estimate in humans
(ICRP60). The kidney sub-region absorbed doses were calculated based on
the multi-region kidney model phantom.
Dose limit values in the organs for radiation exposure were taken from
the norms set by the European nuclear society. (http://www.euronuclear.org
/info/encyclopedia/r/radiation-exposure-dose-limit.htm)
Statistical Analysis
Unpaired student t-test
x Study I: [68Ga]Exendin-4 uptake in the pancreas of non-diabetic and
diabetic rat.
x Study III: [68Ga]Exendin-4 biodistribution studies in normal, INS-1,
and PANC-1 mice at baseline and blocking dose.
One-way ANOVA
x Study I: [68Ga]Exendin-4’s pancreatic uptake in the dose escalation
studies, rats.
Mann-Whitney test
x Study II: To compare the changes in the tissue perfusion and kinetic
parameters between the non-diabetic and diabetic pigs.
SYDOXH”ZDVFRQVLGHUHGVLJQLILFDQW>S”S”S”]
34
Results
[68Ga]Ga-DO3A-VS-Cys40-Exendin-4 Synthesis
Figure 9. Schematic representation of [68Ga]Ga-DO3A-VS-Cys40-Exendin-4 synthesis
Buffers (pH: 4.2, 4.6, and 5.0), temperature (60, 75, and 94ºC), and radical
scavengers were optimized in the 68Ga labeling of the DO3A-VS-Cys40Exendin-4 in order to suppress the radiolysis and ensure high radioactivity
incorporation and specific radioactivity (SRA) (figure 9). In order to suppress the radiolysis, post labeling addition of ascorbic acid was investigated.
However, the addition of ethanol to the reaction mixture prior to the synthesis demonstrated more robust and higher yields.
The decay-uncorrected radiochemical yield was 80 ± 5%. [68Ga]Exendin4 was produced with a radiochemical purity of over 95% and with SRA of
78 ± 19 MBq/nmol. The tracer was stable in the formulation buffer for at
least 3 hours at room temperature. The product was purified, formulated in
sterile phosphate buffer (pH 7.4), and passed through a 0.22 μm filter into a
sterile injection vial.
Molecular weight of [68Ga]Exendin-4 is 4818 g/mol.
35
Beta cell Imaging and quantification
Rat Model (I and V)
Specificity of the tracer to GLP-1R
In healthy rats, the tracer uptake in the pancreas was significantly decreased
from baseline (0.1μg/kg) by approximately 42, 74, and 90% upon coadministration of 1, 2, and 100μg/kg DO3A-Exendin4, at 60 min p.i. At 80
min, the respective decrease was 31, 46, and 82% (figure 10A).This indicates
that the tracer uptake was mediated by the specific binding to the GLP-1R
expressed in the pancreas.
Figure 10. (A). Dose escalation studies in healthy rats. (%'HVWUXFWLRQRIYLDEOHȕ-cells by
STZ decreased in vivo uptake in a diabetic pancreas.
In diabetic rats, the tracer uptake in the pancreas was decreased by >80%,
compared to healthy controls at baseline dose (figure 10B). These results
further support that pancreatic uptake of [68Ga]Exendin-4 was mediated by
the specific binding to the GLP-1R expressed on the beta cells.
From an ex vivo autoradiography study, injected with 0.1μg/kg of
[68Ga]Exendin-4 tracer, the radioactive distribution in the autoradiogram of
the pancreas was highly correlated to the location of the islets of Langerhans, as shown by the autoradiogram/IF stain fusion, figure 11.
A
B
C
Figure 11. (A) Hotspot uptake of [68Ga]Exendin-4was around 50 times higher than the weak
background in the tissue section. (B)The corresponding IF staining for the insulin of the same
section shows the location of the islets of Langerhans. (C) autoradiogram/IF stain fusion.
36
In dynamic PET studies with [68Ga]Exendin-4, the tracer was rapidly excreted through the kidneys and reached a SUV of 10 within 5 min from the start
of the scan, and accumulation continued until the end of the scan, 60 min.
The pancreas was nearly impossible to delineate from the CT or in the PET
scan considering its close proximity to the kidney, spill over; and further
limitation from PET spatial resolution.
NHP model (I)
Specificity of the tracer to the GLP-1R
Tracer uptake in the cynomolgus pancreas was progressively reduced from
baseline (0.05 μg/kg) by 50, 66, 71, and 97% by co-injection of escalating
doses (0.15, 1, 3, and 20 μg/kg) of unlabeled DO3A-Exendin4. Uptake in
the GLP-1R negative tissues such as the liver was not influenced by coadministration of the unlabeled DO3A-Exendin4. This strongly suggests that
the pancreatic uptake of the tracer is mediated by the specific binding to the
GLP-1R in the pancreas.
GLP-1R imaging
At baseline dose (0.05 μg/kg), a distinct uptake of the tracer in the pancreas
was observed within 10 min from the start of the scan. Apart from the pancreas and the renal excretory organs (kidney and urinary bladder), no other
organs showed considerable uptake in the whole body scan (figure 12).
A
0.05 μg/kg
0.15 μg/kg
1 μg/kg
3 μg/kg
20 μg/kg
7
SUV
0
B
MIP
Figure 12. (A) Trans-axial PET/CT images of
dose escalation studies in NHP (upper row) are
summations of the dynamic sequences 30–90
min after the tracer administration. An increasing concentration of the unlabeled peptide resulted in a competition for the GLP-1R between
the tracer and the unlabeled peptide. The tracer
uptake was almost completely abolished by coadministration of the unlabeled peptide of 20
μg/kg.
(B) Whole-body maximum-intensity projections
(of PET) were acquired 90–120 min after the
tracer administration.
administration Pancreas (white arrow);
kidneys (green arrow).
37
GLP-1R Quantification
Tracer activity in the blood samples was not influenced by the coadministration of the unlabeled tracer (figure 13A). The GLP-1R negative
organs such as the kidneys, liver, or muscle demonstrated a similar pattern.
Absolute uptake in the GLP-1R positive pancreas was decreased by the coadministration of increasing amounts of unlabeled DO3A-Exendin-4, and
modulated kinetics from gradual increase (0.05 μg/kg), “steady state” (1 –3
μg/kg) to washout (10–20 μg/kg), (figure 13B).
Results from the 1TCM (figure 13C) showed that the rate constant K1 representing the total tracer uptake in the tissue (free, specific, and nonspecific)
from the plasma decreased in the pancreas with increasing doses of competing DO3A-Exendin-4. This can be interpreted as that the increasing amounts
of unlabeled DO3A-Exendin-4 would compete with the radiolabeled tracer
for binding to the GLP-1R, indicating the receptor-specific tissue uptake in
vivo. The rate constant k2, representing the washout from the tissue, was low
and not influenced by dose escalation, indicating a very slow clearance from
the GLP-1R positive pancreas.
A
Blood
SUV
Time (Min)
Time (Min)
Dose
vT
(μg/kg)
K1
k2
0.05
0.071
0.009
8.390
0.15
0.041
0.010
4.242
(K1/k2)
1
0.031
0.010
3.203
3
0.048
0.020
2.443
10
0.023
0.029
0.774
20
0.010
0.033
0.287
D
In vivo GLP-1R occupancy (%)
C
Pancreas
SUV
B
IC50, 0,15 μg/kg
Occupancy(%) 100 QT ( Pg / kg )
*100
QTbaseline
Dose (μg/kg)
Figure 13. (A)Time activity curve (TAC) of the tracer in the blood. (B) TAC of the tracer in
the pancreas at different doses. (C) Tracer kinetic parameters, as estimated from 1TCM. (D)
In vivo GLP-1R occupancy at different doses as determined from the parameters of 1TCM.
GLP-1R occupancy (figure 13D) in the pancreas was estimated from changes in the vT relative to the baseline. At peptide dose of 0.15 μg/kg, receptor
occupancy of 50% was observed and nearly 100% occupancy at 20 μg/kg.
38
Pig model (II)
STZ-diabetic pig model
Beta cell ablation and onset of T1D in pigs treated with STZ was confirmed
by blood glucose values, clinical examinations and from insulin staining of
pancreatic sections (duodenal, spleenic and connective), post mortem.
Diabetic pigs had mean arterial blood pressure values that ranged between
52 and 61 mmHg and non-diabetics between 88 and 115 mmHg, at the time
of the scans.
Specificity of tracer to GLP-1R
[68Ga]-Exendin-4’s uptake in the pancreas of both non-diabetic and diabetic
pigs was almost completely abolished by co-injection of unlabeled Exendin4 peptide but, tracer uptake did not differ between non-diabetic and diabetic
pigs at baseline dose (0.025 μg/kg).
GLP-1R imaging
At baseline dose, non-diabetic pancreas and diabetic pancreas displayed a
distinct uptake of tracer within 10 min p.i. and different lobes of the pancreas showed similar intensity of tracer uptake until the last frame of the scan.
Competition study with excess of unmodified Exendin-4 (~4 μg/kg), abolished the tracer uptake in pancreas almost completely in both non-diabetic
and diabetic pancreas (figure 14A & B). Competition studies were performed
75 min after baseline scan for all the subjects.
Figure 14. Trans-axial PET/CT
images (summed 2–60 min) of
[68Ga]Exendin-4 at baseline and
blocking dose, in different lobes
of the non-diabetic pancreas(A)
and diabetic pancreas(B). (dduodenal; c-connective; s-spleenic
lobes).Coronal PET- MIP images
(C,D &E) of whole body biodistribution. L-lungs; P-pancreas; KKidneys; B-Bladder.
39
From the whole body scans in the non-diabetic pigs, apart from the pancreas,
tracer accumulation was observed only in the excretory organs, namely the
kidneys and the urinary bladder (figure 14C). In diabetic pigs (figure 14D
&E), a similar pattern of tracer uptake was observed, with the exception of
high (SUV 8.9 and 12.2) uptake in the lungs, which was only partially reduced by competition by Exendin-4 (SUV 5.1 and 7.9). This effect is attributed to the anesthesia used for the diabetic pigs.
GLP-1R quantification
From the dynamic PET analysis, only the pancreas showed a specific uptake
of the tracer in both the groups. At baseline dose, the pancreatic tracer uptake increased with time and was nearly abolished in the competition studies
(figure 15A and B).
A
B
Baseline (0.025 μg/kg)
Block (4 μg/kg)
Pancreas (DB; n=3)
SUV
SUV
Pancreas (NDB; n=4)
Time (min)
C
Baseline (0.025 μg/kg)
Block (4 μg/kg)
Time (min)
D
Figure 15.Time-activity curves describing the tracer uptake in the pancreas of non-diabetic
(A) and diabetic (B) pigs at baseline and during competition with the unmodified Exendin-4.
(C)Perfusion in the pancreas was reduced in the DB pigs. (D) Total volume of distribution
(vT) of [68Ga]Exendin-4, as determined by a single tissue compartment model. Change in the
vT was negligible between the non-diabetic and diabetic pigs (ns).vT was reduced by competition with native Exendin-4 in excess, 86% (*p<0.05) and 80% (*p<0.05).
ns-no significant difference
40
The perfusion in the pancreas and the kidneys of the diabetic pigs was decreased by 46% (*p<0.05) and 40% (*p<0.05), respectively, compared to the
non-diabetic animals (figure 15C).
The concentration of the tracer in the plasma, which was used as input for
the modeling, was similar in amplitude between the individuals, except in
one diabetic pig, which exhibited slightly lower plasma levels throughout the
baseline examination. At baseline dose, a high accumulation of the tracer in
the lungs and the reduced arterial blood pressure were expected to affect the
rate constant K1 [total tracer uptake in the tissue (free, specific, and nonspecific) from plasma] estimated from 1TCM. However, the K1 did not differ
between the non-diabetic and the diabetic pigs (p=0.57, 0.036±0.006, compared to 0.032±0.006, respectively). Similarly, no difference was found in
the k2 (tracer washout from the pancreas) (p=0.57, 0.016±0.002, compared to
0.014±0.004, respectively), (figure 14D).
Inability to obtain arterial blood samples for kinetic modeling of PET data
is a drawback. It is conceivable that a reference tissue model could have
been used to avoid the lack of arterial blood samples and metabolite analysis. Since, no suitable reference tissue is present in the abdominal region, we
opted for venous input.
Insulinoma Imaging
Murine model (III)
Tracer Specificity to GLP-1R
In vitro autoradiography
[68Ga]Exendin- 4 binding to the INS-1 sections was displaceable by both the
native Exendin-4 and the tracer precursor DO3A-VS-Cys40-Exendin-4 at in
vitro autoradiography studies. This indicates that the introduction of the
DO3A chelator has a negligible effect on the biocompatibility and its affinity
to GLP1-R, compared to the non-modified peptide.
Saturable tracer binding was observed in the INS-1 xenograft samples
with a Kd of 3.13 nM. The affinity in INS-1 xenograft was 3.1 nM, and the
specificity was >92% at concentrations below Kd. The GLP1-R density was
estimated to be 175.8 pmol/mg tissues.
.
Ex vivo biodistribution studies
Specific uptake of the tracer was observed in the GLP-1R positive tissues such
as the lungs, pancreas, and INS-1 tumor, and lower in GLP-1R negative tissues
such as the PANC-1 (exocrine tumor), liver, and muscle (figure 16A &B). In
INS-1 xenografts, at baseline dose, the tumor showed a significantly high up-
41
take, compared to the liver and the muscle (figure 16B). The low uptake in the
liver is noteworthy since the liver is a major site for potential metastasis.
Figure 16. Biodistribution profile of [68Ga]Exendin-4 in normal balb c nu/nu mice (A),INS1, and PANC-1 xenografts (B)
42
GLP-1R imaging
The imaging with small animal PET/CT showed concordant results with the
biodistribution studies. [68Ga]Exendin-4 showed a prominent uptake in the
INS-1 tumor and had a better tumor-to-background ratio (figure 17), compared to the clinical neuroendocrine marker, [11C]5-HTP to the background,
which is essential for detecting small lesions in vivo. Similar to the rat PET
image, the pancreas was difficult to visualize and delineate due to its proximity to the kidney. The uptake in the OXQJVLVOLNHO\DUWL¿FLDOO\ORZLQWKH
PET images due to the color scale used for the images, which is dominated
by the kidneys and the tumor.
The liver and the muscle are sites of clinical importance for islet transplantation, and the low uptake in these organs suggests that the visualization
of insulinoma metastasis and islet grafts may be possible in these tissues.
[68Ga]Exendin-4
[68Ga]Exendin-4
Figure 17. Coronal MIP PET/CT image of [68Ga]Exendin-4 in the INS-1 and the PANC-1
xeongraft and compared with the uptake of a clinical marker [11C]HTP in INS-1 xenograft.
T-Tumor, K-Kidney and B-Bladder.
Clinical Study (IV)
[68Ga]Exendin-4-PET/CT scan revealed occult hepatic metastasis and a
small metastasis in the para-aortal lymph node (figure 18), untraceable by
current state-of the-art imaging [11C]5-HTP-PET/CT or the [18F]FDGPET/CT. Pancreatic uptake of the tracer was clearly visible. Apart from the
GLP-1R expressing tissues such as the pancreas and the insulinoma tumor,
only the excretory organs, that is, the kidney and the urinary bladder,
showed a remarkable uptake, within FOV.
43
[11C]5-HTP
[18F]FDG
[68Ga]Exendin 4
1
SUV
0
MIP
Figure 18. Representative PET/CT images of the insulinoma patient with [11C]5-HTP (left
panel), [18F]FDG (middle panel), and [68Ga]Exendin-4 (right panel). Hepatic metastasis was
not evident in the [11C]5-HTP-PET or the [18F] FDG-PET. [68Ga]Exendin-4 revealed metastasis in the para-aortal lymph node and the liver (orange arrow) with better tumor-tobackground ratio, and showed a notable uptake in the pancreas (white arrow). Tumor burden
in the liver (orange arrows) could be visualized by the whole body [68Ga]Exendin-4 scan,
bottom row (right panel).
[68Ga]Exendin-4 -PET/CT scan results provided the basis for the patient’s
continued systemic treatment. Treatment with everolimus was interrupted
several times because of pneumonitis, a known side effect of this drug. Hyperinsulinemia then recurred and a bland emolization with embospheres of
the right hepatic artery supplying most of the liver was performed.
Since the levels of insulin and pro-insulin increased again after a few
months and the patient’s [68Ga]-DOTATATE scan for the determination of
the somatostatin receptor expression was positive, she is currently under
peptide receptor radionuclide therapy with [177Lu]-DOTATATE and responding well.
44
Dosimetry (V)
[68Ga]Exendin-4 in different animal models
The pancreas of the larger animal models and in the human showed a distinct uptake of [68Ga]Exendin-4 and was easy to delineate in the PET image.
This pattern was observable within a few minutes after the start of the scan,
at their respective baseline dose (figure 19, upper row). At the end of the
scan, the highest uptake of the tracer was observed in the kidneys, followed
by the pancreas and the insulinoma tumor.
In a normal biodistribution of the tracer in the whole body scan, the uptake in the kidney was dominant, and within the kidneys, the accumulation
was predominantly observed in the cortex region (figure 19, bottom row). It
is fairly evident that the kidneys may be the critical organ in absorbed dose
estimation.
Pig
NHP
Human
4
S
U
V
E
G
F
H
0
1
M
I
P
K
L
0
Figure 19. Trans-axial PET/CT images of [68Ga]Exendin-4 show that the pancreas (white
arrow) was easily delineated within 10 min post injection in the PET images (upper row). The
MIP coronal images demonstrate that the highest uptake of the tracer was observed in the
kidney (green arrow).
Quantification of [68Ga]Exendin-4 (decay corrected-dc and decay
uncorrected-ndc) in the organs of diferent species are summarized in figure
20. The tracer showed a higher uptake in the kidneys, pancreas, and
insulinoma, increasing with time in the pigs, NHP, and in the human.
45
Figure 20.The left panel shows decay corrected [68Ga]Exendin-4 kinetics in the organs of
different species at baseline doses. Rapid clearance of the tracer from the blood, liver, and
muscle can be observed. The right panel shows the tracer’s decay- uncorrected data, used for
residence time estimation.
46
Residence time and estimated absorbed dose
The residence time for [68Ga]Exendin-4 in the different species, extrapolated
to human, using adult male and female phantom models are summarized in
table 5, along with the estimated absorbed dose from OLINDA/EXM 1.1.
Data are presented as (average ± standard deviation) between the male and
female.
The organs are presented in decreasing order of absorbed dose, as observed in the patient, with the kidney first followed by the pancreas, bone
marrow, spleen, liver, muscle, and lungs.
The local dose to the kidney was the limiting factor rather than the estimated whole body effective dose. The estimated whole body effective dose
was similar, regardless of the differences in the species. Among the different
species, the highest kidney absorbed dose was observed in the NHP, 0.648
mGy/MBq.
Given a yearly limiting kidney dose of 150mGy, this corresponds to 231
MBq of administered tracer per year based on the extrapolation from the
NHP.
Assuming a reproducible specific radioactivity of 50MBq/nmol, at least
50–100 MBq of [68Ga]Exendin-4 (mol wt=4818 g/mol) can be administered
to a subject weighing 73 kg in order to keep below 0.1 μg/kg. We observed
partial saturation of the receptors in the NHP model at dose above 0.1μg/kg.
Thus, at least 2–4 (NHP extrapolation) examinations can be performed yearly in humans.
In the patient study, the most relevant for the clinical situation, the absorbed dose in the kidney was 0.28 mGy/MBq. This corresponds to 536
MBq before reaching the yearly kidney limiting dose of 150 mGy. Assuming similar calculation as performed for the NHP extrapolation, at least 5–10
examinations can potentially be performed yearly in humans.
Administration of 50–100 MBq of [68Ga]Exendin-4 would yield a dose of
1–2 mSv, for the whole body effective dose of 0.02mSv/MBq (extrapolation
from the rat data). A CT examination for anatomical information and attenuation correction amounts to approximately 1 mSv per bed position, and
when added to the PET dose, it gives a total of 2–3 mSv per examination.
This corresponds to 3–5 PET/CT examinations yearly before reaching the
acceptable whole body dose of 10 mSv.
From both critical organs’ dose estimation and the whole body effective
dose, atleast 2-4 PET/CT examinations can be performed annually in an
adult human. This enables longitudinal clinical [68Ga]Exendin-4- PET/CT
imaging studies of the GLP-1R in the pancreas, transplanted islets, or insulinoma, as well as in healthy volunteers enrolled in the early phase of antidiabetic drug development studies.
47
48
0.173 ± 0.001
NA*
NA*
Liver
Muscle
Lungs
0.013 ± 0.002
0.012 ± 0.001
0.059 ± 0.01
0.068 ± 0.011
0.028 ± 0.001
0.013 ± 0.002
-
-
0.336 ± 0.056
Organ Dose
(mSv/MBq)
0.013 ± 0.002
0.007 ± 0.001
0.017 ± 0.003
0.014 ± 0.002
0.022 ± 0.001
0.046 ± 0.008
0.115 ± 0.013
0.282 ± 0.03
0.281 ± 0.047
Organ Dose
(mSv/MBq)
#
0.014 ± 0.004
NA#
0.114 ± 0.021
0.039 ± 0.002
0.002 ± 0.000
0.04 ± 0.004
0.008 ± 0.001
0.012 ± 0.001
0.142 ± 0.015
0.173 ± 0.019
Residence time
(MBq-h/MBq)
PIG
0.027 ± 0.002
0.008 ± 0.001
0.020 ± 0.003
0.03 ± 0.002
0.022 ± 0
0.091 ± 0.008
0.334 ± 0.07
0.575 ± 0.218
0.648 ± 0.109
Organ Dose
(mSv/MBq)
0.016 ± 0.004
0.045 ± 0.000
0.177 ± 0.032
0.045 ± 0.002
0.007 ± 0.000
0.052 ± 0.005
0.016 ± 0.001
0.046 ± 0.006
0.285 ± 0.110
0.402 ± 0.046
Residence time
(MBq-h/MBq)
NHP
NA* - Not measured during ex vivo biodistribution study; NA - Organ was out of the PET field of view
0.017 ± 0.004
0.021 ± 0.001
Spleen
Effective Dose
(mSv/MBq)
0.001 ± 0.00
0.102 ± 0.011
-
Red Marrow
-
Medulla
Pancreas
0.206 ± 0.023
Cortex
Residence time
(MBq-h/MBq)
Kidney
Organ
RAT
0.014
0.389
0.047
0.005
0.055
0.008
0.021
0.189
0.163
0.016
0.013
0.015
0.022
0.023
0.026
0.05
0.181
0.378
0.276
Organ Dose
(mSv/MBq)
PATIENT
Residence time
(MBq-h/MBq)
Table 5. Estimated residence times and absorbed dose of [68Ga]Exendin-4, extrapolated to human from different species.
Discussion
The following discussion section is divided into four parts:
x [68Ga]Exendin-4 for diabetes
x [68Ga]Exendin-4 for cancer
x [68Ga]Exendin-4 dosimetry
x Stability of [68Ga]Exendin-4
Results from all the animal models and from the human PET/CT scan have
been used in each of the discussion categories.
[68Ga]Exendin-4 for diabetes
Several PET/ SPECT agents aiming for many different molecular targets
have been investigated in studies for imaging of focal clusters of beta cells,
such as insulinoma and transplanted islets. Yet, progress in the field of imaging and quantification of the native pancreatic beta cells has been more difficult in the following areas and reasons:
1. Lower density of beta cells in the pancreas than in the islet graft or
insulinoma.
2. Few cell membrane bound beta cell specific markers.
3. Expression or density of receptors (markers) on the beta cells.
4. Species differences in beta cell biology and receptor expression.
5. Limitations from the technology and instrumentation (partial volume
effects and resolution).
6. Use of radioactive ligands and risk of radiation overdose to critical
organs.
In this research work, we tried to answer the above challenges with
[68Ga]Exendin-4 PET/CT.
Importance of specific radioactivity
SRA is defined as the amount of radioactive nuclide incorporated per unit of
peptide mass used in the radiosynthesis. It is expressed as radioactivity per
molecule amount (Bq/mol). High specific radioactivity plays an increasingly
important role when the target (beta cells) is of low density and the imaging
49
agent (GLP-1 analogues) has a high affinity to beta cell biomarker (GLP1R). Thereby, few micrograms of radiolabeled imaging agents can be administered without masking or saturating the available receptors on the target cells. We have shown that the administration of more than 0.1 μg/kg of
[68Ga]Exendin-4 peptide induces a partial saturation of the GLP-1R in the
NHP pancreas (figure 13).
The injected peptide dose of a highly potent tracer such as Exendin-4 is of
significant emphasis, as merely 5– ȝJ VXEFXWDQHRXV ([HQGLQ-4 (~0.1
ȝJNJLVVXIILFLHQWIRUHOLFLWLQJDJOXFRVHORZHULQJHIIHFWLQhumans. Another side effect includes tachycardia, which is an abnormal rise in the heartbeat
(over 100 beats per minute) in some animals. In pigs studies (II), all subjects
showed an increase in the heartrate (HR) upon administration of both the
WUDFHUDORQH“ȝJ/kg) and co-injection of Exendin-4 (3.98±1.33
ȝJ/kg), resulting in an increase in the HR from 92±14 beats/min to 102±10
beats/min (p<0.01) and 115±17 beats/min to 217±35 beats/min (p<0.001),
respectively. The effect on the heart rate has to be considered if this tracer
compound is to be used in the clinical setting. However, cardiac effects of
this magnitude have not been observed in clinical studies using radiolabeled
Exendin-4 analogues [20].
Given the consistent production of [68Ga]Exendin-4(4818 g/mol) tracer
batches of 80 MBq/nmol on average and in combination with the increase in
the body size from NHP (7–9 kg) or pigs (30–40 kg) to human (60–80 kg),
we estimate that administration of 50 MBq of [68Ga]Exendin-4, the peptide
dosage given in the clinical setting will decrease to <ȝJNJ. At such low
doses, we expect low occupancy of available GLP-1R in the pancreas and no
such severe physiological side effects, thereby falling under the microdosing
concept.
Choice of animal model
Rodent animal models (genetic or chemically induced diabetic models) are
the most commonly used in the diabetes research [72]. Though these animal
models may aid in the initial characterization of the novel tracers for in vivo
biodistribution, translation of the results from these small animals to humans
has always had obstacles.
First, the size itself. Relatively small sizes of the organs in the rodents,
especially the pancreas and its anatomical position close to the kidneys have
several disadvantages for in vivo imaging. It is difficult to outline the rodents’ pancreas in the CT. Small peptides such as Exendin-4 (< 45 kDa) and
its radiolabeled derivatives are excreted through the kidneys and reach the
SUV >50, compared to SUV<2 in the pancreas (from ex vivo results). Due to
the spillover of the tracer signal from the kidney onto the pancreas and the
partial volume effect, it is nearly impossible to delineate the pancreas from
50
the PET or SPECT image. Their small organ sizes also miniaturize the vascularization thus affecting the tracer distribution.
Second, morphology and composition of the islets. The rodent pancreas
may consist of approximately 3,000–8,000 islets based on the strain, compared to a million islets in the human. In contrast to the rodent islets, the
population of islet cell types varies considerably in the human islets [1]. A
major proportion of rodent islets is beta cells (~75%), which are concentrated in the center of the islets, surrounded by alpha cells (~19%), delta cells,
and others (~6%). In contrast, in the human islets (figure 1), the beta cells
(~54%) are often on the surface thus more reachable than in the rodent, and
heterogeneously distributed alpha cells (~35%) and delta cells (~11%).
Third, the difficulty in blood sampling. It is difficult to place catheters in
the rodent models for longitudinal blood sampling to study the correlation
between BCM and BCF such as C-peptide or insulin. The amount of blood
required for repeated sampling may reach too large a fraction (>20%) of the
total blood volume.
We conclude that the in vivo imaging of the rodent pancreatic islets with
[68Ga]Exendin-4 or any other small peptides is difficult, and that extrapolation to humans should be done with caution. Larger animal models, such as
NHPs or pigs, could be explored for comparison.
In large animal PET/CT scan, the entire pancreas was possible to delineate in the CT image and hence, partly alleviates the spillover and the PVE
from the kidneys to the pancreas in the fused PET images. Delineation of
distal parts of the pancreas, close to the kidneys must be drawn with extra
caution. Since the NHPs and the pig’s pancreas share similar physiological
characteristics to the human pancreas, results from these animal models may
be considered reliable in this respect.
Since the NHP models are highly expensive, rare, and ethically sensitive
to induce T1D by STZ, pigs can be considered as a suitable animal model
for diabetes. From the current investigation, we showed the preparation of
the diabetic model, its similar functional characteristics to the diabetic human upon insulin treatment, and that it could be properly anaesthetized for
several hours to perform PET/CT scans and normal recovery after experimentations; possibility for frequent blood sampling makes the pig an ideal
animal model for further human diabetic research using PET or SPECT.
Uptake of [68Ga]Exendin-4 in the pancreas of different species
The pancreatic uptake of the tracer in the different animal models is presented as SUV, decay corrected to 60 minutes p.i. in figure 21. A significant
difference in the uptake of the tracer can be observed between the rodent
models and the large animal models. In rodents, the SUV reaches <2 on
average, while in pigs, NHPs, and human; the SUV is more than 4. Moreo-
51
ver, the use of the same PET/CT camera for the pigs, NHPs, and the human
study makes the results more comparable in large animal models and human.
The tracer uptake on INS-1 tumor autoradiography showed a proportional
increase with increase in tracer concentration, and was almost abolished in
the blocking studies. In addition, tracer uptake in pancreas showed same
pattern at blocking studies, thus strongly indicating that the [68Ga]Exendin-4
uptake in the pancreas is mediated by the specific binding to the GLP-1R.
Pancreatic uptake (SUV), 60 min
Baseline
Block
Mice
Rat
Pig
NHP
Human
68
Figure 21. Comparison of [ Ga]Exendin-4 uptake in the pancreas of different animal models.
Asterisks represent the significance, compared within the species at baseline and block dose,
and between the species at baseline dose by unpaired student t-test.
Ex vivo autoradiography with [177Lu]Exendin-4 in the rats showed a focal
uptake similar to the [68Ga]Exendin-4 in the pancreas (figure 11), whereas in
the pig, the uptake was throughout the pancreatic section. Ex vivo autoradiography was performed with [177Lu]-Exendin-4 at baseline dose of 0.035
μg/kg (SRA= 73 MBq/nmol) in a pig.
177
Lu (t1/2 = 6.67 days), a trivalent metal has a similar labeling chemistry
to DO3A-VS-Cys40-Exendin-4 and a stability compared to 68Ga, also a trivalent metal. The longer half-life of 177Lu made the post mortem analysis of
the pig pancreas and the ex vivo autoradiography practical.
Autoradiography studies in human pancreatic samples are yet to be investigated. Ideally, ex vivo autoradiography with [68Ga]Exendin-4 in patients
undergoing pancreatectomy will provide the answer if Exendin-4/GLP-1R
combination is specific to native pancreatic beta cells.
52
Figure 22. [177Lu]Exendin-4 autoradiograms of the rat pancreas and the pig pancreas.
Our results, thus far, show striking divergences in the pancreatic uptake of
Exenin-4 between the rodent models and the large animal models.
Is GLP-1R a BCM biomarker?
[68Ga]Exendin-4’s kinetics in different animal models shows specific binding to the GLP-1R positive organs. Since the tracer is a small peptide
(4.8kDa), it was expected to be excreted through the kidneys, in vivo.
Evaluation of the [68Ga]Exendin-4 in the non-diabetic rat model demonstrated that the tracer uptake is mediated by specific binding to the GLP-1R
expressed in the pancreas. The absolute tissue uptake was relatively constant
over time at all levels of blocking, likely because of the rapid tissue extraction in combination with the slow washout; hence, the tracer uptake values
are presented as tissue-to-blood ratio. The reduction of tracer uptake by
>80% in the diabetic rats, also correlated to the amount of the BCM loss
from the STZ treatment, in general. This demonstrated that the tracer uptake
is mediated by the specific binding to the GLP-1R expressed in the pancreatic beta cells. So, the in vivo estimation of the tracer concentration in the pancreas may correspond to the pancreatic BCM. However, due to the small
size of the animal, its organs, and the close proximity to the kidney, it was
difficult to delineate the pancreas in the PET/CT studies.
[68Ga]Exendin-4 PET/CT studies in the NHP and the pigs showed the
prospect of real-time imaging the GLP-1R in the pancreas for the first time
in a large animal model and a possible methodology for the tracer quantification by compartmental modeling. The large size of the animals and the
68
Ga labeling chemistry made it possible to administer the [68Ga]Exendin-4
at peptide doses < 0.1μg/kg. The NHP dose escalation studies were in
agreement with the results in the rat model, and the NHP model confirmed
the potential of the radiolabeled Exendin-4 for quantifying GLP-1R in the
pancreas in vivo. However, due to the lack of a diabetic model, we could not
confirm that the specific binding of the tracer to the GLP-1R is restricted to
only the pancreatic beta cells.
53
Studies in the pig model, diabetic and non-diabetic, confirmed that the
[68Ga]Exendin-4 uptake is specific to the GLP-1R in the pancreas; however,
we observed conflicting results with the GLP-1R being specific only to the
pancreatic beta cell. Since ablation of the beta cells and onset of T1D was
confirmed in the diabetic pigs, a high pancreatic uptake of the tracer at baseline dose in these animals despite the reduced blood pressure, tissue perfusion, and tracer depletion due to a high uptake in the lungs, shows that the
GLP-1Rs are expressed in other cell types within the pancreas. It remains to
be seen which animal model translates most accurately to the human situation.
A recent clinical study comparing the [111In]Exendin uptake and the retention in the pancreas of non-diabetic subjects and subjects with T1D
showed a decrease of approximately 65% in the subjects with T1D [20].
However, there was an overlap between the two groups, which was not expected since the subjects with long standing T1D should have negligible
remaining BCM.
The expression of the GLP-1R in the EHWDFHOOVLVLQÀXHQFHGE\the extracellular factors such as glucose levels. Thus, additional studies are required
to clarify whether quantitative measurements of the BCM with the GLP-1R
can be achieved with the Exendin-4 PET.
[68Ga]Exendin-4 for islet transplantation.
For the most severe forms of T1D, islet transplantation is an established
treatment approach [73]. The most common site of transplantation is the
liver, followed by the muscle. The viability and function of the transplanted
islets are known to be affected during the procedure and in the long-term.
Currently, there are no well-established methodologies for non-invasive and
longitudinal assessment of the transplanted islet. The liver and muscle uptake of the [68Ga]Exendin-4 tracer was very low (SUV<0.1). Hence, high
contrast in transplanted islets, compared to the hepatic and muscle background, is potentially achievable. This was evident from the uptake of
[68Ga]Exendin-4 in insulinoma metastases in the liver of the patient, comparable to the focal uptake of the tracer in transplanted islet, which was easy to
delineate. Such non-invasive assessment (imaging and semi-quantification)
of the transplanted islets would enable longitudinal monitoring of the islet
viability and could potentially improve patient management.
54
[68Ga]Exendin-4 for Cancer
Insulinomas are slow growing, well-encapsulated, functional tumors of the
pancreatic beta cells. Negative [18F]FDG-PET scan (figure 18) testifies to
the low proliferation rate of the insulinoma. Hence, diagnoses in patients are
overlooked for years. The mean age of patients at diagnosis is 45 years, and
the disease is rare in adolescence. Insulinoma, being elusive and of great
diagnostic challenge, requires an accurate biochemical diagnosis and precise
pre-localization to avoid extensive surgery.
[68Ga]Exendin-4 for insulinoma preoperative localization
As described earlier, prelocalization of these tumors are of paramount importance, since major cases are benign (>90%), and curative surgery is consider to be the golden standard. But the small sizes of these tumors demand
the need for sensitive, reproducible, and highly accurate method of diagnosis.
[68Ga]Exendin-4-PET/CT has shown to match the above-mentioned qualities. First, Exendin-4 is an agonist, a dose of 5–8μg (50–80 kg patient; dose0.1 μg/kg) can lower the blood glucose levels in diabetic patients.
[68Ga]Exendin-4 batches were consistently reproducible (study I–V) at high
specific activity >50 MBq/nmol, and with the increasing size of the patients,
the dosage given at the clinical settings shall be around 0.04–0.06 μg/kg
(mol wt=4818 g/mol; 50–70 MBq of [68Ga]Exendin-4; 50–70 kg patient
weight), thereby falling under the microdosing concept.
Second, compared to the cyclotron produced [11C]5-HTP or 111In/64Cu labeled Exendin-4, the generator produced [68Ga]Exendin-4 offers a simpler
labeling chemistry and lower cost of production, thus, making
[68Ga]Exendin-4 affordable at any clinical center.
Third, tracer kinetics. The time activity curves of the [68Ga]Exendin-4
from the different species models have shown its faster clearance from the
blood, and the GLP-1R negative organs such as the liver but, higher retention in the GLP-1R positive organs such as the pancreas, insulinoma tumor,
and unspecific accumulation in the kidneys. Thus, [68Ga]Exendin-4 has a
favorable pharmacokinetics of a tumor targeting ligand for better tumor-tobackground ratio, except for a higher retention in the kidneys, which was
expected of the small sized peptide’s clearance, in vivo.
The fourth feature is the use of 68Ga-PET imaging methodology.68Ga’s
decay property of the positron emission by 89% can yield high quality functional images than 111In (SPECT) or 64Cu (18% by positron emission) labeled Exendin-4 derivatives [47, 74], which can aid in the precise localization of the tumor upon fusion with CT. Delineation of a metastasis of size
~0.5 mm, given that the current PET camera’s resolution is limited to 1–2
mm is its evidence. Since high quality images can be acquired in a short time
55
period due to a faster clearance from the blood and the liver, the duration of
the PET acquisitions can be reduced. [68Ga]Exendin-4-PET/CT scan can also
assist in the postoperative imaging.
Fifth, the possibility of in vivo quantification of GLP-1R by
[68Ga]Exendin-4-PET. Such possibility for quantification can potentially be
used for the stratification of the insulinoma malignancy, as it has been observed that GLP-1R is over-expressed in the benign insulinoma but reduced
as malignancy develops [75].
Thus, [68Ga]Exendin-4 for the insulinoma may assist in better patient
management, according to the norms of personalized medicine and may
SURYLGHWKHEDVLVIRUWKHVWUDWL¿FDWLRQRISDWLHQWVLQthe planning of potential
future radiotherapy targeted toward GLP-1R.
Potential use of [177Lu]-Exendin-4 for radiotherapy
Promising results from the [68Ga]Exendin-4 PET/CT scan for accurate localization of the metastasis in the insulinoma patient motivates further translation of this tracer for internal radiotherapy. The estimated residence time for
the [68Ga]Exendin-4 was based on a few time points. Hence, the calculation
from the last time point to infinity, from [68Ga]Exendin-4’s time activity
curve, by single mono-exponential fit may be over-estimated and thus, the
estimated absorbed dose.
We investigated the organ distribution of Exendin-4 labeled with ([177Lu],
t1/2 = 6.67 d) in rats. Similarity in the distribution pattern of [68Ga]Exendin-4
(1μg/ kg) and [177Lu]Exendin-4 (0.7 μg/ kg) was evaluated and thus the potential of using [68Ga]Exendin-4 for the pre-therapeutic dosimetry. Ideally,
the long-lived radioisotope of 68Ga with therapeutic properties should have
been tried. Due to limitations of such tracer availability, clinically used 177Lu
was chosen for the pilot study [76]. Although 177Lu gamma emissions [0.208
MeV (11%) and 0.113 MeV (6.4%)] may be used for SPECT imaging and
dosimetry, the inability of SPECT to provide accurate quantification of the
tracer as well as the poor spatial resolution may underestimate the absorbed
doses in smaller lesions. The quantification accuracy, higher spatial resolution, and dynamic scanning with 68Ga-PET would overcome these disadvantages. Conversely, the major problem is the mismatch of the half-lives of
68
Ga and 177Lu.
The comparison between the biodistribution of [68Ga]Exendin-4 and
177
[ Lu] Exendin-4 in the blood, liver, spleen, pancreas, and kidney demonstrated a similar pattern. Accumulation was not observed in the red bone
marrow for either agent. These results indicate that the biodistribution data
obtained from the [68Ga]Exendin-4 may be extrapolated for the dosimetric
calculations of [177Lu] Exendin-4. However, more investigations are required
in order to prove this model. We observed that the estimated absorbed doses
56
of the [68Ga]Exendin-4 were lower in the pigs, NHPs, and human as compared to the rat (table 5).
From the dosimetric data of [177Lu]Exendin-4, the highest absorbed dose
was observed in the kidney, followed by the lungs, bone marrow, pancreas,
spleen, and the liver. The results indicated that the kidney was the doselimiting organ with an absorbed dose of ~6 mGy/MBq. Hence, a maximum
of 4.8 GBq of [177Lu]Exendin-4 can be administered for one radiotherapy
before reaching a kidney tolerable absorbed dose of 29 Gy [77]. This tolerated radioactivity amount is lower, compared to the commonly used radioactivity dose of 7.4 GBq in 177Lu-Somatostatin analogue based Peptide receptor radionuclide therapy [77].
Recently, various renal uptake reducing agents such as polyglutamic acid
(PGA) and gelofusine (GF) have been investigated [78-81]. Using the above
combinations, the renal uptake of [111In]Exendin was reduced by 48% in the
rat model [81]. Assuming a reduction of the residence time by 48%, we estimated that the absorbed dose of [177Lu]Exendin-4 to the kidney in a human,
using the rat extrapolated residence times, could be reduced to approximately 3.1 mGy/MBq. This would potentially allow for administration of up to
9.3 GBq of [177Lu]Exendin-4 and thereby permitting one cycle of radiotherapy.
From the [68Ga]Exendin-4-PET/CT scan in the insulinoma patient, a tumor SUV of 5 was observed at the end of 120 min p.i. Assuming a similar
tracer kinetics of [177Lu]Exendin-4 in the tumor as in pancreas and a further
decay is due to the physical half-live of the radionuclide, the preliminary
estimation of the absorbed dose to the tumor of 1cm3 sphere was 0.7
mGy/MBq. For 4.8GBq, this would have given an absorbed dose to the tumor of 3.4 Gy, which might not be sufficient for tumor control.
The role of neutral endopeptidase (NEP) for the catabolism of glucagon
like peptides has been identified in vitro [82]. Up to 50% of GLP-1 in the
circulation might be degraded by NEP, and metabolic stability was improved
upon inhibition of NEP and DPPIV in vivo in pigs [83]. Recent studies have
demonstrated a significant improvement of the in vivo stability and the xenografted tumor uptake of somatostatin, gastrin, and bombesin radiopeptides
by co-injection of phosphoramidon (PA) acting as an inhibitor to NEP [84].
The biological half-life of [177Lu]Exendin-4 might be prolonged by the
NEP inhibition. This would increase the agent availability for the tumor
uptake and consequently, the tumor absorbed dose.
The approaches for kidney protection and peptidase inhibition may allow
for a considerable increase of the tumor absorbed doses and improve the
radiotherapeutic efficacy. This motivates for further investigation of
[177Lu]Exendin-4 for internal radiotherapy.
57
[68Ga]Exendin-4 dosimetry
[68Ga]Exendin-4 versus other Exendin-4 derivatives
In comparison with the other reported Exendin-4 analogues, labeled with
other radionuclides, we observed that the extrapolated absorbed dose of
[68Ga]Exendin-4 is lower (table 6).
Table 6.Comparison of absorbed doses of other exendin-4 radiotracers with [68Ga]Exendin-4
(human data, table 5). Doses are given as mGy/MBq. N/A: information not available.
[68Ga]
[111In]
[64Cu]
[99mTc]
Organs
[85]
[60]
Exendin-4
Exendin-4
Exendin-4
Exendin-4[85]
Kidney
0.276
4.48
1.48
0.083
Liver
0.022
0.20
N/A
0.0046
Lungs
0.013
0.13
N/A
0.0046
Pancreas
0.050
0.70
N/A
0.020
Muscles
0.015
0.12
N/A
0.0024
Bone marrow
0.026
0.14
N/A
0.0030
Spleen
Effective dose
(mSv/MBq)
0.023
0.37
N/A
0.068
0.016
0.155
0.074
0.0037
The absorbed dose in the kidney for 111In and 64Cu labeled Exendin-4 was
approximately 17 and 5 folds higher than [68Ga]Exendin-4, respectively.
Furthermore, the absorbed doses in all the other organs were relatively low
for the [68Ga]Exendin-4, except for the Technicium-99m (99mTc) labeled
Exendin-4. Of the residualizing nuclides, we also show here that the
[68Ga]Exendin-4 yields the lowest kidney dose in comparison .
The extrapolated effective doses were also lower, compared to the other
Exendin-4 labels. For instance, the effective dose for 111In and 64Cu labeled
Exendn-4 was 10 and 5 times higher, respectively.
Recently, 18F- labeled analogues of Exendin-4 have been developed [59,
64, 87, 88]. Some have a significantly lower renal retention with a correspondingly lower kidney radiation dose [88]. The potential drawback may
instead be a higher whole body effective dose due to the relative longer halflife of 18F (109.8 minutes) and are non-residualizing, compared to 68Ga.The
dosimetry of [18F] labeled Exendin-4 has not yet been reported to the author’s knowledge.
Pancreatic model for internal dosimetry
Due to the small size of the islets (~150 μm) and the limitations of the PET
resolution (1–2 mm), it is not possible to visualize individual islets in the
58
pancreas. Hence, it is assumed that a cumulative uptake of BCI tracer in the
pancreas correlates with the total islets, specific to the BCM.
At present, in doses estimated by the internal dosimetry in OLINDA, the
pancreas is not compartmentalized to its composition, and the tracer uptake
is “assumed” to be homogenously distributed throughout the organ, which is
a major pitfall!
In an ideal situation, as the ligand labeled with the radionuclide
(PET/SPECT) binds to/internalized in the islets of small size, the energy
deposition during the decay per unit mass, in other words, the absorbed dose
imparted in the islets is significantly higher in comparison with the absorbed
dose to the whole pancreas. This may have harmful effects on the islet viability and can profoundly affect the glucose homeostasis in clinical settings
in the long run.
It is evident from the above that the calculated absorbed dose to the islets
of Langerhans is highly underestimated with the current dosimetry methodology. Thus, it is wise to estimate the dose to each compartment of the pancreas; hence, there is need for a pancreatic model for internal dosimetry estimation.
Parameters that would influence the absorbed dose estimation to a single
islet are:
1.
2.
3.
4.
SUV estimation for the islets and in exocrine tissue
Approximate size of an islet
Radionuclide properties: positron range and half-life
Percentage of annihilation that may occur in an islet
1. SUV estimation for the islet and in exocrine
The pancreas can be divided into two simple compartments known
by volume: endocrine/islets (2%) and exocrine (98%). Thus, the
SUV of the whole pancreas can be written as equation.6:
SUV pancreas = 0.02 SUVislets + 0.98 SUVexocrine
(6)
The ratio of the beta cell specific tracer in the islet to the endocrine can be
estimated from autoradiography:
SUVisets / SUVexocrine = X or SUVislets = X * SUVexocrine
(7)
From Equation (6) and (7),
(8)
SUVislets = X*SUVpancreas/ (X *0.02 + 0.98)
68
So for example, in the case of [ Ga]Exendin4 with an X=48 and a pancreatic SUV of 0.6, then the SUVislets is in reality closer to 15
59
2. Approximate size of an islet
The SUV islets from equation (8) correspond to the complete islet
mass. Since we are interested in the absorbed dose estimation in a
single islet and the size of an islet varies from 50–500 μm in diameter, choosing the approximate size of an islet is of vital importance.
Assuming an islet to be spherical and of density (1 g/cm3), the
weight of a single islet can be estimated from its volume. This can
be used for the residence time estimation according to a sphere
model available in the OLINDA software.
3. Radionuclide properties: positron range and half-life
Based on the islet size approximation, the estimated absorbed dose
may differ as some of the radionuclide’s positron range may lie
within its approximation [89-91]. Thereby, the percentage of annihilation within the islet will vary. Since the residence time is proportional to the half-life, the tracer labeled with a long lived radionuclide will impart a higher absorbed dose to the islets.
Table 7.Commonly used PET radionuclides and their positron properties.
Positron range at
Radionuclide Half-life (min) Positron Energy (MeV)
FWHM (μm)
15
O
2.3
1.723
501
13
N
C
68
Ga
11
18
F
9.96
20.4
68.3
1.190
0.961
1.899
282
188
766
109.7
0.635
102
In summary, we propose that new methods for internal dosimetry of islets in
the pancreas should be developed with the above mentioned criteria’s as
input information.
Stability of [68Ga]Exendin-4
Metabolite analysis of [68Ga]Exendin-4 is not reported in this study. This is a
limitation of these studies, especially for the kinetic modeling quantification
performed in pigs and NHP. We posit that the emitted photons are from 68Ga
chelated with intact DO3A-VS-Cys40-Exendin-4 and hence tracing the parent compound rather than free 68Ga or other metabolites, for the following
reasons:
60
SUV
SUV
SUV
First, measurements of blood and plasma samples taken during the PET
scanning period in clinically relevant animal models (figure 23) namely,
NHPs and pigs, suggested that radioactivity was present in plasma throughout the examination. Thus tracer was freely available for tissue extraction.
Though blood sampling was not performed in human study (n=1), we expect
similar tracer distribution pattern.
Second, stability of
68
[ Ga]Exendin-4
in
Non-human primate
plasma can be speculated
from its rapid blood
clearance and no uptake
Plasma
in liver and spleen, clearBlood
ly indicated the absence
of
free
or
weak
68
Gachelate for transchelation
to
large
amounts of serum proteins such as transferrin
Time (min)
and nearly no possibility
for radiocolloid formation
Non-diabetic pig
in the subjects with time.
It has been shown that
Exendin-4, radiolabeled
with Gallium-67/68 by a
Plasma
NODAGA chelator at a
Blood
lysine residue at the 40
position, is highly stable
with >97% of the plasma
radioactivity originating
from intact radiotracer
even after 3h [92]. In this
Time (min)
study, the chelator DO3A
is used instead of
Diabetic pig
NODAGA, but we foresee no reason for any
significant change in
Plasma
stability based on chelaBlood
tor.
However, a reliable
method for metabolite
analysis in plasma should
be developed for future
clinical testing and devel
Time (min)
-opment
of kinetic model
Figure 23. Kinetics of [68Ga]Exendin-4 in blood and
for clinical use.
plasma of large animal models.
61
Conclusions
In this thesis work, the potential of the PET tracer [68Ga]Ga-DO3A-VSCys40-Exendin-4 ([68Ga]Exendin-4) was evaluated for non-invasive imaging
and quantification of GLP-1R in the pancreatic BCM and in the insulinoma,
in vivo. Experiments were performed in mice, rats, pigs, NHPs, and in one
insulinoma patient. Also, the dosimetry of [68Ga]Exendin-4 was estimated
based on the biodistribution data from the above-mentioned, except mice, at
their respective baseline dose, to determine the possibility of repeated
PET/CT examinations in humans.
The major conclusions from this research work are:
x [68Ga]Exendin-4 binds to the GLP-1R, in all the species models,
with high affinity and specificity
x [68Ga]Exendin-4 can be used to selectively image the GLP-1R rich
tissues such as the pancreas
x Dose escalation studies in rats, pigs and NHPs showed that the tracer
uptake in the pancreas is mediated by specific binding to the pancreatic GLP-1R
x [68Ga]Exendin-4 PET/CT studies in the NHP and pigs showed the
prospect of real-time imaging the GLP-1R in the pancreas for the
first time in a large animal model and a possible methodology for
tracer quantification by simple 1-TCM
x Pancreatic uptake of [68Ga]Exendin-4 was not reduced by selective
destruction of the beta cells in the STZ-diabetic pigs. This conflicts
with the notion of GLP-1R being specific to the pancreatic beta cell
x We observed a striking divergence in the pancreatic [68Ga]Exendin-4
uptake between different species
x [68Ga]Exendin-4-PET/CT has proven to be more sensitive than a
clinical neuroendocrine marker, [11C]5-HTP-PET/CT for imaging
insulinoma in mice
x Before reaching a yearly kidney limiting dose of 150mGy and whole
body effective dose of 10 mSv, 2–4 [68Ga]Exendin-4-PET/CT scans
can be performed in an adult human, which enables longitudinal
clinical PET imaging studies of the GLP-1R in the pancreas, transplanted islets, insulinoma, as well as in healthy volunteers enrolled
in the early phase of anti-diabetic drug development studies
62
Acknowledgements
This thesis work has been carried out at the Division of Preclinical PET Platform (PPP), Department of Medicinal Chemistry, Uppsala University, with
financial support from Barndiabetesfonden, Diabetesfonden, Exodiab, and
the Swedish Research Council.
I would like to convey my sincere gratitude to everyone who has generously
contributed to the work presented in this thesis.
Special mention goes to my enthusiastic supervisor, Associate Professor,
Olof Eriksson. My Ph.D. has been an amazing experience and I thank Olof
wholeheartedly, not only for his tremendous academic support, but also for
giving me so many wonderful opportunities. Not many Ph.Ds involve working with different species models. Thank you very much for your patience
and for teaching me everything I learnt about molecular imaging.
Similar, profound gratitude goes to Professor Olle Korsgren and Lars Johansson, my co-supervisors, who have been truly dedicated mentors. I am
particularly grateful to them for their constant faith in my lab work, and for
their support.
Irina Velikyan, the Gallium lady. My thesis work would not have been possible without your help. Your interest in research among your busy scheduled at work, always inspires me.
Professor Barbro Eriksson, Head ENETs Centre of Excellence, Department
of Endocrine Oncology Uppsala University Hospital, for introducing me to
the PET research work. I consider my Master’s thesis project provided by
you, to be a turning point in my research career. Thank you very much!
Professor Mats Larhed, Head of the Division PPP, for all the care and support at work.
Division of Biomedical radiation Sciences (BMS), the reason for me being
here in Sweden. If it wasn’t for the Master’s course, Medical Nuclide Techniques, I wonder what I will be working at now. 1000 tack!
63
Senyor Sergio Estrada, for creating such a calm working (who am I kidding), such a cheerful and playful atmosphere at work. It was always a
pleasure to have a chat with you.
Veronika Asplund, for your care, enthusiasm, and sharing all the lab techniques with me. Thank you for all the lunch trips in and out of Uppsala and
the special trip to Ulva for picking strawberries. Ola Åberg, a good chemist, a good cook and a good friend. Thank you for
suggesting such a “bombastic” title for my thesis work.
Dr.Zohreh (habibiss) my good friend, thanks for tolerating all my poor
jokes. You were always there for me to cheer up, when my days in lab were
dull.Dr.Maria (Maria Rosestedt), a person with very kind and warm personality!
Your “urge” for cleanliness, concern about calorie counting, meeep, I mean
healthy diet and love for pugs always make me smile. I hope you will find
your dream job soon. My best wishes and support, cheers! and Dyakuyu!
(Courtesy: google translator)
Dr. Mitran (BoDGanMitran) the mighty! Hail Dr.Mitran! haha… You sir, I
see a promising dancer in you, jokes apart, I see a promising scientist in you.
I have learnt a lot from our discussion and working with our triumph machine. To my memory, our experiment with an orange in the CT scan was
the most innovative one in the history of PPP.
Marie Berglund, good luck with your research work and take care of your
health.
Thank you Professor Marianne Jensen-Waern, LovisaNalin, Anneli Rydén,
Görel Nyman, Susanne Andréasson, Anna Wikstrand and Elin Manell for
your interest, excellent assistance with large animal models and fun discussion. Lovisa, for an amazing “Beny Lava” evening at GTB and in park lane.
I see you as veterinarian at daytime and a party animal in the evening, haha!
Members of PET centrum Mimmi, Annie, Maj, Marie, Mirtha, Megan, Helena, Thomas, Lasse, Gunnar, Patrik and others, for being such a nice and
easiest people to work with. I appreciate your hospitality, patience and the
services you provide for research work and at clinic.
City of hope’s group, USA, Professor Fouad Kandeel, Jack Shively, Ivan
Todorov and Zhanhong Wu for the precursor and for a fruitful collaboration.
64
Professor Anna Orlova and Professor Vladimir Tolmachev for providing the
opportunities to work with your research group. Unquestionably it improved
my skill in image analysis. Of course, for the BBQ and fun times at conferences, spasibo! Marika Nestor and Diana Spiegelberg my first supervisors at Uppsala University. Thanks for teaching the basic and good lab practices. It feels good
be sharing research ideas with your group again.
Thanks to all co-authors and collaborators, Professor Per-Ola Carlson, Daniel Espes, Marie karlsson, Professor Christer Halldin, Lina Carlbom, Håkan
Ahlström, Gunnar Tufvesson, Torbjörn Lundgren, Mahabuba Jahan and,
Professor Lena Claesson-Welsh and team for providing the opportunity to
work in other disciplines.
People from Nuclear Medicine Department, Anders Sundin, Jens Sörensen,
Lief, Jonas Eriksson, Mattias Sandstrom, Torsten Danfors, Ezgi Ilan, Karolina Lindskog, My Jonsson, Naresh Chowdary and others for sharing your
ideas, for your time and fika.
My master students Thomas, Mariam, Tonja and Martin. Thank you guys
for being a part of my research career. I feel that, I learnt a lot from you guys
than I did for you people.
Thanks to Olof, Meena, Kailash, Maria, Bogdan, Mahesh, Ezgi and Karolina for proofreading my thesis, correcting my Indian English to sound more
scientific and further finalize it to a good shape.
The past and present people from BMS, Jennie, Nazila, Diana, Dr Altai,
Hadis, Joanna, Anja, Andris, Javad, DJ Jonas, Marika, Hanna, kalle, kicki,
Jos, Bosse, Ann-Sofie, Sara, Ulrika and others for all the fun activities and
the best movies making experience.
BioVIS neighbors, Sara, Jermy, Pacho (Namaste), Matyas for sharing the
corridor, coffee machine, dining hall and sometimes Science. We should
soon start the “fika” that we planned for.
People from Rudbeck and BMC, Stina, Dag, Tsong, Sofia, Jessica for the
adventures with the Triumph machine.
My friends from other divisions of Medicinal Chemistry Mari, Bobo, Karin,
Linda, Ahmed, Erik, Ashkan, Jonas, Mark, Henrik, Andrea, Magnus, Sara,
Syun and other for all the fun talks and parties at ULLA.
65
Thanks to all the friends from different nationalities that I met here at Sweden. It was a great experience to know the people from different culture.
Special mention goes to Anja, Hanif, Harshad, Taj bhai, Rajiv, Mahesh,
Lucy, Sofia, Vasila, Vicky, Loffe, Cissi (shakka hum!), Tobias, Heidi, Edit,
Katja, Bish, Nelson, Alicia, Erik, Hazar, Georgie, Filip, Marwan and all
other for the wonderful time spent with you guys.
Mohamed Intikaf and Family. I will never forget the help you did for me
when I arrived to Sweden. It was a pleasure meeting you nice people.
Kailash bhai. My brother and good friend. You are such an inspiration. You
dedication to Science is second to none. My best regards to Bhabhiji.
Mari De Rosa and Ankur. You guys are amazing! The time we spent together in Uppsala and at your grand wedding in Italy will remain in my memory
for long long time.
My friends in India and NRIs (non-returning Indians), Vignesh, Sures, Kai
Harish, Bro Mark, Maggi, Atulya Iyengargaru, Nikhil Biotek, Champion
Avinash, Shashank, Choo, Chaluvadi, Mathur, Puneeth etc. Miss you guys a
lot!
Manoj and Antonia. My family in Sweden. You’ve been there for me
through the good times and bad. Thank you for everything you have done
for me.
Finally, but by no means least, thanks go to my family for almost unbelievable support. My brother Dr. Raj Kumar (B.D.S), my first friend in life. It was
your suggestion to give it a try in Sweden for my higher education. Until
then, for me, Sweden was known only for IKEA and Nobel Prize. Thanks! I
am happy to see that you have settled in your life with Dr. Saranya (B.D.S)
and the little prince, Master Pranav. More the merrier! My Mother Tamilarasi, my chief consultant-. There are no words that can explain how much
you mean to me. You were the inspiration for me to pursue this research
work. To my Father, Selvaraju, my hero! I see you as a true example for the
quote, “work hard in silence and let the success make the noise.” For all the
sacrifices you made for us to provide good education and lift our family
name to a respectable status in the society, I hope we will stand up to your
expectation to be a good researchers, and above all, good sons. All my publications have your name appa (Selvaraju.et al.) and I’m proud of it. Now
you can reply to the people, beaming with pride, “My eldest son is dentist,
and younger one is a scientist!
You are the most important people in my world and I dedicate this thesis
to you.
66
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72
Acta Universitatis Upsaliensis
Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Pharmacy 202
Editor: The Dean of the Faculty of Pharmacy
A doctoral dissertation from the Faculty of Pharmacy, Uppsala
University, is usually a summary of a number of papers. A few
copies of the complete dissertation are kept at major Swedish
research libraries, while the summary alone is distributed
internationally through the series Digital Comprehensive
Summaries of Uppsala Dissertations from the Faculty of
Pharmacy. (Prior to January, 2005, the series was published
under the title “Comprehensive Summaries of Uppsala
Dissertations from the Faculty of Pharmacy”.)
Distribution: publications.uu.se
urn:nbn:se:uu:diva-261629
ACTA
UNIVERSITATIS
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UPPSALA
2015