Transcript
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.
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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.
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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.
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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-
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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-
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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.
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Exendin-4 GLP-1 has a short biological half-OLIH PLQ GXHWRFOHDYDJHE\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 ୵ୣ୧୦୲
(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\FLQ 8PO P0VRGLXPS\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 WZRGLIIHUHQWGRVHVRISHSWLGH ȝJNJ EDVHOLQH LQQRUPDO Q 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. SYDOXHZDVFRQVLGHUHGVLJQLILFDQW> 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 ȝJNJ LVVXIILFLHQWIRUHOLFLWLQJDJOXFRVHORZHULQJHIIHFWLQhumans. 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.
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[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.
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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!
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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.
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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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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”.)
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ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2015
