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1.  Quantification of β-Cell Mass in Intramuscular Islet Grafts Using Radiolabeled Exendin-4 
Transplantation Direct  2016;2(8):e93.
There is an increasing interest in alternative implantation sites to the liver for islet transplantation. Intramuscular implantation has even been tested clinically. Possibilities to monitor β-cell mass would be of huge importance not only for the understanding of islet engraftment but also for the decision of changing the immunosuppressive regime. We have therefore evaluated the feasibility of quantifying intramuscular β-cell mass using the radiolabeled glucagon like peptide-1 receptor agonist DO3A-VS-Cys40-Exendin-4.
One hundred to 400 islets were transplanted to the abdominal muscle of nondiabetic mice. After 3 to 4 weeks, 0.2 to 0.5 MBq [177Lu]DO3A-VS-Cys40-Exendin-4 was administered intravenously. Sixty minutes postinjection abdominal organs and graft bearing muscle were retrieved, and the radioactive uptake measured in a well counter within 10 minutes. The specific uptake in native and transplanted islets was assessed by autoradiography. The total insulin-positive area of the islet grafts was determined by immunohistochemistry.
Intramuscular islet grafts could easily be visualized by this tracer, and the background uptake was very low. There was a linear correlation between the radioactivity uptake and the number of transplanted islets, both for standardized uptake values and the total radiotracer uptake in each graft (percentage of injected dose). The quantified total insulin area of surviving β cells showed an even stronger correlation to both standardized uptake values (R = 0.96, P = 0.0002) and percentage of injected dose (R = 0.88, P = 0.0095). There was no correlation to estimated α cell mass.
[177Lu]DO3A-VS-Cys40-Exendin-4 could be used to quantify β-cell mass after experimental intramuscular islet transplantation. This technique may well be transferred to the clinical setting by exchanging Lutetium-177 radionuclide to a positron emitting Gallium-68.
PMCID: PMC5082995  PMID: 27819034
2.  Feasibility of Multiple Examinations Using 68Ga-Labelled Collagelin Analogues: Organ Distribution in Rat for Extrapolation to Human Organ and Whole-Body Radiation Dosimetry 
Pharmaceuticals  2016;9(2):31.
Objectives: Fibrosis is involved in many chronic diseases. It affects the functionality of vital organs, such as liver, lung, heart and kidney. Two novel imaging agents for positron emission tomography (PET) imaging of fibrosis have previously pre-clinically demonstrated promising target binding and organ distribution characteristics. However, the relevant disease monitoring in the clinical setup would require multiple repetitive examinations per year. Thus, it is of paramount importance to investigate the absorbed doses and total effective doses and thus, the potential maximum number of examinations per year. Methods: Two cyclic peptide (c[CPGRVMHGLHLGDDEGPC]) analogues coupled via an ethylene glycol linker (EG2) to either 2-(4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)acetic acid (NO2A-Col) or 4-(4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazacyclononan-1-yl)-5-(tert-butoxy)-5-oxopentanoic acid (NODAGA-Col) were labelled with 68Ga. The resulting agents, [68Ga]Ga-NO2A-Col and [68Ga]Ga-NODAGA-Col, were administered in the tail vein of male and female Sprague–Dawley rats (N = 24). An ex vivo organ distribution study was performed at the 5-, 10-, 20-, 40-, 60- and 120-min time points. The resulting data were extrapolated for the estimation of human organ and total body absorbed and total effective doses using Organ Level Internal Dose Assessment Code software (OLINDA/EXM 1.1) assuming a similar organ distribution pattern between the species. Time-integrated radioactivity in each organ was calculated by trapezoidal integration followed by a single-exponential fit to the data points extrapolated to infinity. The resulting values were used for the residence time calculation. Results: Ex vivo organ distribution data revealed fast blood clearance and washout from most of the organs. Although the highest organ absorbed dose was found for kidneys (0.1 mGy/MBq), this organ was not the dose-limiting one and would allow for the administration of over 1460 MBq per year for both [68Ga]Ga-NO2A-Col and [68Ga]Ga-NODAGA-Col. The total effective dose was the limiting parameter with 0.0155/0.0156 (female/male) mSv/MBq and 0.0164/0.0158 (female/male) mSv/MBq, respectively, for [68Ga]Ga-NO2A-Col and [68Ga]Ga-NODAGA-Col. This corresponded to the total amount of radioactivity that could be administered per year of 643 and 621 MBq before reaching the annual limit of 10 mSv. Thus, up to six examinations would be possible. The residence time and organ absorbed doses in liver and spleen were higher for [68Ga]Ga-NODAGA-Col as compared to [68Ga]Ga-NO2A-Col. Conclusion: The limiting parameter for the administered dose was the total effective dose that would allow for at least six examinations per year that might be sufficient for adequate disease monitoring in longitudinal studies and a routine clinical setup.
PMCID: PMC4932549  PMID: 27275825
gallium-68; collagelin; dosimetry; fibrosis; imaging
3.  Evaluation of a novel type of imaging probe based on a recombinant bivalent mini-antibody construct for detection of CD44v6-expressing squamous cell carcinoma 
International Journal of Oncology  2015;48(2):461-470.
We have developed the CD44v6-targeting human bivalent antibody fragment AbD19384, an engineered recombinant human bivalent Fab antibody formed via dimerization of dHLX (synthetic double helix loop helix motif) domains, for potential use in antibody-based molecular imaging of squamous cell carcinoma in the head and neck region. This is a unique construct that has, to the best of our knowledge, never been assessed for molecular imaging in vivo before. The objective of the present study was to evaluate for the first time the in vitro and in vivo binding properties of radio-iodinated AbD19384, and to assess its utility as a targeting agent for molecular imaging of CD44v6-expressing tumors. Antigen specificity and binding properties were assessed in vitro. In vivo specificity and biodistribution of 125I-AbD19384 were next evaluated in tumor-bearing mice using a dual-tumor setup. Finally, AbD19384 was labeled with 124I, and its imaging properties were assessed by small animal PET/CT in tumor bearing mice, and compared with 2-deoxy-2-[18F]fluoro-D-glucose (18F-FDG). In vitro studies demonstrated CD44v6-specific binding with slow off-rate for AbD19384. A favorable biodis-tribution profile was seen in vivo, with tumor-specific uptake. Small animal PET/CT images of 124I-AbD19384 supported the results through clearly visible high CD44v6-expressing tumors and faintly visible low expressing tumors, with superior imaging properties compared to 18F-FDG. Tumor-to-blood ratios increased with time for the conjugate (assessed up to 72 h p.i.), although 48 h p.i. proved best for imaging. Biodistribution and small-animal PET studies demonstrated that the recombinant Fab-dHLX construct AbD19384 is a promising tracer for imaging of CD44v6 antigen expression in vivo, with the future aim to be used for individualized diagnosis and early detection of squamous cell carcinomas in the head and neck region. Furthermore, this proof-of-concept research established the feasibility of using recombinant Fab-dHLX constructs for in vivo imaging of tumor biomarkers.
PMCID: PMC4725455  PMID: 26676731
molecular imaging; CD44v6; Fab-dHLX; F(ab′)2; recombinant antibodies; immuno-PET; head and neck squamous cell carcinoma
4.  Molecular imaging of EGFR and CD44v6 for prediction and response monitoring of HSP90 inhibition in an in vivo squamous cell carcinoma model 
Heat shock protein 90 (HSP90) is essential for the activation and stabilization of numerous oncogenic client proteins. AT13387 is a novel HSP90 inhibitor promoting degradation of oncogenic proteins upon binding, and may also act as a radiosensitizer. For optimal treatment there is, however, the need for identification of biomarkers for patient stratification and therapeutic response monitoring, and to find suitable targets for combination treatments. The aim of this study was to assess the response of surface antigens commonly expressed in squamous cell carcinoma to AT13387 treatment, and to find suitable biomarkers for molecular imaging and radioimmunotherapy in combination with HSP90 inhibition.
Cancer cell proliferation and radioimmunoassays were used to evaluate the effect of AT13387 on target antigen expression in vitro. Inhibitor effects were then assessed in vivo in mice-xenografts. Animals were treated with AT13387 (5 × 50 mg/kg), and were imaged with PET using either 18F-FDG or 124I-labelled tracers for EGFR and CD44v6, and this was followed by ex-vivo biodistribution analysis and immunohistochemical staining.
AT13387 exposure resulted in high cytotoxicity and possible radiosensitization with IC50 values below 4 nM. Both in vitro and in vivo AT13387 effectively downregulated HSP90 client proteins. PET imaging with 124I-cetuximab showed a significant decrease of EGFR in AT13387-treated animals compared with untreated animals. In contrast, the squamous cell carcinoma-associated biomarker CD44v6, visualized with 124I-AbD19384 as well as 18F-FDG uptake, were not significantly altered by AT13387 treatment.
We conclude that AT13387 downregulates HSP90 client proteins, and that molecular imaging of these proteins may be a suitable approach for assessing treatment response. Furthermore, radioimmunotherapy targeting CD44v6 in combination with AT13387 may potentiate the radioimmunotherapy outcome due to radiosensitizing effects of the drug, and could potentially lead to a lower dose to normal tissues.
Electronic supplementary material
The online version of this article (doi:10.1007/s00259-015-3260-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4819754  PMID: 26627081
HSP90 inhibitor; AT13387; 124I; PET; EGFR; CD44v6
5.  Dosimetry of [68Ga]Ga-DO3A-VS-Cys40-Exendin-4 in rodents, pigs, non-human primates and human - repeated scanning in human is possible 
Quantitative PET imaging with [68Ga]Ga-DO3A-VS-Cys40-Exendin-4 has potential use in diabetes and cancer. However, the radiation dose to the kidneys has been a concern for the possibility of repeated imaging studies in humans. Therefore, we investigated the dosimetry of [68Ga]Ga-DO3A-VS-Cys40-Exendin-4 based on the biodistribution data in rats, pigs, non-human primates (NHP) and a human.Organ distribution of [68Ga]Ga-DO3A-VS-Cys40-Exendin-4 in rats (Male Lewis; n=12; 30, 60, and 80 min) was measured ex vivo. The dynamic uptake of [68Ga]Ga-DO3A-VS-Cys40-Exendin-4 in the abdomen was assessed by PET/CT scanning of pigs (male; n = 4, 0-60 min), NHP (Female; cynomolgus; n=3; 0-90 min), and human (female; n=1; 0-40, 100, 120 min).The organ distribution data in each species were extrapolated to those of a human, assuming similar distribution between the species. Residence times were assessed by trapezoidal approximation of the kinetic data. Organ doses (mGy/MBq) and the whole body effective dose (mSv/MBq), was extrapolated by using the OLINDA/EXM 1.1 software. The extrapolated human whole body effective dose was 0.017 ± 0.004 (rats), 0.014 ± 0.004 (pigs), 0.017 ± 0.004 (NHP), and 0.016 (human) mSv/MBq. The absorbed dose to the kidneys was limiting:0.33 ± 0.06 (rats), 0.28±0.05 (pigs), 0.65 ± 0.11 (NHP), and 0.28 (human) mGy/MBq, which corresponded to the maximum yearly administered amounts of 455 (rat), 536 (pig), 231 (NHP), and 536 (human) MBq before reaching the yearly kidney limiting dose of 150 mGy. More than 200 MBq of [68Ga]Ga-DO3A-VS-Cys40-Exendin-4 can be administered yearly in a human, allowing for repeated (2-4 times) scanning. This potentially enables longitudinal clinical PET imaging studies of the GLP-1R in the pancreas, transplanted islets, or insulinoma.
PMCID: PMC4446394  PMID: 26069859
Dosimetry; [68Ga]Ga-DO3A-VS-Cys40-Exendin-4; GLP-1R; PET; islet imaging; insulinoma
6.  Dosimetry of [177Lu]-DO3A-VS-Cys40-Exendin-4 – impact on the feasibility of insulinoma internal radiotherapy 
[68Ga]-DO3A-VS-Cys40-Exendin-4 has been shown to be a promising imaging candidate for targeting glucagon like peptide-1 receptor (GLP-1R). In the light of radiotheranostics and personalized medicine the 177Lu-labelled analogue is of paramount interest. In this study we have investigated the organ distribution of [177Lu]-DO3A-VS-Cys40-Exendin-4 in rat and calculated human dosimetry parameters in order to estimate the maximal acceptable administered radioactivity, and thus potential applicability of [177Lu]-DO3A-VS-Cys40-Exendin-4 for internal radiotherapy of insulinomas. Nine male and nine female Lewis rats were injected with [177Lu]-DO3A-VS-Cys40-Exendin-4 for ex vivo organ distribution study at nine time points. The estimation of human organ/total body absorbed and total effective doses was performed using Organ Level Internal Dose Assessment Code software (OLINDA/EXM 1.1). Six more rats (male: n = 3; female: n = 3) were scanned by single photon emission tomography and computed tomography (SPECT-CT). The renal function and potential cell dysfunction were monitored by creatinine ISTAT and glucose levels. The fine uptake structure of kidney and pancreas was investigated by ex vivo autoradiography. Blood clearance and washout from most of the organs was fast. The kidney was the dose-limiting organ with absorbed dose of 5.88 and 6.04 mGy/MBq, respectively for female and male. Pancreatic beta cells demonstrated radioactivity accumulation. Renal function and beta cell function remained unaffected by radiation. The absorbed dose of [177Lu]-DO3A-VS-Cys40-Exendin-4 to kidneys may limit the clinical application of the agent. However, hypothetically, kidney protection and peptidase inhibition may allow reduction of kidney absorbed dose and amplification of tumour absorbed doses.
PMCID: PMC4396004  PMID: 25973333
Lu-177; Exendin-4; dosimetry; insulinoma; imaging; radiotherapy
7.  Strategy to develop a MAO-A-resistant 5-hydroxy-l-[β-11C]tryptophan isotopologue based on deuterium kinetic isotope effects 
EJNMMI Research  2014;4:62.
The serotonin precursor 5-hydroxy-l-[β-11C]tryptophan ([11C]HTP) is in clinical use for localization of neuroendocrine tumors and has been suggested as a proxy marker for pancreatic islet cells. However, degradation by monoamine oxidase-A (MAO-A) reduces retention and the contrast to non-endocrine tissue.
A synthesis method was developed for 5-hydroxy-l-[β-11C2H]tryptophan ([11C]DHTP), an isotopologue of [11C]HTP, labeled with 11C and 2H at the β-position adjacent to the carbon involved in MAO-A decarboxylation. MAO-A-mediated degradation of [11C]DHTP was evaluated and compared to non-deuterated [11C]HTP.
[11C]DHTP was synthesized with a radiochemical purity of >98%, radioactivity of 620 ± 190 MBq, and deuterium (2H or 2H2) incorporation at the β-position of 22% ±5%. Retention and resistance to MAO-A-mediated degradation of [11C]DHTP were increased in cells but not in non-human primate pancreas.
Partial deuteration of the β-position yields improved resistance to MAO-A-mediated degradation in vitro but not in vivo.
PMCID: PMC4452635  PMID: 26116123
5-Hydroxy-tryptophan; [11C]HTP; Kinetic isotope effect; Deuterium; Neuroendocrine tumors; Beta cell imaging
8.  Striated Muscle as Implantation Site for Transplanted Pancreatic Islets 
Journal of Transplantation  2011;2011:352043.
Islet transplantation is an attractive treatment for selected patients with brittle type 1 diabetes. In the clinical setting, intraportal transplantation predominates. However, due to extensive early islet cell death, the quantity of islets needed to restore glucose homeostasis requires in general a minimum of two donors. Moreover, the deterioration of islet function over time results in few insulin-independent patients after five-year followup. Specific obstacles to the success of islet transplantation include site-specific concerns for the liver such as the instant blood mediated inflammatory reaction, islet lipotoxicity, low oxygen tension, and poor revascularization, impediments that have led to the developing interest for alternative implantation sites over recent years. Within preclinical settings, several alternative sites have now been investigated and proven favorable in various aspects. Muscle is considered a very promising site and has physiologically properties and technical advantages that could make it optimal for islet transplantation.
PMCID: PMC3235886  PMID: 22174984
9.  Decreased defluorination using the novel beta-cell imaging agent [18F]FE-DTBZ-d4 in pigs examined by PET 
EJNMMI Research  2011;1:33.
Fluorine-18 dihydrotetrabenazine [DTBZ] analogues, which selectively target the vesicular monoamine transporter 2 [VMAT2], have been extensively studied for in vivo quantification of beta cell mass by positron-emission tomography [PET]. This study describes a novel deuterated radioligand [18F]fluoroethyl [FE]-DTBZ-d4, aimed to increase the stability against in vivo defluorination previously observed for [18F]FE-DTBZ.
[18F]FE-DTBZ-d4 was synthesized by alkylation of 9-O-desmethyl-(+)-DTBZ precursor with deuterated [18F]FE bromide ([18F]FCD2CD2Br). Radioligand binding potential [BP] was assessed by an in vitro saturation homogenate binding assay using human endocrine and exocrine pancreatic tissues. In vivo pharmacokinetics and pharmacodynamics [PK/PD] was studied in a porcine model by PET/computed tomography, and the rate of defluorination was quantified by compartmental modeling.
[18F]FE-DTBZ-d4 was produced in reproducible good radiochemical yield in 100 ± 20 min. Radiochemical purity of the formulated product was > 98% for up to 5 h with specific radioactivities that ranged from 192 to 529 GBq/μmol at the end of the synthesis. The in vitro BP for VMAT2 in the islet tissue was 27.0 ± 8.8, and for the exocrine tissue, 1.7 ± 1.0. The rate of in vivo defluorination was decreased significantly (kdefluorination = 0.0016 ± 0.0007 min-1) compared to the non-deuterated analogue (kdefluorination = 0.012 ± 0.002 min-1), resulting in a six fold increase in half-life stability.
[18F]FE-DTBZ-d4 has similar PK and PD properties for VMAT2 imaging as its non-deuterated analogue [18F]FE-DTBZ in addition to gaining significantly increased stability against defluorination. [18F]FE-DTBZ-d4 is a prime candidate for future preclinical and clinical studies on focal clusters of beta cells, such as in intramuscular islet grafts.
PMCID: PMC3284452  PMID: 22214308
beta cell mass; dihydrotetrabenazine; PET; VMAT2
10.  Assessment of receptor occupancy-over-time of two dopamine transporter inhibitors by [11C]CIT and target controlled infusion 
Upsala Journal of Medical Sciences  2011;116(2):100-106.
Occupancy-over-time was determined for two dopamine transporter (DAT) inhibitors through modeling of their ability to displace the PET ligand [11C]CIT. The tracer was held at a pseudo steady state in a reference tissue by target controlled infusion.
Rhesus monkeys (n = 5) were given [11C]CIT and studied with a PET scanner. Tracer uptake in the reference tissue cerebellum was held at a pseudo steady state by use of target controlled infusion. The pharmacokinetics/pharmacodynamics(PK/PD) of [11C]CIT was assessed through the simplified reference tissue model (SRTM). Bupropion (n = 2) and GBR-12909 (n = 2) receptor occupancies were estimated through modeling of their effects on [11C]CIT displacement.
There was a high uptake of [11C]CIT in striatum, which contains a high DAT density. The reference tissue cerebellum had a comparatively low uptake. The modeling of [11C]CIT PK/PD properties in striatum showed high binding potential (BP = 5.34 ± 0.78). Both DAT inhibitors caused immediate displacement of [11C]CIT after administration. The occupancy-over-time was modeled as a mono-exponential function, describing initial maximal occupancy (Occ0) and rate of ligand–receptor dissociation (koff). GBR-12909 showed irreversible binding (koff = 0) after an initial occupancy of 76.1%. Bupropion had a higher initial occupancy (84.5%) followed by a release half-life of 33 minutes (koff = 0.021).
The proposed model can be used for assessment of in-vivo occupancy-over-time of DAT ligands by use of target controlled infusion of [11C]CIT. The concept of assessing drug–receptor interactions by studying perturbations of a PET tracer from a pseudo steady state can be transferred to other CNS systems.
PMCID: PMC3078538  PMID: 21443419
CCIP; [11C]CIT; DAT inhibitor; SRTM; TCI
11.  A computerized Infusion Pump for control of tissue tracer concentration during Positron Emission Tomography in vivo Pharmacokinetic/Pharmacodynamic measurements 
A computer controlled infusion pump (UIPump) for regulation of target tissue concentration of radioactive compounds was developed for use in biological research and tracer development for PET.
Based on observed tissue or plasma kinetics after a bolus injection of the tracer an algorithm calculates the infusion needed to obtain a specified target kinetic curve. A computer feeds this infusion scheme into an infusion pump connected to an animal via a venous catheter. The concept was validated using [11C]Flumazenil administrated to Sprague-Dawley rats where the whole brain distribution and kinetic of the tracer was measured over time using a microPET-scanner. The accuracy and precision of the system was assessed by producing steady-state levels of the tracer and by mimicking kinetics after oral administration.
Various kinetic profiles could be generated, including rapid achievement of constant levels, or step-wise increased levels. The resulting tissue curves had low deviation from the target curves according to the specified criteria: AUC (%): 4.2 ± 2.8, Maximal deviation (%): 13.6 ± 5.0 and R2: 0.95 ± 0.02.
The UIPump-system is suitable for use in PET-research for assessment of PK/PD properties by simulation of different tracer tissue kinetics in vivo.
PMCID: PMC2430701  PMID: 18513382

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