Cardiac amyloidosis is a differential diagnosis in heart failure and is associated with high mortality. There is currently no noninvasive imaging test available for specific diagnosis. N-[methyl-¹¹C]2-(4′-methylamino-phenyl)-6-hydroxybenzothiazole (¹¹C-PIB) PET is used in the evaluation of brain amyloidosis. We evaluated the potential use of ¹¹C-PIB PET in systemic amyloidosis affecting the heart.

Methods:

Patients (n = 10) diagnosed with systemic amyloidosis—including heart involvement of either monoclonal immunoglobulin light-chain (AL) or transthyretin (ATTR) type—and healthy volunteers (n = 5) were investigated with PET/CT using ¹¹C-PIB to study cardiac amyloid deposits and with ¹¹C-acetate to measure myocardial blood flow to study the impact of global and regional perfusion on PIB retention.

Results:

Myocardial ¹¹C-PIB uptake was visually evident in all patients 15–25 min after injection and was not seen in any volunteer. A significant difference in ¹¹C-PIB retention in the heart between patients and healthy controls was found. The data indicate that myocardial amyloid deposits in patients diagnosed with systemic amyloidosis could be visualized with ¹¹C-PIB. No correlation between ¹¹C-PIB retention index and myocardial blood flow as measured with ¹¹C-acetate was found on the global level, whereas a positive correlation on the segmental level was seen in a single patient.

Conclusion:

¹¹C-PIB and PET could be a method to study systemic amyloidosis of type AL and ATTR affecting the heart and should be investigated further both as a diagnostic tool and as a noninvasive method for treatment follow-up.

The cholinergic system is involved in neurodegenerative diseases, and visualization of cholinergic innervations with positron emission tomography (PET) would be a useful tool in understanding these diseases. A ligand for the vesicular acetylcholine transporter (VAChT), acknowledged as a marker for cholinergic neurons, could serve as such a PET tracer. The aim was to find a VAChT PET tracer using a library concept to create a small but diverse library of labeled compounds. From the same precursor and commercially available aryl iodides 6a-f, six potential VAChT PET tracers, [C-11]-(+/-)5a-f, were C-11-labeled by a palladium (0)-mediated aminocarbonylation, utilizing a standard protocol. The labeled compounds [C-11]-(+/-)5a-f were obtained in radiochemical purities >95% with decay-corrected radiochemical yields and specific radioactivities between 4-25% and 124-597 GBq/mu mol, respectively. Autoradiography studies were then conducted to assess the compounds binding selectivity for VAChT. Labeled compounds [C-11]-(+/-)5d and [C-11]-(+/-)5e showed specific binding but not enough to permit further preclinical studies. To conclude, a general method for a facile synthesis and labeling of a small piperazine-based library of potential PET tracers for imaging of VAChT was shown, and in upcoming work, another scaffold will be explored using this approach.

Several peptides comprising Arg-Gly-Asp (RGD) domain and macrocyclic chelator were labeled with ⁶⁸Ga for the imaging of angiogenesis. The analogues varied in peptide constitution, linker and chelator type. The labeling efficiency did not vary with the peptide constitution and linker type, but depended on the chelator type. Four of the compounds containing 2,2',2'',2'''-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA) chelator were labeled at 90 ± 5°C using conventional or microwave heating reaching 90% of ⁶⁸Ga incorporation after 5 and 2 min respectively, when the concentration of the precursor was 2.5 μM. The compound having 2,2',2''-(1,4,7-triazonane-1,4,7-triyl)triacetic acid (NOTA) as the chelator could be labeled at room temperature within 5 min using 2.5 μM peptide precursor. Two of the compounds contained a poly (ethylene glycol) (PEG) linker to the chelator. The biodistribution of the analogues was studied in male rats.

Several peptides comprising Arg-Gly-Asp (RGD) domain and macrocyclic chelator were labeled with Ga-68 for the imaging of angiogenesis. The analogues varied in peptide constitution, linker and chelator type. The labeling efficiency did not vary with the peptide constitution and linker type, but depended on the chelator type. Four of the compounds containing 2,2', 2 '', 2'''-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl) tetraacetic acid (DOTA) chelator were labeled at 90 +/- 5 degrees C using conventional or microwave heating reaching 90% of Ga-68 incorporation after 5 and 2 min respectively, when the concentration of the precursor was 2.5 mu M. The compound having 2,2', 2 ''-(1,4,7-triazonane1,4,7-triyl)triacetic acid (NOTA) as the chelator could be labeled at room temperature within 5 min using 2.5 mu M peptide precursor. Two of the compounds contained a poly (ethylene glycol) (PEG) linker to the chelator. The biodistribution of the analogues was studied in male rats.

A validated imaging marker for beta-cell mass would improve understanding of diabetes etiology and enable new strategies in therapy development. We previously identified the membrane-spanning protein GPR44 as highly expressed and specific to the beta cells of the pancreas. The selective GPR44 antagonist MK-7246 was radiolabeled with carbon-11 and the resulting positron-emission tomography (PET) tracer [C-11]MK-7246 was evaluated in a pig model and in vitro cell lines. The [C-11]MK-7246 compound demonstrated mainly hepatobiliary excretion with a clearly defined pancreas, no spillover from adjacent tissues, and pancreatic binding similar in magnitude to the previously evaluated GPR44 radioligand [C-11]AZ12204657. The binding could be blocked by preadministration of nonradioactive MK-7246, indicating a receptor-binding mechanism. [C-11]MK-7246 showed strong potential as a PET ligand candidate for visualization of beta-cell mass (BCM) and clinical translation of this methodology is ongoing.

INTRODUCTION: 5-Hydroxy-[β-(11)C]-L-tryptophan ([(11)C]HTP) is an established positron emission tomography (PET) imaging agent for neuroendocrine tumors (NETs). It has also been used for other clinical research purposes in neurology and diabetes. However, its widespread use is limited by the short physical half-life of the radionuclide and a difficult radiosynthesis. Therefore, a Fluorine-18 labeled analogue, 5-[(18)F]Fluoro-L-tryptophan ([(18)F]FTRP), has been proposed as a functional analogue. There is no published method for the synthesis of L-[(18)F]FTRP. We have therefore developed a synthesis of 5-fluoro-[β-(11)C]-L-tryptophan ([(11)C]FTRP), based on the existing chemo-enzymatic method for [(11)C]HTP and evaluated the potential usefulness of radiolabeled FTRP as a substitute for [(11)C]HTP.

METHODS: The in vitro and in vivo behavior of [(11)C]FTRP, including the dependence of key enzymes in the serotonergic metabolic pathway, was investigated in NET cell lines, NET xenograft carrying immunodeficient mice, normal rats and in non-human primate. [(11)C]HTP was used for direct comparison.

RESULTS: Uptake of [(11)C]FTRP in NET cell lines in vitro was mediated by enzymes involved in serotonin synthesis and metabolism, similar to [(11)C]HTP. In vivo biodistribution, either in rodent or non-human primate, was not affected by selectively inhibiting enzymatic steps in the serotonergic metabolic pathway.

CONCLUSION: [(11)C]FTRP has in vitro biological function similar to that of [(11)C]HTP. However, this function is not retained in vivo as shown by biodistribution and PET/CT studies. Radiolabeled FTRP is thus not likely to provide an advantage over [(11)C]HTP in PET imaging in oncology, neurology or diabetes.

Introduction: Neutrophils are part of the innate immune system and function as a first line of defense against invading microorganisms. Overactivity of the immune system may result in a devastating immuno-inflammation with extensive damage to tissue leading to organ damage and/or failure. The literature suggests several human diseases in which neutrophil elastase (NE) is postulated to be important in the pathophysiology including inflammatory bowel disease (IBD), chronic obstructive pulmonary disorder (COPD), abdominal aortic aneurysms (AAA), breast and lung cancer, and recently also in Sars-cov-2 virus infection (Covid-19). In particular, the lungs are affected by the destructive power of the protease neutrophil elastase (NE). In this paper, we report the pre-clinical development of a selective and specific positron emission tomography (PET) tracer, [C-11] GW457427, as an in vivo biomarker for the study of NE, now available for human studies.

Methods: [C-11]GW457427 was produced by methylation of GW447631 using [C-11]methyl triflate and GMP validated production and quality control methods were developed. Chemical purity was high with no traces of the precursor GW611437 or other uv-absorbing compounds. A method for the determination of intact [C-11] GW457427 in plasma was developed and the binding characteristics were evaluated in vitro and in vivo. An animal model for lung inflammation was used to investigate the specificity and sensitivity of the [C-11]GW457427 tracer for neutrophil elastase (NE) in pulmonary inflammation, verified by blockade using two structurally different elastase inhibitors.

Results: [C-11]GW457427 was obtained in approximately 45% radiochemical yield and with a radiochemical purity higher than 98%. Molar activity was in the range 130-360 GBq/mu mol. Binding to NE was shown to be highly specific both in vitro and in vivo and a significantly higher uptake of tracer was found in a lipopolysaccharide mouse model of pulmonary inflammation compared with control animals. The uptake in lung tissue measured as standardized uptake value (SUV) strongly correlated with tissue NE content as measured by ELISA. In vitro studies also showed specific tracer binding in aortic tissue of patients with abdominal aorta aneurysm (AAA). The rate of metabolism in rats was appropriate considering the critical balance between available tracer for binding and requirement for blood clearance with about 40% and 20% intact [C-11]GW457427 in plasma at 5 and 40 min, respectively. Radioactivity was cleared from blood and organs in control animals with mainly hepatobiliary excretion with distribution in the intestines and the urinary bladder; but without retention of the tracer in healthy organs of interests such as the lung, liver, kidneys or in the cardiovascular system. A dosimetry study in rat indicated that the whole-body effective dose was 2.2 mu Sv/MBq with bone marrow as the limiting organ. It is estimated that up to five PET-CT investigations could be performed in humans without exceeding a total dose of 10 mSv.

Conclusion: [C-11]GW457427 is a promising in vivo PET-biomarker for NE with high specific binding demonstrated both in vitro and in vivo. A GMP validated production method including quality control has been developed and a microdosing toxicity study performed with no adverse signs. [C-11]GW457427 is currently being evaluated in a First-In-Man PET study.

Introduction: Development of a selective and specific high affinity PET tracer, [C-11]UCB-A, for the in vivo study of SV2A expression in humans. Radiochemistry and preclinical studies in rats and pigs including development of a tracer kinetic model to determine V-T. A method for the measurement of percent intact tracer in plasma was developed and the radiation dosimetry was determined in rats. Results: 3-5 GBq of [C-11]UCB-A could be produced with radiochemical purity exceeding 98% with a specific radioactivity of around 65 GBq/mu mol. In vitro binding showed high selective binding towards SV2A. [C-11]UCB-A displayed a dose-dependent and reversible binding to SV2A as measured with PET in rats and pigs and the V-T could be determined by Logan analysis. The dosimetry was favorable and low enough to allow multiple administrations of [C-11]UCB-A to healthy volunteers, and the metabolite analysis showed no sign of labeled metabolites in brain. Conclusions: We have developed the novel PET tracer, [C-11]UCB-A, that can be used to measure SV2A expression in vivo. The dosimetry allows up to 5 administrations of 400 MBq of [C-11]UCB-A in humans. Apart from measuring drug occupancy, as we have shown, the tracer can potentially be used to compare SV2A expression between individuals because of the rather narrow range of baseline V-T values. This will have to be further validated in human studies.

Two F-18-labeled analogs of vorozole ([F-18]FVOZ and [F-18]FVOO) have been developed as potential tools for the in vivo characterization of aromatase. The pharmacologicalproperties of these radioligands were evaluated using in vitro binding and in vivo distribution studies in the rat and primate. Saturation binding studies using rat ovary gave K-D and B-max values of 0.21 +/- 0.1 nM and 210 +/- 20 fmol/mg, respectively, for [F-18]FVOZ, and 7.6 +/- 1nMand 293 +/- 12fmol/mg, respectively, for [F-18]FVOO. Organ distribution studies in rats showed the highest accumulation in the adrenal glands, with standardized uptake values (SUVs) of 15 to 20, followed by ovaries and liver with SUVs of approximately 5. Ex vivo and in vitro autoradiography of the rat brain showed specific binding of both [F-18]FVOZ and [F-18]FVOO mainly in the amygdala. Positron emission tomography (PET) studies were performed in the Rhesus monkey, and these showed displaceable binding in the amygdala and the hypothalamus preoptic area. The PET images were also analyzed using masked volume-wise principal component analysis. These studies suggest that [F-18]FVOZ might be a suitable tracer for the study of aromatase in vitro and in vivo, and could be an alternative to [C-11]vorozole in human PET studies.

Systemic amyloidosis is caused by extracellular deposition of insoluble fibrillar proteins arranged in beta-pleated sheets. [C-11] PIB has been used in PET studies to assess A beta deposition in brain of patients with Alzheimer's disease (AD). The possibility to visualize other types of amyloid deposits with [C-11] PIB would be of potential clinical importance in early diagnosis and for following therapeutic effects. In the present study, we evaluated in vitro binding of [3 H] PIB to tissues containing transthyretin (ATTR), immunoglobulin light-chain (AL), amyloid protein A (AA) and Ab amyloid. We found significantly higher binding of [H-3] PIB in tissue from systemic amyloidoses than in control tissue, i.e. 4.7 times higher (p<0.05). [H-3] PIB showed the highest affinity to cortex of AD brain (IC50 = 3.84 nM), while IC50 values were much higher for ATTR, AA and AL type of amyloidosis and large variations in affinity were observed even within tissues having the same type of amyloidosis. Extraction with guanidine-HCl, which disrupts the beta-sheet structure, decreased the protein levels and, concomitantly, the binding of [H-3] PIB in all four types of amyloidoses.

Bone morphogenetic proteins (BMP's) are vital for bone and cartilage formation, where bone morphogenetic protein-2 (BMP-2) is acknowledged as a growth factor in osteoblast differentiation. However, uncontrolled delivery may result in adverse clinical effects. In this study we investigated the possibility for longitudinal and non-invasive monitoring of implanted [¹²⁵I]BMP-2 retention and its relation to ossification at the site of implantation. A unilateral critically sized femoral defect was produced in the left limb of rats while the right femur was retained intact as a paired reference control. The defect was filled with a hyaluronan hydrogel with 25% hydroxyapatite alone (carrier control; n = 2) or combined with a mixture of [¹²⁵I]BMP-2 (150 μg/ml; n = 4). Bone formation was monitored using micro computed tomography (μCT) scans at 1, 3, 5, 7, 9 and 12 weeks. The retention of [¹²⁵I]BMP-2 was assessed with single photon emission computed tomography (SPECT), and the bone healing process was followed with sodium fluoride (Na¹⁸F) using positron emission tomography (PET) at day 3 and at week 2, 4, and 6. A rapid burst release of [¹²⁵I]BMP-2 was detected via SPECT. This was followed by a progressive increase in uptake levels of [¹⁸F]fluoride depicted by PET imaging that was confirmed as bone formation via μCT. We propose that this functional, non-invasive imaging method allows tri-modal visualisation of the release of BMP-2 and the following in vivo response. We suggest that the potential of this novel technique could be considered for preclinical evaluation of novel smart materials on bone regeneration.

Phosphodiesterase 10A (PDE10A) plays a key role in the regulation of brain striatal signaling, and several pharmaceutical companies currently investigate PDE10A inhibitors in clinical trials for various central nervous system diseases. A PDE10A PET ligand may provide evidence that a clinical drug candidate reaches and binds to the target. Here we describe the successful discovery and initial validation of the novel radiolabeled PDE10A ligand 5,8-dimethyl-2-[2-((1-C-11-methyl)-4-phenyl-1H-imidazol-2-yl)-ethyl]-[1,2,4]triazolo[1,5-a]pyridine (C-11-Lu AE92686) and its tritiated analog H-3-Lu AE92686. Methods: Initial in vitro experiments suggested Lu AE92686 as a promising radioligand, and the corresponding tritiated and C-11-labeled compounds were synthesized. 3H-Lu AE92686 was evaluated as a ligand for in vivo occupancy studies in mice and rats, and C-11-Lu AE92686 was evaluated as a PET tracer candidate in cynomolgus monkeys and in humans. Results: C-11-Lu AE92686 displayed high specificity and selectivity for PDE10A-expressing regions in the brain of cynomolgus monkeys and humans. Similar results were found in rodents using 3H-Lu AE92686. The binding of C-11-Lu AE92686 and 3H-Lu AE92686 to striatum was completely and dose-dependently blocked by the structurally different PDE10A inhibitor 2-[4-(1-methyl-4-pyridin-4-yl-1H-pyrazol-3-yl)-phenoxymethyl]-quinoline (MP-10) in rodents and in monkeys. In all species, specific binding of the radioligand was seen in the striatum but not in the cerebellum, supporting the use of the cerebellum as a reference region. The binding potentials (BPND) of C-11-Lu AE92686 in the striatum of both cynomolgus monkeys and humans were evaluated by the simplified reference tissue model with the cerebellum as the reference tissue, and BPND was found to be high and reproducible-that is, BP(ND)s were 6.5 +/- 0.3 (n = 3) and 7.5 +/- 1.0 (n = 12) in monkeys and humans, respectively. Conclusion: Rodent, monkey, and human tests of labeled Lu AE92686 suggest that C-11-Lu AE92686 has great potential as a human PET tracer for the PDE10A enzyme.

Introduction

The enzyme β-secretase 1 (BACE-1) is associated with the catalytic cleavage of amyloid precursor protein (APP) which leads to the production of amyloid-β, an amyloidogenic peptide that forms insoluble fibrils and is linked to neurodegeneration and Alzheimer's disease (AD). A PET-radioligand for the quantification of BACE-1 would be useful for the understanding of AD. In this report, we describe the synthesis and carbon-11 radiolabeling of a potent hydroxyethylamine BACE-1 enzyme inhibitor (BSI-IV) and its evaluation in vitro and in vivo.

Methods

¹¹[C]-N¹-((2S,3R)-4-(cyclopropylamino)-3-hydroxy-1-phenylbutan-2-yl)-5-(N-methylmethyl-sulfonamido)-N³-((R)-1-phenylethyl)isophthalamide, a β-secretase inhibitor, denoted here as [¹¹C]BSI-IV was synthesized through a palladium-mediated aminocarbonylation with an aryl halide precursor (I or Br) and [¹¹C]CO. The effect of different palladium/ligand-complexes on radiochemical yield in the carbonylative reaction was investigated. The binding of the labeled compound to BACE-1 enzyme was studied in vitro by frozen section autoradiography from brains of healthy rats. Dynamic small animal PET-CT studies and ex vivo biodistribution were performed in male rats.

Results

The halide precursors were synthesized in six steps starting from methyl-3-nitrobenzoate with an overall yield of 21–26%. [¹¹C]BSI-IV was obtained in 29 ± 12% decay corrected radiochemical yield (n = 12) with a specific activity of 790 ± 155 GBq/μmol at the end of synthesis with a radiochemical purity of > 99%. The preclinical studies showed that [¹¹C]BSI-IV has a rapid metabolism in rat with excretion to the small intestines.

Conclusion

¹¹[C]BSI-IV was obtained in sufficient amount and purity to enable preclinical investigation. The preclinical studies showed low specific binding in vitro and fast clearance in vivo and a low uptake in the brain. These findings suggests that [¹¹C]BSI-IV has limited use as a PET-ligand for the study of BACE-1 or AD.

Introduction: The enzyme beta-secretase 1 (BACE-1) is associated with the catalytic cleavage of amyloid precursor protein (APP) which leads to the production of amyloid-p, an amyloidogenic peptide that forms insoluble fibrils and is linked to neurodegeneration and Alzheimer's disease (AD). A PET-radioligand for the quantification of BACE-1 would be useful for the understanding of AD. In this report, we describe the synthesis and carbon-11 radiolabeling of a potent hydroxyethylamine BACE-1 enzyme inhibitor (BSI-IV) and its evaluation in vitro and in vivo. Methods: (11)[C]-N-1-((2S,3R)-4-(cyclopropylamino)-3-hydroxy-1-phenylbutan-2-y1)-5-(N-methylmethylsulfonamido)-N-3-((R)-1-phenylethyl)isophthalamide, a p-secretase inhibitor, denoted here as [C-11]BSIIV was synthesized through a palladium-mediated aminocarbonylation with an aryl halide precursor (I or Br) and [C-11]CO. The effect of different palladium/ligand-complexes on radiochemical yield in the carbonylative reaction was investigated. The binding of the labeled compound to BACE-1 enzyme was studied in vitro by frozen section autoradiography from brains of healthy rats. Dynamic small animal PET-CT studies and ex vivo biodistribution were performed in male rats. Results: The halide precursors were synthesized in six steps starting from methyl-3-nitrobenzoate with an overall yield of 21-26%. [C-11]BSI-IV was obtained in 29 +/- 12% decay corrected radiochemical yield (n = 12) with a specific activity of 790 +/- 155 GBq/umol at the end of synthesis with a radiochemical purity of >99%. The predinical studies showed that [C-11]BSI-IV has a rapid metabolism in rat with excretion to the small intestines. Conclusion: [C-11]BSI-IV was obtained in sufficient amount and purity to enable predinical investigation. The predinical studies showed low specific binding in vitro and fast clearance in vivo and a low uptake in the brain. These findings suggests that [C-11]BSI-IV has limited use as a PET-ligand for the study of BACE-1 or AD.

PURPOSE: With the ambition of improving the management of pancreatic neuroendocrine tumors (P-NETs), we developed and preliminary validated a novel fluorine-18 labelled HSP₉₀ ligand.

METHODS: A precursor containing methoxymethyl ethers protecting groups and a tosyl as leaving group was synthesized. The target compound was labeled with nucleophilic ¹⁸F-fluoride and the protecting groups was subsequently removed with hydrochloric acid before purification. In vitro cell- and frozen section autoradiography and in vivo animal studies were performed.

RESULTS: The precursor was successfully synthesized and utilized in the ¹⁸F-radiolabeling giving 0.5-1.0 GBq of pure product with a synthesis time of 70 min. In vitro experiments indicated a high specific binding, but in vivo studies showed no tumor uptake due to fast hepatobiliary metabolism and excretion.

CONCLUSIONS: Despite the unfavorable in vivo properties of the tracer, the promising results from in vitro autoradiography experiments in frozen sections of P-NETs from surgical resection encourage us to continue the project aiming the improvement of in vivo properties of the tracer.

Three oligothiophenes were evaluated as PET ligands for the study of local and systemic amyloidosis ex vivo using tissue from patients with amyloid deposits and in vivo using healthy animals and PET-CT. The ex vivo binding studies revealed that all three labeled compounds bound specifically to human amyloid deposits. Specific binding was found in the heart, kidney, liver, and spleen. To verify the specificity of the oligothiophenes toward amyloid deposits, tissue sections with amyloid pathology were stained using the fluorescence exhibited by the compounds and evaluated with multiphoton microscopy. Furthermore, a in vivo monkey PET-CT study showed very low uptake in the brain, pancreas, and heart of the healthy animal indicating low nonspecific binding to healthy tissue. The biological evaluations indicated that this is a promising group of compounds for the visualization of systemic and localized amyloidosis.

Ischemia reperfusion injury is one of the major complications responsible for delayed graft function in kidney transplantation. Applications to reduce reperfusion injury are essential due to the widespread use of kidneys from deceased organ donors where the risk for delayed graft function is especially prominent. We have recently shown that coating of inflamed or damaged endothelial cells with a unique heparin conjugate reduces thrombosis and leukocyte recruitment. In this study we evaluated the binding capacity of the heparin conjugate to cultured human endothelial cells, to kidneys from brain-dead porcine donors, and to murine kidneys during static cold storage. The heparin conjugate was able to stably bind cultured endothelial cells with high avidity, and to the renal vasculature of explanted kidneys from pigs and mice. Treatment of murine kidneys prior to transplantation reduced platelet deposition and leukocyte infiltration 24 hours post-transplantation, and significantly improved graft function. The present study thus shows the benefits of enhanced protection of the renal vasculature during cold storage, whereby increasing the antithrombotic and anti-adhesive properties of the vascular endothelium yields improved renal function early after transplantation.

Today, there is a lack of clinically available imaging techniques to detect and quantify specific immune cell populations. Neutrophils are one of the first immune cells at the site of inflammation, and they secrete the serine protease neutrophil elastase (NE), which is crucial in the fight against pathogens. However, the prolonged lifespan of neutrophils increases the risk that patients will develop severe complications, such as acute respiratory distress syndrome (ARDS). Here, we evaluated the novel radiolabeled NE inhibitor 11C-GW457427 in a pig model of ARDS, for detection and quantification of neutrophil activity in the lungs. Methods: ARDS was induced by intravenous administration of oleic acid to 5 farm pigs, and 4 were considered healthy controls. The severity of ARDS was monitored by clinical parameters of lung function and plasma biomarkers. Each pig was studied with 11C-GW457427 and PET/CT, before and after pretreatment with the NE inhibitor GW311616 to determine in vivo binding specificity. PET image data were analyzed as SUVs and correlated with immunohistochemical staining for NE in biopsies. Results: The binding of 11C-GW457427 was increased in pig lungs with induced ARDS (median SUVmean, 1.91; interquartile range [IQR], 1.67-2.55) compared with healthy control pigs (P < 0.05 and P = 0.03, respectively; median SUVmean, 1.04; IQR, 0.66-1.47). The binding was especially strong in lung regions with high levels of NE and ongoing inflammation, as verified by immunohisto-chemistry. The binding was successfully blocked by pretreatment of an NE inhibitor drug, which demonstrated the in vivo specificity of 11C-GW457427 (P < 0.05 and P = 0.04, respectively; median SUVmean, 0.60; IQR, 0.58-0.77). The binding in neutrophil-rich tissues such as bone marrow (P < 0.05 and P = 0.04, respectively; baseline median SUVmean, 5.01; IQR, 4.48-5.49; block median SUVmean, 1.57; IQR, 0.95-1.85) and spleen (median SUVmean, 2.14; IQR, 1.19-2.36) was also high in all pigs. Conclusion: 11C-GW457427 binds to NE in a porcine model of oleic acid-induced lung inflammation in vivo, with a specific increase in regional lung, bone marrow, and spleen SUV. 11C-GW457427 is a promising tool for localizing, tracking, and quantifying neutrophil-facilitated inflammation in clinical diagnostics and drug development.

Introduction

Pathological formation of fibrosis, is an important feature in many diseases. Fibrosis in liver and pancreas has been associated to metabolic disease including type 1 and 2 diabetes. The current methods for detecting and diagnosing fibrosis are either invasive, or their sensitivity to detect fibrosis in early stage is limited. Therefore, it is crucial to develop non-invasive methods to detect, stage and study the molecular processes that drive the pathology of liver fibrosis. The peptide LRELHLNNN was previously identified as a selective binder to collagen type I with an affinity of 170 nM. Radiolabelled LRELHLNNN thus constitute a potential PET tracer for fibrosis.

Method

LRELHLNNN was conjugated to a DOTA/NOTA moiety via a PEG₂-linker. DOTA-PEG₂-LRELHLNNN was labelled with Gallium-68 and NOTA- PEG₂-LRELHLNNN with aluminium fluoride-18. Biodistribution of [⁶⁸Ga]Ga-DOTA-PEG₂-LRELHLNNN and [¹⁸F]AlF-NOTA-PEG₂-LRELHLNNN was performed in healthy rats ex vivo and in vivo. The ⁶⁸Ga-labelled analogue was evaluated in a mouse model of liver fibrosis by PET/MRI-imaging. The human predicted dosimetry of the tracers was extrapolated from rat ex vivo biodistribution studies at 10, 20, 40, 60, 120, 180 min (only fluoride-18) post-injection.

Results

The peptides were successfully radiolabelled with gallium-68 and aluminium fluoride-18, respectively. The biodistribution of [⁶⁸Ga]Ga-DOTA-PEG₂-LRELHLNNN and [¹⁸F]AlF-NOTA-PEG₂-LRELHLNNN was favorable showing rapid clearance and low background binding in organs where fibrosis may develop. Binding of [⁶⁸Ga]Ga-DOTA-PEG₂-LRELHLNNN to fibrotic liver was higher than surrounding tissues in mice with induced hepatic fibrosis. However, the binding was in the range of SUV 0.3, indicating limited targeting of the tracer to liver. The extrapolated human predicted dosimetric profiles of [⁶⁸Ga]Ga-DOTA-PEG₂-LRELHLNNN and [¹⁸F]AlF-NOTA-PEG₂-LRELHLNNN were beneficial, potentially allowing at least three PET examinations annually.

Conclusions

We describe the modification, radiolabelling and evaluation of the collagen type I binding peptide LRELHLNNN. The resulting radiotracer analogues demonstrated suitable biodistribution and dosimetry. [⁶⁸Ga]Ga-DOTA-PEG₂-LRELHLNNN exhibited binding to hepatic fibrotic lesions and is a promising tool for PET imaging of fibrosis.

Advances in knowledge

Validation of a new collagen targeting PET tracer.

Implications for patient care

Early, non-invasive diagnosis and stratification of fibrosis in order to improve the diagnosis, staging and treatment of patients with diseases involving fibrosis.

PurposePolyvinylalcohol-carbazate (PVAC) is a soluble functionalized polymer acting as a carbonyl scavenger. This study aimed to create a radiolabelled PVAC and investigate the pharmacokinetics and biodistribution of PVAC in naïve animals and ischemia models. 

MethodsPVAC was labelled using radionuclide [18F]FBA to track the substance with PET. Sprague Dawley rats underwent an ischemic event, either to the hind limb or to the kidney, while others served as controls. To study the pharmacokinetics rats were injected with radiolabelled or fluorochrome labelled PVAC. Radiolabelled PVAC was injected, and animals were followed by PET for 90 min, biodistribution ex vivo was finally examined.  Injection of fluorochrome-labelled PVAC was followed by repeated blood sampling to measure the circulating concentration. 

ResultsIn control animals, PVAC was mainly confined to the bloodstream followed by elimination via kidneys and accumulation in the bladder. Ex vivo biodistribution of PVAC confirmed the highest uptake in urine followed by blood, kidneys and other well-perfused organs. The elimination of I.V. administered PVAC was split into a fast phase (t1/2 = 0.2 h) followed by a slow phase (t1/2 = 10.73 h), with near-complete elimination from blood after 48 h. Both the ischemic kidney (fourfold increase, p = <0.001) and limb models (threefold increase, p = <0.001) demonstrated a higher uptake of PVAC in ischemic tissues, ex vivo radioactivity detection.

ConclusionLabelled PVAC, an aldehyde-carbonyl scavenger, is a promising new strategy to detect ischemic tissues. Potential therapeutic effects of PVAC on ischemic injuries should be investigated further.

Introduction: [¹¹C]Metomidate ([¹¹C]MTO), the methyl ester analogue of etomidate, was developed as a positron emission tomography (PET) radiotracer for adrenocortical tumours and has also been suggested for imaging in primary aldosteronism (PA). A disadvantage of [¹¹C]MTO is the rather high non-specific binding in the liver, which impacts both visualization and quantification of the uptake in the right adrenal gland. Furthermore, the short 20-minute half-life of carbon-11 is a logistic challenge in the clinical setting.

Objectives: The aim of this study was to further evaluate the previously published fluorine-18 (T1/2=109.5 min) etomidate analogue, para-chloro-2-[¹⁸F]fluoroethyl etomidate; [¹⁸F]CETO, as an adrenal PET tracer.

Methods: In vitro experiments included autoradiography on human and cynomolgus monkey (non-human primate, NHP) tissues and binding studies on adrenal tissue from NHPs. In vivo studies with [¹⁸F]CETO in mice, rats and NHP, using PET and CT/MRI, assessed biodistribution and binding specificity in comparison to [¹¹C]MTO.

Results: The binding of [¹⁸F]CETO in the normal adrenal cortex, as well as in human adrenocortical adenomas and adrenocortical carcinomas, was shown to be specific, both in vitro (in humans) and in vivo (in rats and NHP) with an in vitro Kd of 0.66 nM. Non-specific uptake of [¹⁸F]CETO in NHP liver was found to be low compared to that of [¹¹C]MTO.

Conclusions: High specificity of [¹⁸F]CETO to the adrenal cortex was demonstrated, with in vivo binding properties qualitatively surpassing those of [¹¹C]MTO. Non-specific binding to the liver was significantly lower than that of [¹¹C]MTO. [¹⁸F]CETO is a promising new PET tracer for imaging of adrenocortical disease and should be evaluated further in humans.

Herein we describe the development of new methodologyfocusing on ¹¹C-labelling of sulfonyl carbamates in a multicomponentreaction comprising a sulfonyl azide, an alkyl alcohol and [¹¹C]CO. Anumber of 11C-labelled sulfonyl carbamates were synthesised andisolated, and the developed methodology was then applied in thepreparation of a biologically active molecule. The target compoundwas obtained in 18±8% isolated radiochemical yield and wasevaluated for binding properties in a tumor cell assay, as well asundergoing in vivo biodistribution and imaging studies. Thisrepresents the first successful radiolabelling of C21, a non-peptideangiotensin II receptor subtype 2 agonist currently in clinical trials.

We describe the development of a new methodology focusing on C-11-labeling of sulfonyl carbamates in a multicomponent reaction comprised of a sulfonyl azide, an alkyl alcohol, and [C-11] CO. A number of C-11-labeled sulfonyl carbamates were synthesized and isolated, and the developed methodology was then applied in the preparation of a biologically active molecule. The target compound was obtained in 24 +/- 10% isolated radiochemical yield and was evaluated for binding properties in a tumor cell assay; in vivo biodistribution and imaging studies were also performed. This represents the first successful radiolabeling of a non-peptide angiotensin II receptor subtype 2 agonist, C21, currently in clinical trials for the treatment of idiopathic pulmonary fibrosis.

Objective. The aetiology and early pathophysiological mechanisms of aortic aneurysm formation are still unknown and challenging to study in vivo. Positron emission tomography (PET) is a potentially valuable instrument for non-invasive in vivo pathophysiological studies. No specific tracer to identify the pathophysiological process of aneurysmal dilatation is yet available, however. The aim of this study was to explore if different PET tracers could be useful to image aneurysmal disease. Methods and results. Human aneurysmal aortic tissue, collected during elective resection of abdominal aortic aneurysm (AAA) of asymptomatic patients, was investigated in vitro by means of autoradiography with [Ga-68]CRP-binder targeting C-reactive protein, [C-11]DAA1106 targeting translocator protein (18 kDa), [C-11]D-deprenyl with unknown target receptor, [C-11] deuterium-L-deprenyl targeting astrocytes, [F-18]fluciclatide targeting integrin alpha(V)beta(3), [Ga-68]IMP461 and bi-specific antibody TF2 052107 targeting carcinoembryonic antigen, [F-18]F-metomidate targeting mitochondrial cytochrome P-450 species in the adrenal cortex, and [F-18]vorozole targeting aromatase. Of the investigated tracers, only [F-18]fluciclatide exhibited specific binding, whereas the other PET tracers failed to show specific uptake in the investigated tissue and are probably not useful for the intended purpose. Conclusion. It seems likely that alpha(V)beta(3) integrin expression in AAA can be visualized with PET and that the alpha(V)beta(3) selective tracer, [F-18]fluciclatide, may be suitable for in vivo molecular imaging of asymptomatic AAA. Additional evaluation of [F-18]fluciclatide and alpha(V)beta(3) integrin expression in AAA will be performed in vitro as well as in vivo.

Correct staging of prostate cancer is an unmet clinical need. Radionuclide targeting of prostate-specific membrane antigen (PSMA) with In-111-labeled capromab pendetide (ProstaScint) is a clinical option for prostate cancer staging. We propose the use of I-124-labeled capromab to decrease the retention of radioactivity in healthy organs (due to the non-residualizing properties of the radiolabel). The use of I-124 as a label should increase imaging sensitivity due to the advantages of PET as an imaging modality. Capromab targets the intracellular domain of PSMA; accumulation of radioactivity in the tumor should not depend on internalization of the antigen/antibody complex. Capromab was iodinated, and its targeting properties were compared with indium labeled counterpart in LNCaP xenografts in dual isotope mode. PSMA-negative xenografts (PC3) were used as a negative control. Radioiodinated capromab bound to PSMA specifically. Biodistribution of I-125/In-111-capromab showed a more rapid clearance of iodine radioactivity from liver, spleen, kidneys, bones, colon tissue, as well as tumors. Maximum tumor uptake (13 +/- 8% ID/g for iodine and 29 +/- 9% ID/g for indium) and tumor-to-non-tumor ratios for both agents were measured 5 days post-injection (pi). High tumor accumulation and low uptake of radioactivity in normal organs were confirmed using microPET/CT 5 days pi of I-124-capromab.

BACKGROUND: Perfusion strategies during aortic surgery usually comprise hypothermic circulatory arrest (HCA), often combined with selective antegrade cerebral perfusion (SACP) or retrograde cerebral perfusion. Cerebral blood flow (CBF) is a fundamental parameter for which the optimal level has not been clearly defined. We sought to determine the CBF at a pump flow level of 6 mL/kg/min, previously shown likely to provide adequate SACP at 20°C in pigs.

METHODS: Repeated positron emission tomography (PET) scans were used to quantify the CBF and glucose metabolism throughout HCA and SACP including cooling and rewarming. Eight pigs on cardiopulmonary bypass were assigned to either HCA alone (n = 4) or HCA+SACP (n = 4). The CBF was measured by repeated [(15)O]water PET scans from baseline to rewarming. The cerebral glucose metabolism was examined by [(18)F]fluorodeoxyglucose PET scans after rewarming to 37°C.

RESULTS: Cooling to 20°C decreased the cortical CBF from 0.31 ± 0.06 at baseline to 0.10 ± 0.02 mL/cm(3)/min (p = 0.008). The CBF was maintained stable by SACP of 6 mL/kg/min during 45 minutes. After rewarming to 37°C, the mean CBF increased to 0.24 ± 0.07 mL/cm(3)/min, without significant differences between the groups at any time-point exclusive of the HCA period. The net cortical uptake (Ki) of [(18)F]fluorodeoxyglucose after rewarming showed no significant difference between the groups.

CONCLUSIONS: Cooling autoregulated the CBF to 0.10 mL/cm(3)/min, and 45 minutes of SACP at 6 mL/kg/min maintained the CBF in the present model. Cerebral glucose metabolism after rewarming was similar in the study groups.

Positron Emission Tomography (PET) and in particular gallium-68 (⁶⁸Ga) applications are growing exponentially worldwide contributing to the expansion of nuclear medicine and personalized management of patients. The significance of ⁶⁸Ga utility is reflected in the implementation of European Pharmacopoeia monographs. However, there is one crucial point in the monographs that might limit the use of the generators and consequently expansion of ⁶⁸Ga applications and that is the limit of 0.001% of Germanium-68 (⁶⁸Ge(IV)) radioactivity content in a radiopharmaceutical. We have investigated the organ distribution of ⁶⁸Ge(IV) in rat and estimated human dosimetry parameters in order to provide experimental evidence for the determination and justification of the ⁶⁸Ge(IV) limit. Male and female rats were injected in the tail vein with formulated [⁶⁸Ge]GeCl₄ in the absence or presence of [⁶⁸Ga]Ga-DOTA-TOC. The tissue radioactivity distribution data was extrapolated for the estimation of human organ equivalent doses and total effective dose using Organ Level Internal Dose Assessment Code software (OLINDA/EXM). ⁶⁸Ge(IV) was evenly distributed among the rat organs and fast renal excretion prevailed. Human organ equivalent dose and total effective dose estimates indicated that the kidneys were the dose-limiting organs (185±54 μSv/MBq for female and 171±38 μSv/MBq for male) and the total effective dose was 15.5±0.1 and 10.7±1.2 μSv/MBq, respectively for female and male. The results of this dosimetry study conclude that the ⁶⁸Ge(IV) limit currently recommended by monographs could be increased considerably (>100 times) without exposing the patient to harm given the small absorbed doses to normal organs and fast excretion.

Imaging and radiotherapy targeting the glucose-dependent insulinotropic polypeptide receptor (GIPR) could potentially benefit the management of neuroendocrine neoplasms (NENs), complementing clinically established radiopharmaceuticals. The aim of this study was to evaluate a GIPR-targeting positron emission tomography (PET) radioligand with receptor-specific binding, fast blood clearance, and low liver background uptake. The peptide DOTA-bioconjugate, C803-GIP, was developed based on the sequence of the endogenous GIP(1-30) and synthetic exendin-4 peptides with selective amino acid mutations to combine their specificity for the GIPR and in vivo stability, respectively. The Ga-68-labeled bioconjugate was evaluated in vitro in terms of binding affinity, specificity, and internalization in HEK293 cells transfected with the human GIPR, GLP1, or GCG receptors and in sections of human insulinoma and NENs. In vivo binding specificity, biodistribution, and tissue background were investigated in mice bearing huGIPR-HEK293 xenografts and in a pig. Ex vivo organ distribution, pharmacokinetics, and dosimetry were studied in normal rats. [Ga-68]Ga-C803-GIP was stable and demonstrated a high affinity to the huGIPR-HEK293 cells. Binding specificity was demonstrated in vitro in frozen sections of NENs and huGIPR-HEK293 cells. No specific uptake was observed in the negative controls of huGLP1R and huGCGR cells. A novel rationally designed PET radioligand, [Ga-68]Ga-C803-GIP, demonstrated promising binding characteristics and specificity towards the GIPR.

Introduction: Early detection of liver fibrosis and monitoring response to treatment crucial for the management of patients are currently not feasible in clinical practice. Platelet derived growth factor receptor beta (PDGER-beta) expression is regarded as a potential biomarker to determine the stages of fibrotic diseases including liver fibrosis. [Ga-68]Ga-BOT5035 comprising a bicyclic peptide was developed for specific targeting of ppGrR-beta overexpressed in pathological fibrosis. The realization of microdosing phase 0 study using [Ga-68]Ga-BOT5035 positron emission tomography required automated good manufacturing practice (GMP) compliant production of [Ga-68]Ga-BOT5035 presented herein. Moreover, the investigation of radiation dosimetry was conducted to ensure possibility of multiple annual examinations for disease monitoring in clinical setup.

Methods: The active pharmaceutical ingredient starting material BOT5035 (GMP grade) was provided by BiOrion Technologies BV. The Ga-68-labelling process was developed and automated using synthesis platform (ModularLab PharmTrace, Eckert & Ziegler), disposable cassettes for Ga-68-labelling, and pharmaceutical grade Ge-68/Ga-68 generator (GalliaPharm (R)) purchased from Eckert & Ziegler. Radiolysis sensitive BOT5035 required development and systematic optimization of the labelling synthesis parameters such as time, temperature, precursor concentration, radical scavenger, buffer concentration and pH. The validation process was conducted with regard to the product quality and quantity, as well as production reproducibility. Human organ equivalent doses and total body effective doses were calculated using Organ Level Internal Dose Assessment Code software (OLINDA/EXM 1.1), based on ex vivo organ distribution in Sprague-Dawley rats.

Results: The GMP compliant automated production of [Ga-68]Ga-BOT5035 with on-line documentation demonstrated high reproducibility. The time for the labelling synthesis and quality control was approximately 60 min. The non-decay corrected radiochemical yield and radiochemical purity of the radiopharmaceutical were 43.7 +/- 7.6% (n = 3, process validation) and 97.7 +/- 0.4% (n = 3, process validation), respectively. Predefined acceptance criteria were met for the sterility, endotoxins level, radionuclidic purity and residual solvent content. The stability at ambient temperature was controlled for 120 min with approved results. Ex vivo organ distribution data revealed fast blood clearance and washout from most of the organs. The dose-limiting organs were kidney and bone marrow. The total effective dose as limiting parameter would allow for up to 3-4 PET scans per annum.

Conclusion: The fully automated and GMP compliant production of [Ga-68]Ga-BOT5035 was developed and thoroughly validated. The radiopharmaceutical was approved by Swedish Medicinal Products Agency and the Ethical Review Authority for the Phase 0 clinical study of the quantitative imaging of liver fibrosis. Human dosimetry calculations extrapolated from animal experiment indicated possibility of 3-4 PET examinations per year.

Objectives: Fibrosis affecting functionality of vital organs such as liver, lung, heart, and kidney, is involved in many chronic diseases. Positron emission tomography (PET) would not only provide precise localization and extent of the affected tissue but also allow the accurate quantification of the fibrotic process for the subsequent prognosis. Methods: A cyclic peptide c[CPGRVMHGLHLGDDEGPC] conjugated either to 2-(4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)acetic acid (NOTA(tBu)2) or 4-(4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazacyclononan-1-yl)-5-(tert-butoxy)-5-oxopentanoic acid (NODAGA(tBu)3) via polyethylene glycol link (PEG2) was synthesized and labeled with Ga-68. Non-specific organ distribution, blood clearance, and excretion were investigated ex vivo in healthy rats. The binding specificity of the radioligands was assessed in vitro using autoradiography on cryosections of dog fibrotic heart tissue. Results: The yield of NOTA-PEG(2)-c[CPGRVMHGLHLGDDEGPC] and NODAGA-PEG(2)-c[CPGRVMHGLHLGDDEGPC] was 56% and 41%, respectively. Non-decay-corrected radiochemical yield was 80 +/- 5% with radiochemical purity of 95 +/- 4%. Pharmacokinetic studies in healthy male Sprague-Dawley rats showed fast blood clearance and renal excretion. Lower uptake in liver, spleen, and kidney was found for [[Ga-68]Ga-NOTA](+1)-PEG(2)-c [CPGRVMHGLHLGDDEGPC] as compared to [[Ga-68]Ga-NODAGA](0)-PEG(2)-c[CPGRVMHGLHLGDDEGPC]. Histologic evaluation of the left ventricle (LV) myocardium from a dog with severe mitral regurgitation (MR), revealed mild to moderate perivascular and subendocardial, and mild diffuse interstitial fibrosis. The tracer binding to the cryosections of the tissue was specific with the equilibrium Kd of 2.3 +/- 0.8 mu M and 2.1 +/- 0.9 mu M, respectively for [Ga-68]Ga-NO2A-Col and [Ga-68]Ga-NODAGA-Col. Conclusions: Two novel peptide based agents for the imaging of fibrosis by PET were developed. Moderation of the biodistribution could be achieved by variation of the charge on the complex moiety of the agents. The combination of the fast clearance from non-target organs as well as organs of interest such as lung, heart, and liver and binding specificity to the target tissue suggests the potential of the analogs for the imaging of fibrosis.