Behorende bij het proefschrift 1. Prodrug research needs more imagination and less dependency on ... more Behorende bij het proefschrift 1. Prodrug research needs more imagination and less dependency on what has been tried in the past. (Valentino J. Stella; Advanced drug delivery reviews, 1996) 2. �-glucuronidase-based prod rug therapy can be applied in the treatment of cancer, but also in inflammatory disorders (this thesis, chapter 2) 3. PET can be an attractive tool to evaluate new enzyme-prodrug combinations and to optimize treatment strategies. (this thesis, chapter 3 and 4) 4. The best parameter to measure �-glucuronidase activity is the distribution volume of the PET tracer (this thesis, chapter 5) 5. The characteristics of a drug can not always be applied to predict the success of a PET tracer that is derived from this drug. (this thesis, chapter 6) 6. The release of �-glucuronidase accompanies the activation of microglia in neuroinflammation. (this thesis, chapter 7) 7. A single dose of a cytostatic drug can increase the release of �-glucuronidase in small tumors, resulting in enhanced extracellular levels of the prodrug-converting enzyme and therefore could improve the efficacy of glucuronide prodrug treatment. (this thesis, chapter 8) 8. All truths are easy to understand once they are discovered; the point is to discover them. (Galileo Galilei) 9. Being a PhD student is like becoming all of the Seven Dwarves. In the beginning you are Dopey and Bashful. In the middle, you are usually sick (Sneezy), tired (Sleepy), and irritable (Grumpy). But at the end, they call you Doc, and then you are Happy 10. I was taught that the way of progress is neither swift nor easy (Marie Curie; Pierre Curie, 1936, 167) 11. Like taxes, radioactivity has long been with us and in increasing amounts .... consider
Inhibition of the sodium−glucose cotransporter 2 (SGLT2) by canagliflozin in type 2 diabetes mell... more Inhibition of the sodium−glucose cotransporter 2 (SGLT2) by canagliflozin in type 2 diabetes mellitus results in large between-patient variability in clinical response. To better understand this variability, the positron emission tomography (PET) tracer [ 18 F]canagliflozin was developed via a Cu-mediated 18 F-fluorination of its boronic ester precursor with a radiochemical yield of 2.0 ± 1.9% and a purity of >95%. The GMP automated synthesis originated [ 18 F]canagliflozin with a yield of 0.5−3% (n = 4) and a purity of >95%. Autoradiography showed [ 18 F]canagliflozin binding in human kidney sections containing SGLT2. Since [ 18 F]canagliflozin is the isotopologue of the extensively characterized drug canagliflozin and thus shares its toxicological and pharmacological characteristics, it enables its immediate use in patients.
Aim: There was continued interest in developing more efficient new chelating agents for metal rad... more Aim: There was continued interest in developing more efficient new chelating agents for metal radionuclides mostly used for positron emission tomography (PET) imaging. We focused on the development of convenient syntheses of acyclic siderophore derivatized with four N-hydroxy-N-methyl succinamide pendant arms called 4HMS and its bifunctional analog, 4HMSA, for zirconium-89 (89 Zr) and gallium-68 (68 Ga) complexation. The aim of this project was to assess the suitability of 4HMS and 4HMSA for 89 Zr and 68 Ga-PET. Methods & Results: 4HMS and 4HMSA were prepared through multiple steps starting with a spermine backbone to offer both chelating agents with high overall yield (72-58%). Both chelating agents exhibited strong selective coordination of 89 Zr and 68 Ga and offered a very fast labelling kinetic at room temperature as compared to DFO and DOTA/NOTA analogs. Achievable molar activity for 68 Ga-4HMSA is almost 10 and 3 times higher compared to 68 Ga-DOTA and NOTA analogs. Molar activity of 89 Zr-4HMS is approximately 16 fold higher compared to 89 Zr-DFO. Both radio-complexes were stable in saline, serum, as well as against transchelation and transmetallation. 68 Ga-4HMSA showed high stability in mouse plasma in vitro and in vivo over 1h and 89 Zr-4HMS chelator also showed high stability in mouse plasma over 7 days. Biodistribution and imaging studies were performed in balb/C mice. The background activity in various tissues was low at 1h post-injection (p.i.) for 68 Ga-4HMSA with a rapid elimination mainly through the kidneys and liver. At the same time point, the activity was largely found in kidneys for 89 Zr-4HMS chelator. At 24 h p.i. most of the 89 Zr-4HMS chelator was cleared from all organs and the low amount of activity in kidneys and bone is consistent with the clearance of the intact complex. Finally, the conjugation of unprotected 4HMSA to peptides of biological interest was complete within~4 h with overall yields of 50-60%. Conclusion: 4HMSA and 4HMS show an outstanding promise as 68 Ga and 89 Zr chelators. In terms of Zr 4+ chelation and stability, 4HMS ligand has proven to be a superior chelator compared to DFO.
Bisphosphonates (BPs) are an important class of drugs used in the treatment of abnormal calcium m... more Bisphosphonates (BPs) are an important class of drugs used in the treatment of abnormal calcium metabolism diseases. The first syntheses of bisphosphonates derived from indazole, substituted at the N-1, N-2 and C-3 positions are reported. The 1-hydroxy-1,1-bisphosphonates were synthesized from the corresponding carboxylic acid or acyl chloride compounds, by two different methods. These BPs have a side chain with different lengths ((CH 2) n , n = 0-5) between the indazole ring and the bisphosphonate group.
The Editorial Board of EJNMMI Radiopharmacy and Chemistry releases a biannual highlight commentar... more The Editorial Board of EJNMMI Radiopharmacy and Chemistry releases a biannual highlight commentary to update the readershipon trends in the field of radiopharmaceuticals development.
The estrogen receptor (ER) is a target for endocrine therapy in breast cancer patients. Individua... more The estrogen receptor (ER) is a target for endocrine therapy in breast cancer patients. Individual quantification of ERa and ERb expression, rather than total ER levels, might enable better prediction of the response to treatment. We recently developed the tracer 2-18 F-fluoro-6-(6-hydroxynaphthalen-2-yl)pyridin-3-ol (18 F-FHNP) for assessment of ERb levels with PET. In the current study, we investigated several pharmacokinetic analysis methods to quantify changes in ERb availability with 18 F-FHNP PET. Methods: Male nude rats were subcutaneously inoculated in the shoulder with ERa/ERb-expressing SKOV3 human ovarian cancer cells. Two weeks after tumor inoculation, a dynamic 18 F-FHNP PET scan with arterial blood sampling was acquired from rats treated with vehicle or various concentrations of estradiol (nonspecific ER agonist) or genistein (ERbselective agonist). Different pharmacokinetic models were applied to quantify ERb availability in the tumor. Results: Irreversible-uptake compartmental models fitted the kinetics of 18 F-FHNP uptake better than reversible models. The irreversible 3-tissue-compartment model, which included both the parent and the metabolite input function, gave results comparable to those of the irreversible 2-tissue-compartment model with only a parent input function, indicating that radioactive metabolites contributed little to the tumor uptake. Patlak graphical analysis gave metabolic rates (K i , the irreversible uptake rate constant) comparable to compartment modeling. The K i values correlated well with ERb expression but not with ERa, confirming that K i is a suitable parameter to quantify ERb expression. SUVs at 60 min after tracer injection also correlated (r 2 5 0.47; P 5 0.04) with ERb expression. A reduction in 18 F-FHNP tumor uptake and K i values was observed in the presence of estradiol or genistein. Conclusion: 18 F-FHNP PET enables assessment of ERb availability in tumor-bearing rats. The most suitable parameter to quantify ERb expression is the K i. However, a simplified static imaging protocol for determining the SUVs can be applied to assess ERb levels.
Arginase hydrolyzes L-arginine and influences levels of polyamines and nitric oxide. Arginase ove... more Arginase hydrolyzes L-arginine and influences levels of polyamines and nitric oxide. Arginase overexpression is associated with inflammation and tumorigenesis. Thus, radiolabeled arginase inhibitors may be suitable PET tracers for staging arginase-related pathophysiologies. We report the synthesis and evaluation of 2 radiolabeled arginase inhibitors, 18 F-FMARS and 18 F-FBMARS, developed from a-substituted-2amino-6-boronohexanoic acid derivatives. Methods: Arylboronic ester-derived precursors were radiolabeled via copper-mediated fluorodeboronation. Binding assays using arginase-expressing PC3 and LNCaP cells were performed. Autoradiography of lung sections from a guinea pig model of asthma overexpressing arginase and dynamic small-animal PET imaging with PC3-xenografted mice evaluated the radiotracers' specific binding and pharmacokinetics. Results: 18 F-fluorinated compounds were obtained with radiochemical yields of up to 5% (decay-corrected) and an average molar activity of 53 GBqÁlmol 21. Cell and lung section experiments indicated specific binding that was blocked up to 75% after pretreatment with arginase inhibitors. Smallanimal PET studies indicated fast clearance of the radiotracers (7.3 6 0.6 min), arginase-mediated uptake, and a selective tumor accumulation (SUV, 3.0 6 0.7). Conclusion: The new 18 F-fluorinated arginase inhibitors have the potential to map increased arginase expression related to inflammatory and tumorigenic processes. 18 F-FBMARS showed the highest arginase-mediated uptake in PET imaging and a significant difference between uptake in control and arginaseinhibited PC3 xenografted mice. These results encourage further research to examine the suitability of 18 F-FBMARS for selecting patients for treatments with arginase inhibitors.
Statins are 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors that are widely used to pr... more Statins are 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors that are widely used to prevent cardiovascular diseases. However, a series of pleiotropic mechanisms have been associated with statins, particularly with atorvastatin. Therefore, the assessment of [18F]atorvastatin kinetics with positron emission tomography (PET) may elucidate the mechanism of action of statins and the impact of sexual dimorphism, which is one of the most debated interindividual variations influencing the therapeutic efficacy. [18F]Atorvastatin was synthesized via a previously optimized 18F-deoxyfluorination strategy, used for preclinical PET studies in female and male Wistar rats (n = 7 for both groups), and for subsequent ex vivo biodistribution assessment. PET data were fitted to several pharmacokinetic models, which allowed for estimating relevant kinetic parameters. Both PET imaging and biodistribution studies showed negligible uptake of [18F]atorvastatin in all tissues compared with the primary target organ (liver), excretory pathways (kidneys and small intestine), and stomach. Uptake of [18F]atorvastatin was 38 ± 3% higher in the female liver than in the male liver. The irreversible 2-tissue compartment model showed the best fit to describe [18F]atorvastatin kinetics in the liver. A strong correlation (R2 > 0.93) between quantitative Ki (the radiotracer’s unidirectional net rate of influx between compartments) and semi-quantitative liver’s SUV (standard uptake value), measured between 40 to 90 min, showed potential to use the latter parameter, which circumvents the need for blood sampling as a surrogate of Ki for monitoring [18F]atorvastatin uptake. Preclinical assays showed faster uptake and clearance for female rats compared to males, seemingly related to a higher efficiency for exchanges between the arterial input and the hepatic tissue. Due to the slow [18F]atorvastatin kinetics, equilibrium between the liver and plasma concentration was not reached during the time frame studied, making it difficult to obtain sufficient and accurate kinetic information to quantitatively characterize the radiotracer pharmacokinetics over time. Nevertheless, the reported results suggest that the SUV can potentially be used as a simplified measure, provided all scans are performed at the same time point. Preclinical PET-studies with [18F]atorvastatin showed faster uptake and clearance in female compared to male rats, apparently related to higher efficiency for exchange between arterial blood and hepatic tissue.
A new series of ligands for the estrogen receptor (ER) based on a three-dimensional structural mo... more A new series of ligands for the estrogen receptor (ER) based on a three-dimensional structural motif consisting of a bridged oxabicyclic core (7-oxabicyclo[2.2.1]heptene or heptadiene) were synthesized and examined for their receptor binding activity and as regulators of transcription through the two ER subtypes, ERR and ER. The prototypical ligands also contain a 1,2-diarylethylene motif, common to many nonsteroidal estrogens, as an embellishment on the oxabicyclic core. Thus, these ligands bear peripheral groups typically found in ER ligands, built here upon an overall three-dimensional core topology that is unusual for these targets. Most of these compounds were conveniently synthesized by a Diels-Alder reaction of various 3,4-diarylfurans with a variety of dienophiles, neat and under mild conditions in the absence of catalysts. Some of the synthesized compounds display good binding affinity for the ER, and in transcription assays, the highest affinity compounds are antagonists on both ERs. Molecular modeling studies suggest a structural basis for the antagonist activity of these compounds. These compounds, based on the bicyclo[2.2.1]core system, expand the structural diversity of ligands that can be antagonists for the estrogen receptors.
The increasing incidence of cancer over the years is one of the most challenging problems in heal... more The increasing incidence of cancer over the years is one of the most challenging problems in healthcare. As cancer progresses, the recruitment of several immune cells is triggered. Infiltration of tumor-associated macrophages (TAMs) is correlated with poor patient prognosis. Since TAMs constitute a big portion of the tumor mass, targeting these cells seems to be an attractive approach for cancer immunotherapy. Additionally, TAM assessment using non-invasive imaging techniques, such as positron emission tomography (PET), might provide a better understanding of the role of TAMs in cancer, and a means for tumor profile characterization, patient selection for individualized immunotherapy and treatment monitoring. Imaging of TAMs using PET tracers is still in its infancy. TAMs have several characteristics that could be exploited as potential targets for imaging. Various PET tracers for these TAM biomarkers have been developed, although often in the context of (neuro)inflammatory diseases...
Background Statins are lipid-lowering agents that inhibit cholesterol synthesis and are clinicall... more Background Statins are lipid-lowering agents that inhibit cholesterol synthesis and are clinically used in the primary and secondary prevention of cardiovascular diseases. However, a considerable group of patients does not respond to statin treatment, and the reason for this is still not completely understood. [18F]Atorvastatin, the 18F-labeled version of one of the most widely prescribed statins, may be a useful tool for statin-related research. Results [18F]Atorvastatin was synthesized via an optimized ruthenium-mediated late-stage 18F-deoxyfluorination. The defluoro-hydroxy precursor was produced via Paal-Knorr pyrrole synthesis and was followed by coordination of the phenol to a ruthenium complex, affording the labeling precursor in approximately 10% overall yield. Optimization and automation of the labeling procedure reliably yielded an injectable solution of [18F]atorvastatin in 19% ± 6% (d.c.) with a molar activity of 65 ± 32 GBq·μmol−1. Incubation of [18F]atorvastatin in hum...
Background The Editorial Board of EJNMMI Radiopharmacy and Chemistry releases a biyearly highligh... more Background The Editorial Board of EJNMMI Radiopharmacy and Chemistry releases a biyearly highlight commentary to describe trends in the field. Results This commentary of highlights has resulted in 19 different topics selected by each member of the Editorial Board addressing a variety of aspects ranging from novel radiochemistry to first in man application of novel radiopharmaceuticals. Conclusion Trends in radiochemistry and radiopharmacy are highlighted demonstrating the progress in the research field being the scope of EJNMMI Radiopharmacy and Chemistry.
Background: Molecular imaging of immune cells might be a potential tool for response prediction, ... more Background: Molecular imaging of immune cells might be a potential tool for response prediction, treatment evaluation and patient selection in inflammatory diseases as well as oncology. Targeting interleukin-2 (IL2) receptors on activated T-cells using positron emission tomography (PET) with N-(4-[ 18 F]fluorobenzoyl)interleukin-2 ([ 18 F]FB-IL2) could be such a strategy. This paper describes the challenging translation of the partly manual labeling of [ 18 F]FB-IL2 for preclinical studies into an automated procedure following Good Manufacturing Practices (GMP), resulting in a radiopharmaceutical suitable for clinical use. Methods: The preclinical synthesis of [ 18 F]FB-IL2 was the starting point for translation to a clinical production method. To overcome several challenges, major adaptations in the production process were executed. The final analytical methods and production method were validated and documented. All data with regards to the quality and safety of the final drug product were documented in an investigational medicinal product dossier. Results: Restrictions in the [ 18 F]FB-IL2 production were imposed by hardware configuration of the automated synthesis equipment and by use of disposable cassettes. Critical steps in the [ 18 F]FB-IL2 production comprised the purification method, stability of recombinant human IL2 and the final formulation. With the GMP compliant production method, [ 18 F]FB-IL2 could reliably be produced with consistent quality complying to all specifications. Conclusions: To enable the use of [ 18 F]FB-IL2 in clinical studies, a fully automated GMP compliant production process was developed. [ 18 F]FB-IL2 is now produced consistently for use in clinical studies.
16b-18 F-fluoro-5a-dihydrotestosterone (18 F-FDHT) is a radiopharmaceutical that has been investi... more 16b-18 F-fluoro-5a-dihydrotestosterone (18 F-FDHT) is a radiopharmaceutical that has been investigated as a diagnostic agent for the assessment of androgen receptor (AR) density in prostate cancer using PET. However, 18 F-FDHT is rapidly metabolized in humans and excreted via the kidneys into the urine, potentially compromising the detection of tumor lesions close to the prostate. Enzalutamide is an AR signaling inhibitor currently used in different stages of prostate cancer. Enzalutamide and its primary metabolite N-desmethylenzalutamide have an AR affinity comparable to that of FDHT but are excreted mainly via the hepatic route. Radiolabeled enzalutamide could thus be a suitable candidate PET tracer for AR imaging. Here, we describe the radiolabeling of enzalutamide with 18 F. Moreover, the in vitro and in vivo behavior of 18 F-enzalutamide was evaluated and compared with the current standard, 18 F-FDHT. Methods: 18 F-enzalutamide was obtained by fluorination of the nitro precursor. In vitro cellular uptake studies with 18 F-enzalutamide and 18 F-FDHT were performed in LNCaP (AR-positive) and HEK293 (AR-negative) cells. Competition assays with both tracers were conducted on the LNCaP (AR-positive) cell line. In vivo PET imaging, ex vivo biodistribution, and metabolite studies with 18 F-enzalutamide and 18 F-FDHT were conducted on athymic nude male mice bearing an LNCaP xenograft in the shoulder. Results: 18 F-enzalutamide was obtained in 1.4% 6 0.9% radiochemical yield with an apparent molar activity of 6.2 6 10.3 GBq/mmol. 18 F-FDHT was obtained in 1.5% 6 0.8% yield with a molar activity of more than 25 GBq/mmol. Coincubation with an excess of 5a-dihydrotestosterone or enzalutamide significantly reduced the cellular uptake of 18 F-enzalutamide and 18 F-FDHT to about 50% in AR-positive LNCaP cells but not in AR-negative HEK293 cells. PET and biodistribution studies on male mice bearing a LnCaP xenograft showed about 3 times higher tumor uptake for 18 F-enzalutamide than for 18 F-FDHT. Sixty minutes after tracer injection, 93% of 18 F-enzalutamide in plasma was still intact, compared with only 3% of 18 F-FDHT. Conclusion: Despite its lower apparent molar activity, 18 F-enzalutamide shows higher tumor uptake and better metabolic stability than 18 F-FDHT and thus seems to have more favorable properties for imaging of AR with PET. However, further evaluation in other oncologic animal models and patients is warranted to confirm these results.
Background: Several P-glycoprotein (P-gp) substrate tracers are available to assess Pgp function ... more Background: Several P-glycoprotein (P-gp) substrate tracers are available to assess Pgp function in vivo, but attempts to develop a tracer for measuring expression levels of P-gp have not been successful. Recently, (Z)-2-(5-fluoro-2-oxoindolin-3-ylidene)-N-(4methoxyphenyl)hydrazine-carbothioamide was described as a potential selective P-gp inhibitor that is not transported by P-gp. Therefore, the purpose of this study was to radiolabel two of its analogues and to assess their potential for imaging P-gp expression using PET. Results: [ 18 F]2-(4-fluoro-2-oxoindolin-3-ylidene)-N-(4-methoxyphenyl)hydrazinecarbothioamide ([ 18 F]5) and [ 18 F]2-(6-fluoro-2-oxoindolin-3-ylidene)-N-(4-methoxyphenyl) hydrazine-carbothioamide ([ 18 F]6) were synthesized and both their biodistribution and metabolism were evaluated in rats. In addition, PET scans were acquired in rats before and after tariquidar (P-gp inhibitor) administration as well as in P-gp knockout (KO) mice. Both [ 18 F]5 and [ 18 F]6 were synthesized in 2-3% overall yield, and showed high brain uptake in ex vivo biodistribution studies. [ 18 F]6 appeared to be metabolically unstable in vivo, while [ 18 F]5 showed moderate stability with limited uptake of radiolabelled metabolites in the brain. PET studies showed that transport of [ 18 F]5 across the bloodbrain barrier was not altered by pre-treatment with the P-gp inhibitor tariquidar, and uptake was significantly lower in P-gp KO than in wild-type animals and indeed transported across the BBB or bound to P-gp in endothelial cells. Conclusion: In conclusion, [ 18 F]5 and [ 18 F]6 were successfully and reproducibly synthesized, albeit with low radiochemical yields. [ 18 F]5 appears to be a radiotracer that binds to P-gp, as showed in P-gp knockout animals, but is not a substrate for P-gp.
Behorende bij het proefschrift 1. Prodrug research needs more imagination and less dependency on ... more Behorende bij het proefschrift 1. Prodrug research needs more imagination and less dependency on what has been tried in the past. (Valentino J. Stella; Advanced drug delivery reviews, 1996) 2. �-glucuronidase-based prod rug therapy can be applied in the treatment of cancer, but also in inflammatory disorders (this thesis, chapter 2) 3. PET can be an attractive tool to evaluate new enzyme-prodrug combinations and to optimize treatment strategies. (this thesis, chapter 3 and 4) 4. The best parameter to measure �-glucuronidase activity is the distribution volume of the PET tracer (this thesis, chapter 5) 5. The characteristics of a drug can not always be applied to predict the success of a PET tracer that is derived from this drug. (this thesis, chapter 6) 6. The release of �-glucuronidase accompanies the activation of microglia in neuroinflammation. (this thesis, chapter 7) 7. A single dose of a cytostatic drug can increase the release of �-glucuronidase in small tumors, resulting in enhanced extracellular levels of the prodrug-converting enzyme and therefore could improve the efficacy of glucuronide prodrug treatment. (this thesis, chapter 8) 8. All truths are easy to understand once they are discovered; the point is to discover them. (Galileo Galilei) 9. Being a PhD student is like becoming all of the Seven Dwarves. In the beginning you are Dopey and Bashful. In the middle, you are usually sick (Sneezy), tired (Sleepy), and irritable (Grumpy). But at the end, they call you Doc, and then you are Happy 10. I was taught that the way of progress is neither swift nor easy (Marie Curie; Pierre Curie, 1936, 167) 11. Like taxes, radioactivity has long been with us and in increasing amounts .... consider
Inhibition of the sodium−glucose cotransporter 2 (SGLT2) by canagliflozin in type 2 diabetes mell... more Inhibition of the sodium−glucose cotransporter 2 (SGLT2) by canagliflozin in type 2 diabetes mellitus results in large between-patient variability in clinical response. To better understand this variability, the positron emission tomography (PET) tracer [ 18 F]canagliflozin was developed via a Cu-mediated 18 F-fluorination of its boronic ester precursor with a radiochemical yield of 2.0 ± 1.9% and a purity of >95%. The GMP automated synthesis originated [ 18 F]canagliflozin with a yield of 0.5−3% (n = 4) and a purity of >95%. Autoradiography showed [ 18 F]canagliflozin binding in human kidney sections containing SGLT2. Since [ 18 F]canagliflozin is the isotopologue of the extensively characterized drug canagliflozin and thus shares its toxicological and pharmacological characteristics, it enables its immediate use in patients.
Aim: There was continued interest in developing more efficient new chelating agents for metal rad... more Aim: There was continued interest in developing more efficient new chelating agents for metal radionuclides mostly used for positron emission tomography (PET) imaging. We focused on the development of convenient syntheses of acyclic siderophore derivatized with four N-hydroxy-N-methyl succinamide pendant arms called 4HMS and its bifunctional analog, 4HMSA, for zirconium-89 (89 Zr) and gallium-68 (68 Ga) complexation. The aim of this project was to assess the suitability of 4HMS and 4HMSA for 89 Zr and 68 Ga-PET. Methods & Results: 4HMS and 4HMSA were prepared through multiple steps starting with a spermine backbone to offer both chelating agents with high overall yield (72-58%). Both chelating agents exhibited strong selective coordination of 89 Zr and 68 Ga and offered a very fast labelling kinetic at room temperature as compared to DFO and DOTA/NOTA analogs. Achievable molar activity for 68 Ga-4HMSA is almost 10 and 3 times higher compared to 68 Ga-DOTA and NOTA analogs. Molar activity of 89 Zr-4HMS is approximately 16 fold higher compared to 89 Zr-DFO. Both radio-complexes were stable in saline, serum, as well as against transchelation and transmetallation. 68 Ga-4HMSA showed high stability in mouse plasma in vitro and in vivo over 1h and 89 Zr-4HMS chelator also showed high stability in mouse plasma over 7 days. Biodistribution and imaging studies were performed in balb/C mice. The background activity in various tissues was low at 1h post-injection (p.i.) for 68 Ga-4HMSA with a rapid elimination mainly through the kidneys and liver. At the same time point, the activity was largely found in kidneys for 89 Zr-4HMS chelator. At 24 h p.i. most of the 89 Zr-4HMS chelator was cleared from all organs and the low amount of activity in kidneys and bone is consistent with the clearance of the intact complex. Finally, the conjugation of unprotected 4HMSA to peptides of biological interest was complete within~4 h with overall yields of 50-60%. Conclusion: 4HMSA and 4HMS show an outstanding promise as 68 Ga and 89 Zr chelators. In terms of Zr 4+ chelation and stability, 4HMS ligand has proven to be a superior chelator compared to DFO.
Bisphosphonates (BPs) are an important class of drugs used in the treatment of abnormal calcium m... more Bisphosphonates (BPs) are an important class of drugs used in the treatment of abnormal calcium metabolism diseases. The first syntheses of bisphosphonates derived from indazole, substituted at the N-1, N-2 and C-3 positions are reported. The 1-hydroxy-1,1-bisphosphonates were synthesized from the corresponding carboxylic acid or acyl chloride compounds, by two different methods. These BPs have a side chain with different lengths ((CH 2) n , n = 0-5) between the indazole ring and the bisphosphonate group.
The Editorial Board of EJNMMI Radiopharmacy and Chemistry releases a biannual highlight commentar... more The Editorial Board of EJNMMI Radiopharmacy and Chemistry releases a biannual highlight commentary to update the readershipon trends in the field of radiopharmaceuticals development.
The estrogen receptor (ER) is a target for endocrine therapy in breast cancer patients. Individua... more The estrogen receptor (ER) is a target for endocrine therapy in breast cancer patients. Individual quantification of ERa and ERb expression, rather than total ER levels, might enable better prediction of the response to treatment. We recently developed the tracer 2-18 F-fluoro-6-(6-hydroxynaphthalen-2-yl)pyridin-3-ol (18 F-FHNP) for assessment of ERb levels with PET. In the current study, we investigated several pharmacokinetic analysis methods to quantify changes in ERb availability with 18 F-FHNP PET. Methods: Male nude rats were subcutaneously inoculated in the shoulder with ERa/ERb-expressing SKOV3 human ovarian cancer cells. Two weeks after tumor inoculation, a dynamic 18 F-FHNP PET scan with arterial blood sampling was acquired from rats treated with vehicle or various concentrations of estradiol (nonspecific ER agonist) or genistein (ERbselective agonist). Different pharmacokinetic models were applied to quantify ERb availability in the tumor. Results: Irreversible-uptake compartmental models fitted the kinetics of 18 F-FHNP uptake better than reversible models. The irreversible 3-tissue-compartment model, which included both the parent and the metabolite input function, gave results comparable to those of the irreversible 2-tissue-compartment model with only a parent input function, indicating that radioactive metabolites contributed little to the tumor uptake. Patlak graphical analysis gave metabolic rates (K i , the irreversible uptake rate constant) comparable to compartment modeling. The K i values correlated well with ERb expression but not with ERa, confirming that K i is a suitable parameter to quantify ERb expression. SUVs at 60 min after tracer injection also correlated (r 2 5 0.47; P 5 0.04) with ERb expression. A reduction in 18 F-FHNP tumor uptake and K i values was observed in the presence of estradiol or genistein. Conclusion: 18 F-FHNP PET enables assessment of ERb availability in tumor-bearing rats. The most suitable parameter to quantify ERb expression is the K i. However, a simplified static imaging protocol for determining the SUVs can be applied to assess ERb levels.
Arginase hydrolyzes L-arginine and influences levels of polyamines and nitric oxide. Arginase ove... more Arginase hydrolyzes L-arginine and influences levels of polyamines and nitric oxide. Arginase overexpression is associated with inflammation and tumorigenesis. Thus, radiolabeled arginase inhibitors may be suitable PET tracers for staging arginase-related pathophysiologies. We report the synthesis and evaluation of 2 radiolabeled arginase inhibitors, 18 F-FMARS and 18 F-FBMARS, developed from a-substituted-2amino-6-boronohexanoic acid derivatives. Methods: Arylboronic ester-derived precursors were radiolabeled via copper-mediated fluorodeboronation. Binding assays using arginase-expressing PC3 and LNCaP cells were performed. Autoradiography of lung sections from a guinea pig model of asthma overexpressing arginase and dynamic small-animal PET imaging with PC3-xenografted mice evaluated the radiotracers' specific binding and pharmacokinetics. Results: 18 F-fluorinated compounds were obtained with radiochemical yields of up to 5% (decay-corrected) and an average molar activity of 53 GBqÁlmol 21. Cell and lung section experiments indicated specific binding that was blocked up to 75% after pretreatment with arginase inhibitors. Smallanimal PET studies indicated fast clearance of the radiotracers (7.3 6 0.6 min), arginase-mediated uptake, and a selective tumor accumulation (SUV, 3.0 6 0.7). Conclusion: The new 18 F-fluorinated arginase inhibitors have the potential to map increased arginase expression related to inflammatory and tumorigenic processes. 18 F-FBMARS showed the highest arginase-mediated uptake in PET imaging and a significant difference between uptake in control and arginaseinhibited PC3 xenografted mice. These results encourage further research to examine the suitability of 18 F-FBMARS for selecting patients for treatments with arginase inhibitors.
Statins are 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors that are widely used to pr... more Statins are 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors that are widely used to prevent cardiovascular diseases. However, a series of pleiotropic mechanisms have been associated with statins, particularly with atorvastatin. Therefore, the assessment of [18F]atorvastatin kinetics with positron emission tomography (PET) may elucidate the mechanism of action of statins and the impact of sexual dimorphism, which is one of the most debated interindividual variations influencing the therapeutic efficacy. [18F]Atorvastatin was synthesized via a previously optimized 18F-deoxyfluorination strategy, used for preclinical PET studies in female and male Wistar rats (n = 7 for both groups), and for subsequent ex vivo biodistribution assessment. PET data were fitted to several pharmacokinetic models, which allowed for estimating relevant kinetic parameters. Both PET imaging and biodistribution studies showed negligible uptake of [18F]atorvastatin in all tissues compared with the primary target organ (liver), excretory pathways (kidneys and small intestine), and stomach. Uptake of [18F]atorvastatin was 38 ± 3% higher in the female liver than in the male liver. The irreversible 2-tissue compartment model showed the best fit to describe [18F]atorvastatin kinetics in the liver. A strong correlation (R2 > 0.93) between quantitative Ki (the radiotracer’s unidirectional net rate of influx between compartments) and semi-quantitative liver’s SUV (standard uptake value), measured between 40 to 90 min, showed potential to use the latter parameter, which circumvents the need for blood sampling as a surrogate of Ki for monitoring [18F]atorvastatin uptake. Preclinical assays showed faster uptake and clearance for female rats compared to males, seemingly related to a higher efficiency for exchanges between the arterial input and the hepatic tissue. Due to the slow [18F]atorvastatin kinetics, equilibrium between the liver and plasma concentration was not reached during the time frame studied, making it difficult to obtain sufficient and accurate kinetic information to quantitatively characterize the radiotracer pharmacokinetics over time. Nevertheless, the reported results suggest that the SUV can potentially be used as a simplified measure, provided all scans are performed at the same time point. Preclinical PET-studies with [18F]atorvastatin showed faster uptake and clearance in female compared to male rats, apparently related to higher efficiency for exchange between arterial blood and hepatic tissue.
A new series of ligands for the estrogen receptor (ER) based on a three-dimensional structural mo... more A new series of ligands for the estrogen receptor (ER) based on a three-dimensional structural motif consisting of a bridged oxabicyclic core (7-oxabicyclo[2.2.1]heptene or heptadiene) were synthesized and examined for their receptor binding activity and as regulators of transcription through the two ER subtypes, ERR and ER. The prototypical ligands also contain a 1,2-diarylethylene motif, common to many nonsteroidal estrogens, as an embellishment on the oxabicyclic core. Thus, these ligands bear peripheral groups typically found in ER ligands, built here upon an overall three-dimensional core topology that is unusual for these targets. Most of these compounds were conveniently synthesized by a Diels-Alder reaction of various 3,4-diarylfurans with a variety of dienophiles, neat and under mild conditions in the absence of catalysts. Some of the synthesized compounds display good binding affinity for the ER, and in transcription assays, the highest affinity compounds are antagonists on both ERs. Molecular modeling studies suggest a structural basis for the antagonist activity of these compounds. These compounds, based on the bicyclo[2.2.1]core system, expand the structural diversity of ligands that can be antagonists for the estrogen receptors.
The increasing incidence of cancer over the years is one of the most challenging problems in heal... more The increasing incidence of cancer over the years is one of the most challenging problems in healthcare. As cancer progresses, the recruitment of several immune cells is triggered. Infiltration of tumor-associated macrophages (TAMs) is correlated with poor patient prognosis. Since TAMs constitute a big portion of the tumor mass, targeting these cells seems to be an attractive approach for cancer immunotherapy. Additionally, TAM assessment using non-invasive imaging techniques, such as positron emission tomography (PET), might provide a better understanding of the role of TAMs in cancer, and a means for tumor profile characterization, patient selection for individualized immunotherapy and treatment monitoring. Imaging of TAMs using PET tracers is still in its infancy. TAMs have several characteristics that could be exploited as potential targets for imaging. Various PET tracers for these TAM biomarkers have been developed, although often in the context of (neuro)inflammatory diseases...
Background Statins are lipid-lowering agents that inhibit cholesterol synthesis and are clinicall... more Background Statins are lipid-lowering agents that inhibit cholesterol synthesis and are clinically used in the primary and secondary prevention of cardiovascular diseases. However, a considerable group of patients does not respond to statin treatment, and the reason for this is still not completely understood. [18F]Atorvastatin, the 18F-labeled version of one of the most widely prescribed statins, may be a useful tool for statin-related research. Results [18F]Atorvastatin was synthesized via an optimized ruthenium-mediated late-stage 18F-deoxyfluorination. The defluoro-hydroxy precursor was produced via Paal-Knorr pyrrole synthesis and was followed by coordination of the phenol to a ruthenium complex, affording the labeling precursor in approximately 10% overall yield. Optimization and automation of the labeling procedure reliably yielded an injectable solution of [18F]atorvastatin in 19% ± 6% (d.c.) with a molar activity of 65 ± 32 GBq·μmol−1. Incubation of [18F]atorvastatin in hum...
Background The Editorial Board of EJNMMI Radiopharmacy and Chemistry releases a biyearly highligh... more Background The Editorial Board of EJNMMI Radiopharmacy and Chemistry releases a biyearly highlight commentary to describe trends in the field. Results This commentary of highlights has resulted in 19 different topics selected by each member of the Editorial Board addressing a variety of aspects ranging from novel radiochemistry to first in man application of novel radiopharmaceuticals. Conclusion Trends in radiochemistry and radiopharmacy are highlighted demonstrating the progress in the research field being the scope of EJNMMI Radiopharmacy and Chemistry.
Background: Molecular imaging of immune cells might be a potential tool for response prediction, ... more Background: Molecular imaging of immune cells might be a potential tool for response prediction, treatment evaluation and patient selection in inflammatory diseases as well as oncology. Targeting interleukin-2 (IL2) receptors on activated T-cells using positron emission tomography (PET) with N-(4-[ 18 F]fluorobenzoyl)interleukin-2 ([ 18 F]FB-IL2) could be such a strategy. This paper describes the challenging translation of the partly manual labeling of [ 18 F]FB-IL2 for preclinical studies into an automated procedure following Good Manufacturing Practices (GMP), resulting in a radiopharmaceutical suitable for clinical use. Methods: The preclinical synthesis of [ 18 F]FB-IL2 was the starting point for translation to a clinical production method. To overcome several challenges, major adaptations in the production process were executed. The final analytical methods and production method were validated and documented. All data with regards to the quality and safety of the final drug product were documented in an investigational medicinal product dossier. Results: Restrictions in the [ 18 F]FB-IL2 production were imposed by hardware configuration of the automated synthesis equipment and by use of disposable cassettes. Critical steps in the [ 18 F]FB-IL2 production comprised the purification method, stability of recombinant human IL2 and the final formulation. With the GMP compliant production method, [ 18 F]FB-IL2 could reliably be produced with consistent quality complying to all specifications. Conclusions: To enable the use of [ 18 F]FB-IL2 in clinical studies, a fully automated GMP compliant production process was developed. [ 18 F]FB-IL2 is now produced consistently for use in clinical studies.
16b-18 F-fluoro-5a-dihydrotestosterone (18 F-FDHT) is a radiopharmaceutical that has been investi... more 16b-18 F-fluoro-5a-dihydrotestosterone (18 F-FDHT) is a radiopharmaceutical that has been investigated as a diagnostic agent for the assessment of androgen receptor (AR) density in prostate cancer using PET. However, 18 F-FDHT is rapidly metabolized in humans and excreted via the kidneys into the urine, potentially compromising the detection of tumor lesions close to the prostate. Enzalutamide is an AR signaling inhibitor currently used in different stages of prostate cancer. Enzalutamide and its primary metabolite N-desmethylenzalutamide have an AR affinity comparable to that of FDHT but are excreted mainly via the hepatic route. Radiolabeled enzalutamide could thus be a suitable candidate PET tracer for AR imaging. Here, we describe the radiolabeling of enzalutamide with 18 F. Moreover, the in vitro and in vivo behavior of 18 F-enzalutamide was evaluated and compared with the current standard, 18 F-FDHT. Methods: 18 F-enzalutamide was obtained by fluorination of the nitro precursor. In vitro cellular uptake studies with 18 F-enzalutamide and 18 F-FDHT were performed in LNCaP (AR-positive) and HEK293 (AR-negative) cells. Competition assays with both tracers were conducted on the LNCaP (AR-positive) cell line. In vivo PET imaging, ex vivo biodistribution, and metabolite studies with 18 F-enzalutamide and 18 F-FDHT were conducted on athymic nude male mice bearing an LNCaP xenograft in the shoulder. Results: 18 F-enzalutamide was obtained in 1.4% 6 0.9% radiochemical yield with an apparent molar activity of 6.2 6 10.3 GBq/mmol. 18 F-FDHT was obtained in 1.5% 6 0.8% yield with a molar activity of more than 25 GBq/mmol. Coincubation with an excess of 5a-dihydrotestosterone or enzalutamide significantly reduced the cellular uptake of 18 F-enzalutamide and 18 F-FDHT to about 50% in AR-positive LNCaP cells but not in AR-negative HEK293 cells. PET and biodistribution studies on male mice bearing a LnCaP xenograft showed about 3 times higher tumor uptake for 18 F-enzalutamide than for 18 F-FDHT. Sixty minutes after tracer injection, 93% of 18 F-enzalutamide in plasma was still intact, compared with only 3% of 18 F-FDHT. Conclusion: Despite its lower apparent molar activity, 18 F-enzalutamide shows higher tumor uptake and better metabolic stability than 18 F-FDHT and thus seems to have more favorable properties for imaging of AR with PET. However, further evaluation in other oncologic animal models and patients is warranted to confirm these results.
Background: Several P-glycoprotein (P-gp) substrate tracers are available to assess Pgp function ... more Background: Several P-glycoprotein (P-gp) substrate tracers are available to assess Pgp function in vivo, but attempts to develop a tracer for measuring expression levels of P-gp have not been successful. Recently, (Z)-2-(5-fluoro-2-oxoindolin-3-ylidene)-N-(4methoxyphenyl)hydrazine-carbothioamide was described as a potential selective P-gp inhibitor that is not transported by P-gp. Therefore, the purpose of this study was to radiolabel two of its analogues and to assess their potential for imaging P-gp expression using PET. Results: [ 18 F]2-(4-fluoro-2-oxoindolin-3-ylidene)-N-(4-methoxyphenyl)hydrazinecarbothioamide ([ 18 F]5) and [ 18 F]2-(6-fluoro-2-oxoindolin-3-ylidene)-N-(4-methoxyphenyl) hydrazine-carbothioamide ([ 18 F]6) were synthesized and both their biodistribution and metabolism were evaluated in rats. In addition, PET scans were acquired in rats before and after tariquidar (P-gp inhibitor) administration as well as in P-gp knockout (KO) mice. Both [ 18 F]5 and [ 18 F]6 were synthesized in 2-3% overall yield, and showed high brain uptake in ex vivo biodistribution studies. [ 18 F]6 appeared to be metabolically unstable in vivo, while [ 18 F]5 showed moderate stability with limited uptake of radiolabelled metabolites in the brain. PET studies showed that transport of [ 18 F]5 across the bloodbrain barrier was not altered by pre-treatment with the P-gp inhibitor tariquidar, and uptake was significantly lower in P-gp KO than in wild-type animals and indeed transported across the BBB or bound to P-gp in endothelial cells. Conclusion: In conclusion, [ 18 F]5 and [ 18 F]6 were successfully and reproducibly synthesized, albeit with low radiochemical yields. [ 18 F]5 appears to be a radiotracer that binds to P-gp, as showed in P-gp knockout animals, but is not a substrate for P-gp.
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