Rolf Zijlma

In Vivo Biodistribution of No-Carrier-Added 6-18F-Fluoro-3,4-Dihydroxy-l-Phenylalanine (18F-DOPA), Produced by a New Nucleophilic Substitution Approach, Compared with Carrier-Added 18F-DOPA, Prepared by Conventional Electrophilic Substitution

A novel synthetic approach to 6-18F-fluoro-3,4-dihydroxy-l-phenylalanine (18F-DOPA), involving the nucleophilic substitution of a diaryliodonium salt precursor with non-carrier-added 18F-fluoride, yielded a product with a specific activity that was 3 orders of magnitude higher than the product of the conventional synthesis method, involving an electrophilic substitution of a trialkylstannane precursor with 18F2. We performed a direct comparison of high- and low-specific-activity 18F-DOPA in a neuroendocrine tumor model to determine whether this difference in specific activity has implications for the biologic behavior and imaging properties of 18F-DOPA. Methods: 18F-DOPA was produced via the novel synthesis method, yielding 18F-DOPA-H with a high specific activity (35,050 ± 4,000 GBq/mmol). This product was compared in several experiments with conventional 18F-DOPA-L with a low specific activity (11 ± 2 GBq/mmol). In vitro accumulation experiments with the human pancreatic neuroendocrine tumor cell line BON-1 were performed at both 0°C and 37°C and at 37°C in the presence of pharmacologic inhibitors of proteins involved in the uptake mechanism of 18F-DOPA. Small-animal PET experiments were performed in athymic nude mice bearing a BON-1 tumor xenograft. Results: At 37°C, the uptake of both 18F-DOPA-H and 18F-DOPA-L did not differ significantly during a 60-min accumulation experiment in BON-1 cells. At 0°C, the uptake of 18F-DOPA-L was significantly decreased, whereas the lower temperature did not alter the uptake of 18F-DOPA-H. The pharmacologic inhibitors carbidopa and tetrabenazine also revealed differential effects between the 2 types of 18F-DOPA in the 60-min accumulation experiment. The small-animal PET experiments did not show any significant differences in distribution and metabolism of 18F-DOPA-H and 18F-DOPA-L in carbidopa-pretreated mice. Conclusion: The advantages of the novel synthesis of 18F-DOPA, which relies on nucleophilic fluorination of a diaryliodonium salt precursor, lie in the simplicity of the synthesis method, compared with the conventional, electrophilic approach and in the reduced mass of administered, pharmacologically active 19F-DOPA. 18F-DOPA-H demonstrated comparable imaging properties in an in vivo model for neuroendocrine tumors, despite the fact that the injected mass of material was 3 orders of magnitude less than 18F-DOPA-L.

Development of [18F]-labeled pyrazolo[4,3-e]-1,2,4- triazolo[1,5-c]pyrimidine (SCH442416) analogs for the imaging of cerebral adenosine A2A receptors with positron emission tomography

Cerebral adenosine A2A receptors (A2ARs) are attractive therapeutic targets for the treatment of neurodegenerative and psychiatric disorders. We developed high affinity and selective compound 8 (SCH442416) analogs as in vivo probes for A2ARs using PET. We observed the A2AR-mediated accumulation of [18F]fluoropropyl ([18F]-10b) and [18F]fluoroethyl ([18F]-10a) derivatives of 8 in the brain. The striatum was clearly visualized in PET and in vitro autoradiography images of control animals and was no longer visible after pretreatment with the A2AR subtype-selective antagonist KW6002. In vitro and in vivo metabolite analyses indicated the presence of hydrophilic (radio)metabolite(s), which are not expected to cross the blood-brain-barrier. [18F]-10b and [18F]-10a showed comparable striatum-to- cerebellum ratios (4.6 at 25 and 37 min post injection, respectively) and reversible binding in rat brains. We concluded that these compounds performed equally well, but their kinetics were slightly different. These molecules are potential tools for mapping cerebral A2ARs with PET.

Synthesis and preclinical evaluation of 2-(2-furanyl)-7-[2-[4-[4-(2-[11C]methoxyethoxy)phenyl]-1-piperazinyl]ethyl]7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine-5-amine ([11C]Preladenant) as a PET tracer for the imaging of cerebral adenosine A2A receptors

2-(2-Furanyl)-7-[2-[4-[4-(2-[(11)C]methoxyethoxy)phenyl]-1-piperazinyl]ethyl]7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine-5-amine [(11)C]-3 ([(11)C]Preladenant) was developed for mapping cerebral adenosine A2A receptors (A2ARs) with PET. The tracer was synthesized in high specific activity and purity. Tissue distribution was studied by PET imaging, ex vivo biodistribution (BD), and in vitro autoradiography (ARG) experiments. Regional brain uptake of [(11)C]-3 was consistent with known A2ARs distribution, with highest uptake in striatum. The results indicate that [(11)C]-3 has favorable brain kinetics and exhibits suitable characteristics as an A2AR PET tracer.

Contribution of neuroinflammation to changes in [11C]flumazenil binding in the rat brain: Evaluation of the inflamed pons as reference tissue

INTRODUCTIONn[11C]Flumazenil is a well-known PET tracer for GABAA receptors and is mainly used as an imaging biomarker for neuronal loss. Recently, GABAA receptors on immune cells have been investigated as target for modulation of inflammation. Since neuronal loss is often accompanied by neuroinflammation, PET imaging with [11C]flumazenil is potentially affected by infiltrating immune cells. This may also compromise the validity of using the pons as reference tissue in quantitative pharmacokinetic analysis. This study aims to evaluate whether inflammatory processes in the brain can influence [11C]flumazenil uptake and affect the outcome of pharmacokinetic modeling when the pons is used as reference tissue.nnnMETHODSnThe herpes simplex encephalitis (HSE) rat model is known to cause neuroinflammation in the brainstem. Dynamic [11C]flumazenil PET scans of 60-min, accompanied by arterial blood sampling and metabolite analysis, were acquired at day 6-7days post-infection of male Wistar rats (HSE, n=5 and control, n=6). Additionally, the GABAA receptor was saturated by injection of unlabeled flumazenil prior to the tracer injection in 4 rats per group. PET data were analyzed by pharmacokinetic modeling.nnnRESULTSnNo statistically significant differences were found in the volume of distribution (VT) or non-displaceable binding potential (BPND) between control and HSE rats in any of the brain regions. Pre-saturation with unlabeled flumazenil resulted in a statistically significant reduction in [11C]flumazenil VT in all brain regions. The BPND obtained from SRTM exhibited a good correlation to DVR - 1 values from the two-tissue compartment model, coupled with some level of underestimation.nnnCONCLUSIONnReliable quantification of [11C]flumazenil binding in rats can be obtained by pharmacokinetic analysis using the pons as a pseudo-reference tissue even in the presence of strong acute neuroinflammation.

In vivo imaging of brain androgen receptors in rats: a ( 18 F)FDHT PET study

INTRODUCTIONnSteroid hormones like androgens play an important role in the development and maintenance of several brain functions. Androgens can act through androgen receptors (AR) in the brain. This study aims to demonstrate the feasibility of positron emission tomography (PET) with 16β-[(18)F]fluoro-5α-dihydrotestosterone ([(18)F]FDHT) to image AR expression in the brain.nnnMETHODSnMale Wistar rats were either orchiectomized to inhibit endogenous androgen production or underwent sham-surgery. Fifteen days after surgery, rats were subjected to a 90-min dynamic [(18)F]FDHT PET scan with arterial blood sampling. In a subset of orchiectomized rats, 1mg/kg dihydrotestosterone was co-injected with the tracer in order to saturate the AR. Plasma samples were analyzed for the presence of radioactive metabolites by radio-TLC. Pharmacokinetic modeling was performed to quantify brain kinetics of the tracer. After the PET scan, the animals were terminated for ex-vivo biodistribution.nnnRESULTSnPET imaging and ex vivo biodistribution studies showed low [(18)F]FDHT uptake in all brain regions, except pituitary. [(18)F]FDHT uptake in the surrounding cranial bones was high and increased over time. [(18)F]FDHT was rapidly metabolized in rats. Metabolism was significantly faster in orchiectomized rats than in sham-orchiectomized rats. Quantitative analysis of PET data indicated substantial spill-over of activity from cranial bones into peripheral brain regions, which prevented further analysis of peripheral brain regions. Logan graphical analysis and kinetic modeling using 1- and 2-tissue compartment models showed reversible and homogenously distributed tracer uptake in central brain regions. [(18)F]FDHT uptake in the brain could not be blocked by endogenous androgens or administration of dihydrotestosterone.nnnCONCLUSIONnThe results of this study indicate that imaging of AR availability in rat brain with [(18)F]FDHT PET is not feasible. The low AR expression in the brain, the rapid metabolism of [(18)F]FDHT in rats and the poor brain penetration of the tracer likely contributed to the poor performance of [(18)F]FDHT PET in this study.

Pharmacokinetic properties of radiolabeled mutant Interleukin-2v: a PET imaging study

Interleukin-2 (IL2) is a cytokine that can stimulate cytotoxic immune cells to attack infected and malignant cells. Unfortunately, IL2 can also cause serious immune-related toxicity. Recently, a mutant of IL2 (IL2v) with abolished CD25 binding, increased plasma half-life and less toxicity was engineered. Unlike wild-type IL2 (wt-IL2), mutant IL2v does not bind to the α-subunit (CD25) of the high affinity IL2αβγ receptor, but only to its β and γ subunit. Here, we investigated the biological properties of IL2v and compared with the wt-IL2 using fluorine-18 and PET. [18F]FB-IL2v binds specifically to IL2 receptors (IL2R) on activated human peripheral blood monocytes (hPBMCs) and is cleared mainly by the kidneys (Balb/c mice). [18F]FB-IL2v PET studies in SCID mice injected with hPBMCs revealed high uptake in the implant (0.85 ± 0.15 SUV), which was significantly reduced after pretreatment with wt-IL2 or mutant IL2v (SUV 0.26 ± 0.1 and 0.46 ± 0.1, p < 0.01). Compartment modeling and Logan graphical analysis in wistar rats inoculated with hPBMCs indicated that the binding of [18F]FB-IL2v to IL2R was reversible. The volume of distribution (VT) and the non-displaceable binding potential (BPnd) of mutant [18F]FB-IL2v in the implant were approximately 3 times lower than those of wild-type [18F]FB-IL2 (p < 0.01). Pretreatment with wt-IL2 significantly reduced the VT and BPnd of mutant [18F]FB-IL2v in the implant (p < 0.001). This demonstrates that wild-type [18F]FB-IL2 binds stronger to IL2R and has faster kinetics than [18F]FB-IL2v, which makes it less suitable as a therapeutic drug. [18F]FB-IL2v, on the other hand, seems to have better properties for use as a therapeutic drug.

PET Imaging with S-[C-11]Methyl-L-Cysteine and L-[Methyl-C-11]Methionine in Rat Models of Glioma, Glioma Radiotherapy, and Neuroinflammation

PurposeS-[11C]-methyl-L-cysteine ([11C]MCYS) has been claimed to offer higher tumor selectivity than L-[methyl-11C]methionine ([11C]MET). We examined this claim in animal models.ProceduresRats with implanted untreated (nxa0=xa010) or irradiated (nxa0=xa07, 1xa0×xa025xa0Gy, on day 8) orthotopic gliomas were scanned after 6, 9, and 12xa0days, using positron emission tomography. Rats with striatal injections of saline (nxa0=xa09) or bacterial lipopolysaccharide (nxa0=xa09) were scanned after 3xa0days.ResultsUptake of the two tracers in untreated gliomas was similar. [11C]MCYS was not accumulated in salivary glands, nasal epithelium, and healing wounds, in contrast to [11C]MET, but showed 40xa0% higher accumulation in the healthy brain. Both tracers showed a reduced tumor uptake 4xa0days after irradiation and minor accumulation in inflamed striatum. [11C]MCYS indicated higher lesion volumes than [11C]MET (untreated tumor +xa047xa0%; irradiated tumor up to +xa0500xa0%; LPS-inflamed striatum +xa0240xa0%).Conclusions[11C]MCYS was less accumulated in some non-tumor tissues than [11C]MET, but showed lower tumor-to-brain contrast.

Improved GMP-compliant multi-dose production and quality control of 6-[18F]fluoro-L-DOPA

Background6-[18F]Fluoro-L-3,4-dihydroxyphenylalanine (FDOPA) is a frequently used radiopharmaceutical for detecting neuroendocrine and brain tumors and for the differential diagnosis of Parkinson’s disease. To meet the demand for FDOPA, a high-yield GMP-compliant production method is required. Therefore, this study aimed to improve the FDOPA production and quality control procedures to enable distribution of the radiopharmaceutical over distances.FDOPA was prepared by electrophilic fluorination of the trimethylstannyl precursor with [18F]F2, produced from [18O]2 via the double-shoot approach, leading to FDOPA with higher specific activity as compared to FDOPA which was synthesized, using [18F]F2 produced from 20Ne, leading to FDOPA with a lower specific activity. The quality control of the product was performed using a validated UPLC system and compared with quality control with a conventional HPLC system. Impurities were identified using UPLC-MS.ResultsThe [18O]2 double-shoot radionuclide production method yielded significantly more [18F]F2 with less carrier F2 than the conventional method starting from 20Ne. After adjustment of radiolabeling parameters substantially higher amounts of FDOPA with higher specific activity could be obtained. Quality control by UPLC was much faster and detected more side-products than HPLC. UPLC-MS showed that the most important side-product was FDOPA-quinone, rather than 6-hydroxydopa as suggested by the European Pharmacopoeia.ConclusionThe production and quality control of FDOPA were significantly improved by introducing the [18O]2 double-shoot radionuclide production method, and product analysis by UPLC, respectively. As a result, FDOPA is now routinely available for clinical practice and for distribution over distances.

UPLC (R)-RAD the new standard in quality control of PET radiopharmaceuticals

Aim: Inflammatory bowel disease (IBD) is defined as a chronic relapsing idiopathic inflammation of the gastrointestinal tract. The two main clinical forms of this disease family are Crohn’s Disease (CD) and Ulcerative Colitis (UC). IBD affects an estimated 3.6 million individuals in Europe and North America. To date it is thought that IBD is the result of continual activation of the mucosal immune system. In order to better understand this disease family an in-house developed animal model was implemented and characterized with [18F]FDG (used to illustrate the increased glucose consumption associated with inflammatory processes) and also with TSPO 18 kDa radioligand [18F]DPA-714, an established radiotracer for the study of inflammation within the central nervous system. Materials and Methods: Colonic inflammation was induced in male Wistar rats weighing between 200-250 g by rectal administration of trinitrobenzenesulfonic acid (TNBS) at 4cm from the anal orifice. Control animals were administered, 0.9% aq. sodium chloride analogously. A Siemens Inveon PET/CT tomograph, dedicated to small animals, was used to acquire [18F]FDG images on day 7 post TNBS administration and [18F]DPA714 images the following day. Rats were then sacrificed by an i.v. injection of pentobarbital, and then the lower intestine was extracted and analyzed by immunohistochemistry to determine macrophage infiltration and the presence of TSPO. Results: PET image analysis clearly shows an important accumulation of both radiotracers within the intestinal walls of treated animals in comparison to control animals. Mean levels of [18F]FDG uptake in treated and control animals were 1.20 ± 0.56 %ID/cc and 0.43 ± 0.18 %ID/cc, respectively. Comparable results were found when using [18F]DPA-714, with mean level of uptake in treated and control animals of 1.21 ± 0.62 %ID/cc and 0.46 ± 0.23 %ID/cc, respectively. Immunohistochemistry analysis revealed a higher presence of macrophages in TNBS treated animals. Expression of TSPO was largely increased in the treated animals, when compared to the controls animals, and mainly localized in macrophages cells. Conclusion: Preliminary results seem to indicate that [18F]DPA-714 is an adapted tracer for the study of inflammation of IBD in our animal model. Beyond this, data demonstrating that [18F]DPA-714 could be used to characterize and quantify the level of inflammation during the disease evolution, within the TNBS treated animals, will also be presented. OP366 Surface displayed SNAP-tag as a novel tool for study of Grampositive bacterial infections. B. Mills, V. Steele, J. C. A. Luckett, R. O. Awais, P. Duncanson, V. Griffiths, A. Cockayne, M. Xu, I. Correa, A. C. Perkins, P. Williams, P. Hill; School of Molecular Medical Sciences, University of Nottingham, Nottingham, UNITED KINGDOM, Radiological and Imaging Sciences, University of Nottingham, Nottingham, UNITED KINGDOM, School of Biological and Chemical Sciences, Queen Mary University of London, London, UNITED KINGDOM, New England Biolabs, Inc, Ipswich, ME, UNITED STATES, School of Biosciences, University of Nottingham, Nottingham, UNITED KINGDOM. Introduction: The design of specific probes for in vivo molecular imaging of microbial infections remains one of the greatest challenges to overcome before useful, functional data can be obtained. An increasingly attractive approach for probe design is to express a ligand-binding protein within a cell, which may then covalently bind specific synthetic ligands with attached imaging moieties. One such labelling system is the commercially available SNAP-tag. SNAP-tag specifically and covalently binds O2-benzylguanine (BG) compounds, which may have fluorophores or other functional elements attached at the 4’ position of their benzyl ring. We have designed a BG ligand labelled with Tc, suitable for SPECT imaging. We propose to utilise this technology for the imaging of Staphylococcal infection in vivo with the view to investigate bacterial pathogenicity and to visualise the effect potential antimicrobials may have on bacterial load. Methods: The SNAP-tag gene was codon optimised for expression in the Gram positive bacterium Staphylococcus aureus and fused with an N-terminal spa secretion leader sequence and a Cterminal spa cell-wall anchoring domain. The N-terminal fusion directs the expressed SNAP-tag towards the cell exterior where the C-terminal domain is recognised by the cell-wall sorting enzyme sortase A, covalently anchoring SNAPtag in such a way that the ligand binding domain decorates the cell surface. A novel 99m Tc-HYNIC–NH-BG ligand for SPECT imaging was prepared by coupling BG to HYNIC and radiolabelling with NaTcO4 in the presence of tricine as co-ligand. Radiochemical yields >99% were obtained. nanoSPECT-CT imaging will be used to assess the functional data produced by using SNAP-tag expressing S. aureus cells in in vivo infection models. Results: We have demonstrated that SNAP-tag was expressed and exported to the cell wall where it was covalently anchored. Deletion of the sortase A enzyme prevented attachment of the SNAP-tag to the cell wall, as determined by Western blot. Once situated within the cell wall, SNAP-tag was functional and able to specifically bind cell-impermeable fluorescent BG ligands and our synthesised precursor HYNIC-NH2-BG ligand, as determined by confocal microscopy and fluorometry assay. Pilot in vivo studies for fluorescence optical imaging and nanoSPECT-CT imaging with the novel 99m Tc-HYNIC-NH-BG ligand are currently under development to visualise S. aureus infections in mouse models. Conclusions: This approach should allow a higher sensitivity to be achieved when investigating bacterial infections in real time compared to current molecular imaging techniques, thus allowing bacterial virulence and the potential effects of new antimicrobials to be assessed. OP367 Dual imaging of lipopolysaccharides (LPS) by SPECT-CT and Confocal Microscopy. M. Moreau, V. Duheron, B. Collin, W. Sali, C. Bernhard, C. Goze, T. Gautier, J. Pais de Barros, V. Deckert, F. Brunotte, L. Lagrost, F. Denat; ICMUB UMR CNRS 6302, Dijon, FRANCE, INSERM UMR866, Dijon, FRANCE, Centre Georges-François Leclerc, Dijon, FRANCE, Centre Hospitalier Universitaire, Dijon, FRANCE. Introduction: Lipopolysaccharides (LPS) or endotoxins are found inserted in the outer membrane of Gram-negative bacterias. Their appearance in blood stream triggers a massive secretion of pro-inflammatory cytokines in mammals. A controlled response allows the neutralization and elimination of LPS, whereas an excessive inflammatory response leads to severe circulatory and respiratory defects. It is the endotoxemic shock or septic shock that can leads to death. Many approaches are used to study LPS, including labeling with radiochemicals (3H, 125I, 99mTc or 51Cr) or with fluorophores (FITC, Alexa488, Bodipy). Bimodality is attracting more and more interest in the field of molecular imaging since the combination of two different techniques may provide complementary information, thus improving the accuracy of diagnosis. Combining nuclear modalities (PET or SPECT) with optical imaging is of particular interest, and the similar sensitivities of the two techniques allows to fuse the signaling moieties into a unique molecule, called monomolecular multimodality imaging agent (MOMIA), ensuring a same biodistribution of the two probes. Method: A recently described bimodal probe, namely DOTA-Bodipy-NCS, has been covalently attached to LPS. The integrity of the LPS after labeling procedure was checked by SDS-PAGE electrophoresis and βhydroxymyristate titration (BHM). Pro-inflammatory activity of LPS was assessed by quantification of cytokines released by differentiated THP-1 cells. This bioconjugate was then radiometallated for SPECT-CT biodistribution imaging. Results: DOTABodipy-LPS was metallated with 111In to yield a high specific activity (600 MBq.mg1), with a radiochemical purity >98 % after purification. Biodistribution of the radiolabeled compound was then evaluated in vivo in WT mice by SPECT-CT imaging. Radiolabeled LPS is rapidly eliminated from the bloodstream and accumulates in spleen and liver. Liver slices were then analyzed by confocal microscopy, and specific fluorescent signals in the cytoplasm of hepatocytes were detected, confirming the accumulation of 111In-DOTA-Bodipy-LPS in the liver. Conclusion: These results demonstrate the efficiency of the conjugation process of our bimodal probe. It made it possible to perform both non-invasive SPECT and ex vivo fluorescence imaging of LPS biodistribution, underlining its liver uptake for further detoxification. The 111In-DOTA-Bodipy-LPS probe arises here as a relevant tool to identify key components of LPS detoxification in vivo paving the way to therapeutic issues in the field of sepsis. Acknowlegement: Support was provided by the CNRS, the University of Burgundy, the Conseil Régional de Bourgogne. O P _ M o nd ay S178 Eur J Nucl Med Mol Imaging (2013) 40 (Suppl 2):S89–S567Affibody molecules constitute a class of small (7 kDa) scaffold proteins that can be engineered to have excellent tumor targeting properties. High reabsorption in kidneys complicates development of ...