Positron emission tomography is an imaging technique with applications in clinical settings as well as in basic research for the study of biological processes. A PET tracer, a biologically active molecule where a positron-emitting radioisotope such as carbon-11 has been incorporated, is used for the studies. Development of robust methods for incorporation of the radioisotope is therefore of the utmost importance. The urea functional group is present in many biologically active compounds and is thus an attractive target for incorporation of carbon-11 in the form of [C-11] carbon monoxide. Starting with amines and [C-11] carbon monoxide, both symmetrical and unsymmetrical C-11-labelled ureas were synthesised via a palladium(II)-mediated oxidative carbonylation and obtained in decay-corrected radiochemical yields up to 65%. The added advantage of using [C-11] carbon monoxide was shown by the molar activity obtained for an inhibitor of soluble epoxide hydrolase (247 GBq/mu mol-319 GBq/mu mol). DFT calculations were found to support a reaction mechanism proceeding through an C-11-labelled isocyanate intermediate.

Neuroinflammation has been suggested as a key determinant of the intrinsic severity of epilepsy. Glial cell activation and associated inflammatory signaling can influence seizure thresholds as well as the pharmacodynamics and pharmacokinetics of antiepileptic drugs. Based on these data, we hypothesized that molecular imaging of microglia activation might serve as a tool to predict drug refractoriness of epilepsy. Brain uptake of (R)-[(11)C]PK11195, a ligand of the translocator protein 18 kDa and molecular marker of microglia activation, was studied in a chronic model of temporal lobe epilepsy in rats with selection of phenobarbital responders and non-responders. In rats with drug-sensitive epilepsy, (R)-[(11)C]PK11195 brain uptake values were comparable to those in non-epileptic controls. Analysis in non-responders revealed enhanced brain uptake of up to 39% in different brain regions. The difference might be related to the fact that non-responders exhibited higher baseline seizure frequencies than responders indicating a more pronounced intrinsic disease severity. In hippocampal sections, ED1 immunostaining argued against a general difference in microglia activation between both groups. Our data suggest that TSPO PET imaging might serve as a biomarker for drug resistance in temporal lobe epilepsy. However, it needs to be considered that our findings indicate that the TSPO PET data might merely reflect seizure frequency. Future experimental and clinical studies should further evaluate the validity of TSPO PET data to predict the response to phenobarbital and other antiepileptic drugs in longitudinal studies with scanning before drug exposure and with a focus on the early phase following an epileptogenic brain insult.

The progressive loss of beta-cell mass is a hallmark of diabetes and has been suggested as a complementary approach to studying the progression of diabetes in contrast to the beta-cell function. Non-invasive nuclear medicinal imaging techniques such as Positron Emission Tomography using radiation emitting tracers have thus been suggested as more viable methodologies to visualize and quantify the beta-cell mass with sufficient sensitivity. The transmembrane G protein-coupled receptor GPR44 has been identified as a biomarker for monitoring beta-cell mass. MK-7246 is a GPR44 antagonist that selectively binds to GPR44 with high affinity and good pharmacokinetic properties. Here, we present the synthesis of MK-7246, radiolabeled with the positron emitter fluorine-18 for preclinical evaluation using cell lines, mice, rats and human pancreatic cells. Here, we have described a synthesis and radiolabeling method for producing [¹⁸F]MK-7246 and its precursor compound. Preclinical assessments demonstrated the strong affinity and selectivity of [¹⁸F]MK-7246 towards GPR44. Additionally, [¹⁸F]MK-7246 exhibited excellent metabolic stability, a fast clearance profile from blood and tissues, qualifying it as a promising radioactive probe for GPR44-directed PET imaging.

BackgroundBeta-cell replacement methods such as transplantation of isolated donor islets have been proposed as a curative treatment of type 1 diabetes, but widespread application is challenging due to shortages of donor tissue and the need for continuous immunosuppressive treatments. Stem-cell-derived islets have been suggested as an alternative source of beta cells, but face transplantation protocols optimization difficulties, mainly due to a lack of available methods and markers to directly monitor grafts survival, as well as their localization and function. Molecular imaging techniques and particularly positron emission tomography has been suggested as a tool for monitoring the fate of islets after clinical transplantation. The integral membrane protein DGCR2 has been demonstrated to be a potential pancreatic islet biomarker, with specific expression on insulin-positive human embryonic stem-cell-derived pancreatic progenitor cells. The candidate Affibody molecule ZDGCR2:AM106 was radiolabeled with fluorine-18 using a novel click chemistry-based approach. The resulting positron emission tomography tracer [18F]ZDGCR2:AM106 was evaluated for binding to recombinant human DGCR2 and cryosections of stem-cell-derived islets, as well as in vivo using an immune-deficient mouse model transplanted with stem-cell-derived islets. Biodistribution of the [18F]ZDGCR2:AM106 was also assessed in healthy rats and pigs.

Results[18F]ZDGCR2:AM106 was successfully synthesized with high radiochemical purity and yield via a pretargeting approach. [18F]ZDGCR2:AM106 retained binding to recombinant human DCGR2 as well as to cryosectioned stem-cell-derived islets, but in vivo binding to native pancreatic tissue in both rat and pig was low. However, in vivo uptake of [18F]ZDGCR2:AM106 in stem-cell-derived islets transplanted in the immunodeficient mice was observed, albeit only within the early imaging frames after injection of the radiotracer.

ConclusionTargeting of DGCR2 is a promising approach for in vivo detection of stem-cell-derived islets grafts by molecular imaging. The synthesis of [18F]ZDGCR2:AM106 was successfully performed via a pretargeting method to label a site-specific covalently bonded fluorine-18 to the Affibody molecule. However, the rapid washout of [18F]ZDGCR2:AM106 from the stem-cell-derived islets graft indicates that dissociation kinetics can be improved. Further studies using alternative binders of similar classes with improved binding potential are warranted.

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.

PURPOSE: The aim of this study was to explore the cerebral distribution of the tau-specific PET tracer [(18)F]THK5317 (also known as (S)-[(18)F]THK5117) retention in different stages of Alzheimer's disease; and study any associations with markers of hypometabolism and amyloid-beta deposition.

METHODS: Thirty-three individuals were enrolled, including nine patients with Alzheimer's disease dementia, thirteen with mild cognitive impairment (MCI), two with non-Alzheimer's disease dementia, and nine healthy controls (five young and four elderly). In a multi-tracer PET design [(18)F]THK5317, [(11)C] Pittsburgh compound B ([(11)C]PIB), and [(18)F]FDG were used to assess tau pathology, amyloid-beta deposition and cerebral glucose metabolism, respectively. The MCI patients were further divided into MCI [(11)C]PIB-positive (n = 11) and MCI [(11)C]PIB-negative (n = 2) groups.

RESULTS: Test-retest variability for [(18)F]THK5317-PET was very low (1.17-3.81 %), as shown by retesting five patients. The patients with prodromal (MCI [(11)C]PIB-positive) and dementia-stage Alzheimer's disease had significantly higher [(18)F]THK5317 retention than healthy controls (p = 0.002 and p = 0.001, respectively) in areas exceeding limbic regions, and their discrimination from this control group (using the area under the curve) was >98 %. Focal negative correlations between [(18)F]THK5317 retention and [(18)F]FDG uptake were observed mainly in the frontal cortex, and focal positive correlations were found between [(18)F]THK5317 and [(11)C]PIB retentions isocortically. One patient with corticobasal degeneration syndrome and one with progressive supranuclear palsy showed no [(11)C]PIB but high [(18)F]THK5317 retentions with a different regional distribution from that in Alzheimer's disease patients.

CONCLUSIONS: The tau-specific PET tracer [(18)F]THK5317 images in vivo the expected regional distribution of tau pathology. This distribution contrasts with the different patterns of hypometabolism and amyloid-beta deposition.

A radical C-11-aminocarbonylation protocol characterized by excellent substrate compatibility was developed to transform alkyl iodides into C-11-labelled amides, including the 11-HSD1 inhibitor [carbonyl-C-11]adamantan-1-yl(piperidin-1-yl)methanone. This protocol serves as a complementary extension of palladium-mediated C-11-aminocarbonylation, which is limited to the preparation of C-11-labelled compounds lacking beta-hydrogen atoms. The use of AIBN as a radical initiator and a low-pressure xenon-[C-11]CO delivery unit represents a simple and convenient alternative to previous radical C-11-carbonylation methodologies burdened with the need for a proprietary high pressure reactor connected to a light source.

Compounds labelled with ¹¹C (β⁺, t_1/2 = 20.4 min) are used in positron emission tomography (PET), which is a quantitative non-invasive molecular imaging technique. It utilizes computerized reconstruction methods to produce time-resolved images of the radioactivity distribution in living subjects.

The feasibility of preparing [¹¹C]methyl iodide from [¹¹C]methane and iodine via a single pass through a non-thermal plasma reactor was explored. [¹¹C]Methyl iodide with a specific radioactivity of 412 ± 32 GBq/µmol was obtained in 13 ± 3% decay-corrected radiochemical yield within 6 min via catalytic hydrogenation of [¹¹C]carbon dioxide (24 GBq) and subsequent iodination, induced by electron impact.

Labelled ethyl-, propyl- and butyl iodide was synthesized, within 15 min, via palladium-mediated carbonylation using [¹¹C]carbon monoxide. The carbonylation products, labelled carboxylic acids, esters and aldehydes, were reduced to their corresponding alcohols and converted to alkyl iodides. [1-¹¹C]Ethyl iodide was obtained via palladium-mediated carbonylation of methyl iodide with a decay-corrected radiochemical yield of 55 ± 5%. [1-¹¹C]Propyl iodide and [1-¹¹C]butyl iodide were synthesized via the hydroformylation of ethene and propene with decay-corrected radiochemical yields of 58 ± 4% and 34 ± 2%, respectively. [1-¹¹C]Ethyl iodide was obtained with a specific radioactivity of 84 GBq/mmol from 10 GBq of [¹¹C]carbon monoxide. [1-¹¹C]Propyl iodide was synthesized with a specific radioactivity of 270 GBq/mmol from 12 GBq and [1-¹¹C]butyl iodide with 146 GBq/mmol from 8 GBq.

Palladium-mediated hydroxycarbonylation of acetylene was used in the synthesis of [1-¹¹C]acrylic acid. The labelled carboxylic acid was converted to its acid chloride and subsequently treated with amine to yield N-[carbonyl-¹¹C]benzylacrylamide. In an alternative method, [carbonyl-¹¹C]acrylamides were synthesized in decay-corrected radiochemical yields up to 81% via palladium-mediated carbonylative cross-coupling of vinyl halides and amines. Starting from 10 ± 0.5 GBq of [¹¹C]carbon monoxide, N-[carbonyl-¹¹C]benzylacrylamide was obtained in 4 min with a specific radioactivity of 330 ± 4 GBq/µmol.

A method to prepare [1-C-11]propyl iodide and [1-C-11]butyl iodide from [C-11]carbon monoxide via a three step reaction sequence is presented. Palladium mediated formylation of ethene with [C-11]carbon monoxide and hydrogen gave [1-C-11]propionaldehyde and [1-C-11]propionic acid. The carbonylation products were reduced and subsequently converted to [1-C-11]propyl iodide. Labelled propyl iodide was obtained in 58 +/- 4% decay corrected radiochemical yield and with a specific radioactivity of 270 +/- 33 GBq/mu mol within 15 min from approximately 12 GBq of [C-11]carbon monoxide. The position of the label was confirmed by C-13-labelling and C-13-NMR analysis. [1-C-11]Butyl iodide was obtained correspondingly from propene and approximately 8 GBq of [C-11]carbon monoxide, in 34 +/- 2% decay corrected radiochemical yield and with a specific radioactivity of 146 +/- 20 GBq/mu mol. The alkyl iodides were used in model reactions to synthesize [O-propyl-1-C-11]propyl and [O-butyl-1-C-11]butyl benzoate. Propyl and butyl analogues of etomidate, a (beta-11-hydroxylase inhibitor, were also synthesized.

The prospects for using carbon-11 labelled compounds in molecular imaging has improved with the development of diverse synthesis methods, including C-11-carbonylations and refined techniques to handle [C-11]carbon monoxide at a nanomole scale. Facilitating biological research and molecular imaging was the driving force when [C-11]carbon monoxide was used in the first in vivo application with carbon-11 in human (1945) and when [C-11]carbon monoxide was used for the first time as a chemical reagent in the synthesis of [C-11]phosgene (1978). This review examines a rich plethora of labelled compounds synthesized from [C-11]carbon monoxide, their chemistry and use in molecular imaging. While the strong development of the C-11-carbonylation chemistry has expanded the carbon-11 domain considerably, it could be argued that the number of C-11-carbonyl compounds entering biological investigations should be higher. The reason for this may partly be the lack of commercially available synthesis instruments designed for C-11-carbonylations. But as this review shows, novel and greatly simplified methods to handle [C-11]carbon monoxide have been developed. The next important challenge is to make full use of these technologies and synthesis methods in PET research. When there is a PET-tracer that meets a more general need, the incentive to implement C-11-carbonylation protocols will increase.

Introduction: MK-7246 is a potent and selective antagonist for chemoattractant receptor-homologous molecule expressed on T_h2 cells (CRTH2). Within the pancreas CRTH2 is selectively expressed in pancreatic β-cells where it is believed to play a role in insulin release. Reduction in β-cell mass and insufficient insulin secretion in response to elevated blood glucose levels is a hallmark for type 1 and type 2 diabetes. Reported here is the synthesis of [¹¹C]MK-7246 and initial preclinical evaluation towards CRTH2 imaging. The aim is to develop a method to quantify β-cell mass with PET and facilitate non-invasive studies of disease progression in individuals with type 2 diabetes.

Methods: The precursor N-desmethyl-O-methyl MK-7246 was synthesized in seven steps and subjected to methylation with [¹¹C]methyl iodide followed by hydrolysis to obtain [¹¹C]MK-7246 labelled in the N-methyl position. Preclinical evaluation included in vitro radiography and immune-staining performed in human pancreatic biopsies. Biodistribution studies were performed in rat by PET-MRI and in pig by PET-CT imaging. The specific tracer uptake was examined in pig by scanning before and after administration of MK-7246 (1 mg/kg). Predicted dosimetry of [¹¹C]MK-7246 in human males was estimated based on the biodistribution in rat.

Results: [¹¹C]MK-7246 was obtained with activities sufficient for the current investigations (270±120 MBq) and a radiochemical purity of 93±2%. The tracer displayed focal binding in areas with insulin positive islet of Langerhans in human pancreas sections. Baseline uptake in pig was significantly reduced in CRTH2-rich areas after administration of MK-7246; pancreas (66% reduction) and spleen (88% reduction). [¹¹C]MK-7246 exhibited a safe human predicted dosimetry profile as extrapolated from the rat biodistribution data.

Conclusions: Initial preclinical in vitro and in vivo evaluation of [¹¹C]MK-7246 show binding and biodistribution properties suitable for PET imaging of CRTH2. Further studies are warranted to assess its potential in β-cell mass imaging and CRTH2 drug development.

A method and an apparatus for preparing [C-11]methyl iodide from [C-11]methane and iodine in a single pass through a non-thermal plasma reactor has been developed. The plasma was created by applying high voltage (400 V/31 kHz) to electrodes in a stream of helium gas at reduced pressure. The [C-11]methane used in the experiments was produced from [C-11]carbon dioxide via reduction with hydrogen over nickel. [C-11]methyl iodide was obtained with a specific radioactivity of 412 +/- 32 GBq/mu mol within 6 min from approximately 24 GBq of [C-11]carbon dioxide. The decay corrected radiochemical yield was 13 +/- 3% based on [C-11]carbon dioxide at start of synthesis. [C-11]Flumazenil was synthesized via a N-alkylation with the prepared [C-11]methyl iodide.

Transition metal mediated carbonylation with [11C]CO has proven a useful method to label a wide array of compounds in the carbonyl position. However, the general use in radiopharmaceutical synthesis has been hampered by the low solubility of carbon monoxide in most solvents and the resulting challenge to confine [11C]CO in low volume reaction vessels. This paper introduces a method that utilises xenon to transfer pre-concentrated [11C]CO to a sealed disposable glass vial containing carbonylation reagents. The high solubility of xenon in the organic solvent made it possible to confine the [11C]CO without utilising a pressure autoclave or chemical trapping additives. The utility of the method in 11C-carbonylation was investigated by conducting three model reactions, where [11C-carbonyl]N-benzylbenzamide, [11C-carbonyl]triclocarban and [11C-carbonyl]methyl nicotinate were afforded in decay corrected radiochemical yields of 71?+/-?6%, 42?+/-?15% and 29?+/-?10%, respectively. These promising results and the straight forward technical implementation suggest that 11C-cabonylation can become a viable mean to provide labelled carbonyl functionalities in routine radiopharmaceutical synthesis. Compounds labelled with short lived positron emitters are used in Positron Emission Tomography, a molecular imaging technology with applications in clinical diagnostics, clinical research and basic biomedical research.

Temozolomide is a chemotherapeutic drug that is mainly used in the treatment of primary glioblastoma multiforme and recurrent high-grade glioma. Here, we report an efficient good manufacturing practice compliant method for the synthesis of [3-N-11C-methyl]temozolomide from 3-N-hydroxymethyl temozolomide that cleaves off formaldehyde in situ and becomes activated towards alkylation with [11C]methyl iodide. The labelling method was developed for an on-going patient study in which the predictive value of [3-N-11C-methyl]temozolomide and positron emission tomography on the outcome of temozolomide treatment is being investigated. The precursor was reacted with [11C]methyl iodide in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene in acetonitrile, heated at stepwise increasing temperature. Purification by semipreparative HPLC with pharmaceutical grade eluent and filtration gave approximately 10 mL sterile product solution ready for injection containing 1.55 ± 0.38 GBq (n = 5), the specific activity was 88 ± 25 GBq/μmol and the radiochemical purity was 98.5 ± 1.9%.13C-NMR spectroscopy confirmed the labelled position after colabelling with 11C and 13C.

Two methods are presented for the synthesis of acrylamides labelled with C-11 (beta(+), t(1/2) = 20.4 min) and C-11 in the carbonyl position. In the first method, [1-C-11]acrylic acid is synthesised from [C-11]carbon monoxide by palladium-mediated hydroxy-carbonylation of acetylene. The labelled carboxylic acid is converted into the acyl chloride and subsequently treated with amine to yield N-benzyl[carbonyl(11)C]acrylamide, The second method utilizes [C-11]carbon monoxide in a palladium-mediated carbonylative cross-coupling of vinyl halides and amines. A higher radiochemical yield is achieved with the latter method and the amount of amine needed is decreased to 1/20. The C-11-labelled acrylamides were isolated in up to 81 % decay-corrected radiochemical yield. Starting from 10 +/- 0.5GBq of [C-11]carbon monoxide, N-benzyl[carbonyl-C-11]acrylamide was obtained in 4 min with a specific radioactivity of 330 +/- 4 GBq mu mol-(1). Co-labelling with C-11 and C-13 enabled confirmation of the labelled position by C-13 NMR spectroscopy.

Background

The serotonin precursor 5-hydroxy-L-[β-¹¹C]tryptophan ([¹¹C]HTP) is in clinical use for localization of neuroendocrine tumors and has been suggested as a proxy marker for pancreatic islet cells. However, degradation by monoamine oxidase-A (MAO-A) reduces retention and the contrast to non-endocrine tissue.

Methods

A synthesis method was developed for 5-hydroxy-L-[β-¹¹C²H]tryptophan ([¹¹C]DHTP), an isotopologue of [¹¹C]HTP, labeled with ¹¹C and ²H at the β-position adjacent to the carbon involved in MAO-A decarboxylation. MAO-A-mediated degradation of [¹¹C]DHTP was evaluated and compared to non-deuterated [¹¹C]HTP.

Results

[¹¹C]DHTP was synthesized with a radiochemical purity of >98%, radioactivity of 620 ± 190 MBq, and deuterium (²H or ²H₂) incorporation at the β-position of 22% ±5%. Retention and resistance to MAO-A-mediated degradation of [¹¹C]DHTP were increased in cells but not in non-human primate pancreas.

Conclusions

Partial deuteration of the β-position yields improved resistance to MAO-A-mediated degradation in vitro but not in vivo.

Alzheimer's disease (AD) is characterized by aggregation of amyloid beta (Aβ) into insoluble plaques. Intermediates, Aβ oligomers (Aβo), appear to be the mechanistic cause of disease. The de facto PET AD ligand, [(11)C]PIB, binds and visualizes Aβ plaque load, which does not correlate well with disease severity. Therefore, finding a dynamic target that changes with pathology progression in AD is of great interest. Aβo alter synaptic plasticity, inhibit long-term potentiation, and facilitate long-term depression; key mechanisms involved in memory and learning. In order to convey these neurotoxic effects, Aβo requires interaction with the metabotropic glutamate 5 receptor (mGluR5). The aim was to investigate in vivo mGluR5 changes in an Aβ pathology model using PET. Wild type C57/BL6 (wt) and AβPP transgenic mice (tg-ArcSwe), 4, 8, and 16 months old, were PET scanned with [(11)C]ABP688, which is highly specific to mGluR5, to investigate changes in mGluR5. Mouse brains were extracted postscan and mGluR5 and Aβ protofibril levels were assessed with immunoblotting and ELISA respectively. Receptor-dense brain regions (hippocampus, thalamus, and striatum) displayed higher [(11)C]ABP688 concentrations corresponding to mGluR5 expression pattern. Mice had similar uptake levels of [(11)C]ABP688 regardless of genotype or age. Immunoblotting revealed general decline in mGluR5 expression and elevated levels of mGluR5 in 16 months old tg-ArcSwe compared with wt mice. [(11)C]ABP688 could visualize mGluR5 in the mouse brain. In conclusion, mGluR5 levels were found to decrease with age and tended to be higher in tg-ArcSwe compared with wt mice, however these changes could not be quantified with PET.

The secondary a- and B-deuterium, the a-carbon, the nucleophile carbon, the nucleophile nitrogen, and the chlorine leaving group kinetic isotope effects forthe SN2 reaction between cyanide ion an dethyl chloride were determined in the very slightly polar solvent THF at 30 C. A comparison of these KIEs with those reported earlier for the same reaction in the polar solvent DMSO shows that the transition state in THF is only sligthly tighter with very slightly shorter NC-Ca-CI bonds. This minor change in transition state structure does not account for the different transition structures that were earlier suggested by interpreting the experimental KIEs and the gas-phase calculations, respectively. It therefore seems unlikely that the different transition states suggested by the two methods are due to the lack of appropriate solvent modeling in the theoretical calculations. Previously it was predicted that the transition state of SN2 reactions where the nucleophile and the leaving group have the same charge would be unaffected by a charge in solvent. The experimental KIEs support this view.

Background: Camelid antibody fragments are interesting for use as radioligands for Positron Emission Tomography (PET), in central nervous system imaging, due to their fast clearance from blood. This study evaluated single variable domain of heavy chain (VHH) antibodies derived from llama, targeting the mouse and human transferrin receptor (TfR) for mediating increased brain uptake. In experiments, VHHs were combined with either a human Fc or with the single chain fragment of the amyloid beta (Aβ) antibody 3D6 (scFv3D6) to investigate intrabrain targeting.

Methods: One novel and one previously disclosed species cross-reactive VHH towards murine TfR (mTfR) and human TfR (hTfR), as well as two VHHs with selective reactivity to mTfR and hTfR, respectively, were compared. The TfR binders were evaluated as recombinant fusion protein (FP) constructs fused with either a human Fc-fragment (FP_Fc) or with the Aβ-binding fragment scFv3D6 (FP_scFv) at either C- or N-terminal positions of scFv3D6. The above FPs were radiolabeled with iodine-125 (¹²⁵I) and biodistribution was studied ex vivo at 2 h, 6 h and 24 h after injection in wild-type (WT) mice and AD mouse model App^NL-G-F. Brain, blood, plasma and organ concentrations of the ¹²⁵I-FPs were measured in a γ-counter. Autoradiography, nuclear track emulsion, and immunohistofluorescence imaging were used to study the brain distribution of the FPs. 

Results: The constructs based on Fc fusions (FP_Fc) with binding affinity to mTfR displayed significantly higher brain uptake (around 1-3% ID/g_brain) in comparison with FP_Fc specific to only hTfR (control; 0.2% ID/gbrain). The VHHs reactive to mTfR fused to a scFv (FP_scFv) showed an increased brain uptake 2 h after injection compared to control (FP_scFv reactive to hTfR only). FP_scFv with VHH linked to the N-terminus of scFv3D6 showed more efficient brain delivery than those fused with the C-terminal of scFv3D6. There was a 17-fold higher brain uptake in App^NL-G-F than WT mice for one of the cross-species  reactive FP_scFv (FP_scFv1B) at 24 h post-injection, and 2.5-fold higher at 6 h, in ex vivo studies. FP_scFv1B  also showed consistently higher relative brain parenchymal localization compared to the other FPs, whether as Fc- or scFv fusion.  

Conclusion: We showed that the novel cross-reactive VHHs tested herein displayed enhanced brain delivery in mice and that these could be successfully fused with an Aβ-binding scFv-fragment, maintaining high brain and preferential parenchymal delivery with increased retention to Aβ in brain. In summary, a FP_scFv construct with affinity towards both  Aβ and mTfR showed differentiated and favorable distribution in AD-mice compared to WT already after 6 h (measured ex vivo); a relevant time point for clinical brain PET.

BACKGROUND: [11C]Flumazenil and positron emission tomography (PET) are used clinically to assess gamma-aminobutyric acid (GABA)-ergic function and to localize epileptic foci prior to resective surgery. Enhanced P-glycoprotein (P-gp) activity has been reported in epilepsy and this may confound interpretation of clinical scans if [11C]flumazenil is a P-gp substrate. The purpose of this study was to investigate whether [11C]flumazenil is a P-gp substrate.

METHODS: [11C]Flumazenil PET scans were performed in wild type (WT) (n = 9) and Mdr1a/1b, (the genes that encode for P-gp) double knockout (dKO) (n = 10) mice, and in naive rats (n = 10). In parallel to PET scanning, [11C]flumazenil plasma concentrations were measured in rats. For 6 of the WT and 6 of the dKO mice a second, [11C]flumazenil scan was acquired after administration of the P-gp inhibitor tariquidar. Cerebral [11C]flumazenil concentrations in WT and Mdr1a/1b dKO mice were compared (genetic disruption model). Furthermore, pre and post P-gp-blocking cerebral [11C]flumazenil concentrations were compared in all animals (pharmacological inhibition model).

RESULTS: Mdr1a/1b dKO mice had approximately 70% higher [11C]flumazenil uptake in the brain than WT mice. After administration of tariquidar, cerebral [11C]flumazenil uptake in WT mice increased by about 80% in WT mice, while it remained the same in Mdr1a/1b dKO mice. In rats, cerebral [11C]flumazenil uptake increased by about 60% after tariquidar administration. Tariquidar had only a small effect on plasma clearance of flumazenil.

CONCLUSIONS: The present study showed that [11C]flumazenil is a P-gp substrate in rodents. Consequently, altered cerebral [11C]flumazenil uptake, as observed in epilepsy, may not reflect solely GABAA receptor density changes but also changes in P-gp activity.

Fluorine-18 labelled N,N-diethyl-2-(2-[4-(2-fluoroethoxy)phenyl]-5,7-dimethylpyrazolo[1,5-α]pyrimidine-3-yl)acetamide ([(18)F]DPA-714) binds to the 18-kDa translocator protein (TSPO) with high affinity. The aim of this initial methodological study was to develop a plasma input tracer kinetic model for quantification of [(18)F]DPA-714 binding in healthy subjects and Alzheimer's disease (AD) patients, and to provide a preliminary assessment whether there is a disease-related signal. Ten AD patients and six healthy subjects underwent a dynamic positron emission tomography (PET) study along with arterial sampling and a scan protocol of 150 minutes after administration of 250 ± 10 MBq [(18)F]DPA-714. The model that provided the best fits to tissue time activity curves (TACs) was selected based on Akaike Information Criterion and F-test. The reversible two tissue compartment plasma input model with blood volume parameter was the preferred model for quantification of [(18)F]DPA-714 kinetics, irrespective of scan duration, volume of interest, and underlying volume of distribution (VT). Simplified reference tissue model (SRTM)-derived binding potential (BPND) using cerebellar gray matter as reference tissue correlated well with plasma input-based distribution volume ratio (DVR). These data suggest that [(18)F]DPA-714 cannot be used for separating individual AD patients from healthy subjects, but further studies including TSPO binding status are needed to substantiate these findings.

(18)F-labeled N,N-diethyl-2-(2-[4-(2-fluoroethoxy)phenyl]-5,7-dimethylpyrazolo[1,5-α]pyrimidine-3-yl)acetamide (DPA-714) is a radioligand for the 18-kDa translocator protein. The purpose of the present study was to identify the best method for generating quantitative parametric images of (18)F-DPA-714 binding.

METHODS: Ninety-minute dynamic (18)F-DPA-714 PET scans with full arterial sampling from 6 healthy subjects and 9 Alzheimer disease (AD) patients were used. Plasma-input-based Logan graphical analysis and spectral analysis were used to generate parametric volume of distribution (VT) images. Five versions of Ichise, reference Logan, and 2 basis function implementations (receptor parametric mapping and simplified reference tissue model 2 [SRTM2]) of SRTM, all using gray matter cerebellum as the reference region, were applied to generate nondisplaceable binding potential (BPND) images.

RESULTS: Plasma-input Logan analysis (r(2) = 0.99; slope, 0.88) and spectral analysis (r(2) = 0.99, slope, 0.93) generated estimates of VT that correlated well with values obtained using nonlinear regression. BPND values generated using SRTM2 (r(2) = 0.83; slope, 0.95) and reference Logan analysis (r(2) = 0.88; slope, 1.01) correlated well with nonlinear regression-based estimates.

CONCLUSION: Both Logan analysis and spectral analysis can be used to obtain quantitatively accurate VT images of (18)F-DPA-714. In addition, SRTM2 and reference Logan analysis can provide accurate BPND images. These parametric images could be used for voxel-based comparisons.

Methods to investigate blood-brain barrier transport and pharmacologically active drug concentrations in the human brain are limited and data translation between species is challenging. Hence, there is a need to further develop the read-out of techniques like positron emission tomography ( PET) for studying neuropharmacokinetics. PET has a high translational applicability from rodents to man and measures total drug concentrations in vivo. The aim of the present study was to investigate the possibility of translating total drug concentrations, acquired through PET, to unbound concentrations, resembling those measured in the interstitial fluid by microdialysis sampling. Simultaneous PET scanning and brain microdialysis sampling were performed in rats throughout a 60 min infusion of [N-methyl-C-11] oxycodone in combination with a therapeutic dose of oxycodone and during a 60 min follow up period after the end of infusion. The oxycodone concentrations acquired with PET were converted into unbound concentrations by compensating for brain tissue binding and brain intracellular distribution, using the unbound volume of distribution in brain (Vu, brain), and were compared to microdialysis measurements of unbound concentrations. A good congruence between the methods was observed throughout the infusion. However, an accumulating divergence in the acquired PET and microdialysis data was apparent and became more pronounced during the elimination phase, most likely due to the passage of radioactive metabolites into the brain. In conclusion, the study showed that PET can be used to translate non-invasively measured total drug concentrations into unbound concentrations as long as the contribution of radiolabelled metabolites is minor or can be compensated for.

Measuring synaptic density in vivo using positron emission tomography (PET) imaging-based biomarkers targeting the synaptic vesicle protein 2A (SV2A) has received much attention recently due to its potential research and clinical applications in synaptopathies, including neurodegenerative and psychiatric diseases. Fluid-based biomarkers in proteinopathies have previously been suggested to provide information on pathology and disease status that is complementary to PET-based measures, and the same can be hypothesized with respect to SV2A. This review provides an overview of the current state of SV2A PET imaging as a biomarker of synaptic density, the potential role of fluid-based biomarkers for SV2A, and related future perspectives.

Aromatase, the enzyme that in the brain converts testosterone and androstenedione to estradiol and estrone, respectively, is a putative key factor in psychoneuroendocrinology. In vivo assessment of aromatase was performed to evaluate tracer kinetic models and optimal scan duration, for quantitative analysis of the aromatase positron emission tomography (PET) ligand [¹¹ C]cetrozole. Anatomical magnetic resonance and 90-min dynamic [¹¹ C]cetrozole PET-CT scans were performed on healthy women. Volume of interest (VOI)-based analyses with a plasma-input function were performed using the single-tissue and two-tissue (2TCM) reversible compartment models and plasma-input Logan analysis. Additionally, the simplified reference tissue model (SRTM), Logan reference tissue model (LRTM), and standardized uptake volume ratio model, with cerebellum as reference region, were evaluated. Parametric images were generated and regionally averaged voxel values were compared with VOI-based analyses of the reference tissue models. The optimal reference model was used for evaluation of a decreased scan duration. Differences between the plasma-input- and reference tissue-based methods and comparisons between scan durations were assessed by linear regression. The [¹¹ C]cetrozole time-activity curves were best described by the 2TCM. SRTM nondisplaceable binding potential (BP_ND ), with cerebellum as reference region, can be used to estimate [¹¹ C]cetrozole binding and generated robust and quantitatively accurate results for a reduced scan duration of 60 min. Receptor parametric mapping, a basis function implementation of SRTM, as well as LRTM, produced quantitatively accurate parametric images, showing BP_ND at the voxel level. As PET tracer, [¹¹ C]cetrozole can be employed for relatively short brain scans to measure aromatase binding using a reference tissue-based approach.

[F-18]THK5317 is a PET tracer for in-vivo imaging of tau associated with Alzheimer's disease (AD). This work aimed to evaluate optimal timing for standardized uptake value ratio (SUVR) measures with [F-18]THK5317 and automated generation of SUVR-1 and relative cerebral blood flow (R-1) parametric images. Nine AD patients and nine controls underwent 90 min [F-18]THK5317 scans. SUVR-1 was calculated at transient equilibrium (TE) and for seven different 20 min intervals and compared with distribution volume ratio (DVR; reference Logan). Cerebellar grey matter (MRI) was used as reference region. A supervised cluster analysis (SVCA) method was implemented to automatically generate a reference region, directly from the dynamic PET volume without the need of a structural MRI scan, for computation of SUVR-1 and R-1 images for a scan duration matching the optimal timing. TE was reached first in putamen, frontal- and parietal cortex at 22 +/- 4 min for AD patients and in putamen at 20 +/- 0 min in controls. Over all regions and subjects, SUVR20-40-1 correlated best with DVR-1, R-2 = 0.97. High correlation was found between values generated using MRI- and SVCA-based reference (R-2 = 0.93 for SUVR20-40-1; R-2 = 0.94 for R-1). SUVR20-40 allows for accurate semi-quantitative assessment of tau pathology and SVCA may be used to obtain a reference region for calculation of both SUVR-1 and R-1 with 40 min scan duration.

Because a correlation between tau pathology and the clinical symptoms of Alzheimer's disease (AD) has been hypothesized, there is increasing interest in developing PET tracers that bind specifically to tau protein. The aim of this study was to evaluate tracer kinetic models for quantitative analysis and generation of parametric images for the novel tau ligand (S)-(18)F-THK5117.

METHODS: 9 subjects (5 with AD, 4 with mild cognitive impairment) received a 90 min dynamic (S)-(18)F-THK5117 PET scan. Arterial blood was sampled for measurement of blood radioactivity and metabolite analysis. VOI-based analysis was performed using plasma-input models; single-tissue and two-tissue (2TCM) compartment models and plasma-input Logan, and reference tissue models; simplified reference tissue model (SRTM), reference Logan and standardised uptake value ratio (SUVr). Cerebellum grey matter was used as reference region. Voxel-level analysis was performed using basis function implementations of SRTM, reference Logan and SUVr. Regionally averaged voxel values were compared to VOI-based values from the optimal reference tissue model and simulations were made to assess accuracy and precision. In addition to 90 min, initial 40 and 60 min data were analysed.

RESULTS: Plasma-input Logan distribution volume ratio (DVR)-1 values agreed well with 2TCM DVR-1 values (R2=0.99, slope=0.96). SRTM binding potential (BPND) and reference Logan DVR-1 values were highly correlated with plasma-input Logan DVR-1 (R2=1.00, slope≈1.00) while SUVr70-90-1 values correlated less well and overestimated binding. Agreement between parametric methods and SRTM was best for reference Logan (R2=0.99, slope=1.03). SUVr70-90-1 values were almost 3 times higher than BPND values in white matter and 1.5 times higher in grey matter. Simulations showed poorer accuracy and precision for SUVr70-90-1 values than for the other reference methods. SRTM BPND and reference Logan DVR-1 values were not affected by a shorter scan duration of 60 min.

CONCLUSION: SRTM BPND and reference Logan DVR-1 values were highly correlated with plasma-input Logan DVR-1 values. VOI-based data analyses indicated robust results for scan durations of 60 min. Reference Logan generated quantitative (S)-(18)F-THK5117 DVR-1 parametric images with the greatest accuracy and precision, and with a much lower white matter signal than seen with SUVr-1 images.

INTRODUCTION:  The accumulation of neurofibrillary tangles, composed of aggregated hyperphosphorylated tau protein, starts spreading early in specific regions in the course of Alzheimer's disease (AD), correlating with the progression of memory dysfunction. The non-invasive imaging of tau could therefore facilitate the early diagnosis of AD, differentiate it from other dementing disorders and allow evaluation of tau immunization therapy outcomes. In this study we characterized the in vitro binding properties of THK5117, a tentative radiotracer for positron emission tomography (PET) imaging of tau brain deposits.

RESULTS: Saturation and competition binding studies of (3)H-THK5117 in post-mortem AD brain tissue showed the presence of multiple binding sites. THK5117 binding was significantly higher in hippocampal (p < 0.001) and temporal (p < 0.01) tissue homogenates in AD compared to controls. Autoradiography studies with (3)H-THK5117 was performed on large frozen brain sections from three AD cases who had been followed clinically and earlier undergone in vivo (18)F-FDG PET investigations. The three AD cases showed distinct differences in regional THK5117 binding that were also observed in tau immunohistopathology as well as in clinical presentation. A negative correlation between in vivo (18)F-FDG PET and in vitro (3)H-THK5117 autoradiography was observed in two of the three AD cases.

CONCLUSIONS: This study supports that new tau PET tracers will provide further understanding on the role of tau pathology in the diversity of the clinical presentation in AD.

In this study, the performance of various methods for generating quantitative parametric images of dynamic (11)C-phenytoin PET studies was evaluated. Methods: Double-baseline 60-min dynamic (11)C-phenytoin PET studies, including online arterial sampling, were acquired for 6 healthy subjects. Parametric images were generated using Logan plot analysis, a basis function method, and spectral analysis. Parametric distribution volume (VT) and influx rate (K1) were compared with those obtained from nonlinear regression analysis of time-activity curves. In addition, global and regional test-retest (TRT) variability was determined for parametric K1 and VT values. Results: Biases in VT observed with all parametric methods were less than 5%. For K1, spectral analysis showed a negative bias of 16%. The mean TRT variabilities of VT and K1 were less than 10% for all methods. Shortening the scan duration to 45 min provided similar VT and K1 with comparable TRT performance compared with 60-min data. Conclusion: Among the various parametric methods tested, the basis function method provided parametric VT and K1 values with the least bias compared with nonlinear regression data and showed TRT variabilities lower than 5%, also for smaller volume-of-interest sizes (i.e., higher noise levels) and shorter scan duration.

PET imaging of amyloid-β (Aβ) has become an important component of Alzheimer disease diagnosis. 11C-Pittsburgh compound B (11C-PiB) and analogs bind to fibrillar Aβ. However, levels of nonfibrillar, soluble, aggregates of Aβ appear more dynamic during disease progression and more affected by Aβ-reducing treatments. The aim of this study was to compare an antibody-based PET ligand targeting nonfibrillar Aβ with 11C-PiB after β-secretase (BACE-1) inhibition in 2 Alzheimer disease mouse models at an advanced stage of Aβ pathology.

Methods: Transgenic ArcSwe mice (16 mo old) were treated with the BACE-1 inhibitor NB-360 for 2 mo, whereas another group was kept as controls. A third group was analyzed at the age of 16 mo as a baseline. Mice were PET-scanned with 11C-PiB to measure Aβ plaque load followed by a scan with the bispecific radioligand 124I-RmAb158-scFv8D3 to investigate nonfibrillar aggregates of Aβ. The same study design was then applied to another mouse model, AppNL-G-F. In this case, NB-360 treatment was initiated at the age of 8 mo and animals were scanned with 11C-PiB-PET and 125I-RmAb158-scFv8D3 SPECT. Brain tissue was isolated after scanning, and Aβ levels were assessed.

Results: 124I-RmAb158-scFv8D3 concentrations measured with PET in hippocampus and thalamus of NB-360–treated ArcSwe mice were similar to those observed in baseline animals and significantly lower than concentrations observed in same-age untreated controls. Reduced 125I-RmAb158-scFv8D3 retention was also observed with SPECT in hippocampus, cortex, and cerebellum of NB-360–treated AppNL-G-F mice. Radioligand in vivo concentrations corresponded to postmortem brain tissue analysis of soluble Aβ aggregates. For both models, mice treated with NB-360 did not display a reduced 11C-PiB signal compared with untreated controls, and further, both NB-360 and control mice tended, although not reaching significance, to show higher 11C-PiB signal than the baseline groups.

Conclusion: This study demonstrated the ability of an antibody-based radioligand to detect changes in brain Aβ levels after anti-Aβ therapy in ArcSwe and AppNL-G-F mice with pronounced Aβ pathology. In contrast, the decreased Aβ levels could not be quantified with 11C-PiB PET, suggesting that these ligands detect different pools of Aβ.

To self-archive is to make research publications freely accessible by depositing them in an open digital archive or on a public website. The aim of this thesis is to explore what Swedish scientists think of self-archiving, what they know about it and how they make use of it, in order to understand why they do not self-archive more actively.

A web survey was conducted which was answered by 296 scientists at the Swedish University of Agricultural Sciences (SLU). Results show that only 24 % have previous experience of self-archiving, but that a majority is willing to self-archive if there are no legal objections or if it is demanded by the research funder. The most important reasons why many scientists do not self-archive are that they feel uncertain about copyright issues and/or lack knowledge about self-archiving.

The radiosynthesis and GMP validation of [¹¹ C]AMT for human use are described. Three consecutive batches were produced giving 940-3790 MBq (4%-17% RCY, decay corrected, based on [¹¹ C]CO₂ ) of the tracer. The molar activity at the end of synthesis was 19 to 35 GBq/μmol, the radiochemical purity was ≥98%, and the enantiomeric purity was >99%. While the synthesis method was automated using a new generation of synthesis equipment, tracer production system developed in house, the method should be readily applicable to other synthesis platforms with minor modifications.

PURPOSE: To investigate whether time-trends of enhanced [(18)F]Fluoromethylcholine ([(18)F]FCH) in lymph nodes (LN) of prostate cancer (PCa) patients can help to discriminate reactive from malignant ones, and whether single time point standardized uptake value (SUV) measurements also suffice.

PROCEDURES: 25 PCa patients with inguinal (presumed benign) and enlarged pelvic LN (presumed malignant) showing enhanced [(18)F]FCH uptake at dual-phase PET-CT were analyzed. Associations between LN status (benign versus malignant) and SUV(max) and SUV(meanA50), determined at 2 min (early) and 30 min (late) post injection, were assessed. We considered two time-trends of [(18)F]FCH uptake: type A (SUV early > SUV late) and type B (SUV late ≥ SUV early). Histopathology and/or follow-up were used to confirm the assumption that LN with type A pattern are benign, and LN with type B pattern malignant.

RESULTS: Analysis of 54 nodes showed that LN status, time-trends, and 'late' (30 min p.i.) SUV(max) and SUV(meanA50) parameters were strongly associated (P<0.0001). SUV(max) relative difference was the best LN status predictor. All but one inguinal LN showed a decreasing [(18)F]FCH uptake over time (pattern A), while 95% of the pelvic nodes presented a stable or increasing uptake (pattern B) type.

CONCLUSIONS: Time-trends of enhanced [(18)F]FCH uptake can help to characterize lymph nodes in prostate cancer patients. Single time-point SUV measurements, 30 min p.i., may be a reasonable alternative for predicting benign versus malignant status of lymph nodes, but this remains to be validated in non-enlarged pelvic lymph nodes.

Deposition of amyloid beta (Aβ) into plaques is a major hallmark of Alzheimer’s disease (AD). Different amyloid precursor protein (APP) mutations cause early-onset AD by altering the production or aggregation properties of Aβ. We recently identified the Uppsala APP mutation (APPUpp), which causes Aβ pathology by a triple mechanism: increased β-secretase and altered α-secretase APP cleavage, leading to increased formation of a unique Aβ conformer that rapidly aggregates and deposits in the brain. The aim of this study was to further explore the effects of APPUpp in a transgenic mouse model (tg-UppSwe), expressing human APP with the APPUpp mutation together with the APPSwe mutation. Aβ pathology was studied in tg-UppSwe brains at different ages, using ELISA and immunohistochemistry. In vivo PET imaging with three different PET radioligands was conducted in aged tg-UppSwe mice and two other mouse models; tg-ArcSwe and tg-Swe. Finally, glial responses to Aβ pathology were studied in cell culture models and mouse brain tissue, using ELISA and immunohistochemistry. Tg-UppSwe mice displayed increased β-secretase cleavage and suppressed α-secretase cleavage, resulting in AβUpp42 dominated diffuse plaque pathology appearing from the age of 5–6 months. The γ-secretase cleavage was not affected. Contrary to tg-ArcSwe and tg-Swe mice, tg-UppSwe mice were [¹¹C]PiB-PET negative. Antibody-based PET with the 3D6 ligand visualized Aβ pathology in all models, whereas the Aβ protofibril selective mAb158 ligand did not give any signals in tg-UppSwe mice. Moreover, unlike the other two models, tg-UppSwe mice displayed a very faint glial response to the Aβ pathology. The tg-UppSwe mouse model thus recapitulates several pathological features of the Uppsala APP mutation carriers. The presumed unique structural features of AβUpp42 aggregates were found to affect their interaction with anti-Aβ antibodies and profoundly modify the Aβ-mediated glial response, which may be important aspects to consider for further development of AD therapies.

PURPOSE: Several individual clinical and preclinical studies have shown the possibility of evaluating tumor hypoxia by using noninvasive positron emission tomography (PET). The current study compared 3 hypoxia PET tracers frequently used in the clinic, [(18)F]FMISO, [(18)F]FAZA, and [(18)F]HX4, in a preclinical tumor model. Tracer uptake was evaluated for the optimal time point for imaging, tumor-to-blood ratios (TBR), spatial reproducibility, and sensitivity to oxygen modification.

METHODS AND MATERIALS: PET/computed tomography (CT) images of rhabdomyosarcoma R1-bearing WAG/Rij rats were acquired at multiple time points post injection (p.i.) with one of the hypoxia tracers. TBR values were calculated, and reproducibility was investigated by voxel-to-voxel analysis, represented as correlation coefficients (R) or Dice similarity coefficient of the high-uptake volume. Tumor oxygen modifications were induced by exposure to either carbogen/nicotinamide treatment or 7% oxygen breathing.

RESULTS: TBR was stabilized and maximal at 2 hours p.i. for [(18)F]FAZA (4.0 ± 0.5) and at 3 hours p.i. for [(18)F]HX4 (7.2 ± 0.7), whereas [(18)F]FMISO showed a constant increasing TBR (9.0 ± 0.8 at 6 hours p.i.). High spatial reproducibility was observed by voxel-to-voxel comparisons and Dice similarity coefficient calculations on the 30% highest uptake volume for both [(18)F]FMISO (R = 0.86; Dice coefficient = 0.76) and [(18)F]HX4 (R = 0.76; Dice coefficient = 0.70), whereas [(18)F]FAZA was less reproducible (R = 0.52; Dice coefficient = 0.49). Modifying the hypoxic fraction resulted in enhanced mean standardized uptake values for both [(18)F]HX4 and [(18)F]FAZA upon 7% oxygen breathing. Only [(18)F]FMISO uptake was found to be reversible upon exposure to nicotinamide and carbogen.

CONCLUSIONS: This study indicates that each tracer has its own strengths and, depending on the question to be answered, a different tracer can be put forward.