Chelator impact: investigating the pharmacokinetic behavior of copper-64 labeled PD-L1 radioligands.

BACKGROUND: Programmed cell death ligand 1 (PD-L1) plays a critical role in the tumor microenvironment and overexpression in several solid cancers has been reported. This was associated with a downregulation of the local immune response, specifically of T-cells. Immune checkpoint inhibitors showed a potential to break this localized immune paralysis, but only 30% of patients are considered responders. New diagnostic approaches are therefore needed to determine patient eligibility. Small molecule radiotracers targeting PD-L1, may serve as such diagnostic tools, addressing the heterogeneous PD-L1 expression between and within tumor lesions, thus aiding in therapy decisions. RESULTS: Four biphenyl-based small-molecule PD-L1 ligands were synthesized using a convergent synthetic route with a linear sequence of up to eleven steps. As a chelator NODA-GA, CB-TE2A or DiAmSar was used to allow radiolabeling with copper-64 ([64Cu]Cu-14-[64Cu]Cu-16). In addition, a dimeric structure based on DiAmSar was synthesized ([64Cu]Cu-17). All four radioligands exhibited high proteolytic stability (> 95%) up to 48 h post-radiolabeling. Saturation binding yielded moderate affinities toward PD-L1, ranging from 100 to 265 nM. Real-time radioligand binding provided more promising KD values around 20 nM for [64Cu]Cu-14 and [64Cu]Cu-15. In vivo PET imaging in mice bearing both PC3 PD-L1 overexpressing and PD-L1-mock tumors was performed at 0-2, 4-5 and 24-25 h post injection (p.i.). This revealed considerably different pharmacokinetic profiles, depending on the substituted chelator. [64Cu]Cu-14, substituted with NODA-GA, showed renal clearance with low liver uptake, whereas substitution with the cross-bridged cyclam chelator CB-TE2A resulted in a primarily hepatobiliary clearance. Notably, the monomeric DiAmSar radioligand [64Cu]Cu-16 demonstrated a higher liver uptake than [64Cu]Cu-15, but was still renally cleared as evidenced by the lack of uptake in gall bladder and intestines. The dimeric structure [64Cu]Cu-17 showed extensive accumulation and trapping in the liver but was also cleared via the renal pathway. Of all tracer candidates and across all timepoints, [64Cu]Cu-17 showed the highest accumulation at 24 h p.i. in the PD-L1-overexpressing tumor of all timepoints and all radiotracers, indicating drastically increased circulation time upon dimerization of two PD-L1 binding motifs. CONCLUSIONS: This study shows that chelator choice significantly influences the pharmacokinetic profile of biphenyl-based small molecule PD-L1 radioligands. The NODA-GA-conjugated radioligand [64Cu]Cu-14 exhibited favorable renal clearance; however, the limited uptake in tumors suggests the need for structural modifications to the binding motif for future PD-L1 radiotracers.

Design, Synthesis, and Biological Evaluation of Small-Molecule-Based Radioligands with Improved Pharmacokinetic Properties for Imaging of Programmed Death Ligand 1.

Small molecules offer some advantages for developing positron emission tomography (PET) tracers and are therefore a promising approach for imaging and therapy monitoring of programmed death ligand 1 (PD-L1) positive tumors. Here, we report six biphenyl PD-L1 radioligands using the NODA-GA-chelator for efficient copper-64 complexation. These radioligands contain varying numbers of sulfonic and/or phosphonic acid groups, serving as hydrophilizing units to lower the log D7.4 value down to -4.28. The binding affinities of compounds were evaluated using saturation binding and a real-time binding assay, with a highest binding affinity of 21 nM. Small-animal PET imaging revealed vastly different pharmacokinetic profiles depending on the quantity and type of hydrophilizing units. Of the investigated radioligands, [64Cu]Cu-3 showed the most favorable kinetics in vitro. This was also found in vivo, with a predominantly renal clearance and a specific uptake in the PD-L1-overexpressing tumor. With further modifications, this compound could be a promising candidate for the imaging of PD-L1 in the clinical setting.

Preparation of 18F-Labeled Tracers Targeting Fibroblast Activation Protein via Sulfur [18F]Fluoride Exchange Reaction.

Early detection and treatment of cancers can significantly increase patient prognosis and enhance the quality of life of affected patients. The emerging significance of the tumor microenvironment (TME) as a new frontier for cancer diagnosis and therapy may be exploited by radiolabeled tracers for diagnostic imaging techniques such as positron emission tomography (PET). Cancer-associated fibroblasts (CAFs) within the TME are identified by biomarkers such as fibroblast activation protein alpha (FAPα), which are expressed on their surfaces. Targeting FAPα using small-molecule 18F-labeled inhibitors (FAPIs) has recently garnered significant attention for non-invasive tumor visualization using PET. Herein, two potent aryl-fluorosulfate-based FAPIs, 12 and 13, were synthetically prepared, and their inhibition potency was determined using a fluorimetric FAP assay to be IC50 9.63 and 4.17 nM, respectively. Radiofluorination was performed via the sulfur [18F]fluoride exchange ([18F]SuFEx) reaction to furnish [18F]12 and [18F]13 in high activity yields (AY) of 39-56% and molar activities (Am) between 20-55 GBq/µmol. In vitro experiments focused on the stability of the radiolabeled FAPIs after incubation with human serum, liver microsomes and liver cytosol. Preliminary PET studies of the radioligands were performed in healthy mice to investigate the in vivo biodistribution and 18F defluorination rate. Fast pharmacokinetics for the FAP-targeting tracers were retained and considerable bone uptake, caused by either 18F defluorination or radioligand accumulation, was observed. In summary, our findings demonstrate the efficiency of [18F]SuFEx as a radiolabeling method as well as its advantages and limitations with respect to PET tracer development.

Exploring Hydrophilic PD-L1 Radiotracers Utilizing Phosphonic Acids: Insights into Unforeseen Pharmacokinetics.

Immune checkpoint inhibitor therapy targeting the PD-1/PD-L1 axis in cancer patients, is a promising oncological treatment. However, the number of non-responders remains high, causing a burden for the patient and the healthcare system. Consequently, a diagnostic tool to predict treatment outcomes would help with patient stratification. Molecular imaging provides said diagnostic tool by offering a whole-body quantitative assessment of PD-L1 expression, hence supporting therapy decisions. Four PD-L1 radioligand candidates containing a linker-chelator system for radiometalation, along with three hydrophilizing units-one sulfonic and two phosphonic acids-were synthesized. After labeling with 64Cu, log D7.4 values of less than -3.03 were determined and proteolytic stability confirmed over 94% intact compound after 48 h. Binding affinity was determined using two different assays, revealing high affinities up to 13 nM. µPET/CT imaging was performed in tumor-bearing mice to investigate PD-L1-specific tumor uptake and the pharmacokinetic profile of radioligands. These results yielded an unexpected in vivo distribution, such as low tumor uptake in PD-L1 positive tumors, high liver uptake, and accumulation in bone/bone marrow and potentially synovial spaces. These effects are likely caused by Ca2+-affinity and/or binding to macrophages. Despite phosphonic acids providing high water solubility, their incorporation must be carefully considered to avoid compromising the pharmacokinetic behavior of radioligands.

Design and Biological Evaluation of Small-Molecule PET-Tracers for Imaging of Programmed Death Ligand 1.

Noninvasive molecular imaging of the PD-1/PD-L1 immune checkpoint is of high clinical relevance for patient stratification and therapy monitoring in cancer patients. Here we report nine small-molecule PD-L1 radiotracers with solubilizing sulfonic acids and a linker-chelator system, designed by molecular docking experiments and synthesized according to a new, convergent synthetic strategy. Binding affinities were determined both in cellular saturation and real-time binding assay (LigandTracer), revealing dissociation constants in the single digit nanomolar range. Incubation in human serum and liver microsomes proved in vitro stability of these compounds. Small animal PET/CT imaging, in mice bearing PD-L1 overexpressing and PD-L1 negative tumors, showed moderate to low uptake. All compounds were cleared primarily through the hepatobiliary excretion route and showed a long circulation time. The latter was attributed to strong blood albumin binding effects, discovered during our binding experiments. Taken together, these compounds are a promising starting point for further development of a new class of PD-L1 targeting radiotracers.

Traumatic Brain Injury Leads to Alterations in Contusional Cortical miRNAs Involved in Dementia.

There is compelling evidence that head injury is a significant environmental risk factor for Alzheimer's disease (AD) and that a history of traumatic brain injury (TBI) accelerates the onset of AD. Amyloid-ß plaques and tau aggregates have been observed in the post-mortem brains of TBI patients; however, the mechanisms leading to AD neuropathology in TBI are still unknown. In this study, we hypothesized that focal TBI induces changes in miRNA expression in and around affected areas, resulting in the altered expression of genes involved in neurodegeneration and AD pathology. For this purpose, we performed a miRNA array in extracts from rats subjected to experimental TBI, using the controlled cortical impact (CCI) model. In and around the contusion, we observed alterations of miRNAs associated with dementia/AD, compared to the contralateral side. Specifically, the expression of miR-9 was significantly upregulated, while miR-29b, miR-34a, miR-106b, miR-181a and miR-107 were downregulated. Via qPCR, we confirmed these results in an additional group of injured rats when compared to naïve animals. Interestingly, the changes in those miRNAs were concomitant with alterations in the gene expression of mRNAs involved in amyloid generation and tau pathology, such as ß-APP cleaving enzyme (BACE1) and Glycogen synthase-3-ß (GSK3ß). In addition increased levels of neuroinflammatory markers (TNF-α), glial activation, neuronal loss, and tau phosphorylation were observed in pericontusional areas. Therefore, our results suggest that the secondary injury cascade in TBI affects miRNAs regulating the expression of genes involved in AD dementia.

CRISPR/Cas9 Mediated Knockout of Cyclooxygenase-2 Gene Inhibits Invasiveness in A2058 Melanoma Cells.

The inducible isoenzyme cyclooxygenase-2 (COX-2) is an important hub in cellular signaling, which contributes to tumor progression by modulating and enhancing a pro-inflammatory tumor microenvironment, tumor growth, apoptosis resistance, angiogenesis and metastasis. In order to understand the role of COX-2 expression in melanoma, we investigated the functional knockout effect of COX-2 in A2058 human melanoma cells. COX-2 knockout was validated by Western blot and flow cytometry analysis. When comparing COX-2 knockout cells to controls, we observed significantly reduced invasion, colony and spheroid formation potential in cell monolayers and three-dimensional models in vitro, and significantly reduced tumor development in xenograft mouse models in vivo. Moreover, COX-2 knockout alters the metabolic activity of cells under normoxia and experimental hypoxia as demonstrated by using the radiotracers [18F]FDG and [18F]FMISO. Finally, a pilot protein array analysis in COX-2 knockout cells verified significantly altered downstream signaling pathways that can be linked to cellular and molecular mechanisms of cancer metastasis closely related to the enzyme. Given the complexity of the signaling pathways and the multifaceted role of COX-2, targeted suppression of COX-2 in melanoma cells, in combination with modulation of related signaling pathways, appears to be a promising therapeutic approach.

Annexin A1 restores cerebrovascular integrity concomitant with reduced amyloid-ß and tau pathology.

Alzheimer's disease, characterized by brain deposits of amyloid-ß plaques and neurofibrillary tangles, is also linked to neurovascular dysfunction and blood-brain barrier breakdown, affecting the passage of substances into and out of the brain. We hypothesized that treatment of neurovascular alterations could be beneficial in Alzheimer's disease. Annexin A1 (ANXA1) is a mediator of glucocorticoid anti-inflammatory action that can suppress microglial activation and reduce blood-brain barrier leakage. We have reported recently that treatment with recombinant human ANXA1 (hrANXA1) reduced amyloid-ß levels by increased degradation in neuroblastoma cells and phagocytosis by microglia. Here, we show the beneficial effects of hrANXA1 in vivo by restoring efficient blood-brain barrier function and decreasing amyloid-ß and tau pathology in 5xFAD mice and Tau-P301L mice. We demonstrate that young 5xFAD mice already suffer cerebrovascular damage, while acute pre-administration of hrANXA1 rescued the vascular defects. Interestingly, the ameliorated blood-brain barrier permeability in young 5xFAD mice by hrANXA1 correlated with reduced brain amyloid-ß load, due to increased clearance and degradation of amyloid-ß by insulin degrading enzyme (IDE). The systemic anti-inflammatory properties of hrANXA1 were also observed in 5xFAD mice, increasing IL-10 and reducing TNF-α expression. Additionally, the prolonged treatment with hrANXA1 reduced the memory deficits and increased synaptic density in young 5xFAD mice. Similarly, in Tau-P301L mice, acute hrANXA1 administration restored vascular architecture integrity, affecting the distribution of tight junctions, and reduced tau phosphorylation. The combined data support the hypothesis that blood-brain barrier breakdown early in Alzheimer's disease can be restored by hrANXA1 as a potential therapeutic approach.

In Vitro and In Vivo Characterization of Dibenzothiophene Derivatives [125I]Iodo-ASEM and [18F]ASEM as Radiotracers of Homo- and Heteromeric α7 Nicotinic Acetylcholine Receptors.

The α7 nicotinic acetylcholine receptor (α7 nAChR) is involved in several cognitive and physiologic processes; its expression levels and patterns change in neurologic and psychiatric diseases, such as schizophrenia and Alzheimer's disease, which makes it a relevant drug target. Development of selective radioligands is important for defining binding properties and occupancy of novel molecules targeting the receptor. We tested the in vitro binding properties of [125I]Iodo-ASEM [(3-(1,4-diazabycyclo[3.2.2]nonan-4-yl)-6-(125I-iododibenzo[b,d]thiopentene 5,5-dioxide)] in the mouse, rat and pig brain using autoradiography. The in vivo binding properties of [18F]ASEM were investigated using positron emission tomography (PET) in the pig brain. [125I]Iodo-ASEM showed specific and displaceable high affinity (~1 nM) binding in mouse, rat, and pig brain. Binding pattern overlapped with [125I]α-bungarotoxin, specific binding was absent in α7 nAChR gene-deficient mice and binding was blocked by a range of α7 nAChR orthosteric modulators in an affinity-dependent order in the pig brain. Interestingly, relative to the wild-type, binding in ß2 nAChR gene-deficient mice was lower for [125I]Iodo-ASEM (58% ± 2.7%) than [125I]α-bungarotoxin (23% ± 0.2%), potentially indicating different binding properties to heteromeric α7ß2 nAChR. [18F]ASEM PET in the pig showed high brain uptake and reversible tracer kinetics with a similar spatial distribution as previously reported for α7 nAChR. Blocking with SSR-180,711 resulted in a significant decrease in [18F]ASEM binding. Our findings indicate that [125I]Iodo-ASEM allows sensitive and selective imaging of α7 nAChR in vitro, with better signal-to-noise ratio than previous tracers. Preliminary data of [18F]ASEM in the pig brain demonstrated principal suitable kinetic properties for in vivo quantification of α7 nAChR, comparable to previously published data.

Radiation dosimetry of the α4ß2 nicotinic receptor ligand (+)-[18F]flubatine, comparing preclinical PET/MRI and PET/CT to first-in-human PET/CT results.

BACKGROUND: Both enantiomers of [18F]flubatine are new radioligands for neuroimaging of α4ß2 nicotinic acetylcholine receptors with positron emission tomography (PET) exhibiting promising pharmacokinetics which makes them attractive for different clinical questions. In a previous preclinical study, the main advantage of (+)-[18F]flubatine compared to (-)-[18F]flubatine was its higher binding affinity suggesting that (+)-[18F]flubatine might be able to detect also slight reductions of α4ß2 nAChRs and could be more sensitive than (-)-[18F]flubatine in early stages of Alzheimer's disease. To support the clinical translation, we investigated a fully image-based internal dosimetry approach for (+)-[18F]flubatine, comparing mouse data collected on a preclinical PET/MRI system to piglet and first-in-human data acquired on a clinical PET/CT system. Time-activity curves (TACs) were obtained from the three species, the animal data extrapolated to human scale, exponentially fitted and the organ doses (OD), and effective dose (ED) calculated with OLINDA. RESULTS: The excreting organs (urinary bladder, kidneys, and liver) receive the highest organ doses in all species. Hence, a renal/hepatobiliary excretion pathway can be assumed. In addition, the ED conversion factors of 12.1 µSv/MBq (mice), 14.3 µSv/MBq (piglets), and 23.0 µSv/MBq (humans) were calculated which are well within the order of magnitude as known from other 18F-labeled radiotracers. CONCLUSIONS: Although both enantiomers of [18F]flubatine exhibit different binding kinetics in the brain due to the respective affinities, the effective dose revealed no enantiomer-specific differences among the investigated species. The preclinical dosimetry and biodistribution of (+)-[18F]flubatine was shown and the feasibility of a dose assessment based on image data acquired on a small animal PET/MR and a clinical PET/CT was demonstrated. Additionally, the first-in-human study confirmed the tolerability of the radiation risk of (+)-[18F]flubatine imaging which is well within the range as caused by other 18F-labeled tracers. However, as shown in previous studies, the ED in humans is underestimated by up to 50 % using preclinical imaging for internal dosimetry. This fact needs to be considered when applying for first-in-human studies based on preclinical biokinetic data scaled to human anatomy.

Evaluation of the Enantiomer Specific Biokinetics and Radiation Doses of [(18)F]Fluspidine-A New Tracer in Clinical Translation for Imaging of σ1 Receptors.

The enantiomers of [(18)F]fluspidine, recently developed for imaging of σ1 receptors, possess distinct pharmacokinetics facilitating their use in different clinical settings. To support their translational potential, we estimated the human radiation dose of (S)-(-)-[(18)F]fluspidine and (R)-(+)-[(18)F]fluspidine from ex vivo biodistribution and PET/MRI data in mice after extrapolation to the human scale. In addition, we validated the preclinical results by performing a first-in-human PET/CT study using (S)-(-)-[(18)F]fluspidine. Based on the respective time-activity curves, we calculated using OLINDA the particular organ doses (ODs) and effective doses (EDs). The ED values of (S)-(-)-[(18)F]fluspidine and (R)-(+)-[(18)F]fluspidine differed significantly with image-derived values obtained in mice with 12.9 µSv/MBq and 14.0 µSv/MBq (p < 0.025), respectively. A comparable ratio was estimated from the biodistribution data. In the human study, the ED of (S)-(-)-[(18)F]fluspidine was calculated as 21.0 µSv/MBq. Altogether, the ED values for both [(18)F]fluspidine enantiomers determined from the preclinical studies are comparable with other (18)F-labeled PET imaging agents. In addition, the first-in-human study confirmed that the radiation risk of (S)-(-)-[(18)F]fluspidine imaging is within acceptable limits. However, as already shown for other PET tracers, the actual ED of (S)-(-)-[(18)F]fluspidine in humans was underestimated by preclinical imaging which needs to be considered in other first-in-human studies.

Comparison of in†Silico, Electrochemical, in†Vitro and in†Vivo Metabolism of a Homologous Series of (Radio)fluorinated σ1 Receptor Ligands Designed for Positron Emission Tomography.

The imaging of σ1 receptors in the brain by fluorinated radiotracers will be used for the validation of σ1 receptors as drug targets as well as for differential diagnosis of diseases in the central nervous system. The biotransformation of four homologous fluorinated PET tracers 1'-benzyl-3-(ω-fluoromethyl to ω-fluorobutyl)-3H-spiro[2]benzofuran-1,4'-piperidine] ([18 F]1-4) was investigated. In silico studies using fast metabolizer (FAME) software, electrochemical oxidations, in vitro studies with rat liver microsomes, and in vivo metabolism studies after application of the PET tracers [18 F]1-4 to mice were performed. Combined liquid chromatography and mass spectrometry (HPLC-MS) analysis allowed structural identification of non-radioactive metabolites. Radio-HPLC and radio-TLC provided information about the presence of unchanged parent radiotracers and their radiometabolites. Radiometabolites were not found in the brain after application of [18 F]2-4, but liver, plasma, and urine samples contained several radiometabolites. Less than 2 % of the injected dose of [18 F]4 reached the brain, rendering [18 F]4 less appropriate as a PET tracer than [18 F]2 and [18 F]3. Compounds [18 F]2 and [18 F]3 possess the most promising properties for imaging of σ1 receptors in the brain. High σ1 affinity (Ki =0.59 nm), low lipophilicity (logD7.4 =2.57), high brain penetration (4.6 % of injected dose after 30 min), and the absence of radiometabolites in the brain favor the fluoroethyl derivative [18 F]2 slightly over the fluoropropyl derivative [18 F]3 for human use.

Changes in Binding of [(123)I]CLINDE, a High-Affinity Translocator Protein 18 kDa (TSPO) Selective Radioligand in a Rat Model of Traumatic Brain Injury.

After traumatic brain injury (TBI), secondary injuries develop, including neuroinflammatory processes that contribute to long-lasting impairments. These secondary injuries represent potential targets for treatment and diagnostics. The translocator protein 18 kDa (TSPO) is expressed in activated microglia cells and upregulated in response to brain injury and therefore a potential biomarker of the neuroinflammatory processes. Second-generation radioligands of TSPO, such as [(123)I]CLINDE, have a higher signal-to-noise ratio as the prototype ligand PK11195. [(123)I]CLINDE has been employed in human studies using single-photon emission computed tomography to image the neuroinflammatory response after stroke. In this study, we used the same tracer in a rat model of TBI to determine changes in TSPO expression. Adult Sprague-Dawley rats were subjected to moderate controlled cortical impact injury and sacrificed at 6, 24, 72 h and 28 days post surgery. TSPO expression was assessed in brain sections employing [(123)I]CLINDE in vitro autoradiography. From 24 h to 28 days post surgery, injured animals exhibited a marked and time-dependent increase in [(123)I]CLINDE binding in the ipsilateral motor, somatosensory and parietal cortex, as well as in the hippocampus and thalamus. Interestingly, binding was also significantly elevated in the contralateral M1 motor cortex following TBI. Craniotomy without TBI caused a less marked increase in [(123)I]CLINDE binding, restricted to the ipsilateral hemisphere. Radioligand binding was consistent with an increase in TSPO mRNA expression and CD11b immunoreactivity at the contusion site. This study demonstrates the applicability of [(123)I]CLINDE for detailed regional and quantitative assessment of glial activity in experimental models of TBI.

Imaging of α7 nicotinic acetylcholine receptors in brain and cerebral vasculature of juvenile pigs with [(18)F]NS14490.

BACKGROUND: The α7 nicotinic acetylcholine receptor (nAChR) is an important molecular target in neuropsychiatry and oncology. Development of applicable highly specific radiotracers has been challenging due to comparably low protein expression. To identify novel ligands as candidates for positron emission tomography (PET), a library of diazabicyclononane compounds was screened regarding affinity and specificity towards α7 nAChRs. From these, [(18)F]NS14490 has been shown to yield reliable results in organ distribution studies; however, the radiosynthesis of [(18)F]NS14490 required optimization and automation to obtain the radiotracer in quantities allowing dynamic PET studies in piglets. METHODS: Automated radiosynthesis of [(18)F]NS14490 has been performed by [(18)F]fluorination with the tosylate precursor in the TRACERlab™ FX F-N synthesis module (Waukesha, WI, USA). After optimization, the radiochemical yield of [(18)F]NS14490 was consistently approximately 35%, and the total synthesis time was about 90 min. The radiotracer was prepared with >92% radiochemical purity, and the specific activity at the end of the synthesis was 226 ± 68 GBq µmol(-1). PET measurements were performed in young pigs to investigate the metabolic stability and cerebral binding of [(18)F]NS14490 without and with administration of the α7 nAChR partial agonist NS6740 in baseline and blocking conditions. RESULTS: The total distribution volume relative to the metabolite-corrected arterial input was 3.5 to 4.0 mL g(-1) throughout the telencephalon and was reduced to 2.6 mL g(-1) in animals treated with NS6740. Assuming complete blockade, this displacement indicated a binding potential (BPND) of approximately 0.5 in the brain of living pigs. In addition, evidence for specific binding in major brain arteries has been obtained. CONCLUSION: [(18)F]NS14490 is not only comparable to other preclinically investigated PET radiotracers for imaging of α7 nAChR in brain but also could be a potential PET radiotracer for imaging of α7 nAChR in vulnerable plaques of diseased vessels.

Distinctive in vivo kinetics of the new σ1 receptor ligands (R)-(+)- and (S)-(-)-18F-fluspidine in porcine brain.

UNLABELLED: Because of their involvement in growth and survival signaling cascades, the σ(1) receptors (σ(1)Rs) represent a novel target for the treatment of cancer and several brain diseases such as depression and neurodegeneration. From a series of σ1R-specific (18)F-fluoroalkylated spirocyclic piperidines, we have chosen (18)F-fluspidine for detailed investigation of the in vivo kinetics of the (R)-(+)- and (S)-(-)-enantiomers to identify their potential for imaging in humans. METHODS: Enantiopure tosylate precursors for radiolabeling were obtained using chiral preparative high-performance liquid chromatography and used for radiosynthesis of both (18)F-fluspidine enantiomers by nucleophilic substitution with K-(18)F-F-Kryptofix 222-carbonate complex in a synthesis module. Brain pharmacokinetics were investigated by dynamic PET studies in piglets under baseline and blocking conditions using the highly selective σ1R agonist SA4503. Standardized uptake values (SUVs) were calculated for 24 MR-defined brain regions. Total distribution volume (V(T)) and binding potentials (k3'/k4) of (S)-(-)- and (R)-(+)-(18)F-fluspidine were estimated. Furthermore, V(T) values were estimated by graphical analysis using Logan plots. RESULTS: The (S)- and (R)-tosylates were obtained in excellent enantiomeric purities (>98% and >96% enantiomeric excess, respectively). (S)-(-)- and (R)-(+)-(18)F-fluspidine were synthesized within approximately 70 min (radiochemical yield, 35%-45%; specific activity, 650-870 GBq/µmol; radiochemical purity, >99%). Both radiotracers displayed different brain uptake kinetics. Although the initial brain uptake was similar, the SUV at the end of the study differed significantly (P < 0.05), with (R)-(+)-(18)F-fluspidine showing about 60%-150% higher values. Administration of SA4503 reduced SUV almost equally for both radiotracers by approximately 65%. Furthermore, k(3)' was significantly decreased under blocking conditions in almost all regions ((S)-(-)-(18)F-fluspidine, -90%-95%; (R)-(+)-(18)F-fluspidine, -70%-90%) whereas effects on k(4) differed according to the particular brain region. V(T) estimated by both graphical analysis using Logan plots and full nonlinear kinetic analysis revealed significant inhibition for both radiotracers under blocking conditions. CONCLUSION: Both (S)-(-)- and (R)-(+)-(18)F-fluspidine appear to be suitable for σ1R imaging in humans. The different pharmacokinetics of (S)-(-)-(18)F-fluspidine and (R)-(+)-(18)F-fluspidine may have the potential for application in the diagnostics of different pathologic conditions.

Asymmetric Synthesis of Spirocyclic 2-Benzopyrans for Positron Emission Tomography of σ1 Receptors in the Brain.

Sharpless asymmetric dihydroxylation of styrene derivative 6 afforded chiral triols (R)-7 and (S)-7, which were cyclized with tosyl chloride in the presence of Bu2SnO to provide 2-benzopyrans (R)-4 and (S)-4 with high regioselectivity. The additional hydroxy moiety in the 4-position was exploited for the introduction of various substituents. Williamson ether synthesis and replacement of the Boc protective group with a benzyl moiety led to potent σ1 ligands with high σ1/σ2-selectivity. With exception of the ethoxy derivative 16, the (R)-configured enantiomers represent eutomers with eudismic ratios of up to 29 for the ester (R)-18. The methyl ether (R)-15 represents the most potent σ1 ligand of this series of compounds, with a Ki value of 1.2 nM and an eudismic ratio of 7. Tosylate (R)-21 was used as precursor for the radiosynthesis of [18F]-(R)-20, which was available by nucleophilic substitution with K[18F]F K222 carbonate complex. The radiochemical yield of [18F]-(R)-20 was 18%-20%, the radiochemical purity greater than 97% and the specific radioactivity 175-300 GBq/µmol. Although radiometabolites were detected in plasma, urine and liver samples, radiometabolites were not found in brain samples. After 30 min, the uptake of the radiotracer in the brain was 3.4% of injected dose per gram of tissue and could be reduced by coadministration of the σ1 antagonist haloperidol. [18F]-(R)-20 was able to label those regions of the brain, which were reported to have high density of σ1 receptors.

Impact of 5-lipoxygenase inhibitors on the spatiotemporal distribution of inflammatory cells and neuronal COX-2 expression following experimental traumatic brain injury in rats.

The inflammatory response following traumatic brain injury (TBI) contributes to neuronal death with poor outcome. Although anti-inflammatory strategies were beneficial in the experimental TBI, clinical translations mostly failed, probably caused by the complexity of involved cells and mediators. We recently showed in a rat model of controlled cortical impact (CCI) that leukotriene inhibitors (LIs) attenuate contusion growth and improve neuronal survival. This study focuses on spatiotemporal characteristics of macrophages and granulocytes, typically involved in inflammatory processes, and neuronal COX-2 expression. Effects of treatment with LIs (Boscari/MK-886), started prior trauma, were evaluated by quantifying CD68(+), CD43(+) and COX-2(+) cells 24h and 72 h post-CCI in the parietal cortex (PC), CA3 region, dentate gyrus (DG) and visual/auditory cortex (v/aC). Correlations were applied to identify intercellular relationships. At 24h, untreated animals showed granulocyte invasion in all regions, decreasing towards 72 h. Macrophages increased from 24h to 72 h post-CCI in PC and v/aC. COX-2(+) neurones showed no temporal changes, except of an increase in the CA3 region at 72 h. Treatment reduced granulocytes at 24h in the pericontusional zone and hippocampus, and macrophages at 72 h in the PC and v/aC. COX-2 expression remained unaffected by LIs, except of time-specific changes in the DG (increase/decrease at 24/72 h). Interrelations confirmed concomitant cellular reactions beyond the initial trauma site. In conclusion, LIs attenuated the cellular inflammatory response following CCI. Future studies have to clarify region-specific effects and explore the potential of a clinically more relevant therapeutic approach applying LIs after CCI.