Efficient Reductive N-11C-Methylation Using Arylamines or Alkylamines and In Situ-Generated [11C]Formaldehyde From [11C]Methyl Iodide.

Reductive N-11C-methylation using [11C]formaldehyde and amines has been used to prepare N-11C-methylated compounds. However, the yields of the N-11C-methylated compounds are often insufficient. In this study, we developed an efficient method for base-free reductive N-11C-methylation that is applicable to a wide variety of substrates, including arylamines bearing electron-withdrawing and electron-donating substituents. A 2-picoline borane complex, which is a stable and mild reductant, was used. Dimethyl sulfoxide was used as the primary reaction solvent, and glacial acetic acid or aqueous acetic acid was used as a cosolvent. While reductive N-11C-methylation efficiently proceeded under anhydrous conditions in most cases, the addition of water to the reductive N-11C-methylation generally increased the yield of the N-11C-methylated compounds. Substrates with hydroxy, carboxyl, nitrile, nitro, ester, amide, and phenone moieties and amine salts were applicable to the reaction. This proposed method for reductive N-11C-methylation should be applicable to a wide variety of substrates, including thermo-labile and base-sensitive compounds because the reaction was performed under relatively mild conditions (70°C) without the need for a base.

Validation of a good manufacturing practice procedure for the production of [11C]AZD4747, a CNS penetrant KRASG12c inhibitor.

AZD4747 is a KRASG12C inhibitor recently shown to cross the non-human primate blood-brain barrier efficiently. In the current study, a GMP-compliant production of [11C]AZD4747 was developed to enable PET studies in human subjects. The validated procedure afforded [11C]AZD4747 as an injectable solution in good radioactivity yield (1656 ± 532 MBq), excellent radiochemical purity (100%), and a molar activity of 77 ± 13 GBq/µmol at the end of the synthesis, which took 46 ± 1 min from the end of the bombardment. Quality control on the final product was performed satisfactorily and met all acceptance criteria.

A novel antioxidant ergothioneine PET radioligand for in vivo imaging applications.

Ergothioneine (ERGO) is a rare amino acid mostly found in fungi, including mushrooms, with recognized antioxidant activity to protect tissues from damage by reactive oxygen species (ROS) components. Prior to this publication, the biodistribution of ERGO has been performed solely in vitro using extracted tissues. The aim of this study was to develop a feasible chemistry for the synthesis of an ERGO PET radioligand, [11C]ERGO, to facilitate in vivo study. The radioligand probe was synthesized with identical structure to ERGO by employing an orthogonal protection/deprotection approach. [11C]methylation of the precursor was performed via [11C]CH3OTf to provide [11C]ERGO radioligand. The [11C]ERGO was isolated by RP-HPLC with a molar activity of 690 TBq/mmol. To demonstrate the biodistribution of the radioligand, we administered approximately 37 MBq/0.1 mL in 5XFAD mice, a mouse model of Alzheimer's disease via the tail vein. The distribution of ERGO in the brain was monitored using 90-min dynamic PET scans. The delivery and specific retention of [11C]ERGO in an LPS-mediated neuroinflammation mouse model was also demonstrated. For the pharmacokinetic study, the concentration of the compound in the serum started to decrease 10 min after injection while starting to distribute in other peripheral tissues. In particular, a significant amount of the compound was found in the eyes and small intestine. The radioligand was also distributed in several regions of the brain of 5XFAD mice, and the signal remained strong 30 min post-injection. This is the first time the biodistribution of this antioxidant and rare amino acid has been demonstrated in a preclinical mouse model in a highly sensitive and non-invasive manner.

Abnormal neuroinflammation in fibromyalgia and CRPS using [11C]-(R)-PK11195 PET.

PURPOSE: Fibromyalgia (FM) and complex regional pain syndrome (CRPS) share many pathological mechanisms related to chronic pain and neuroinflammation, which may contribute to the multifactorial pathological mechanisms in both FM and CRPS. The aim of this study was to assess neuroinflammation in FM patients compared with that in patients with CRPS and healthy controls. METHODS: Neuroinflammation was measured as the distribution volume ratio (DVR) of [11C]-(R)-PK11195 positron emission tomography (PET) in 12 FM patients, 11 patients with CRPS and 15 healthy controls. RESULTS: Neuroinflammation in FM patients was significantly higher in the left pre (primary motor cortex) and post (primary somatosensory cortex) central gyri (p < 0.001), right postcentral gyrus (p < 0.005), left superior parietal and superior frontal gyri (p < 0.005), left precuneus (p < 0.01), and left medial frontal gyrus (p = 0.036) compared with healthy controls. Furthermore, the DVR of [11C]-(R)-PK11195 in FM patients demonstrated decreased neuroinflammation in the medulla (p < 0.005), left superior temporal gyrus (p < 0.005), and left amygdala (p = 0.020) compared with healthy controls. CONCLUSIONS: To the authors' knowledge, this report is the first to describe abnormal neuroinflammation levels in the brains of FM patients compared with that in patients with CRPS using [11C]-(R)-PK11195 PET. The results suggested that abnormal neuroinflammation can be an important pathological factor in FM. In addition, the identification of common and different critical regions related to abnormal neuroinflammation in FM, compared with patients with CRPS and healthy controls, may contribute to improved diagnosis and the development of effective medical treatment for patients with FM.

Biodistribution and radiation dosimetry of the positron emission tomography probe for AMPA receptor, [11C]K-2, in healthy human subjects.

[11C]K-2, a radiotracer exhibiting high affinity and selectivity for α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs), is suitable for the quantification of AMPARs in living human brains and potentially useful in the identification of epileptogenic foci in patients. This study aimed to estimate the radiation doses of [11C]K-2 in various organs and calculate the effective dose after injection of [11C]K-2 in healthy human subjects. Twelve healthy male subjects were registered and divided into two groups (370 or 555 MBq of [11C]K-2), followed by 2 h whole-body scans. We estimated the radiation dose of each organ and then calculated the effective dose for each subject. The highest uptake of [11C]K-2 was observed in the liver, while the brain also showed relatively high uptake. The urinary bladder exhibited the highest radiation dose. The kidneys and liver also showed high radiation doses after [11C]K-2 injections. The effective dose of [11C]K-2 ranged from 5.0 to 5.2 µSv/MBq. Our findings suggest that [11C]K-2 is safe in terms of the radiation dose and adverse effects. The injection of 370-555 MBq (10 to 15 mCi) for PET studies using this radiotracer is applicable in healthy human subjects and enables serial PET scans in a single subject.

A novel carbon-11 radiolabeled maternal embryonic leucine zipper kinase inhibitor for PET imaging of triple-negative breast cancer.

Maternal embryonic leucine zipper kinase (MELK) plays an important role in the regulation of tumor cell growth. It is abundant in triple-negative breast cancers (TNBC), making it a promising target for molecular imaging and therapy. Based on the structure of a potent MELK inhibitor (OTSSP167) with high affinity, we developed a novel carbon-11 radiolabeled molecular probe 11C-methoxy-OTSSP167, and evaluated its application in positron emission tomography (PET) imaging of TNBC. 11C-methoxy-OTSSP167 was successfully synthesized and was identical to its non-radiolabeled compound methoxy-OTSSP167 in high-pressure liquid chromatography (HPLC) chromatogram. The obtained tracer had 10 ± 2% radiolabeling yield with a total synthesis time of 40 min. The radiochemical purity of the tracer was more than 95%. The maximum uptake (9.97 ± 0.70%) of 11C-methoxy-OTSSP167 in MELK-overexpressing MDA-MB-231 cells was at 60 min in vitro. On PET, MDA-MB-231 tumors were clearly visible at 30, 60, and 90 min after injection of 11C-methoxy-OTSSP167, while no obvious radioactivity accumulation was found in the low-MELK MCF-7 tumors. In vivo biodistribution data were consistent with the findings of the PET images. However, the radioactive tracer showed high uptake in normal organs such as liver and intestine, which may limit the application of the tracer. In addition, a markedly different MELK expression level in MDA-MBA-231 and MCF-7 tumors was verified via IHC staining. In conclusion, 11C-methoxy-OTSSP167 was successfully developed and exhibited elevated uptake in MELK overexpressed tumor, indicating its potential for noninvasively imaging of MELK overexpressed TNBC.

Colchicine treatment early after infarction attenuates myocardial inflammatory response demonstrated by 14C-methionine imaging and subsequent ventricular remodeling by quantitative gated SPECT.

OBJECTIVE: Colchicine has been used as an anti-inflammatory agent and may be cardioprotective after acute myocardial infarction (AMI). We investigated how colchicine administration after AMI affects the myocardial inflammatory response using 14C-methionine and subsequent ventricular remodeling using single-photon emission computed tomography (SPECT) in a rat model of AMI. METHODS: The left coronary artery (LCA) was occluded for 30 min followed by reperfusion. 14C-methionine was injected at 20 min before sacrifice. The LCA was re-occluded at 1 min before sacrifice and 99mTc-methoxyisobutylisonitrile (99mTc-MIBI) was injected. Colchicine was administered intraperitoneally from day 1 to the day before 14C-methionine injection. Dual-tracer autoradiography of the left ventricular short-axis slices was performed. The methionine uptake ratio in an ischemic area was calculated. 99mTc-MIBI gated SPECT assessed end-diastolic volume (EDV), end-systolic volume (ESV) and left ventricular ejection fraction (LVEF). On Cluster of Differentiation 68 with 4',6-diamidino-2-phenylindole (CD68/DAPI) staining the positive myocardial cell percentage in an ischemic area was calculated. RESULTS: In control rats, 14C-methionine uptake ratios on day 3 and 7 were 1.87 ± 0.15 and 1.39 ± 0.12, respectively. With colchicine, the uptake was reduced on days 3 (1.56 ± 0.26, p = 0.042) and 7 (1.23 ± 0.10, p = 0.030). Colchicine treated rats showed smaller EDV, ESV, and higher LVEF compared with control rats. At 8 weeks, those in control rats were 864 ± 115 µL, 620 ± 100 µL, 28.4 ± 2.5%, and in colchicine rats 665 ± 75 µL, 390 ± 97 µL, 42.2 ± 8.5% (p = 0.012, 0.0061, 0.0083), respectively. In control rats, CD68/DAPI positive myocardial cell percentages on days 3 and 7 were 38.4 ± 1.9% and 24.0 ± 2.4%, respectively. With colchicine, the percentages were reduced significantly on both days 3 (31.5 ± 2.0%, p < 0.0001) and 7 (12.0 ± 1.6%, p < 0.0001) as compared with the control. CONCLUSIONS: Short-term colchicine treatment after AMI attenuated the post-AMI inflammatory response and subsequent ventricular remodeling and dysfunction. 14C-methionine imaging and gated 99mTc-MIBI SPECT would be feasible to monitor the effectiveness of anti-inflammatory therapy and left ventricular function.

Clinical value of PET/CT with carbon-11 4DST in the evaluation of malignant and benign lung tumors.

OBJECTIVES: The aim of this study was to assess the clinical value of [11C]4DST uptake in patients with lung nodules, including benign and malignant tumors, and to assess the correlation between [11C]4DST uptake and proliferative activity of tumors in comparison with [18F]FDG uptake. METHODS: Twenty-six patients (22 males and 4 females, mean age of 65.5-year-old) were analyzed in this prospective study. Patients underwent [11C]4DST and [18F]FDG PET/CT imaging on the same day. Diagnosis of each lung nodule was confirmed by histopathological examination of tissue specimens at surgery, or during clinical follow-up after the PET/CT studies. To assess the utility of the semi-quantitative evaluation method, the SUVmax was calculated of [11C]4DST and [18F]FDG uptake by the lesion. Proliferative activities of each tumor as indicated by the immunohistochemical Ki-67 index was also estimated using surgical specimens of patients. Then the relationship between the SUVmax of both PET/CT and the Ki-67 index was examined. Furthermore, the relationship between the uptake of [11C]4DST or [18F]FDG and the histopathological findings, the clinical stage, and the clinical outcome of patients were also assessed. RESULTS: There was a positive linear relationship between the SUVmax of [11C]4DST images and the Ki-67 index (Correlation coefficients = 0.68). The SUVmax of [11C]4DST in the 26 lung nodules were 1.65 ± 0.40 for benign lesions, 3.09 ± 0.83 for adenocarcinomas (P < 0.001 between benign and adenocarcinoma), and 2.92 ± 0.58 for SqCCs (P < 0.001 between benign and SqCC). Whereas, the SUVmax of [18F]FDG were 2.38 ± 2.27 for benign lesions, 6.63 ± 4.24 for adenocarcinomas (n.s.), and 7.52 ± 2.84 for SqCCs (n.s.). The relationship between TNM tumor stage and the SUVmax of [11C]4DST were 2.54 ± 0.37 for T1, 3.48 ± 0.57 for T2, and 4.17 ± 0.72 for T3 (P < 0.005 between T1 and T2, and P < 0.001 between T1 and T3). In comparison with the TNM pathological stage, SUVmax of [11C]4DST were 2.63 ± 0.49 for stage I, 3.36 ± 0.23 for stage II, 3.40 ± 1.12 for stage III, and 4.65 for stage IV (P < 0.05 between stages I and II). In comparison of the clinical outcome, the SUVmax of [11C]4DST were 2.72 ± 0.56 for the no recurrence (No Rec.) group, 3.10 ± 0.33 for the recurrence-free with adjuvant chemotherapy after the surgery (the No Rec. Adjv. CTx. group) and 4.66 ± 0.02 for the recurrence group (Rec. group) (P < 0.001 between the No Rec and Rec. groups, and P < 0.005 between the No Rec. Adjv. CTx. and Rec. groups). CONCLUSIONS: PET/CT with [11C]4DST is as feasible for imaging of lung tumors as [18F]FDG PET/CT. For diagnosing lung tumors, [11C]4DST PET is useful in distinguishing benign nodules from malignancies. [11C]4DST uptake in lung carcinomas is correlated with the proliferative activity of tumors, indicating a promising noninvasive PET imaging of DNA synthesis in malignant lung tumors.

Carbon-11: Radiochemistry and Target-Based PET Molecular Imaging Applications in Oncology, Cardiology, and Neurology.

The positron emission tomography (PET) molecular imaging technique has gained its universal value as a remarkable tool for medical diagnosis and biomedical research. Carbon-11 is one of the promising radiotracers that can report target-specific information related to its pharmacology and physiology to understand the disease status. Currently, many of the available carbon-11 (t1/2 = 20.4 min) PET radiotracers are heterocyclic derivatives that have been synthesized using carbon-11 inserted different functional groups obtained from primary and secondary carbon-11 precursors. A spectrum of carbon-11 PET radiotracers has been developed against many of the upregulated and emerging targets for the diagnosis, prognosis, prediction, and therapy in the fields of oncology, cardiology, and neurology. This review focuses on the carbon-11 radiochemistry and various target-specific PET molecular imaging agents used in tumor, heart, brain, and neuroinflammatory disease imaging along with its associated pathology.

Metallaphotoredox aryl and alkyl radiomethylation for PET ligand discovery.

Positron emission tomography (PET) radioligands (radioactively labelled tracer compounds) are extremely useful for in vivo characterization of central nervous system drug candidates, neurodegenerative diseases and numerous oncology targets1. Both tritium and carbon-11 radioisotopologues are generally necessary for in vitro and in vivo characterization of radioligands2, yet there exist few radiolabelling protocols for the synthesis of either, inhibiting the development of PET radioligands. The synthesis of such radioligands also needs to be very rapid owing to the short half-life of carbon-11. Here we report a versatile and rapid metallaphotoredox-catalysed method for late-stage installation of both tritium and carbon-11 into the desired compounds via methylation of pharmaceutical precursors bearing aryl and alkyl bromides. Methyl groups are among the most prevalent structural elements found in bioactive molecules, and so this synthetic approach simplifies the discovery of radioligands. To demonstrate the breadth of applicability of this technique, we perform rapid synthesis of 20 tritiated and 10 carbon-11-labelled complex pharmaceuticals and PET radioligands, including a one-step radiosynthesis of the clinically used compounds [11C]UCB-J and [11C]PHNO. We further outline the direct utility of this protocol for preclinical PET imaging and its translation to automated radiosynthesis for routine radiotracer production in human clinical imaging. We also demonstrate this protocol for the installation of other diverse and pharmaceutically useful isotopes, including carbon-14, carbon-13 and deuterium.

Preclinical evaluation of [11 C]L-235 as a radioligand for Positron Emission Tomography cathepsin K imaging in bone.

The cathepsin K (CatK) enzyme is abundantly expressed in osteoclasts, and CatK inhibitors have been developed for the treatment of osteoporosis. In our effort to support discovery and clinical evaluations of a CatK inhibitor, we sought to discover a radioligand to determine target engagement of the enzyme by therapeutic candidates using positron emission tomography (PET). L-235, a potent and selective CatK inhibitor, was labeled with carbon-11. PET imaging studies recording baseline distribution of [11 C]L-235, and chase and blocking studies using the selective CatK inhibitor MK-0674 were performed in juvenile and adult nonhuman primates (NHP) and ovariectomized rabbits. Retention of the PET tracer in regions expected to be osteoclast-rich compared with osteoclast-poor regions was examined. Increased retention of the radioligand was observed in osteoclast-rich regions of juvenile rabbits and NHP but not in the adult monkey or adult ovariectomized rabbit. Target engagement of CatK was observed in blocking studies with MK-0674, and the radioligand retention was shown to be sensitive to the level of MK-0674 exposure. [11 C]L-235 can assess target engagement of CatK in bone only in juvenile animals. [11 C]L-235 may be a useful tool for guiding the discovery of CatK inhibitors.

Radiosynthesis challenges of 11C and 18F-labeled radiotracers in the FX2C/N tracerlab and their validation through PET-MR imaging.

Glucose is the renowned source of the energy for the cancer growth, that's the reason for [18F]FDG success and make it widely used radiotracer. Though [18F]FDG has its own inherent limitations therefore many tracers have been developed to target specific receptors, and other metabolic routes. We have used FX2C and FX2N Tracerlab modules for the synthesis of the [11C]methionine, [18F]choline and [18F]fluorodopa via nucleophilic pathway in FX2C/N module. [11C]methionine was standardized in FX2C module using two different precursors, and purified using C18 cartridge based technique. [18F]methylcholine was synthesized using dimethylaminoethanol precursor and purified using cartridge-based method. [18F]fluorodopa was synthesized using nucleophilic precursor and purified using in-built preparative HPLC on FX2N module. All radioactive intermediates and chemical impurities were evaluated by analytical HPLC. The radiochemical purity of D and L-[11C]methionine were 4.6 ± 3.2% and 95.4 ± 3.6% while other chemical impurities were less than prescribed limits with yield of 20 ± 5%. [18F]fluoromethylcholine was prepared with high radiochemical purity of 97.3 ± 2.6% with yield of 8 ± 3%. [18F]fluorodopa was synthesized with high radiochemical purity of 95.8 ± 1.4% with 15 ± 3% yield. The adaptation of [18F]fluorodopa synthesis to FX2N module via designing synthesis sequence and purified through on-line HPLC has provided high radiochemical purity. PET-MR imaging was done using these tracers which have validated the synthesis and their availability for future clinical applications.

11C-choline positron emission tomography/computed tomography for detection of disease relapse in patients with history of biochemically recurrent prostate cancer and prostate-specific antigen ≤0.1 ng/ml.

OBJECTIVES: The objective was to evaluate the diagnostic performance of surveillance11 C-choline positron emission tomography/computed tomography (PET/CT) for the detection of disease relapse in patients with a history of biochemically recurrent (BCR) prostate cancer (PCa) and prostate-specific antigen (PSA) ≤0.1 ng/ml. MATERIALS AND METHODS: We included patients who had been treated for BCR PCa and had a surveillance11 C-choline PET/CT at serum PSA ≤0.1 ng/ml. Positive surveillance PET/CT was defined as a study that identified a new tracer-avid lesion or new tracer uptake in a previously treated lesion or both. Findings were confirmed against a composite radiologic-pathologic gold standard. Time to recurrence association analyses were performed for disease relapse risk with the use of Cox proportional hazards regression. RESULTS: In total, 13 (12.1%) of the 107 patients had positive surveillance PET/CT scans, confirmed on pathologic assessment (n = 5) and subsequent imaging (n = 8). Among these 13 patients, ten had distant metastases, two had local recurrence, and one had both. Nine of the ten patients with metastases had oligometastatic disease defined as the presence of ≤3 metastases. Serum PSA became detectable again in only seven patients with positive surveillance PET/CT, after a mean interval from surveillance PET/CT of 292 days (range: 105-543 days). We identified an association of N stage with increased risk of recurrence (hazard ratio = 3.85; P = 0.036) although this was not significant after accounting for multiple testing. CONCLUSIONS: Surveillance11 C-choline PET/CT can detect early disease relapse at serum PSA ≤0.1 ng/ml in patients with a history of BCR PCa.

Radiosynthesis of a Bruton's tyrosine kinase inhibitor, [11 C]Tolebrutinib, via palladium-NiXantphos-mediated carbonylation.

Bruton's tyrosine kinase (BTK) is a key component in the B-cell receptor signaling pathway and is consequently a target for in vivo imaging of B-cell malignancies as well as in multiple sclerosis (MS) with positron emission tomography (PET). A recent Phase 2b study with Sanofi's BTK inhibitor, Tolebrutinib (also known as [a.k.a.] SAR442168, PRN2246, or BTK'168) showed significantly reduced disease activity associated with MS. Herein, we report the radiosynthesis of [11 C]Tolebrutinib ([11 C]5) as a potential PET imaging agent for BTK. The N-[11 C]acrylamide moiety of [11 C]5 was labeled by 11 C-carbonylation starting from [11 C]CO, iodoethylene, and the secondary amine precursor via a novel palladium-NiXantphos-mediated carbonylation protocol, and the synthesis was fully automated using a commercial carbon-11 synthesis platform (TracerMaker™, Scansys Laboratorieteknik). [11 C]5 was obtained in a decay-corrected radiochemical yield of 37 ± 2% (n = 5, relative to starting [11 C]CO activity) in >99% radiochemical purity, with an average molar activity of 45 GBq/µmol (1200 mCi/µmol). We envision that this methodology will be generally applicable for the syntheses of labeled N-acrylamides.

Methods to Enhance the Metabolic Stability of Peptide-Based PET Radiopharmaceuticals.

The high affinity and specificity of peptides towards biological targets, in addition to their favorable pharmacological properties, has encouraged the development of many peptide-based pharmaceuticals, including peptide-based positron emission tomography (PET) radiopharmaceuticals. However, the poor in vivo stability of unmodified peptides against proteolysis is a major challenge that must be overcome, as it can result in an impractically short in vivo biological half-life and a subsequently poor bioavailability when used in imaging and therapeutic applications. Consequently, many biologically and pharmacologically interesting peptide-based drugs may never see application. A potential way to overcome this is using peptide analogues designed to mimic the pharmacophore of a native peptide while also containing unnatural modifications that act to maintain or improve the pharmacological properties. This review explores strategies that have been developed to increase the metabolic stability of peptide-based pharmaceuticals. It includes modifications of the C- and/or N-termini, introduction of d- or other unnatural amino acids, backbone modification, PEGylation and alkyl chain incorporation, cyclization and peptide bond substitution, and where those strategies have been, or could be, applied to PET peptide-based radiopharmaceuticals.

The Acute and Early Effects of Whole-Brain Irradiation on Glial Activation, Brain Metabolism, and Behavior: a Positron Emission Tomography Study.

PURPOSE: Radiotherapy is a frequently applied treatment modality for brain tumors. Concomitant irradiation of normal brain tissue can induce various physiological responses. The aim of this study was to investigate whether acute and early-delayed effects of brain irradiation on glial activation and brain metabolism can be detected with positron emission tomography (PET) and whether these effects are correlated with behavioral changes. PROCEDURES: Rats underwent 0-, 10-, or 25-Gy whole-brain irradiation. At 3 and 31 days post irradiation, 1-(2-chlorophenyl)-N-[11C]methyl-(1-methylpropyl)-3-isoquinoline carboxamide ([11C]PK11195) and 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) PET scans were acquired to detect changes in glial activation (neuroinflammation) and glucose metabolism, respectively. The open-field test (OFT) was performed on days 6 and 27 to assess behavioral changes. RESULTS: Twenty-five-gray-irradiated rats showed higher [11C]PK11195 uptake in most brain regions than controls on day 3 (striatum, hypothalamus, accumbens, septum p < 0.05), although some brain regions had lower uptake (cerebellum, parietal association/retrosplenial visual cortex, frontal association/motor cortex, somatosensory cortex, p < 0.05). On day 31, several brain regions in 25-Gy-irradiated rats still showed significantly higher [11C]PK11195 uptake than controls and 10-Gy-irradiated group (p < 0.05). Within-group analysis showed that [11C]PK11195 uptake in individual brain regions of 25-Gy treated rats remained stable or slightly increased between days 3 and 31. In contrast, a significant reduction (p < 0.05) in tracer uptake between days 3 and 31 was found in all brain areas of controls and 10-Gy-irradiated animals. Moreover, 10-Gy treatment led to a significantly higher [18F]FDG uptake on day 3 (p < 0.05). [18F]FDG uptake decreased between days 3 and 31 in all groups; no significant differences between groups were observed anymore on day 31, except for increased uptake in the hypothalamus in the 10-Gy group. The OFT did not show any significant differences between groups. CONCLUSIONS: Non-invasive PET imaging indicated that brain irradiation induces neuroinflammation and a metabolic flare, without causing acute or early-delayed behavioral changes.

Effect of Cholesterol on the Organic Cation Transporter OCTN1 (SLC22A4).

The effect of cholesterol was investigated on the OCTN1 transport activity measured as [14C]-tetraethylamonium or [3H]-acetylcholine uptake in proteoliposomes reconstituted with native transporter extracted from HeLa cells or the human recombinant OCTN1 over-expressed in E. coli. Removal of cholesterol from the native transporter by MßCD before reconstitution led to impairment of transport activity. A similar activity impairment was observed after treatment of proteoliposomes harboring the recombinant (cholesterol-free) protein by MßCD, suggesting that the lipid mixture used for reconstitution contained some cholesterol. An enzymatic assay revealed the presence of 10 µg cholesterol/mg total lipids corresponding to 1% cholesterol in the phospholipid mixture used for the proteoliposome preparation. On the other way around, the activity of the recombinant OCTN1 was stimulated by adding the cholesterol analogue, CHS to the proteoliposome preparation. Optimal transport activity was detected in the presence of 83 µg CHS/ mg total lipids for both [14C]-tetraethylamonium or [3H]-acetylcholine uptake. Kinetic analysis of transport demonstrated that the stimulation of transport activity by CHS consisted in an increase of the Vmax of transport with no changes of the Km. Altogether, the data suggests a direct interaction of cholesterol with the protein. A further support to this interpretation was given by a docking analysis indicating the interaction of cholesterol with some protein sites corresponding to CARC-CRAC motifs. The observed direct interaction of cholesterol with OCTN1 points to a possible direct influence of cholesterol on tumor cells or on acetylcholine transport in neuronal and non-neuronal cells via OCTN1.

Synthesis of imatinib, a tyrosine kinase inhibitor, labeled with carbon-14.

Imatinib (Gleevec) is a multiple tyrosine kinase inhibitor that decreases the activity of the fusion oncogene called BCR-ABL (breakpoint cluster region protein-Abelson murine leukemia viral oncogene homolog) and is clinically used for the treatment of chronic myelogenous leukemia and acute lymphocytic leukemia. Small molecule drugs, such as imatinib, can bind to several cellular proteins including the target proteins in the cells, inducing undesirable effects along with the effects against the disease. In this study, we report the synthetic optimization for 14 C-labeling and radiosynthesis of [14 C]imatinib to analyze binding with cellular proteins using accelerator mass spectroscopy. 14 C-labeling of imatinib was performed by the synthesis of 14 C-labeld 2-aminopyrimidine intermediate using [14 C]guanidine·HCl, which includes an in situ reduction of an inseparable byproduct for easy purification by HPLC, followed by a cross-coupling reaction with aryl bromide precursor. The radiosynthesis of [14 C]imatinib (specific activity, 631 MBq/mmol; radiochemical purity, 99.6%) was achieved in six steps with a total chemical yield of 29.2%.

Radiosynthesis of the 11 C-methyl derivative of LBQ657 for PET investigation of the neprilysin inhibitor sacubitril.

Neprilysin, also known as neutral endopeptidase, is a cell surface membrane metalo-endopeptidase that cleaves various peptides. Altered neprilysin expression has been correlated with various cancers and cardiovascular diseases. In this work, we present the radiosynthesis of the novel O-11 C-methylated derivative of LBQ657 (a potent neprilysin inhibitor). (2R,4S)-5-(Biphenyl-4-yl)-4-[(3-carboxypropionyl)amino]-2-methylpentanoic acid [11 C]methyl ester ([11 C]MeOLBQ) is an analog of sacubitril where the alkyl ester is a 11 C-methyl instead of an ethyl. [11 C]MeOLBQ was produced in a one-pot two-step synthesis. The O-11 C-methylation of the pentanoic acid part was done with [11 C]methyl triflate followed by the deprotection of the tert-butyl ester precursor in acidic conditions. [11 C]MeOLBQ ([11 C]7) was produced in 9.5 ± 2.5% RCY (25 ± 6% decay-corrected from [11 C]CO2 , n = 3) high molar activity 348 ± 100 GBq/µmol (9425 ± 2720 mCi/µmol) at EOS, in high chemical (>95%) and radiochemical (>99%) purities. The total synthesis time including HPLC purification and reformulation was 29 minutes. To our knowledge, this is the first PET-labeled analog of the clinically used NEP inhibitor sacubitril.

Visualization of AMPA receptors in living human brain with positron emission tomography.

Although aberrations in the number and function of glutamate AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors are thought to underlie neuropsychiatric disorders, no methods are currently available for visualizing AMPA receptors in the living human brain. Here we developed a positron emission tomography (PET) tracer for AMPA receptors. A derivative of 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro-phenoxyacetamide radiolabeled with 11C ([11C]K-2) showed specific binding to AMPA receptors. Our clinical trial with healthy human participants confirmed reversible binding of [11C]K-2 in the brain according to Logan graphical analysis (UMIN000020975; study design: non-randomized, single arm; primary outcome: dynamics and distribution volumes of [11C]K-2 in the brain; secondary outcome: adverse events of [11C]K-2 during the 4-10 d following dosing; this trial met prespecified endpoints). In an exploratory clinical study including patients with epilepsy, we detected increased [11C]K-2 uptake in the epileptogenic focus of patients with mesial temporal lobe epilepsy, which was closely correlated with the local AMPA receptor protein distribution in surgical specimens from the same individuals (UMIN000025090; study design: non-randomized, single arm; primary outcome: correlation between [11C]K-2 uptake measured with PET before surgery and AMPA receptor protein density examined by biochemical study after surgery; secondary outcome: adverse events during the 7 d following PET scan; this trial met prespecified endpoints). Thus, [11C]K-2 is a potent PET tracer for AMPA receptors, potentially providing a tool to examine the involvement of AMPA receptors in neuropsychiatric disorders.