Synthesis and preclinical evaluation of a novel fluorine-18 labeled small-molecule PET radiotracer for imaging of CXCR3 receptor in mouse models of atherosclerosis.

BACKGROUND: CXCR3 is a chemokine receptor and is expressed in innate and adaptive immune cells. It promotes the recruitment of T-lymphocytes and other immune cells to the inflammatory site in response to the binding of cognate chemokines. Upregulation of CXCR3 and its chemokines has been found during atherosclerotic lesion formation. Therefore, detection of CXCR3 by positron emission tomography (PET) radiotracer can be a useful tool for detecting the development of atherosclerosis in a noninvasive manner. Herein, we report the synthesis, radiosynthesis, and characterization of a novel fluorine-18 (F-18, 18F) labeled small-molecule radiotracer for the imaging of the CXCR3 receptor in mouse models of atherosclerosis. RESULTS: The reference standard 1 and its precursor 9 were synthesized over 5 steps from starting materials in good to moderate yields. The measured Ki values of CXCR3A and CXCR3B were 0.81 ± 0.02 nM and 0.31 ± 0.02 nM, respectively. [18F]1 was prepared by a two-step radiosynthesis with a decay-corrected radiochemical yield of 13 ± 2%, radiochemical purity > 99%, and specific activity of 44.4 ± 3.7 GBq/µmol at the end of synthesis (n = 6). The baseline studies showed that [18F]1 displayed high uptake in the atherosclerotic aorta and brown adipose tissue in Apolipoprotein E (ApoE) knockout (KO) mice fed with a high-fat diet over 12 weeks. The uptake of [18F]1 in these regions was reduced significantly in self-blocking studies, demonstrating CXCR3 binding specificity. Contrary to this, no significant differences in uptake of [18F]1 in the abdominal aorta of C57BL/6 control mice fed with a normal diet were observed in both baseline and blocking studies, indicating increased CXCR3 expression in atherosclerotic lesions. Immunohistochemistry studies demonstrated that [18F]1-positive regions were correlated with CXCR3 expression, but some atherosclerotic plaques with significant size were not detected by [18F]1, and their CXCR3 expressions were minimal. CONCLUSION: [18F]1 was synthesized with good radiochemical yield and high radiochemical purity. In PET imaging studies, [18F]1 displayed CXCR3-specific uptake in the atherosclerotic aorta in ApoE KO mice. [18F]1 visualized CXCR3 expression in different regions in mice aligned with the tissue histology studies. Taken together, [18F]1 is a potential PET radiotracer for imaging CXCR3 in atherosclerosis.

Synthesis and preclinical evaluation of a novel fluorine-18 labeled small-molecule PET radiotracer for imaging of CXCR3 receptor in mouse models of atherosclerosis.

Background: CXCR3 is a chemokine receptor and is expressed on innate and adaptive immune cells. It promotes the recruitment of T-lymphocytes and other immune cells to the inflammatory site in response to the binding of cognate chemokines. Upregulation of CXCR3 and its chemokines has been found during atherosclerotic lesion formation. Therefore, the detection of CXCR3 by positron emission tomography (PET) radiotracer may be a useful tool to detect atherosclerosis development noninvasively. Herein, we report the synthesis, radiosynthesis, and characterization of a novel fluorine-18 (F-18, 18 F) labeled small-molecule radiotracer for the imaging of the CXCR3 receptor in mouse models of atherosclerosis. Methods: The reference standard ( S )-2-(5-chloro-6-(4-(1-(4-chloro-2-fluorobenzyl)piperidin-4-yl)-3-ethylpiperazin-1-yl)pyridin-3-yl)-1,3,4-oxadiazole ( 1 ) and its corresponding precursor 9 were synthesized using organic syntheses. The radiotracer [ 18 F] 1 was prepared in one-pot, two-step synthesis via aromatic 18 F-substitution followed by reductive amination. Cell binding assays were conducted using 1 , [ 125 I]CXCL10, and CXCR3A- and CXCR3B-transfected human embryonic kidney (HEK) 293 cells. Dynamic PET imaging studies over 90 min were performed on C57BL/6 and apolipoprotein E (ApoE) knockout (KO) mice that were subjected to a normal and high-fat diet for 12 weeks, respectively. Blocking studies were conducted with preadministration of the hydrochloride salt of 1 (5 mg/kg) to assess the binding specificity. Time-activity curves (TACs) for [ 18 F] 1 in both mice were used to extract standard uptake values (SUVs). Biodistribution studies were performed on C57BL/6 mice, and the distribution of CXCR3 in the abdominal aorta of ApoE KO mice was assessed by immunohistochemistry (IHC). Results: The reference standard 1 and its precursor 9 were synthesized over 5 steps from starting materials in good to moderate yields. The measured K i values of CXCR3A and CXCR3B were 0.81 ± 0.02 nM and 0.31 ± 0.02 nM, respectively. [ 18 F] 1 was prepared with decay-corrected radiochemical yield (RCY) of 13 ± 2%, radiochemical purity (RCP) >99%, and specific activity of 44.4 ± 3.7 GBq/µmol at the end of synthesis (EOS) ( n =6). The baseline studies showed that [ 18 F] 1 displayed high uptake in the atherosclerotic aorta and brown adipose tissue (BAT) in ApoE KO mice. The uptake of [ 18 F] 1 in these regions was reduced significantly in self-blocking studies, demonstrating CXCR3 binding specificity. Contrary to this, no significant differences in uptake of [ 18 F] 1 in the abdominal aorta of C57BL/6 mice were observed in both baseline and blocking studies, indicating increased CXCR3 expression in atherosclerotic lesions. IHC studies demonstrated that [ 18 F] 1 -positive regions were correlated with CXCR3 expression, but some atherosclerotic plaques with significant size were not detected by [ 18 F] 1 , and their CXCR3 expressions were minimal. Conclusion: The novel radiotracer, [ 18 F] 1 was synthesized with good RCY and high RCP. In PET imaging studies, [ 18 F] 1 displayed CXCR3-specific uptake in the atherosclerotic aorta in ApoE KO mice. [ 18 F] 1 visualized CXCR3 expression in different regions in mice is in line with the tissue histology studies. Taken together, [ 18 F] 1 is a potential PET radiotracer for the imaging of CXCR3 in atherosclerosis.

Preclinical evaluation of new C-11 labeled benzo-1,4-dioxane PET radiotracers for brain α2C adrenergic receptors.

As one of the nine subtypes of adrenergic receptors (ARs) in the brain, α2C-ARs play essential roles in emotion and memory, and are implicated in neuropsychiatric disorders, including depression, Alzheimer's disease, substance use disorder, and schizophrenia. A recently developed α2C-AR specific positron emission tomography (PET) radiotracer, [11C]ORM-13070, showed promise for imaging α2C-ARs in the brain. Herein, we prepared highly potent C-11 labeled benzo-1,4-dioxane derivatives and compared them with [11C]ORM-13070, aiming to improve the specific binding signal, as evaluated by in vivo rodent brain PET imaging.© 2022 Elsevier Inc. All rights reserved.

PET Imaging Radiotracers of Chemokine Receptors.

Chemokines and chemokine receptors have been recognized as critical signal components that maintain the physiological functions of various cells, particularly the immune cells. The signals of chemokines/chemokine receptors guide various leukocytes to respond to inflammatory reactions and infectious agents. Many chemokine receptors play supportive roles in the differentiation, proliferation, angiogenesis, and metastasis of diverse tumor cells. In addition, the signaling functions of a few chemokine receptors are associated with cardiac, pulmonary, and brain disorders. Over the years, numerous promising molecules ranging from small molecules to short peptides and antibodies have been developed to study the role of chemokine receptors in healthy states and diseased states. These drug-like candidates are in turn exploited as radiolabeled probes for the imaging of chemokine receptors using noninvasive in vivo imaging, such as positron emission tomography (PET). Recent advances in the development of radiotracers for various chemokine receptors, particularly of CXCR4, CCR2, and CCR5, shed new light on chemokine-related cancer and cardiovascular research and the subsequent drug development. Here, we present the recent progress in PET radiotracer development for imaging of various chemokine receptors.

PET Radiotracers for CNS-Adrenergic Receptors: Developments and Perspectives.

Epinephrine (E) and norepinephrine (NE) play diverse roles in our body's physiology. In addition to their role in the peripheral nervous system (PNS), E/NE systems including their receptors are critical to the central nervous system (CNS) and to mental health. Various antipsychotics, antidepressants, and psychostimulants exert their influence partially through different subtypes of adrenergic receptors (ARs). Despite the potential of pharmacological applications and long history of research related to E/NE systems, research efforts to identify the roles of ARs in the human brain taking advantage of imaging have been limited by the lack of subtype specific ligands for ARs and brain penetrability issues. This review provides an overview of the development of positron emission tomography (PET) radiotracers for in vivo imaging of AR system in the brain.

Assessment of a 18F-Phenylboronic Acid Radiotracer for Imaging Boron in Maize.

Boron (B) is an essential plant micronutrient. Deficiencies of B have drastic consequences on plant development leading to crop yield losses and reductions in root and shoot growth. Understanding the molecular and cellular consequences of B deficiency is challenging, partly because of the limited availability of B imaging techniques. In this report we demonstrate the efficacy of using 4-fluorophenylboronic acid (FPBA) as a B imaging agent, which is a derivative of the B deficiency mimic phenylboronic acid (PBA). We show that radioactively labelled [18F]FPBA (t½=110 m) accumulates at the root tip, the root elongation zone and at lateral root initiation sites in maize roots, and also translocates to the shoot where it accumulates along the leaf edges. Treatment of maize seedlings using FPBA and PBA causes a shortened primary root phenotype with absence of lateral roots in a dose-dependent manner. The primary root defects can be partially rescued by the addition of boric acid indicating that PBA can be used to induce B deficiency in maize and that radioactively labelled FPBA can be used to image sites of B demand on a tissue level.

Synthesis and evaluation of N-(methylthiophenyl)picolinamide derivatives as PET radioligands for metabotropic glutamate receptor subtype 4.

In recent years, mGlu4 has received great research attention because of the potential benefits of mGlu4 activation in treating numerous brain disorders, such as Parkinson's disease (PD). A specific mGlu4 PET radioligand could be an important tool in understanding the role of mGlu4 in both healthy and disease conditions, and also for the development of new drugs. In this study, we synthesized four new N-(methylthiophenyl)picolinamide derivatives 11-14. Of these ligands, 11 and 14 showed high in vitro binding affinity for mGlu4 with IC50 values of 3.4nM and 3.1nM, respectively, and suitable physicochemical parameters. Compound 11 also showed enhanced metabolic stability and good selectivity to other mGluRs. [(11)C]11 and [(11)C]14 were radiolabeled using the [(11)C]methylation of the thiophenol precursors 20a and 20c with [(11)C]CH3I in 19.0% and 34.8% radiochemical yields (RCY), and their specific activities at the end of synthesis (EOS) were 496±138GBq/µmol (n=6) and 463±263GBq/µmol (n=4), respectively. The PET studies showed that [(11)C]11 accumulated fast into the brain and had higher uptake, slower washout and 25% better contrast than [(11)C]2, indicating improved imaging characteristics as PET radiotracer for mGlu4 compared to [(11)C]2. Therefore, [(11)C]11 will be a useful radioligand to investigate mGlu4 in different biological applications.

Functional modulation of G-protein coupled receptors during Parkinson disease-like neurodegeneration.

G-protein coupled dopamine and metabotropic glutamate receptors (mGlu) can modulate neurotransmission during Parkinson's disease (PD)-like neurodegeneration. PET imaging studies in a unilateral dopamine denervation model (6-OHDA) showed a significant inverse correlation of presynaptic mGlu4 and postsynaptic mGlu5 expression in the striatum and rapidly declining mGlu4 and enhanced mGlu5 expression in the hippocampus during progressive degeneration over time. Immunohistochemical studies verified the decreased mGlu4 expression in the hippocampus on the lesion side but did not show difference in mGlu5 expression between lesion and control side. Pharmacological MRI studies showed enhanced hemodynamic response in several brain areas on the lesion side compared to the control side after challenge with mGlu4 positive allosteric modulator or mGlu5 negative allosteric modulator. However, mGlu4 response was biphasic having short enhancement followed by negative response on both sides of brain. Studies in mGlu4 expressing cells demonstrated that glutamate induces cooperative increase in binding of mGlu4 ligands - especially at high glutamate levels consistent with in vivo concentration. This suggests that mGlu allosteric modulators as drug candidates will be highly sensitive to changes in glutamate concentration and hence metabolic state. These experiments demonstrate the importance of the longitudinal imaging studies to investigate temporal changes in receptor functions to obtain individual response for experimental drugs.

PET imaging studies show enhanced expression of mGluR5 and inflammatory response during progressive degeneration in ALS mouse model expressing SOD1-G93A gene.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative motor neuron disorder. Genetic studies have linked mutation of the gene SOD1 to ALS pathology as well as several other pathological processes including modulation of glutamatergic function and inflammatory processes. Since therapeutic approaches for ALS are focused on glutamatergic function, we investigated modulation of glutamate transport based on its receptor function as well as excitotoxicity-induced inflammatory response. METHODS: In vivo positron emission tomography (PET) imaging studies of metabotropic glutamate receptor subtype 5 (mGluR5) using [(18)F]FPEB ([(18)F]3-fluoro-5-(2-pyridylethynyl)benzonitrile) and inflammatory response using [(11)C]PBR28 (peripheral benzodiazepine receptor ligand 28) were done in an early and a late phase of neurodegeneration in four ALS mice expressing SOD1-G93A gene and four control base mice (C57/BL6). Accumulation of [(18)F]FPEB and [(11)C]PBR28 were quantitated in several brain areas and spinal cord to determine degeneration-induced modulation. The studies were completed with immunohistochemical analyses of mGluR5 and inflammatory response. RESULTS: These studies showed enhanced binding potential of [(18)F]FPEB in several brain areas including striatum, hippocampus, and frontal cortex. In the whole brain, the binding potential increased 49 ± 9 % from base mice to ALS-type mice and further enhanced 23 ± 4 % during disease progression. Also, in the spinal cord 6-22 %, enhanced accumulation of [(18)F]FPEB was observed during progression of the disease. The accumulation of [(11)C]PBR28 increased by 110 ± 33 % in the whole brain during progression of the disease indicating significant inflammatory process. [(11)C]PBR28 accumulation enhanced 89-264 % in the spinal cord and 204 % in the lungs. The end point immunohistochemical analyses verified the enhanced mGluR5 expression and inflammation. CONCLUSIONS: These results confirm the role of glutamate and inflammation in ALS-type pathology. These data also support the hypothesis that excessive glutamate may contribute to inflammation in the chronic neurodegenerative processes in ALS.

Loss of Metabotropic Glutamate Receptor 5 Function on Peripheral Benzodiazepine Receptor in Mice Prenatally Exposed to LPS.

Parental microglial induced neuroinflammation, triggered by bacterial- or viral infections, can induce neuropsychiatric disorders like schizophrenia and autism to offspring in animal models. Recent investigations suggest that microglia, the resident immune cells of the brain, provides a link between neurotransmission, immune cell activation, brain inflammation and neuronal dysfunction seen with the offspring. Relatively little is known about how reduction of brain inflammation and restoration of glial function are associated with diminution of brain degeneration and behavioral deficits in offspring. Increased mGluR5 expression and the long-lasting excitotoxic effects of the neurotoxin during brain development are associated with the glial dysfunctions. We investigated the relationship of mGluR5 and PBR and how they regulate glial function and inflammatory processes in mice prenatally exposed to LPS (120µg/kg, between gestational days 15 and 17), an inflammatory model of a psychiatric disorder. Using PET imaging, we showed that pharmacological activation of mGluR5 during 5 weeks reduced expression of classic inflammation marker PBR in many brain areas and that this molecular association was not present in LPS-exposed offspring. The post-mortem analysis revealed that the down regulation of PBR was mediated through activation of mGluR5 in astrocytes. In addition, we demonstrated that this interaction is defective in a mouse model of the psychiatric deficit offering a novel insight of mGluR5 involvement to brain related disorders and PBR related imaging studies. In conclusion, mGluR5 driven glutamatergic activity regulates astrocytic functions associated with PBR (cholesterol transport, neurosteroidogenesis, glial phenotype) during maturation and could be associated with neuropsychiatric disorders in offspring.

Combined behavioral studies and in vivo imaging of inflammatory response and expression of mGlu5 receptors in schnurri-2 knockout mice.

Schnurri-2 (Shn-2) knockout (KO) mice have been proposed as a preclinical neuroinflammatory schizophrenia model. We used behavioral studies and imaging markers that can be readily translated to human populations to explore brain effects of inflammation. Shn-2 KO mice and their littermate control mice were imaged with two novel PET ligands; an inflammation marker [(11)C]PBR28 and the mGluR5 ligand [(18)F]FPEB. Locomotor activity was measured using open field exploration with saline, methamphetamine or amphetamine challenge. A significantly increased accumulation of [(11)C]PBR28 was found in the cortex, striatum, hippocampus and olfactory bulb of Shn-2 KO mice. Increased mGluR5 binding was also observed in the cortex and hippocampus of the Shn-2 KO mice. Open field locomotor testing revealed a large increase in novelty-induced hyperlocomotion in Shn-2 KO mice with abnormal (decreased) responses to either methamphetamine or amphetamine. These data provide additional support to demonstrate that the Shn-2 KO mouse model exhibits several behavioral and pathological markers resembling human schizophrenia making it an attractive translational model for the disease.

Re-exploring the N-phenylpicolinamide derivatives to develop mGlu4 ligands with improved affinity and in vitro microsomal stability.

In recent years, mGlu4 has received great attention and research effort because of the potential benefits of mGlu4 activation in treating numerous brain disorders, such as Parkinson's disease (PD). Many positive allosteric modulators of mGlu4 have been developed. To better understand the role of mGlu4 in healthy and disease conditions, we are interested in developing an mGlu4 selective radioligand for in vivo studies. Thus, we had synthesized and studied [(11)C]2 as a PET tracer for mGlu4, which demonstrated some promising features as a PET radioligand as well as the limitation need to be improved. In order to develop an mGlu4 ligand with enhanced affinity and improved metabolic stability, we have modified, synthesized and evaluated a series of new N-phenylpicolinamide derivatives. The SAR study has discovered a number of compounds with low nM affinity to mGlu4. The dideuteriumfluoromethoxy modified compound 24 is identified as a very promising mGlu4 ligand, which has demonstrated enhanced affinity, improved in vitro microsomal stability, good selectivity and good permeability.

Co-operative binding assay for the characterization of mGlu4 allosteric modulators.

The interest in the role of metabotropic glutamate receptor 4 (mGlu4) in CNS related disorders has increased the need for methods to investigate the binding of allosteric drug candidates. Our aim is to present the first fully characterized in vitro binding assay of mGlu4 positive allosteric modulators (PAMs). Results suggest that mGlu4 PAMs have characteristic co-operative binding with orthosteric glutamate, which offers a notable insight to the further development of mGlu4 targeted therapies.

Hypo-anxious phenotype of adolescent offspring prenatally exposed to LPS is associated with reduced mGluR5 expression in hippocampus.

Many studies have reported long-term modulation of metabotropic glutamate receptor 5 (mGluR5) by inflammatory processes and a pharmacological modulation of mGluR5 is known to regulate anxiety level. However, it is not known if non-pharmacological modulation of mGluR5 by inflammation impaired the unconditional level of anxiety. In this study, we investigated this relation in LPS prenatal immune challenge (120µg/kg, 3x i.p. injection in late gestation), a developmental model of neuroinflammation in which some studies have reported hypo-anxious phenotype. Using positron emission tomographic imaging (PET) approaches, we have demonstrated a decrease in the binding potential of [18F]fluoro-5-(2-pyridinylethynyl)benzonitrile ([18F]FPEB, a radioligand for mGluR5) in hippocampus of adolescent offspring prenatally exposed to LPS, without significant change in the binding of [11C]peripheral benzodiazepine receptor 28 ([11C]PBR28), an inflammatory marker. In addition, dark-light box emergence test revealed a lower level of anxiety in LPS-exposed offspring and this behavioural phenotype was associated with the binding potential of [18F]FPEB in hippocampus. These results confirm that neuroinflammation during developmental phase modulates the physiology of mGluR5 and this alteration can be associated with behavioural phenotype related to anxiety. In addition, this study supports a hypotheses that mGluR5 could be used as a diagnostic target in anxiety.

Radiosynthesis and evaluation of an 18F-labeled positron emission tomography (PET) radioligand for metabotropic glutamate receptor subtype 4 (mGlu4).

Four 4-phthalimide derivatives of N-(3-chlorophenyl)-2-picolinamide were synthesized as potential ligands for the PET imaging of mGlu4 in the brain. Of these compounds, N-(3-chloro-4-(4-fluoro-1,3-dioxoisoindolin-2-yl)phenyl)-2-picolinamide (3, KALB001) exhibited improved binding affinity (IC50 = 5.1 nM) compared with ML128 (1) and was subsequently labeled with (18)F. When finally formulated in 0.1 M citrate buffer (pH 4) with 10% ethanol, the specific activity of [(18)F]3 at the end of synthesis (EOS) was 233.5 ± 177.8 GBq/µmol (n = 4). The radiochemical yield of [(18)F]3 was 16.4 ± 4.8% (n = 4), and the purity was over 98%. In vivo imaging studies in a monkey showed that the radiotracer quickly penetrated the brain with the highest accumulation in the brain areas known to express mGlu4. Despite some unfavorable radiotracer properties like fast washout in rodent studies, [(18)F]3 is the first (18)F-labeled mGlu4 radioligand, which can be further modified to improve pharmacokinetics and brain penetrability for future human studies.

Development of [(123)I]IPEB and [(123)I]IMPEB as SPECT Radioligands for Metabotropic Glutamate Receptor Subtype 5.

mGlu5 play an important role in physiology and pathology to various central nervous system (CNS) diseases. Several positron emission tomography (PET) radiotracers have been developed to explore the role of mGlu5 in brain disorders. However, there are no single photon emission computed tomography (SPECT) radioligands for mGlu5. Here we report development of [(123)I]IPEB ([(123)I]1) and [(123)I]IMPEB ([(123)I]2) as mGlu5 radioligands for SPECT. [(123)I]1 and [(123)I]2 were produced by copper(I) mediated aromatic halide displacement reactions. The SPECT imaging using mouse models demonstrated that [(123)I]1 readily entered the brain and accumulated specifically in mGlu5-rich regions of the brain such as striatum and hippocampus. However, in comparison to the corresponding PET tracer [(18)F]FPEB, [(123)I]1 showed faster washout from the brain. The binding ratios of the striatum and the hippocampus compared to the cerebellum for [(123)I]1 and [(18)F]FPEB were similar despite unfavorable pharmacokinetics of [(123)I]1. Further structural optimization of 1 may lead to more viable SPECT radiotracers for the imaging of mGlu5.

Radiosynthesis of N-(4-chloro-3-[(11)C]methoxyphenyl)-2-picolinamide ([(11)C]ML128) as a PET radiotracer for metabotropic glutamate receptor subtype 4 (mGlu4).

N-(Chloro-3-methoxyphenyl)-2-picolinamide (3, ML128, VU0361737) is an mGlu4 positive allosteric modulator (PAM), which is potent and centrally penetrating. 3 is also the first mGlu4 PAM to show efficacy in a preclinical Parkinson disease model upon systemic dosing. As a noninvasive medical imaging technique and a powerful tool in neurological research, positron emission tomography (PET) offers a possibility to investigate mGlu4 expression in vivo under physiologic and pathological conditions. We synthesized a carbon-11 labeled ML128 ([(11)C]3) as a PET radiotracer for mGlu4, and characterized its biological properties in Sprague Dawley rats. [(11)C]3 was synthesized from N-(4-chloro-3-hydroxyphenyl)-2-picolinamide (2) using [(11)C]CH3I. Total synthesis time was 38±2.2min (n=7) from the end of bombardment to the formulation. The radioligand [(11)C]3 was obtained in 27.7±5.3% (n=5) decay corrected radiochemical yield based on the radioactivity of [(11)C]CO2. The radiochemical purity of [(11)C]3 was >99%. Specific activity was 188.7±88.8GBq/mol (n=4) at the end of synthesis (EOS). PET images were conducted in 20 normal male Sprague Dawley rats including 11 control studies, 6 studies blocking with an mGlu4 modulator (4) to investigate specificity and 3 studies blocking with an mGlu5 modulator (MTEP) to investigate selectivity. These studies showed fast accumulation of [(11)C]3 (peak activity between 1-3min) in several brain areas including striatum, thalamus, hippocampus, cerebellum, and olfactory bulb following with fast washout. Blocking studies with the mGlu4 modulator 4 showed 22-28% decrease of [(11)C]3 accumulation while studies of selectivity showed only minor decrease supporting good selectivity over mGlu5. Biodistribution studies and blood analyses support fast metabolism. Altogether this is the first PET imaging ligand for mGlu4, in which the labeled ML128 was used for imaging its in vivo distribution and pharmacokinetics in brain.

Synthesis, radiolabeling and initial in vivo evaluation of [(11)C]KSM-01 for imaging PPAR-α receptors.

Peroxisome proliferator-activated receptor alpha (PPAR-α) is a ligand-activated nuclear receptor transcription factor that regulates the fatty acid ß-oxidation. An in vitro assay identified the p-methoxy phenyl ureido thiobutyric acid derivative KSM-01 (IC(50)=0.28±0.09nM) having a higher affinity to activate PPAR-α than the PPAR-α agonist GW7647 (IC(50)=0.46±0.19nM). In this study, we report the synthesis and initial in vivo evaluation of [(11)C]KSM-01. The radiosynthesis was carried out by first alkylating the corresponding p-phenol precursor with [(11)C]MeI in DMF using NaOH, followed by deprotection of the t-butyl ester group by TFA, yielding [(11)C]KSM-01. SUV analysis of dynamic micro PET/CT imaging data showed that [(11)C]KSM-01 accumulation was ∼2.0-fold greater in cardiac-specific PPAR-α overexpressing transgenic mice compared to wild-type littermates. The post-PET biodistribution studies were consistent with these results and demonstrated 2.5-fold greater radiotracer uptake in the heart of transgenic mice compared to the wild-type littermates. These results demonstrate the potential utility of PPAR-α agonists as PET radiopharmaceuticals.

Synthesis and PET studies of [(11)C-cyano]letrozole (Femara), an aromatase inhibitor drug.

INTRODUCTION: Aromatase, a member of the cytochrome P450 family, converts androgens such as androstenedione and testosterone into estrone and estradiol, respectively. Letrozole (1-[bis-(4-cyanophenyl)methyl]-1H-1,2,4-triazole; Femara) is a high-affinity aromatase inhibitor (K(i)=11.5 nM) that has Food and Drug Administration approval for breast cancer treatment. Here we report the synthesis of carbon-11-labeled letrozole and its assessment as a radiotracer for brain aromatase in the baboon. METHODS: Letrozole and its precursor (4-[(4-bromophenyl)-1H-1,2,4-triazol-1-ylmethyl]benzonitrile) were prepared in a two-step synthesis from 4-cyanobenzyl bromide and 4-bromobenzyl bromide, respectively. The [(11)C]cyano group was introduced via tetrakis(triphenylphosphine)palladium(0)-catalyzed coupling of [(11)C]cyanide with the bromo precursor. Positron emission tomography (PET) studies in the baboon brain were carried out to assess regional distribution and kinetics, reproducibility of repeated measures and saturability. Log D, the free fraction of letrozole in plasma and the [(11)C-cyano]letrozole fraction in arterial plasma were also measured. RESULTS: [(11)C-cyano]Letrozole was synthesized in 60 min with a radiochemical yield of 79-80%, with a radiochemical purity greater than 98% and a specific activity of 4.16+/-2.21 Ci/mumol at the end of bombardment (n=4). PET studies in the baboon revealed initial rapid and high uptake and initial rapid clearance, followed by slow clearance of carbon-11 from the brain, with no difference between brain regions. Brain kinetics was not affected by coinjection of unlabeled letrozole (0.1 mg/kg). The free fraction of letrozole in plasma was 48.9%, and log D was 1.84. CONCLUSION: [(11)C-cyano]Letrozole is readily synthesized via a palladium-catalyzed coupling reaction with [(11)C]cyanide. Although it is unsuitable as a PET radiotracer for brain aromatase, as revealed by the absence of regional specificity and saturability in brain regions such as amygdala, which are known to contain aromatase, it may be useful in measuring letrozole distribution and pharmacokinetics in the brain and peripheral organs.

Synthesis and positron emission tomography studies of carbon-11-labeled imatinib (Gleevec).

INTRODUCTION: Imatinib mesylate (Gleevec) is a well known drug for treating chronic myeloid leukemia and gastrointestinal stromal tumors. Its active ingredient, imatinib ([4-[(4-methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridyl)-2-pyrimidinyl]amino]phenyl]benzamide), blocks the activity of several tyrosine kinases. Here we labeled imatinib with carbon-11 as a tool for determining the drug distribution and pharmacokinetics of imatinib, and we carried out positron emission tomography (PET) studies in baboons. METHODS: [N-(11)C-methyl]imatinib was synthesized from [(11)C]methyl iodide and norimatinib was synthesized by the demethylation of imatinib (isolated from Gleevec tablets) according to a patent procedure [Collins JM, Klecker RW Jr, Anderson LW. Imaging of drug accumulation as a guide to antitumor therapy. US Patent 20030198594A1, 2003]. Norimatinib was also synthesized from the corresponding amine and acid. PET studies were carried out in three baboons to measure pharmacokinetics in the brain and peripheral organs and to determine the effect of a therapeutic dose of imatinib. Log D and plasma protein binding were also measured. RESULTS: [N-(11)C-methyl]imatinib uptake in the brain is negligible (consistent with P-glycoprotein-mediated efflux); it peaks and clears rapidly from the heart, lungs and spleen. Peak uptake and clearance occur more slowly in the liver and kidneys, followed by accumulation in the gallbladder and urinary bladder. Pretreatment with imatinib did not change uptake in the heart, lungs, kidneys and spleen, and increased uptake in the liver and gallbladder. CONCLUSIONS: [N-(11)C-methyl]imatinib has potential for assessing the regional distribution and kinetics of imatinib in the human body to determine whether the drug targets tumors and to identify other organs to which the drug or its labeled metabolites distribute. Paired with tracers such as 2'deoxy-2'-[(18)F]fluoro-D-glucose ((18)FDG) and 3'deoxy-3'-[(18)F]fluorothymidine ((18)FLT), [N-(11)C-methyl]imatinib may be a useful radiotracer for planning chemotherapy, for monitoring response to treatment and for assessing the role of drug pharmacokinetics in drug resistance.