p75NTR,but Not proNGF,Is Upregulated Following Status Epilepticus in Mice

ProNGF and p75^NTR are upregulated and induce cell death following status epilepticus (SE) in rats. However, less is known about the proneurotrophin response to SE in mice, a more genetically tractable species where mechanisms can be more readily dissected. We evaluated the temporal- and cell-specific induction of the proneurotrophins and their receptors, including p75^NTR, sortilin, and sorCS2, following mild SE induced with kainic acid (KA) or severe SE induced by pilocarpine. We found that mature NGF, p75^NTR, and proBDNF were upregulated following SE, while proNGF was not altered, indicating potential mechanistic differences between rats and mice. ProBDNF was localized to mossy fibers and microglia following SE. p75^NTR was transiently induced primarily in axons and axon terminals following SE, as well as in neuron and astrocyte cell bodies. ProBDNF and p75^NTR increased independently of cell death and their localization was different depending on the severity of SE. We also examined the expression of proneurotrophin co-receptors, sortilin and sorCS2. Following severe SE, sorCS2, but not sortilin, was elevated in neurons and astrocytes. These data indicate that important differences exist between rat and mouse in the proneurotrophin response following SE. Moreover, the proBDNF and p75^NTR increase after seizures in the absence of significant cell death suggests that proneurotrophin signaling may play other roles following SE.     

[11C]BCTC: Radiosynthesis and in vivo binding to transient receptor potential vanilloid subfamily member 1 (TRPV1) receptor in the mouse trigeminal nerve

The purpose of this study was to synthesize a new positron emission tomography radiotracer, N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-[¹¹C]carboxamide ([¹¹C]BCTC, [¹¹C]1), and assess its in vivo binding to the transient receptor potential vanilloid subfamily member 1 (TRPV1) receptor in mice. [¹¹C]BCTC was synthesized by reacting the hydrochloride of 4-tertiarybutylaniline (2·HCl) with [¹¹C]phosgene ([¹¹C]COCl₂) to give isocyanate [¹¹C]4, followed by reaction with 4-(3-chloropyridin-2-yl)tetrahydropyrazine (3). [¹¹C]BCTC was obtained at a 16–20% radiochemical yield, based on the cyclotron-produced [¹¹C]CO₂ (decay-corrected to the end of bombardment), with >98% radiochemical purity and 65–110 GBq/μmol specific activity at the end of synthesis. An ex vivo biodistribution study in mice confirmed the specific binding of [¹¹C]BCTC to TRPV1 in the trigeminal nerve, which is a tissue with high TRPV1 expression.     

Synthesis and preliminary biological evaluation of [11C]methyl (2-amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-yl)-d-leucinate for the fractalkine receptor (CX3CR1)

The reference standard methyl (2-amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-yl)-d-leucinate (5) and its precursor 2-amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-yl)-d-leucine (6) were synthesized from 6-amino-2-mercaptopyrimidin-4-ol and BnBr with overall chemical yield 7% in five steps and 4% in six steps, respectively. The target tracer [¹¹C]methyl (2-amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-yl)-d-leucinate ([¹¹C]5) was prepared from the acid precursor with [¹¹C]CH₃OTf through O-[¹¹C]methylation and isolated by HPLC combined with SPE in 40–50% radiochemical yield, based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB). The radiochemical purity was >99%, and the specific activity (SA) at EOB was 370–1110 GBq/μmol with a total synthesis time of ～40-min from EOB. The radioligand depletion experiment of [¹¹C]5 did not display specific binding to CX₃CR1, and the competitive binding assay of ligand 5 found much lower CX₃CR1 binding affinity.     

3-(Cyclopropylmethyl)-7-((4-(4-[11C]methoxyphenyl)piperidin-1-yl)methyl)-8-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine: Synthesis and preliminary evaluation for PET imaging of metabotropic glutamate receptor subtype 2

Selective metabotropic glutamate receptor 2 (mGluR2) inhibitors have been demonstrated to show therapeutic effects by improving alleviating symptoms of schizophrenic patients in clinical studies. Herein we report the synthesis and preliminary evaluation of a ¹¹C-labeled positron emission tomography (PET) tracer originating from a mGluR2 inhibitor, 3-(cyclopropylmethyl)-7-((4-(4-methoxyphenyl)piperidin-1-yl)methyl)-8-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine (CMTP, 1a). [¹¹C]CMTP ([¹¹C]1a) was synthesized by O-[¹¹C]methylation of desmethyl precursor 1b with [¹¹C]methyl iodide in 19.7 ± 8.9% (n = 10) radiochemical yield (based on [¹¹C]CO₂) with >98% radiochemical purity and >74 GBq/μmol molar activity. Autoradiography study showed that [¹¹C]1a possessed moderate in vitro specific binding to mGluR2 in the rat brain, with a heterogeneous distribution of radioactive accumulation in the mGluR2-rich brain tissue sections, such as the cerebral cortex and striatum. PET study indicated that [¹¹C]1a was able to cross the blood–brain barrier and enter the brain, but had very low specific binding in the rat brain. Further optimization for the chemical structure of 1a is necessary to increase binding affinity to mGluR2 and then improve in vivo specific binding in brain.     

The first radiosynthesis of [11C]AZD8931 as a new potential PET agent for imaging of EGFR,HER2 and HER3 signaling

The reference standard AZD8931{2-(4-((4-((3-chloro-2-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)piperidin-1-yl)-N-methylacetamide} (11a) was synthesized from methyl 4,5-dimethoxy-2-nitrobenzoate or ethyl 4,5-dimethoxy-2-nitrobenzoate and 2-chloro-N-methylacetamide in 11 steps with 2–5% overall chemical yield. The precursor N-desmethyl-AZD8931{2-(4-((4-((3-chloro-2-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)piperidin-1-yl)acetamide} (11b) was synthesized from methyl 4,5-dimethoxy-2-nitrobenzoate or ethyl 4,5-dimethoxy-2-nitrobenzoate and 2-bromoacetamide in 11 steps with 2–4% overall chemical yield. The target tracer [¹¹C]AZD8931 {2-(4-((4-((3-chloro-2-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)piperidin-1-yl)-N-[¹¹C]methylacetamide} ([¹¹C]11a) was prepared from N-desmethyl-AZD8931 (11b) with [¹¹C]CH₃OTf under basic condition (NaH) through N-[¹¹C]methylation and isolated by HPLC combined with solid-phase extraction (SPE) in 40–50% radiochemical yield based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB) with 370–1110 GBq/μmol specific activity at EOB.     

Towards tropomyosin-related kinase B (TrkB) receptor ligands for brain imaging with PET: Radiosynthesis and evaluation of 2-(4-[18F]fluorophenyl)-7,8-dihydroxy-4H-chromen-4-one and 2-(4-([N-methyl-11C]-dimethylamino)phenyl)-7,8-dihydroxy-4H-chromen-4-one

The interaction of tropomyosin-related kinase B (TrkB) with the cognate ligand brain-derived neurotrophic factor (BDNF) mediates fundamental pathways in the development of the nervous system. TrkB signaling alterations are linked to numerous neurodegenerative diseases and conditions. Herein we report the synthesis, biological evaluation and radiosynthesis of the first TrkB radioligands based on the recently identified 7,8-dihydroxyflavone chemotype. 2-(4-[¹⁸F]fluorophenyl)-7,8-dihydroxy-4H-chromen-4-one ([¹⁸F]10b) was synthesized in high radiochemical yields via an efficient S_NAr radiofluorination involving a para-Michael acceptor substituted aryl followed by BBr₃-promoted double demethylation. Selective N-[¹¹C]methylation afforded 2-(4-([N-methyl-¹¹C]-dimethylamino)phenyl)-7,8-dihydroxy-4H-chromen-4-one ([¹¹C]10c) from the fully deprotected catechol-bearing normethyl precursor 13 with [¹¹C]MeOTf. In vitro autoradiography of [¹⁸F]10b with transverse rat brain sections revealed high specific binding in the cortex, striatum, hippocampus and thalamus in accordance with expected TrkB distribution. Blockade experiments with both 7,8-dihydroxyflavone (1a) and TrkB cognate ligand, BDNF, led to decreases of 80% and 85% of radioligand binding strongly supporting the hypothesis that 7,8-dihydroxyflavones exert their effect on TrkB phosphorylation via direct TrkB extracellular domain (ECD) binding. Positron emission tomography (PET) studies revealed that [¹⁸F]10b and [¹¹C]10c brain uptake is minimal and that they are rapidly eliminated from the plasma (effective plasma half-life 5–10 min) via hepatic secretion. Nevertheless, the high specific binding and TrkB specificity derived from in vitro experiments suggests that the 7,8-disubstituted flavone chemotype represents a promising scaffold for the development of TrkB radiotracers for PET.     

Synthesis and in vitro evaluation of small-molecule [18F] labeled gonadotropin-releasing hormone (GnRH) receptor antagonists as potential PET imaging agents for GnRH receptor expression

Two novel small molecule gonadotropin-releasing hormone (GnRH) receptor antagonists (12 and 13) of the furamide-class were synthesized and evaluated in vitro for their receptor binding affinities for the rat GnRH receptor. Radiolabeling with no carrier added fluorine-18 of the appropriate precursors was investigated in a one-step reaction. Log P (Octanol/PBS pH 7.4) and serum stability of the compounds were investigated. The antagonists showed low nM affinity for the rat GnRH receptor. ¹⁸F-radiolabled compounds were obtained in high radiochemical purity (>95%) and specific activity (>75 GBq/μmol). These findings suggest this class of compounds holds promise as potential probes for PET targeting of GnRH-receptor expression.     

Synthesis and evaluation of 4-(2-fluoro-4-[11C]methoxyphenyl)-5-((2-methylpyridin-4-yl)methoxy)picolinamide for PET imaging of the metabotropic glutamate receptor 2 in the rat brain

Metabotropic glutamate receptor 2 (mGluR2) has been suggested as a therapeutic target for treating schizophrenia-like symptoms arising from increased glutamate transmission in the human forebrain. However, no reliable positron emission tomography (PET) radiotracer allowing for in vivo visualization of mGluR2 in the human brain is currently available. In this study, we synthesized 4-(2-fluoro-4-[¹¹C]methoxyphenyl)-5-((2-methylpyridin-4-yl)methoxy)picolinamide ([¹¹C]1) and evaluated its potential as a PET tracer for imaging mGluR2 in the rodent brain. Compound 1, a negative allosteric modulator (NAM) of mGluR2, showed high in vitro binding affinity (IC₅₀: 26?nM) for mGluR2 overexpressed in human cells. [¹¹C]1 was synthesized by O-[¹¹C]methylation of the phenol precursor 2 with [¹¹C]methyl iodide. After the reaction, HPLC purification and formulation, [¹¹C]1 of 7.4?±?2.8?GBq (n?=?8) was obtained from [¹¹C]carbon dioxide of 22.5?±?4.8?GBq (n?=?8) with >99% radiochemical purity and 70?±?32?GBq/μmol (n?=?8) molar activity at the end of synthesis. In vitro autoradiography for rat brains showed that [¹¹C]1 binding was heterogeneously distributed in the cerebral cortex, striatum, hippocampus, and cerebellum. This pattern is consistent with the regional distribution pattern of mGluR2 in the rodent brain. The radioactivity was significantly reduced by self- or MNI-137 (a mGluR2 NAM) blocking. Small-animal PET studies indicated a low in vivo specific binding of [¹¹C]1 in the rat brain. The brain uptake was increased in a P-glycoprotein and breast cancer resistant protein double knockout mouse, when compared to a wild-type mouse. While [¹¹C]1 presented limited potential as an in vivo PET tracer for mGluR2, we suggested that it can be used as a lead compound for developing new radiotracers with improved in vivo brain properties.     

Increasing synaptic noradrenaline, serotonin and histamine enhances in vivo binding of phosphodiesterase-4 inhibitor (R)-[11C]rolipram in rat brain, lung and heart

Phosphodiesterase-4 (PDE4) is one of the main enzymes that specifically terminate the action of cAMP, thereby contributing to intracellular signaling following stimulation of various G protein-coupled receptors. PDE4 expression and activity are modulated by agents affecting cAMP levels. The selective PDE4 inhibitor (R)-rolipram labeled with C-11 was tested in vivo in rats to analyze changes in PDE4 levels following drug treatments that increase synaptic noradrenaline (NA), serotonin (5HT), histamine (HA) and dopamine (DA) levels. We hypothesized that increasing synaptic neurotransmitter levels and subsequent cAMP-mediated signaling would significantly enhance (R)-[(11)C]rolipram retention and specific binding to PDE4 in vivo. Pre-treatments were performed 3 h prior to tracer injection, and rats were sacrificed 45 min later. Biodistribution studies revealed a dose-dependent increase in (R)-[(11)C]rolipram uptake following administration of the monoamine oxidase (MAO) inhibitor tranylcypromine, NA and 5HT reuptake inhibitors (desipramine [DMI], maprotiline; and fluoxetine, sertraline, respectively), and the HA H(3) receptor antagonist (thioperamide), but not with DA transporter blockers GBR 12909, cocaine or DA D(1) agonist SKF81297. Significant increases in rat brain and heart reflect changes in PDE4 specific binding (total-non-specific binding [coinjection with saturating dose of (R)-rolipram]). These results demonstrate that acute treatments elevating synaptic NA, 5HT and HA, but not DA levels, significantly enhance (R)-[(11)C]rolipram binding. Use of (R)-[(11)C]rolipram and positron emission tomography as an index of PDE4 activity could provide insight into understanding disease states with altered NA, 5HT and HA concentrations.     

The synthesis and biodistribution of [11C]metformin as a PET probe to study hepatobiliary transport mediated by the multi-drug and toxin extrusion transporter 1 (MATE1) in vivo

In order to develop a new positron emission tomography (PET) probe to study hepatobiliary transport mediated by the multi-drug and toxin extrusion transporter 1 (MATE1), ¹¹C-labelled metformin was synthesized and then evaluated as a PET probe. [¹¹C]Metformin ([¹¹C]4) was synthesized in three steps, from [¹¹C]methyl iodide. Evaluation by small animal PET of [¹¹C]4 showed that there was increased concentrations of [¹¹C]4 in the livers of mice pre-treated with pyrimethamine, a potential inhibitor of MATEs, inhibiting the hepatobiliary excretion of metformin. Radiometabolite analysis showed that [¹¹C]4 was not degraded in vivo during the PET scan. Biodistribution studies were undertaken and the organ distributions were extrapolated into a standard human model. In conclusion, [¹¹C]4 may be useful as a PET probe to non-invasively study the in vivo function of hepatobiliary transport and drug–drug interactions, mediated by MATE1 in future clinical investigations.