Synthesis, bioconjugation and stability studies of [18 F]ethenesulfonyl fluoride.

Fluorine-18 labelled prosthetic groups (PGs) are often necessary for radiolabelling sensitive biological molecules such as peptides and proteins. Several shortcomings, however, often diminish the final yield of radiotracer. In an attempt to provide higher yielding and operationally efficient tools for radiolabelling biological molecules, we describe herein the first radiochemical synthesis of [18 F]ethenesulfonyl fluoride ([18 F]ESF) and its Michael conjugation with amino acids and proteins. The synthesis of [18 F]ESF was optimised using a microfluidic reactor under both carrier-added (c.a.) and no-carrier-added (n.c.a.) conditions, affording, in a straightforward procedure, 30-50% radiochemical yield (RCY) for c.a. [18 F]ESF and 60-70% RCY for n.c.a. [18 F]ESF. The conjugation reactions were performed at room temperature using 10 mg/mL precursor in aqueous/organic solvent mixtures for 15 min. The radiochemical stability of the final conjugates was evaluated in injectable formulation and rat serum, and resulted strongly substrate dependent and generally poor in rat serum. Therefore, in this work we have optimised a straightforward synthesis of [18 F]ESF and its Michael conjugation with model compounds, without requiring chromatographic purification. However, given the general low stability of the final products, further studies will be required for improving conjugate stability, before assessing the use of this PG for PET imaging.

Neutron Capture Enhances Dose and Reduces Cancer Cell Viability in and out of Beam During Helium and Carbon Ion Therapy.

PURPOSE: Neutron capture enhanced particle therapy (NCEPT) is a proposed augmentation of charged particle therapy that exploits thermal neutrons generated internally, within the treatment volume via nuclear fragmentation, to deliver a biochemically targeted radiation dose to cancer cells. This work is the first experimental demonstration of NCEPT, performed using both carbon and helium ion beams with 2 different targeted neutron capture agents (NCAs). METHODS AND MATERIALS: Human glioblastoma cells (T98G) were irradiated by carbon and helium ion beams in the presence of NCAs [10B]-BPA and [157Gd]-DOTA-TPP. Cells were positioned within a polymethyl methacrylate phantom either laterally adjacent to or within a 100 × 100 × 60 mm spread out Bragg peak (SOBP). The effect of NCAs and location relative to the SOBP on the cells was measured by cell growth and survival assays in 6 independent experiments. Neutron fluence within the phantom was characterized by quantifying the neutron activation of gold foil. RESULTS: Cells placed inside the treatment volume reached 10% survival by 2 Gy of carbon or 2 to 3 Gy of helium in the presence of NCAs compared with 5 Gy of carbon and 7 Gy of helium with no NCA. Cells placed adjacent to the treatment volume showed a dose-dependent decrease in cell growth when treated with NCAs, reaching 10% survival by 6 Gy of carbon or helium (to the treatment volume), compared with no detectable effect on cells without NCA. The mean thermal neutron fluence at the center of the SOBP was approximately 2.2 × 109 n/cm2/Gy (relative biological effectiveness) for the carbon beam and 5.8 × 109 n/cm2/Gy (relative biological effectiveness) for the helium beam and gradually decreased in all directions. CONCLUSIONS: The addition of NCAs to cancer cells during carbon and helium beam irradiation has a measurable effect on cell survival and growth in vitro. Through the capture of internally generated neutrons, NCEPT introduces the concept of a biochemically targeted radiation dose to charged particle therapy. NCEPT enables the established pharmaceuticals and concepts of neutron capture therapy to be applied to a wider range of deeply situated and diffuse tumors, by targeting this dose to microinfiltrates and cells outside of defined treatment regions. These results also demonstrate the potential for NCEPT to provide an increased dose to tumor tissue within the treatment volume, with a reduction in radiation doses to off-target tissue.

Ascertaining the suitability of aryl sulfonyl fluorides for [18F]radiochemistry applications: a systematic investigation using microfluidics.

Optimization of [(18)F]radiolabeling conditions and subsequent stability analysis in mobile phase, PBS buffer, and rat serum of 12 aryl sulfonyl chloride precursors with various substituents (electron-withdrawing groups, electron-donating groups, increased steric bulk, heterocyclic) were performed using an Advion NanoTek Microfluidic Synthesis System. A comparison of radiochemical yields and reaction times for a microfluidics device versus a conventional reaction vessel is reported. [(18)F]Radiolabeling of sulfonyl chlorides in the presence of competing nucleophiles, H-bond donors, and water was also assessed and demonstrated the versatility and potential utility of [(18)F]sulfonyl fluorides as synthons for indirect radiolabeling.

Synthesis and pharmacological evaluation of [18F]PBR316: a novel PET ligand targeting the translocator protein 18 kDa (TSPO) with low binding sensitivity to human single nucleotide polymorphism rs6971.

Radiopharmaceuticals that target the translocator protein 18 kDa (TSPO) have been investigated with positron emission tomography (PET) to study neuroinflammation, neurodegeneration and cancer. We have developed the novel, achiral, 2-phenylimidazo[1,2-a]pyridine, PBR316 that targets the translocator protein 18 kDa (TSPO) that addresses some of the limitations inherent in current TSPO ligands; namely specificity in binding, blood brain barrier permeability, metabolism and insensitivity to TSPO binding in subjects as a result of rs6971 polymorphism. PBR316 has high nanomolar affinity (4.7-6.0 nM) for the TSPO, >5000 nM for the central benzodiazepine receptor (CBR) and low sensitivity to rs6971 polymorphism with a low affinity binders (LABs) to high affinity binders (HABs) ratio of 1.5. [18F]PBR316 was prepared in 20 ± 5% radiochemical yield, >99% radiochemical purity and a molar activity of 160-400 GBq µmol-1. Biodistribution in rats showed high uptake of [18F]PBR316 in organs known to express TSPO such as heart (3.9%) and adrenal glands (7.5% ID per g) at 1 h. [18F]PBR316 entered the brain and accumulated in TSPO-expressing regions with an olfactory bulb to brain ratio of 3 at 15 min and 7 at 4 h. Radioactivity was blocked by PK11195 and Ro 5-4864 but not Flumazenil. Metabolite analysis showed that radioactivity in adrenal glands and the brain was predominantly due to the intact radiotracer. PET-CT studies in mouse-bearing prostate tumour xenografts indicated biodistribution similar to rats with radioactivity in the tumour increasing with time. [18F]PBR316 shows in vitro binding that is insensitive to human polymorphism and has specific and selective in vivo binding to the TSPO. [18F]PBR316 is suitable for further biological and clinical studies.

A rapid MS/MS method to assess the deuterium kinetic isotope effect and associated improvement in the metabolic stability of deuterated biological and pharmacological molecules as applied to an imaging agent.

The deuterium kinetic isotope effect has been known for a period of 40 years, but it is only relatively recently that new drug entities (NDEs) incorporating deuterium demonstrating beneficial pharmacokinetics, pharmacodynamics, and toxicology have arrived to market. Determination of the precise location to deuterate and subsequently any evaluation for a kinetic isotope effect (KIE) is challenging. Typically, such an evaluation would be performed in an in vitro metabolic assay (e.g. liver microsomes) in separate reaction media for both the deuterated and non-deuterated analogues. Here, we have devised an approach whereby we incubate a 1:1 ratio of both the deuterated and protio-form of an imaging agent together in the same liver microsomal assay and determine the relative rate of consumption of both moieties, based upon specific MS-MS transitions unique to both molecules without the need for liquid chromatography-mass spectrometry (LC-MS) separation and quantification. Any deviation of the ratio of the MS transitions from the initial starting point indicated an observable KIE. A site specific deuteration of PBR111, a neuroinflammation imaging agent, was chosen for a proof-of-concept study. Based upon prior mechanistic knowledge of PBR111, two locations were selected for deuteration; an active and inactive site, to corroborate that there was no significant KIE for the inactive site and confirm the efficacy of the developed methodology.

Comparative analysis of novel decynium-22 analogs to inhibit transport by the low-affinity, high-capacity monoamine transporters, organic cation transporters 2 and 3, and plasma membrane monoamine transporter.

Growing evidence supports involvement of low-affinity/high-capacity organic cation transporters (OCTs) and plasma membrane monoamine transporter (PMAT) in regulating clearance of monoamines. Currently decynium-22 (D22) is the best pharmacological tool to study these transporters, however it does not readily discriminate among them, underscoring a need to develop compounds with greater selectivity for each of these transporters. We developed seven D22 analogs, and previously reported that some have lower affinity for α1-adrenoceptors than D22 and showed antidepressant-like activity in mice. Here, we extend these findings to determine the affinity of these analogs for OCT2, OCT3 and PMAT, as well as serotonin, norepinephrine and dopamine transporters (SERT, NET and DAT) using a combination of uptake competition with [3H]methyl-4-phenylpyridinium acetate in overexpressed HEK cells and [3H]citalopram, [3H]nisoxetine and [3H]WIN 35428 displacement binding in mouse hippocampal and striatal preparations. Like D22, all analogs showed greater binding affinities for OCT3 than OCT2 and PMAT. However, unlike D22, some analogs also showed modest affinity for SERT and DAT. Dual OCT3/SERT and/or OCT3/DAT actions of certain analogs may help explain their ability to produce antidepressant-like effects in mice and help account for our previous findings that D22 lacks antidepressant-like effects unless SERT function is either genetically or pharmacologically compromised. Though these analogs are not superior than D22 in discriminating among OCTs/PMAT, our findings point to development of compounds with combined ability to inhibit both low-affinity/high-capacity transporters, such as OCT3, and high-affinity/low-capacity transporters, such as SERT, as therapeutics with potentially improved efficacy for treatment of psychiatric disorders.

Evaluation of the antidepressant therapeutic potential of isocyanine and pseudoisocyanine analogues of the organic cation decynium-22.

Herein we describe the synthesis and evaluation of antidepressant properties of seven analogues (1-7) of the low affinity/high capacity transporter blocker decynium-22 (D-22). All analogues (1-7) were synthesized via base promoted coupling reactions between N-alkylated-2-methylquinolinium iodides or N-alkylated-4-methylquinolinium iodides and electrophilic N-alkylated-2-iodoquinolinium iodides. All final compounds were purified by re-crystallization or preparative HPLC and initial evaluation studies included; 1) screening for in vitro α1-adrenoceptor activity (a property that can lead to unwanted side-effects), 2) measuring antidepressant-like activity in a mouse tail suspension test (TST), and 3) measuring effects upon mouse locomotion. The results showed some analogues have lower affinities at α1-adrenoceptors compared to D-22 and showed antidepressant-like activity without the need for co-administration of SSRIs. Additionally, many analogues did not affect mouse locomotion to the same extent as D-22. Plans for additional evaluations of these promising analogues, including measurement of antidepressant-like activity with co-administration of selective serotonin re-uptake inhibitors (SSRIs), are outlined.

Synthesis and in Vivo Evaluation of [123I]Melanin-Targeted Agents.

This study reports the synthesis, [(123)I]radiolabeling, and biological profile of a new series of iodinated compounds for potential translation to the corresponding [(131)I]radiolabeled compounds for radionuclide therapy of melanoma. Radiolabeling was achieved via standard electrophilic iododestannylation in 60-90% radiochemical yield. Preliminary SPECT imaging demonstrated high and distinct tumor uptake of all compounds, as well as high tumor-to-background ratios compared to the literature compound [(123)I]4 (ICF01012). The most favorable compounds ([(123)I]20, [(123)I]23, [(123)I]41, and [(123)I]53) were selected for further biological investigation. Biodistribution studies indicated that all four compounds bound to melanin containing tissue with low in vivo deiodination; [(123)I]20 and [(123)I]53 in particular displayed high and prolonged tumor uptake (13% ID/g at 48 h). [(123)I]53 had the most favorable overall profile of the cumulative uptake over time of radiosensitive organs. Metabolite analysis of the four radiotracers found [(123)I]41 and [(123)I]53 to be the most favorable, displaying high and prolonged amounts of intact tracer in melanin containing tissues, suggesting melanin specific binding. Results herein suggest that compound [(123)I]53 displays favorable in vivo pharmacokinetics and stability and hence is an ideal candidate to proceed with further preclinical [(131)I] therapeutic evaluation.

Hardware and software modifications on the Advion NanoTek microfluidic platform to extend flexibility for radiochemical synthesis.

Microfluidic systems are currently receiving a lot of attention in the PET radiochemistry field, due to their demonstrated ability to obtain higher incorporation yields with reduced total processing time and using a decreased amount of precursors. The Advion NanoTek LF was the first commercial microfluidic system available for radiochemistry that allows basic parameter optimization to be performed. In this paper we report hardware and software modifications that would allow better performing procedures, higher product throughput and flexibility to utilize the system. In particular, HPLC purification and SPE formulation have been fully integrated.

Prenatal poly I:C age-dependently alters cannabinoid type 1 receptors in offspring: a longitudinal small animal PET study using [(18)F]MK-9470.

Evidence suggests that there is a link between the endocannabinoid system (ECS) and neuropsychiatric illnesses, including schizophrenia. Whilst the ECS has been shown to be involved in immune system regulation in various ways, it is known that infections during pregnancy can modulate the immune system of the mother and increase the risk for schizophrenia in offspring. In animal studies, maternal immune activation following administration of viral or bacterial mimics has been shown to reproduce many key structural, behavioural, and pharmacological abnormalities in offspring that resemble schizophrenia. In the present study, we used Positron Emission Tomography (PET) and [(18)F]MK-9470, a selective high-affinity inverse agonist radioligand for cannabinoid type 1 receptors (CB1R), to longitudinally assess CB1R expression in the progeny of female rats exposed to the viral mimic polyriboinosinic-polyribocytidilic acid (poly I:C) (4mg/kg i.v.) or vehicle at gestational day 15 (GD 15). PET scans were performed in offspring at postnatal days (PND) 32-42 (adolescence) and in the same animals again at PNDs 75-79 (adulthood). Sixteen regions of interest were assessed, encompassing the whole rat brain. At adolescence, offspring exposed prenatally to poly I:C had significantly lower CB1R relative Standard Uptake Values (rSUV) compared to controls in the globus pallidus (p=0.046). In adulthood, however, poly I:C exposed offspring had higher levels of CB1R rSUV in sensory cortex (p=0.034) and hypothalamus (p=0.032) compared to controls. Our results suggest that prenatal poly I:C leads to long term alterations in the integrity of the ECS that are age and region-specific. The increased CB1R expression in adulthood following poly I:C mirrors the increased CB1R observed in patients with schizophrenia in post-mortem and in vivo PET studies.

Optimization of nucleophilic ¹8F radiofluorinations using a microfluidic reaction approach.

Microfluidic techniques are increasingly being used to synthesize positron-emitting radiopharmaceuticals. Several reports demonstrate higher incorporation yields, with shorter reaction times and reduced amounts of reagents compared with traditional vessel-based techniques. Microfluidic techniques, therefore, have tremendous potential for allowing rapid and cost-effective optimization of new radiotracers. This protocol describes the implementation of a suitable microfluidic process to optimize classical (18)F radiofluorination reactions by rationalizing the time and reagents used. Reaction optimization varies depending on the systems used, and it typically involves 5-10 experimental days of up to 4 h of sample collection and analysis. In particular, the protocol allows optimization of the key fluidic parameters in the first tier of experiments: reaction temperature, residence time and reagent ratio. Other parameters, such as solvent, activating agent and precursor concentration need to be stated before the experimental runs. Once the optimal set of parameters is found, repeatability and scalability are also tested in the second tier of experiments. This protocol allows the standardization of a microfluidic methodology that could be applied in any radiochemistry laboratory, in order to enable rapid and efficient radiosynthesis of new and existing [(18)F]-radiotracers. Here we show how this method can be applied to the radiofluorination optimization of [(18)F]-MEL050, a melanoma tumor imaging agent. This approach, if integrated into a good manufacturing practice (GMP) framework, could result in the reduction of materials and the time required to bring new radiotracers toward preclinical and clinical applications.

High levels of intravenous mephedrone (4-methylmethcathinone) self-administration in rats: neural consequences and comparison with methamphetamine.

Mephedrone (MMC) is a relatively new recreational drug that has rapidly increased in popularity in recent years. This study explored the characteristics of intravenous MMC self-administration in the rat, with methamphetamine (METH) used as a comparator drug. Male Sprague-Dawley rats were trained to nose poke for intravenous MMC or METH in daily 2 h sessions over a 10 d acquisition period. Dose-response functions were then established under fixed- and progressive-ratio (FR and PR) schedules over three subsequent weeks of testing. Brains were analyzed ex vivo for striatal serotonin (5-HT) and dopamine (DA) levels and metabolites, while autoradiography assessed changes in the regional density of 5-HT and serotonin transporter (SERT) and DA transporter (DAT) and induction of the inflammation marker translocator protein (TSPO). Results showed that MMC was readily and vigorously self-administered via the intravenous route. Under a FR1 schedule, peak responding for MMC was obtained at 0.1 mg/kg/infusion, versus 0.01 mg/kg/infusion for METH. Break points under a PR schedule peaked at 1 mg/kg/infusion MMC versus 0.3 mg/kg/infusion for METH. Final intakes of MMC were 31.3 mg/kg/d compared to 4 mg/kg/d for METH. Rats self-administering MMC, but not METH, gained weight at a slower rate than control rats. METH, but not MMC, self-administration elevated TSPO receptor density in the nucleus accumbens and hippocampus, while MMC, but not METH, self-administration decreased striatal 5-hydroxyindolacetic acid (5-HIAA) concentrations. In summary, MMC supported high levels of self-administration, matching or exceeding those previously reported with other drugs of abuse.

Mephedrone in adolescent rats: residual memory impairment and acute but not lasting 5-HT depletion.

Mephedrone (4-methylmethcathinone, MMC) is a popular recreational drug, yet its potential harms are yet to be fully established. The current study examined the impact of single or repeated MMC exposure on various neurochemical and behavioral measures in rats. In Experiment 1 male adolescent Wistar rats received single or repeated (once a day for 10 days) injections of MMC (30 mg/kg) or the comparator drug methamphetamine (METH, 2.5 mg/kg). Both MMC and METH caused robust hyperactivity in the 1 h following injection although this effect did not tend to sensitize with repeated treatment. Striatal dopamine (DA) levels were increased 1 h following either METH or MMC while striatal and hippocampal serotonin (5-HT) levels were decreased 1 h following MMC but not METH. MMC caused greater increases in 5-HT metabolism and greater reductions in DA metabolism in rats that had been previously exposed to MMC. Autoradiographic analysis showed no signs of neuroinflammation ([(125)I]CLINDE ligand used as a marker for translocator protein (TSPO) expression) with repeated exposure to either MMC or METH. In Experiment 2, rats received repeated MMC (7.5, 15 or 30 mg/kg once a day for 10 days) and were examined for residual behavioral effects following treatment. Repeated high (30 mg/kg) dose MMC produced impaired novel object recognition 5 weeks after drug treatment. However, no residual changes in 5-HT or DA tissue levels were observed at 7 weeks post-treatment. Overall these results show that MMC causes acute but not lasting changes in DA and 5-HT tissue concentrations. MMC can also cause long-term memory impairment. Future studies of cognitive function in MMC users are clearly warranted.