In vitro and in vivo characterisation of [3H]ANSTO-14 binding to the sigma 1 binding sites

N-(4-phenylbutyl)-3-hydroxy-4-azahexacyclo[5.4.1.0(2,6).0(3, 10).0(5,9) .0(8,11)]dodecane (ANSTO-14) showed the highest activity for the sigma 1 site (Ki = 9.4 nM) and 19-fold sigma 1/sigma 2 selectivity. The present study showed that [3H]ANSTO-14 binds to a single high-affinity site in guinea pig brain membranes with an equilibrium Ki of 8.0 +/- 0.3 nM, in good agreement with the kinetic studies (Kd = 13.3 +/- 5.4 nM, n = 4), and a Bmax of 3.199 +/- 105 fmol/mg protein (n = 4). The in vivo biodistribution of [3H]ANSTO-14 showed a high uptake in the diencephalon. Pretreatment of rats with sigma ligands including (+)-pentazocine (sigma 1), ANSTO-14 (sigma 1), and DTG (sigma 1 and sigma 2) did not significantly reduce radiotracer uptake in the brain, but did in the spleen. A labelled metabolite was found in the liver and brain. Due to its insensitivity to sigma ligands, the accumulation of [3H]ANSTO-14 in the brain indicates high nonspecific binding. Therefore, [3H]ANSTO-14 is a suitable ligand for labelling sigma 1 sites in vitro but is not suitable for brain imaging of sigma binding sites in vivo.     

The role of positron emission tomography in the discovery and development of new drugs; as studied in laboratory animals.

Drug discovery and development is time consuming and a costly procedure. The challenges for the pharmaceutical industry range from the evaluation of potential new drug candidates, the determination of drug pharmacokinetics/pharmacodynamics, the measurement of receptor occupancy as a determinant of drug efficacy, and the pharmacological characterisation of mechanisms of action. Positron emission tomography (PET) is a powerful quantitative imaging technique for looking at biochemical pathways, molecular interactions, drug pharmacokinetics and pharmacodynamics. Recent advances in emission tomography, particularly the development of small animal PET scanners, image reconstruction and animal models of disease have led to the development of extremely sensitive and specific tools for imaging biochemical processes in vivo, therefore representing a new means of providing information for drug development and evaluation. Many human genes have a related mouse gene, allowing mice to be used as a platform for mimicking human disease, using knock-out and knock-in gene technology. Consequently PET imaging of rodents is emerging as a cost effective means of screening new pharmaceuticals and decreasing the time required for new drug development.     

New-generation azaindole-adamantyl-derived synthetic cannabinoids

Purpose

This work reports the synthesis and pharmacological and analytical data for a new series of recently identified azaindole-adamantyl-derived synthetic cannabinoids (SCs).

Methods

Each SC was synthesised using an efficient and divergent synthesis, and assessed by electron ionisation mass spectrometry (EIMS). The cannabimimetic activity of each compound was conducted using a fluorometric imaging plate reader (FLIPR) assay.

Results

The described EIMS method and retention time by gas chromatography were able to effectively differentiate each of the analogues regardless of the bicyclic core. For the first time in these SC structures, the bicyclic ring system was shown to have an impact on the cannabimimetic activities in the fluorometric assay of membrane potential. Analogues ranged from moderately potent at both CB₁ and CB₂ (e.g., AP4AIC EC₅₀?=?160 nM and EC₅₀?=?64 nM, respectively) to not active at either cannabinoid receptor (AP4AICA, AP5AICA, and APIC).

Conclusions

Further investigation into receptor selectivity surrounding these bicyclic cores could prove useful for future therapeutic applications.

     

The chemistry and pharmacology of synthetic cannabinoid SDB‐006 and its regioisomeric fluorinated and methoxylated analogs

Synthetic cannabinoids are the largest and most structurally diverse class of new psychoactive substances, with manufacturers often using isomerism to evade detection and circumvent legal restriction. The regioisomeric methoxy‐ and fluorine‐substituted analogs of SDB‐006 (N‐benzyl‐1‐pentyl‐1H‐indole‐3‐carboxamide) were synthesized and could not be differentiated by gas chromatography–mass spectrometry (GC–MS), but were distinguishable by liquid chromatography–quadrupole time‐of‐flight–MS (LC–QTOF–MS). In a fluorescence‐based plate reader membrane potential assay, SDB‐006 acted as a potent agonist at human cannabinoid receptors (CB₁ EC₅₀ = 19 nM). All methoxy‐ and fluorine‐substituted analogs showed reduced potency compared to SDB‐006, although the 2‐fluorinated analog (EC₅₀ = 166 nM) was comparable to known synthetic cannabinoid RCS‐4 (EC₅₀ = 146 nM). Using biotelemetry in rats, SDB‐006 and RCS‐4 evoked comparable reduction in body temperature (~0.7°C at a dose of 10 mg/kg), suggesting lower potency than the recent synthetic cannabinoid AB‐CHMINACA (>2°C, 3 mg/kg).     

[18F]DPA‐C5yne,a novel fluorine‐18‐labelled analogue of DPA‐714: radiosynthesis and preliminary evaluation as a radiotracer for imaging neuroinflammation with PET

DPA‐C5yne, the lead compound of a novel series of DPA‐714 derivatives in which the fluoroethoxy chain linked to the phenylpyrazolopyrimidine scaffold has been replaced by a fluoroalkyn‐1‐yl moiety, is a high affinity (K_i: 0.35 nM) and selective ligand targeting the translocator protein 18 kDa. In the present work, DPA‐C5yne was labelled with no‐carrier‐added [¹⁸F]fluoride based on a one‐step tosyloxy‐for‐fluorine nucleophilic substitution reaction, purified by cartridge and HPLC, and formulated as an i.v. injectable solution using a TRACERLab FX N Pro synthesizer. Typically, 4.3–5.2 GBq of [¹⁸F]DPA‐C5yne, ready‐to‐use, chemically and radiochemically pure (> 95%), was obtained with specific radioactivities ranging from 55 to 110 GBq/µmol within 50–60 min, starting from a 30 GBq [¹⁸F]fluoride batch (14–17%). LogP and LogD of [¹⁸F]DPA‐C5yne were measured using the shake‐flask method and values of 2.39 and 2.51 were found, respectively. Autoradiography studies performed on slices of ((R,S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolopropionique (AMPA)‐lesioned rat brains showed a high target‐to‐background ratio (1.9 ± 0.3). Selectivity and specificity of the binding for the translocator protein was demonstrated using DPA‐C5yne (unlabelled), PK11195 and Flumazenil (central benzodiazepine receptor ligand) as competitors. Furthermore, DPA‐C5yne proved to be stable in plasma at 37°C for at least 90 min.     

Radiosynthesis of (E)-N-(2-[11C]methoxybenzyl)-3-phenyl-acrylamidine, a novel subnanomolar NR2B subtype-selective NMDA receptor antagonist.

Recently, a novel series of amidines has been described, exhibiting high NR2B-subtype selective N-methyl-D-aspartate (NMDA) antagonist activity with nanomolar or subnanomolar affinity. Within the styrylamidine subclass, (E)-N-(2-methoxybenzyl)-3-phenyl-acrylamidine (1), displayed the highest affinity (Ki=0.7 nM versus [(3)H]ifenprodil) and was considered an appropriate candidate for isotopic labelling with carbon-11 (T(1/2): 20.38 min) at its methoxy group for imaging of NMDA receptors with PET. Derivative 1 has been labelled from the corresponding nor-analogue using [(11)C]methyl triflate and the following experimental conditions : (1) trapping at -10 degrees C of [(11)C]methyl triflate in 300 microL of acetone containing 0.6-0.8 mg of precursor 5 (2.4-3.2 micromol) and 5 microL of a 3M solution of NaOH in water (about 5 eq.); (2) concentration to dryness of the reaction mixture (at 110 degrees C, using a helium stream for 1-2 min); (3) taking up the residue with 0.5 mL of the HPLC mobile phase and (4) purification using semi-preparative HPLC (SymmetryPrep) C-18, Waters, 300 x 7.8 mm). Typically, starting from a 1.5 Ci (55.5 GBq) [(11)C]CO(2) production batch, 120-240 m Ci (4.44-8.88 GBq) of [(11)C]-1 (20-40% decay-corrected radiochemical yield, n=5) was obtained within a total synthesis time of 25-30 min. Specific radioactivities ranged from 0.8 to 1.2 Ci/micromol (29.6-44.4 GBq/micromol) at the end of radiosynthesis. No attempts were made to further optimise these reactions, as sufficient material was obtained to allow for preliminary pharmacological characterisation.     

Pharmacological evaluation of a Br-76 analog of epibatidine: a potent ligand for studying brain nicotinic acetylcholine receptors

[(76)Br]-Norchlorobromoepibatidine ([(76)Br]BrPH) is a specific and high affinity radioligand for the nicotinic acetylcholine receptors (nAChRs). In vitro, on rat thalamus membranes [(76)Br]BrPH bound to two sites with apparent affinities of 8 pM and 3 nM. The density of binding sites were 1.9 and 70 fmol/mg protein, respectively. In vivo, in biodistribution and autoradiographic studies in rats the regional distribution of [(76)Br]BrPH paralleled the neuroanatomical localization of nAChRs. Two hours postinjection, the highest concentration in the brain was found in thalamus and colliculi (4% ID/g). Competition experiments with specific nicotinic, muscarinic, dopaminergic, and serotoninergic drugs confirmed that the in vivo binding of [(76)Br]BrPH was consistent with neuronal nicotinic receptors. PET imaging of [(76)Br]BrPH in baboon demonstrated a rapid and high uptake in the brain. Peak uptake occurred at 30-40 min for the thalamus. Due to the constant washout in the cerebellum, the thalamus to cerebellum ratio was 5 at 2 h postinjection. Subcutaneous injection of cytisine (1 mg/kg), 3 h postinjection of [(76)Br]BrPH reduced the radioactivity concentration in thalamus and cortex by 58 and 50%, respectively, as observed 1 h later. Cytisine pretreatment (5 mg/kg s.c.) inhibited completely the radioligand accumulation in the thalamus. Chronic MPTP pretreatment resulted in reduction of [(76)Br]BrPH uptake in all brain regions except in cerebellum. These preliminary results suggest that [(76)Br]BrPH has the potential to be a useful radioligand for studying the pharmacology of nicotinic acetylcholine receptors in preclinical experiments.     

In vivo imaging of nicotinic receptor upregulation following chronic (-)-nicotine treatment in baboon using SPECT

To quantify changes in neuronal nAChR binding in vivo, quantitative dynamic SPECT studies were performed with 5-[(123)I]-iodo-A-85380 in baboons pre and post chronic treatment with (-)-nicotine or saline control. Infusion of (-)-nicotine at a dose of 2.0 mg/kg/24h for 14 days resulted in plasma (-)-nicotine levels of 27.3 ng/mL. This is equivalent to that found in an average human smoker (20 cigarettes a day). In the baboon brain the regional distribution of 5-[(123)I]-iodo-A-85380 was consistent with the known densities of nAChRs (thalamus > frontal cortex > cerebellum). Changes in nAChR binding were estimated from the volume of distribution (V(d) ) and binding potential (BP) derived from 3-compartment model fits. In the (-)-nicotine treated animal V(d) was significantly increased in the thalamus (52%) and cerebellum (50%) seven days post cessation of (-)-nicotine treatment, suggesting upregulation of nAChRs. The observed 33% increase in the frontal cortex failed to reach significance. A significant increase in BP was seen in the thalamus. In the saline control animal no changes were observed in V(d) or BP under any experimental conditions. In this preliminary study, we have demonstrated for the first time in vivo upregulation of neuronal nAChR binding following chronic (-)-nicotine treatment.     

Behavioural effects of trishomocubanes in rats with unilateral 6-hydroxydopamine lesions

Whilst dopamine replacement improves cardinal features of Parkinson's disease, chronic levodopa administration is associated with dose-related side effects and not all symptoms are ameliorated, necessitating the development of new treatments. Studies of trishomocubanes, a novel group of sigma ligands, have shown enhanced amphetamine-stimulated striatal release of dopamine and a potentially neuroprotective action in vitro and reversal of reserpine-induced catalepsy in vivo. Such effects warrant investigation in animal models of parkinsonism. Our study therefore examines two novel trishomocubane compounds, N-(3'-fluorophenyl)methyl-4-azahexacyclo[5.4.1.0(2,6).0(3,10).0(5,9).0(8,11)]dodecan-3-ol (1) and, N-(3'-fluorophenyl)ethyl-4-azahexacyclo[5.4.1.0(2,6).0(3,10).0(5,9).0(8,11)]dodecan-3-ol (2) in the 6-hydroxydopamine (6-OHDA) rat model of Parkinson's disease. A variety of motor behaviours were studied in rats given 6-OHDA lesions. Groups of lesioned rats were given either (1) or (2) or vehicle solution i.p. Acute administration of 3 mg/kg (1) resulted in a decrease in locomotor activity. Twenty-five milligrams per kilogram (2) caused a decrease in locomotor activity at t=10 and t=20 min of the locomotor test but this was not found when (2) was co-administered with either apomorphine or amphetamine. The decreased locomotor activity indicates that (1) and (2) may have sedative/anxiolytic effect(s). However, elevated plus maze data failed to demonstrate anxiolysis with (2). Quantification of dopaminergic neurons did not demonstrate any significant difference in the magnitude of cell loss between drug-treated vs. vehicle treated rats so no neuroprotective effect was demonstrated in this model at the doses utilised.