Karine Mardon

Halogenated 4-(phenoxymethyl)piperidines as potential radiolabeled probes for σ-1 receptors : In vivo evaluation of [123I]-1-(iodopropen-2-yl)-4-[(4-cyanophenoxy)methyl]piperidine

Several halogenated 4-(4-phenoxymethyl)piperidines were synthesized as potential sigma receptor ligands. The affinity and selectivity of these compounds were determined using in vitro receptor binding assays, and their log P values were estimated using HPLC analysis. The effect of various N-substituents on the sigma-1 and sigma-2 dissociation constants was examined. These substituents included fluoroalkyl, hydroxyalkyl, iodopropenyl, and selected ortho-, meta-, and para-substituted benzyl groups. Also determined were the effects of various moieties on the phenoxy ring; specifically 4-iodo, 4-bromo, 4-nitro, 4-cyano, 3-bromo, and pentafluoro substituents were examined. The ranges in the dissociation constants of these compounds for sigma-1 and sigma-2 receptors were 0.38-24.3 and 3.9-361 nM, respectively. The ratio of Ki (sigma-2/sigma-1) ranged from 1.19 to 121. One of the most promising of the iodinated ligands, 1-(trans-iodopropen-2-yl)-4-[(4-cyanophenoxy)methyl]piperidi ne (4), was labeled with 123I and studied in vivo in adult male rats. High uptake and good retention of radioactivity was observed in the brain and many other organs known to possess sigma receptors. Blocking studies revealed high specific binding of [123I]-4 to sigma receptors in the brain, lung, kidney, heart, muscle, and other organs known to possess these sites. These results indicate that [123I]-4 and other halogenated 4-(phenoxymethyl)piperidines of this series may provide useful probes for in vivo tomographic studies of sigma receptors.

Preparation and pharmacological characterization of [76Br]-meta-bromobenzylguanidine ([76Br]MBBG)

[76Br]-meta-Bromobenzylguanidine ([76Br]MBBG) was prepared from the iodinated analog (MIBG) and [76Br]NH4 using a Cu(+)-assisted halogen exchange reaction. [76Br]MBBG was produced in a 60-65% radiochemical yield with a specific activity of 20 MBq/nmol. In rats, biodistribution kinetic studies showed a high uptake of [76Br]MBBG in heart tissues with its maximum of 5% ID/S at 2 h p.i.; whereas 4 h p.i., the maximum of the heart-to-lung concentration ratio of 8 was observed. Metabolic studies in rats indicated that [76Br]MBBG was rapidly metabolized in plasma. However in heart tissue, 25 h p.i., 85% of the radioactivity still represented unchanged radiotracer. Pharmacological studies in rats showed that the myocardial uptake of [76Br]MBBG was similar to that of norepinephrine. After pretreatment of the rats, the uptake of [76Br]MBBG was reduced 4 h p.i. to the following values: after desipramine (DMI) to 37%, after dexamethasone (DXM) to 88% and after 6-hydroxydopamine (6-OHDA) to 16%. These preliminary results suggest that [76Br]MBBG can be useful for the assessment of heart catecholamine reuptake disorders with PET.

Prenatal pharmacotherapy rescues brain development in a Down’s syndrome mouse model

Intellectual impairment is a strongly disabling feature of Downs syndrome, a genetic disorder of high prevalence (1 in 700-1000 live births) caused by trisomy of chromosome 21. Accumulating evidence shows that widespread neurogenesis impairment is a major determinant of abnormal brain development and, hence, of intellectual disability in Downs syndrome. This defect is worsened by dendritic hypotrophy and connectivity alterations. Most of the pharmacotherapies designed to improve cognitive performance in Downs syndrome have been attempted in Downs syndrome mouse models during adult life stages. Yet, as neurogenesis is mainly a prenatal event, treatments aimed at correcting neurogenesis failure in Downs syndrome should be administered during pregnancy. Correction of neurogenesis during the very first stages of brain formation may, in turn, rescue improper brain wiring. The aim of our study was to establish whether it is possible to rescue the neurodevelopmental alterations that characterize the trisomic brain with a prenatal pharmacotherapy with fluoxetine, a drug that is able to restore post-natal hippocampal neurogenesis in the Ts65Dn mouse model of Downs syndrome. Pregnant Ts65Dn females were treated with fluoxetine from embryonic Day 10 until delivery. On post-natal Day 2 the pups received an injection of 5-bromo-2-deoxyuridine and were sacrificed after either 2 h or after 43 days (at the age of 45 days). Untreated 2-day-old Ts65Dn mice exhibited a severe neurogenesis reduction and hypocellularity throughout the forebrain (subventricular zone, subgranular zone, neocortex, striatum, thalamus and hypothalamus), midbrain (mesencephalon) and hindbrain (cerebellum and pons). In embryonically treated 2-day-old Ts65Dn mice, precursor proliferation and cellularity were fully restored throughout all brain regions. The recovery of proliferation potency and cellularity was still present in treated Ts65Dn 45-day-old mice. Moreover, embryonic treatment restored dendritic development, cortical and hippocampal synapse development and brain volume. Importantly, these effects were accompanied by recovery of behavioural performance. The cognitive deficits caused by Downs syndrome have long been considered irreversible. The current study provides novel evidence that a pharmacotherapy with fluoxetine during embryonic development is able to fully rescue the abnormal brain development and behavioural deficits that are typical of Downs syndrome. If the positive effects of fluoxetine on the brain of a mouse model are replicated in foetuses with Downs syndrome, fluoxetine, a drug usable in humans, may represent a breakthrough for the therapy of intellectual disability in Downs syndrome.

Iodine 123-labeled metaiodobenzylguanidine imaging in heart disease

Scintigraphic images of myocardial iodine 123-labeled metaiodobenzylguanidine (MIBG) reflect the relative distribution of adrenergic neurodensity and function in the myocardium. In patients with hypertrophic cardiomyopathy or after infarction, MIBG uptake in hypertrophied myocardium and the infarct-related myocardium was found to be decreased in comparison to blood flow distribution, delineated with thallium 201. Most intriguingly, semiquantitative measurements in patients with congestive heart failure demonstrated reduced myocardial MIBG uptake. This reduction correlated directly with indexes of left ventricular function. Decreases in neuronal density, dysfunction of adrenergic neurons, or chronically elevated circulating norepinephrine levels may account for this diminished myocardial uptake, which, as demonstrated in a pilot study of 90 patients with congestive heart failure, was found to be of predictive value for survival.

Synthesis and preliminary evaluation of [123I]1-(4-cyanobenzyl)-4-[[(trans-iodopropen-2-yl)oxy]-methyl]piperidine : A novel high affinity sigma receptor radioligand for SPECT

1-(4-Cyanobenzyl)-4-[[(trans-iodopropen-2-yl)oxy]methyl]pipe ridine (2) has been synthesized as a novel iodinated ligand for sigma receptors. This new compound exhibited high affinity (Ki = 0.38 nM) for the sigma-1 receptor and selectivity for sigma-1 vs. sigma-2 receptors (Ki sigma-1/sigma-2 = 0.02) using in vitro receptor binding assays. Moderate affinity for muscarinic M1 (Ki = 322 nM) and M2 (Ki = 178 nM) receptors and low affinity (Ki = 1,460 nM) for dopamine D2 receptors was also noted. The lipophilicity of 2 (log P7.5 = 3.24) is moderate, indicating that the ligand should readily cross the blood/brain barrier. The corresponding radioiodinated ligand, 123I-2, was synthesized from the appropriate trans vinyl tributylstannane precursor under acidic conditions using oxidative iododestannylation methods. HPLC purification provided the radioligand in yields ranging between 63 and 75% EOS (n = 5) and with > 99% radiochemical purity and a specific activity > 77,000 MBq/mumol. Preliminary in vivo distribution and pharmacological blocking studies using 123I-2 were performed in male Australian Albino Wistar rats. 123I-2 exhibited good brain uptake (1.11 +/- 0.14% ID at 20 min PI) with no significant loss of radioactivity from the brain over the course of the study (4 h). The gross regional brain distribution of the radioligand showed highest uptake in the posterior cortex and frontal cortex, with slightly lower uptake in other brain regions examined. Most of the uptake of radioactivity in the brain, lung, heart, muscle, and kidney was prevented by pre-administration of compounds with affinity for sigma receptors, indicating the in vivo specificity of the ligand. In view of these results, 123I-2 shows promise for the in vivo tomographic evaluation of sigma receptor densities.

Synthesis of a multimodal molecular imaging probe based on a hyperbranched polymer architecture

Molecular imaging is utilised in modern medicine to aid in the diagnosis and treatment of disease by allowing its spatiotemporal state to be examined in vivo. This study focuses on the development of novel multimodal molecular imaging agents based on hyperbranched polymers that combine the complementary capabilities of optical fluorescence imaging and positron emission tomography-computed tomography (PET/CT) into one construct. RAFT-mediated polymerisation was used to prepare two hydrophilic hyperbranched polymers that were differentiated by their size and level of branching. The multiple functional end-groups facilitated covalent attachment of both near infrared fluorescent dyes for optical imaging, as well as a copper chelator allowing binding of 64Cu as a PET radio nuclei. In vivo multimodal imaging of mice using PET/CT and planar optical imaging was first used to assess the biodistribution of the polymeric materials and it was shown that the larger and more branched polymer had a significantly longer circulation time. The larger constructs were also shown to exhibit enhanced accumulation in solid tumours in a murine B16 melanoma model. Importantly, it was demonstrated that the PET modality gave rise to high sensitivity immediately after injection of the agent, while the optical modality facilitated extended longitudinal studies, thus highlighting how the complementary capabilities of the molecular imaging agents can be useful for studying various diseases, including cancer.

Rorα deficiency and decreased adiposity are associated with induction of thermogenic gene expression in subcutaneous white adipose and brown adipose tissue.

The Rar-related orphan receptor-α (Rorα) is a nuclear receptor that regulates adiposity and is a potential regulator of energy homeostasis. We have demonstrated that the Rorα-deficient staggerer (sg/sg) mice display a lean and obesity-resistant phenotype. Adaptive Ucp1-dependent thermogenesis in beige/brite and brown adipose tissue serves as a mechanism to increase energy expenditure and resist obesity. DEXA and MRI analysis demonstrated significantly decreased total fat mass and fat/lean mass tissue ratio in male chow-fed sg/sg mice relative to wt mice. In addition, we observed increased Ucp1 expression in brown adipose and subcutaneous white adipose tissue but not in visceral adipose tissue from Rorα-deficient mice. Moreover, this was associated with significant increases in the expression of the mRNAs encoding the thermogenic genes (i.e., markers of brown and beige adipose) Pparα, Errα, Dio2, Acot11/Bfit, Cpt1β, and Cidea in the subcutaneous adipose in the sg/sg relative to WT mice. These changes in thermogenic gene expression involved the significantly increased expression of the (cell-fate controlling) histone-lysine N-methyltransferase 1 (Ehmt1), which stabilizes the Prdm16 transcriptional complex. Moreover, primary brown adipocytes from sg/sg mice displayed a higher metabolic rate, and further analysis was consistent with increased uncoupling. Finally, core body temperature analysis and infrared thermography demonstrated that the sg/sg mice maintained greater thermal control and cold tolerance relative to the WT littermates. We suggest that enhanced Ucp1 and thermogenic gene expression/activity may be an important contributor to the lean, obesity-resistant phenotype in Rorα-deficient mice.

The Evolution of Fangs, Venom, and Mimicry Systems in Blenny Fishes

Venom systems have evolved on multiple occasions across the animal kingdom, and they can act as key adaptations to protect animals from predators [1]. Consequently, venomous animals serve as models for a rich source of mimicry types, as non-venomous species benefit from reductions in predation risk by mimicking the coloration, body shape, and/or movement of toxic counterparts [2-5]. The frequent evolution of such deceitful imitations provides notable examples of phenotypic convergence and are often invoked as classic exemplars of evolution by natural selection. Here, we investigate the evolution of fangs, venom, and mimetic relationships in reef fishes from the tribe Nemophini (fangblennies). Comparative morphological analyses reveal that enlarged canine teeth (fangs) originated at the base of the Nemophini radiation and have enabled a micropredatory feeding strategy in non-venomous Plagiotremus spp. Subsequently, the evolution of deep anterior grooves and their coupling to venom secretory tissue provide Meiacanthus spp. with toxic venom that they effectively employ for defense. We find that fangblenny venom contains a number of toxic components that have been independently recruited into other animal venoms, some of which cause toxicity via interactions with opioid receptors, and result in a multifunctional biochemical phenotype that exerts potent hypotensive effects. The evolution of fangblenny venom has seemingly led to phenotypic convergence via the formation of a diverse array of mimetic relationships that provide protective (Batesian mimicry) and predatory (aggressive mimicry) benefits to other fishes [2, 6]. Our results further our understanding of how novel morphological and biochemical adaptations stimulate ecological interactions in the natural world.

Effects of streptozotocin-induced diabetes on neuronal sigma receptors in the rat brain

To study the effect of diabetes mellitus on the density of sigma receptors, in vitro binding experiments were conducted in whole brain homogenate membranes of 5-week and 10-week control rats and streptozotocin (STZ)-induced diabetic rats. sigma-1 Receptors were labelled with [3H](+)-pentazocine while sigma-2 receptors were labelled with [3H] 1,3-di-o-tolylguanidine (DTG) in the presence of 0.5 microM (+)-pentazocine to mask sigma-1 sites. Non-specific binding was determined in the presence of 20 microM haloperidol. Scatchard analysis revealed a 27% (p<0.01) decreased in sigma-1 receptor density and a 33% (p<0.01) decreased in sigma-2 receptor density in whole brain of 10-week STZ-diabetic rats. No statistically significant difference was found in the sigma receptor content of 5-week STZ-diabetic rats. These results provide evidence that neuronal sigma receptors are reduced in 10-week STZ-diabetic rats and suggest that changes in sigma receptors may play a role in diabetes related abnormalities. Further evaluation is required to determine whether changes observed in the brain are homogeneous for either or both sigma receptor subtypes as well as potential links between other CNS receptor changes previously observed in STZ-induced diabetic rats.

(+)-[76Br]A-69024: a non-benzazepine radioligand for studies of dopamine D1 receptors using PET.

(+)-[76Br]A-69024 is a specific and enantioselective dopamine D1 receptor radioligand. The Bmax of (+)-[76Br]A-69024 measured in vitro on rat striatum membranes was 320 +/- 25 fmoles/mg protein with an apparent dissociation constant of Kd = 0.6 +/- 0.1 nM. The inactive enantiomer, (-)-[76Br]A-69024, displayed no affinity in the same assay. In vivo, the biodistribution (+)-[76Br]A-69024 in rats showed a rapid and high uptake in the striatum (1% ID/g), followed by a slow wash out. The striatum/cerebellum concentration ratio (index of specific binding) reached a maximum value of 10 at 60 minutes post injection. A tissue to cerebellum ratio of 2.8 and 1.5 was also observed for frontal and posterior cortex respectively. With the pharmacologically inactive enantiomer, (-)-[76Br]A-69024, the brain uptake was determined to be non specific since a striatum/cerebellum ratio of approximately 1 was observed throughout the time course of the experiment. The selectivity of (+)-[76Br]A-69024 uptake was demonstrated in competition experiments. The specific uptake in the striatum and cortical regions was completely prevented after administration of the D1 antagonist SCH 23390. Pre-treatment of rats with unlabelled (+)A-69024 also displayed the same regional inhibition of (+)-[76Br]A-69024 uptake. Pre-administration of rats with spiperone (D2) and ketanserin (5-HT2/5-HT2C) showed no inhibitory effect on (+)-[76Br]A-69024 uptake in any brain region. Using (+)-[76Br]A-69024, PET study in baboon demonstrated a preferential accumulation of the radioactivity in the striatum, frontal and posterior cortex which was displaced to the level of the cerebellum by SCH 23390. These results suggest that (+)-[76Br]A-69024 may deserve further investigation as a potential radioligand for studying striatal and cortical dopamine D1 receptors using PET.