In Pursuit of an Allosteric Human Tropomyosin Kinase A (hTrkA) Inhibitor for Chronic Pain.

Human ß-nerve growth factor (ß-NGF) and its associated receptor, human tropomyosin receptor kinase A (hTrkA), have been demonstrated to be key factors in the perception of pain. However, efficacious small molecule therapies targeting the intracellularly located hTrkA kinase have not been explored thoroughly for pain management. Herein, we report the pharmacological properties of a selective hTrkA allosteric inhibitor, 1. 1 was shown to be active against the full length hTrkA, showing preferential binding for the inactive kinase, and was confirmed through the X-ray of hTrkA···1 bound complex. 1 was also found to inhibit ß-NGF induced neurite outgrowth in rat PC12 cells. Daily oral administration of 1 improved the joint compression threshold of rats injected intra-articularly with monoiodoacetate over a 14-day period. The efficacy of 1 in a relevant chronic pain model of osteoarthritis coupled with in vitro confirmation of target mediation makes allosteric hTrkA inhibitors potential candidates for modulating pain.

Automated detection of electroencephalography artifacts in human, rodent and canine subjects using machine learning.

BACKGROUND: Electroencephalography (EEG) invariably contains extra-cranial artifacts that are commonly dealt with based on qualitative and subjective criteria. Failure to account for EEG artifacts compromises data interpretation. NEW METHOD: We have developed a quantitative and automated support vector machine (SVM)-based algorithm to accurately classify artifactual EEG epochs in awake rodent, canine and humans subjects. An embodiment of this method also enables the determination of 'eyes open/closed' states in human subjects. RESULTS: The levels of SVM accuracy for artifact classification in humans, Sprague Dawley rats and beagle dogs were 94.17%, 83.68%, and 85.37%, respectively, whereas 'eyes open/closed' states in humans were labeled with 88.60% accuracy. Each of these results was significantly higher than chance. COMPARISON WITH EXISTING METHODS: Other existing methods, like those dependent on Independent Component Analysis, have not been tested in non-human subjects, and require full EEG montages, instead of only single channels, as this method does. CONCLUSIONS: We conclude that our EEG artifact detection algorithm provides a valid and practical solution to a common problem in the quantitative analysis and assessment of EEG in pre-clinical research settings across evolutionary spectra.

FGF signaling facilitates postinjury recovery of mouse hematopoietic system.

Previous studies have shown that fibroblast growth factor (FGF) signaling promotes hematopoietic stem and progenitor cell (HSPC) expansion in vitro. However, it is unknown whether FGF promotes HSPC expansion in vivo. Here we examined FGF receptor 1 (FGFR1) expression and investigated its in vivo function in HSPCs. Conditional knockout (CKO) of Fgfr1 did not affect phenotypical number of HSPCs and homeostatic hematopoiesis, but led to a reduced engraftment only in the secondary transplantation. When treated with 5-fluorouracil (5FU), the Fgfr1 CKO mice showed defects in both proliferation and subsequent mobilization of HSPCs. We identified megakaryocytes (Mks) as a major resource for FGF production, and further discovered a novel mechanism by which Mks underwent FGF-FGFR signaling dependent expansion to accelerate rapid FGF production under stress. Within HSPCs, we observed an up-regulation of nuclear factor κB and CXCR4, a receptor for the chemoattractant SDF-1, in response to bone marrow damage only in control but not in Fgfr1 CKO model, accounting for the corresponding defects in proliferation and migration of HSPCs. This study provides the first in vivo evidence that FGF signaling facilitates postinjury recovery of the mouse hematopoietic system by promoting proliferation and facilitating mobilization of HSPCs.

Effects of the nicotinic receptor partial agonists varenicline and cytisine on the discriminative stimulus effects of nicotine in rats.

The nicotinic partial agonist varenicline (VCL) is a recently approved medication for the treatment of tobacco dependence, yet very little preclinical research on this drug has been published. The present experiment examined the nicotinic partial agonist properties of VCL and its parent compound, cytisine (CYT), in a nicotine discrimination assay. Rats were trained to discriminate nicotine (0.4 mg/kg, s.c.) from saline using a two-lever discrimination procedure, followed by generalization and antagonism tests with VCL and CYT. Antagonism was examined across a range of nicotine doses. In generalization tests, VCL produced a maximum of 63% responding on the nicotine-appropriate lever, indicating partial generalization. In antagonism tests, VCL decreased the % responding on the nicotine-appropriate lever at 0.2 and 0.4 mg/kg nicotine, indicating antagonism of nicotine's discriminative stimulus effects. No dose of VCL produced significant effects on response rate. The two highest doses of CYT weakly substituted for nicotine, producing a maximum of 23% nicotine-appropriate responding. CYT produced a weak antagonism of the discrimination of moderate nicotine doses, but not of the training dose. These results demonstrate that VCL and CYT partially generalize to and partially antagonize nicotine's discriminative stimulus effects, consistent with a partial agonist mechanism of action.

N-cadherin expression level distinguishes reserved versus primed states of hematopoietic stem cells.

Osteoblasts expressing the homophilic adhesion molecule N-cadherin form a hematopoietic stem cell (HSC) niche. Therefore, we examined how N-cadherin expression in HSCs relates to their function. We found that bone marrow (BM) cells highly expressing N-cadherin (N-cadherin(hi)) are not stem cells, being largely devoid of a Lineage(-)Sca1(+)cKit(+) population and unable to reconstitute hematopoietic lineages in irradiated recipient mice. Instead, long-term HSCs form distinct populations expressing N-cadherin at intermediate (N-cadherin(int)) or low (N-cadherin(lo)) levels. The minority N-cadherin(lo) population can robustly reconstitute the hematopoietic system, express genes that may prime them to mobilize, and predominate among HSCs mobilized from BM to spleen. The larger N-cadherin(int) population performs poorly in reconstitution assays when freshly isolated but improves in response to overnight in vitro culture. Their expression profile and lower cell-cycle entry rate suggest N-cadherin(int) cells are being held in reserve. Thus, differential N-cadherin expression reflects functional distinctions between two HSC subpopulations.

Effects of two novel D3-selective compounds, NGB 2904 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-9H-fluorene-2-carboxamide] and CJB 090 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-4-(pyridin-2-yl)benzamide], on the reinforcing and discriminative stimulus effects of cocaine in rhesus monkeys.

The present study examined the effects of two novel dopamine D3 receptor compounds, NGB 2904 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-9H-fluorene-2-carboxamide], an antagonist, and CJB 090 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-4-(pyridin-2-yl)benzamide], a partial agonist, in two models of cocaine abuse in rhesus monkeys. To establish a dose range and time course of effects, both compounds were shown to block quinpirole-induced yawning when administered i.m. 15, 30, or 120 min before quinpirole. Next, rhesus monkeys were trained to discriminate i.m. injections of saline (0.5 ml) and cocaine (0.3 mg/kg). Neither D3 compound (0.03-3.0 mg/kg; n=3) substituted for cocaine in any monkey. When given in combination with cocaine, CJB 090 but not NGB 2904 attenuated the discriminative stimulus effects of cocaine, shifting the cocaine dose-response curve to the right. In a separate group of monkeys, responding was maintained under a second-order schedule of either food (1.0-g pellets; n=3) or cocaine (0.1 mg/kg/injection; n=4) presentation. When responding was stable, a dose of NGB 2904 (1.0-5.6 mg/kg i.v.) or CJB 090 (0.3-3.0 mg/kg i.v.) was administered for 5 consecutive days, immediately before the session. CJB 090, but not NGB 2904, decreased cocaine- and food-maintained responding. These data indicate that compounds with relatively high affinity and selectivity for the D3 receptor can attenuate the discriminative and reinforcing stimulus effects of cocaine while not producing cocaine-like effects. The present findings support the continued examination of D3 compounds as pharmacological tools for better understanding the role of this receptor subtype in cocaine addiction and as potential lead compounds for novel therapeutic agents.

Effects of the selective dopamine D3 receptor antagonist SB-277011A on the reinforcing effects of nicotine as measured by a progressive-ratio schedule in rats.

The dopamine D3 receptor is primarily localized within the mesocorticolimbic system, and may therefore have potential as a pharmacotherapeutic target for the treatment of drug dependence. Studies have shown that the selective dopamine D3 receptor antagonist SB-277011A reduces a variety of dependence-related behavioral effects of cocaine, alcohol and heroin. A previous study examining SB-277011A on nicotine self-administration using relatively low doses of the antagonist and a low response requirement for nicotine found no effect on drug-taking behavior per se, whereas reinstatement of nicotine-seeking was reduced. The purpose of the present study was to further examine the effects of higher doses of SB-277011A on nicotine self-administration in rats under a progressive-ratio (PR) schedule, which imposes relatively high response requirements for nicotine. Rats were trained to respond under a PR schedule of either nicotine or food reinforcement. Once responding was stable, SB-277011A (3-56 mg/kg) or vehicle was administered i.p. 1 h prior to the operant session. The highest dose tested significantly decreased the mean number of reinforcers and mean response rates in the nicotine self-administration group, but had no effect on either the mean number of reinforcers or response rate in the food group. In a separate set of experiments, the effects of SB-277011A on locomotor activity were measured. At the dose that significantly decreased nicotine self-administration, total distance traveled was also significantly decreased, suggesting that the effect on operant responding at the high dose of SB-277011A is at a threshold for motor effects and may not be directly mediated by an action at dopamine D3 receptors.

PTEN maintains haematopoietic stem cells and acts in lineage choice and leukaemia prevention.

Haematopoietic stem cells (HSCs) must achieve a balance between quiescence and activation that fulfils immediate demands for haematopoiesis without compromising long-term stem cell maintenance, yet little is known about the molecular events governing this balance. Phosphatase and tensin homologue (PTEN) functions as a negative regulator of the phosphatidylinositol-3-OH kinase (PI(3)K)-Akt pathway, which has crucial roles in cell proliferation, survival, differentiation and migration. Here we show that inactivation of PTEN in bone marrow HSCs causes their short-term expansion, but long-term decline, primarily owing to an enhanced level of HSC activation. PTEN-deficient HSCs engraft normally in recipient mice, but have an impaired ability to sustain haematopoietic reconstitution, reflecting the dysregulation of their cell cycle and decreased retention in the bone marrow niche. Mice with PTEN-mutant bone marrow also have an increased representation of myeloid and T-lymphoid lineages and develop myeloproliferative disorder (MPD). Notably, the cell populations that expand in PTEN mutants match those that become dominant in the acute myeloid/lymphoid leukaemia that develops in the later stages of MPD. Thus, PTEN has essential roles in restricting the activation of HSCs, in lineage fate determination, and in the prevention of leukaemogenesis.

A comparison of the reinforcing efficacy of 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") with cocaine in rhesus monkeys.

The purpose of the present study was to compare the reinforcing efficacy of 3,4-methylenedioxymethamphetamine (MDMA, 'ecstasy') to cocaine. Rhesus monkeys (n=4) responded under a within-session, exponentially increasing, progressive-ratio (PR) schedule of cocaine reinforcement. Breaking point (BP) for the PR schedule was defined as the final response requirement completed before 2 h had elapsed without an injection delivered. Saline and doses of cocaine (0.003-0.3 mg/kg/injection) and MDMA (0.01-0.56 mg/kg/injection) were substituted for the training dose of cocaine for at least five consecutive sessions. Both cocaine and MDMA functioned as reinforcers, but self-administration of MDMA occurred at fewer doses and a significantly lower peak BP was obtained for MDMA. These data demonstrate that MDMA functions as a reinforcer, although its reinforcing efficacy appears to be less than that of cocaine.