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The discovery of disease-modifying therapies for Parkinson’s Disease (PD) represents a critical need in neurodegenerative medicine. Genetic mutations in LRRK2 are risk factors for the development of PD, and some of these mutations have been linked to increased LRRK2 kinase activity and neuronal toxicity in cellular and animal models. As such, research towards brain-permeable kinase inhibitors of LRRK2 has received much attention. In the course of a program to identify structurally diverse inhibitors of LRRK2 kinase activity, a 5-azaindazole series was optimized for potency, metabolic stability and brain penetration. A key design element involved the incorporation of an intramolecular hydrogen bond to increase permeability and potency against LRRK2. This communication will outline the structure-activity relationships of this matched pair series including the challenge of obtaining a desirable balance between metabolic stability and brain penetration.

The modulation of LRRK2 kinase activity by a selective small molecule inhibitor has been proposed as a potentially viable treatment for Parkinson's disease. By using aminopyrazoles as aniline bioisosteres, we discovered a novel series of LRRK2 inhibitors. Herein, we describe our optimization effort that resulted in the identification of a highly potent, brain-penetrant aminopyrazole LRRK2 inhibitor (18) that addressed the liabilities (e.g., poor solubility and metabolic soft spots) of our previously disclosed anilino-aminopyrimidine inhibitors. In in vivo rodent PKPD studies, 18 demonstrated good brain exposure and engendered significant reduction in brain pLRRK2 levels post-ip administration. The strategies of bioisosteric substitution of aminopyrazoles for anilines and attenuation of CYP1A2 inhibition described herein have potential applications to other drug discovery programs.

Aim: Histamine H3 receptor antagonists have been proposed as a novel therapeutic approach for the symptomatic treatment of Alzheimer's disease (AD). However, it is unclear whether there is a neurochemical basis for extending their potential use in vascular and mixed dementias. In this study, we measured cortical H3 receptors in patients with subcortical ischemic vascular dementia (SIVD) and mixed SIVD/AD (MIX). Materials and methods: Radioligand binding assays using [3H]GSK189254 were used to measure H3 receptors in the postmortem frontal cortex, anterior cingulate gyrus and hippocampus of a cohort of longitudinally assessed SIVD, MIX and age-matched controls. Results: H3 receptor levels were unchanged in SIVD and MIX in all areas studied. Furthermore, frontal H3 receptor densities negatively correlated with predeath assessment of cognition using Mini-Mental State Examination (MMSE) scores. Conclusion: Our data suggest that H3 receptors are preserved in SIVD and MIX, thus supporting further assessments of H3 antagonists as potential therapeutics in these dementias. © 2011 Elsevier B.V. All rights reserved.

After oral administration, the nonimidazole histamine H3 receptor antagonist, 6-[(3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride (GSK189254), increased histamine release from the tuberomammillary nucleus, where all histaminergic somata are localized, and from where their axons project to the entire brain. To further understand functional histaminergic circuitry in the brain, dual-probe microdialysis was used to pharmacologically block H3 receptors in the tuberomammillary nucleus, and monitor histamine release in projection areas. Perfusion of the tuberomammillary nucleus with GSK189254 increased histamine release from the tuberomammillary nucleus, nucleus basalis magnocellularis, and cortex, but not from the striatum or nucleus accumbens. Cortical acetylcholine (ACh) release was also increased, but striatal dopamine release was not affected. When administered locally, GSK189254 increased histamine release from the nucleus basalis magnocellularis, but not from the striatum. Thus, defined by their sensitivity to GSK189254, histaminergic neurons establish distinct pathways according to their terminal projections, and can differentially modulate neurotransmitter release in a brain region-specific manner. Consistent with its effects on cortical ACh release, systemic administration of GSK189254 antagonized the amnesic effects of scopolamine in the rat object recognition test, a cognition paradigm with important cortical components.

To further understand the procognitive actions of GSK189254, a histamine H(3) receptor antagonist, we determined its influence on the modulation of hippocampal neural cell adhesion molecule (NCAM) polysialylation (PSA) state, a necessary neuroplastic mechanism for learning and memory consolidation. A 4-day treatment with GSK189254 significantly increased basal expression of dentate polysialylated cells in rats with the maximal effect being observed at 0.03-0.3 mg/kg. At the optimal dose (0.3 mg/kg), GSK189254 enhanced water maze learning and the associated transient increase in NCAM-polysialylated cells. The increase in dentate polysialylated cell frequency induced by GSK189254 was not attributable to enhanced neurogenesis, although it did induce a small, but significant, increase in the survival of these newborn cells. GSK189254 (0.3 mg/kg) was without effect on polysialylated cell frequency in the entorhinal and perirhinal cortex, but significantly increased the diffuse PSA staining observed in the anterior, ventromedial, and dorsomedial aspects of the hypothalamus. Consistent with its ability to enhance the learning-associated, post-training increases in NCAM PSA state, GSK189254 (0.3 mg/kg) reversed the amnesia induced by scopolamine given in the 6-h post-training period after training in an odor discrimination paradigm. Moreover, GSK189254 significantly improved the performance accuracy of a delayed match-to-position paradigm, a task dependent on the prefrontal cortex and degree of cortical arousal, the latter may be related to enhanced NCAM PSA-associated plasticity in the hypothalamus. The procognitive actions of H3 antagonism combined with increased NCAM PSA expression may exert a disease-modifying action in conditions harboring fundamental deficits in NCAM-mediated neuroplasticity, such as schizophrenia and Alzheimer's disease.

Cellular interactions between activated microglia and degenerating neurons in in vivo models of Parkinson's disease are not well defined. This time course study assesses the dynamics of morphological and immunophenotypic properties of activated microglia in a 6-hydroxydopamine (6-OHDA) model of Parkinson's disease. Neurodegeneration in the substantia nigra pars compacta (SNc) was induced by unilateral injection of 6-OHDA into the medial forebrain bundle. Activated microglia, identified using monoclonal antibodies: clone of antibody that detects major histocompatibility complex (MHC) class II antigens (OX6) for MHC class II, clone of antibody that detects cell surface antigen-cluster of differentiation 11b - anti-complement receptor 3, a marker for complement receptor 3 and CD 68 for phagocytic activity. Activation of microglia in the lesioned SNc was rapid with cells possessing amoeboid or ramified morphology appeared on day 1, whilst antibody clone that detects macrophage-myeloid associated antigen immunoreactivity was observed at day 3 post-lesion when there was no apparent loss of tyrosine hydroxylase (TH)+ve dopaminergic (DA) SNc neurons. Thereafter, OX6 and antibody clone that detects macrophage-myeloid associated antigen activated microglia selectively adhered to degenerating axons, dendrites and apoptotic (caspase 3+ve) DA neurons in the SNc were observed at day 7. This was followed by progressive loss of TH+ve SNc neurons, with the peak of TH+ve cell loss (51%) being observed at day 9. This study suggests that activation of microglia precedes DA neuronal cell loss and neurons undergoing degeneration may be phagocytosed prematurely by phagocytic microglia.

Background and purpose: Histamine H3 receptor antagonists are currently being evaluated in clinical trials for a number of central nervous system disorders including narcolepsy. These agents can increase wakefulness (W) in cats and rodents following acute administration, but their effects after repeat dosing have not been reported previously.

Chronic inflammation is known to occur in the brains of Alzheimer's Disease (AD) patients, including the presence of activated microglia close to amyloid plaques. We utilised real time autoradiography and immunohistochemistry to investigate microglial activation and the potential anti-inflammatory effects of PPARγ agonists in the Thy-1 APP695swe/Thy-1 PS-1.M146V (TASTPM) overexpressing transgenic mouse model of AD. An age dependent increase in specific [ 3 H]( R )-PK11195 binding to peripheral benzodiazepine receptors (PBR)/translocator protein (18 kDa) (TSPO) was observed in the cortex of TASTPM mice compared to wild type mice, indicative of microglial activation. This was consistent with immunohistochemical data showing age-dependent increases in CD68 immunoreactivity co-localised with amyloid β (Aβ) deposits. In 10 month old TASTPM mice, pioglitazone (20 mg/kg) and ciglitazone (50 mg/kg) significantly reduced [ 3 H]( R )-PK11195 and [ 3 H]DPA-713 binding in cortex and hippocampus, indicative of reduced microglial activation. In AD brain, significant [ 3 H]( R )-PK11195 and [ 3 H]DPA-713 binding was observed across all stages of the disease. These results support the use of PBR/TSPO autoradiography in TASTPM mice as a functional readout of microglial activation to assess anti-inflammatory drugs prior to evaluation in AD patients.

Background and purpose: Histamine H3 receptor antagonists are currently being evaluated for their potential use in a number of central nervous system disorders including Alzheimer's Disease (AD). To date, little is known about the state of H3 receptors in AD. Experimental approach: In the present study we used the radiolabelled H3 receptor antagonist [3H]GSK189254 to investigate H3 receptor binding in the amyloid over-expressing double mutant APPswe × PSI.MI46V (TASTPM) transgenic mouse model of AD and in post-mortem human AD brain samples. Key results: No significant differences in specific H3 receptor binding were observed between wild type and TASTPM mice in the cortex, hippocampus or hypothalamus. Specific [3H]GSK189254 binding was detected in sections of human medial frontal cortex from AD brains of varying disease severity (Braak stages I–VI). With more quantitative analysis in a larger cohort, we observed that H3 receptor densities were not significantly different between AD and age-matched control brains in both frontal and temporal cortical regions. However, within the AD group, [3H]GSK189254 binding density in frontal cortex was higher in individuals with more severe dementia prior to death. Conclusions and implications: The maintenance of H3 receptor integrity observed in the various stages of AD in this study is important, given the potential use of H3 antagonists as a novel therapeutic approach for the symptomatic treatment of AD.

Background and purpose: The peroxisome proliferator-activated receptor-g (PPARg) agonist pioglitazone has previously been shown to attenuate dopaminergic cell loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson’s disease, an effect attributed to its anti-inflammatory properties. In the present investigation, we provide evidence that pioglitazone is effective in the MPTP mouse model, not via an anti-inflammatory action, but through inhibition of MAO-B, the enzyme required to biotransform MPTP to its active neurotoxic metabolite 1-methyl-4-phenylpyridinium (MPP þ ). Experimental approach: Mice were treated with pioglitazone (20 mg kg � 1 b.i.d. (twice a day), p.o., for 7 days), prior and post or post-MPTP (30 mg kg � 1 s.c.) treatment. Mice were then assessed for motor impairments on a beam-walking apparatus and for reductions in TH immunoreactivity in the substantia nigra and depletions in striatal dopamine. The effects of pioglitazone on striatal MPP þ levels and MAO-B activity were also assessed. Key results: Mice treated with MPTP showed deficits in motor performance, marked depletions in striatal dopamine levels and a concomitant reduction in TH immunoreactivity in the substantia nigra. Pretreatment with pioglitazone completely prevented these effects of MPTP. However, pretreatment with pioglitazone also significantly inhibited the MPTP-induced production of striatal MPP þ and the activity of MAO-B in the striatum. Conclusions and implications: The neuroprotection observed with pioglitazone pretreatment in the MPTP mouse model was due to the blockade of the conversion of MPTP to its active toxic metabolite MPP þ , via inhibition of MAO-B. British Journal of Pharmacology (2008) 154, 226–233; doi:10.1038/bjp.2008.78; published online 10 March 2008

GSK207040 (5-[(3-cyclobutyl-2,3,4,5-tetrahydro-1 H -3-benzazepin-7-yl)oxy]- N -methyl-2-pyrazinecarboxamide) and GSK334429 (1-(1-methylethyl)-4-({1-[6-(trifluoromethyl)-3-pyridinyl]-4-piperidinyl}carbonyl)hexahydro-1 H -1,4-diazepine) are novel and selective non-imidazole histamine H 3 receptor antagonists from distinct chemical series with high affinity for human (p K i = 9.67 ± 0.06 and 9.49 ± 0.09, respectively) and rat (p K i = 9.08 ± 0.16 and 9.12 ± 0.14, respectively) H 3 receptors expressed in cerebral cortex. At the human recombinant H 3 receptor, GSK207040 and GSK334429 were potent functional antagonists (pA 2 = 9.26 ± 0.04 and 8.84 ± 0.04, respectively versus H 3 agonist-induced changes in cAMP) and exhibited inverse agonist properties (pIC 50 = 9.20 ± 0.36 and 8.59 ± 0.04 versus basal GTPγS binding). Following oral administration, GSK207040 and GSK334429 potently inhibited cortical ex vivo [ 3 H]- R -α-methylhistamine binding (ED 50 = 0.03 and 0.35 mg/kg, respectively). Functional antagonism of central H 3 receptors was demonstrated by blockade of R -α-methylhistamine-induced dipsogenia in rats (ID 50 = 0.02 and 0.11 mg/kg p.o. for GSK207040 and GSK334429, respectively). In more pathophysiologically relevant pharmacodynamic models, GSK207040 (0.1, 0.3, 1 and 3 mg/kg p.o.) and GSK334429 (0.3, 1 and 3 mg/kg p.o.) significantly reversed amnesia induced by the cholinergic antagonist scopolamine in a passive avoidance paradigm. In addition, GSK207040 (0.1, 0.3 and 1 mg/kg p.o.) and GSK334429 (3 and 10 mg/kg p.o.) significantly reversed capsaicin-induced reductions in paw withdrawal threshold, suggesting for the first time that blockade of H 3 receptors may be able to reduce tactile allodynia. Novel H 3 receptor antagonists such as GSK207040 and GSK334429 may therefore have therapeutic potential not only in dementia but also in neuropathic pain.

Starting from the potent and selective but poorly brain penetrant 5-HT6 receptor antagonist SB-271046, a successful strategy for improving brain penetration was adopted involving conformational constraint with concomitant reduction in hydrogen bond count. This provided a series of bicyclic heteroarylpiperazines with high 5-HT6 receptor affinity. 5-Chloroindole 699929 combined high 5-HT6 receptor affinity with excellent brain penetration and also had good oral bioavailability in both rat and dog.

5-HT(4) receptors are widely distributed in both peripheral and central nervous systems where they couple, via a G-protein, to the activation of adenylate cyclase. In the brain, the highest 5-HT(4) receptor densities are found in the limbic system, including the hippocampus and frontal cortex. It has been suggested that activation of these receptors may be of therapeutic benefit in diseases that produce cognitive deficits such as Alzheimer's disease (AD). Previous electrophysiological studies have shown that the 5-HT(4) agonist, Zacopride, can increase population spike amplitude recorded in region CA1 of rat hippocampal slices in a cyclic AMP (cAMP)/cAMP-dependent protein kinase A-dependent manner. We report here that the 5-HT(4) agonist, Prucalopride, and the 5-HT(4) partial agonist, SL65.0155, produce a similar effect in rat hippocampal slices and that the specific 5-HT(4) antagonist, GR113808, blocks these effects. To investigate the potential use of 5-HT(4) agonists in the treatment of AD, Prucalopride was applied to hippocampal slices from a transgenic mouse line that overexpresses the Abeta peptide. Despite the deficit in synaptic transmission present in these mice, the percentage increase of the CA1 population spike induced by Prucalopride was the same as that observed in wild-type mice. These data support 5-HT(4) receptors as a target for cognitive enhancement and suggest that a partial agonist would be sufficient to produce benefits, while reducing potential peripheral side effects. In addition, we show that 5-HT(4) receptors remain functional in the presence of excess Abeta peptide and may therefore be a useful target in AD.

Apelin peptides have recently been identified to be the endogenous ligands for the G protein-coupled receptor APJ. However, little is known about the physiological roles of this ligand-receptor pairing. In the present study we investigated the pharmacology of several apelin analogues at the human recombinant APJ receptor using radioligand binding and functional assays. This has led to the identification of key residues in the apelin peptide required for functional potency and binding affinity through structure-activity studies. In particular, we have identified that replacement of leucine in position 5, or arginine in position 2 and 4 of the C-terminal apelin peptide, apelin-13, resulted in significant changes in pharmacology. We also investigated the detailed localization of pre-proapelin and APJ receptor mRNA in a wide range of human, rat and mouse tissues using quantitative RT-PCR, and carried out a detailed immunohistochemical study of the distribution of the APJ receptor in rat brain and spinal cord. Interestingly, the APJ receptor was not only co-localized in white matter with GFAP in the spinal cord, but was also clearly localized on neurones in the brain, suggesting that this receptor and its peptide may be involved in a wide range of biological process yet to be determined.

The mammalian homologues of the Drosophila transient receptor potential (TRP) channel are plasma membrane proteins involved in the regulation of cellular Ca(2+) influx. These ion channels can be activated subsequent to either depletion of Ca(2+) from internal stores or through receptor-mediated processes. The mRNA expression patterns of several individual mammalian short transient receptor potential channels (TRPCs) have been described. Cross-comparisons between these data, however, are at best difficult predominantly due to the non-quantitative methods used. Furthermore there is limited data on the expression of TRPC family members in human tissues. In the present study we used a single technique, namely TaqMan real-time quantitative RT-PCR, to investigate the mRNA distribution of human TRPC1, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7 (hTRPCs) in discrete human brain areas, peripheral tissues as well as a panel of cell-lines. All hTRPCs studied were widely expressed within CNS and significant peripheral expression was often observed. Despite this, each channel exhibited a distinctive hallmark distribution profile. hTRPC1 was widely expressed in CNS and peripheral tissues, whereas hTRPC3 and hTRPC5 were predominantly expressed in tissues of CNS. hTRPC4 mRNA was detected in CNS and certain peripheral tissues such as bone, heart and prostate. hTRPC6 was homogeneously expressed throughout the CNS and peripheral tissues with the highest levels in placenta and lung. hTRPC7 mRNA was also broadly expressed in CNS as well as some peripheral tissues. The pattern of expression of the TRPCs was quite different in the various cell lines examined. TRPC3 and TRPC6 were selectively present in HEK-293 cells whilst TRPC1 was broadly distributed in the cell lines analyzed. In contrast TRPC4 and TRPC5 mRNAs were predominantly expressed in HK-2 and HEK-293 cell lines respectively. TRPC7 was selectively expressed in COS-1, COS-7 and HK-2 cell lines. These results show tissue- and cell-specific co-expression of multiple TRPC forms indicating widespread potential for formation of heteromeric channels. These data will be useful in the complex task of relating channel subunit composition to function in native cells.

The regulation and control of plasma membrane Ca(2+) fluxes is critical for the initiation and maintenance of a variety of signal transduction cascades. Recently, the study of transient receptor potential channels (TRPs) has suggested that these proteins have an important role to play in mediating capacitative calcium entry. In this study, we have isolated a cDNA from human brain that encodes a novel transient receptor potential channel termed human TRP7 (hTRP7). hTRP7 is a member of the short TRP channel family and is 98% homologous to mouse TRP7 (mTRP7). At the mRNA level hTRP7 was widely expressed in tissues of the central nervous system, as well as some peripheral tissues such as pituitary gland and kidney. However, in contrast to mTRP7, which is highly expressed in heart and lung, hTRP7 was undetectable in these tissues. For functional analysis, we heterologously expressed hTRP7 cDNA in an human embryonic kidney cell line. In comparison with untransfected cells depletion of intracellular calcium stores in hTRP7-expressing cells, using either carbachol or thapsigargin, produced a marked increase in the subsequent level of Ca(2+) influx. This increased Ca(2+) entry was blocked by inhibitors of capacitative calcium entry such as La(3+) and Gd(3+). Furthermore, transient transfection of an hTRP7 antisense expression construct into cells expressing hTRP7 eliminated the augmented store-operated Ca(2+) entry. Our findings suggest that hTRP7 is a store-operated calcium channel, a finding in stark contrast to the mouse orthologue, mTRP7, which is reported to enhance Ca(2+) influx independently of store depletion, and suggests that human and mouse TRP7 channels may fulfil different physiological roles.

International audience; 5-HT4 receptors mediate several physiological effects of 5-HT, particularly in the central nervous system (CNS), heart and gut. Recently, several C-terminal splice variants of the human 5-HT4 (h5-HT4) receptor have been described, namely h5-HT4(a), h5-HT4(b), h5-HT4(c), h5-HT4(d) and h5-HT4(g). Previous tissue distribution data suggest some degree of specificity in the mRNA expression patterns of the different h5-HT4 receptor splice variants. However, comparison of the mRNA expression profiles of these splice variants is difficult due to the non-quantitative methods used, and in addition, there is very limited data on the expression of each splice variant in human CNS subregions. In the present study we used a single technique, TaqMan real time quantitative RT-PCR, to investigate the mRNA distribution of 5-HT4 receptor C-terminal splice variants in multiple human CNS and peripheral tissues. Using a primer/probe set that amplified all 5-HT4 splice variants (5-HT4pan), the highest CNS expression of 5-HT4 receptor mRNA was observed in basal ganglia, amygdala and hippocampus, consistent with previous studies. h5-HT4(a), h5-HT4(b), h5-HT4(c) and h5-HT4(g) were predominantly expressed in various CNS tissues, compared to most peripheral tissues, but there were differences in expression levels and distribution patterns of each variant. The distribution profile and expression levels observed for the 5-HT4(b) splice variant were virtually identical to that obtained with the 5-HT4pan primer/probe set, whilst the other splice variants were expressed at much lower levels and with different expression patterns obtained with both 5-HT4(b) and 5-HT4pan primer/probe sets. Highest levels of 5-HT4(g) were observed in the hypothalamus and cortex, whilst the 5-HT4(a) variant was highest in the amygdala. 5-HT4(c) expression was highest in the pituitary gland whilst 5-HT4(d) mRNA was only detected in the small intestine at very low levels and not in the CNS. In conclusion, we have shown quantitative differences in the mRNA distribution profiles of the 5-HT4 receptor C-terminal splice variants in human CNS subregions as well as peripheral tissues. In addition, our data suggests that the h5-HT4(b) variant is the most predominant form of the 5-HT4 receptor in humans.

Leucine-rich repeat kinase 2 (LRRK2) has drawn significant interest in the neuroscience research community because it is one of the most compelling targets for a potential disease-modifying Parkinson's disease therapy. Herein, we disclose structurally diverse small molecule inhibitors suitable for assessing the implications of sustained in vivo LRRK2 inhibition. Using previously reported aminopyrazole 2 as a lead molecule, we were able to engineer structural modifications in the solvent-exposed region of the ATP-binding site that significantly improve human hepatocyte stability, rat free brain exposure, and CYP inhibition and induction liabilities. Disciplined application of established optimal CNS design parameters culminated in the rapid identification of GNE-0877 (11) and GNE-9605 (20) as highly potent and selective LRRK2 inhibitors. The demonstrated metabolic stability, brain penetration across multiple species, and selectivity of these inhibitors support their use in preclinical efficacy and safety studies.

Orphan transporters form a growing subfamily of genes related by sequence similarity to the Na+/Cl- -dependent neurotransmitter superfamily. Using a combination of database similarity searching and cloning methods, we have identified and characterized two novel human orphan transporter genes, v7-3 and NTT5. Similar to other known orphan transporters, v7-3 and NTT5 contain 12 predicted transmembrane domains, intracellular N- and C-terminal domains, and large extracellular loops between transmembrane (TM) domains 3 and 4 and between TM domains 7 and 8. Residues within the extracellular loops are also predicted to contain sites for N-linked glycosylation. Human v7-3, the species orthologue of rat v7-3, contains an open reading frame (ORF) of 730 amino acids. Human NTT5 is a new member of the orphan transporter family and has an ORF of 736 amino acids. The amino acid sequences of human v7-3 and NTT5 are greater than 50% similar to other known orphan neurotransmitter transporters and also show sequence similarity to the human serotonin and dopamine transporters. Radiation hybrid mapping located the human v7-3 and NTT5 genes on chromosomes 12q21.3-q21.4 and 19q13.1-q13.4, respectively. Human mRNA distribution analysis by TaqMan reverse transcription-polymerase chain reaction showed that v7-3 mRNA is predominantly expressed in neuronal tissues, particularly amygdala, putamen, and corpus callosum, with low-level expression in peripheral tissues. In contrast, NTT5 mRNA was highly expressed in peripheral tissues, particularly in testis, pancreas, and prostate. Transient transfection with epitope-tagged transporter constructs demonstrated v7-3 to be expressed at the cell surface, whereas NTT5 was predominantly intracellular, suggestive of a vesicular location. Although the substrates transported by these transporters remain unknown, their specific but widespread distribution suggests that they may mediate distinct and important functions within the brain and the periphery.

Capsaicin, resiniferatoxin, protons or heat have been shown to activate an ion channel, termed the rat vanilloid receptor-1 (rVR1), originally isolated by expression cloning for a capsaicin sensitive phenotype. Here we describe the cloning of a human vanilloid receptor-1 (hVR1) cDNA containing a 2517 bp open reading frame that encodes a protein with 92% homology to the rat vanilloid receptor-1. Oocytes or mammalian cells expressing this cDNA respond to capsaicin, pH and temperature by generating inward membrane currents. Mammalian cells transfected with human VR1 respond to capsaicin with an increase in intracellular calcium. The human VR1 has a chromosomal location of 17p13 and is expressed in human dorsal root ganglia and also at low levels throughout a wide range of CNS and peripheral tissues. Together the sequence homology, similar expression profile and functional properties confirm that the cloned cDNA represents the human orthologue of rat VR1.

The novel 5-HT7 receptor antagonist, SB-269970-A, potently displaced [3H]-5-CT from human 5-HT7(a) (pKi 8.9±0.1) and 5-HT7 receptors in guinea-pig cortex (pKi 8.3±0.2). 5-CT stimulated adenylyl cyclase activity in 5-HT7(a)/HEK293 membranes (pEC50 7.5±0.1) and SB-269970-A (0.03–1 μM) inhibited the 5-CT concentration-response with no significant alteration in the maximal response. The pA2 (8.5±0.2) for SB-269970-A agreed well with the pKi determined from [3H]-5-CT binding studies. 5-CT-stimulated adenylyl cyclase activity in guinea-pig hippocampal membranes (pEC50 of 8.4±0.2) was inhibited by SB-269970-A (0.3 μM) with a pKB (8.3±0.1) in good agreement with its antagonist potency at the human cloned 5-HT7(a) receptor and its binding affinity at guinea-pig cortical membranes. 5-HT7 receptor mRNA was highly expressed in human hypothalamus, amygdala, thalamus, hippocampus and testis. SB-269970-A was CNS penetrant (steady-state brain : blood ratio of ca. 0.83 : 1 in rats) but was rapidly cleared from the blood (CLb=ca. 140 ml min−1 kg−1). Following a single dose (3 mg kg−1) SB-269970 was detectable in rat brain at 30 (87 nM) and 60 min (58 nM). In guinea-pigs, brain levels averaged 31 and 51 nM respectively at 30 and 60 min after dosing, although the compound was undetectable in one of the three animals tested. 5-CT (0.3 mg kg−1 i.p.) induced hypothermia in guinea-pigs was blocked by SB-269970-A (ED50 2.96 mg kg−1 i.p.) and the non-selective 5-HT7 receptor antagonist metergoline (0.3–3 mg kg−1 s.c.), suggesting a role for 5-HT7 receptor stimulation in 5-CT induced hypothermia in guinea-pigs. SB-269970-A (30 mg kg−1) administered at the start of the sleep period, significantly reduced time spent in Paradoxical Sleep (PS) during the first 3 h of EEG recording in conscious rats. British Journal of Pharmacology (2000) 130, 539–548; doi:10.1038/sj.bjp.0703357

Quantitative reverse transcription and polymerisation chain reaction (RT-PCR) using Taqman¿trade mark omitted¿ fluorogenic probes has been used to measure changes in gene expression in the cerebral cortex of rats in the permanent middle cerebral artery occlusion (pMCAO) model of focal ischemia. The mRNA levels of three housekeeping genes have been analysed in this model to determine which gene showed least change following experimental insult. In the lesioned cortex, beta-actin mRNA increased at 24 h, while the levels of cyclophilin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) did not change. We have also used this methodology to examine modulations in the level of caspase-3 mRNA during focal ischemia in the rat. Caspase-3 mRNA showed a 41% increase at 6 h post-MCAO, which was specific to the lesioned cortex. This change became more pronounced with time, showing an increase of 220% at 24 h. This methodology enables changes in mRNA expression to be analysed more sensitively and quantitatively than other available techniques and highlights the need for careful choice of control or housekeeping genes used for RNA comparisons.

Partial sciatic nerve injury, a model of neuropathic pain, elicits a variety of neurochemical, electrophysiological and neuroanatomical changes in primary sensory neurons. We have used the technique of messenger RNA differential display to identify genes with altered expression in these neurons which may contribute to the development of aberrant sensation following such peripheral nerve damage. This approach identified 14 distinct complementary DNA clones, representing transcripts with increased ipsilateral expression in L4/5 dorsal root ganglia, two weeks after unilateral partial ligation of the rat sciatic nerve. Both Zucker diabetic fatty rats and their lean counterparts were used in this study but none of the transcripts identified showed an induction that was confined to one of the two groups. The majority of the clones did not show significant sequence similarity to previously reported genes and therefore may represent novel messenger RNA sequences or, alternatively, unknown regions of partially characterised messenger RNAs. Two of the clones represented transcripts for the known proteins muscle LIM protein and acidic epididymal glycoprotein, neither of which had previously been associated with expression in the nervous system. Reverse transcriptase-polymerase chain reaction analysis and in situ hybridization confirmed that the messenger RNA expression of both muscle LIM protein and acidic epididymal glycoprotein was induced in an ipsilateral-specific manner. Their localisations, examined with in situ hybridization in L5 dorsal root ganglia, were limited in each case to a sub-population of neuronal profiles. Those neuronal profiles that demonstrated muscle LIM protein hybridization were distributed across the profile size range, whereas the distribution of acidic epididymal glycoprotein-positive profiles appeared to be skewed towards smaller profiles. The induction of muscle LIM protein and acidic epididymal glycoprotein in dorsal root ganglia may play an important functional role in the adaptive response of primary sensory neurons following partial sciatic nerve injury.

A functional tachykinin NK3 receptor was cloned from the rabbit iris sphincter muscle and its distribution investigated in ocular tissues. Standard polymerase chain reaction (PCR) techniques were used to clone a full length rabbit NK3 receptor cDNA consisting of 1404 nucleotides. This cDNA encoded a protein of 467 amino acids with 91 and 87% homology to the human and rat NK3 receptors respectively. In CHO-K1 cells transiently expressing the recombinant rabbit NK3 receptor, the relative order of potency of NKB>>NKASP to displace [125I]-[MePhe7]-NKB binding and to increase intracellular calcium, together with the high affinity of NK3 selective agonists (e.g. senktide, [MePhe7]-NKB) and antagonists (e.g. SR 142801, SB 223412) in both assays was consistent with NK3 receptor pharmacology. In binding and functional experiments, agonist concentration response curves were shallow (0.7–0.8), suggesting the possibility of multiple affinity states of the receptor. Quantitative real time PCR analysis revealed highest expression of rabbit NK3 receptor mRNA in iris sphincter muscle, lower expression in retina and iris dilator muscle, and no expression in lens and cornea. In situ hybridization histochemistry revealed discrete specific localization of NK3 receptor mRNA in the iris muscle and associated ciliary processes. Discrete specific labelling of NK3 receptors with the selective NK3 receptor agonist [125I]-[MePhe7]-NKB was also observed in the ciliary processes using autoradiography. Our study reveals a high molecular similarity between rabbit and human NK3 receptor mRNAs, as predicted from previous pharmacological studies, and provide the first evidence that NK3 receptors are precisely located on ciliary processes in the rabbit eye. In addition, there could be two affinity states of the receptor which may correspond to the typical and ‘atypical’ NK3 receptor subtypes previously reported. British Journal of Pharmacology (1999) 128, 627–636; doi:10.1038/sj.bjp.0702854

Starting from a benzazepine sulfonamide 5-HT(6) receptor antagonist lead with limited brain penetration, application of a strategy of conformational constraint and reduction of hydrogen bond donor count led to a novel series of tricyclic derivatives with high 5-HT(6) receptor affinity and excellent brain:blood ratios.

1. Inhibition of NK3 receptor agonist-induced contraction in the rabbit isolated iris sphincter muscle was used to assess the in vitro functional activity of three 2-phenyl-4-quinolinecarboxamides, members of a novel class of potent and selective non-peptide NK3 receptor antagonists. In addition, an in vivo correlate of this in vitro response, namely NK3 receptor agonist-induced miosis in conscious rabbits, was characterized with some of these antagonists. 2. In vitro senktide (succinyl-[Asp9,MePhe8]-substance P (6-11) and [MePhe7]-neurokinin B ([MePhe7]-NKB) were potent contractile agents in the rabbit iris sphincter muscle but exhibited quite different profiles. Senktide produced monophasic log concentration-effect curves with a mean pD2=9.03+/-0.06 and mean nH=1.2+/-0.02 (n=14). In contrast, [MePhe7]-NKB produced shallow log concentration-effect curves which often appeared biphasic (nH=0.54+/-0.04, n=8), preventing the accurate determination of pD2 values. 3. The contractile responses to the NK3 receptor agonist senktide were antagonized in a surmountable and concentration-dependent manner by SB 223412 ((-)-(S)-N-(alpha-ethylbenzyl)-3-hydroxy-2-phenylquinoline-4-ca rboxamide; 3-30 nM, pA2=8.4, slope=1.8+/-0.3, n=4). SB 222200 ((-)-(S)-N-(alpha-ethylbenzyl)-3-methyl-2-phenylquinoline-4-car box amide; 30-300 nM, pA2=7.9, slope=1.4+/-0.06, n=4) and SB 218795 ((-)-(R)-N-(alpha-methoxycarbonylbenzyl)-2-phenylquinoline-4-carboxamide; 0.3 and 3 microM apparent pKB=7.4+/-0.06, n=6). 4. Contractile responses to the NK3 receptor agonist [MePhe7]-NKB in the rabbit iris sphincter muscle were unaffected by SB 218795 (0.3 and 3 microM, n=8). In contrast, SB 223412 (30 and 300 microM n=4) and SB 222200 (0.3 and 3 microM, n=4) inhibited responses to low concentrations ( 1 nM) of [MePhe7]-NKB. Furthermore, log concentration-effect curves to [MePhe7]-NKB became steeper and monophasic in the presence of each antagonist. 5. SB 218795 (3 microM, n=4) had no effect on contractions induced by transmural nerve stimulation (2 Hz) or substance P, exemplifying the selectivity of this class of antagonist for functional NK3 receptors over NK1 receptors in the rabbit. 6. In vivo, senktide (1, 10 and 25 microg i.v., i.e. 1.2, 11.9 and 29.7 nmol, respectively) induced concentration-dependent bilateral miosis in conscious rabbits (maximum pupillary constriction=4.25+/-0.25 mm; basal pupillary diameter 7.75+/-0.48 mm; n=4). The onset of miosis was within 2-5 min of application of senktide and responses lasted up to 30 min. Responses to two i.v. administrations of 25 microg senktide given 30 min apart revealed no evidence of tachyphylaxis. Topical administration of atropine (1%) to the eye enhanced pupillary responses to 25 microg senktide. This was probably due to the mydriatic effect of atropine since it significantly increased baseline pupillary diameter from 7.0+/-0.4 mm to 9.0+/-0.7 mm (n=4), thereby increasing the maximum capacity for miosis. Senktide-induced miosis was inhibited by SB 222200 (1 and 2 mg kg[-1], i.v., i.e. 2.63 and 5.26 micromol kg[-1]; maximum inhibition 100%; n=3-4), SB 223412 (0.5 and 1 mg kg[-1], i.v., i.e. 1.31 and 2.61 micromol kg[-1]; maximum inhibition 100%; n=3), SB 218795 (0.5 and 1 mg kg[-1] i.v., i.e. 1.26 and 2.52 micromol kg-1; maximum inhibition 78%; n=3), and the structurally distinct NK3 receptor antagonist SR 142801 ((S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl)propyl)-4-phenylepipiperidin-4-yl)-N-methylacetamide; 1.5mg kg-1, i.v., i.e. 2.47micromol kg-1, maximum inhibition 92%; n=3). 7. Topical administration of senktide (25microg; 29.7nmol) to the eye induced unilateral miosis in the treated eye only. At this dose there was no significant difference (P

A novel class of potent and selective non-peptide neurokinin-3 (NK-3) receptor antagonists, featuring the 4-quinolinecarboxamide framework, has been designed based upon chemically diverse NK-1 receptor antagonists. The novel compounds 33-76, prompted by chemical modifications of the prototype 4, have been characterized by binding analysis using a membrane preparation of chinese hamster ovary (CHO) cells expressing the human neurokinin-3 receptors (hNK-3-CHO), and clear structure-activity relationships (SARs) have been established. From SARs, (R)-N-[alpha-(methoxycarbonyl)benzyl]-2-phenylquinoline-4-carboxamide (65, SB 218795, hNK-3-CHO binding Ki = 13 nM) emerged as one of the most potent compounds of this novel class. Selectivity studies versus the other neurokinin receptors (hNK-2-CHO and hNK-1-CHO) revealed that 65 is about 90-fold selective for hNK-3 versus hNK-2 receptors (hNK-2-CHO binding Ki = 1221 nM) and over 7000-fold selective versus hNK-1 receptors (hNK-1-CHO binding Ki =100 microM). In vitro functional studies in rabbit isolated iris sphincter muscle preparation demonstrated that 65 is a competitive antagonist of the contractile response induced by the potent and selective NK-3 receptor agonist senktide with a Kb = 43 nM. Overall, the data indicate that 65 is a potent and selective hNK-3 receptor antagonist and a useful lead for further chemical optimization.

Using a tissue bath system which allowed the simultaneous measurement of electrically-induced [3H]noradrenaline release and neurogenic contraction under identical conditions, we investigated the prejunctional inhibitory activity of the selective 5-HT(1D/1B) receptor agonists BRL 56905 ((+/-)-3-amino-6-carboxamido-1,2,3,4-tetrahydrocarbazole) and SKF 99101H (3-(2-dimethylaminoethyl)-4-chloro-5-propoxyindole hemifumarate), compared to sumatriptan and 5-HT. Transmural electrical stimulation (2 Hz) of dog saphenous vein induced consistent increases in [3H]noradrenaline release as well as reproducible contractile responses (

Tachykinin NK3 receptors were characterized in the rabbit isolated iris sphincter muscle by use of autoradiography and in vitro functional studies. [125I]-[MePhe7]-neurokinin B (NKB) (1 nm), a selective NK3 receptor agonist, specifically labelled a population of NK3 receptors that were uniformly distributed throughout the rabbit iris sphincter muscle. This labelling was inhibited by unlabelled [MePhe7]-NKB (1 μm) but not by the NK1 receptor antagonist CP 99994 (1 μm). In the presence of CP 99994 (1 μm), the selective NK3 receptor agonists senktide (n=14) and [Pro7]-NKB (n=4), and the natural preferred ligand for the NK3 receptor, NKB (n=8), were potent contractile agents in the rabbit iris sphincter muscle. They all produced monophasic concentration-effect curves with pD2 values of 9.53±0.08, 8.56±0.09 and 9.75±0.09, and nH values of 0.93±0.03, 1.53±0.17 and 0.76±0.06, respectively. [MePhe7]-NKB (n=12) was also a potent agonist, but produced shallow concentration-effect curves which appeared biphasic (nH=0.45±0.04). Contractile responses to senktide were surmountably antagonized in a concentration-dependent manner by the selective non-peptide NK3 receptor antagonist, SR 142801 (3–30 nm; pA2= 8.9; slope = 0.99) and the non-peptide NK2/NK3 receptor antagonist, SR 48968 (3–30 μm; pA2=6.1; slope=1.5). These pA2 values were consistent with functional rabbit NK3 receptors more closely resembling guinea-pig and human NK3 receptors, than rat NK3 receptors. SR 142801 (10–100 nm) and SR 48968 (3 and 30 μm) inhibited responses to low (1 nm) but not higher (>1 nm) concentrations of [MePhe7]-NKB, and concentration-effect curves to [MePhe7]-NKB became steeper and monophasic in the presence of either antagonist. SR 142801 (3–30 nm) and SR 48968 (3–30 μm) also surmountably antagonized concentration-effect curves to [Pro7]-NKB and NKB, although results were more difficult to interpret, since the relationship between log concentration-ratios and the concentration of antagonist used did not adhere to the Schild equation. However, analysis of data with the lowest concentration of SR 142801 (3 nm) tested against NKB, and SR 48968 (3 μm) tested against [Pro7]-NKB and NKB, yielded apparent pA2 estimates of 9.3, 6.8 and 6.4, respectively, consistent with blockade of NK3 receptors. SR 142801 (100 nm) had no effect on contractions induced by transmural nerve stimulation (2 Hz, 0.3 ms, 20 V for 30 s), whereas CP 99994 (1 μm) abolished these responses. Phenoxybenzamine pretreatment (20 μm, 10 min) markedly reduced maximum responses to [MePhe7]-NKB (from 101±6.2% to 38±9.5% reference contraction, n=4) and induced a marked (10 fold) rightward shift in the concentration-effect curve. The residual responses to [MePhe7]-NKB after phenoxybenzamine pretreatment were unaffected by 1 μm CP 99994 (maximum response=41±9.4%, n=4). These results demonstrate autoradiographically and functionally, the presence of NK3 receptors in rabbit iris sphincter muscle that mediate contractile responses to NK3 receptor agonists, but not to sensory trigeminal nerve stimulation. The present data with senktide and selective NK3 receptor antagonists suggest that functional rabbit NK3 receptors more closely resemble human and guinea-pig NK3 receptors than rat NK3 receptors. However, the pharmacological profiles of [MePhe7]-NKB, SR 142801 and SR 48968 suggest the presence of an ‘atypical’ NK3 receptor or a heterogeneous population of NK3 receptors in this tissue. British Journal of Pharmacology (1997) 120, 93–101; doi:10.1038/sj.bjp.0700867

This Letter describes the discovery of GSK189254 and GSK239512 that were progressed as clinical candidates to explore the potential of H3 receptor antagonists as novel therapies for the treatment of Alzheimer's disease and other dementias. By carefully controlling the physicochemical properties of the benzazepine series and through the implementation of an aggressive and innovative screening strategy that employed high throughput in vivo assays to efficiently triage compounds, the medicinal chemistry effort was able to rapidly progress the benzazepine class of H3 antagonists through to the identification of clinical candidates with robust in vivo efficacy and excellent developability properties.

This Letter describes the discovery of a novel series of H3 receptor antagonists. The initial medicinal chemistry strategy focused on deconstructing and simplifying an early screening hit which rapidly led to the discovery of a novel series of H3 receptor antagonists based on the benzazepine core. Employing an H3 driven pharmacodynamic model, the series was then further optimised through to a lead compound that showed robust in vivo functional activity and possessed overall excellent developability properties.

Pharmacological and molecular biological studies indicate the existence of at least three human tachykinin receptor subtypes, designated neurokinin-1 (NK-1), neurokinin-2 (NK-2), and neurokinin-3 (NK-3),1-3 which belong to the superfamily of G-protein-coupled receptors possessing seven transmembrane domains.4 The endogenous ligands for these receptors constitute a family of small neuropeptides, named tachykinins or neurokinins, which share the common carboxy-terminal region Phe-X-Gly-Leu-MetNH2. The main mammalian tachykinins, substance P, neurokinin A (NKA), and neurokinin B (NKB), interact with all three tachykinin receptors, although there is a defined agonist rank order of potency for NK-1, NK-2, and NK-3 receptors, respectively; for example, for the NK-3 receptor the rank potency order is NKB > NKA > substance P. Over the past few years potent and selective peptide and non-peptide antagonists for the NK-1 and NK-2 receptors have been identified.5-8 These pharmacological tools accelerated the clarification of physiological and pathophysiological roles of these receptors9 and the potential therapeutic indications for NK-1 and NK-2 receptor antagonists.10-13 In contrast to the NK-1 and NK-2 receptor research area, there is limited information on the biology and potential pathophysiological significance of the NK-3 receptor. This was, to a large extent, due to the lack of sufficient potency and selectivity of the peptide NK-3 antagonists described thus far14 and to the absence of non-peptide NK-3 receptor ligands until recently. However, the recent disclosure of the “peptoid” NK-3 antagonist PD 15767215 and the peptidederived PD 16118216 has provided improved reagents for these studies and stimulated the search for more potent and metabolically stable non-peptide NK-3 receptor antagonists. The human NK-3 (hNK-3) receptor mRNA has been detected, using polymerase chain reaction (PCR), to various regions in the central nervous system (CNS) and also, albeit to a lesser extent, in some peripheral tissues, including kidney, placenta, lung, and colon.17 Activation of NK-3 receptors modulates the release of various transmitters in the CNS and periphery,18-20 suggesting that they may have a neuromodulatory role. Recently, (S)-(+)-N-{{3-[1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl]prop-1-yl}-4-phenylpiperidin-4-yl}-Nmethylacetamide, SR 142801 (1, Chart 1), was reported as the first potent non-peptide NK-3 receptor antagonist.21,22 Chemically, SR 142801 derives from a constrained analog of SR 48968, a potent NK-2 receptor antagonist (Ki ) 0.51 nM, for displacement of [125I]NKA) in rat duodenum membranes,7 which has moderate affinity for NK-3 receptors (IC50 ) 320 nM, for displacement of [3H]senktide {succinyl-[Asp9MePhe8]SP(6-13)}) in guinea pig cerebral cortex membranes.23 We now report on the discovery of a novel class of potent and selective non-peptide NK-3 receptor antagonistssstructurally unrelated to the piperidine derivatives SR 142801 and SR 48968swhich are based on the 2-phenylquinoline backbone.24 Chemical synthesis (Scheme 1), radioligand binding affinities for the cloned human neurokinin receptors stably expressed in CHO cell lines (hNKs-CHO),17,25,26 in vitro functional activity in the rabbit isolated iris sphincter muscle preparation (antagonism of senktide-induced contraction),27 and structure-activity relationships (SARs) of the novel † Italy. ‡ USA. § England. © Copyright 1996 by the American Chemical Society

There is a high demand for potent, selective, and brain-penetrant small molecule inhibitors of leucine-rich repeat kinase 2 (LRRK2) to test whether inhibition of LRRK2 kinase activity is a potentially viable treatment option for Parkinson's disease patients. Herein we disclose the use of property and structure-based drug design for the optimization of highly ligand efficient aminopyrimidine lead compounds. High throughput in vivo rodent cassette pharmacokinetic studies enabled rapid validation of in vitro-in vivo correlations. Guided by this data, optimal design parameters were established. Effective incorporation of these guidelines into our molecular design process resulted in the discovery of small molecule inhibitors such as GNE-7915 (18) and 19, which possess an ideal balance of LRRK2 cellular potency, broad kinase selectivity, metabolic stability, and brain penetration across multiple species. Advancement of GNE-7915 into rodent and higher species toxicity studies enabled risk assessment for early development.

Background:Allosteric modulation of γ-secretase is an attractive therapeutic approach for the treatment of Alzheimer’s disease. We recently identified a novel γ-secretase modulator, GSM-10h, which effectively lowers Aβ42 production in cells and in amyloid precursor protein transgenic mice. Objective: Here, we describe the in vivo characterization of GSM-10h in a model of endogenous Aβ production. Methods: Rats were administered orally with GSM-10h, and the effect on Aβ levels in peripheral and central compartments was determined. In addition, the effect of GSM-10h on Notch processing was assessed. Results: Acute administration of GSM-10h to rats causes a dose-dependent decrease in the level of Aβ42 in plasma, CSF and brain, with little effect on the level of Aβ40 in these compartments. The magnitude of Aβ42 lowering in the CSF and brain was further enhanced upon sub-chronic administration of GSM-10h. No deleterious effect on Notch processing was evident in either of these studies. To further explore the dynamics of Aβ42 reduction in peripheral and CNS compartments, a time course study was conducted. In all compartments, the decrease in Aβ42 was greatest at 6 h after administration of GSM-10h. This decrease in Aβ42 was maintained for 9–15 h, after which time Aβ42 levels returned to baseline levels. Encouragingly, no rebound in Aβ42 levels beyond baseline levels was observed. Conclusions: These findings support the γ-secretase modulator profile of GSM-10h, and highlight the utility of the rat for assessing the pre-clinical efficacy of γ-secretase modulators.

Neuroinflammation and the activation of inducible nitric oxide synthase (iNOS) have been proposed to play a role in the pathogenesis of Parkinson disease (PD). In this study we investigated the effects of the selective iNOS inhibitor GW274150 in the 6-OHDA model of PD. 6-OHDA administration was associated with increased numbers of cells expressing iNOS. Administration of the iNOS inhibitor twice daily for 7 days, beginning 2 days after the 6-OHDA lesioning, led to a significant neuroprotection as shown by assessment of the integrity of the nigrostriatal system by tyrosine hydroxylase immunocytochemistry and HPLC assessment of striatal dopamine content. However, GW274150 displayed a bell-shaped neuroprotective profile, being ineffective at high doses. 6-OHDA lesioning was associated with an increase in microglial activation as assessed by the MHC II antigen OX-6 and the number of matrix metalloproteinase 9 (MMP-9)-immunopositive cells. NO is a known modulator of MMP-9, and iNOS inhibition was associated with decreased numbers of MMP-9-immunopositive cells, culminating in a reduction in the numbers of reactive microglia. Withdrawal of GW274150 for a further 7 days negated any neuroprotective effects of iNOS inhibition, suggesting that the damaging effects of inflammation last beyond 7 days in this model and the continued administration of the drug may be required.

Background Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear hormone receptor that has been shown to have anti-inflammatory and matrix metalloproteinase (MMP) inhibitor properties. PPARγ agonists have been shown to have neuroprotective effects in various neurodegeneration models where inflammation is implicated, including models of Parkinson's disease. However, no studies have looked at the effects of partial PPARγ agonists. Experimental approach The neuroprotective effects of the PPARγ full agonist, pioglitazone (20 mg/kg), partial PPARγ agonist GW855266X (15 mg/kg) and PPAR-δ full agonist GW610742X (10 mg/kg) were investigated in the 6-hydroxydopamine (6-OHDA) model of Parkinson's disease when administered prior to or post 6-OHDA lesioning. The integrity of the nigrostriatal system was assessed by assessing the numbers dopaminergic neurons in the substantia nigra (SN) and by assessing striatal dopamine content. The degree of microglia activation in the SN was also immunohistochemistry assessed utilizing the marker OX-6 for activated microglia and CD-68 a marker for phagocytic microglia. Additionally we performed immunocytochemistry for MMP3 in the SN. Finally, we investigated whether a period of drug withdrawal for a further 7 days affected the neuroprotection produced by the PPARγ agonists. Key results Both Pioglitazone and GW855266X protected against 6-OHDA induced loss of dopaminergic neurons in the substantia nigra and depletion of striatal dopamine when administered orally twice daily for either 1) 7 day prior to and 7 days post lesioning or 2) for 7 days starting 2 days post lesioning when neurons will be severely traumatized. 6-OHDA lesioning was associated with an increase in microglia activation and in numbers of MMP-3 immunoreactive cells which was attenuated by pioglitazone and GW855266X. Neuroprotective effects were not replicated using the PPARδ agonist GW610742X. Subsequent withdrawal of both Pioglitazone and GW855266X, for a further 7 days negated any neuroprotective effect suggesting that long-term administration may be required to attenuate the inflammatory response. Conclusions and implications For the first time a partial PPAR-γ agonist has been shown to be neuroprotectory when administered post lesioning in a Parkinsonian model. Effects may be via the inhibition of microglial and MMP activation and support further research.

The histamine H3 receptor is a G-protein–coupled presynaptic auto- and heteroreceptor whose activation leads to a decrease in the release of several neurotransmitters including histamine, acetycholine, noradrenaline, and dopamine. H3 receptor antagonists such as 6-[(3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride (GSK189254) can increase the release of these neurotransmitters and thus may offer potential therapeutic benefits in diseases characterized by disturbances of neurotransmission. The aim of this study was to synthesize and evaluate 11C-labeled GSK189254 (11C-GSK189254) for imaging the histamine H3 receptor in vivo by PET. Methods: GSK189254 exhibits high affinity (0.26 nM) and selectivity for the human histamine H3 receptor. Autoradiography experiments were performed using 3H-GSK189254 to evaluate its in vitro binding in porcine brain tissues. GSK189254 was labeled by N-alkylation using 11C-methyl iodide in good yields, radiochemical purity, and specific activity. A series of PET experiments was conducted to investigate 11C-GSK189254 binding in the porcine brain. Results: In vitro autoradiography demonstrated specific 3H-GSK189254 binding in the porcine brain; therefore, 11C-GSK189254 was evaluated in vivo in pigs and showed good brain penetration and high uptake in regions such as the striatum and cortices, known to contain high densities of the histamine H3 receptors. The radioligand kinetics were reversible, and quantitative analysis was achieved with a 2-tissue-compartmental model yielding the distribution volume as the outcome measure of interest. The distribution volume was reduced to a homogeneous level in all regions after blocking by the coadministration of either unlabeled GSK189254 or ciproxifan, a structurally distinct histamine H3 antagonist. Further coadministration studies allowed for the estimation of the radioligand affinity (0.1 nM) and the density of histamine H3 receptor sites in the cerebellum (0.74 nM), cortex (2.05 nM), and striatum (2.65 nM). Conclusion: These findings suggest that 11C-GSK189254 possesses appropriate characteristics for the in vivo imaging of the histamine H3 receptor by PET.

Histaminergic neurons of the hypothalamic tuberomammillary nuclei (TMN) send projections to the whole brain. Early anatomical studies described histaminergic neurons as a homogeneous cell group, but recent evidence indicates that histaminergic neurons are heterogeneous and organized into distinct circuits. We addressed this issue using the double-probe microdialysis in freely moving rats to investigate if two compounds acting directly onto histaminergic neurons to augment cell firing [thioperamide and bicuculline, histamine H(3)- and gamma-aminobutyric acid (GABA)(A)-receptor (R) antagonists, respectively] may discriminate groups of histaminergic neurons impinging on different brain regions. Intra-hypothalamic perfusion of either drug increased histamine release from the TMN and cortex, but not from the striatum. Thioperamide, but not bicuculline, increased histamine release from the nucleus basalis magnocellularis (NBM), bicuculline but not thioperamide increased histamine release from the nucleus accumbens (NAcc). Intra-hypothalamic perfusion with thioperamide increased the time spent in wakefulness. To explore the local effects of H(3)-R blockade in the histaminergic projection areas, each rat was implanted with a single probe to simultaneously administer thioperamide and monitor local changes in histamine release. Thioperamide increased histamine release from the NBM and cortex significantly, but not from the NAcc or striatum. The presence of H(3)-Rs on histaminergic neurons was assessed using double-immunofluorescence with anti-histidine decarboxylase antibodies to identify histaminergic cells and anti-H(3)-R antibodies. Confocal analysis revealed that all histaminergic somata were immunopositive for the H(3)-R. This is the first evidence that histaminergic neurons are organized into functionally distinct circuits that influence different brain regions, and display selective control mechanisms.

To test the novel nonimidazole histamine H3 receptor antagonist 5-[(3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazapin-7-yl)oxy]-N-methyl-2-pyrazinecarboxamide (GSK207040) in a series of behavioral and neurochemical paradigms designed to evaluate its antipsychotic potential.Acute orally administered GSK207040 was investigated for its capacity to reverse a 24-h-induced deficit in novel object recognition memory, deficits in prepulse inhibition (PPI) induced by isolation rearing, and hyperlocomotor activity induced by amphetamine. The acute neurochemical effects of GSK207040 were explored by analyzing rat anterior cingulate cortex microdialysates for levels of dopamine, noradrenaline, and acetylcholine and by c-fos immunohistochemistry. The potential for interaction with the antipsychotic dopamine D2 receptor antagonist haloperidol was explored behaviorally (spontaneous locomotor activity and catalepsy), biochemically (plasma prolactin), and via ex vivo receptor occupancy determinations.GSK207040 significantly enhanced object recognition memory (3 mg/kg) and attenuated isolation rearing-induced deficits in PPI (1.0 and 3.2 mg/kg) but did not reverse amphetamine-induced increases in locomotor activity. There was no evidence of an interaction of GSK207040 with haloperidol. GSK207040 (3.2 mg/kg) raised extracellular concentrations of dopamine, noradrenaline, and acetylcholine in the anterior cingulate cortex and c-fos expression in the core of the nucleus accumbens was increased at doses of 3.2 and 10.0 mg/kg.The behavioral and neurochemical profile of GSK207040 supports the potential of histamine H3 receptor antagonism to treat the cognitive and sensory gating deficits of schizophrenia. However, the failure of GSK207040 to reverse amphetamine-induced locomotor hyperactivity suggests that the therapeutic utility of histamine H(3) receptor antagonism versus positive symptoms is less likely, at least following acute administration.

The human 5-hydroxytryptamine (5-HT(4)) receptor is encoded by a highly complex gene which gives rise to at least 10 distinct splice variants. However, the functional relevance of these variants is unknown. In rat, only three such variants have been identified, 5-HT(4a) (r5-HT(4a)), 5-HT(4b) (r5-HT(4b)) and 5-HT(4e) (r5-HT(4e)). In the current study we identify and characterise the pharmacology of a novel rat splice variant (r5-HT(4c1)) and present the first comprehensive analysis of 5-HT(4) splice variant mRNA expression levels throughout the rat gastrointestinal tract. In addition, we describe preliminary characterisation of the first 5-HT(4) splice variant specific antibodies. In transfected cells, r5-HT(4c1) receptor exhibited similar binding properties to r5-HT(4a) and r5-HT(4b). Functional studies showed that 5-HT(4) agonists prucalopride (4-amino-5-chloro-2,3-dihydro-N-[1-(3-methoxypropyl)-4-piperidinyl]-7-benzofuran carboxamide monohydrochloride and renzapride (+/-)-endo-4-amino-5-chloro-2-methoxy-N-(1-azabicyclo[3.3.1]non-4-yl)benzamide monohydrochloride) acted as partial agonists at r5-HT(4c1), but full agonists at r5-HT(4a) and r5-HT(4b). Moreover, in contrast to r5-HT(4a) and r5-HT(4b), r5-HT(4c1) was not constitutively active. TaqMan mRNA analysis showed that r5-HT(4a) expression in brain and dorsal root ganglion exceeded that in the gastrointestinal tract, whilst the reverse was true for r5-HT(4b) and r5-HT(4c1). mRNA expression of each variant also increased distally throughout the gastrointestinal tract with the highest levels in the colon. r5-HT(4a) and r5-HT(4b) specific immunoreactivity was abundant on enteric neurons in jejunum, ileum and colon as well as neurons and satellite cells of the dorsal root ganglion. Only r5-HT(4b) immunoreactivity was observed on endocrine cells in the duodenum. These data could have implications in rat models and aid understanding of 5-HT(4) splice variant function.

The possible neuroprotective role of a novel and highly selective cyclooxygenase-2 inhibitor GW637185X was studied in a model of acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced injury of nigrostriatal dopaminergic (DA) neurons in the mouse. Stereological and microdensitometrical analysis of nigral tyrosine hydroxylase-immunoreactive cell bodies and striatal tyrosine hydroxylase-immunoreactive terminals, respectively, showed that GW637185X exerted a full protection against MPTP-induced degeneration of the nigro-striatal pathway. In contrast to earlier studies, these findings demonstrate that acute inhibition of cyclooxygenase-2 can result in a full neuroprotective effect not only on nigral DA cell bodies, but also on striatal DA terminals in the mouse MPTP model.

The possible neuroprotective role of a novel and highly selective cyclooxygenase-2 inhibitor GW637185X was studied in a model of acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced injury of nigrostriatal dopaminergic (DA) neurons in the mouse. Stereological and microdensitometrical analysis of nigral tyrosine hydroxylase-immunoreactive cell bodies and striatal tyrosine hydroxylase-immunoreactive terminals, respectively, showed that GW637185X exerted a full protection against MPTP-induced degeneration of the nigro-striatal pathway. In contrast to earlier studies, these findings demonstrate that acute inhibition of cyclooxygenase-2 can result in a full neuroprotective effect not only on nigral DA cell bodies, but also on striatal DA terminals in the mouse MPTP model.

Several studies have implicated a potential role for histamine H 3 receptors in pain processing, although the data are somewhat conflicting. In the present study we investigated the effects of the novel potent and highly selective H 3 receptor antagonists GSK189254 (6-[(3-cyclobutyl-2,3,4,5-tetrahydro-1 H -3-benzazepin-7-yl)oxy]- N -methyl-3-pyridinecarboxamide hydrochloride) and GSK334429 (1-(1-methylethyl)-4-({1-[6-(trifluoromethyl)-3-pyridinyl]-4-piperidinyl}carbonyl)hexahydro-1 H -1,4-diazepine) in two rat models of neuropathic pain, namely the chronic constriction injury (CCI) model and the varicella-zoster virus (VZV) model. Both GSK189254 (0.3, 3 and/or 10 mg/kg p.o.) and GSK334429 (1, 3 and 10 mg/kg p.o.) significantly reversed the CCI-induced decrease in paw withdrawal threshold (PWT) measured using an analgesymeter and/or von Frey hairs. In addition, GSK189254 (3 mg/kg p.o.) and GSK334429 (10 mg/kg p.o.) both reversed the VZV-induced decrease in PWT using von Frey hairs. We also investigated the potential site of action of this analgesic effect of H 3 antagonists using autoradiography. Specific binding to H 3 receptors was demonstrated with [ 3 H]-GSK189254 in the dorsal horn of the human and rat spinal cord, and in human dorsal root ganglion (DRG), consistent with the potential involvement of H 3 receptors in pain processing. In conclusion, we have shown for the first time that chronic oral administration of selective H 3 antagonists is effective in reversing neuropathic hypersensitivity in disease-related models, and that specific H 3 receptor binding sites are present in the human DRG and dorsal horn of the spinal cord. These data suggest that H 3 antagonists such as GSK189254 and GSK334429 may be useful for the treatment of neuropathic pain.

6-[(3-Cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride (GSK189254) is a novel histamine H(3) receptor antagonist with high affinity for human (pK(i) = 9.59 -9.90) and rat (pK(i) = 8.51-9.17) H(3) receptors. GSK189254 is >10,000-fold selective for human H(3) receptors versus other targets tested, and it exhibited potent functional antagonism (pA(2) = 9.06 versus agonist-induced changes in cAMP) and inverse agonism [pIC(50) = 8.20 versus basal guanosine 5'-O-(3-[(35)S]thio)triphosphate binding] at the human recombinant H(3) receptor. In vitro autoradiography demonstrated specific [(3)H]GSK189254 binding in rat and human brain areas, including cortex and hippocampus. In addition, dense H(3) binding was detected in medial temporal cortex samples from severe cases of Alzheimer's disease, suggesting for the first time that H(3) receptors are preserved in late-stage disease. After oral administration, GSK189254 inhibited cortical ex vivo R-(-)-alpha-methyl[imidazole-2,5(n)-(3)H]histamine dihydrochloride ([(3)H]R-alpha-methylhistamine) binding (ED(50) = 0.17 mg/kg) and increased c-Fos immunoreactivity in prefrontal and somatosensory cortex (3 mg/kg). Microdialysis studies demonstrated that GSK189254 (0.3-3 mg/kg p.o.) increased the release of acetylcholine, noradrenaline, and dopamine in the anterior cingulate cortex and acetylcholine in the dorsal hippocampus. Functional antagonism of central H(3) receptors was demonstrated by blockade of R-alpha-methylhistamine-induced dipsogenia in rats (ID(50) = 0.03 mg/kg p.o.). GSK189254 significantly improved performance of rats in diverse cognition paradigms, including passive avoidance (1 and 3 mg/kg p.o.), water maze (1 and 3 mg/kg p.o.), object recognition (0.3 and 1 mg/kg p.o.), and attentional set shift (1 mg/kg p.o.). These data suggest that GSK189254 may have therapeutic potential for the symptomatic treatment of dementia in Alzheimer's disease and other cognitive disorders.

The mGluR5 antagonist MPEP was used to study the role of mGluR5 in MPTP-induced injury of the nigrostriatal DA neurons. The findings indicate that acute blockade of mGluR5 may result in neuroprotective actions against MPTP neurotoxicity on nigral DA cell bodies and striatal DA terminals using stereological analysis of TH immunoreactivity and microdensitometry. Biochemical analysis showed no restoration of DA levels and metabolism indicating a maintained reduction of DA transmission.