Brian Lockhart

Neuroprotective and anti-amnesic potentials of sigma (σ) receptor ligands

Abstract 1. 1. Although the physical nature of sigma (σ) receptors have not yet been fully defined, several classes of selective ligands have been characterised, demonstrating a plethora of physiological actions. In the present review, the authors have set out to highlight two important aspects of the biological activities of σ ligands, their neuroprotective and anti-amnesic effects. 2. 2. The σ ligands present a therapeutic potential as neuroprotective agents in brain ischemia. The neuroprotective activity of many non-selective σ ligands is primarily a result of their affinity for the NMDA receptor complex. However, selective σ ligands are also neuroprotective, possibly by inhibition of the ischemic-induced presynaptic release of excitotoxic amino acids. 3. 3. The σ 1 ligands prevent the experimental amnesia induced by muscarinic cholinergic antagonists at either the learning, consolidation or retention phase of the mnesic process. This effect involves a potentation of acetylcholine release induced by σ 1 ligands selectively in the hippocampal formation and cortex. 4. 4. The σ 1 receptor ligands also attenuate the learning impairment induced by dizocilpine, a non-competitive antagonist of the NMDA receptor, and may relate to the potentiating effect of σ 1 ligands on several NMDA receptor-mediated responses previously described in vitro and in vivo in the hippocampus. This effect is shared by NPY- and CGRP-related peptides and by neuroactive steroids, confirming the in vitro evidences of functional interactions between the σ 1 receptors and these different systems. 5. 5. Additional amnesia models also seem to be alleviated by σ 1 ligands, such as phencyclidine-induced cognitive dysfunctions, and amnesia induced by the calcium channel blocker nimodipine, or by exposure to carbon monoxide. Furthermore, a preliminary study in an animal model of age-related memory deficits, the senescence-accelerated mouse, strengthened the therapeutic potentials of selective σ 1 receptor ligands in aging-related pathologies.

Distinct neuroprotective profiles for σ ligands against N-methyl-d-aspartate (NMDA), and hypoxia-mediated neurotoxicity in neuronal culture toxicity studies

Substantiating evidence has raised the possibility that sigma ligands may have therapeutic potential as neuroprotective agents in brain ischemia. It has been suggested that the neuroprotective capacity of sigma ligands is related primarily to their affinity for the NMDA receptor complex and not to any selective action at the sigma binding site. However, sigma specific ligands, devoid of significant affinity for the NMDA receptor, are also neuroprotective via an inhibition of the ischemic-induced presynaptic release of excitotoxic amino acids. In the present study, we have investigated the potential neuroprotective effect of a comprehensive series of sigma ligands, with either significant (sigma/PCP) or negligible (sigma) affinity for the PCP site of the NMDA receptor, in order to delineate a selective sigma site-dependent neuroprotective effect. For this aim, we have employed two different neuronal culture toxicity paradigms implicating either postsynaptic-mediated neurotoxicity, (brief exposure of cultures to a low concentration of NMDA or Kainate) or pre- and postsynaptic mechanisms (exposure to hypoxic/hypoglycemic conditions). Only sigma ligands with affinity for the NMDA receptor [(+) and (-) cyclazocine, (+) pentazocine, (+) SKF-10047, ifenprodil and haloperidol] were capable of attenuating NMDA-induced toxicity whereas the sigma [(+)BMY-14802, DTG, JO1784, JO1783, and (+)3-PPP] and kappa-opioid [CI-977, U-50488H] ligands, with very low affinity for the NMDA receptor, were inactive. The rank order of potency, based on the 50% protective concentration (PC50) value, of sigma/PCP ligands against NMDA-mediated neurotoxicity correlates with their affinity for the PCP site of the NMDA receptor, and not with their affinity for the sigma site. In addition sigma/PCP, sigma or kappa-opioid ligands failed to attenuate kainate-mediated neurotoxicity. On the other hand, sigma/PCP, sigma and kappa-opioid ligands were potent inhibitors of hypoxia/hypoglycemia-induced neurotoxicity, although their neuroprotective potency did not correlate with their affinity for either the sigma or PCP binding sites. In conclusion, the ability of sigma and kappa-opioid ligands to attenuate hypoxia/hypoglycemia, but not NMDA or kainate-induced toxicity, suggests that these drugs exert their neuroprotective role by a predominantly presynaptic mechanism possibly by inhibiting ischemic-mediated glutamate release.

Reversion of β25–35-amyloid peptide-induced amnesia by NMDA receptor-associated glycine site agonists

The effects of D-cycloserine (DCS), a N-methyl-D-aspartate receptor-associated glycine site agonist, and milacemide (MIL), a glycine prodrug, were examined on learning impairments induced by administration of beta 25-35-amyloid peptide (3 nmol i.c.v.). Mice were examined for spontaneous alternation and step-down passive avoidance, 7 and 14 days after beta 25-35, respectively. The beta 25-35-induced deficits were reversed by DCS, 1-30 mg/kg i.p., or MIL, 3-100 mg/kg i.p., each drug being ineffective on control mice behaviours. These observations strengthen the therapeutic potential of glycine site agonists against the memory impairments induced by beta-amyloid peptides.

Nox4 mediates the expression of plasminogen activator inhibitor-1 via p38 MAPK pathway in cultured human endothelial cells

INTRODUCTION Plasminogen Activator Inhibitor-1 (PAI-1) is the most potent endogenous inhibitor of fibrinolysis which is implicated in the pathogenesis of myocardial infarction and metabolic syndrome. The formation of reactive oxygen species (ROS) plays an important role in the pathology of vascular disorders and has been shown to increase PAI-1 expression by endothelial cells. Growing evidence indicates that NADPH oxidase and in particular the constitutively active Nox4-p22(phox) complexes are major sources of ROS in endothelial cells. The aim of the present study was to characterize the role of NADPH oxidase and in particular Nox4 in the regulation of PAI-1 expression in cultured Human Umbilical Venous Endothelial Cells (HUVECs). METHODS AND RESULTS N-acetylcysteine (NAC, scavenger of ROS), diphenylene iodonium chloride (DPI, inhibitor of flavoproteins), M40403 (superoxyde dismutase mimic) and S17834 (inhibitor of NADPH oxidase) inhibited PAI-1 release and promoter activity in HUVECs. Specific knock down of Nox4 mRNA by siRNA caused a decrease in ROS production and NADPH oxidase activity. Moreover, Nox4 silencing decreased PAI-1 expression, release and activity as well as p38 MAPK pathways and NFkappaB activation. These signalling pathways are also involved in PAI-1 release. CONCLUSIONS The NADPH oxidase inhibitors DPI and S 17834 as well as Nox4 silencing decreased PAI-1 synthesis in human cultured endothelial cells demonstrating the involvement of the constitutively active Nox4-containing NADPH oxidase in ROS-mediated PAI-1 transcription via p38 MAPK pathways. NADPH oxidase targeting with inhibitors such as S17834 could be an interesting strategy to decrease both oxidative stress and PAI-1 synthesis.

S49076 is a Novel Kinase Inhibitor of MET, AXL and FGFR with Strong Preclinical Activity Alone and in Association with Bevacizumab

Aberrant activity of the receptor tyrosine kinases MET, AXL, and FGFR1/2/3 has been associated with tumor progression in a wide variety of human malignancies, notably in instances of primary or acquired resistance to existing or emerging anticancer therapies. This study describes the preclinical characterization of S49076, a novel, potent inhibitor of MET, AXL/MER, and FGFR1/2/3. S49076 potently blocked cellular phosphorylation of MET, AXL, and FGFRs and inhibited downstream signaling in vitro and in vivo. In cell models, S49076 inhibited the proliferation of MET- and FGFR2-dependent gastric cancer cells, blocked MET-driven migration of lung carcinoma cells, and inhibited colony formation of hepatocarcinoma cells expressing FGFR1/2 and AXL. In tumor xenograft models, a good pharmacokinetic/pharmacodynamic relationship for MET and FGFR2 inhibition following oral administration of S49076 was established and correlated well with impact on tumor growth. MET, AXL, and the FGFRs have all been implicated in resistance to VEGF/VEGFR inhibitors such as bevacizumab. Accordingly, combination of S49076 with bevacizumab in colon carcinoma xenograft models led to near total inhibition of tumor growth. Moreover, S49076 alone caused tumor growth arrest in bevacizumab-resistant tumors. On the basis of these preclinical studies showing a favorable and novel pharmacologic profile of S49076, a phase I study is currently underway in patients with advanced solid tumors. Mol Cancer Ther; 12(9); 1749–62. ©2013 AACR.

(S)-2,3-dihydro-[3,4]cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide: (S18986-1) a positive modulator of AMPA receptors enhances (S)-AMPA-mediated [3H]noradrenaline release from rat hippocampal and frontal cortex slices

The present study describes the effect of (S)-2,3-dihydro-[3, 4]cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide (S18986-1), a positive allosteric modulator of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors with cognitive-enhancing effects, on (S)-AMPA-induced [3H]noradrenaline release in rat hippocampal and frontal cortex slices. (S)-AMPA significantly increased [3H]noradrenaline release in rat hippocampus and frontal cortex slices, whereas S18986-1 (3-1000 microM) alone, was inactive. However, S18986-1 between 30 and 1000 microM potently enhanced (+200%) (S)-AMPA-mediated [3H]noradrenaline release in both hippocampal and frontal cortex slices. The capacity of S18986-1 to potentiate [3H]noradrenaline release was specific for AMPA receptors as S18986-1 failed to potentiate either kainate and N-methyl-D-aspartate (NMDA)-mediated release of [3H]noradrenaline in rat hippocampal slices. Moreover, 1, 2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX) and 1-(4-aminophenyl)-3-methylcarbamoyl-4-methyl-3, 4-dihydro-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI-53655) but not (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine ((+)-MK-801), inhibited (S)-AMPA and S18986-induced stimulation of (S)-AMPA-mediated [3H]noradrenaline release. In addition, S18986-1-induced stimulation of (S)-AMPA-evoked [3H]noradrenaline release was markedly attenuated in the presence of tetrodotoxin (1 microM) and in Ca(2+)-free buffer. S18986-1 enhanced (S)-AMPA-mediated [3H]noradrenaline release to a greater extent than its corresponding (R)-enantiomer S19024-1 and racemic mixture S17951-1. However, positive allosteric modulators of AMPA receptors such as aniracetam failed to potentiate AMPA-mediated noradrenaline release in hippocampal slices, whereas cyclothiazide potently enhanced (S)-AMPA-mediated [3H]noradrenaline release. These results suggest that the capacity of S18986-1 to enhance AMPA receptor-mediated release of noradrenaline in rat hippocampus and frontal cortex, could contribute to the cognition enhancing mechanisms of S18986-1.

Synthesis and pharmacological evaluation of new 1,2-dithiolane based antioxidants

Molecules containing a dithiolane moiety are widely investigated due to their antioxidant properties. The archetypal representative of this class of compounds is lipoic acid and indeed the lipoic acid-dihydrolipoic acid couple is part of the antioxidant defence system of the cell. In the course of a program aiming to find improved antioxidants effective in vivo, we designed, synthesised and pharmacologically investigated new lipoic acid analogs. The salient feature of these structures is the connection, via a thioamide or a thiocarbamate, of a 1,2-dithiolane moiety bearing a carbon chain and a N-alkyl-substituted morpholine ring. It was expected that the antioxidant and chelating properties of these functional groups combined with the basicity of the morpholine ring will impact on the antioxidant as well as on the partition and solubility characteristics of the compounds. Indeed in vitro and in vivo pharmacological investigation showed that these new molecules and especially those containing a thiocarbamate linker possess superior antioxidant properties compared with alpha-lipoic acid and to the amide or carbamate linker analogs. In particular, some of these compounds efficiently cross the blood brain barrier (BBB) thus providing efficient protection from lethality in a situation of induced oxidative stress. Moreover the absence of the 1,2-dithiolane moiety does not completely abolish antioxidant effects thus demonstrating that these compounds are distinct new chemical entities and not merely lipoic acid prodrugs. The chemical and pharmacological features of these new antioxidants are presented and discussed in the following paper.

RhoA guanine exchange factor expression profile in arteries: evidence for a Rho kinase-dependent negative feedback in angiotensin II-dependent hypertension

Sustained overactivation of RhoA is a common component for the pathogenesis of several cardiovascular disorders, including hypertension. Although activity of Rho proteins depends on Rho exchange factors (Rho-GEFs), the identity of Rho-GEFs expressed in vascular smooth muscle cells (VSMC) and participating in the control of Rho protein activity and Rho-dependent functions remains unknown. To address this question, we analyzed by quantitative RT-PCR the expression profile of 28 RhoA-GEFs in arteries of normotensive (saline-treated) and hypertensive (ANG II-treated) rats. Sixteen RhoA-GEFs were downregulated in mesenteric arteries of hypertensive rats, among which nine are also downregulated in cultured VSMC stimulated by ANG II (100 nM, 48 h), suggesting a direct effect of ANG II. Inhibition of type 1 ANG II receptors (losartan, 1 μM) or Rho kinase (fasudil, 10 μM) prevented ANG II-induced RhoA-GEF downregulation. Functionally, ANG II-induced downregulation of RhoA-GEFs is associated with decreased Rho kinase activation in response to endothelin-1, norepinephrine, and U-46619. This work thus identifies a group of RhoA-GEFs that controls RhoA and RhoA-dependent functions in VSMC, and a negative feedback of RhoA/Rho kinase activity on the expression of these RhoA-GEFs that may play an adaptative role to limit RhoA/Rho kinase activation.

Targeting Bcl-2/Bcl-XL Induces Antitumor Activity in Uveal Melanoma Patient-Derived Xenografts

Purpose Uveal melanoma (UM) is associated with a high risk of metastases and lack of efficient therapies. Reduced capacity for apoptosis induction by chemotherapies is one obstacle to efficient treatments. Human UM is characterized by high expression of the anti-apoptotic protein Bcl-2. Consequently, regulators of apoptosis such as Bcl-2 family inhibitors may constitute an attractive approach to UM therapeutics. In this aim, we have investigated the efficacy of the Bcl-2/Bcl-XL inhibitor S44563 on 4 UM Patient-Derived Xenografts (PDXs) and derived-cell lines. Experimental Design Four well characterized UM PDXs were used for in vivo experiments. S44563 was administered alone or combined with fotemustine either concomitantly or after the alkylating agent. Bcl-2, Bcl-XL, and Mcl-1 expressions after S44563 administration were evaluated by immunohistochemistry (IHC). Results S44563 administered alone by at 50 and 100 mg/kg i.p. induced a significant tumour growth inhibition in only one xenograft model with a clear dose effect. However, when S44563 was concomitantly administered with fotemustine, we observed a synergistic activity in 3 out of the 4 tested models. In addition, S44563 administered after fotemustine induced a tumour growth delay in 2 out of 3 tested xenografts. Finally, IHC analyses showed that Bcl-2, Bcl-XL, and Mcl-1 expression were not modified after S44563 administration. Conclusion The novel anti-apoptotic experimental compound S44563, despite a relative low efficacy when administered alone, increased the efficacy of fotemustine in either concomitant or sequential combinations or indeed subsequent to fotemustine. These data support further exploration of potential therapeutic effect of Bcl-2/Bcl-xl inhibition in human UM.

Protective effect of the antioxidant 6-ethoxy-2,2-pentamethylen-1,2-dihydroquinoline (S 33113) in models of cerebral neurodegeneration.

In a previous study Dorey et al. [Bio. Org. Chem. Lett., 10 (2000) 935] a series of novel dihydroquinoline compounds were developed, based on the potent antioxidant 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline (ethoxyquin), and permitted the selection of the analogue 6-ethoxy-2,2-pentamethylen-1,2-dihydroquinoline (S 33113) lacking the hypothermic effects associated with ethoxyquin at equivalent doses. Herein, an extensive investigation of the neuroprotective capacity of S 33113 in different in vitro and in vivo paradigms of oxidative stress-mediated cellular degeneration was undertaken. In vitro S 33113 was a potent inhibitor (IC(50) = 0.29 microM) of Fenton-reaction-induced lipid peroxidation in mouse cortical membranes. Administration of S 33113 either intraperitoneally (< or =150 mg/kg i.p.) or orally (< or =600 mg/kg p.o.) did not significantly modify body temperature in NMRI mice. Furthermore, S 33113 (150 mg/kg i.p. or 600 mg/kg p.o.) markedly reduced the lethality induced by an intracerebroventricular injection of t-butylhydroperoxide in NMRI (naval medical research institute) mice for up to 5 h. Oral administration of S 33113, significantly attenuated alloxan-mediated hyperglycaemia in NMRI mice at 400 and 600 mg/kg (60%; P < 0.001). Administration of S 33113 (150 mg/kg i.p.) 30 min before transient global ischaemia significantly prevented delayed neuronal cell death in the CA1 region of the rat hippocampal formation, 7 days post-ischaemia (33% cell loss vs. 88% in ischaemia controls; P < 0.001). Similarly, a single pre-administration of S 33113 (150 mg/kg i.p.) prevented kainic acid-induced cell death in the CA3 hippocampal region at 7 days post-exposure (17% cell loss vs. 52% in kainate-treated controls; P < 0.01). Furthermore, D-methamphetamine-mediated dopamine depletion in the striatum of C57BL/6 mice (39-46%) was significantly prevented with S 33113 administered at either (2 x 150mg/kg i.p.) (11%; P < 0.01) or (2x150 mg/kg p.o.) (17%; P < 0.001). In conclusion, S 33113 represents a novel dihydroquinoline compound with potential for the treatment of cerebral pathologies implicating chronic neurodegeneration.