mGlu1 Receptor (IC50 = 160 nM); mGluR1a (IC50 = 280 nM); mGluR2 (IC50 = 140 nM); mGluR 5 (IC50 = 240 nM)

At 10 μM, BAY 36-7620 (0.1-10 μM) totally prevents mGlu1 capture in HEK 293 cells [1]. BAY 36-7620 (0.1-10 μM, 4 days) decreases tumor growth and A549 cell engraftment. The growth and proliferation of MCF-7, T-47D, BT-474, MDA-MB-231, Hs578T, and BT-549 cells are inhibited by BAY 36-7620 (72 hours), and their IC50s are related protein expressions [2]. In T-47D, BT-474, MDA-MB-231, and BT-549 cells, BAY 36-7620 (25-50 μM, 24-72 h) causes 27.7, 37.1, 20.8, 41.0, 21.0, and 15.7μM [3]. significant G2/M phase arrest and DNA damage [3].

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L-Glutamate (Glu) activates at least eight different G protein-coupled receptors known as metabotropic glutamate (mGlu) receptors, which mostly act as regulators of synaptic transmission. These receptors consist of two domains: an extracellular domain in which agonists bind and a transmembrane heptahelix region involved in G protein activation. Although new mGlu receptor agonists and antagonists have been described, few are selective for a single mGlu subtype. Here, we have examined the effects of a novel compound, BAY 36-7620 [(3aS,6aS)- 6a-Naphtalen-2-ylmethyl-5-methyliden-hexahydro-cyclopental[c]furan-1-on], on mGlu receptors (mGlu1-8), transiently expressed in human embryonic kidney 293 cells. BAY 36-7620 is a potent (IC(50) = 0.16 microM) and selective antagonist at mGlu1 receptors and inhibits >60% of mGlu1a receptor constitutive activity (IC(50) = 0.38 microM). BAY36-7620 is therefore the first described mGlu1 receptor inverse agonist. To address the mechanism of action of BAY36-7620, Glu dose-response curves were performed in the presence of increasing concentrations of BAY36-7620. The results show that BAY36-7620 largely decreases the maximal effect of Glu. Moreover, BAY36-7620 did not displace the [(3)H]quisqualate binding from the Glu-binding pocket, further indicating that BAY36-7620 is a noncompetitive mGlu1 antagonist. Studies of chimeric receptors containing regions of mGlu1 and regions of DmGluA, mGlu2, or mGlu5, revealed that the transmembrane region of mGlu1 is necessary for activity of BAY36-7620. Transmembrane helices 4 to 7 are shown to play a critical role in the selectivity of BAY36-7620. This specific site of action of BAY36-7620 differs from that of competitive antagonists and indicates that the transmembrane region plays a pivotal role in the agonist-independent activity of this receptor. BAY36-7620 will be useful to further delineate the functional importance of the mGlu1 receptor, including its putative agonist-independent activity. [1]

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Role of mGlu1 receptor/AKT in A549 cells [2]
A549 cells were treated with BAY 36-7620 (10 and 25 μM) or vehicle (0.05% DMSO, control). Treatment with BAY36-7620 reduced cellular proliferation of A549 cells when compared to control group (Fig. 4(A)). Similar result was also found in SK-MES-1 cells (Fig. S3, shown in the supplementary data). In addition, incubation with BAY36-7620 had no significant effect on cell viability of 16HBE.
A549 cells were treated with BAY 36-7620 (an mGlu1 receptor-specific inhibitor, 25 μM), L-quisqualate (a potent mGlu1 receptor agonist, 10 μM) or MK2206 (an AKT inhibitor, 5 μM), either alone or in combination. Following 4 days of incubation, treatment of A549 cells with BAY36-7620 enhanced cleaved PARP levels and reduced bcl-2 protein expression. Treatment of A549 cells with L-quisqualate reduced cleaved PARP levels and enhanced bcl-2 protein expression; Co-incubation with MK2206 blocked the effect of L-quisqualate on bcl-2, but did not affect its effect on cleaved PARP (Fig. 4(B)). Under condition of hypoxia, A549 cells were treated with BAY36-7620 (25 μM), L-quisqualate (10 μM) or MK2206 (5 μM), either alone or in combination. Following 4 days incubation, treatment of A549 cells with BAY36-7620 reduced HIF-1α (Fig. 4(C)) protein expression and HIF activity (Fig. 4(D)) and secretion of VEGF (Fig. 4(E)) and IL-8 (Fig. 4(F)) into supernatants. Treatment of A549 cells with L-quisqualate enhanced HIF-1α protein expression and HIF activity and secretion of VEGF and IL-8 in supernatants, which was abolished by co-incubation with MK2206.

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Role of mGlu1 receptor/AKT in HUVECs [2]
HUVECs were pre-treated for 30 min with BAY 36-7620 (25 μM) or not, then they were stimulated with VEGF (100 ng/ml). VEGF stimulation led to increased AKT phosphorylation levels. However, pre-treatment with BAY36-7620 prior to VEGF stimulation blocked this increase in AKT phosphorylation levels (Fig. 5(A)), indicating that BAY36-7620 treatment blocked VEGF/AKT signaling in HUVECs.
HUVECs were plated onto Matrigel coated 24 well plates at 2×105 cells per well and incubated in the presence of VEGF (100 ng/ml) and treated with BAY 36-7620 (25 μM), L-quisqualate (10 μM) or MK2206 (5 μM), either alone or in combination. About 48 h later, it was found that treatment with BAY36-7620 led to a reduction in capillary tube formation. Treatment of HUVECs with L-quisqualate resulted in enhancement of capillary tube formation, which was blocked by co-incubation with MK2206 (Fig. 5(B)).

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In this study, the anti-cancer effects of riluzole were explored in a panel of breast cancer cell lines in comparison to the metabotropic glutamate receptor 1-specific inhibitor BAY 36-7620. While both drugs inhibited breast cancer cell proliferation, there were distinct functional effects suggesting that riluzole action may be metabotropic glutamate receptor 1-independent. Riluzole induced mitotic arrest independent of oxidative stress while BAY 36-7620 had no measurable effect on mitosis. BAY 36-7620 had a more pronounced and significant effect on DNA damage than riluzole. Riluzole altered cellular metabolism as demonstrated by changes in oxidative phosphorylation and cellular metabolite levels. These results provide a better understanding of the functional action of riluzole in the treatment of breast cancer. [3]

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Riluzole and BAY 36-7620 inhibit breast cancer cell growth [3]
GRM1 has previously been reported to play a role in breast cancer cell growth and proliferation. To determine the effects of these drugs on cell growth, ER+ and ER- breast cancer cell lines were treated with either riluzole or BAY 36-7620 for 72 h. Both drugs inhibited the number of viable cells in all cell lines (Figure 2A and 2B). IC50 values for riluzole and BAY 36-7620 ranged from 19.0-62.4 μM and 15.7-41.0 μM, respectively (Table 1). BT-474, Hs578T, and BT-549 cells were the most sensitive to both drugs while MDA-MB-231 cells were the least sensitive. BAY 36-7620 at the highest concentrations completely inhibited cell growth. At the highest concentrations evaluated, riluzole inhibited cell growth by 70-90% compared to control.

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Treatment with riluzole or BAY 36-7620 inhibits cell proliferation [3]
Since both drugs reduced cell number, their effect on proliferation was determined as measured by 5-ethynyl-2´-deoxyuridine (EdU) incorporation. The percentage of proliferating cells was decreased in each breast cancer cell line by riluzole or BAY 36-7620 (Figure 3). However, no association between the anti-proliferative effect of riluzole or BAY 36-7620 and GRM1 levels was observed. T-47D, BT-474, and BT-549 cells were significantly more sensitive to BAY 36-7620 than to riluzole at the concentration evaluated suggesting that BAY 36-7620 has a more potent effect on cell proliferation. Notably, the low GRM1-expressors were still very sensitive to riluzole or BAY 36-7620 implying that there may be off-target effects for each drug.

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Riluzole and BAY 36-7620 alter gene expression signatures in cell cycle and oncogenic pathways [3]
Gene expression analysis of MCF-7, BT-474, and BT-549 cells was done to identify gene sets in pathways altered by either drug to better understand their mechanism of action. These cell lines were included to compare cells with a range of sensitivity to riluzole or BAY 36-7620 with BT-549 being the most sensitive and MCF-7 being the least sensitive. As BT-549 and BT-474 cells are more sensitive to cell death by both drugs, these cell lines were treated for 24 h, whereas the less sensitive MCF-7 cells were treated for 48 h. Overall, riluzole or BAY 36-7620 induced similar gene signature profiles for each of the three cell lines as compared to dimethyl sulfoxide (DMSO) control (Figure 4A). However, differential expression signatures were observed for riluzole as compared to BAY 36-7620. For example, BAY 36-7620, but not riluzole, induced the cholesterol biosynthesis gene signature in MCF-7 and BT-474 cells.

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Riluzole more strongly induces G2/M cell cycle arrest than BAY 36-7620 [3]
Effects of riluzole and BAY 36-7620 on cell cycle distribution were investigated. Each breast cancer cell line treated with riluzole showed a significant dose- and time-dependent induction of G2/M arrest (Figure 5). BT-474 and BT-549 cells were most sensitive to riluzole with an increase in the sub G1 population as early as 48 h and 24 h respectively (Figure 5E and 5I). BAY 36-7620 induced a more modest G2/M arrest in T-47D, BT-474, MDA-MB-231, and BT-549 cell lines (Figure 5D-5J) but had no effect in MCF-7 cells (Figure 5A and 5B) as compared to riluzole. Although both riluzole and BAY 36-7620 inhibited proliferation, more pronounced G2/M arrest by riluzole may implicate other targets beyond those of BAY 36-7620.

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Riluzole but not BAY 36-7620 induces mitotic arrest in breast cancer cells [3]
To distinguish whether riluzole or BAY 36-7620 treatment induces G2 arrest or mitotic arrest in breast cancer cells, the fraction of cells with phosphorylation of histone H3 was utilized as a marker of mitosis. Riluzole significantly increased the number of phospho-H3 stained cells compared to control in all cell lines suggesting that riluzole induced mitotic arrest (Figure 6A and 6B). In contrast, BAY 36-7620 exhibited variable cell line-dependent effects. BAY 36-7620 significantly decreased the number of phospho-H3 stained cells in MCF-7, T-47D and BT-549 cells while a modest increase was observed in MDA-MB-231 cells (Figure 6A and 6B).
Breast cancer cells treated with riluzole or BAY 36-7620 were also investigated for changes in known markers for mitosis. Riluzole significantly decreased phospho-cdc2 in MCF-7, T-47D, Hs578T, and BT-549 cells and significantly increased cyclin B1 in Hs578T cells with a trend toward increase in T-47D, BT-474, and BT-549 cells (Figure 6C, Supplementary Figure 2). This supports a role for riluzole in induction of mitotic arrest. BAY 36-7620 only decreased phospho-cdc2 in MCF-7, T-47D, Hs578T, and BT-549 cells at higher, more cytotoxic concentrations and did not increase cyclin B levels (Figure 6C, Supplementary Figure 2). The effects of riluzole on phospho-H3, phospho-cdc2, and cyclin B1 suggest that riluzole induces mitotic arrest within G2/M arrest whereas BAY 36-7620 had a minimal effect on both G2/M arrest and more specifically mitotic arrest.

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DNA damage is observed after treatment with BAY 36-7620 [3]
DNA damage is known to result in G2/M arrest within the cell cycle. To determine whether riluzole or BAY 36-7620 induces DNA damage as a potential cause of G2/M arrest, phosphorylation of histone H2AX (γ-H2AX) was evaluated as a well-described marker of DNA damage, specifically DNA double strand breaks. All breast cancer cell lines treated with either riluzole or BAY 36-7620 had an increased percentage of cells positive for γ-H2AX foci as detected by immunofluorescence (Figure 7A). However, BAY 36-7620 induced a significantly more robust H2AX phosphorylation than riluzole. Increased γ-H2AX nuclear foci after drug treatment can be seen in representative images from an ER+ (MCF-7) and ER- (MDA-MB-231) cell line (Figure 7B).

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Riluzole-induced cell cycle arrest is independent of oxidative stress [3]
It has been hypothesized that riluzole induces oxidative stress due to reduced antiport of glutamate and cystine via xCT, leading to depletion of glutathione stores then DNA damage in melanoma cells. To evaluate if riluzole or BAY 36-7620 increased oxidative stress in breast cancer cells, levels of reactive oxygen species (ROS) and total intracellular glutathione (GSH) were evaluated. BAY 36-7620 significantly increased ROS in T-47D and BT-474 ER+ breast cancer cell lines, while riluzole resulted in significantly increased ROS only in BT-474 cells (Figure 8A). Although the increase in ROS by BAY 36-7620 was not statistically significant in MCF-7 cells, the trend was similar to the other two ER+ cell lines. When comparing the two drugs, the increase in ROS was significantly higher with BAY 36-7620 as compared with riluzole. Neither drug significantly increased ROS in ER- cells. Interestingly, there was a modest decrease in ROS in MDA-MB-231 treated with BAY 36-7620 and Hs578T ER- cell lines treated with either riluzole or BAY 36-7620. Total glutathione (GSH) levels decreased after riluzole or BAY 36-7620 treatment in BT-474 and Hs578T cells while no significant effect was observed in the other cell lines (Figure 8B). Both cell lines have relatively low GRM1 protein levels suggesting that drug treatment may affect other glutamate receptor targets.

BAY 36-7620 (5-10 mg/kg, intraperitoneal injection; once daily for 24 days) reduced tumor growth in the thymic nude mice model and extended the formation of lung cancer tumors [2]. BAY 36-7620 (0.01-0.03 mg/kg, injection; 4 hours) had neuroprotective effects in an acute subdural hematoma model [4].

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The role of mGlu1 receptor in NSCLC [2]
Athymic nude mice were injected with 5×105 cells of A549 or H1299 into the flank, and treated with BAY 36-7620 (an mGlu1 receptor-specific inhibitor, 5, 10 mg/kg/day, i.p.). 24 days after implantation, volumes of both A549 (Fig. 2(A)) and H1299 tumors (Fig. 2(B)) were lower in BAY36-7620-treated group than that in control group, indicating that mGlu1 receptor inhibition by BAY36-7620 suppressed tumor growth in athymic mice with lung tumors. According to Kaplan–Meier analysis, the survival curves for the control and BAY36-7620-treated mice differed significantly. Inhibition of mGlu1 receptor by BAY36-7620 treatment markedly prolonged the survival (Fig. 2(C) and (D)) of inoculated mice when compared to control group.

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mGlu1 receptor/AKT signaling [2]
Athymic nude mice were injected with 5×105 cells of A549 into the flank, and treated with BAY 36-7620 (an mGlu1 receptor-specific inhibitor, 5, 10 mg/kg/day, i.p.). It was found that treatment with BAY36-7620 lowered the phosphorylated AKT (Ser-473) levels in A549 tumors (Fig. 3(A)), but had no significant effect on phosphorylation of JNK, ERK, and p38 (Fig. S1, shown in the supplementary data). In SK-MES-1 tumors, treatment with BAY36-7620 also suppressed phosphorylation of AKT (Fig. S2, shown in the supplementary data).

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This study characterized the neuroprotective and behavioral effects of (3aS,6aS)-6a-naphtalen-2-ylmethyl-5-methyliden-hexahydro-cyclopenta[c]furan-1-on (BAY 36-7620), a novel, selective and systemically active metabotropic glutamate (mGlu)(1) receptor antagonist. In the rat, neuroprotective effects were obtained in the acute subdural hematoma model (efficacy of 40-50% at 0.01 and 0.03 mg/kg/h, i.v. infusion during the 4 h following surgery); whereas in the middle cerebral artery occlusion model, a trend for a neuroprotective effect was obtained after triple i.v. bolus application of 0.03-3 mg/kg, given immediately, 2 and 4 h after occlusion. Hypothermic effects were mild and only obtained at doses which were considerably higher than those at which maximal neuroprotective efficacy was obtained, indicating that the neuroprotective effects are not a consequence of hypothermia. BAY 36-7620 protected against pentylenetetrazole-induced convulsions in the mouse (MED: 10 mg/kg, i.v.). As assessed in rats, BAY 36-7620 was devoid of the typical side-effects of the ionotropic glutamate (iGlu) receptor antagonists phencyclidine and (+)-5-methyl-10,11-dihydroxy-5H-dibenzo(a,d)cyclohepten-5,10-imine (MK-801). Thus, BAY 36-7620 did not disrupt sensorimotor gating, induce phencyclidine-like discriminative effects or stereotypical behavior, or facilitate intracranial self-stimulation behavior. Although behavioral stereotypies and disruption of sensorimotor gating induced by amphetamine or apomorphine were not affected by BAY 36-7620, the compound attenuated some behavioral effects of iGlu receptor antagonists, such as excessive grooming or licking, and their facilitation of intracranial self-stimulation behavior. It is concluded that mGlu(1) receptor antagonism results in neuroprotective and anticonvulsive effects in the absence of the typical side-effects resulting from antagonism of iGlu receptors. [4]

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Neuroprotective effects [4]
In the subdural hematoma model of traumatic brain injury, 4-h post-surgery infusion of BAY 36-7620 induced neuroprotective effects [F(5,59)=3.86, P<0.01; Fig. 2, upper panel]. The dose–response curve, however, was inverted U-shaped, with a maximal efficacy of about 40–50% obtained in the 0.01–0.03 mg/kg dose range. In the middle cerebral artery occlusion model of ischemic stroke, triple i.v. bolus application tended to induce neuroprotective effects in the tested dose range of 0.03 to 3 mg/kg, but the effect failed to reach statistical significance [F(3,31)=2.55, P=0.07; Fig. 2, lower panel].

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Anticonvulsive effects [4]
In the pentylenetetrazol model, BAY 36-7620 induced an increase in the convulsion threshold, with an MED of 10 mg/kg, i.v. and about 35% efficacy at 20 mg/kg [F(3,58)=8.17, P<0.001; Fig. 3].

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Hypothermic effects [4]
BAY 36-7620 induced hypothermic effects after both i.v. administration [F(3,24)=2.84, P=0.059] and i.p. administration [F(2,18)=4.28, P<0.05; Fig. 4]. Thus, after i.v. administration, the compound induced mild and short-lasting hypothermic effects with an MED of 3 mg/kg and a maximal effect of −0.72 °C obtained at 10 mg/kg. At each dose tested, the hypothermic effect was maximal around 15 min, and was no longer present at 60 min post-application. Although the compound was only slightly less potent after i.p. administration (MED: 10 mg/kg), the hypothermic effect appeared to be more pronounced (maximal effect of −1.48 °C at 30 mg/kg, at 20 min post-application), as compared to i.v. administration. Effects on body temperature were time-dependent [i.v.: F(4,96)=89.27, P<0.001; i.p.: F(6,108)=21.04, P<0.001] and the time course was similar after both routes of administration.

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Behavioral stimulation/stereotypies induced by MK-801, amphetamine and apomorphine [4]
Pretreatment with BAY 36-7620 (0.1–10 mg/kg, i.v.) attenuated licking [F(3,36)=7.97, P<0.001] and facial grooming [F(3,36)=27.77, P<0.001] induced by MK-801 (Fig. 5, upper panel) with an MED of 10 mg/kg; whereas it did not affect other behavioral symptoms induced by MK-801, such as sniffing, ataxia and tongue rolling (data not shown). Interestingly, when tested in the same dose range, BAY 36-7620 failed to affect behavioral stimulation/stereotypies induced by amphetamine or apomorphine (such as, sniffing, exploration and biting; Fig. 5, middle and lower panels), suggesting that the interaction between BAY 36-7620 and MK-801 is behaviorallly specific and not merely the result of a drug-induced suppression of behavior. When tested alone, BAY 36-7620 (0.1–10 mg/kg, i.v.) did not induce behavioral symptoms (data not shown).

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PCP drug discrimination [4]
Rats trained to discriminate PCP (2 mg/kg) showed complete generalization when tested with PCP [ED50 value (95% confidence limits): 1.10 (0.69–1.74) mg/kg, i.p.] or MK-801 [0.11 (0.06–0.21) mg/kg, i.p.] (Fig. 6, upper panel). BAY 36-7620 did not induce generalization to the PCP cue (maximal level of generalization: 20% drug lever selections at 30 mg/kg, i.p.; Fig. 6, upper panel). There was no indication for the occurrence of behavioral disruption in the tested dose range, as all rats selected a lever after each test dose (except for one out of five rats which failed to select a lever at 0.3 mg/kg MK-801). Pretreatment with 1–30 mg/kg BAY 36-7620 failed to antagonize the PCP cue (Fig. 6, lower panel) and, again, no behavioral disruption was observed (all rats selected a lever; except at the 3 mg/kg dose, where one out of five rats failed to select a lever).

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Startle-threshold and prepulse inhibition [4]
After i.v. administration, BAY 36-7620 reduced the acoustic startle magnitude in rats [Factor BAY 36-7620: F(3,224)=6.85, P<0.001; Factor Trial: F(7,224)=35.13, P<0.001], with an MED of 3 mg/kg and a maximal effect of about 50% obtained at 10 mg/kg (efficacy tended to be more pronounced at the higher pulse intensities; Fig. 7, upper panel). The rightward shift of the startle curve indicates that BAY 36-7620 increases the startle threshold. In mice, however, i.v. administration of the same dose range of BAY 36-7620 did not affect startle responding (Fig. 7, lower panel). BAY 36-7620 did not affect prepulse inhibition in rats (data not shown) and did not reverse the disruption of prepulse inhibition induced by either MK-801 [F(1,112)=41.32, P<0.001], PCP [F(1,112)=33.41, P<0.001], or apomorphine [F(1,112)=53.13, P<0.001; data not shown].

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Intracranial self-stimulation [4]
ANOVA indicated that the intracranial self-stimulation threshold was affected by Factor MK-801 [F(1,24)=6.03, P<0.05] and by Factor BAY 36-7620 [F(1,24)=5.62, P<0.05]. While BAY 36-7620 (10 mg/kg, i.p.) failed to affect the threshold when tested in combination with vehicle pretreatment, the compound completely prevented the facilitation of intracranial self-stimulation behavior induced by MK-801 (0.025 mg/kg, i.p.; Fig. 8).

Membrane preparation and [3H]-Quisqualate Binding Assay [1]
[3H]QA binding was performed in HEK293 cells cultured and transiently transfected with mGlu1as receptor as described above. Membranes were recovered 24 hours after transfection in KREBS-Tris buffer (Tris 20mM, NaCl 118mM, KH2PO4 1.2mm, MgSO4 1.2 mM, KCl 4.7mM, CaCl2 1.8mM, glucose 5.6mM, pH 7.4), homogenized, pooled and centrifuged at 40,000g for 20 min. The resulting pellet was resuspended in the same buffer, homogenized and stored as pellets at −20°C until use (< 1 month). Protein levels were determined using a bicinchoninic acid assay (Smith et al., 1985) with bovine serum albumin as a standard.
Binding conditions used were performed and modified as follows. Binding experiments were carried out in a buffer containing HEPES 40mM and CaCl2 2.5mM, adjusted to pH 7.4 with NaOH. Membrane pellets were resuspended in the above buffer plus protease inhibitor cocktail and aliquots of 50μg protein were incubated in the presence of [3H]QA (specific activity 25Ci/mmol), in a final volume of 100μl at room temperature, for one hour. Competition experiments were performed with 600nM [3H]QA and varying concentrations of cold QA and BAY 36-7620. Incubation was terminated by rapid filtration through Whatmann QF/C filters, (pre-soaked for 45 min in 3% powdered milk), using a Brandel Harvester. Filters were rinsed in HEPES/CaCl2 buffer and counted in 3ml PCS scintillant. Non-specific binding was determined in the presence of 1mM Glu.

Western Blot Analysis[2]
Cell Types: A549 cell line
Tested Concentrations: 10, 25 μM
Incubation Duration: Overnight
Experimental Results: cleaved PARP expression was enhanced, and bcl diminished -2 protein expression. Reduce HIF-1α protein expression and HIF activity. diminished secretion of VEGF and IL-8 in the supernatant.

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Cell proliferation assay[3]
Cell Types: T MCF-7, T-47D, BT-474, MDA-MB-231, Hs578T and BT-549 Cell Line
Tested Concentrations: 50 μM
Incubation Duration: 72 hrs (hours)
Experimental Results: Reduction of all breast cancers Proliferating cells in cell lines.

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Measurement of HIF activity [2]
Reverse transfection into A549 cells was made with a mixture of a transcription factorresponsive firefly luciferase construct ‘HIF Reporter’and constitutively expressing Renilla luciferase construct. A mixture of non-inducible firefly luciferase construct and constitutively expressing Renilla luciferase construct used as negative control and a mixture of constitutively expressing firefly and Renilla luciferase constructs used as positive control were also transfected. At 24 h after transfection, A549 cells were treated with BAY 36-7620 (25 μM), L-quisqualate (10 μM) or MK2206 (5 μM), either alone or in combination under condition of hypoxia, and 4 days later, the activities of HIF was measured with the Dual-Glo luciferase assay.

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Experiment 4: A549 cells were pre-treated for 30 min with BAY 36-7620 (25 μM) or not, then they were stimulated with L-quisqualate (10 μM). At various time points after treatment, cells were collected for determination of AKT phosphorylation (Ser-473). [2]
Experiment 5: A549 cells were treated with BAY 36-7620 (10 and 25 μM) or vehicle (0.05% DMSO, control). At various time points after treatment, cell proliferation was determined. [2]
Experiment 6: A549 cells were treated with BAY 36-7620 (25 μM), L-quisqualate (10 μM) or MK2206 (5 μM), either alone or in combination. Following 4 days of incubation, cells were collected for determination of cleaved-PARP, PARP, and bcl-2 protein expression. [2]
Experiment 7: Under condition of hypoxia, A549 cells were treated with BAY 36-7620 (25 μM), L-quisqualate (10 μM) or MK2206 (5 μM), either alone or in combination. Following 4 days incubation, cells were collected for determination of HIF-1α protein expression and HIF activity and supernatants were collected for determination of VEGF and IL-8 protein levels. [2]
Experiment 8: HUVECs were pre-treated for 30 min with BAY 36-7620 (25 μM) or not, then they were stimulated with VEGF (100 ng/ml). At various time points after treatment, cells were collected for determination of AKT phosphorylation (Ser-473). [2]
Experiment 9: HUVECs were plated onto Matrigel coated 24 well plates at 2×105 cells per well and incubated in the presence of VEGF (100 ng/ml) and treated with BAY36-7620 (25 μM), L-quisqualate (10 μM) or MK2206 (5 μM), either alone or in combination. About 48 h later, capillary tubes formed were evaluated. [2]

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Gene expression analysis [3]
MCF-7 cells were treated with 50 μM riluzole or BAY 36-7620 for 48h. BT-474 and BT-549 cells were treated for 24h due to rapid entry of cells into subG1. Total RNA was purified with RNeasy mini kit following manufacturer's protocol. RNA was subjected to DNase treatment to remove contaminating DNA. The Human Genome U133A 2.0 Array was used to measure gene expression changes from drug treatment compared to DMSO control. Three independent replicates were used for each condition. For analysis, raw CEL files were processed using the justRMA function in R Bioconductor, obtaining log2 expression values. Gene expression signatures were analyzed using Gene Set Enrichment Analysis, obtaining a quantification of the statistical significance for upregulation (P+) or downregulation (P-) for each signature and sample pair. A sample was said to have a signature significantly upregulated if P+ < 0.05 (red), significantly downregulated if P- < 0.05 (blue), and no significant change otherwise (black). For microarray validation, RNA from cells treated with DMSO, 50 μM riluzole, or 50 μM BAY 36-7620 was reverse transcribed using the Taqman Reverse Transcription kit following manufacturer's protocol (Thermo Fisher Scientific, Waltham, MA). Pre-designed Taqman assays for genes validated were used to perform quantitative PCR on the complementary DNA. The RPLP0 gene was used as a housekeeping gene control. Results are shown as relative fold change of gene expression compared to DMSO control treatment using the delta delta Ct method.

Animal/Disease Models: Lung tumor mouse model [2]
Doses: 5, 10 mg/kg
Route of Administration: intraperitoneal (ip) injection; one time/day for 24 days
Experimental Results: Inhibition of tumor growth in athymic mice with lung tumors . The survival time of the vaccinated mice was extended compared with the control group. diminished AKT phosphorylation levels in A549 tumors.

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Animal/Disease Models: Subdural hematoma rat model [4]
Doses: 0-3 mg/kg
Route of Administration: intravenous (iv) (iv)injection; 4 hrs (hrs (hours))
Experimental Results: Neuroprotective, efficacy at 0.01 and 0.03 mg/kg 40-50%.

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Experiment 2: Athymic nude mice were injected with 5×105 cells of A549 or H1299 into the flank, and treated with BAY 36-7620 (5, 10 mg/kg/day, i.p.). Tumor volumes were measured every six days. Overall survival was calculated from the day of inoculation to end of survival.
Experiment 3: Athymic nude mice were injected with 5×105 cells of A549 into the flank, and treated with BAY36-7620 (5, 10 mg/kg/day, i.p.). 24 days after implantation, the tumors were removed for determination of AKT phosphorylation (Ser-473) and microvessel density. [2]

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Middle cerebral artery occlusion [4]
The left middle cerebral artery was occluded via a subtemporal approach under general anesthesia with the inhalational anesthetic isoflurane (Forene®, Abbott, Wiesbaden, Germany) mixed with compressed air or 80% styckoxydul:30% oxygen to 4–1.5% v/v (Bederson et al., 1986). The left temporal–parietal region of the head was shaved and the skin was disinfected and opened between the orbit and the external ear canal. A midline incision was made, the temporal muscle was divided and pulled aside to expose the lateral aspect of the skull. The middle cerebral artery was exposed under an operating microscope, without damaging the facial nerve, major facial arteries and veins, the lateral eye muscle, the lacrimal glands and the zygomatic bone. The dura was carefully opened and the middle cerebral artery and its branches were permanently occluded between the olfactory tract and the inferior cerebral vein by microbipolar electrocoagulation. To avoid recanalization, the occluded vessels were removed. The muscle and skin wounds were closed in layers using cyanacrylate tissue glue. BAY 36-7620 (0.03–3 mg/kg) or vehicle was administered as a triple i.v. bolus injection, immediately, 2 and 4 h after the insult. After recovery from anesthesia the animals were returned to their home cage.

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Acute subdural hematoma [4]
Rats were anesthetized with isoflurane (Forene®, see middle cerebral artery occlusion method) and a subdural hematoma was induced according to a standard surgical procedure (Miller et al., 1990) with some modifications. Briefly, the top of the head was shaved, the skin was disinfected and opened with a longitudinal midline cut. A small part of the periosteum was removed and a burr hole was drilled into the skull, according to the stereotaxic coordinates: −1.0 mm caudal, −2.8 mm lateral to the bregma (Paxinos and Watson, 1986). The dura was carefully opened and a specially designed plastic cannula was inserted into the subdural space between the dorsal surface of the brain and the dura. Thereafter, the cannula was fixed in position with a tissue glue. Nonheparinized autologous blood was collected by puncture of the tail vein and injected directly via the prefixed cannula into the subdural space (total volume of 200 μl within 4 min). Thereafter, the probe was shortened and closed with the cyanacrylate tissue glue. The skin wound was closed with suture clips. BAY 36-7620 (0.003–0.03 mg/kg/h) or vehicle was administered as a 4-h continuous i.v. infusion, starting immediately after the surgery. During the surgery and the infusion of BAY 36-7620 or vehicle, the body temperature was monitored and maintained in physiological range (37.0±0.5 °C) with a warming pad and by covering the rats with some layers of tissue. After recovery from anesthesia the animals were returned to their home cage.

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Pentylenetetrazol convulsion test [4]
After a food-deprivation period of 16–24 h, mice (n=10 per group) received an i.v. bolus injection of BAY 36-7620 (3–20 mg/kg) or vehicle, immediately followed by an injection of a pentylenetetrazol solution (5 mg/ml) into the tail vein at a rate of 0.3 ml/min. The pentylenetetrazol injection was stopped as soon as the mouse showed a clonic seizure. The amount of pentylenetetrazol needed to induce such a seizure was considered to be the convulsion threshold dose. For graphical presentation, the mean threshold dose obtained after pretreatment with BAY 36-7620 was expressed as percentage increase as compared with the mean threshold dose obtained after pretreatment with vehicle. Individual threshold doses were analyzed by one-way ANOVA, followed by Tukey's post hoc comparisons. BAY 36-7620 was considered to have an anticonvulsive effect if the drug induced a statistically significant increase in the threshold dose, as compared to vehicle control (P<0.05).

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Body temperature [4]
Different groups of rats (n=7 per group) were treated with vehicle or various doses of BAY 36-7620 (1–10 mg/kg, i.v.; 10–30 mg/kg, i.p.) and their body temperature was oesophagally measured repeatedly at fixed time points. Time points measured included: 5 min before, and 7.5, 15, 30 and 60 min (i.v. dose–response determination), or 5, 10, 20, 40 and 80 min (i.p.) after drug administration. For graphical presentation, results were expressed as temperature change in °C relative to baseline value, and corrected for the temperature change observed in the vehicle-treated control group. Absolute body temperature data were analyzed by one-way ANOVA with repeated measures, followed by Tukey's post hoc comparisons.

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Behavioral stimulation/stereotypies induced by MK-801, amphetamine and apomorphine [4]
Male Wistar rats were treated with BAY 36-7620 (0.1–10 mg/kg, i.v.) 5 min before administration of MK-801 (0.2 mg/kg, i.p.; n=10 per group), amphetamine (3 mg/kg, i.p.; n=5 per group) or apomorphine (0.1 mg/kg, s.c.; n=5 per group) and observed in individual standard Makrolon® (type 3) cages for the occurrence of particular behavioral symptoms. Animals were observed during 60 min (amphetamine test) or 30 min (MK-801 and apomorphine test), starting immediately after the second administration. The behavioral check lists for the MK-801 test included the following symptoms: licking, biting, genital grooming, facial grooming, sniffing, exploration, ataxia, “wet dog” shakes and tongue rolling; whereas for the amphetamine and the apomorphine test it included: licking, biting, genital grooming, sniffing, exploration and yawning. Symptoms were scored by means of a time sampling method. Thus, for the MK-801 and apomorphine test, rats were observed each 2.5th min, and for the amphetamine test each 5th min of the observation period, for the occurrence of each of the behavioral symptoms (value “1” if present, value “0” if absent; in the case of the apomorphine test, scores included value “0” if symptom was absent, “1” if weakly present and “2” if clearly present).

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Phencyclidine (PCP) drug discrimination [4]
The animals were tested with different doses of the training compound (0.5–2 mg/kg, i.p.) before being submitted to further generalization tests with MK-801 (0.03–0.3 mg/kg, i.p.) and BAY 36-7620 (10–30 mg/kg, i.p.), or antagonism tests with BAY 36-7620 (0, 1–30 mg/kg, i.p.). Generalization tests were performed 15 min after application of the test compound. In the antagonism study, pretreatment with BAY 36-7620 (or vehicle) occurred 15 min before treatment with PCP (2 mg/kg, i.p.). Test results were expressed as the percentage of rats that selected the drug lever (% Drug Lever Selections). In addition, the percentage of animals that selected a lever (either drug or vehicle lever) was determined as an index of behavioral disruption (i.e., % Lever Selections). Least-square linear regression analysis was used to estimate ED50 values and the corresponding 95% confidence limits after log-probit conversion of the data. Generalization was considered to be complete if at least 80% drug lever selections was obtained.

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Acoustic startle experiments: startle-threshold and prepulse inhibition [4]
Before drug testing, each animal was placed in a startle chamber with 70-dB background noise and 5 min later exposed to 20 120-dB, 40-ms broad-band bursts, with a 15-s intertrial interval. Subsequently, animals were divided in groups matched for mean amplitude on these trials. Testing occurred 2 to 3 days after matching. The effects of BAY 36-7620 were tested alone (1, 3 and 10 mg/kg, administered i.v. 5 min before test), or after pretreatment with MK-801 (0 and 0.5 mg/kg, s.c.), PCP (0 and 1.5 mg/kg, s.c.), and apomorphine (0 and 1 mg/kg, s.c.), administered 15 min prior to BAY 36-7620. In the combination experiments with MK-801 and PCP, BAY 36-7620 was tested at a dose of 10 mg/kg, i.v.; whereas in the combination experiments with apomorphine, BAY 36-7620 was tested at a dose of 3 mg/kg, i.v. In all combination experiments, testing took place 5 min after administration of BAY 36-7620.

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Intracranial self-stimulation experiments [4]
Therefore, it can be considered as a threshold value for the rewarding efficacy of intracranial self-stimulation, and a left- or rightward shift can be interpreted as a decrease or increase of the rewarding efficacy, respectively. MK-801 (0 or 0.025 mg/kg, i.p.) was administered 20 min, and BAY 36-7620 (0 or 10 mg/kg, i.p.) 5 min before test.

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BAY 36-7620 was suspended in a solvent containing 2.5–5% Solutol® HS 15 (12-hydroxystearic acid ethoxilate) and 2.5–5% ethanol (ethanol absolute, 99.8%), or a solvent containing 5–10% cremophor (Cremophor EL®), and deionisated water or 0.9% NaCl.

[1]. BAY36-7620: a potent non-competitive mGlu1 receptor antagonist with inverse agonist activity. Mol Pharmacol. 2001 May;59(5):965-73.

[2]. Inhibition of metabotropic glutamate receptor 1 suppresses tumor growth and angiogenesis in experimental non-small cell lung cancer. Eur J Pharmacol. 2016 Jul 15;783:103-11.

[3]. Riluzole exerts distinct antitumor effects from a metabotropic glutamate receptor 1-specific inhibitor on breast cancer cells. Oncotarget. 2017 Jul 4;8(27):44639-44653.

[4]. Neuroprotective and behavioral effects of the selective metabotropic glutamate mGlu(1) receptor antagonist BAY 36-7620. Eur J Pharmacol. 2001 Oct 5;428(2):203-14.

Our data using chimeric mGlu receptors revealed that the TM region of mGlu1 was necessary for the inhibitory action of BAY 36-7620. Moreover, its apparent affinity was the same whether the ECD was that of either mGlu5, mGlu2 or DmGluA receptors even though these later two ECDs share only 41% sequence identity with that of mGlu1 receptor. Within the TM regions, we found that TM4 to TM7 of mGlu1 receptor were sufficient to create a BAY36-7620 site in mGlu5 receptor. This shows that in contrast to what has been observed for the selectivity of action of MPEP (Pagano et al., 2000), TM3 does not play a role in the selective recognition of BAY36-7620 in the mGlu1 receptor. Amongst the TM4 to 7, TM6 is identical in all mGlu receptors, and as such cannot play a role in the specific recognition of BAY36-7620, but a few residues are different between mGlu1 and mGlu5 receptor in TM4, TM5 and TM7. Preliminary experiments have revealed that indeed, as observed with the other mGlu1 selective non-competitive antagonist, CPCCOEt (Litschig et al., 1999), TM7 of mGlu1 appears critical for the action of BAY36-7620 (Carroll, Kuhn, Pin and Prézeau, unpublished data). It has recently been proposed that CPCCOEt and the mGlu5 selective non-competitive antagonist MPEP interact within a similar cavity found in both mGlu1 and mGlu5 receptors (Litschig et al., 1999; Pagano et al., 2000). Indeed, it has been shown that only few residues within this binding pocket are responsible for the high selectivity of these two molecules. According to our data, it is likely that BAY36-7620 binds in the same cavity as these other non-competitive mGlu receptor antagonists. Although more work is necessary to further characterize the BAY36-7620 binding site, our data are sufficient to demonstrate the pivotal role played by the TM region of mGlu1 receptor for the antagonistic action of BAY36-7620. Of interest, not only BAY 36-7620, but also MPEP have been found to be inverse agonists. Moreover, although no inverse agonist activity of CPCCOEt could be detected on native mGlu1 receptors expressed alone (Litschig et al., 1999), we recently found that this compound is able to significantly inhibit 10-15% of the agonist-independent activity of mGlu1 receptors boosted by co-expression with the Gαq subunit (F. Carroll, unpublished data), indicating that CPCCOEt is a very partial inverse agonist at mGlu1 receptors. Taken together, these data show that amongst all the non-competitive antagonists which have been shown to interact within the TM region of either mGlu1 or mGlu5 receptors, all have inverse agonist activity. This is in contrast with all competitive antagonists known to bind on the ECD of these receptors. Accordingly, we propose that the natural constitutive activities of mGlu1 and mGlu5 receptors originate more from their TM region than from their ECD. Indeed, it may be possible that the TM region, like that of the rhodopsin-like GPCRs, oscillates between active and inactive states, even when the ECD remains in an inactive (open) conformation. The binding of the non-competitive antagonists within the TM region would stabilize an inactive state as observed with the rhodopsin-like receptors. In contrast, the binding of a competitive antagonist within the ECD would prevent the binding of an agonist, and maintain it in an inactive open state, but would not be able to prevent the equilibrium between active and inactive states of the TM region of these receptors. Taken together, our study identified a new highly selective mGlu1 receptor antagonist that will be useful to further elucidate the action of this receptor subtype in the brain. Moreover, our data showed that BAY 36-7620 is an inverse agonist on mGlu1. As the constitutive activity of mGlu1 is under the control of alternative splicing (Prezeau et al., 1996), this compound will be useful to help discriminate between some of the actions of mGlu1 variants. Our study further documents the observation that antagonists acting within the TM region of mGlu1 and mGlu5 receptors can inhibit their agonist-independent activity. Such an activity of these compounds shed some light on the specific role of the TM region of these receptors in their natural constitutive activity. Moreover, such an activity of BAY36-7620 and MPEP will be useful to elucidate the possible physiological relevance of the mGlu receptor constitutive activity. Of interest, inverse agonists of several family 1 receptors have been reported to have specific properties not shared by neutral antagonists, as for example in the case of 5HT2C (Barker et al., 1994) or 5HT1A (Albert et al., 1999) receptors. [1]

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Metabotropic glutamate receptor 1 (mGlu1 receptor) is expressed in many cancer cell types as compared to normal counterparts underscoring its potential role in tumor behavior. The aim of present study was to test the role of mGlu1 receptor in experimental non-small cell lung cancer (NSCLC). First, protein expression of mGlu1 receptor was higher in human NSCLC cell lines, including both adenocarcinoma and squamous carcinoma subtypes, when compared to normal bronchial epithelial cells. Inhibition of mGlu1 receptor by BAY 36-7620 (an mGlu1 receptor-specific inhibitor) inhibited tumor growth and prolonged survival of mice with tumors of A549 or H1299. Treatment with BAY36-7620 suppressed AKT phosphorylation in A549 tumors and pre-treatment with BAY36-7620 blocked the L-quisqualate (a potent mGlu1 receptor agonist)-induced AKT phosphorylation in A549 cells. Treatment with BAY36-7620 reduced cellular proliferation of A549 cells. Treatment with BAY36-7620 enhanced cleaved PARP levels and reduced protein expression of bcl-2, HIF-1α, and VEGF. In contrast, treatment with L-quisqualate reduced cleaved PARP levels and enhanced protein expression of bcl-2, HIF-1α, VEGF, and IL-8, which was reversed by co-incubation with MK2206 (an AKT inhibitor). Pre-treatment with BAY36-7620 blocked the VEGF-induced AKT phosphorylation in HUVECs. Treatment of HUVECs with L-quisqualate resulted in enhancement of capillary tube formation, which was reversed by co-incubation with MK2206. Furthermore, mGlu1 receptor knockdown suppressed tumor growth and prolonged survival of mice with tumors of A549 or H1299. Collectively, inhibition of mGlu1 receptor suppressed tumor growth and angiogenesis in experimental NSCLC. [2]

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Incorporation of riluzole into breast cancer treatment paradigms has been hampered by a limited understanding of its mechanism of action. In this study, a pharmacologic approach was undertaken to investigate the antitumor effects of riluzole in a panel of human breast cancer cell lines and compared to the effects of the known glutamate receptor antagonist BAY 36-7620. Treatment with either drug produced cell line-dependent effects on markers for proliferation, cell cycle, and DNA damage. Both drugs inhibited cell growth and cell number while altering expression of genes involved in cell cycle regulation and oncogenic pathways. While riluzole and BAY 36-7620 both induce cell death, they have differential effects within cell cycle. Whereas riluzole induced cell death with mitotic arrest, BAY 36-7620 caused cell death without substantial effect on cell cycle. Riluzole induced significant metabolic changes in the cell including decreased oxidative phosphorylation and alteration of cellular metabolite levels suggesting a novel role for riluzole in cell metabolism. Some cell lines showed differential sensitivity to either riluzole or BAY 36-7620. These data support distinct drug-induced mechanisms of cell cycle inhibition leading to cell death. [3]

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Interestingly, it was found that BAY 36-7620 was even able to attenuate some behavioral effects of noncompetitive NMDA receptor antagonists, such as stereotypic grooming or licking, and their facilitation of intracranial self-stimulation. When tested under similar conditions, behavioral stereotypies induced by amphetamine or apomorphine were not affected by BAY 36-7620. Therefore, it can be concluded that the behavioral interactions between BAY 36-7620 and noncompetitive NMDA receptor antagonists are specific and not merely the result of a drug-induced suppression of (stimulated) behavior. The lack of effect of BAY 36-7620 on amphetamine-induced behavioral stimulation/stereotypies, suggests that the previously found modulatory effects of mGlu receptors on dopaminergic neurotransmission, as observed in behavioral studies, involve other subtypes of mGlu receptors than the mGlu1 receptor (e.g., Kim and Vezina, 1998a, Kim and Vezina, 1998b, Kronthaler and Schmidt, 1996). The finding that BAY 36-7620 was able to completely block facilitation of intracranial self-stimulation induced by MK-801 suggests that mGlu1 receptor antagonists may reduce the abuse potential of noncompetitive NMDA receptor antagonists. Therefore, further experiments directly aimed at investigating the interaction of BAY 36-7620 with the positive reinforcing stimulus properties of noncompetitive NMDA receptor antagonists, as assessed in self-administration paradigms, seem to be warranted. The presently obtained behavioral interactions between an mGlu1 receptor antagonist and an NMDA receptor antagonist are compatible with the previously reported facilitation of NMDA receptor function by mGlu1 receptor (or group I mGlu receptor) activation, as observed in different in vitro assays (e.g., Fitzjohn et al., 1996, Martin et al., 1997, Pisani et al., 1997, Rahman and Neuman, 1996). Nevertheless, as observed in the present study, not all behavioral effects induced by the noncompetitive NMDA receptor antagonists were affected similarly by BAY 36-7620, suggesting that the modulatory effects of mGlu1 receptors depend on the brain location of the NMDA receptors underlying the particular behavioral effect of PCP or MK-801. In the light of this differential interaction and the finding that cotreatment of mGlu1 receptor antagonists with MK-801 showed additive neuroprotective effects in glutamate injured cultures (Faden et al., 2001), it should be of interest to investigate to what extent neuroprotective and anticonvulsive effects of NMDA receptor antagonists are affected by co-treatment with BAY 36-7620. [4]

C19H18O2

278.345025539398

278.131

C, 74.24; H, 6.89; N, 13.67; O, 5.20

232605-26-4

9903757

Typically exists as solid at room temperature

471.861ºC at 760 mmHg

199.799ºC

0mmHg at 25°C

1.633

3.891

0

2

2

21

455

2

O1C([C@]2(CC3C=CC4C=CC=CC=4C=3)CC(=C)C[C@@H]2C1)=O

CVIRWLJKDBYYOG-MJGOQNOKSA-N

InChI=1S/C19H18O2/c1-13-8-17-12-21-18(20)19(17,10-13)11-14-6-7-15-4-2-3-5-16(15)9-14/h2-7,9,17H,1,8,10-12H2/t17-,19+/m1/s1

(3aS,6aS)-5-methylidene-3a-(naphthalen-2-ylmethyl)-1,4,6,6a-tetrahydrocyclopenta[c]furan-3-one

BAY-36-7620; BAY367620; BAY 367620; 232605-26-4; BAY-367620; BAY367620; 1H-Cyclopenta(C)furan-1-one, hexahydro-5-methylene-6a-(2-naphthalenylmethyl)-, (3aS,6aS)-; (3aS,6aS)-5-methylidene-3a-(naphthalen-2-ylmethyl)-1,4,6,6a-tetrahydrocyclopenta[c]furan-3-one; 0P934RSF8B; CHEMBL254372; BAY 36-7620

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)