σ1receptor （IC50 = 17.4 nM, guinea pig brain membranes)

In vitro activity: SA4503 protects motor neuron NSC34 cells against superoxide dismutase 1 and serum free neurotoxicity. It upregulates the phosphorylation levels of Akt and extracellular signal-regulated kinase (ERK) 1/2.The sigma receptor might be involved in several diseases in the central nervous system. SA4503, a potent σ1receptor agonist, has 103-fold higher affinity for σ1 (IC50=17.4 nM) than σ2 (IC50=1,784 nM) sites in guinea pig brain membranes. SA4503 is 14-fold selective for σ1 (Ki=4.6 nM) over σ2 (Ki=63.1 nM) sites in guinea pig brain homogenates.
Kinase Assay: SA4503, a potent sigma(1) receptor agonist, is reported as having 103-fold higher affinity for sigma(1) (IC(50) = 17.4 nM) than sigma(2) (IC(50) = 1,784 nM) sites in guinea pig brain membranes. Modest structural changes appear to have major effects on sigma(1)/sigma(2) selectivity. The fluoroethyl analog, FE-SA4503, is described as having high primary affinity for sigma(2) sites (IC(50) = 2.11 nM) and a weaker interaction with sigma(1) sites (IC(50) = 6.48 nM). SA4503 and FE-SA4503 have been radiolabeled for PET studies, and both bind selectively to sigma(1) receptors in animal and human brain in vivo. We prepared SA4503 and FE-SA4503 as reference compounds for radioligand development efforts. In our hands, SA4503 is 14-fold selective for sigma(1) (K(i) = 4.6 nM) over sigma(2) (K(i) = 63.1 nM) sites in guinea pig brain homogenates. Further, FE-SA4503 exhibits the same 14-fold selectivity for sigma(1) (K(i) = 8.0 nM) over sigma(2) (K(i) = 113.2 nM) receptors. The main differences from previously reported values stem from sigma(2) affinity determinations. This protocol, displacement of [(3)H]DTG binding to sigma(2) sites using (+)-pentazocine (200 nM) to mask sigma(1) sites, was validated by the proper rank order of sigma(2) inhibitory potencies shown by a panel of additional ligands: ifenprodil > haloperidol > DTG >> (+)-pentazocine. Robust Pearson correlation (r = 1.0, P = 0.002; slope = 0.97) was observed for our pK(i) values against those from a prior study by others. The findings have bearing on structure-activity relationships for this active series, and on conclusions that might be drawn from experiments relying upon defined sigma(1)/sigma(2) binding selectivity.
Cell Assay:The NSC34 cells are seeded at a density of 7000 cells per well into 96-well plates with D-MEM and transfected using Lipofectamine 2000 mixed with 2 μg /mL of plasmid vector in D-MEM for 6 h. After 6 h, the cell-culture medium is replaced with fresh D-MEM and culture and allowed to proceed for a further 42 h. The cells are then transferred to serum-free D-MEM and immediately treated with SA4503 at a final concentration of 1, 3, or 10 nM.

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cutamesine/SA4503, a potent sigma(1) receptor agonist, is reported as having 103-fold higher affinity for sigma(1) (IC(50) = 17.4 nM) than sigma(2) (IC(50) = 1,784 nM) sites in guinea pig brain membranes. Modest structural changes appear to have major effects on sigma(1)/sigma(2) selectivity. The fluoroethyl analog, FE-SA4503, is described as having high primary affinity for sigma(2) sites (IC(50) = 2.11 nM) and a weaker interaction with sigma(1) sites (IC(50) = 6.48 nM). SA4503 and FE-SA4503 have been radiolabeled for PET studies, and both bind selectively to sigma(1) receptors in animal and human brain in vivo. We prepared SA4503 and FE-SA4503 as reference compounds for radioligand development efforts. In our hands, cutamesine/SA4503 is 14-fold selective for sigma(1) (K(i) = 4.6 nM) over sigma(2) (K(i) = 63.1 nM) sites in guinea pig brain homogenates. Further, FE-SA4503 exhibits the same 14-fold selectivity for sigma(1) (K(i) = 8.0 nM) over sigma(2) (K(i) = 113.2 nM) receptors. The main differences from previously reported values stem from sigma(2) affinity determinations. This protocol, displacement of [(3)H]DTG binding to sigma(2) sites using (+)-pentazocine (200 nM) to mask sigma(1) sites, was validated by the proper rank order of sigma(2) inhibitory potencies shown by a panel of additional ligands: ifenprodil > haloperidol > DTG >> (+)-pentazocine. Robust Pearson correlation (r = 1.0, P = 0.002; slope = 0.97) was observed for our pK(i) values against those from a prior study by others. The findings have bearing on structure-activity relationships for this active series, and on conclusions that might be drawn from experiments relying upon defined sigma(1)/sigma(2) binding selectivity.[1]

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Many studies suggest that antidepressants act as neuroprotective agents in the central nervous system (CNS), though the underlying mechanism has not been fully elucidated. In the present study, we examined the effect of cutamesine/SA4503, which is a sigma-1 receptor agonist and a novel antidepressant candidate, on oxidative stress-induced cell death in cultured cortical neurons. Exposure of the neurons to H(2)O(2) induced cell death, while pretreatment with SA4503 inhibited neuronal cell death. The SA4503-dependent survival effect was reversed by co-application with BD1047 (an antagonist of sigma-1/2 receptors). Previously we found that H(2)O(2) triggers a series of events including over-activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and intracellular Ca(2+) accumulation via voltage-gated Ca(2+) channels and ionotropic glutamate receptors, resulting in neuronal cell death (Numakawa et al. (2007) [20]). Importantly, we found in this study that SA4503 reduced the activation of the MAPK/ERK pathway and down-regulated the ionotropic glutamate receptor, GluR1. Taking these findings together, it is possible that cutamesine/SA4503 blocks neuronal cell death via repressing activation of the MAPK/ERK pathway and, consequently, expression levels of glutamate receptors. [2]

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cutamesine reduced cell damage induced by light exposure in 661W cells [3]
We examined whether the cutamesine protected the 661W cells against light-induced cell death. Representative photographs of the Hoechst 33342 and PI staining of the 661W cells pretreated with cutamesine and/or BD-1047 are shown in Fig. 1A. Hoechst 33342 stains all cells (both live and dead), whereas PI stains only dead cells. Pretreatment with 10 μM cutamesine protected against light-induced cell death (Fig. 1B), and the protective effect significantly disappeared after treatment with BD-1047, a sigma-1 receptor antagonist, at 1 μM (Fig. 1C).

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Sigma-1 receptor expression was decreased by light exposure, and cutamesine recovered the expression in 661W cells [3]
To investigate the expression of sigma-1 receptor in 661W cells and the effect of cutamesine, we examined the western blot analysis. Sigma-1 receptor protein expressed in 661W cells, and the expression was significantly decreased by light exposure. Cutamesine at 10 μM significantly prevented the decreased expression of sigma-1 receptor protein (Fig. 2).

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cutamesine prevented the disruption of mitochondrial membrane potential [3]
We investigated whether cutamesine might prevent the disruption of the mitochondrial membrane potential. Healthy cells were detected with JC-1 J-aggregates (red fluorescent cells) and apoptotic or unhealthy cells were detected with JC-1 monomers (green fluorescent cells) (Fig. 3A). Light irradiation increased the numbers of green fluorescent 661W cells, indicating a disruption of the mitochondrial membrane potential. Cutamesine significantly reduced the number of green fluorescent cells, and BD-1047 significantly inhibited the effect of cutamesine (Fig. 3B).

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cutamesine prevented caspase-3/7 activation induced by light exposure [3]
To investigate the effect of cutamesine on caspase-3/7 activation induced by light irradiation, we measured caspase-3/7 activity using the Caspase-Glo 3/7 Assay System. Light irradiation significantly increased caspase-3/7 activity (Fig. 4). Cutamesine at 10 μM significantly reduced the activation of caspase 3/7, and this effect was attenuated by BD-1047 at 1 μM (Fig. 4).

In SOD1G93A mice, cutamesine prolongs their survival period[2].

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In in vivo studies, cutamesine suppressed the light-induced retinal dysfunction and thinning of the outer nuclear layer in the mouse retina. These findings indicate that cutamesine protects against retinal cell death in vitro and in vivo by the agonistic effect of sigma-1 receptor. Therefore, sigma-1 receptor may have a potential as a therapeutic target in retinal diseases mediated by photoreceptor degeneration.[3]

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cutamesine suppressed light-induced damage to retinal function and histological changes in mice [3]
The effects of cutamesine on light-induced retinal dysfunction were examined by electrophysiological analysis. The a-wave shows the function of the photoreceptors and the b-wave reflects the function of bipolar cells and Müller cells (Fig. 5A). Amplitudes of the a- and b-waves were significantly reduced five days after 8000 lux light exposure for 3 h. The decreases in the a- and b-wave amplitudes were significantly recovered by intravitreal injection of cutamesine (50 or 500 μM, 2 μL) compared with the vehicle-treated group (Fig. 5B).

Representative entire retinal images were obtained five days after light exposure for histological evaluation (Fig. 6A). The ONL was remarkably thinner in the light-irradiated retina than in the normal retina (Fig. 6A). cutamesine prevented the damage induced by light exposure (Fig. 6B). The thickness of the ONL was measured in 240-μm steps; the data were averaged and are shown in Fig. 6C. Cutamesine significantly protected the retinal damage in a dose-dependent manner. Furthermore, the protective effect of cutamesine (500 μM, 2 μL) on light-induced ONL thinning was significantly eliminated by co-administration with BD-1047 (500 μM), a sima-1 receptor antagonist (Fig. 7). On the other hand, BD-1047 alone did not show any effects on the light damages.

SA4503, a potent sigma(1) receptor agonist, is reported as having 103-fold higher affinity for sigma(1) (IC(50) = 17.4 nM) than sigma(2) (IC(50) = 1,784 nM) sites in guinea pig brain membranes. Modest structural changes appear to have major effects on sigma(1)/sigma(2) selectivity. The fluoroethyl analog, FE-SA4503, is described as having high primary affinity for sigma(2) sites (IC(50) = 2.11 nM) and a weaker interaction with sigma(1) sites (IC(50) = 6.48 nM). SA4503 and FE-SA4503 have been radiolabeled for PET studies, and both bind selectively to sigma(1) receptors in animal and human brain in vivo. We prepared SA4503 and FE-SA4503 as reference compounds for radioligand development efforts. In our hands, SA4503 is 14-fold selective for sigma(1) (K(i) = 4.6 nM) over sigma(2) (K(i) = 63.1 nM) sites in guinea pig brain homogenates. Further, FE-SA4503 exhibits the same 14-fold selectivity for sigma(1) (K(i) = 8.0 nM) over sigma(2) (K(i) = 113.2 nM) receptors. The main differences from previously reported values stem from sigma(2) affinity determinations. This protocol, displacement of [(3)H]DTG binding to sigma(2) sites using (+)-pentazocine (200 nM) to mask sigma(1) sites, was validated by the proper rank order of sigma(2) inhibitory potencies shown by a panel of additional ligands: ifenprodil > haloperidol > DTG >> (+)-pentazocine. Robust Pearson correlation (r = 1.0, P = 0.002; slope = 0.97) was observed for our pK(i) values against those from a prior study by others. The findings have bearing on structure-activity relationships for this active series, and on conclusions that might be drawn from experiments relying upon defined sigma(1)/sigma(2) binding selectivity[1].

NSC34 cells are seeded at a density of 7000 cells per well into 96-well plates with D-MEM and transfected using Lipofectamine 2000 mixed with 2 μg /mL of plasmid vector in D-MEM for 6 h. After 6 h, the cell-culture medium is replaced with fresh D-MEM and culture and allowed to proceed for a further 42 h. The cells are then transferred to serum-free D-MEM and immediately treated with Cutamesine at a final concentration of 1, 3, or 10 nM[2].

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The effects of cutamesine against white light-induced retinal photoreceptor damage were evaluated in vitro by measuring cell death. The expression of sigma-1 receptor after the light exposure was examined by immunoblot analysis. The disruption of the mitochondrial membrane potential and caspase-3/7 activation after excessive light exposure were also examined. In addition, retinal damage in mice induced by irradiation to white light was evaluated using histological staining and electroretinography. Cutamesine reduced the cell death rate induced by light exposure, and the protective effect was prevented by N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine (BD-1047) dihydrobromide, a sigma-1 receptor antagonist. Sigma-1 receptor expression was decreased by light exposure, and cutamesine suppressed the decreased expression of sigma-1 receptor protein. Cutamesine also reduced the mitochondrial damage and reduced the elevated level of caspase 3/7 activity; this effect was attenuated by BD-1047.[3]

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Light-induced cell death model in 661W cell cultures [3]
The 661W cells were seeded at a density of 3 × 103 cells per well in 96-well plates, and then incubated for 24 h under a humidified atmosphere of 5% CO2 at 37 °C. Then, they were treated with 1 or 10 μM cutamesine and/or 1 μM N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine (BD-1047) dihydrobromide and incubated for 1 h under a humidified atmosphere of 5% CO2 at 37 °C. The cells were exposed to 2500 lux of white fluorescent light for 24 h with each agent under a humidified atmosphere of 5% CO2 at 37 °C. The dark control cells and light-irradiated 661W cells were all from the same stock, eliminating any preexisting bias (such as light and temperature), as previously described by Kanan et al. (2007).

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Western blotting analysis [3]
The 661W cells were seeded at a density of 3 × 104 cells per well in 12-well plates, and then incubated for 24 h under a humidified atmosphere of 5% CO2 at 37 °C. Then, they were treated with 10 μM cutamesine and incubated for 1 h. The cells were exposed to 2500 lux of white fluorescent light for 24 h, then washed with PBS, lysed in RIPA buffer supplemented with 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktails 2 and 3, and harvested. The lysates were centrifuged at 12,000 g for 15 min at 4 °C. Protein concentrations were measured by comparison with a known concentration of bovine serum albumin, using a BCA Protein Assay Kit.

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Primary cortical cultures were prepared from postnatal 2-day-old rats as previously reported. The culture medium consisted of 5% fetal bovine serum, 5% heated-inactivated horse serum, 90% of a 1:1 mixture of Dulbecco's modified Eagle's medium, and Ham's F-12 medium. Dissociated cortical neurons were cultured for 4 or 5 days before cutamesine/SA4503 was applied. Twenty-four hours after SA4503 addition, H2O2 (final 50 μM) was applied for 12 h. Then, the cell viability was analyzed. To determine the cell viability, we carried out a mitochondrial-dependent conversion of the tetrazolium salt (MTT) assay. The metabolic activity of mitochondria was estimated with the MTT assay as previously reported. BD1047 (1 μM), an antagonist of sigma-1/2 receptors, was applied 20 min before adding SA4503. [2]

SOD1G93A mice: Transgenic female mice overexpressing mutated human SOD1G93A are used in the study. Cutamesine is dissolved in saline and subcutaneously administered at a dose of 1 mg/kg once daily to 5-week-old SOD1G93A mice to the time of death. In a control group, vehicle (saline) is subcutaneously administered at 10 ml/kg[2].

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Exposure to visible light [3]
The mice were kept in dark conditions for 24 h prior to light exposure for dark adaptation. The pupils of the mice were dilated using 1% cyclopentolate hydrochloride eye drops 30 min prior to exposure to light. The mice, which were not anesthetized, were exposed to visible light (8000 lux) emitted by white fluorescent lamps for 3 h in cages with reflective interiors. The temperature during exposure to light was maintained at 25 ± 1.5 °C. After the exposure to light, all of the mice were returned to darkness for 24 h and then kept under normal light/dark cycling conditions. cutamesine (50 or 500 μM, injected volume; 2 μL), BD-1047 (500 μM), cutamesisne (500 μM) plus BD-1047 (500 μM), or PBS was injected into the intravitreal space 1 h prior to the light exposure. The concentrations of cutamesine in the vitreous body just after the intravitreal administration at 50 and 500 μM were estimated to be approximately 10 and 100 μM, respectively.

[1]. Sigma1 and sigma2 receptor binding affinity and selectivity of SA4503 and fluoroethyl SA4503. Synapse. 2006 May;59(6):350-8.
[2]. SA4503, a sigma-1 receptor agonist, prevents cultured cortical neurons from oxidative stress-induced cell death via suppression of MAPK pathway activation and glutamate receptor expression. Neurosci Lett. 2010 Jan 29;469(3):303-8.
[3]. Effect of a sigma-1 receptor agonist, cutamesine dihydrochloride (SA4503), on photoreceptor cell death against light-induced damage. Exp Eye Res. 2015 Mar;132:64-72.

Drug Indication
Investigated for use/treatment in strokes and depression.

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Many studies suggest that antidepressants act as neuroprotective agents in the central nervous system (CNS), though the underlying mechanism has not been fully elucidated. In the present study, we examined the effect of SA4503, which is a sigma-1 receptor agonist and a novel antidepressant candidate, on oxidative stress-induced cell death in cultured cortical neurons. Exposure of the neurons to H2O2 induced cell death, while pretreatment with SA4503 inhibited neuronal cell death. The SA4503-dependent survival effect was reversed by co-application with BD1047 (an antagonist of sigma-1/2 receptors). Previously we found that H2O2 triggers a series of events including over-activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and intracellular Ca2+ accumulation via voltage-gated Ca2+ channels and ionotropic glutamate receptors, resulting in neuronal cell death (Numakawa et al. (2007) [20]). Importantly, we found in this study that SA4503 reduced the activation of the MAPK/ERK pathway and down-regulated the ionotropic glutamate receptor, GluR1. Taking these findings together, it is possible that SA4503 blocks neuronal cell death via repressing activation of the MAPK/ERK pathway and, consequently, expression levels of glutamate receptors.[2]

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Cutamesine dihydrochloride is an agonist of sigma-1 receptor, which is a ligand-operated receptor chaperone at the mitochondrion-associated endoplasmic reticulum (ER) membrane. ER stress plays a pivotal role in light irradiation-induced retinal damage. In the present study, we examined whether cutamesine is effective against experimental degenerative retinal damages in vitro and in vivo. [3]

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Nootropic Agents: Drugs used to specifically facilitate learning or memory, particularly to prevent the cognitive deficits associated with dementias. These drugs act by a variety of mechanisms.

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To clarify the mechanisms underlying the cutamesine/SA4503-dependent survival, we examined the change in activation of intracellular signaling. PI3K, phosphatidylinositol 3-kinase, and MAPK/ERK pathways are essential for neuronal survival in the CNS. On the other hand, we previously reported that over-activation of ERK1/2 caused by H2O2 is involved in cell death. Thus, to examine the activation of MAPK/ERK pathway to the downstream effects of cutamesine/SA4503, we applied an inhibitor of the MAPK/ERK pathway, U0126, to the cortical cultures. U0126 (10 μM, 3 h before H2O2 stimulation) significantly inhibited H2O2-dependent cell death (Fig. 2A), suggesting that the MAPK/ERK pathway is involved in cell death by H2O2. No additional or synergistic effect by co-application of U0126 and SA4503 as compared with solo SA4503 or U0126 application was observed (Fig. 2A), suggesting that reduction in the activation of the MAPK/ERK pathway contributes to cell protection by SA4503. We confirmed that a decrease in activation (phosphorylation) of p44/42 MAPK (ERK1/2) after U0126 exposure (3 h) occurred in a dose-dependent manner, though the total expression of ERK1/2 was not changed (Fig. 2B). Next, we examined activation of ERK1/2 after SA4503 exposure. SA4503 treatment for 0.5–3 h reduced levels of pERK1/2 (Fig. 2C). The total ERK1/2 was not changed by SA4503 (Fig. 2C). SA4503 depressed the pERK1/2 levels in a dose-dependent manner (Fig. 2D). We checked the activation of Akt (a component of the PI3K pathway). When the time- or dose-dependency of SA4503 on Akt activation was determined, the activation levels were not influenced by SA4503 (Fig. 2E and F). Total Akt expression was intact after SA4503 application (Fig. 2E and F). Furthermore, both total JNK1/2 (c-JunNH2-terminal kinase1/2, an another member of MAPKs) and pJNK1/2, which regulate apoptosis, were examined. After the time- or dose-dependency of SA4503 was determined, it was revealed that total JNK1/2 and pJNK1/2 were not changed by SA4503 (Fig. 2G and H). These results suggest that SA4503 has a protective effect on cortical neurons via repressing activation of the MAPK/ERK pathway.[2]
In summary, SA4503, a sig-1R agonist, stimulates survival-promoting effects on cultured cortical neurons. Previously, we found that antidepressants (imipramine, and fluvoxamine) potentiate BDNF-induced intracellular signaling for release of glutamate via stimulation of sig-1R. Recently, up-regulation of BDNF protein in the rat hippocampus by chronic treatment with SA4503 has been reported. Collectively, these results, including our present study, suggest that SA4503 plays various functions in the CNS. In addition to the potential as a novel antidepressant agent, SA4503 may be valuable to study as a therapeutic agent in the treatment of neurodegenerative diseases of the CNS, although further studies concerning intracellular mechanisms are needed.[2]

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cutamesine dihydrochloride is an agonist of sigma-1 receptor, which is a ligand-operated receptor chaperone at the mitochondrion-associated endoplasmic reticulum (ER) membrane. ER stress plays a pivotal role in light irradiation-induced retinal damage. In the present study, we examined whether cutamesine is effective against experimental degenerative retinal damages in vitro and in vivo. The effects of cutamesine against white light-induced retinal photoreceptor damage were evaluated in vitro by measuring cell death. The expression of sigma-1 receptor after the light exposure was examined by immunoblot analysis. The disruption of the mitochondrial membrane potential and caspase-3/7 activation after excessive light exposure were also examined. In addition, retinal damage in mice induced by irradiation to white light was evaluated using histological staining and electroretinography. Cutamesine reduced the cell death rate induced by light exposure, and the protective effect was prevented by N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine (BD-1047) dihydrobromide, a sigma-1 receptor antagonist. Sigma-1 receptor expression was decreased by light exposure, and cutamesine suppressed the decreased expression of sigma-1 receptor protein. Cutamesine also reduced the mitochondrial damage and reduced the elevated level of caspase 3/7 activity; this effect was attenuated by BD-1047. In in vivo studies, cutamesine suppressed the light-induced retinal dysfunction and thinning of the outer nuclear layer in the mouse retina. These findings indicate that cutamesine protects against retinal cell death in vitro and in vivo by the agonistic effect of sigma-1 receptor. Therefore, sigma-1 receptor may have a potential as a therapeutic target in retinal diseases mediated by photoreceptor degeneration. [3]

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In the present study, we investigated whether cutamesine, a sigma-1 receptor agonist, would play a protective role against light-induced retinal damage in vitro and in vivo.
To examine the effect of cutamesine against light-induced cell death in 661W cells (murine photoreceptor-derived cells), we measured the cell death rate by nuclear staining assay. Cutamesine at 10 μM significantly decreased the cell death rate against light-induced damage. This protective effect was inhibited by BD-1047, a sigma-1 receptor antagonist. These results suggest that cutamesine has a protective effect on light-induced photoreceptor cell death via sigma-1 receptor in vitro.
The protein expression of the sigma-1 receptor in photoreceptor cells was previously demonstrated in an in vivo model (Mavlyutov et al., 2011). The present results demonstrated the expression of sigma-1 receptor in 661W cells. The expression was decreased by light irradiation and attenuated by cutamesine. The sigma-1 receptor has been reported to downregulate in several models. For example, tunicamycin, an ER stress inducer, decreased in the expression of sigma-1 receptor protein in HT22 cells, and imipramine inhibited this downregulation (Ono et al., 2012). In another example, downregulation of the sigma-1 receptor in the heart induced by transverse aortic constriction was attenuated by fluvoxamine, a sigma-1 receptor agonist (Tagashira et al., 2010). These reports show that sigma-1 receptor agonists have stabilization or upregulation effects on the sigma-1 receptor. Thus, cutamesine might stabilize or upregulate the sigma-1 receptor in the present study.
Next, we investigated the effect of cutamesine on the disruption of the mitochondrial membrane potential. Cutamesine recovered the disruption of the mitochondrial membrane potential, and BD-1047 inhibited this effect of cutamesine. These results suggest that cutamesine activated the sigma-1 receptor and enhanced its function to restore abnormal mitochondrial Ca2+ as well, as previously reported by another group (Tagashira et al., 2013). Therefore, the protective effect of cutamesine against light-induced damage might be dependent on recovering mitochondrial function. The activation of caspase-3, a cell death effector, was reported at the photoreceptors in retinal degeneration model animals (Jomary et al., 2001). Abnormal mitochondrial Ca2+ also mediates caspase-3 activation via cytochrome c release (He et al., 2000). In the present study, irradiation of white fluorescent light caused the caspase 3/7 activation in the 661W cells. Cutamesine reduced this elevated level, and this effect was prevented by BD-1047. These reports suggest that cutamesine inhibits caspase activity by recovering mitochondrial function.
Finally, we investigated the protective effect of cutamesine against light-induced damage in an in vivo murine model. The intravitreal injection of 50 and 500 μM of cutamesine suppressed the light-induced damage to retinal function and histological changes in mice. The concentrations of cutamesine in the vitreous body just after the intravitreal administration were estimated to be approximately 10 and 100 μM, respectively. The protective effect was similar to the effective concentration at 10 μM in vitro. Furthermore, the protective effect of cutamesine on the light-induced damage was eliminated by co-administration with BD-1047, a sigma-1 receptor antagonist. Therefore, this effect might depend on the activation of sigma-1 receptor protein, which leads to normalized mitochondrial function and the prevention of caspase activities, as in the present in vitro study.
We recently reported that light exposure causes excessive ER stress and induces photoreceptor cell death via activation of the C/EBP-homologous protein (CHOP)-dependent apoptotic pathway (Nakanishi et al., 2013). Li et al. (2009) also reported that ER stress leads to CHOP-mediated macrophage apoptosis through a pathway involving the release of ER calcium and the mitochondrial release of apoptogens, suggesting that CHOP-dependent apoptosis is related to mitochondrial dysfunction. In addition, in our previous study, imipramine inhibited hippocampal HT22 cell death via normalization of the downregulated sigma-1 receptor, without altering the elevated expression of CHOP (Ono et al., 2012). In the present study, cutamesine might have protected 661W cell death by normalizing the downregulated sigma-1 receptor. As other possible mechanisms, we reviously reported that cutamesine activates both extracellular signal-regulated kinase (ERK)1/2 and Akt, survival factors, in mouse motor neuron (NSC34) cells (Ono et al., 2014). Excessive exposure of visible light also induces ERK inactivation through its dephosphorylation (Kuse et al., 2014). Furthermore, Fujimoto et al. (2012) have been reported that cutamesine potentiates the posttranslational processing of neurotrophins such as BDNF and GDNF thorough prevention of the misfolding of secretary proteins in ER: The sigma-1 receptor is considered to function as a molecular chaperone regulating protein folding at the ER. Therefore, the above mechanisms may be also involved in the protective effect of cutamesine.
In conclusion, cutamesine protected against light-induced photoreceptor cell death by recovering the expression of sigma-1 receptor protein from downregulation and by increasing stimulation of the sigma-1 receptor. Therefore, these findings might provide evidence of a novel effect of cutamesine against light-induced retinal damage, suggesting that cutamesine has medicinal potential in retinal diseases mediated by photoreceptor degeneration. [3]

C₂₃H₃₂N₂O₂

368.51

368.246

C, 74.96; H, 8.75; N, 7.60; O, 8.68

165377-43-5

Cutamesine dihydrochloride;165377-44-6

9907323

Typically exists as solid at room temperature

3.372

0

4

9

27

392

0

COC1=CC=C(CCN2CCN(CCCC3=CC=CC=C3)CC2)C=C1OC

UVSWWUWQVAQPJR-UHFFFAOYSA-N

InChI=1S/C23H32N2O2/c1-26-22-11-10-21(19-23(22)27-2)12-14-25-17-15-24(16-18-25)13-6-9-20-7-4-3-5-8-20/h3-5,7-8,10-11,19H,6,9,12-18H2,1-2H3

1-[2-(3,4-dimethoxyphenyl)ethyl]-4-(3-phenylpropyl)piperazine

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)

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.)