AMPA receptor

Sunifiram is a novel pyrrolidone nootropic drug structurally related to piracetam, which was developed for neurodegenerative disorder like Alzheimer's disease. To address question whether sunifiram affects N-methyl-D-aspartate receptor (NMDAR)-dependent synaptic function in the hippocampal CA1 region, we assessed the effects of sunifiram on NMDAR-dependent long-term potentiation (LTP) by electrophysiology and on phosphorylation of synaptic proteins by immunoblotting analysis. In mouse hippocampal slices, sunifiram at 10-100 nM significantly enhanced LTP in a bell-shaped dose-response relationship which peaked at 10 nM. Sunifiram treatments at 1-1000 nM increased the slope of field excitatory postsynaptic potentials (fEPSPs) in a dose-dependent manner. The enhancement was associated with an increase in phosphorylation of AMPAR receptor through activation of CaMKII. Interestingly, under the basal condition, sunifiram treatments increased PKCα (Ser-657) and Src family (Tyr-416) activities with the same bell-shaped dose-response curve as that of LTP peaking at 10 nM. The increase in phosphorylation of PKCα (Ser-657) and Src (Tyr-416) induced by sunifiram was inhibited by 7-ClKN treatment. The LTP enhancement by sunifiram was significantly inhibited by PP2, a Src family inhibitor. Finally, when pretreated with a high concentration of glycine (300 μM), sunifiram treatments failed to potentiate LTP in the CA1 region. Taken together, sunifiram stimulates the glycine-binding site of NMDAR with concomitant PKCα activation through Src kinase. Enhancement of PKCα activity triggers to potentiate hippocampal LTP through CaMKII activation.[2]

OBX mice were administered once a day for 7-12 days with sunifiram (0.01-1.0mg/kg p.o.) from 10 days after operation with or without gavestinel (10mg/kg i.p.), which is glycine-binding site inhibitor of N-methyl-d-aspartate receptor (NMDAR). The spatial reference memory assessed by Y-maze and short-term memory assessed by novel object recognition task were significantly improved by sunifiram treatment in OBX mice. Sunifiram also restored hippocampal LTP injured in OBX mice without treatment with gavestinel. By contrast, sunifiram treatment did not ameliorate the depressive behaviors assessed by tail suspension task in OBX mice. Notably, sunifiram treatment restored CaMKIIα (Thr-286) autophosphorylation and GluR1 (Ser-831) phosphorylation in the hippocampal CA1 region from OBX mice to the levels of control mice. Likewise, sunifiram treatment improved PKCα (Ser-657) autophosphorylation and NR1 (Ser-896) phosphorylation to the control levels. Stimulation of CaMKII and PKC autophosphorylation by sunifiram was significantly inhibited by pre-treatment with gavestinel. However, sunifiram treatment did not affect the phosphorylation of CaMKIV (Thr-196) and ERK. Taken together, sunifiram ameliorates OBX-induced deficits of memory-related behaviors and impaired LTP in the hippocampal CA1 region via stimulation of glycine-binding site of NMDAR.[3]

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DM 232 and DM 235/sunifiram are novel antiamnesic compounds structurally related to ampakines. The involvement of AMPA receptors in the mechanism of action of DM 232 and DM 235 was, therefore, investigated in vivo and in vitro. Both compounds (0.1 mg/kg(-1) i.p.) were able to reverse the amnesia induced by the AMPA receptor antagonist NBQX (30 mg/kg(-1) i.p.) in the mouse passive avoidance test. At the effective doses, the investigated compounds did not impair motor coordination, as revealed by the rota rod test, nor modify spontaneous motility and inspection activity, as revealed by the hole board test. DM 232 and DM 235 reversed the antagonism induced by kynurenic acid of the NMDA-mediated release of [(3)H]NA in the kynurenate test performed in rat hippocampal slices. This effect was abolished by NBQX. DM 232 increases, in a concentration dependent manner, excitatory synaptic transmission in the rat hippocampus in vitro. These results suggest that DM 232 and DM 235 act as cognition enhancers through the activation of the AMPA-mediated neurotransmission system. [1]

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Alzheimer's disease (AD) shows degeneration of the cholinergic system in the medial septum, thereby eliciting down-regulation of the olfactory function in patients. We have previously reported that olfactory bulbectomized (OBX) mice show hippocampus-dependent memory impairment as assessed by memory-related behavioral tasks and hippocampal long-term potentiation (LTP). In the present study, we focused whether novel pyrrolidone nootropic drug sunifiram improves both memory impairment and depression observed in OBX mice. OBX mice were administered once a day for 7-12 days with sunifiram (0.01-1.0mg/kg p.o.) from 10 days after operation with or without gavestinel (10mg/kg i.p.), which is glycine-binding site inhibitor of N-methyl-d-aspartate receptor (NMDAR). The spatial reference memory assessed by Y-maze and short-term memory assessed by novel object recognition task were significantly improved by sunifiram treatment in OBX mice. Sunifiram also restored hippocampal LTP injured in OBX mice without treatment with gavestinel. By contrast, sunifiram treatment did not ameliorate the depressive behaviors assessed by tail suspension task in OBX mice. Notably, sunifiram treatment restored CaMKIIα (Thr-286) autophosphorylation and GluR1 (Ser-831) phosphorylation in the hippocampal CA1 region from OBX mice to the levels of control mice. Likewise, sunifiram treatment improved PKCα (Ser-657) autophosphorylation and NR1 (Ser-896) phosphorylation to the control levels. Stimulation of CaMKII and PKC autophosphorylation by sunifiram was significantly inhibited by pre-treatment with gavestinel. However, sunifiram treatment did not affect the phosphorylation of CaMKIV (Thr-196) and ERK. Taken together, sunifiram ameliorates OBX-induced deficits of memory-related behaviors and impaired LTP in the hippocampal CA1 region via stimulation of glycine-binding site of NMDAR [3].

Biochemical Analysis [2]
Biochemical analysis was performed as described previously (Laemmli, 1970; Moriguchi et al., 2008). We used antibody as listed below: anti-phospho CaMKII, (1:5,000; Fukunaga et al., 2002), anti-CaMKII, (1:5,000; Fukunaga et al., 1995), anti-phospho-PKCα (Ser-657) (1:2,000), anti-PKCα (1:2,000), anti-phospho-MAP kinase (diphosphorylated ERK 1/2) (1:2,000), anti-ERK (1:2,000), anti-phospho-GluR1 (Ser-831) (1:1,000), anti-GluR1 (1:1,000), anti-phospho-synapsin I (Ser-603) (1:2,000), anti-phospho-NR1 (Ser-896) (1:2,000; Millipore), anti-NR1 (1:2,000), anti-phospho-Src family (Tyr-416) (1:2,000), and anti-β-tubulin (1:5,000). Bound antibodies were visualized using the enhanced chemiluminescence detection system and analyzed semiquantitatively using the National Institutes of Health Image program.

Drugs [DM 232 (unifiram) and DM 235 (sunifiram)] were dissolved in isotonic (NaCl 0.9%) saline solution immediately before use. Drug concentrations were prepared so that the necessary dose could be administered in a volume of 10 ml/kg−1 by i.p. injection for mice. For electrophysiological experiments DM 232 was dissolved in dimethylsulfoxide (DMSO) and stock solutions were made to obtain concentrations of DMSO of 0.05% and 0.01% in aCSF, respectively. Control experiments, carried out in parallel for an unrelated project, showed that this concentration of DMSO did not affect the amplitude of synaptic potential. [1]

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Electrophysiology [2]
Hippocampal slices were prepared as described previously (Moriguchi et al., 2008). Transverse hippocampal slices (400 μm thick) prepared using a vibratome were incubated for 2 h in continuously oxygenized (95% O2, 5% CO2) artificial cerebrospinal fluid (ACSF) at room temperature. After a 2-h recovery period, slices were transferred to an interface recording chamber and perfused at a flow rate of 2 ml/min with ACSF warmed to 34°C. Field excitatory postsynaptic potentials (fEPSPs) were evoked by a 0.05-Hz test stimulus through a bipolar stimulating electrode placed on the Schaffer collateral/commissural pathway and recorded from the stratum radiatum of CA1 region using a glass electrode filled with 3 M NaCl. High-frequency stimulation (HFS) of 100 Hz with a 1-s duration was applied twice with a 10-s interval and test stimulation was continued for the indicated periods. After recording, slices were transferred to a plastic plate cooled on ice to dissect out the CA1 areas. CA1 regions were frozen in liquid nitrogen and stored at –80°C until biochemical analysis was performed.

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Operation [3]
OBX mice were prepared as describe previously [30]. Mice were treated once a day for 7–12 days with sunifiram or sunifiram plus gavestinel starting at 10 days after OBX operation. Behavioral tests were performed at 7–8 days treatment with sunifiram (p.o.) or sunifiram plus gavestinel (i.p.) and electrophysiological and biochemical experiments were performed 9–12 days after sunifiram or sunifiram plus gavestinel treatments. OBX-operated mice had no stereotype killing behavior at least until 3 weeks after operation. However, aggressive behavior without killing behavior was observed in several mice. In the present study, we did not use the aggressive mice separated after OBX surgery for experiment. We breed 3 or 4 mice in each cage and treated the same drug. All animals were sacrificed at the end of experiment and the lesions were verified histologically.

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Drug treatment [3]
Sunifiram was dissolved in (CMC) and administered orally (p.o.) using metal gastric zonde. The dose or volume of injection of sunifiram was 0.1 or 1.0 mg/kg in a volume of 1 ml/100 g body weight. We compared both sunifiram treatment mice and CMC treatment mice (vehicle). Gavestinel was dissolved in tap water and administered intraperitoneal injection (i.p.). The dose or volume of injection of gavestinel was 10 mg/kg. We compared both gavestinel treatment and tap water treatment (vehicle).

[1]. AMPA-receptor activation is involved in the antiamnesic effect of DM 232 (unifiram) and DM 235 (sunifiram). Naunyn Schmiedebergs Arch Pharmacol. 2003 Dec;368(6):538-45.

[2]. Novel nootropic drug sunifiram enhances hippocampal synaptic efficacy via glycine-binding site of N-methyl-D-aspartate receptor. Hippocampus. 2013 Oct;23(10):942-51.

[3]. Novel nootropic drug sunifiram improves cognitive deficits via CaM kinase II and protein kinase C activation in olfactory bulbectomized mice. Behav Brain Res. 2013 Apr 1;242:150-7.

Sunifiram is a novel pyrrolidone nootropic drug structurally related to piracetam, which was developed for neurodegenerative disorder like Alzheimer's disease. Sunifiram is known to enhance cognitive function in some behavioral experiments such as Morris water maze task. To address question whether sunifiram affects N-methyl-D-aspartate receptor (NMDAR)-dependent synaptic function in the hippocampal CA1 region, we assessed the effects of sunifiram on NMDAR-dependent long-term potentiation (LTP) by electrophysiology and on phosphorylation of synaptic proteins by immunoblotting analysis. In mouse hippocampal slices, sunifiram at 10-100 nM significantly enhanced LTP in a bell-shaped dose-response relationship which peaked at 10 nM. The enhancement of LTP by sunifiram treatment was inhibited by 7-chloro-kynurenic acid (7-ClKN), an antagonist for glycine-binding site of NMDAR, but not by ifenprodil, an inhibitor for polyamine site of NMDAR. The enhancement of LTP by sunifilam was associated with an increase in phosphorylation of α-amino-3-hydroxy-5-methylisozazole-4-propionate receptor (AMPAR) through activation of calcium/calmodulin-dependent protein kinase II (CaMKII) and an increase in phosphorylation of NMDAR through activation of protein kinase Cα (PKCα). Sunifiram treatments at 1-1000 nM increased the slope of field excitatory postsynaptic potentials (fEPSPs) in a dose-dependent manner. The enhancement was associated with an increase in phosphorylation of AMPAR receptor through activation of CaMKII. Interestingly, under the basal condition, sunifiram treatments increased PKCα (Ser-657) and Src family (Tyr-416) activities with the same bell-shaped dose-response curve as that of LTP peaking at 10 nM. The increase in phosphorylation of PKCα (Ser-657) and Src (Tyr-416) induced by sunifiram was inhibited by 7-ClKN treatment. The LTP enhancement by sunifiram was significantly inhibited by PP2, a Src family inhibitor. Finally, when pretreated with a high concentration of glycine (300 μM), sunifiram treatments failed to potentiate LTP in the CA1 region. Taken together, sunifiram stimulates the glycine-binding site of NMDAR with concomitant PKCα activation through Src kinase. Enhancement of PKCα activity triggers to potentiate hippocampal LTP through CaMKII activation. [2]

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This is a first report showing that nootropic drug, sunifiram-enhanced hippocampal LTP via stimulation of the glycine-binding site of NMDAR. The glycine-binding site is present in the NR1 subunit (Wafford et al., 1995) and it is an attractive target site for AD drugs. For example, the ligands for glycine-binding site of NMDAR such as milacemide, a glycine prodrug, and D-cycloserine, a partial glycine agonist, have been clinically tested for use in AD patients (Schwartz et al., 1991, 1996; Dysken et al., 1992). In addition, a partial glycine agonist, D-cycloserine, and a glycine prodrug, milacemide, are known to improve memory deficits in aged rat (Hanndelmann et al., 1989; Baxter et al., 1994). We have previously reported that the other nootropic drug, nefiracetam, enhances synaptic transmission through the glycine-binding site of NMDAR in rat cortical neurons (Moriguchi et al., 2003). We hypothesized that activation of the glycine-binding site of NMDAR improves cognitive function and memory deficits seen in AD patients. In this study, we have demonstrated that the precise molecular mechanism underlying LTP enhancement by sunifiram via the glycine-binding site of NMDAR.
The sunifiram-enhanced LTP showed a bell-shaped dose-response relationship that peaked at 10 nM. Bell-shaped increases in PKCα (Ser-657) autophosphorylation and NR1 (Ser-896) phosphorylation were closely associated with the bell-shaped enhancement of LTP by sunifiram in the CA1. Increase in NR1 phosphorylation by PKC are critically important for the LTP enhancement, because NR1 phosphorylation accounts for upregulation of NMDAR function (Tingley et al., 1993). In addition, the enhanced Tyr-416 phosphorylation of Src kinase and the concomitant increased NR2B (Tyr-1472) phosphorylation were also associated with the bell-shaped enhancement of LTP by sunifiram in the CA1. Site of NR2B (Try-1472) is phosphorylated by Src family tyrosine kinases such as Fyn (Nakazawa et al., 2001). Src kinase is activated by NMDAR stimulation via PKC-dependent tyrosine kinases (Kelso et al., 1992; Luttrell et al., 1996; Della Rocca et al., 1997) and is sufficient for LTP induction (Lu et al., 1998). Interestingly, when treated with the Src family inhibitor PP2 in hippocampal CA1 slices, autophosphorylation of PKCα (Ser-657) and phosphorylation of Src family (Tyr-416) failed to be potentiated by sunifiram. In addition, PP2 treatment also inhibited the enhancement of LTP by sunifiram in the CA1. Our data suggested that activation of PKC and Src should be a downstream event following stimulation of the glycine site of NMDARs.
We found that sunifiram potentiates fEPSPs in the CA1 region through activation of AMPAR in a dose-dependent manner (1–1,000 nM). Galeotti et al. (2003) reported that sunifiram improves cognitive function via AMPAR activation. In fact, other nootropic drugs also directly activate AMPAR function (Ito et al., 1990; Galeotti et al., 2003; Moriguchi et al., 2003). L-type Ca2+ channels and AMPAR in a high concentration of nootropic compounds are likely mediated by CaMKII activity (Yoshii et al., 1997). Indeed, high concentration of nefiracetam enhanced AMPAR functions through increased CaMKII activity in the cortex (Moriguchi et al., 2003). In this study, sunifiram-enhanced AMPAR function likely through increased CaMKII activity.
We also found that PKC-dependent phosphorylation of NR1 contributed to the enhancement of NMDAR function potentiated by sunifiram in the CA1. The bell-shaped increases in PKCα (Ser-657) autophosphorylation and phosphorylation of NR1 (Ser-896) were closely associated with the bell-shaped enhancement of LTP by sunifiram. The activation of PKCα (Ser-657) by sunifiram triggers enhancement of NMDAR functions, because PKCα (Ser-657) phosphorylates the Ser-896 of NMDAR NR1 subunit (Tingley et al., 1997). However, the mechanisms underlying the bell-shaped dose-response curve in activation of NMDAR function and in LTP enhancement by sunifiram are unclear at present.
AD is associated with downregulation of brain cholinergic and glutamatergic activities (Greenamyre et al., 1987; Giacobini, 2000). Recently, clinical approaches tried to enhance the cholinergic system using anti-cholinesterases such as donepezil, galantamine, and rivastigmine and neuroprotective action using anti-NMDAR such as memantine. In this study, sunifiram potently enhances NMDAR-dependent LTP via the glycine-binding site of NMDAR in the hippocampus. In addition, nootropics including sunifiram increase acetylcholine release in rat brain (Manetti et al., 2000). Our hypothesis is that enhancement of both cholinergic and glutamatergic activities by sunifiram seems to improve cognitive deficits in AD patients.
In conclusion, the present study demonstrated that sunifiram treatment markedly enhances hippocampal LTP via glycine-binding site of NMDAR. Sunifiram stimulates the glycine-binding site of NMDAR through PKCα and Src kinase activation. Enhancement of PKCα activity triggers to potentiate hippocampal LTP through CaMKII-mediated NMDAR activation. Enhancement of hippocampal LTP by sunifiram forms the basis of the improvement of cognitive and memory deficits seen in AD patients. [2]

C14H18N2O2

246.31

246.136

C, 68.27; H, 7.37; N, 11.37; O, 12.99

314728-85-3

4223812

White to off-white solid powder

1.2±0.1 g/cm3

442.0±38.0 °C at 760 mmHg

205.0±19.1 °C

0.0±1.1 mmHg at 25°C

1.561

0.26

0

2

2

18

303

0

DGOWDUFJCINDGI-UHFFFAOYSA-N

InChI=1S/C14H18N2O2/c1-2-13(17)15-8-10-16(11-9-15)14(18)12-6-4-3-5-7-12/h3-7H,2,8-11H2,1H3

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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

Solubility in Formulation 1: 50 mg/mL (203.00 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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