AMPA receptors (EC50 = 0.15, 1.44, 1.66, 0.21 µM, 5.65 for GluR2i, GluR2o, GluR3i, GluR4i, GluR1i, respectively)

Human GluR4-transfected HEK293 cells exhibit enhanced glutamate-evoked inward current in response to LY-404187 (3-10 nM) [2]. In acutely isolated pyramidal neurons, LY-404187 (0.03-10 µM) selectively increases glutamate-evoked currents through AMPA receptors/channels with notable potency (EC50=1.3±0.3 µM) and efficacy (Emax = 45.3±8.0 times increase) [3]. At a dosage of 10 µM, LY-404187 has no effect on the amplitude or time course of whole-cell K+ or Na+ currents in pyramidal neurons of the prefrontal cortex (PFC) [3].

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LY404187 is a selective, potent and centrally active positive allosteric modulator of AMPA receptors. LY404187 preferentially acts at recombinant human homomeric GluR2 and GluR4 versus GluR1 and GluR3 AMPA receptors. In addition, LY404187 potentiates the flip splice variant of these AMPA receptors to a greater degree than the flop splice variant. In both recombinant and native AMPA receptors, potentiation by LY404187 displays a unique time-dependent growth that appears to involve a suppression of the desensitization process of these ion channels. LY404187 has been shown to enhance glutamatergic synaptic transmission both in vitro and in vivo. This augmentation of synaptic activity is due to the direct potentiation of AMPA receptor function, as well as an indirect recruitment of voltage-dependent NMDA receptor activity. Enhanced calcium influx through NMDA receptors is known to be a critical step in initiating long-term modifications in synaptic function (e.g., long-term potentiation, LTP). These modifications in synaptic function may be substrates for certain forms of memory encoding. [1]

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The present study describes the activity of two novel potent and selective AMPA receptor potentiator molecules LY392098 and LY404187. LY392098 and LY404187 enhance glutamate (100 μM) stimulated ion influx through recombinant homomeric human AMPA receptor ion channels, GluR1-4, with estimated EC50 values of 1.77 μM (GluR1i), 0.22 μM (GluR2i), 0.56 μM (GluR2o), 1.89 μM (GluR3i) and 0.20 μM (GluR4i) for LY392098 and EC50 values of 5.65 μM (GluR1i), 0.15 μM (GluR2i), 1.44 μM (GluR2o), 1.66 μM (GluR3i) and 0.21 μM (GluR4i) for LY404187. Neither compound affected ion influx in untransfected HEK293 cells or GluR transfected cells in the absence of glutamate. Both compounds were selective for activity at AMPA receptors, with no activity at human recombinant kainate receptors. Electrophysiological recordings demonstrated that glutamate (1 mM)-evoked inward currents in human GluR4 transfected HEK293 cells were potentiated by LY392098 and LY404187 at low concentrations (3–10 nM). In addition, both compounds removed glutamate-dependent desensitization of recombinant GluR4 AMPA receptors. These studies demonstrate that LY392098 and LY404187 allosterically potentiate responses mediated by human AMPA receptor ion channels expressed in HEK 293 cells in vitro. [2]

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Positive modulators of glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors can enhance cognitive function in several species. The present experiments compared the actions of a novel biarylpropylsulfonamide compound, LY404187, with the prototypical benzoylpiperidine, 1-(quinoxalin-6-ylcarbonyl)-piperidine (CX516), on AMPA receptors of prefrontal cortex (PFC) pyramidal neurons. LY404187 (0.03-10 microM) selectively enhanced glutamate-evoked currents through AMPA receptor/channels of acutely isolated pyramidal neurons with considerably greater potency (EC50 = 1.3 +/- 0.3 microM) and efficacy (Emax = 45.3 +/- 8.0-fold increase) than did CX516 (EC50 = 2.8 +/- 0.9 mM; Emax = 4.8 +/- 1.4-fold increase). Both LY404187 and CX516 increased the potency of the glutamate concentration-response profile by 6- and 3-fold, respectively. Rapid perfusion experiments demonstrated that LY404187 produced a marked suppression in the magnitude but no change in the kinetics of receptor desensitization; whereas CX516 produced little change in the degree and a modest deceleration of the desensitization process. In PFC slices, both spontaneous and stimulus-evoked AMPA receptor-mediated excitatory postsynaptic potentials were enhanced by nanomolar concentrations of LY404187. Voltage-sensitive N-methyl-D-aspartate (NMDA) receptor-dependent synaptic responses also were indirectly augmented as a consequence of greater postsynaptic depolarization. Consistent with the in vitro data, LY404187 was 1000-fold more potent than CX516 in enhancing the probability of discharge of PFC neurons in response to stimulation of glutamatergic afferents from hippocampus in vivo. This potentiation by LY404187 was reduced by both selective AMPA (LY300168, 1 mg/kg, i.v.) and NMDA (LY235959, 5 mg/kg, i.v.) receptor antagonists. Collectively, these results demonstrate that LY404187 is an extremely potent and centrally active potentiator of native AMPA receptors and has a unique mechanism of action. The therapeutic implications of AMPA receptor potentiators are discussed. [3]

Mice exposed to 0.5 mg/kg of LY404187 subcutaneously for 11 days are protected against MPTP-induced neurotoxicity [4]. When administered subcutaneously for 28 days at a dose of 0.5 mg/kg, LY-404187 significantly prevents the loss of tyrosine hydroxylase-positive substantia nigra cell bodies and reduces apomorphine-induced counterrotation [4]. The substantia nigra of rats infused with 6-hydroxydopamine is protected functionally, neurochemically, and histologically by LY-404187 (0.1 or 0.5 mg/kg; subcutaneously for 14 days) [4]. Improvements in both function and histology were observed with LY-404187 (0.5 mg/kg) applied subcutaneously for 14 days after therapy was delayed. This suggests that the drug may have a nutritional impact when given after cell death [4]. LY-404187 raises GAP-43 immunoreactivity in the striatum in a dose-dependent manner when administered subcutaneously for 14 days at 0.1 and 0.5 mg/kg [4].

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Consistent with a recruitment of NMDA receptor activity, LY404187 has been shown to enhance performance in animal models of cognitive function requiring different mnemonic processes. These data suggest that AMPA receptor potentiators may be therapeutically beneficial for treating cognitive deficits in a variety of disorders, particularly those that are associated with reduced glutamatergic signaling such as schizophrenia. In addition, LY404187 has been demonstrated to be efficacious in animal models of behavioral despair that possess considerable predictive validity for antidepressant activity. Although the therapeutic efficacy of AMPA receptor potentiators in these and other diseases will ultimately be determined in the clinic, evidence suggests that the benefit of these compounds will be mediated by multiple mechanisms of action. These mechanisms include direct enhancement of AMPA receptor function, secondary mobilization of intracellular signaling cascades, and prolonged modulation of gene expression. [1]

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Recent developments in the molecular biology and pharmacology of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors has led to the discovery of selective, potent and systemically active AMPA receptor potentiators. These molecules enhance synaptic transmission and evidence suggests that they play important roles in plasticity and cognitive processes. Activation of AMPA receptors also increases neuronal activation and activity-dependent signalling, which may increase brain-derived neurotrophic factor (BDNF) expression and enhance cell proliferation in the brain. We therefore hypothesised that an AMPA receptor potentiator may provide neurotrophic effects in rodent models of Parkinson's disease. In the present studies we report that the potent and selective AMPA receptor potentiator, R,S-N-2-(4-(4-Cyanophenyl)phenyl)propyl 2-propanesulfonamide (LY404187), provides both functional, neurochemical and histological protection against unilateral infusion of 6-hydroxydopamine into the substantia nigra or striatum of rats. The compound also reduced 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced toxicity in mice. Interestingly, we were also able to observe large functional and histological effects when we delayed treatment until after cell death had occurred (3 or 6 days after 6-hydroxydopamine infusion), supporting a neurotrophic mechanism of action. In addition, LY404187 provided a dose-dependent increase in growth-associated protein-43 expression in the striatum. Therefore, we propose that AMPA receptor potentiators offer the potential of a new therapy to halt the progression and perhaps repair the degeneration in Parkinson's disease. [4]

Calcium influx measurements [2]
96-well plates containing confluent monolayers of HEK293 cells stably expressing human AMPA receptors were prepared. Cells were incubated in buffer solution (10 mM glucose, 138 mM sodium chloride, 1 mM magnesium chloride, 5 mM potassium chloride, 5 mM calcium chloride, 10 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, to pH 7.1 to 7.3) containing 20 μM Fluo3-AM dye for 60 min. Cells were washed with buffer solution and fluorescence measurements made using a FLUOROSKAN II fluorimeter that indicated changes in fluorescence upon influx of calcium into cells upon stimulation by glutamate (100 μM) in the presence of cyclothiazide (100 μM) or compound. Compound applications preceded glutamate additions by 5 min and fluorescent measurements were made immediately prior to addition of glutamate and 3 min following glutamate addition. Data are expressed relative to fluorescent changes produced by cyclothiazide (100 μM) for GluR1-4 (i) and for GluR2 flop data is expressed relative to the maximal fluorescence obtained with either LY392098 or LY404187.

Animal/Disease Models: Male C57BL/6J mice (20-25 g) were challenged with MPTP on day 8 [4]
Doses: 0.5 mg/kg
Route of Administration: Sc; twice (two times) daily on weekdays and one time/day on weekends for 11 days
Experimental Results: Attenuated loss of tyrosine hydroxylase immunoreactivity in the substantia nigra. There were no significant changes in dorsal and ventral striatal tyrosine hydroxylase immunoreactivity.

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MPTP neurotoxicity in mice [4]
Male C57BL/6J mice weighing 20–25 g were used. They were housed in groups of five mice per cage under a 12:12-h light/dark cycle (lights on 7:00 a.m. to 7:00 p.m.) with food and water available ad libitum. LY404187 was administered at 0.5 mg/kg s.c for 11 days. On day 8 the animals received 4×20 mg/kg MPTP at 2 h intervals.

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Drug studies [4]
For all studies, LY404187 was dissolved in 12.5% β-cyclodextrin and sonicated prior to administration and was administered subcutaneously (s.c.) twice daily on weekdays and once daily at weekends.
For studies using the striatal lesion model, LY404187 was administered for 28 days at 0.5 mg/kg s.c. starting 1 day after 6-hydroxydopamine lesion.
For nigral lesion studies, LY404187 was administered for 14 days at either 0.1 or 0.5 mg/kg s.c. starting 1 day after 6-hydroxydopamine lesion. In additional studies treatment with LY404187 was delayed until 3, or 6 days after 6-hydroxydopamine infusion.

[1]. LY404187: a novel positive allosteric modulator of AMPA receptors. CNS Drug Rev. Fall 2002; 8(3): 255-82.

[2]. Novel AMPA receptor potentiators LY392098 and LY404187: effects on recombinant human AMPA receptors in vitro. Neuropharmacology. 2001 Jun; 40(8): 976-83.

[3]. Positive modulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors in prefrontal cortical pyramidal neurons by a novel allosteric potentiator. J Pharmacol Exp Ther. 2001 Jul; 298(1): 86-102.

[4]. Neurotrophic actions of the novel AMPA receptor potentiator, LY404187, in rodent models of Parkinson's disease. Eur J Pharmacol. 2004 Feb 20; 486(2): 163-74.

Multiple intracellular signaling pathways regulate brain-derived neurotrophic factor (BDNF) levels. Antidepressants that increase norepinephrine release may enhance BDNF levels through the CREB pathway. Activation of the MAP kinase pathway can also increase BDNF mRNA levels. Lyn, a member of the Src family of protein tyrosine kinases, can activate the MAP kinase pathway. Studies have shown that Lyn physically interacts with the AMPA receptor GluR2 and GluR3 subunits and can be activated by potent stimulation of the AMPA receptor. Furthermore, primary cerebellar culture studies have shown that cyclothiazide alone has no effect on Lyn activation, but its activation is enhanced when used in combination with AMPA. Therefore, it is hypothesized that Lyn activation can increase BDNF levels by enhancing AMPA receptor activity. Neuron culture studies have confirmed this hypothesis, showing that adding LY392098 in the presence of AMPA or glutamate increases BDNF mRNA and protein levels. In summary, these data suggest that positively modulating AMPA receptors by increasing BDNF levels through the MAP kinase pathway may be beneficial for the treatment of depression. We have preliminarily explored the application of AMPA receptor enhancers in the treatment of depression. The activity of LY404187 was tested in a forced swimming test model, which can be used to detect antidepressant compounds (P. Skolnick, personal communication). The results showed that LY404187 reduced immobility time in mice and rats during the forced swimming test, with minimum effective doses (MEDs) of 0.05 and 0.025 mgu0001kg (oral), respectively (Figure 13). In this study, imipramine (15 mgu0001kg, intraperitoneal injection) served as a positive control. LY404187 did not affect motor activity, indicating that the efficacy of this compound in the forced swimming test is independent of its motor excitatory effect. Similar results were reported for LY392098. Furthermore, systemic administration of LY300168 blocked the activity of LY392098 but not the activity of imipramine, indicating that the action of this enhancer is mediated by AMPA receptors. In summary, these findings suggest that positive regulators of AMPA receptors are active in animal models capable of detecting clinically effective antidepressants. This supports the hypothesis that such compounds may represent a new class of antidepressants. [1]

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Electrophysiological recordings of glutamate-induced currents in human GluR4-transfected HEK293 cells showed that the detected AMPA receptor enhancers significantly enhanced the inward current. The observed enhancement was reversible, and no effect of the compounds was observed in the absence of agonists. The potency order of LY392098 and LY404187 was similar in both fluorescence assays and electrophysiological studies. Both compounds were more potent in enhancing the glutamate response than cyclothiazide or CX516. As shown by GluR4i (Fig. 3b), the desensitization of the AMPA receptor response to glutamate was rapid. Cyclothiazide significantly reduced the observed desensitization, although the current continued to decrease after sustained application of glutamate. Conversely, in the presence of LY392098 and LY404187, the amplitude of the glutamate-induced current increased during a 10-second glutamate application. The maximum rate of increase in current observed during the 10-second glutamate application appears to depend on the concentration of the AMPA receptor enhancer. The mechanism of this apparent agonist-dependent enhancement is currently under investigation. Shorter glutamate applications lasting 5 milliseconds are closer to the synaptic state (Clements et al., 1992). We investigated recovery after AMPA receptor desensitization using short-duration agonist applications. In this study, the AMPA receptor-mediated response recovery time progression approximates a single exponential time constant t = 48 milliseconds. LY404187 (1 μM), LY392098 (1 μM), and cyclothiazide (10 μM) all accelerated the recovery rate after desensitization. Possible mechanisms for reduced AMPA receptor desensitization include alterations in AMPA receptor inactivation mechanics. Cyclothiazide and aniracetam have both been shown to modulate the inactivation kinetics of recombinant and native AMPA receptors (Hestrin, 1992; Patneau et al., 1993; Partin et al., 1996). In order to study inactivation kinetics, in vitro experiments on the membrane are required. Due to the rapid decrease in current observed in in vitro hGluR receptor membranes, we have been unable to study the potential activity of these compounds in vitro. Current studies have confirmed that LY392098 and LY404187 are potent and selective enhancers of the glutamate response of human AMPA receptors in vitro. It will be important to study the activity of these AMPA receptor enhancers in heterologous oligomeric human recombinant ion channel complexes and neurons. [2] In summary, we have provided strong evidence that AMPA receptor enhancers can provide functional, neurochemical and histological protection in rodent models of Parkinson's disease. Such molecules have been reported to play a positive role in models of depression (Li et al., 2001; Quirk and Nisenbaun, 2002) and cognitive impairment (Staubli et al., 1994; Hampson et al., 1998a; Hampson et al., 1998b; Quirk and Nisenbaun, 2002), both of which are comorbidities in Parkinson's disease. Furthermore, in this study, these protective effects were maintained even after delayed administration until cell death, and the increased expression of GAP-43 in the striatum provided encouraging evidence for neurotrophic effects. These results suggest that LY404187 or its analogues are ideal molecules that could serve as clinical candidates to prevent or potentially reverse the degenerative changes observed in Parkinson's disease. [4]

C19H22N2O2S

342.455183506012

348.187

C, 66.64; H, 6.48; N, 8.18; O, 9.34; S, 9.36

211311-95-4

211311-95-4;

9928016

White to off-white solid powder

1.1±0.1 g/cm3

498.4±47.0 °C at 760 mmHg

255.2±29.3 °C

0.0±1.3 mmHg at 25°C

1.546

4.19

1

4

6

24

527

0

S(C(C)C)(NCC(C)C1C=CC(C2C=CC(C#N)=CC=2)=CC=1)(=O)=O

HOQAVGZLYRYHSO-UHFFFAOYSA-N

InChI=1S/C19H22N2O2S/c1-14(2)24(22,23)21-13-15(3)17-8-10-19(11-9-17)18-6-4-16(12-20)5-7-18/h4-11,14-15,21H,13H2,1-3H3

2-Propanesulfonamide, N-(2-(4'-cyano(1,1'-biphenyl)-4-yl)propyl)-

LY-404187; LY 404187; 211311-95-4; N-(2-(4'-Cyano-[1,1'-biphenyl]-4-yl)propyl)propane-2-sulfonamide; LY404187; LY-404,187; LY 404,187; N-[2-[4-(4-cyanophenyl)phenyl]propyl]propane-2-sulfonamide; 75W6I8W6OU; CHEMBL435582; LY404187.

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)

DMSO : ~100 mg/mL (~292.00 mM)

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.)