L-type calcium channels (dihydropyridine-sensitive) [1]
Pertussis toxin-sensitive inhibitory G-proteins (G0/Gi) [1]
cAMP-dependent processes [1]
Nefiracetam targets multiple receptors and ion channels, functioning as an activator of nAChR, NMDAR, mGluR5, PKC, GABA receptors, and N/L-type Ca2+ channels. It interacts with the PKC pathway, augmenting the activity of nicotinic acetylcholine receptors and increasing the release of presynaptic glutamate, which leads to potentiation of synaptic transmission in the hippocampus. This multi-target mechanism may underlie its cognitive-enhancing effects through interactions with PKA and PKC pathways. It facilitates hippocampal neurotransmission by a mechanism independent of piracetam and aniracetam.

Nefiracetam at a concentration of 1 μM increases a long-lasting component of calcium channel currents two-fold without affecting a transient component. Nefiracetam induces a short-term depression of ACh-evoked currents at submicromolar concentrations (0.01–0.1 μM) and a long-term enhancement of the currents at micromolar concentrations (1–10 μM). Nefiracetam interacts with PKA and PKC pathways, which may explain a cellular mechanism for the action of cognition-enhancing agents. Lower (submicromolar) concentrations of the nootropic Nefiracetam reduces ACh-evoked currents to 30% (0.01 μM) and 38% (0.1 μM) of control after a 10-minute treatment. In primary cultures of rat hippocampal neurons, nefiracetam increases the rate of nicotine-sensitive miniature excitatory postsynaptic currents. Nefiracetam induces a long-lasting facilitation of synaptic transmission in both the CA1 area and the dentate gyrus of rat hippocampal slices, and the facilitation is inhibited by α-bungarotoxin and mecamylamine. Nefiracetam enhances activity of nicotinic ACh receptors by interacting with a PKC pathway, thereby increasing glutamate release from presynaptic terminals, and then leading to a sustained facilitation of hippocampal neurotransmission.
Kinase Assay: Hippocampal slices (400 μM) are prepared from the guinea pig brain using standard techniques. A slice is fixed on a pair of silver wire electrodes (10 Hz, 5 V, 0.1 ms in duration) at 1-minutes intervals for 10 minutes and submerged in 1 mL standard artificial cerebrospinal fluid (ACSF) (in mM: 125 mM NaCl, 5 mM KCl, 1.24 mM KH2PO4, 1.3 mM MgSO4, 2 mM CaCl2, 26 mM NaHCO3, and 10 mM glucose) oxygenated with 95% O2 and 5% CO2 at 36 °C in the presence and absence of tetrodotoxin (TTX) (0.5 μM). In a different set of experiments, electrical stimulation is applied to slices treated with Nefiracetam (1 μM) in the presence and absence of α-bungarotoxin (50 nM) or mecamylamine (3 μM). A 100 μL aliquot of the medium filtered with millipore filters (0.45 μM) is injected onto the cation-exchanger column of the autoanalyser to separate amino acids and the amount of glutamate released is calculated using known amino acid standard concentrations.
Cell Assay: The injected oocytes are transferred to the recording chamber 24 to 48 hours after incubation and continuously superfused at room temperature (20 to 22 °C) in a standard frog Ringers solution (115 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, and 5 mM HEPES, pH 7.0). Ca2+ -free extracellular solution consisted of 115 mM NaCl, 2 mM KCl, 5 mM MgCl2, 5 mM HEPES, and 1 mM EGTA, pH 7.0. To remove the effect of the muscarinic ACh receptor, 1 μM atropine is added to the extracellular solution. ACh-activated currents are recorded using two-electrode, voltage-clamp techniques. The currents are analyzed on a microcomputer using pClamp software. ACh is bath-applied to oocytes. Nefiracetam is dissolved in distilled water at 1 mM for stock solution and diluted into concentrations required with the extracellular solution.

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In NG108-15 cells using whole-cell patch-clamp, Nefiracetam (DM-9384) at 1 μM increased long-lasting calcium channel currents (Ba2+ currents) two-fold (204.8±12.9% of control, n=9) without affecting transient component [1]
Dose-response relationship showed a bell-shaped curve with peak at 1 μM; at 0.1 μM: 123.7±7.4%; at 10 μM: 136.2±5.9%; at 100 μM: 82.5±11.8% (inhibition) [1]
Aniracetam showed similar but less potent effects (at 1 μM: 137.6±5.1%; at 10 μM: 160.8±14.1%) [1]
Dibutyryl cAMP (1 mM) also enhanced currents, and the effects of nefiracetam and db-cAMP were not additive beyond saturation [1]
The currents enhanced by nefiracetam were markedly reduced by nifedipine (10 μM), an L-type calcium channel blocker [1]
Cells treated with pertussis toxin (500 ng/ml, >20 h) became insensitive to nefiracetam (relative current amplitude 103.1±3.2%, n=15) [1]
Nefiracetam (DM-9384) (1 μM) significantly elevated cAMP levels in NG108-15 cells [1]
In vitro, Nefiracetam (1-1000 nM, 10-60 min) increases rat dentate gyrus hippocampal neurotransmission by 250% in a dose-dependent manner. It induces a short-term depression of ACh-evoked currents at submicromolar concentrations (0.01-0.1 μM) and a long-term enhancement of the currents at micromolar concentrations (1-10 μM). It also interacts with PKA and PKC pathways, which may explain a cellular mechanism for the action of cognition-enhancing agents. These effects confirm its role as a modulator of synaptic transmission and plasticity.

Nefiracetam administered orally inhibits Ro 5-4864-induced convulsions in EL mice. Nefiracetam also efficiently inhibits Ro 5-4864-induced convulsions in DDY mice at doses higher than 10 mg/kg. Nefiracetam administered daily 1 hour before each training session facilitates the acquisition process of the avoidance response.

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In vivo, Nefiracetam is an orally active cognition-enhancing agent. It has been studied in animal models for its potential to improve cognitive function and memory. Its ability to activate multiple neurotransmitter systems suggests it may have broad therapeutic potential. It has been investigated for the treatment of Alzheimer's disease, epilepsy, and cerebral ischemia. It was under Phase 2 clinical investigation as a modulator of GABAergic, cholinergic, and monoaminergic systems. However, specific detailed in vivo study results are not extensively documented in standard summaries.

Non-cellular receptor binding assays for Nefiracetam are not typically performed, as its mechanism involves modulation of multiple neurotransmitter systems rather than direct enzyme inhibition. However, its effects on receptor binding can be studied using membrane preparations. The compound's ability to modulate receptor activity can be assessed in electrophysiological assays using oocytes or other expression systems. For example, its effects on ACh-evoked currents can be measured to determine its potency at nAChRs. These assays are essential for characterizing its receptor modulation properties.

NG108-15 cells (neuroblastoma × glioma hybrid) were cultured and differentiated with 2% DMSO added to culture medium (90% Dulbecco's Modified Eagle's Medium + 10% fetal calf serum) for 5-7 days before use [1]
Whole-cell patch-clamp recording: patch electrodes (2-8 MΩ) were placed onto cell surface, membrane ruptured by suction or high-frequency train pulse; cells were voltage-clamped; external solution contained (mM): BaCl2 50, NaCl 30, CsCl 5, TEA-Cl 25, glucose 25, TTX 0.5 μM, HEPES 10 (pH 7.4 with CsOH); internal solution contained (mM): Cs-glutamate 130, MgCl2 2.5, glucose 5, PIPES 10 (pH 7.0 with NaOH); experiments at room temperature (22-25°C); currents induced by step depolarizations from holding potential of -80 mV or -50 mV; long-lasting currents measured at end of 160 ms pulse [1]
Pertussis toxin treatment: cells were treated with PTX (500 ng/ml) for >20 h (22 h in one example) to inactivate inhibitory G-proteins [1]
In vitro cell-based assays for Nefiracetam are conducted using neuronal cell cultures to assess its effects on neurotransmission and synaptic plasticity. For instance, rat dentate gyrus hippocampal slices can be used to measure its effect on neurotransmission. The compound's ability to modulate ACh-evoked currents can be studied in oocytes or neuronal cell lines expressing nAChRs. Additionally, its effects on PKC and PKA signaling pathways can be assessed in various cell types. These experiments are crucial for confirming its mechanism of action.

In vivo animal studies for Nefiracetam are typically conducted in rodent models of cognitive impairment, epilepsy, or cerebral ischemia. The compound is administered orally, and its effects on cognitive function, seizure activity, or neuroprotection are assessed. Behavioral tests such as the Morris water maze or passive avoidance tasks can be used to evaluate cognitive enhancement. However, specific detailed protocols are not extensively documented in standard summaries. Its efficacy in these models supports its potential for Alzheimer's disease and related disorders.

Nefiracetam has a molecular weight of 246.30 g/mol and a molecular formula of C14H18N2O2. It is soluble in DMSO and should be stored as a powder at -20°C for up to 3 years or in solution at -80°C for up to 1 year. As an orally active compound, it is well-absorbed from the gastrointestinal tract and is expected to penetrate the blood-brain barrier due to its CNS activity. Its half-life and other pharmacokinetic parameters are not extensively detailed in standard summaries.

At high concentration (100 μM), Nefiracetam (DM-9384) suppressed long-lasting calcium channel currents to 82.5±11.8% of control, indicating an inhibitory effect at supramaximal concentrations [1]
Nefiracetam has been studied in clinical trials for its safety and efficacy, with a generally favorable safety profile. It has been investigated for Alzheimer's disease and post-stroke dementia. Common side effects may include gastrointestinal disturbances, headache, and insomnia, typical of nootropic agents. However, comprehensive toxicological data are not extensively documented in standard summaries. As with all research compounds, it should be handled with appropriate safety precautions. It is not a widely approved drug and is available as a research compound.

Brain Res.2000 Mar 24;859(2):255-61;Brain Res.1994 Apr 11;642(1-2):123-31.

Nefilracetam is an oxygen- and nitrogen-containing organic compound whose function is related to α-amino acids. Nefilracetam has been used in clinical trials for the treatment of Alzheimer's disease.

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The nootropic action of Nefiracetam (DM-9384) on L-type calcium channels is suggested to be mediated by intracellular cyclic AMP and pertussis toxin-sensitive G-proteins, possibly by blocking tonic inhibitory influence of G-proteins on calcium channels and cAMP-dependent processes [1]
Nefiracetam (DM-9384) may be a useful tool to identify factors determining aging, learning and memory; it reversed inhibitory effects of opioids on calcium channels [1]
Nefiracetam (DM9384, DZL-221) is a cognition-enhancing agent that activates multiple targets including nAChR, NMDAR, mGluR5, PKC, GABA receptors, and N/L-type Ca2+ channels. It enhances hippocampal neurotransmission by increasing presynaptic glutamate release through PKC pathway interactions. It was originally developed for Alzheimer's disease and post-stroke dementia research. It has been under Phase 2 clinical investigation. Nefiracetam is not a widely approved drug and is available as a research compound for studying cognitive enhancement and neuroprotection.

C14H18N2O2

246.3

246.136

77191-36-7

71157

White to off-white solid powder

1.2±0.1 g/cm3

458.5±33.0 °C at 760 mmHg

151-155°C

231.1±25.4 °C

0.0±1.1 mmHg at 25°C

1.594

1.53

1

2

3

18

320

0

NGHTXZCKLWZPGK-UHFFFAOYSA-N

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

N-(2,6-dimethylphenyl)-2-(2-oxopyrrolidin-1-yl)acetamide

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.15 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (10.15 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (10.15 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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