Nefiracetam

Catalog No.S1969 Batch:S196901

Technical Data

Formula	C₁₄H₁₈N₂O₂
Molecular Weight	246.3	CAS No.	77191-36-7
Solubility (25°C)*	In vitro	DMSO	49 mg/mL (198.94 mM)
		Ethanol	49 mg/mL (198.94 mM)
		Water	3 mg/mL (12.18 mM)
* <1 mg/ml means slightly soluble or insoluble. * Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations. * Room temperature shipping (Stability testing shows this product can be shipped without any cooling measures.)

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Biological Activity

Description

Nefiracetam (DZL 221) is a GABAergic, cholinergic, and monoaminergic neuronal system enhancer for Ro 5-4864-induced convulsions. Phase 2.

Targets

GABA receptor ^[1]

In vitro

Nefiracetam at a concentration of 1 μM increases a long-lasting component of calcium channel currents two-fold without affecting a transient component. ^[2] 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.^[3] 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. ^[4]

In vivo

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. ^[1] Nefiracetam administered daily 1 hour before each training session facilitates the acquisition process of the avoidance response. ^[5]

Protocol (from reference)

Kinase Assay:^{^[4]}	Assay of glutamate released 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:^{^[3]}	Cell lines Oocytes Concentrations ~1 μM Incubation Time 24 hours - 48 hours Method 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 Ringer's 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.

Kinase Assay:^{^[4]}

Assay of glutamate released
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:^{^[3]}

Cell lines
Oocytes
Concentrations
~1 μM
Incubation Time
24 hours - 48 hours
Method
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 Ringer's 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.

References

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