Abstract
Neurounina-1 [chemical name: 7-nitro-5-phenyl-1-(pyrrolidin-1-ylmethyl)-1H-benzo[e][1,4]diazepin-2(3H)-one] is a new compound provided with relevant neuroprotective effect during stroke and in neonatal hypoxia by increasing the Na+/Ca2+ exchanger (NCX) isoforms NCX1 and NCX2 activity. This study shows for the first time, the development and validation of a sensitive and selective method for analysis of neurounina-1 in beagle dog plasma by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The sample preparation consisted of extraction of the analyte and the internal standard (IS) (ropivacaine) from plasma (50 μL) by liquid-liquid extraction using acetonitrile (100 μL). The selected reaction monitoring mode of the positive ion was performed and the precursor to the product ion transitions of m/z 365 > 83 and m/z 275 > 126 were used to measure the derivative of neurounina-1 and ropivacaine. The chromatographic separation was achieved using a Phenomenex C18 Luna (150 mm × 4.6 mm × 5 μm) analytical column with an isocratic mobile phase composed of methanol/acetonitrile/water (50/40/10, v/v/v) + 0.1% formic acid + 1 M ammonium formate. The method was linear over a concentration range of 1–500 ng/mL. The method was applied to evaluate the pharmacokinetics of neurounina-1 after a single intravenous administration of three different doses (0.1 mg/kg, 0.3 mg/kg, and 1 mg/kg) to beagle dogs (n = 5). The mean AUC0-tlast values were 26.10, 115.81, and 257.28 ng∗h/mL following intravenous administration of 0.1, 0.3, and 1 mg/kg, respectively. Linear pharmacokinetics was observed up to 1.0 mg/kg. The neurounina-1 was rapidly eliminated, with mean CL values of 46.24, 47.57, and 69.15 L/h, Vd of 130.31, 154.15, and 210.79 L and t1/2 of 2.14, 2.54, and 2.04 h after intravenous administration of 0.1, 0.3, and 1 mg/kg, respectively. This new analytical method allows the rapid determination of the neurounina-1, a new developed compound, able to exert a remarkable neuroprotective effect in the low nanomolar range.
Highlights
Stroke is a leading cause of long-lasting injury, disability, and death
This study presents for the first time, the development and validation of a sensitive and selective method to quantify neurounina-1 in beagle dog plasma using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), with ropivacaine as internal standard (IS)
The spectrum for neurounina-1 showed a protonated molecular ion at m/z 365.3 and its collision-induced dissociation formed a distinctive product at m/z 83.9, corresponding to the 1methylenepyrrolidinium ion
Summary
Stroke is a leading cause of long-lasting injury, disability, and death. Early treatment and preventive measures can reduce the brain damage that occurs as a result of a stroke. In recent years, the interest has been focused on the identification of NCX activator aiming to have new therapeutic tools able to limit the extension of ischemic brain damage (Annunziato et al, 2004). With this purpose, the structure of one of the most potent inhibitors, SM15811, was modified obtaining 7-nitro-5-phenyl-1-(pyrrolidin1-ylmethyl)-1H-benzo[e][1,4]diazepin-2(3H)-one, a new small molecule named neurounina-1 that was patented in 2012 (Pignataro et al, 2012). Neurounina-1 presents a high lipophilicity index and low toxicity (Molinaro et al, 2013)
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