title: Determination of 1-(4'-aminophenyl)-4-methyl-7,8-methylene-dioxy-2,3-benzodiazepine by high-performance liquid chromatography-diode array detection in plasma and brain in healthy and hypoxic-ischaemic rats.
abstract: Previously we showed that 1-(4'-aminophenyl)-4-methyl-7,8-methylene-dioxy-2,3-benzodiazepine (GYKI-52466), an ionotropic AMPA receptor antagonist, can trigger strong, presumably metabotropic, protection against seizures and stroke at very low doses. To date, no study has determined brain and plasma concentrations of GYKI-52466 following subcutaneous administration in animals with or without brain damage. Here we developed and validated a rapid method of high-performance liquid chromatography with diode array detection. Chromatographic separation was achieved by a Luna C18 column using a mixture of 25 mM phosphate buffer (pH 7.0)-methanol-acetonitrile (40:37.5:22.5, v/v/v) as the mobile phase at a flow rate of 1.2 mL/min. The method showed acceptable precision and accuracy and allowed a precise quantification of 25 ng/mL GYKI-52466 in the plasma and brain. Recovery of GYKI-52466 from the plasma and brain was >87%, and GYKI was stable at room temperature and during prolonged storage at -20 °C. The method was successfully applied in measuring levels of GYKI-52466 following administration of 3 and 20 mg/kg of GYKI-52466 in control and brain damaged rats. A low brain concentration of 0.56 μM GYKI-52466 was observed with 3mg/kg compared to 10.7 μM with 20 mg/kg at 90 min post drug administration. Severe ataxia was observed with the 20mg/kg dose for up to 90 min. Furthermore, in ischaemic animals, there was no evidence of a 'surge' in brain GYKI concentrations at the injury site, confirming the integrity of the blood-brain barrier in the region of infarct. Taken together, our findings support a metabotropic mode of action underlying the low-dose neuroprotective efficacy of GYKI-52466.
authors.affiliation: Department of Pharmacology and Toxicology, University of Otago School of Medical Sciences, Dunedin, New Zealand.
authors.forename: P K
authors.initials: PK
authors.lastname: Nayak
authors.affiliation: New Zealand's National School of Pharmacy, University of Otago, Dunedin, New Zealand.
authors.forename: H
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Pharmacology and Toxicology, University of Otago School of Medical Sciences, Dunedin, New Zealand. Electronic address: steve.kerr@otago.ac.nz.
authors.forename: D S
authors.initials: DS
authors.lastname: Kerr
mesh_terms: D000818:Animals; D001259:Ataxia / Q000188:drug therapy / Q000378:metabolism; D001569:Benzodiazepines / Q000493:pharmacokinetics* / Q000494:pharmacology; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D018691:Excitatory Amino Acid Antagonists / Q000493:pharmacokinetics* / Q000494:pharmacology; D020925:Hypoxia-Ischemia, Brain / Q000188:drug therapy / Q000378:metabolism*; D008297:Male; D018696:Neuroprotective Agents / Q000493:pharmacokinetics* / Q000494:pharmacology; D011897:Random Allocation; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity
chemical_list: D018691:Excitatory Amino Acid Antagonists; D018696:Neuroprotective Agents; D001569:Benzodiazepines; C079692:GYKI 53655