Lars Nordholm

AMPA, but not NMDA, receptor antagonism is neuroprotective in gerbil global ischaemia, even when delayed 24 h

The selective alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX) and the selective N-methyl-D-aspartate (NMDA) receptor antagonists MK 801 and ifenprodil were administered to Mongolian gerbils following a 5 min period of bilateral carotid artery occlusion. NBQX when given 4, 6 or 24 h after ischaemia gave a reduced loss of hippocampal CA1 neurones compared to control animals receiving vehicle only. Dizocilipine (MK 801) (1-10 mg/kg i.p.) and ifenprodil (a total of 45 mg/kg i.p.) gave no protection. The peak levels of NBQX obtained in the cerebrospinal fluid of gerbils receiving the neuroprotective dose (3 x 30 mg/kg i.p.) was 1 microM. In gerbil cortex slices, this concentration had no effect on NMDA-evoked depolarization, but had a moderate effect on kainate and gave a total blockade of AMPA depolarizations. It is concluded that antagonists of non-NMDA glutamate receptor subtypes, possibly AMPA, may be a useful therapeutic approach for cerebral ischaemia-related brain damage following global ischaemia.

Enzymatic post-column cleavage and electrochemical detection of glycosides separated by high-performance liquid chromatography

Abstract Crude extract of Helix pomatia, commercially available as β-glucuronidase, was immobilized on porous glass and packed in a column (50 × 3 mm I.D.). Similarly, β-glucuronidase from bovine liver, immobilized on agarose beads, was used as a post-column reactor in the high-performance liquid chromatography of phenolic glycosides. Electrochemical oxidation by glassy carbon electrodes was used for the detection of the phenolic products formed by the enzymatic reaction and proved to be useful for the identification and sensitive detection of phenolic glycosides. Enzymatic activities were in the range 0.1–1 I.U. The detection limits of various phenolic glycosides were 3–23 pmol. Peak broadening and the linearity of the system were evaluated. The reactor containing immobilized Helix pomatia crude extract was also shown to posses enzymatic activity towards cyanogenic glycosides.

Respiratory effects of glutamate receptor antagonists in neonate and adult mammals.

We determined the conditions (immaturity, species, anesthesia, receptor blockade selectivity) under which glutamate receptor blockade produces respiratory depression in mammals. In unrestrained 0- to 2-day-old neonate and adult mice and cats, ventilation was measured by the barometric method, before and after separate or sequential administration of a non-NMDA receptor antagonist, NBQX (2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline, 2-200 mg kg(-1) in mice, 10-40 mg kg(-1) in cats), and a NMDA receptor antagonist, dizocilpine (3 mg kg(-1) in mice, 0.15-1.0 mg kg(-1) in cats). NBQX or dizocilpine alone did not decrease ventilation in awake adults, but NBQX strongly depressed ventilation in neonate awake mice and in adult anesthetized animals. Given together, dizocilpine and NBQX always profoundly depressed ventilation by producing a lethal apnea in neonate mice, and an apneustic pattern of breathing in adults of both species and in neonate cats. We conclude that blockade of either NMDA or non-NMDA receptors is innocuous in awake adults. The factors which may potentiate respiratory depression are (1) anesthesia, (2) immaturity, and (3) combined blockade of both receptors types. The mechanism of depression is species-dependent and age-dependent.

Determination of trimethoprim metabolites including conjugates in urine using high-performance liquid chromatography with combined ultraviolet and electrochemical detection

A high-performance liquid chromatographic method for the determination of trimethoprim metabolites in pig urine was developed. The metabolites-glucuronic acid and sulphuric acid conjugates of phenolic metabolites formed by demethylation of trimethoprim-were quantitated after treatment of urine with beta-glucuronidase (Escherichia coli). The sulphuric acid conjugate was not susceptible to enzymatic hydrolysis and was therefore assayed as the conjugate by use of ion-pair chromatography on the reversed-phase column. In order to find suitable conditions for enzymatic hydrolysis of the glucuronides, the conjugates were obtained by gel chromatography of urine from a [14C] trimethoprim-treated pig.

The NBQX Story

Publisher Summary This chapter discusses pharmacology of 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline (NBQX). NBQX is the first selective α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) antagonist to demonstrate neuroprotective properties in animal models of focal and global cerebral ischemia. NBQX potently increased the focal seizure threshold and inhibited seizure spread from the focus. It protected against seizure induced by maximal electroshock (MES) and pentylenetetrazol. NBQX treatment did not reverse the Parkinsonism nor changed the responses to the selective D 2 dopamine receptor agonist (+)-PHNO or the partial dopamine D 1 receptor agonist CY208-243 in two Parkinsonian monkeys. NBQX can reduce the hippocampal CA1 neuronal loss observed following severe cerebral trauma, suggesting a further potential clinical use for AMPA antagonists in head injury. It is suggested that NBQX be administered in relatively high doses to get an anti-ischemic effect. It is observed that NBQX is as efficacious as the N -methyl- D -aspartate (NMDA) antagonists in most models of focal ischemia and does not seem to have the psychotomimetic side-effect problem.

Interaction of the competitive AMPA receptor antagonist NBQX with hexobarbital

IP administration of hexobarbital to rats caused a mean sleeping time of 93.6 min (SD 21.5). IV infusion of 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX) at a dose rate of 0.11 mg/kg/min starting 30 min after administration of hexobarbital prolonged the mean sleeping time to 132.2 min (SD 14.9). Dose rates of 0.33 and 1.10 mg/kg/min prolonged the mean sleeping times to 176.4 min (SD 33.3) and 444.1 min (SD 72.0), respectively. Measured 180 and 450 min after the start of the study, there were no differences in the plasma concentrations of hexobarbital in groups receiving hexobarbital alone compared to groups receiving the high-dose rate of NBQX starting 30 min after administration of hexobarbital. The present results demonstrate that by IV infusion NBQX dose dependently prolonged the sleeping time of hexobarbital. There were no indications of interactions on hexobarbital elimination of either isomer. It is therefore likely that NBQX acts synergistically with hexobarbital to depress the central nervous activity.

Determination of the neuroprotective agent 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione in plasma using high-performance liquid chromatography

A rapid, sensitive and selective high-performance liquid chromatographic method for the determination of the neuroprotectant 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione in rat plasma has been established and validated. Samples of 0.5 ml of plasma are extracted by elution from a Bond-Elut column with methanol and analysed on a reversed-phase column. The wavelength of UV detection is 254 nm. The method is linear at least up to 30 micrograms/ml, with a lowest reliable determination level of 4 mg/ml. The assays has a coefficient of variation of 13% at 10 ng/ml and 4% at 1000 ng/ml. Small variations in the extraction procedure and the liquid chromatographic conditions have minimal or no influence on the assay.

Determination of an oxadiazole-substituted 1,4-benzodiazepine in plasma by high-performance liquid chromatography with ultraviolet detection and by a radioreceptor assay

A high-performance liquid chromatographic (HPLC) method for the determination of an oxadiazole-substituted 1,4-benzodiazepine [3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-5,6-dihydro-5-methyl-6-oxo -4H-imidazo[1,5a] [1,4]benzodiazepine] in plasma has been developed and compared with a radioreceptor assay. The results given by the two methods were in good agreement, with detection limits of ca. 1 ng/ml (signal-to-noise ratio = 3). The radioreceptor method is preferred for the monitoring of toxicological and other well controlled studies, while HPLC is preferred where greater specificity is essential. Further, the HPLC assay is applicable over a much wider concentration range.

Identification of the major metabolites of [3H]-(+)-8-chloro-5-(2,3-dihydrobenzofuran-7-yl)-7-methoxymethyloxy-3-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine in rats

1. The in vivo urinary metabolites of 3H(+)-8-chloro-5(2,3- dihydrobenzofuran-7-yl)-7-methoxymethyloxy-3-methyl-2,3,4,5-tetrah ydro-1H-3- benzazepine isolated from Wistar rats have been characterized by mass spectrometry and n.m.r. spectroscopy. 2. Metabolites are formed by N-demethylation, hydroxylation of the dihydrobenzofuran moiety, and elimination of the methoxymethyl group followed by glucuronidation. All combinations of these metabolic pathways were found in the metabolites in urine. 3. In the faeces metabolites formed by hydroxylation of the dihydrobenzofuran moiety and elimination of the methoxymethyl moiety dominate, while N-demethylated metabolites are present only in small amounts. 4. The major plasma metabolite was formed by elimination of the methoxymethyl group followed by glucuronidation. Only minute amounts of other metabolites were present.