John W. Ferkany

Dextromethorphan inhibits NMDA-induced convulsions

Dextromethorphan, its metabolite dextrorphan, phencyclidine, ketamine, MK-801, 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid and DL-2-amino-7-phosphonoheptanoic acid were evaluated for potency to antagonize N-methyl-D-aspartate-induced convulsions following intraperitoneal administration using male CF-1 mice. Whereas reference anticonvulsants (e.g., phenytoin) were ineffective in this model, dextromethorphan and all competitive and noncompetitive N-methyl-D-aspartate antagonists blocked seizures. The results are consistent with the interpretation that dextromethorphan elicits some of its pharmacological responses via an interaction with receptors for excitatory amino acids.

Excitatory Amino Acid Receptors

The potent neuroexcitatory effects of acidic amino acids were first described over 25 years ago (Hayashi, 1954). In the interim, through the sustained efforts of several neurophysiologic laboratories, evidence has accrued that glutamic acid and possibly aspartic acid may serve as excitatory neurotransmitters in the mammalian central nervous system (Curtis et al., 1972; Curtis and Johnston, 1974; Krnjevic, 1974; McLennan, 1975). Prompted by these neurophysiologic observations, neurochemists over the last several years have attempted to identify neuronal pathways in the mammalian CNS that may utilize the acidic amino acids as their neurotransmitters; and neuropharmacologists have begun to explore interactions between drugs and the excitatory amino acids. An overview of this research suggests that the excitatory amino acids may play a fundamental role in brain function that is only beginning to gain wide appreciation. Thus, circumstantial evidence has implicated L-glutamic acid as the neurotransmitter for cortical pyramidal cells (Divak et al., 1977; Lund-Karlsen and Fonnum, 1978), cerebellar granule cells (Young et al., 1974) and primary sensory afferents (Johnson, 1972), in the mechanisms of action of sedatives and anticonvulsants (Richards and Smaje, 1976; MacDonald and Barker, 1979), and in the pathophysiology of certain neurodegenerative disorders (Perry et al., 1977; Coyle, 1979).

N-methyl-D-aspartate antagonists and working memory performance : comparison with the effects of scopolamine, propranolol, diazepam, and phenylisopropyladenosine

The effects of the competitive N-methyl-D-aspartate (NMDA) antagonists CPP (5 & 10 mg/kg) and NPC 12626 (25 & 40 mg/kg) and the noncompetitive NMDA antagonists phencyclidine (1, 3, & 6.25 mg/kg) and MK 801 (0.1 & 0.2 mg/kg) on performance of rats on a nonspatial delayed matching-to-sample working memory task were evaluated. At the highest dose, each NMDA antagonist reduced choice accuracy at all retention intervals. In contrast, the reference anticholinergic agent scopolamine selectively reduced accuracy at long retention intervals, suggesting that scopolamine but not the NMDA antagonists directly interfered with time-dependent working memory retention. Propranolol, diazepam, and phenylisopropyladenosine had little or no effect on choice accuracy, suggesting that noradrenergic, gamma-aminobutyric acid-diazepam, and adenosine receptors may be relatively unimportant for working memory performance as assessed in this task. The NMDA antagonists also differed from scopolamine in that doses of NMDA antagonists that reduced response accuracy also reduced response probability, altered bias (competitive antagonists only), and increased intertrial interval responding (noncompetitive antagonists only). It was concluded that NMDA antagonists disrupt cognitive functions including, but not limited to, those required for accurate working memory performance.

Measurement of gamma-aminobutyric acid (GABA) in blood

Abstract Blood GABA levels can be readily determined using a radioreceptor assay or gas chromatography-mass spectrometry. After withdrawal of blood, GABA levels remain stable with 25–50% of the GABA in whole blood found in the plasma fraction. Whole blood GABA concentrations range from 500 pmoles/ml to 1200 pmoles/ml in 8 mammalian species with human values being about 900 pmoles/ml. in vivo administration of aminooxyacetic acid increases both blood and brain GABA levels to a similar extent.

Effect of drugs on rat brain, cerebrospinal fluid and blood GABA content.

Acute administration of GABA transaminase inhibitors to rats results in a dose‐dependent increase in both brain and blood GABA content and administration of isonicotinic acid hydrazide (INH), at a dose which decreases the amount of brain GABA, also lowers blood levels of this amino acid. Chronic treatment (10 days) with INH (20mg/kg), y‐acetylenic‐GABA (10 mg/kg) or aminooxyacetic acid (AOAA) (10 mg/kg) results in a significant elevation in both rat brain and blood GABA concentrations. At the doses studied, only AOAA caused a significant elevation in CSF GABA content. Co‐administration of pyridoxal phosphate (2 mg/kg) blocks the chronic INH‐induced rise in blood GABA but does not affect the increase in brain content of this amino acid. Chronic administration of di‐n‐propylacetate (20 mg/kg) did not significantly alter brain, blood or CSF GABA levels. The results suggest that, under the proper conditions, changes in blood GABA levels after administration of inhibitors of GABA synthesis or degradation may be an indirect indicator of changes in the brain content of this amino acid. Blood GABA determinations may be useful for studying the biochemical effectiveness of GABA transaminase inhibitors in man.

Possible cerebroprotective and in vivo NMDA antagonist activities of sigma agents

The recent finding that ifenprodil binds with high affinity to sigma sites suggests that other sigma agents may have ifenprodil-like cerebroprotectant and functional N-methyl-D-aspartate (NMDA) antagonist effects. The present study, compared the in vivo effects of ifenprodil and the sigma agents, BMY 14802, caramiphen and haloperidol, in three tests sensitive to NMDA antagonists and purported cerebroprotectant drugs. When administered at or below the rotorod TD50 dose, all four compounds significantly increased survival time in an hypoxic environment (4% O2 in nitrogen). Caramiphen and ifenprodil (ED50 = 52 and 61 mg/kg, respectively) also blocked maximal electroshock-induced seizures, whereas BMY 14802 and haloperidol were ineffective. Finally, caramiphen (ED50 = 95 mg/kg) antagonized seizures and lethality induced by administration of NMDA (250 mg/kg, IP). BMY 14802, haloperidol and ifenprodil only partially antagonized NMDA-induced seizures, but did enhance the anticonvulsant potency of the noncompetitive NMDA antagonist, MK-801. Together, these findings suggest that sigma agents may have cerebroprotective effects.

Development of rat brain uptake and calcium-dependent release of GABA.

Summary High-affinity uptake of GABA in the developing rat brain is greater in 7-day-old rats than in adults, which is quite different from the developmental profile observed for biogenic amine uptake in brain, where the magnitude of uptake increases in a linear fashion. In contrast to uptake, the neuronal calcium-dependent release system for GABA develops in a progressive fashion, similar to that observed for the biogenic amines, suggesting that the enhanced GABA uptake observed in the younger animals is not stored in a functionally releaseable pool. This conclusion is based on the findings that the lack of correlation between the magnitude of GABA release and uptake in the developing brain is not due to the enhanced uptake itself, to the presence of some identifiable subcellular constituent, or to a change in calcium sensitivity for the release process at the earlier time. Furthermore, uptake of β-alanine and leucine is also greater in the 7-day-old rat forebrain than adult, whereas other biogenic amines such as norepinephrine and dopamine are similar to serotonin with regard to the development of their uptake and release processes. These results suggest that high-affinity GABA uptake is a questionable marker for studying neuronal development of this transmitter system, and that calcium-dependent release is probably a more valid indicator of the functional state of GABA terminals.

A Small Molecule Inhibitor of Enterocytic Microsomal Triglyceride Transfer Protein; SLx-4090, Biochemical, Pharmacodynamic, Pharmacokinetic and Safety Profile

First-generation microsomal triglyceride transfer protein (MTP) inhibitors were designed to inhibit hepatic MTP and provide a novel treatment of dyslipidemia. Effective at lowering low-density lipoprotein-cholesterol (LDL-C), these inhibitors also elevate liver enzymes and induce hepatic steatosis in animals and humans. MTP is highly expressed in the enterocytes, lining the lumen of the jejunum, and is critical in the production of chylomicrons assembled from lipid/cholesterol and their transfer into systemic circulation. 6-(4′-Trifluoromethyl-6-methoxy-biphenyl-2-ylcarboxamido)-1,2,3,4-tetrahydroisoquinoline-2-carboxylic acid phenyl ester (SLx-4090) (IC50 value ∼8 nM) was designed to inhibit only MTP localized to enterocytes. In Caco-2 cells SLx-4090 inhibited apolipoprotein B (IC50 value ∼9.6 nM) but not apolipoprotein A1 secretion. Administered orally to rats SLx-4090 reduced postprandial lipids by >50% with an ED50 value ∼7 mg/kg. SLx-4090 was not detected in the systemic or portal vein serum of the animals (lower limit of quantitation ∼5 ng/ml) after single or multiple oral doses in fasted rodents. When coadministered with tyloxapol, SLx-4090 did not inhibit the secretion of hepatic triglycerides (TG), consistent with the absence of systemic exposure. Chronic treatment with SLx-4090 in mice maintained on a high-fat diet decreased LDL-C and TG and resulted in weight loss without the elevation of liver enzymes or an increase in hepatic fat. The compound did not result in toxicity when administered to rats for 90 days at a dose of 1000 mg/kg per day. These data support the concept that the inhibition of enterocytic MTP could serve as a useful strategy in the treatment of metabolic disorders.

Subcellular distribution of GABA receptors in bovine retina

Abstract Two synaptosomal fractions were isolated from bovine retina and analyzed for GABA receptors using a [3H]GABA binding assay. Two receptor sites were identified: one with an affinity constant of 345 n m , which was uniformly distributed between the two fractions, and a second site with an affinity constant of 38 n m , which was primarily associated with the synaptosomal fraction enriched in photoreceptor cell terminals. Both sites demonstrated a high degree of pharmacologic specificity. The GABA receptor agonist, muscimol, was a more potent displacer than imidazolacetic acid or bicuculline; uptake blockers, diaminobutyric acid and β-alanine were poor displacers. These data provide further support for the role of GABA as a neurotransmitter in retina. It also suggests the presence of a significant GABAnergic system in the outer plexiform layer of the bovine retina.

Properties of 3H-cimetidine binding in rat brain membrane fractions

Abstract In an attempt to characterize the brain histamine H 2 receptor, experiments were undertaken to study the binding properties of (N-methyl- 3 H) -cimetidine, an H 2 receptor antagonist, in rat brain membranes. Using a centrifugation assay, 3 H-cimetidine binding having a K d of 0.40μM and a Bmax of 3.9 pmoles/mg protein was detected. Of fourteen anions and cations tested, one, Cu ++ , dramatically increased specific 3 H-cimetidine binding, the increase being due mainly to a change in Bmax. Studies of substrate specificity for 3 H-cimetidine binding revealed that Cu ++ , while not significantly affecting the potency of H 2 receptor agonists and antagonists, dramatically decreases the potency of H 1 receptor substances on the 3 H-cimetidine binding site. In addition, both the relative and absolute potencies of various H 2 receptor agonistsv and antagonists in displacing the ligand in the presence of Cu ++ parallels their potencies in biological systems. These findings suggest that, under these conditions, 3 H-cimetidine may be labelling a biologically relevant H 2 binding site in brain and that Cu ++ may regulate the substrate specificity for this site. The brain regional distribution and kinetic analysis of the binding suggest that it is not localized solely to the synaptic receptor for histamine, but may also be associated with histamine receptors at other neuronal, glial or vascular sites.