Deborah Murphy

Safety and Tolerability of the Glutamate Antagonist CGS 19755 (Selfotel) in Patients With Acute Ischemic Stroke Results of a Phase IIa Randomized Trial

Background and Purpose CGS 19755 is a competitive N-methyl-d-aspartate (NMDA) receptor antagonist that limits neuronal damage in animal stroke models. The objectives of this multicenter (7 centers), randomized, double-blind, placebo-controlled, ascending-dose phase IIa study were to evaluate the safety and tolerability of CGS 19755 and obtain pharmacokinetic and preliminary data on its efficacious dose range in patients treated within 12 hours of hemispheric ischemic stroke. Methods At each dose level, 6 patients were randomized to one or two intravenous bolus doses of CGS 19755, and 2 patients were randomized to placebo. An unblinded safety and monitoring committee evaluated results at each dose before ascending to the next level. All patients at the first level (1 mg/kg) received two doses separated by 12 hours. The first 2 patients at 2 mg/kg received two doses, but adverse experiences occurred in both; subsequent patient groups received single doses of 2.0, 1.75, or 1.5 mg/kg. Results Adverse experien...

Characterization of the binding of [3H]-CGS 19755: a novel N-methyl-d-aspartate antagonist with nanomolar affinity in rat brain

1 CGS 19755 (cis‐4‐phosphonomethyl‐2‐piperidine carboxylic acid), a rigid analogue of 2‐amino‐5‐phosphonopentanoic acid (AP5), is one of the most potent competitive N‐methyl‐d‐aspartate (NMDA) antagonists described. Using Triton‐treated crude synaptic membranes from rat brain, binding studies indicated that [3H]‐CGS 19755 bound with high affinity and selectivity to the NMDA‐type excitatory amino acid receptor. 2 [3H]‐CGS 19755 binding was saturable, reversible, heat‐labile, pH‐dependent and linear with protein concentration. Specific binding represented 80–85% of the total amount bound. 3 Using a centrifugation assay, saturation experiments revealed two distinct binding components with Kd values of 9 and 200 nM, and corresponding Bmax values of 0.55 and l.00 pmol mg−1 protein. In contrast, a single binding component with a Kd value of 24 nM and an apparent value of 0.74 pmol mg−1 protein was observed with a filtration assay. 4 Competition experiments in which both assay techniques were used, showed that [3H]‐CGS 19755 selectively labels the NMDA receptor. The most active inhibitors of [3H]‐CGS 19755 binding were l‐glutamate and CGS 19755 (IC50 values = 100 nm). 5 In the centrifugation assay, a number of excitatory amino acids were found to generate shallow inhibition curves, and computer analysis indicated the presence of two binding components. The quisqualate receptor ligand AMPA (D,L‐α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate), kainic acid and the non‐competitive NMDA antagonists, such as phencyclidine, tiletamine and MK‐801, were without activity. 6 The high affinity binding obtained with [3H]‐CGS 19755 by use of filtration techniques thus permits the more rapid evaluation of compounds as potential NMDA antagonists and agonists. Therefore, this rigid analogue of AP5 is a more suitable radioligand for NMDA receptors than [3H]‐CPP (3‐(±)−(2‐carboxypiperazin‐4‐yl)propyl‐l‐phosphonic acid), the corresponding analogue of 2‐amino‐7‐phosphonoheptanoic acid (AP7).

Characterization of quisqualate recognition sites in rat brain tissue using DL-[3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and a filtration assay.

AbstractThe binding of [3H]AMPA (Dl-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid), a ligand for the putative quisqualate excitatory amino acid receptor subtype, was evaluated using centrifugation and filtration receptor binding techniques in rat brain crude synaptosomal membrane preparations. Maximal specific binding of [3H]AMPA occurred in Triton X-100 treated membranes in the presence of the chaotropic agent potassium thiocyanate (KSCN). The effects of KSCN on binding were reversible and optimal at 100 mM. Supernatant obtained from detergent-treated membranes inhibited specific [3H]AMPA and [3H]kainic acid binding, suggesting the presence of an inhibitory agent which was tentatively identified as glutamate. Using centrifugation, saturation analysis revealed two distinct binding sites in both the absence and presence of KSCN. The chaotrope was most effective in increasing binding at the low affinity binding site, enhancing the affinity (Kd) without a concommitant change in the total number of binding sites. Using filtration, a single binding site was detected in Triton-treated membranes. Like the data obtained by centrifugation, KSCN enhanced the affinity of the receptor (Kd value=10 nM) without altering the number of binding sites (Bmax=1.2 pmol/mg protein). The rank order of potency of various glutamate analogs in the [3H]AMPA binding assay was quisqualate > AMPA > l-glutamate > kainate > d-glutamate, consistent with the labeling of a quisqualate-type excitatory amino acid receptor subtype.l-glutamic acid diethylester, and 2-amino-7-phosphonoheptanoic acid (AP7) were inactive. The present technique provides a rapid, reliable assay for the evaluation of quisqualate-type excitatory amino acid agonists and/or antagonists that may be used to discover more potent and selective agents.

Quantitative autoradiographic localization of NMDA receptors in rat brain using [3H]CPP: comparison with [3H]TCP binding sites.

The regional distribution of N-methyl-D-aspartate (NMDA) receptors in rat brain using the selective NMDA receptor ligand [3H]3-[+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) has been quantitated by in vitro autoradiography. [3H]CCP binding was highest in the CA1 region of the hippocampus. Relatively high levels of binding were also observed in the cerebral cortex, while moderate binding was obtained in the thalamus, striatum and granule cell layer of the cerebellum. Concurrent studies examining the phencyclidine receptor ligand [3H]1-[1-(2-thienyl)cyclohexyl]-piperidine (TCP), revealed a similar pattern of binding that correlated well with the localization of [3H]CPP-labeled NMDA receptors (r = 0.88, P less than 0.01).

The N-Methyl-D-Aspartate Receptor Complex

The effects of agonists of the N-methyl-D-aspartate (NMDA) receptor can be blocked by dissociative anesthetics such as phencyclidine (PCP) in a non-competitive manner. This finding together with the fact that ligand binding to the PCP receptor is dependent on the presence of L-glutamate has led to the suggestion that there may exist an NMDA/PCP receptor complex in mammalian brain tissue. This concept has been extended to the inclusion of a cation channel based on the inhibitory actions of the divalent cation, magnesium. Evaluation of the binding of tritiated TCP (thienylcyclohexylpiperidine) a high affinity ligand for the PCP receptor, under four conditions: in basal, well washed rat cortical membranes; in the presence of L-glutamate; in the presence of magnesium; and in the presence of both magnesium and L-glutamate, with NMDA antagonists and dissociative anesthetics showed that these agents had distinct profiles of activity at the PCP receptor. Furthermore, while both classes of compound could modulate TCP binding, only NMDA receptor antagonists inhibited the binding of tritiated CPP (3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid) which labels central NMDA recognition sites. The present data support the existence of an NMDA/PCP receptor complex in mammalian brain tissue. The data currently available would suggest however, that the interface is sequentially NMDA to PCP with the latter site affecting NMDA-mediated responses at a step intermediate between receptor activation and physiological response.

Safety and tolerability of the glutamate antagonist CGS 19755 (Selfotel) in patients with acute ischemic stroke. Results of a phase IIa randomized trial

BACKGROUND AND PURPOSE CGS 19755 is a competitive N-methyl-D-aspartate (NMDA) receptor antagonist that limits neuronal damage in animal stroke models. The objectives of this multicenter (7 centers), randomized, double-blind, placebo-controlled, ascending-dose phase IIa study were to evaluate the safety and tolerability of CGS 19755 and obtain pharmacokinetic and preliminary data on its efficacious dose range in patients treated within 12 hours of hemispheric ischemic stroke. METHODS At each dose level, 6 patients were randomized to one or two intravenous bolus doses of CGS 19755, and 2 patients were randomized to placebo. An unblinded safety and monitoring committee-evaluated results at each dose before ascending to the next level. All patients at the first level (1 mg/kg) received two doses separated by 12 hours. The first 2 patients at 2 mg/kg received two doses, but adverse experiences occurred in both; subsequent patient groups received single doses of 2.0, 1.75, or 1.5 mg/kg. RESULTS Adverse experiences (agitation, hallucinations, confusion, paranoia, and delirium) occurred in all 6 patients treated with 2 mg/kg, and in 3 of 5 at 1.75 mg/kg. Similar but milder adverse experiences were noted in 4 of 7 patients at 1.5 mg/kg and 1 of 6 patients at 1.0 mg/kg. Adverse experiences began between 20 minutes and 22 hours (mean, 8 hours) after treatment and lasted 2 to 60 hours (mean, 24 hours). Mortality was 1 of 8 in patients receiving placebo and 3 of 24 in treated patients. In treated survivors, median and mean percent improvement in National Institutes of Health Stroke Scale scores from baseline to terminal visit (mean, 86 days) was comparable at all doses, and 95% of treated patients had Barthel Index scores of > or = 70 at the terminal visit. CONCLUSIONS We conclude that a single intravenous dose of 1.5 mg/kg CGS 19755 is safe and tolerable in patients with acute ischemic stroke. An efficacy trial is indicated.