Dennis M. Zimmerman

AMPA Receptor Potentiators for the Treatment of CNS Disorders

Glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors mediate most of the excitatory neurotransmission in the mammalian central nervous system and also participate in forms of synaptic plasticity thought to underlie memory and learning, and the formation of neural networks during development. Molecular cloning techniques have shown that the AMPA receptor family is composed of four different subunits named GluR1-4 or GluRA-D (newly termed as Glu(A1)-Glu(A4)) and native AMPA receptors are most likely tetramers generated by the assembly of one or more of these subunits, yielding homomeric or heteromeric receptors. Additional complexity among AMPA receptors is conferred by alternative splicing of RNA for each subunit giving rise to flip and flop variants. Clinical and experimental data have suggested that positive modulation of AMPA receptors may be therapeutically effective in the treatment of cognitive deficits. Several classes of AMPA receptor potentiators have been reported, including pyrroliddones (piracetam, aniracetam), benzothiazides (cyclothiazide), benzylpiperidines (CX-516, CX-546) and more recently biarylpropylsulfonamides (LY392098, LY404187 and LY503430). These molecules enhance cognitive function in rodents, which appears to correlate with increased hippocampal activity. In addition, clinical studies have suggested that AMPA receptor modulators enhance cognitive function in elderly subjects, as well as patients suffering from neurological and psychiatric disorders. Several independent studies have suggested that AMPA receptors can increase BDNF expression by both calcium-dependent and independent pathways. For example, recent studies have shown that AMPA receptors interact with the protein tyrosine kinase, Lyn. Activation of Lyn can recruit the mitogen-activated protein kinase (MAPK) signalling pathway and increase the expression of BDNF. Therefore, in addition to directly enhancing glutamatergic synaptic transmission, AMPA receptor activation can increase the expression of BDNF in vitro and in vivo. This may account for activity of AMPA receptor potentiators in rodent models predictive of antidepressant activity (forced swim and tail suspension tests). The increase in neurotrophin expression also may contribute to the functional, neuroprotective and neurotrophic actions of LY404187 and LY503430 after infusion of 6-OHDA into the substantia nigra. In conclusion, several potent, selective and systemically active AMPA receptor potentiators have been reported. Data indicate that these molecules modulate glutamatergic transmission, enhance synaptic transmission, long-term potentiation (LTP) and increase neurotrophin expression. Therefore, these AMPA receptor potentiators offer an exciting new class of drugs with potential for treating (1) cognitive impairment associated with Alzheimers disease and schizophrenia, (2) depression, (3) slowing the progression and potentially enhancing recovery from Parkinsons disease.

Anticonvulsant effects of phencyclidine-like drugs: relation to N-methyl-d-aspartic acid antagonism

Various compounds that have been identified in the literature as binding to the [3H]phencyclidine receptor site and as producing behavioral effects similar to phencyclidine (phencyclidine-like) protected mice from maximal electric shock-induced tonic-extensor seizures. These anticonvulsant effects appear to be due to blockade of the N-methyl-D-aspartic acid receptor, as recently reported for phencyclidine-like compounds. Phencyclidine-like compounds produced their anticonvulsant effects at doses that were also neurologically impairing.

N-Methyl-d-aspartic acid-induced lethality in mice: selective antagonism by phencylidine-like drugs

Abstract N- Methyl- d -aspartic acid (NMDA) produced a dose-related increase in lethality in mice, with 200 mg/kg (i.p.) effecting 100% lethality. Upon daily dosing, acutely sublethal doses of NMDA produced deaths. This NMDA-induced lethality was stereoselective: N- methyl- l -aspartic acid had no effects at doses as high as 400 mg/kg. Moderate doses of phencyclidine (PCP) and drugs having PCP-like behavioral effects blocked the NMDA-induced lethality. Other classes of psychoactive drugs, including opioids, anticonvulsants and antipsychotics, were ineffective in preventing NMDA-induced lethality. The potency of PCP-like drugs to block the NMDA-induced lethality correlates highly with the dose necessary to produce PCP-like catalepsy and PCP-like discrimination in pigeons. These data support the hypothesis that PCP-like drugs produce many of their effects by impairing the normal functioning of the NMDA-defined excitatory neurotransmitter receptor in the central nervous system.

Phencyclidine receptors in rat brain cortex

The binding of [3H]phencyclidine (PCP) to receptors in rat brain cortex has been studied. Two receptors have been detected, a high affinity receptor site with a KD of 23.5 +/- 7.4 nM and a low affinity site with a KD of 7.6 +/- 1.8 microM. The binding of [3H]PCP to its receptors was pH and temperature dependent and was destroyed by heat-denaturation. The binding of [3H]PCP was inhibited by compounds which produce PCP-like behavioral effects including dexoxadrol, etoxadrol and ketamine as well as a novel series of benz(f)isoquinolines. The low affinity site was blocked by PCP, etoxadrol and (+)-SKF-10,047 but not morphine or leu-enkephalin, suggesting that it also represents a specific PCP site. Stereoselective displacement of PCP at the high affinity receptor was observed with the isomers of cyclazocine, cyclorphan, SKF-10,047 and dioxadrol (dexoxadrol and levoxadrol). Naloxone, 4,5,6,7-tetrahydroisoxazolo(S,4-C)pyridin-3-ol (THIP) hydrate and haloperidol inhibited binding poorly (Ki greater than 1 microM), suggesting that these compounds do not interact significantly with the high affinity PCP receptor in vivo. The affinity of ligands for the phencyclidine receptor was highly correlated (r = 0.714, P less than 0.01) with their potency to produce catalepsy in pigeons.

Structure-activity relationship of a series of diaminoalkyl substituted benzimidazole as neuropeptide Y Y1 receptor antagonists

A series of benzimidazoles (4) was synthesized and evaluated in vitro as potent and selective NPY Y1 receptor antagonists. Substitution of the piperidine nitrogen of 4 with appropriate R groups resulted in compounds with more than 80-fold higher affinity at the Y receptor compared to the parent compound 5 (R = H). The most potent benzimidazole in this series was 21 (Ki = 0.052 nM).

Novel AMPA receptor potentiators LY392098 and LY404187: effects on recombinant human AMPA receptors in vitro.

The present study describes the activity of two novel potent and selective AMPA receptor potentiator molecules LY392098 and LY404187. LY392098 and LY404187 enhance glutamate (100 microM) stimulated ion influx through recombinant homomeric human AMPA receptor ion channels, GluR1-4, with estimated EC(50) values of 1.77 microM (GluR1(i)), 0.22 microM (GluR2(i)), 0.56 microM (GluR2(o)), 1.89 microM (GluR3(i)) and 0.20 microM (GluR4(i)) for LY392098 and EC(50) values of 5.65 microM (GluR1(i)), 0.15 microM (GluR2(i)), 1.44 microM (GluR2(o)), 1.66 microM (GluR3(i)) and 0.21 microM (GluR4(i)) for LY404187. Neither compound affected ion influx in untransfected HEK293 cells or GluR transfected cells in the absence of glutamate. Both compounds were selective for activity at AMPA receptors, with no activity at human recombinant kainate receptors. Electrophysiological recordings demonstrated that glutamate (1 mM)-evoked inward currents in human GluR4 transfected HEK293 cells were potentiated by LY392098 and LY404187 at low concentrations (3-10 nM). In addition, both compounds removed glutamate-dependent desensitization of recombinant GluR4 AMPA receptors. These studies demonstrate that LY392098 and LY404187 allosterically potentiate responses mediated by human AMPA receptor ion channels expressed in HEK 293 cells in vitro.

Molecular Cloning, Expression, and Pharmacological Characterization of humEAA1, a Human Kainate Receptor Subunit

Abstract: Kainate is a potent neuroexcitatory agent; its neurotoxicity is thought to be mediated by an ionotropic receptor with a nanomolar affinity for kainate. In this report, we describe the cloning of a cDNA encoding a human glutamate ionotropic receptor subunit protein from a human hippocampal library. This cDNA, termed humEAA1, is most closely related to rat and human cDNAs for kainate receptor proteins and, when expressed in COS or Chinese hamster ovary cells, is associated with high‐affinity kainate receptor binding. We have successfully established cell lines stably expressing humEAA1. This is the first report of establishment of stable cell lines expressing a glutamate receptor subunit. The relative potency of compounds for displacing [3H] kainate binding of humEAA1 receptors expressed in these stable cell lines was kainate > quisqualate > domoate > L‐glutamate > (RS)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid > dihydro‐kainate > 6, 7‐dinitroquinoxaline‐2, 3‐dione > 6‐cyano‐7‐nitroquinoxaline‐2, 3‐dione. Homooligomeric expression of humEAA1 does not appear to elicit ligand‐gated ion channel activity. Nevertheless, the molecular structure and pharmacological characterization of high‐affinity kainate binding of the humEAA1 expressed in the stable cell line (ppEAA1–16) suggest that the humEAA1 is a subunit protein of a human kainate receptor complex.

Central administration of the opioid antagonist, LY255582, decreases short- and long-term food intake in rats

A variety of opioid antagonists have been reported to decrease short-term food intake, but few appear to reduce long-term intake. In the present study we evaluated the effect of a relatively new class of opioid antagonists, 3,4-dimethyl-4-phenylpiperidines, on short-term and long-term food intake after central administration. We also evaluated their affinities for the mu and kappa opioid receptor sites in synaptosomal membranes derived from rat whole brain tissue (minus cerebellum) and guinea-pig cortex, respectively. The affinities for the mu receptor sites were LY255582 greater than LY217273 greater than LY256897 greater than naloxone greater than LY227444. The affinities for the kappa receptor sites were LY255582 greater than LY256897 = LY217273 greater than LY227444. LY255582 reduced food intake for up to 24 h after a single intraventricular injection. Doses as low as 1 microgram of LY255582 decreased food intake for up to 4 h. All other drugs were much less powerful. Naloxone and LY256897 only decreased food intake after injection of the 100 microgram dose. LY227444 and LY217273 failed to decrease intake at all doses tested. LY255582 (100 micrograms) decreased food intake over a 7 day period when injected intraventricularly once per day. The body weight of the rats also decreased during the 7 day period. Upon cessation of drug administration body weights and food intake approached control levels. Thus, LY255582 appears to be a very potent and long-acting anorectic agent which may be useful in the treatment of obesity. The mu and kappa binding profile of the phenylpiperidines does not seem to clearly correlate with their anorectic activity.

Novel phenylpiperidine opioid antagonists and partial agonists: Effects on fluid consumption

The effects of five opioid antagonists, a racemate partial agonist and its agonist and antagonist optical isomers were studied on deprivation-induced drinking. All compounds had a phenylpiperidine nucleus. The antagonists produced dose-related decreases in drinking, and the potencies for decreasing drinking correlated with morphine-antagonist doses. The racemic partial agonist and its agonist isomer decreased drinking at doses higher than those which produced marked analgesia. Within the class of phenylpiperidine drugs studied, some had less specificity than naloxone for the mu-receptors as compared to the delta-receptor, but the suppression of drinking was not related to changes in mu-to-delta ratios.

Structure-activity relationships of a series of 1-substituted-4-methylbenzimidazole neuropeptide Y-1 receptor antagonists.

The characterization of a novel series of NPY-1 receptor antagonists derived from the 4-methylbenzimidazole 4 is described. Appropriate substitution on the piperidyl nitrogen of 4 led to systematic increases in Y-1 receptor affinity, to approximately 50-fold, and to the discovery of the importance of a second basic substituent.