Polyamine Antagonist Research Articles

Effect of N1-dansylspermine and Ro25,6981 on locomotor activity in naive mice and in the reserpinized mouse model of Parkinson's disease.

The effect of N1-dansylspermine, a polyamine analogue and competitive polyamine antagonist, and Ro25,6981, a noncompetitive polyamine antagonist with good affinity and selectivity for the GluN2B subunit, on locomotor activity in naive mice was investigated. Furthermore, the ability of the polyamine antagonists to reverse reserpine-induced hypokinesia was assessed, 24 h after injection of a catecholamine-depleting dose of reserpine (5 mg/kg, subcutaneous), to investigate the therapeutic potential of polyamine antagonists in Parkinson's disease. N1-dansylspermine significantly decreased locomotor activity in naive animals (P<0.001) but caused a mild, but significant increase in locomotor activity in reserpinized mice at the highest dose tested (P<0.05). Ro25,6981 significantly stimulated locomotor activity in naive animals (P<0.001) and had a slight significant stimulatory effect on reserpine-induced hypokinesia (P=0.05). N1-dansylspermine and Ro25,6981 had opposite effects on locomotor activity in naive mice, but both had a mild antiparkinsonian effect in the reserpine model. These findings suggest that antagonism of the polyamine binding site on the GluN2B subunit can reduce hypokinesia, albeit to a limited extent.

Polyamine Antagonist Therapies Inhibit Neuroblastoma Initiation and Progression.

Deregulated MYC drives oncogenesis in many tissues yet direct pharmacologic inhibition has proven difficult. MYC coordinately regulates polyamine homeostasis as these essential cations support MYC functions, and drugs that antagonize polyamine sufficiency have synthetic-lethal interactions with MYC Neuroblastoma is a lethal tumor in which the MYC homologue MYCN, and ODC1, the rate-limiting enzyme in polyamine synthesis, are frequently deregulated so we tested optimized polyamine depletion regimens for activity against neuroblastoma. We used complementary transgenic and xenograft-bearing neuroblastoma models to assess polyamine antagonists. We investigated difluoromethylornithine (DFMO; an inhibitor of Odc, the rate-limiting enzyme in polyamine synthesis), SAM486 (an inhibitor of Amd1, the second rate-limiting enzyme), and celecoxib (an inducer of Sat1 and polyamine catabolism) in both the preemptive setting and in the treatment of established tumors. In vitro assays were performed to identify mechanisms of activity. An optimized polyamine antagonist regimen using DFMO and SAM486 to inhibit both rate-limiting enzymes in polyamine synthesis potently blocked neuroblastoma initiation in transgenic mice, underscoring the requirement for polyamines in MYC-driven oncogenesis. Furthermore, the combination of DFMO with celecoxib was found to be highly active, alone, and combined with numerous chemotherapy regimens, in regressing established tumors in both models, including tumors harboring highest risk genetic lesions such as MYCN amplification, ALK mutation, and TP53 mutation with multidrug resistance. Given the broad preclinical activity demonstrated by polyamine antagonist regimens across diverse in vivo models, clinical investigation of such approaches in neuroblastoma and potentially other MYC-driven tumors is warranted. Clin Cancer Res; 22(17); 4391-404. ©2016 AACR.

Polyaminergic agents modulate contextual fear extinction in rats

Polyamines, such as spermidine and spermine, have been reported to improve memory retention through the activation of N-methyl- d-aspartate receptors (NMDAr). However whether polyamine agonists and antagonists alter extinction remains unclear. In the current study, we investigated whether spermidine and polyamine antagonists that selectively block the NR2B subunit at the NMDAr alter the extinction of contextual conditioned fear in male Wistar rats. The bilateral intra-hippocampal administration of exogenous spermidine (2 nmol/site) immediately after, but not 6 h after extinction training, facilitated the extinction of fear conditioning. The injection of the NMDAr antagonists arcaine (0.2 nmol/site), ifenprodil (20 nmol/site) and traxoprodil (0.2 nmol/site), disrupted fear extinction and, at doses that had no effect per se, reversed the facilitatory effect of spermidine on fear extinction. These results suggest that exogenous and endogenous polyamines facilitate the extinction of contextual conditioned fear through activation of NR2B subunit-containing NMDAr in the hippocampus. Since extinction-based exposure therapy is widely used as treatment for a number of anxiety-related disorders, including phobias and post-traumatic stress, the currently reported facilitation of extinction by polyaminergic agents suggest these compounds as putative candidates for drug development.

The effects of polyamine agonists and antagonists on N-methyl- d-aspartate-induced depolarizations of amphibian motoneurons in situ

Polyamines have been found to reduce proton inhibition of isolated N-methyl- d-aspartate (NMDA) channels recorded in vitro. This study examines the role of polyamine modulation of motoneuronal excitation in situ, with an emphasis on possible interactions with NMDA-mediated depolarization of motoneurons and receptor mediated modulation of NMDA receptors by l-glutamate and serotonin (5-HT). Motoneuron membrane potential changes were electrotonically recorded in situ from the ventral root of isolated, hemisected amphibian spinal cords using sucrose gap techniques. The methods provided highly stable recordings (< 1.0%) of membrane potential changes upon application of NMDA. Spermine, but not spermidine, enhanced NMDA-induced depolarization of motoneurons with and without Mg 2+ present in the superfusate but had no significant effect on either (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD, an mGLU receptor agonist) or 5-HT2B-G-protein receptor mediated enhancement of Mg 2+ blocked NMDA-induced activity. The polyamine antagonist arcaine or the allosteric modulator ifenprodil had no effect on NMDA-induced changes in motoneuron membrane potentials recorded in situ but blocked the effects of spermine. Synthalin did not block spermine enhancement of NMDA-induced depolarization of motoneurons but mimicked Mg 2+ block of the NMDA channel. The data provide evidence that the proton block of the NMDA receptor is maximized in frog motoneurons in situ and also for a spermine specific polyamine site on native NMDA receptors of motoneurons that can enhance NMDA-induced depolarization when activated. Polyamines do not appear to be constitutively active at the motoneurons recorded since polyamine antagonists had no effect on either membrane depolarization or modulation of NMDA receptors.

Histamine Potentiates N-Methyl-d-aspartate Receptors by Interacting with an Allosteric Site Distinct from the Polyamine Binding Site

Histamine potentiates activation of native and recombinant N-methyl-d-aspartate receptors (NMDARs), but its mechanisms of action and physiological functions in the brain remain controversial. Using four different models, we have further investigated the histamine-induced potentiation of various NMDAR-mediated responses. In single cultured hippocampal neurons, histamine potentiated NMDA currents. It also potentiated the NMDA-induced increase in intracellular calcium in the absence, as well as with saturating concentrations, of exogenous d-serine, indicating both glycine-dependent and glycine-independent components of its effect. In rat hippocampal synaptosomes, histamine strongly potentiated NMDA-induced [(3)H]noradrenaline release. The profile of this response contained several signatures of the histamine-mediated effect at neuronal or recombinant NMDARs. It was NR2B-selective, being sensitive to micromolar concentrations of ifenprodil. It was reproduced by tele-methylhistamine, the metabolite of histamine in brain, and it was antagonized by impromidine, an antagonist/inverse agonist of histamine on NMDA currents. Up to now, histamine was generally considered to interact with the polyamine site of the NMDAR. However, spermine did not enhance NMDA-induced [(3)H]noradrenaline release from synaptosomes, and the potentiation of the same response by tele-methylhistamine was not antagonized by the polyamine antagonist arcaine. In hippocampal membranes, like spermine, tele-methylhistamine enhanced [(3)H]dl-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid (CGP39653) binding to the glutamate site. In contrast, spermine increased nonequilibrium [(3)H]5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate; MK-801) binding, and suppressed [(3)H]ifenprodil binding, whereas histamine and tele-methylhistamine had no effect. In conclusion, the histamine-induced potentiation of NMDARs occurs in the brain under normal conditions. Histamine does not bind to the polyamine site, but to a distinct entity, the so-called histamine site of the NMDAR.

Polyamines in the Brain: Distribution, Biological Interactions, and their Potential Therapeutic Role in Brain Ischaemia

The endogenous polyamines (spermine, spermidine, and putrescine) are present at relatively high concentrations in the mammalian brain and play crucial roles in a variety of aspects of cell functioning. Stroke is the third most common cause of death and the leading cause of disability among adults in the western world. Brain polyamine levels change dramatically following cerebral ischaemia. Polyamines may be involved in the pathophysiological processes underlying brain ischaemia through several possible mechanisms. These include direct effects on ion channels and receptors modulating potassium, and most importantly calcium trafficking, or through the production of toxic metabolites. Considerable evidence shows that the non-competitive polyamine antagonists, ifenprodil and eliprodil, are neuroprotective. Interestingly, novel polyamine analogues, such as N(1)-dansylspermine, BU36b, and BU43b, have also recently been shown to have neuroprotective potential. The exact mechanisms of the neuroprotection afforded by the polyamine antagonists and their clinical applicability is worthy of further study.

Visual Improvement in a Patient Taking Ginkgo Biloba Extract: A Case Study

J S O N M J F J J F F F NTRODUCTION laucoma is a progressive optic neuropathy characterized by a pecific pattern of optic nerve head and visual field damage, the auses of which are multifold. Elevated intraocular pressure IOP) is the most important known risk factor for glaucoma, but s not the disease itself. The many non–IOP-dependent risk facors that have been associated with glaucoma and may induce amage by decreasing vascular perfusion at the level of the optic erve head include cardiovascular disease, vasospasm, autommune disease, and cerebral microvascular ischemia. Other otential risk factors for decreased optic nerve head perfusion nclude low blood pressure, orthostatic hypotension, nocturnal ypotension, migraine, Raynaud’s phenomenon, autoimmune henomena, and hemorheologic abnormalities. With the discovery of these risk factors, a new focus has been laced on alternative treatments for glaucoma, those that depart rom the traditional IOP-lowering effect of drug, laser, and oprative therapies. Glaucoma often progresses despite lowering of OP to normal levels, or develops at normal or even low IOP. herefore, a need exists for therapies that prevent or limit the amage due to glaucoma—independent of therapies that simply ower IOP. Neuroprotection, the protection of undamaged neuons from secondary degeneration and subsequent apoptosis, is n important but still unachieved goal for the treatment of glauoma. There are many compounds, both natural and synthetic, eported to have neuroprotective effects, including antioxidants, -methyl-D-aspartate receptor antagonists, inhibitors of glutaate release, polyamine antagonists, and nitric oxide synthase nhibitors, cannabinoids, aspirin, melatonin, and vitamin B12. alcium channel blockers, although improving ocular blood ow, are also believed to have neuroprotective properties. heir use in normal-tension glaucoma has been limited, howver, due to a reported increase in morbidity. The lack of availability of specific neuroprotectant comounds and the lack of clinical trials examining the benefits of europrotective agents for glaucoma limit the current use of

Effect of modulators of the polyamine site on the development of seizures induced by systemic and intracerebral administration of N-methyl-D-aspartate in albino mice

Systemic intraperitoneal administration of polyamine agonist IEM-1460 containing the Me3N(+) group with a stable positive charge preventing permeation of this substance through the blood-brain barrier and polyamine antagonist arcaine had no effect on the development of seizures caused by intracerebral injection of N-methyl-D-aspartate in mice. Intraperitoneal injection of IEM-40 potentiated, while arcaine decreased the severity of seizures induced by intraperitoneal treatment with N-methyl-D-aspartate. This effect was related to modulation of the permeability of the blood-brain barrier for N-methyl-D-aspartate probably due to modulating effects of IEM-40 and arcaine on the polyamine site of N-methyl-D-aspartate receptors in the blood-brain barrier.

Effect of spermine and N 1-dansyl-spermine on epileptiform activity in mouse cortical slices

N 1-dansyl-spermine is a novel polyamine analogue, which has been demonstrated to have an antagonist action at the stimulatory polyamine site on the N-methyl- d-aspartate (NMDA) receptor macrocomplex. Cortical wedges from genetically epilepsy-prone DBA/2 mice demonstrate spontaneous epileptiform activity when perfused with Mg 2+-free artificial cerebrospinal fluid (aCSF). This epileptiform activity has been demonstrated to be primarily mediated through the NMDA receptor. N 1-dansyl-spermine reduced the spontaneous epileptiform activity at a high dose (100 μM) but had no effect at a lower dose (50 μM). The polyamine, spermine (300 μM) caused an increase in the rate of the spontaneous epileptiform discharges. This effect of spermine was antagonised by administration of the low dose of N 1-dansyl-spermine (50 μM). This further demonstrates the role of the NMDA receptor in the production of spontaneous epileptiform discharges in the cortical wedge preparation and clearly illustrates both the facilitatory action of spermine and the polyamine antagonist action of N 1-dansyl-spermine at the stimulatory polyamine site on the NMDA receptor.

1167 PUBLICATION Adjuvant chemotherapy (CT) and patient compliance in non-small cell lung cancer (NSCLC). a multivariate analysis of 356 consecutively treated patients

Among over 60 polyamine derivatives tested, onlyN-(3-aminopropyl)octanediamine and bis-(3-aminopropyl)nonanediamine (TE393) markedly inhibited [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) binding at equilibrium in the presence of added spermidine (SPD) in “non-washed” rat brain synaptic membranes, without affecting that in the absence of added SPD. Although TE393 significantly potentiated [3H]MK-801 binding before equilibrium in the presence ofl-glutamic acid (Glu) alone or both Glu and glycine (Gly) added in “Triton-treated” membranes, the putative polyamine antagonists 1,10-decanediamine (DA10) and arcaine invariably inhibited binding irrespective of the addition of agonists. In the absence of added SPD, in addition, TE393 markedly enhanced abilities of both Glu and Gly to potentiate [3H]MK-801 binding before equilibrium. However, TE393 induced a rightward shift of the concentration-response curve of SPD for [3H]MK-801 binding before equilibrium. Moreover, TE393 was effective in potentiating binding of an antagonist but not an agonist radioligand to the NMDA domain and in inhibiting binding of an antagonist but not an agonist radioligand to the Gly domain. The potentiation of NMDA antagonist binding by TE393 occurred in a manner sensitive to prevention by arcaine but not by DA10. These results suggest that TE393 may be a novel ligand at the polyamine domain with an ability to interact with both the NMDA and Gly recognition domains in antagonist-preferring forms.

1165 PUBLICATION Second-line treatment with pemetrexed following platinum based doublet chemotherapy in Malignant Pleural Mesothelioma

Among over 60 polyamine derivatives tested, onlyN-(3-aminopropyl)octanediamine and bis-(3-aminopropyl)nonanediamine (TE393) markedly inhibited [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) binding at equilibrium in the presence of added spermidine (SPD) in “non-washed” rat brain synaptic membranes, without affecting that in the absence of added SPD. Although TE393 significantly potentiated [3H]MK-801 binding before equilibrium in the presence ofl-glutamic acid (Glu) alone or both Glu and glycine (Gly) added in “Triton-treated” membranes, the putative polyamine antagonists 1,10-decanediamine (DA10) and arcaine invariably inhibited binding irrespective of the addition of agonists. In the absence of added SPD, in addition, TE393 markedly enhanced abilities of both Glu and Gly to potentiate [3H]MK-801 binding before equilibrium. However, TE393 induced a rightward shift of the concentration-response curve of SPD for [3H]MK-801 binding before equilibrium. Moreover, TE393 was effective in potentiating binding of an antagonist but not an agonist radioligand to the NMDA domain and in inhibiting binding of an antagonist but not an agonist radioligand to the Gly domain. The potentiation of NMDA antagonist binding by TE393 occurred in a manner sensitive to prevention by arcaine but not by DA10. These results suggest that TE393 may be a novel ligand at the polyamine domain with an ability to interact with both the NMDA and Gly recognition domains in antagonist-preferring forms.

The pre-ischaemic neuroprotective effect of a novel polyamine antagonist, N1-dansyl-spermine in a permanent focal cerebral ischaemia model in mice

The polyamine sites on the NMDA receptor complex offer a therapeutic target for focal ischaemia, potentially devoid of most side effects associated with NMDA antagonists. In this study, we investigated the effect of a novel polyamine antagonist, N 1-dansyl-spermine (0.5–10 mg kg −1) in a permanent focal cerebral ischaemia model in mice, and compared its effect to that of MK-801 (0.3–3 mg kg −1) following administration 30 min prior to ischaemia. A battery of histological and behavioural tests was employed following permanent middle cerebral artery occlusion to assess any neuroprotective effect. Following middle cerebral artery occlusion, N 1-dansyl-spermine (1–5 mg kg −1) and MK-801 (1 or 3 mg kg −1) caused a comparable and significant reduction in the percentage hemisphere lesion volume. Similarly, both drugs significantly reduced oedema and neurological deficit score to a similar extent. Locomotor activity in MCAO mice was not significantly improved by MK-801 or N 1-dansyl-spermine, although N 1-dansyl-spermine induced a trend towards significant improvement. Significant improvement in rotarod performance was observed at neuroprotective doses with both drugs. Upon comparison of the profile of effects, N 1-dansyl-spermine at least matched the effectiveness of MK-801 as a neuroprotective agent in this model. In addition, in sham-operated control mice, N 1-dansyl-spermine was well tolerated, in contrast to the pronounced adverse effects of MK-801 on locomotor activity and rotarod performance. In conclusion, this study has shown that N 1-dansyl-spermine is as effective a neuroprotective drug as MK-801 in this model. Moreover, in contrast to MK-801, N 1-dansyl-spermine could be a promising therapeutic candidate for stroke as it is well tolerated at neuroprotective doses in sham-operated animals.

N1-Dansyl-spermine: a potent polyamine antagonist

The potential polyamine antagonist action of N 1-dansyl-spermine (a potent NMDA antagonist) was assessed in two in vivo mouse models of polyamine action. Co-administration of N 1-dansyl-spermine (2–10 μg, i.c.v.) with spermine (100 μg, i.c.v.) resulted in a dose-dependent antagonism of the spermine-induced CNS excitation (body tremor and fatal tonic convulsions). In addition, the same dose of N 1-dansyl-spermine antagonised spermine's enhancement of NMDA-induced convulsions. These results suggest that N 1-dansyl-spermine is in vivo a potent antagonist of the CNS effects of spermine and of its action at the positive polyamine modulatory site on the NMDA receptor.

The neuroprotective effects of N1-dansyl-spermine in the gerbil model of cerebral ischaemia

The effects of N 1-dansyl-spermine, a polyamine antagonist, and ifenprodil, a non-competitive N-methyl- d-aspartate (NMDA) receptor antagonist, were investigated in the gerbil model of global cerebral ischaemia. Transient forebrain ischaemia was induced by 5-min bilateral occlusion of the common carotid arteries. N 1-dansyl-spermine (2, 5 and 10 mg/kg) and ifenprodil (30 mg/kg) were administered intraperitoneally 30 min after bilateral carotid artery occlusion. On histological examination, 4 days (96 h) after ischaemia, there was a significant decrease in neuronal density of the hippocampal CA1 subfield. This reduction in neuronal density was attenuated in those animals treated with the 5 or 10 mg/kg dose of N 1-dansyl-spermine and those treated with 30 mg/kg ifenprodil. However, unlike ifenprodil, N 1-dansyl-spermine failed to attenuate the ischaemia-induced increase in locomotor activity. This demonstrates that polyamines play a significant role in the neuronal damage produced after cerebral ischaemia, while casting doubt on the suggestion that increased locomotor activity correlates with CA1 pyramidal cell damage.

Polyamine synthesis and interconversion by the Microsporidian Encephalitozoon cuniculi.

Polyamines are small cationic molecules necessary for growth and differentiation in all cells. Although mammalian cells have been studied extensively, particularly as targets of polyamine antagonists, i.e. antitumor agents, polyamine metabolism has also been studied as a potential drug target in microorganisms. Since little is known concerning polyamine metabolism in the microsporidia, we investigated it in Encephalitozoon cuniculi, a microspordian associated with disseminated infections in humans. Organisms were grown in RK-13 cells and harvested using Percoll gradients. Electron microscopy indicated that the fractions banding at 1.051-1.059/g/ml in a microgradient procedure, and 1.102-1.119/g/ml in a scaled-up procedure were nearly homogenous, consisting of pre-emergent (immature) spores which showed large arrays of ribosomes near polar filament coils. Intact purified pre-emergent spores incubated with [1H] ornithine and methionine synthesized putrescine, spermidine, and spermine, while [14C]spermine was converted to spermidine and putrescine. Polyamine production from ornithine was inhibitable by DL-alpha-difluoromethylornithine (DFMO) but not by DL-alpha-difluoromethylarginine (DFMA). Cell-free extracts from mature spores released into the growth media had ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetdc), and spermidine/spermine N1-acetyltransferase (SSAT) activities. ODC activity was inhibited by DFMO, but not by DFMA. AdoMetdc was putrescine-stimulated and inhibited by methylglyoxal-bis(guanylhydrazone); arginine decarboxylase activity could not be detected. It is apparent from these studies that Encephalitozoon cuniculi pre-emergent spores have a eukaryotic-type polyamine biosynthetic pathway and can interconvert exogenous polyamines. Pre-emergent spores were metabolically active with respect to polyamine synthesis and interconversion, while intact mature spores harvested from culture supernatants had little metabolic activity.

Neuroprotection: is it already applicable to glaucoma therapy?

Many categories of both natural and synthetic compounds have been reported to have neuroprotective activity. These include not only antioxidants, N-methyl-D-aspartate receptor antagonists, inhibitors of glutamate release, calcium channel blockers, polyamine antagonists, and nitric oxide synthase inhibitors, but cannabinoids, aspirin, melatonin, and vitamin B-12. The lack of availability of specific neuroprotectant compounds in the United States and the lack of clinical trials examining the benefits of neuroprotective agents for glaucoma currently limit the use of these agents. This article provides a short overview of the concept of neuroprotection as it applies to glaucoma and suggests the possibility of neuroprotective activity that might be provided by compounds that are presently easily available.

Mutation of a glutamate receptor motif reveals its role in gating and delta2 receptor channel properties.

Despite its importance in the cerebellum, the functions of the orphan glutamate receptor delta2 are unknown. We examined a mutant delta2 receptor channel in lurcher mice that was constitutively active in the absence of ligand. Because this mutation was within a highly conserved motif (YTANLAAF), we tested its effect on several glutamate receptors. Mutant delta2 receptors showed distinct channel properties, including double rectification of the current-voltage relationship, sensitivity to a polyamine antagonist and moderate Ca 2+ permeability, whereas other constitutively active mutant glutamate channels resembled wild-type channels in these respects. Moreover, the kinetics of ligand-activated currents were strikingly altered. We conclude that the delta2 receptor has a functional ion channel pore similar to that of glutamate receptors. The motif may have a role in the channel gating of glutamate receptors.

Antiparkinsonian Actions of Blockade of NR2B-Containing NMDA Receptors in the Reserpine-Treated Rat

Current symptomatic treatment for Parkinson's disease is based largely on dopamine-replacing agents. The fact that long-term treatment with these drugs is characterized by many side effects has lead to widespread interest in nondopaminergic therapies. To date, however, it has proved difficult to devise a nondopaminergic therapy with significant antiparkinsonian efficacy when administered as monotherapy. Overactivity of the striatolateral pallidal pathway, the “indirect” striatal output pathway, is thought be responsible for the generation of parkinsonian symptoms. Indeed, it has been suggested that selective reduction in the activity of the “indirect” pathway may be achieved by blockade of NR2B-containing NMDA receptors. In the present study, we demonstrate that selective blockade of NR2B-containing NMDA receptors with the polyamine antagonists ifenprodil and eliprodil causes a significant increase in locomotor activity in the reserpine-treated rat model of Parkinson's disease (30 mg/kg ifenprodil, 221.2±54 mobile counts compared to vehicle, 19.6±6.87,P<0.001). Additionally, we show that, subsequent to dopamine depletion, the ability of ifenprodil to bind to the polyamine site and inhibit binding of the NMDA channel blocker [3H] MK-801 is increased fourfold (IC503.7±0.4 μM compared to vehicle, IC5014.3±2.34 μM,P<0.01). We suggest that ifenprodil selectively targets the polyamine site on overactive NR2B-containing NMDA receptors. Thus, we propose that NR2B-selective NMDA receptor antagonists may prove useful in the treatment of Parkinson's disease.

Complex polyamine effects on [ 3H]MDL 105,519 binding to the NMDA receptor glycine site

Several studies have suggested that polyamines modulate the interaction of glycine with the NMDA receptor. We have further investigated the effects of polyamines using the NMDA receptor glycine site antagonist [(E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1H-indole-2-carbox ylic acid] ([3H]MDL 105,519). [3H]MDL 105,519 binding assays were performed using well washed membranes prepared from frozen rat brains. The polyamines spermine and spermidine increased the fraction of non-specific binding (determined by the addition of 1 mM glycine) in the [3H]MDL 105,519 binding assay from 40-60% when spermine or spermidine concentration was increased from 1 to 100 microM. Polyamine agonists spermine, spermidine and 1,5-(diethylamino)piperidine (30 microM) did not have a significant effect on displacement of [3H]MDL 105,519 binding by glycine or glycine site antagonists. Similarly, the polyamine antagonist arcaine did not have a significant effect on displacement of [3H]MDL 105,519 binding by glycine or glycine site antagonists. However, spermidine significantly depressed the potency of MDL 105,519 in displacing [3H]dizocilpine binding. These data suggest that [3H]MDL 105,519 may preferentially bind to a polyamine insensitive form of the NMDA receptor.

Investigation of the actions and antagonist activity of some polyamine analogues in vivo.

1. The ability of three putative polyamine antagonists to antagonize behavioural changes induced by spermine was assessed. 2. Injection of an excitotoxic dose of spermine (100 microg, i.c.v.) in mice results in the development of a characteristic behavioural profile, which has two temporally distinct phases. The early events include clonic convulsions, and the later, more general excitation, includes tremor and culminates in the development of a fatal tonic convulsion. 3. Co-administration of arcaine (25 microg, i.c.v.) potentiated the early phase effects after spermine injection, but antagonized the development of spermine-induced tonic convulsions. A larger dose of arcaine (50 microg, i.c.v.) given alone resulted in the development of spermine-like body tremor and convulsions. It therefore appears that arcaine is not a pure polyamine antagonist in vivo, but may be a partial agonist. 4. Similarly, 1,10-diaminodecane appeared to act as a partial agonist in vivo, although it was less potent than arcaine. 5. In contrast, diethylenetriamine (DET) effectively inhibited the development of the early effects of spermine, but was ineffective against the spermine-induced CNS excitation and tonic convulsions. 6. It is concluded that none of the putative polyamine antagonists tested behaved as effective polyamine antagonists in vivo, although each produced some antagonism.