Gene C. Palmer

Biological profile of the metabolites and potential metabolites of the anticonvulsant remacemide.

Remacemide hydrochloride ((+/-)-2-amino-N-(1-methyl-1,2-diphenylethyl)- acetamide hydrochloride or FPL 1292AA) is a novel compound undergoing clinical trials for patients with generalized tonic/clonic and complex partial epilepsy. Remacemide exhibits efficacy against maximal electroconvulsive shock (MES) in rodents and seizures elicited by N-methyl-D,L-aspartate (NMDLA) in mice. Using rat synaptic membrane fractions, remacemide was shown to possess relatively weak noncompetitive binding to the ionic channel site of the NMDA (N-methyl-D-aspartic acid) receptor complex. With the hypothesis that activity against NMDLA-elicited seizures might be reflected by transformation to a more active metabolic species, the aim of the present study was to evaluate potential pharmacological effects of the 9 identified metabolites of remacemide which were all found in human and dog urine. Moreover, specific entities were recognized in plasma (including the rats), as well as dog and rat cerebrospinal fluid. Five putative metabolites were also examined. A major route of metabolic transformation of remacemide in rats yields the formation of a pharmacologically active more potent desglycine derivative, namely FPL 12495 (+/-). Potency over the parent compound is revealed in the MES test in mice and rats, the NMDA-induced convulsions/mortality test in mice, and especially involving in vitro displacement of MK801 binding to the channel subsite of the NMDA receptor. The S isomer (FPL 12859) of this desglycinate is even more potent, while the R isomer is less potent than the corresponding racemate. Unlike the non-competitive NMDA antagonist, MK801, these desglycinates did not prevent kindled seizures. Three other identified metabolites show efficacy in the mouse and rat in vivo tests, namely the N-hydroxy-desglycinate (FPL 15053) and the p-hydroxy-desglycinates (FPL 14331 and FPL 14465). FPL 15053 exhibited modest activity in all tests. The only in vivo activity exhibited by the 2 p-hydroxy-desglycinates was evidenced in the MES test following i.p. and i.v. dosing. However, FPL 14331 was active in the MK801 binding assay. An oxoacetate metabolite, PFL 15455, failed to demonstrate any biological activity. Of potential metabolites tested 2 beta-hydroxy-desglycinates (FPL 14991 and FPL 14981) displayed modest activity in the MES test, however, only FPL 14981 prevented NMDLA-induced convulsions/mortality in mice and was 2-fold more active regarding MK801 binding. The hydroxy-methyl derivative of remacemide (FPL 13592) and its desglycinate (FPL 15112) prevented MES-induced convulsions only after i.v. administration; only the desglycine derivative displaced MK801 binding.(ABSTRACT TRUNCATED AT 400 WORDS)

The Future of Neuroprotection

W. SLIKKKER,a,g M. YOUDIM,b G.C. PALMER,c E. HALL,d C. WILLIAMS,e AND B. TREMBLYf aDivision of Neurotoxicology, National Center for Toxicological Research/FDA, Jefferson, Arkansas, USA bFogarty International Center, National Institute of Mental Health/NIH, Bethesda, Maryland, USA cAstra Arcus USA, Rochester, New York, USA dNeuroscience Therapeutics, Parke-Davis Pharmaceutical Research, Ann Arbor, Michigan, USA eDepartment of Paediatrics, University of Aukland, Aukland, New Zealand fSection of Neurosugery, Veterans Affairs Medical Center, Togus, Maine, USA

Effects of anticonvulsants in a novel operant learning paradigm in rats: Comparison of remacemide hydrochloride and FPL 15896AR to other anticonvulsant agents

One of the primary undesired effects of anticonvulsant medication is an impairment in cognitive function, such as new learning ability. The purpose of the present study was to compare the effects of remacemide hydrochloride [(+/-)-2-amino-N-(1-methyl-1,2,-diphenylethyl)acetamide monohydrochloride] and FPL 15896AR [(+)-alpha-phenyl-2-pyridine-ethanamide] to a number of anticonvulsant agents on an operant acquisition baseline. Remacemide hydrochloride is currently in clinical trials for epilepsy and FPL 15896AR is under development. In the present procedure, fasted, experimentally naive rats were placed into operant chambers in which food pellets were initially available under a Fixed-Ratio 1 (FR1) schedule of food presentation, and as lever pressing progressed, the FR value incremented. All drugs were tested in multiples of three and ten times their respective ED50 values against maximal electroshock-induced seizure (MES) following p.o. administration. The drugs tested varied widely in their ability to disrupt acquisition of the lever-pressing task. Remacemide hydrochloride and a structurally related analog, FPL 15896AR, did not disrupt acquisition. Clonazepam, lamotrigine, MK-801, phenobarbital, felbamate, phenytoin, and carbamazepine increased the number of hours required to achieve FR3 (emit more than 100 responses) with respect to vehicle control performance. Of these, clonazepam, MK-801 and phenytoin produced robust enough disruption to result in significantly fewer reinforcers delivered over the 14-h operant session.

The low-affinity, use-dependent NMDA receptor antagonist AR-R 15896AR. An update of progress in stroke.

ABSTRACT: Use‐dependent N‐methyl‐d‐aspartate (NMDA) receptor antagonists protect neurons from the lethal consequences of excessive stimulation by excitatory amino acids. Clinical development of high‐affinity compounds such as MK801 have been limited due to untoward side effects. Toward this end, the lower‐affinity use‐dependent NMDA antagonists have greater margins of safety and have advanced to clinical trials for stroke, epilepsy, head trauma and chronic neurodegenerative disorders. AR‐R 15896AR is currently in Phase II trials for stroke and has been repeatedly demonstrated to afford neuroprotection in a variety of in vivo and in vitro models associated with ischemia/excitotoxic conditions.

Low‐Affinity NMDA Receptor Antagonists

Antagonists of the NMDA (N-methyh-aspartic acid) subtype of the glutamate (GLU) receptor in the brain have been shown to be highly effective as both neuroprotective agents following stroke or neurotrauma and as anticonvulsants.1.2 A persistent problem associated with highly potent competitive and uncompetitive NMDA receptor antagonists is their propensity to elicit motor impairment, abuse liability, and disruption of cognitive processes. However, these untoward side effects appear to be minimized with compounds exhibiting low or modest affinity for the NMDA receptor: and several such compounds have recently been introduced into clinical trials for stroke, head trauma, Huntington’s disease and ep i lep~y.~-~ The single enantiomeric species, ARL 15896AR ([ +]-a-phenyl-2-pyridine-ethanamine diHC1) (FIG. l), has been profiled preclinically for acute efficacyhafety evaluations and has currently completed phase I trials to assess tolerability in human volunteers.6 ARL 15896AR might offer inherent advantages for the acute treatment of stroke because of its predictable pattern of pharmacokinetics and rapid penetration into the brain. Moreover, preclinical data indicate high degrees of safety when evaluated in animal models for learning, memory deficits, motor impairment, or abuse liability.6-g

Free radicals generated by xanthine oxidase-hypoxanthine damage adenylate cyclase and ATPase in gerbil cerebral cortex

The generation of superoxide radicals from xanthine oxidase-hypoxanthine in a particulate fraction of gerbil cerebral cortex influenced the activity of the synaptic enzyme adenylate cyclase, as well as Mn2+- and Na+,K+-sensitive forms of ATPase. Low concentrations of xanthine oxidase actually elevated the sensitivity of adenylate cyclase to GTP, GTP + norepinephrine (NE), and forskolin but not significantly to Mn2+. Higher levels of xanthine oxidase elicited a marked inhibition of these responses. The stimulation of adenylate cyclase mechanisms requiring GTP (GTP, forskolin, and NE) was more susceptible than was Mn2+, suggesting that the guanine nucleotide stimulatory protein was more vulnerable to free radical attack than the catalytic site of adenylate cyclase. superoxide dismutase (SOD), but not catalase, partially protected the forskolin-sensitive enzyme from the action of xanthine oxidase-hypoxanthine. A combination of SOD plus catalase preserved enzyme responses to forskolin. In comparison, additions of SOD plus mannitol or catalase plus flunarizine were less effective. The sensitivity of the particulate ATPase to Mn2+ was more labile to the consequence of superoxide formation than Na+, K+-ATPase. In this regard the Ca2+, Mg2+ sensitivity of the enzyme was reduced only to a marginal extent. The findings might be analogous toin vivo data in which cerebral adenylate cyclase and Na+, K+-ATPase are damaged following postischemic reperfusion in gerbils, a process thought to be mediated by free radicals.

Acute heat stress model of seizures in weanling rats: Influence of prototypic anti-seizure compounds

The present study tested the therapeutic potential for prototype anti-epilepsy drugs using an animal model of infantile febrile seizures. The model consisted of immersion of weanling rats (21 days old) in a 45 degrees C water bath for a maximum of 4 min (four exposures over a 2 week period) and observing for the progression to stage-5 seizures. All compounds were administered orally at the respective ED50 for prevention of seizures in the maximal electroshock (MES) test. Clonazepam effectively lowered the score for seizure grade, shortened the duration of seizures, as well as reduced the number of animals experiencing seizures during three of the four testing periods. MK801 reduced both the maximum seizure grade, and the number of animals experiencing seizures during sessions two and three. However, the dose of MK801 caused behavioral side effects. Valproate actively decreased seizure grade, while it modestly acted to attenuate seizure duration, extended the time to seizure onset, and reduced the number of animals experiencing seizures on testing day 1. Remacemide hydrochloride and phenobarbital were not effective. The method appears useful for evaluating the potential of agents to prevent acute febrile seizures.

Preclinical and Clinical Aspects of Remacemide Hydrochloride

Publisher Summary Remacemide hydrochloride was discovered from a chemical series employing a rational synthetic approach based on molecular modeling. Remacemide hydrochloride was effective orally, regarding prevention of seizures evoked by maximal electroshock (MES) in rodents. Remacemide hydrochloride and ARL12495AA inhibit seizures induced by N-methyl- D -aspartate (NMDA), whereas remacemide hydrochloride alone is active against kainic acid-induced seizures. Neuronal protective properties of remacemide hydrochloride and ARL12495AA were examined in primary cultures from rat cerebral cortex. Treatment of cells with ARL12495AA also reduced the NMDA-elicited loss in membrane-associated protein kinase C activity and the magnitude in the NMDA-triggered Ca2+ surge into the cells. Potential neuroprotective properties for remacemide hydrochloride were demonstrated when pretreatment of mice exposed to anoxic conditions led to an extension of the time to death. Remacemide hydrochloride appeared free of genotoxicity when evaluated in two in vitro tests for mutagenicity.

Efficacy of AR-R15896AR in the Rat Monofilament Model of Transient Middle Cerebral Artery Occlusion

The monofilament technique of transient middle cerebral artery occlusion (MCAO) was used in 3 separate studies to evaluate the efficacy of the low-affinity, use-dependent N-methyl-d-aspartate receptor antagonist, AR-R15896AR. First, a dose-response curve was attempted. Wister Kyoto rats received 2 hours of MCAO. Five minutes later, a 30-minute intravenous infusion of AR-R15896AR was given, followed by subcutaneous implantation of Alzet minipumps that were calibrated to maintain specified plasma levels (approximately 682, 1885, or 2682 ng/mL) of AR-R15896 (free base) for 1 week. The highest plasma level attained significantly decreased the percentage of damage to the subcortex, cortex, and total brain. Second, the high-dose, 1-week treatment regimen was repeated to determine if neuroprotection would extend to 8 weeks after MCAO. Indeed, in separate groups of animals, significant reduction in the percentage of damage, which was generally confined to the cortex and subcortex, was observed at 1, 2, 4, and 8 weeks. Third, verification was achieved in another laboratory. Lister Hooded rats received 60 minutes of transient MCAO. At 70 minutes, an acute dose of AR-R15896AR (20.3 mg/kg) was injected intraperitoneally and the rats were killed 23 hours later. This treatment group also exhibited significant reduction in the volume of infarction in the subcortex, cortex, and total brain. The outcome of these investigations supports the ongoing Phase II clinical trials in patients with acute stroke.

Lack of evidence for direct involvement of NMDA receptors or polyamines in blood–brain barrier injury after cerebral ischemia in rats

It is hypothesized that after various types of brain injury, blood-brain barrier (BBB) opening and vasogenic edema result from excessive neuronal release of glutamate and stimulation of capillary N-methyl-d-aspartate (NMDA) receptors linked to polyamine (putrescine) synthesis in endothelial cells. We produced cerebral ischemia in rats and measured BBB opening 6 h later as the increase in regional transfer constants (Ki) for blood to brain diffusion of [3H]sucrose. Such BBB opening was not mitigated by drugs which block NMDA receptors (MK801 or AR-R 15896AR) or polyamine synthesis (difluoromethylornithine). These results question generality of the capillary NMDA receptor/polyamine hypothesis.