Ernst Wülfert

The novel antiepileptic drug levetiracetam (ucb L059) appears to act via a specific binding site in CNS membranes.

Levetiracetam ((S)-alpha-ethyl-2-oxo-pyrrolidine acetamide, ucb L059) is a novel potential antiepileptic agent presently in clinical development with unknown mechanism of action. The finding that its anticonvulsant activity is highly stereoselective (Gower et al., 1992) led us to investigate the presence of specific binding sites for [3H]levetiracetam in rat central nervous system (CNS). Binding assays, performed on crude membranes, revealed the existence of a reversible, saturable and stereoselective specific binding site. Results obtained in hippocampal membranes suggest that [3H]levetiracetam labels a single class of binding sites (nH = 0.92 +/- 0.06) with modest affinity (Kd = 780 +/- 115 nM) and with a high binding capacity (Bmax = 9.1 +/- 1.2 pmol/mg protein). Similar Kd and Bmax values were obtained in other brain regions (cortex, cerebellum and striatum). ucb L060, the (R) enantiomer of levetiracetam, displayed about 1000 times less affinity for these sites. The binding of [3H]levetiracetam is confined to the synaptic plasma membranes in the central nervous system since no specific binding was observed in a range of peripheral tissues including heart, kidneys, spleen, pancreas, adrenals, lungs and liver. The commonly used antiepileptic drugs carbamazepine, phenytoin, valproate, phenobarbital and clonazepam, as well as the convulsant agent t-butylbicyclophosphorothionate (TBPS), picrotoxin and bicuculline did not displace [3H]levetiracetam binding. However, ethosuximide (pKi = 3.5 +/- 0.1), pentobarbital (pKi = 3.8 +/- 0.1), pentylenetetrazole (pKi = 4.1 +/- 0.1) and bemegride (pKi = 5.0 +/- 0.1) competed with [3H]levetiracetam with pKi values comparable to active drug concentrations observed in vivo. Structurally related compounds, including piracetam and aniracetam, also displaced [3H]levetiracetam binding. (S) Stereoisomer homologues of levetiracetam demonstrated a rank order of affinity for [3H]levetiracetam binding in correlation with their anticonvulsant activity in the audiogenic mouse test (r2 = 0.84, n = 12, P < 0.0001). These results support a possible role of this binding site in the anticonvulsant activity of levetiracetam and substantiate the singular pharmacological profile of this compound. This site remains however to be further characterised.

ucb L059, a novel anti-convulsant drug : pharmacological profile in animals

The anticonvulsant activity of ucb L059 ((S)-alpha-ethyl-2-oxo-pyrrolidine acetamide) was evaluated in a range of animal models. ucb L059 was active after oral and intraperitoneal administration in both rats and mice, with a unique profile of action incorporating features in common with several different types of antiepileptic drugs. The compound was active, with ED50 values generally within the range of 5.0-30.0 mg/kg, in inhibiting audiogenic seizures, electrically induced convulsions and convulsions induced chemically by pentylenetetrazole (PTZ), bicuculline, picrotoxin and N-methyl-D-aspartate (NMDA). ucb L059 retarded the development of PTZ-induced kindling in mice and reduced PTZ-induced EEG spike wave discharge in rats. The R enantiomer, ucb L060, had low intrinsic anticonvulsant activity, showing the stereospecificity of action of the molecule although the actual mechanism of action remains unknown. Neurotoxicity, evaluated with an Irwin-type observation test, the rotarod test and open-field exploration, was minimal, with only mild sedation being observed, even at doses 50-100 times higher than the anticonvulsant doses; at pharmacologically active doses, the animals appeared calm but slightly more active. ucb L059 thus presents as an orally active, safe, broad-spectrum anticonvulsant agent, with potential antiepileptogenic and anti-absence actions.

7‐Hydroxylated epiandrosterone (7‐OH‐EPIA) reduces ischaemia‐induced neuronal damage both in vivo and in vitro

Recent evidence suggests that steroids such as oestradiol reduce ischaemia‐induced neurodegeneration in both in vitro and in vivo models. A cytochrome P450 enzyme termed cyp7b that 7‐hydroxylates many steroids is expressed at high levels in brain, although the role of 7‐hydroxylated steroids is unknown. We have tested the hypothesis that the steroid‐mediated neuroprotection is dependent on the formation of 7‐hydroxy metabolites. Organotypic hippocampal slice cultures were prepared from Wistar rat pups and maintained in vitro for 14 days. Cultures were then exposed to 3 h hypoxia and neuronal damage assessed 24 h later using propidium iodide fluorescence as a marker of cell damage. Neurodegeneration occurred primarily in the CA1 pyramidal cell layer. The steroids oestradiol, dehydroepiandrosterone and epiandrosterone (EPIA) were devoid of neuroprotective efficacy when present at 100 nm pre‐, during and post‐hypoxia. The 7‐hydroxy metabolites of EPIA, 7α‐OH‐EPIA and 7β‐OH‐EPIA significantly reduced neurotoxicity at 100 nm and 10 nm. 7β‐OH‐EPIA was also neuroprotective in two in vivo rat models of cerebral ischaemia: 0.1 mg/kg 7β‐OH‐EPIA significantly reduced hippocampal cell loss in a model of global forebrain ischaemia, whereas 0.03 mg/kg was neuroprotective in a model of focal ischaemia even when administration was delayed until 6 h after the onset of ischaemia. Taken together, these data demonstrate that 7‐hydroxylation of steroids confers neuroprotective efficacy, and that 7β‐OH‐epiandrosterone represents a novel class of neuroprotective compounds with potential for use in acute neurodegenerative diseases.

Behavioural and histological effects of low concentrations of intraventricular AF64A

The effects of ethylcholine mustard aziridinium ion (AF64A; 0.3, 1.0 and 3.0 nmol), injected into each lateral ventricle in the rat, were determined in a range of behavioural tests, each involving a learning component. Effects were observed at 1.0 and 3.0 nmol/side and, to a lesser extent, at 0.3 nmol/side. Habituation of locomotor activity was impaired and deficits in learning were obtained using a variety of mazes including the Morris swimming maze. Slight, non-significant impairments occurred in shock reinforced behaviours. Histologically, marginal effects were observed at 0.3 nmol/side, and slight ventricular dilatation with necrosis of the hippocampus, restricted to the site of injection at 1.0 nmol/side; at 3.0 nmol/side more widespread necrosis was apparent. Biochemical efficacy of the lesions in terms of cholinergic changes was confirmed by analysis of acetylcholinesterase (AChE) levels showing decreases in the hippocampus and the cortex; no studies were carried out with respect to other neurotransmitters. Cognitive deficits can therefore be obtained by i.c.v. injection of AF64A at doses which cause significant cholinergic changes with minimal histological disturbances.

Anti-inflammatory effects and changes in prostaglandin patterns induced by 7β-hydroxy-epiandrosterone in rats with colitis

High dose levels of dehydroepiandrosterone and its 7-hydroxylated derivatives have been shown to reduce oxidative stress and inflammatory responses in dextran sodium sulfate (DSS)-induced colitis in rats. Another endogenous steroid, 7beta-hydroxy-epiandrosterone (7beta-hydroxy-EpiA) has been shown to exert neuroprotective effects at much smaller doses. Our aims were to evaluate whether 7beta-hydroxy-EpiA pre-treatment prevents DSS-induced colitis and to determine whether the effects involve changes in anti-inflammatory prostaglandin (PG) D(2) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) levels. Rats were administered 0.01, 0.1 and 1mg/kg 7beta-hydroxy-EpiA i.p. once a day for 7 days. Thereafter, colitis was induced by administration of 5% DSS in drinking water for 7 days. Levels of the PGs and the expression of cyclooxygenase (COX-2) and PG synthases were assessed during the course of the experiment. Administration of 7beta-hydroxy-EpiA caused a transient increase in COX-2 and PGE synthase expression within 6-15h and augmented colonic tissue levels of 15d-PGJ(2) levels starting at day 2. Treatment with DSS resulted in shortened colon length, depleted mucus in goblet cells and induced oxidative stress. COX-2 and mPGES-1 synthase expression were enhanced and accompanied by increased PGE(2), D(2) and 15d-PGJ(2) production. Although all dose levels of 7beta-hydroxy-EpiA reduced PGE(2) production, only the lowest dose (0.01mg/kg) of the steroid completely prevented colitis damage and tissue inflammation. 7beta-Hydroxy-EpiA pre-treatment prevents the occurrence of DSS-induced colitis through a shift from PGE(2) to PGD(2) production, associated with an early but transient increase in COX-2 expression and a sustained increase in the production of the anti-inflammatory prostaglandin 15d-PGJ(2).

Inhibition by levetiracetam of a non‐GABAA receptor‐associated epileptiform effect of bicuculline in rat hippocampus

1 Extracellular recording of field potentials, evoked by commissural stimulation in hippocampal area CA3 of anaesthetized rats, was performed in order to study the mode of action of the novel antiepileptic drug levetiracetam (ucb LO59). 2 The amplitude of orthodromic field population spike (PS2) markedly increased and repetitive population spikes appeared when the recording micropipette contained either bicuculline methiodide (BMI), or the specific GABAA antagonist gabazine (SR‐95531). 3 BMI‐induced increases in PS2 were reduced in a dose‐dependent manner by 1 to 320 μmol kg−1 levetiracetam i.v., with a U‐shape dose‐response relationship. However, levetiracetam did not reduce the increases in PS2 produced by gabazine. 4 Clonazepam (1 mg kg−1, i.p.), carbamazepine (20 mg kg−1, i.p.) and valproate (200 mg kg−1, i.v.) were ineffective in preventing BMI‐induced increases in PS2, while the calcium channel antagonist flunarizine, 50 μmol kg−1, i.p., reduced PS2 increments caused by BMI. The L‐type calcium channel blocker nifedipine, 100 μmol kg−1, i.p., was without effect. Similar to levetiracetam, flunarizine did not reduce the increases in PS2 induced by gabazine. 5 These data suggest that the increased excitability of CA3 neurones, caused by BMI administered in situ, involves calcium‐dependent processes not associated with blockade of GABAA receptors. The inhibition by levetiracetam of this calcium‐dependent effect of BMI might contribute to the antiepileptic effects of the drug.

Mivazerol, a novel compound with high specificity for α2 adrenergic receptors : binding studies on different human and rat membrane preparations

Mivazerol, 3-[1(H-imidazol-4-yl)methyl]-2-hydroxybenzamide hydrochloride, a new potential anti-ischemic drug designed by UCB S.A. Pharma Sector, has been studied in binding experiments on adrenergic, dopaminergic, serotoninergic, muscarinic and idazoxan binding sites. Our results indicate that this compound displays high affinity and marked specificity for alpha 2 adrenoceptors. Mivazerol displaced the binding of the alpha 2 adrenoceptor antagonist [3H]RX 821002 to the alpha 2A adrenoceptors in human frontal cortex membranes with an apparent Ki value of 37 nM. The competition curve was shallow (nH = 0.55), suggesting that this compound acts as an alpha 2 adrenergic agonist. Mivazerol was also a potent competitor for [3H]RX 821002 binding to human platelet membranes (containing alpha 2A adrenoceptors) and rat kidney membranes (75% of the alpha 2 adrenoceptors of the alpha 2B subtype), indicating that this compound is not alpha 2 adrenoceptor subtype selective. Equilibrium dissociation constants for alpha 1 adrenoceptors (displacement of [3H]prazosin) and 5-HT1A receptors (displacement of [3H]rauwolscine) were respectively about 120 times (Ki = 4.4 microM) and 14 times (Ki = 530 nM) higher than that for the alpha 2 adrenoceptors. Equilibrium dissociation constants were approximately 1000 times higher for all other receptors tested in this study; namely beta 1 and beta 2 adrenoceptors, D1- and D2-dopamine receptors, M1-, M2- and M3-muscarinic receptors, 5-HT2 receptors and non-adrenergic idazoxan binding sites.

ucb L059, a novel anticonvulsant, reduces bicuculline-induced hyperexcitability in rat hippocampal CA3 in vivo

Local diffusion of bicuculline from a bicuculline-containing recording micropipette increased the orthodromic field population spike (PS2), elicited upon commissural stimulation in the hippocampal CA3 region of anaesthetized rats. This increase in PS2 was consistently reduced by 5.4 mg/kg of the novel anticonvulsant drug ucb L059 ((S)-alpha-ethyl-2-oxo-pyrrolidine acetamide), when injected i.v. 10 min prior to lowering in place the bicuculline-containing micropipette. ucb L059 had no effect on PS2 in the absence of bicuculline. Paired-pulse stimulation produced marked inhibition of the second PS2 at low interstimulus interval, an effect which was significantly reduced by bicuculline. Although ucb L059 reduced the effect of bicuculline on both PS2S elicited by paired stimulations, the drug did not alter the reduction by bicuculline of paired-pulse inhibition at low interstimulus interval. These results suggest that ucb L059 prevents increases in CA3 neuronal excitability by bicuculline through a non gamma-aminobutyric acid-ergic mechanism.

Reversible cholinergic changes induced by AF64A in rat hippocampus and possible septal compensatory effect

Ethylcholine aziridinium (AF64A) was injected intracerebroventricularly in rats, and the dose- and time-dependent effect of this treatment was measured on cholinergic markers in different areas of the brain. Choline acetyltransferase (ChAT) in the hippocampus was reduced by 0, 25, 40 and 50%, 4 days after administration of 0.25, 0.5, 1.0 and 2.0 nmol/side of AF64A. This effect was paralleled by a 0, 21, 38 and 48% increase in septal ChAT, at the same time and dose, respectively. Hippocampal and septal ChAT returned to normal by 14 days, following the two smaller doses of AF64A. Normal values of ChAT activity in the hippocampus were measured by 3 months post-1.0 nmol/side and by 12 months after 2.0 nmol AF64A/side. A transient secondary increase in septal ChAT was measured at 28 and 42 days after 1.0 nmol/side, after which no further changes were measured in this area. High affinity choline transport (HAChT) in the hippocampus showed a 35 and 25% decrease but only 14 and 7 days, after injection of 0.25 and 0.5 nmol AF64A/side, respectively. However, significant reductions by 45% at 14 days and by 65% at 4 days, were measured after the infusion of 1.0 and 2.0 nmol/side, respectively. This effect was significantly attenuated by 3 months and returned to normal by 12 months after treatment. Acetylcholinesterase (AChE) activity in the hippocampus was most severely affected by AF64A. Specifically, significant inhibition of 20% (by 7 days), 35% (by 7 days), 25% (by 2 days) and 30% (by 2 days) was observed at 0.25, 0.5, 1.0 and 2.0 nmol AF64A/side, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Protection against inflammatory neurodegeneration and glial cell death by 7β-hydroxy epiandrosterone, a novel neurosteroid

It has been demonstrated that neuroprotective effects of dehydroepiandrosterone (DHEA) may be mediated by its 7alpha- and 7beta-hydroxy derivatives. Epiandrosterone is also converted to 7beta-hydroxy epiandrosterone (7beta-OH EPIA) in numerous tissues. The aim of the present study was to establish whether treatment with 7beta-hydroxy epiandrosterone has a neuroprotective effect in animal models of Alzheimers disease (AD) lesions. Intra-amygdaloid administration of amyloid beta [Abeta(25-35)] increased the number of tau-positive cells in the ipsilateral hippocampus. Intracerebroventricular administration of ethylcholine aziridinium (AF64A) caused cholinergic damage in the septum, and glial lesions in the lateral septal nucleus and in the lateral zones of the hippocampus. These effects were almost completely prevented when animals were treated subcutaneously (b.i.d.) for 10 days with 0.1 mg/kg 7beta-hydroxy epiandrosterone. These findings indicate that 7beta-hydroxy epiandrosterone has powerful cytoprotective effects suggesting that (a) this neurosteroid may have therapeutic potential in various neurodegenerative conditions such as Alzheimers disease, and (b) 7beta-hydroxy steroids may constitute a novel class of endogenous neuroprotective agents.