WO2001035939A2 - Oleamide in epilepsy - Google Patents

Abstract

There is described a method of treating a convulsant disorder which comprises altering the levels of oleamide in the brain of a patient suffering from such a disorder. The method may comprise altering the oleamide levels by administering a therapeutically effective amount of an amido hydrolase inhibitor, thus preventing the degradation of endogenous oleamide. Alternatively, the method may comprise administering a therapeutically effective amount of oleamide itself.

Description

OLEAMIDE IN EPILEPSY

This invention relates to a new use of known compounds, to new formulations of such compounds and to new methods of treatment.

Epilepsy is prevalent in between 4 and 10 people per 1000 at any given time. In the UK, 65 people suffer from their first seizure each day and about 250,000 people take anti-epileptic drugs such as phenytoin or carbamezepine. Furthermore seizures are resistant to treatment with currently available anticonvulsant drugs about 1 out of every 3 patients treated for epilepsy continue to have seizures despite drug treatment such patients may suffer from both the seizures themselves and the side effects of the antiepileptic drug treatment.

More recent studies suggest that newer drugs like lamotrigine, vigabatrin, gabapentin and topiramate have a place in the treatment of refractory patients. Nevertheless, only 17% of such patients became seizure free following the addition of these molecules to existing ineffective drug drug regimes. These recently developed drugs may have benefits in terms of side effects and tolerability (older molecules were noted for sedation, confusion, metabolic toxicity and rare but severe blood dyscrasia?^ but the more effective molecules, e.g. lamotrigine and topimarate, still produce t mal and other problems.

It is known that most effective antiepileptic drugs either enhance inhibitory neurotransmission (by binding to allosteric sites on the GABA_A complex or altering GABA turnover and re-uptake at inhibitory terminals) or they block paroxysmal firing (the cellular hallmark of epilepsy) by binding in a voltage-, frequency- [and state-] dependent manner to voltage-gated Na⁺ and or Ca^ channels. Historically, drug designers have recognised that "maximal electroshock" tests primarily identify drugs acting on sodium channels and thus on high frequency repetitive firing. Oleamide (cis- 9,10-Octadecenamide) is the primary amide of oleic acid and is a putative sleep hormone. Oleamide was originally isolated from sleep-deprived cats and an enzyme specific for its hydrolysis is known to be present in rat brain (Cravatt et al 1995, Science 268:1506-9). Synthetic oleamide has been found to induce physiological sleep when injected into rats but little is known about its cellular mode of action. High-affinity interactions with recombinant 5HT receptors have been reported (Huidobro-Toro and Harris, 1996, Proc Natl Acad Sci USA, 93:8078-8082), but others conclude that the hypnogenic effects are probably not secondary to activation of G-proteins (Boring et al, 1996, Prostaglandins, Leukotrienes and Essential Fatty-acids, 55(3): 207-210). More recent studies demonstrate that oleamide may bind to cannabinoid sites (IC₅₀ of lOμM) in rodent CNS, that it induces hypolocomotion in vivo which is sensitive to the CB1 antagonist SRI 41716A but that the brain lipid cannot elicit the same second messenger responses as acknowledged CB1 cannabinoid agonists (Cheer et al, 1999, Neuropharmacology, 38, 533-541). At high μM concentrations cw-oleamide can deconvolute gap junctions in glia (Guan et al, 1997, J. Cell Biology, 139, 1785-1792). To date, none of these putative receptor interactions have been shown to contribute to a net depressant effect for oleamide at a systems level and biologically active concentrations of oleamide in CSF are uncertain due to the apolar nature of the molecule and the presence of fatty acid amide hydrolases (FAAH) enzymes.

However, sedation is an undesirable feature of current treatments for epilepsy. Thus the reported hypnogenic effects are clearly a potential problem. Oleamide is structurally and physicochemically similar to endcannabinoids: both classes of molecule are cleaved in vivo by fatty acid amide hydrolases. Some fatty acid amide hydrolases inhibitors have been shown to be hypnogenic. US Patent No. 5,856,537, Lerner et al, describes such enzyme inhibitors. Lerner implies that some of the amide hydrolase inhibitors may concurrently agonise the "oleamide receptor" as well as blocking the enzyme (although this is ambiguous as the authors have cited both 5HT]_/2 receptors and gap junctions as CNS targets). Blocking the breakdown of endogenous fatty acid amides mimics the hypnogenic effects of administration of synthetic oleamide (Boger et al, 1998, Current Pharmaceutical Design, 4, 303-314). By analogy with drugs targeting GABAergic transmission it is well known that chronic therapy with direct receptor modulators can lead to tolerance and reduced anticonvulsant efficacy (e.g. diazepam is used but only acutely in status epilepticus). This is in contrast to novel drugs which target GABA turnover via GABA- transaminase and GABA re-uptake which are licensed as chronic therapies.

Synthetic depressant drugs are acknowledged as stereoselective allosteric modulators of ion channel targets like the GABA_A receptor (a crucial/widespread inhibitory channel) and the voltage-gated Na⁺ channel (important for generating action potentials in excitable membranes) (reviewed by Lees, 1998, Br. J. of Anaesthesia, 81, (4), 491-493). We have examined the potential of oleamide isomers to modulate these key signalling molecules in an attempt to further delineate molecular mode of action and to identify targets contributing to the stereo selectivity of the oleamides in vivo, where the cis isomer is demonstrably more potent (Cravatt et al, 1995, Science, 268, 1506-9).

Oleamide has been reported to induce physiological sleep when injected into laboratory rats. Also, since oleamide accumulates in the cerebrospinal fluid (CSF) of sleep-derived cats it may represent a novel signalling molecule. We have therefore assessed the cellular mode of action of oleamide in vitro.

Thus oleamide may represent an endogenous ligand for allosteric modulatory sites on isoforms of GABA_A receptors which are crucial for the regulation of arousal and have recently been implicated in the circadian control of physiological sleep (Wagner et al, 1997, Nature, 387, 598-603).

The basis for the rationale is, inter alia,

• In all rat cultured cortical neurones (pyramidal cells) examined, the synthetic brain lipid (3.2-64 μM) enhanced the responses to subsaturating GABA concentrations (up to circa 2x) in a concentration-dependent manner (EC ₅₀, circa 15 μM).

• In the absence of GABA, cw-oleamide directly evoked small inhibitory currents in a subpopulation (<7%) of sensitive cells.

• 20 μM s-oleamide reversibly enhanced the duration of spontaneous inhibitory post synaptic currents (circa 2 fold) without significantly altering their amplitude.

• At 32-64 μM, cw-oleamide reversibly reduced the incidence and amplitude of both inhibitory post synaptic currents (i.p.s.cs) and excitatory post synaptic currents (e.p.s.cs) in the cultured neuronal circuits in common with other depressant drugs acting at the GABA_A receptor.

• 32 μM Oleic acid did not modulate exogenous GABA currents or synaptic activity suggesting that cw-oleamide's actions are mediated through a specific receptor.

• A specific, protein-dependent interaction with GABA_A receptors was confirmed in Xenopus oocytes. Recombinant human receptors were modulated by 10 μM s-oleamide (and diazepam) only when a γ subunit was co-expressed with ctiβ?: the cw-oleamide response was not sensitive to the specific benzodiazepine antagonist flumazenil (1 μM).

We have shown that oleamide stereoselectively inhibits sustained repetitive firing (paroxysmal burst firing is the hallmark of epilepsy in single cells) in primary neural cells. The molecule was inactive as a blocker of unitary action potentials at concentrations as high as 64 micromolar. This selective burst suppression is the hallmark of several clinically useful anticonvulsants and we hypothesised that, like the specified anticonvulsant drugs, cis oleamide was producing this effect by interacting with the voltage-gated sodium channels. We have subsequently confirmed this by isolating sodium currents in voltage-clamped neuroblastoma.

We would expect that synthetic fatty acid amides, which are amido hydrolase inhibitors and which block degradation of endogenous oleamide, would mimic this action.

Thus, accordingly we provide a method of treating a convulsant disorder which comprises altering the levels of oleamide in the brain of a patient suffering from such a disorder.

Thus the levels of oleamide may be altered either by the direct exogenous administration of oleamide, e.g. cis-oleamide, to a patient. Alternatively, levels of oleamide may be altered by administration of an amido hydrolase inhibitor which will act to inhibit the amido hydrolase enzymes. The amido hydrolase enzymes are degradative enzymes which enzymes act to reduce levels of endogenous oleamide and thus the administration of an amido hydrolase inhibitor will act to prevent the degradation and depletion of oleamide levels. Such amido hydrolase inhibitors are generally fatty acid amides.

Therefore, one feature of the invention comprises a method of treating a convulsant disorder which comprises administering a therapeutically effective amount of oleamide to a patient suffering from such a disorder.

In an alternative feature of the invention we provide a method of treating a convulsant disorder which comprises administering a therapeutically effective amount of an amido hydrolase inhibitor to a patient suffering from such a disorder.

The method of the invention is especially advantageous in the treatment or alleviation of epilepsy. According to a further feature of the invention we provide the use of oleamide in the manufacture of an anticonvulsant medicament.

Thus according to a yet further feature of the invention we provide the use of an amido hydrolase inhibitor, such as a fatty acid amide, in the manufacture of an anticonvulsant medicament.

Molecules with the capacity to block burst firing selectively (i.e. those which produce a state-dependent, or voltage-dependent block of voltage-gated Na⁺ channels as demonstrated here) have a manageable therapeutic ratio in clinical use because they selectively target diseased tissue which is ischaemic or ectopic (e.g. cardiac arrhythmias ) or tissues exhibiting paroxysmal high frequency bursts (as in epilepsy or pain). Local anaesthetics, anticonvulsants and Class 1 anti-arryhthmics can all be safely used because they do not impair firing patterns in physiologically normal cells or are restricted to a localised site of action (local anaesthetics are injected directly onto nerve trunks often with a pressor agent to restrict systemic distribution). Interestingly, such molecules are finding new applications which are important in both clinical and commercial terms. For example the anticonvulsant carbamezepine is used for intractable pain states or neuropathic pain which responds poorly to existing analgesics like NSAIDS.

High frequency bursting causes the release of glutamate which is excitotoxic in CNS. This leads to conditions of hyperalgesia. Lamotrigine, a glutamate release inhibitor which blocks bursting and exerts a state-dependent effect on Na⁺ channels, is currently in human trials for post-operative pain. Excessive glutamate release is triggered in stroke by a similar mechanisms (anoxic cells fire rapidly and release the excitotoxic 1-glutamate onto neighbouring cells causing an outwardly spreading penumbra of cell loss sometimes known as a "stroke in evolution"). Molecules like BW619C89, a close structural congener of lamotrigine with a very similar mode of action is neuroprotective in animal models for stroke and has reached Phase 3 in human clinical trials for acute thrombotic stroke. Other candidate drugs which inhibit glutamate release by blocking Na^"*" channels in this way include lifarizine and riluzole (already licensed as a neuroprotective agent in motorneurone disease or ALS). Both are in stroke trials.

It is hoped that by blocking glutamate release presynaptically via the Na⁺ chaimel will result in fewer side effects than glutamate receptor antagonists (e.g. nightmares, hallucinations, CNS vacuolation and cardiovascular pitfalls).

In addition to being useful in the treatment of epilepsy, the medicament of the invention may also be useful in the treatment or alleviation of CNS disorders, pain and ischaemia.

Thus, when oleamide itself is used in the manufacture of the medicament of the invention or the method of the invention, cw-oleamide is preferred.

When the medicament of the invention comprises an oleamide hydrolase inhibitor we prefer the oleamide hydrolase inhibitors described in US Patent No 5,856,537. Such oleamide hydrolase inhibitors comprise a head group and a hydrocarbon tail covalently linked to said head group, wherein said head group includes an electrophilic carbonyl and is selected from a group consisting of radicals represented by the following structures:

and wherein said hydrocarbon tail is selected from a group consisting of radicals represented by the following structures:

Specific amido hydrolase inhibitors may be mentioned, such amido hydrolase inhibitors are those selected from the group: l-chloro-10Z-nonadecen-2-one, 8Z- heptadecenal, 2-oxo-9Z-octadecenamide, 2-oxo-10Z-nonadecenamide, ethyl 2- oxo-9Z-octadecenoate, ethyl 2-oxo-9Z-octadecenoate, ethyl 2-oxo-10Z- nonadecenoate, ethyl 2-oxo-nonadecanoate, tert-butyl 3-oxo-2, 2- dihydroxyoctadecanoate, 1,1,1-trifluoro-lOZ nonadecon-2-one, l,l,l-trifluoro-9Z- octadecen-2-one, 1,1,1 -trifluoro- 1 OE-nonadecen-2-one, 2-hydroxy-9Z- octadecenamide, 2-chloro-9Z-octadecenamide, l-diazo-10Z-nonadecen-2-one, N- amino-9Z-octadecenamide, methyl 1 OZ-nonadecenoate, 1 OZ-nonadecenoic acid, 2- hydroxy-lOZ-nonadecenoic acid, 2-hydroxy-lOZ-nonadecenamide, 8Z- heptadecenoic, 3-Oxo-2-(triphenylphosphoranylidene) octadecanoate.

Such compounds may be prepared using the methods described in US Patent No. 5,856,537.

Synthetic molecules enhance the titre of oleamide in CNS and so will exert indirect therapeutic actions. We have found that 20μM c/s-oleamide significantly enhances the affinity of exogenous GABA for its receptor without changing Hill slope or the maximal response. These effects were not voltage-dependent or secondary to shifts in E_d- We have also found that 20μM of c/s-oleamide reversibly enhanced the duration of spontaneous inhibitory post synaptic currents (circa 2 fold) without significantly altering their amplitude. At 32-64 μM cw-oleamide reversibly reduced the incidence and amplitude of both inhibitory post synaptic currents (IPSCs) and excitatory post synaptic currents (EPSCs) in the cultured neuronal circuits in common with other depressant drugs acting at the GABA_A receptor. Also 32 μM oleic acid did not modulate exogenous GABA currents or synaptic activity suggesting that oleamide 's actions are receptor mediated.

We especially provide a method of treatment of a patient suffering from epilepsy which comprises administering an amount of an amido hydrolase inhibitor as hereinbefore described of from 1 nM to 13 μM.

Alternatively, we provide a method of treatment of a patient suffering from epilepsy which comprises administering an amount of cis oleamide sufficient to provide a cerebrospinal fluid concentration of oleamide of from 3.2 to 64 μM, preferably 5 to 20 μM and especially 20 μM.

We further provide a formulation of an anticonvulsant medicament which comprises a therapeutically effective amount of an amido hydrolase inhibitor in combination with a pharmaceutically acceptable adjuvant, diluent or carrier.

The preferred amido hydrolase inhibitors in the formulation of the invention are selected from those described in US Patent No 5,856,537.

We also provide a formulation of an anticonvulsant medicament which comprises a therapeutically effective amount of oleamide in combination with a pharmaceutically acceptable adjuvant, diluent or carrier.

The invention will now be illustrated by way of example only. MATERIALS AND METHODS

Cell-culture

Neuronal cultures were prepared from cerebral cortices of 17-18 day old rat embryos (Lees & Leach, 1993). Cells were plated onto poly-D-lysine coated coverslips (200,000 cells.ml^"1) in Dulbecco's Modified Eagle Medium supplemented with 10% foetal calf serum and 100 u its/μg.ml^"1 penicillin/streptomycin. After 12-24 hr the plating medium was replaced by a maintenance medium comprising, Neurobasal medium, with 2% B27 supplement, 1% glutamax (Gibco, Paisley, UK) and 100 units/μg.ml^"1 penicillin/streptomycin. Cells were used in experiments after 14-28 days in vitro.

Chemical synthesis and suppliers

Details of the synthesis and purification of cw-oleamide have been published (Lees et al, 1998, Br. J. Pharmacology, 124, 873-882). tOA: (e)-9-Octadecenoic acid amide was synthesised as described in J Amer.Chem. Soc., 11, pp 2215-2218 (1949). White crystalline sold was recrystallised from ethanol and its melting point of 92-92.5°C was consistent with literature values. Unless otherwise stated, all chemicals and drugs were obtained from Sigma Aldrich Chemical Co. (Poole, Dorset, UK) or (Merck Ltd, Poole, Dorset, UK).

Electrophysiology

GABA_A currents and spontaneous synaptic activity: cultured neurones on polylysine coated coverslips were placed in a 5mm Perspex trench on the stage of an inverted microscope. The extracellular saline contained (mM): NaCl (142), KC1(5), CaCl (2), MgCl₂ (2), Hepes (5), D-glucose (10), pH 7.4. Whole-cell pipette saline contained (mM): K-gluconate (142), CaCl₂ (1), MgCl₂ (2), Hepes (10), EGTA (11), pH 7.4. All potentials cited are those based on the preamplifier null potential and take no account of the liquid junction offset (circa +14mV by direct measurement) inherent in the use of these asymmetrical solutions. Borosilicate patch pipettes (3-5 MΩ) were used. 60-80% series resistance compensation was applied at the List EP7 pre-amplifier. Whole cell currents were filtered at 1-3 KHz prior to digitisation and monitored on a Gould chart recorder or analysed using WCP (John Dempster, Strathclyde University) or Spike 2 software (Cambridge Electronic Design, Cambridge, U.K).

Isolation of Na⁺ spikes or SRF: pyramidal cells were whole cell clamped in a physiological saline consisting of (mM): NaCl (142), KC1 (2.5), CaCl₂ (1), MgCl₂ (2), CoCl (2), Hepes (5), D-glucose (10), pH 7.4. The gluconate based pipette solution given above was used. Some data were obtained in amphotericin perforated mode (240 μg/ml in above pipette solution; 0.4% DMSO) which did not qualitatively alter modulatory effects. 3-10 MΩ pipettes were fabricated on a Mecanex BBCH puller. An Axoclamp 2B pre-amp was used in bridge mode to monitor cellular excitability and current pulses were delivered from a Grass S66 stimulator (see text for details of stimulus protocols). Cells were held at their resting membrane potential (only those with E_m more negative than -50 mV were selected for study). Analogue data were filtered at 1 KHz and digitised (CED 1401 plus) for analysis using Win WCP software (courtesy of John Dempster, University of Strathclyde).

Pharmacology

C/s-oleamide was dissolved in dimethylsulfoxide (DMSO) then diluted lOOOx into saline: 0.1% DMSO produced no effect on the parameters reported here and was present in pre- and post-treatment phases of all experiments. To further facilitate dissolution of the modulator, all extracellular salines were supplemented with 0.1% bovine serum albumin (fraction V, Sigma) which was present during all phases of the reported experiments. Cw-oleamide was formulated daily and perfused from glass reservoirs via teflon lines. GABA was rapidly (10-90% rise times <50 ms) and quantitatively delivery to cultured cells using the Y-tube technique (Murase et al, 1989, 1989, Neuroscience Letters, 103, 53-63) either alone or with modulatory drugs (previously equilibrated with the cell by superfusion). Log concentration-response curves were fit to the Hill equation by non-linear regression (Graphpad Prism software, San Diego, CA.). Logarithmic values of EC₅₀ and associated standard error were used for statistical comparisons. Student's t-tests (two-tailed throughout, and paired where appropriate) or ANOVA were used as indicated in the text. Significant effects are indicated by p<0.05 throughout. Data are expressed as mean ± standard error (s.e.) of the mean.

RESULTS

Stereoselective modulation of GABAA receptors and reversible depression of spontaneous synaptic activity in cultured cortical neurones

As we have previously reported, c/s-oleamide (3.2-64 μM) has the capacity to positively modulate GABA_A receptors in whole cell patch-clamped cultured rat neurones and human cloned GABA_A isoforms (Lees et al, 1988, Br. J. Pharmacology, 124, 873-882). Here, in rat neocortical pyramidal cells, 20 μM cis- oleamide reversibly enhanced the amplitude of current evoked by Y-tube application of 3.2 μM GABA, reaching peak steady state values between 8-14 minutes (Fig. la). This elevation was significant (Fig. lc: paired student's test, n = 5, P = 0.002). In contrast, the trans isomer did not significantly modulate GABA-evoked current over a period of 14 min (Fig. lb and lc: n = 5; P - 0.41). Modulation of the evoked GABA current by 20 μM cw-oleamide was invariably accompanied by a progressive decrease in the frequency of both IPSCs and EPSCs (Fig. la). The reduced incidence of post-synaptic currents was significant after a 10 minute exposure (IPSCs, P = 0.044, n = 7; EPSCs, P = 0.037, n = l; Fig. Id). However, tOA (Fig. lb and Id) had no effect on the rates of incidence of either inhibitory or excitatory synaptic currents (P = 0.22 and 0.3 respectively, n = 4).

Selective Depression of Sustained Repetitive Firing

The functional significance of these biochemical data was examined in patch clamped cortical neurones in culture. Pyramidal cells were current-clamped at their resting membrane potential, and unitary action potentials were generated by the application of current pulses of 2 ms duration at a frequency of 1 Hz, and of sufficient amplitude to induce all-or-none (TTX-sensitive) events. For analysis, the waveforms of 50 consecutive action potentials were averaged and the parameters of amplitude, overshoot, rise-time, time to 50% depolarisation and resting membrane potential were measured pre- and post-treatment. Cz^'s-oleamide had no significant effect on any of the parameters measured from the TTX-sensitive low frequency evoked spikes at either 20 μM (n = 4) or 64 μM (n = 7) (Fig. 6a and 6b). In contrast, cw-oleamide (cOA) strongly depressed sustained repetitive firing (SRF) elicited by protracted current passage (500 ms duration, 0.1 Hz) through the recording microelectrode (Fig. 6c). At all concentrations tested, c/s-oleamide had no effect on the initial action potential elicited by the depolarising current pulse. However, SRF (defined here as the train of potentials occurring subsequent to this primary action potential) was inhibited in a concentration-dependent manner (Fig. 6d). 64 μM cis- oleamide completely and rapidly abolished SRF (4/4) (Fig. 6c), with an approximate time to block 50% of pre-treatment response (t₅o) of 39 ± 3 seconds (n = 4). 10 μM cw-oleamide was found to abolish SRF in some cases (4/10) but this occurred over a longer time course (t₅₀ = 174 ± 49 seconds). The extent of the steady-state block at intermediate doses was reduced by hyperpolarising the impaled cell (not shown) indicating a voltage-dependent suppression of burst firing. Collation of data interpolated from dose responses in individual replicates yielded an apparent EC₅₀ of 4.39 ± 0.47 μM (n = 5). Data pooled from all experiments were fitted by non-linear regression to a sigmoid curve with an EC₅₀ of 4.12 μM ± 0.024 (mean ± log s.e.) and a Hill slope of 2.9 (95% C.I. 2.1-3.6) (Fig. 6d). Both oleic acid (32 μM) and trans- oleamide (20 μM) were very weak antagonists of SRF (Fig. 6e).

VOLTAGE CLAMP Methods

Neuroblastoma cell culture

Murine NIE115 neuroblastoma cells were kindly supplied by Dr D E Ray, MRC Toxicology Unit, University of Leicester UK. Confluent cells were subcultured twice weekly and grown on small glass coverslips in Dulbecco's modified Eagle Medium (containing Glutamax- 1 : Gibco, Paisley, Renfrewshire, U.K.), supplemented with 10%o foetal calf serum and 50 μg/ml gentamicin. Cells were incubated at 37°C in 5% CO₂ in triple vented 35mm culture dishes. In some experiments 2%> DMSO was added to the growth medium which increased Na⁺ current density in the clamped somata but did not qualitatively alter oleamide sensitivity. Cells were selected for electrophysiological experiments 24-36 hours after plating.

Voltage-clamp Electrophysiology

Cells adhering to glass coverslips were transferred to a Perspex recording chamber

rigidly mounted on the stage of a Nikon diaphot TMD inverted microscope. Cells

were continually superfused with saline containing (mM) 140 NaCl, 5 KC1, 1.8

CaCl₂, 0.8 MgCl₂, 10 HEPES, pH 7.3 and supplemented with 0.1% DMSO and 0.1% BSA. TTX and cOA were applied quantitatively in this superfusing solution (flow

rate c5ml/min; bath volume <0.5ml). The whole cell patch-clamp technique was used to voltage clamp the cells with a single micro-electrode. A Burleigh

(Harpenden, Herts, UK) PCS-5000 micromanipulator was used. 3-4 MΩ pipettes

were fabricated from thin-walled borosilicate glass (1.6mm OD; Hilgenberg-GMBH, Malsfeld, Germany) on a Mecanex BBCH electrode puller. The pipette solution

consisted of (mM) lO NaCl, 20 TEA-C1, 110 CsCl, 2 MgCl₂, 1 CaCl₂, 11 EGTA, 10

HEPES pH 7.4. To reduce stray capacitance, the tips of the electrodes were silanised

by immersion in "Sigmacote" (Sigma, Poole, Dorset UK) after backfilling. Only

spherical and unclumped cells were used in the study and 75-90%) series resistance

compensation was applied directly from the Axopatch 200 pre-amplifier (Axon Instruments Ltd, San Diego, USA). Because I/V curves for the sodium channel shift to more negative potentials in the first few minutes after beginning cell dialysis, at least 6 minutes was allowed after membrane rupture before taking data. Analogue

data were filtered at 5 KHz and were digitised at 20-40 KHz using a "CED1401 plus" laboratory interface for analysis using patch and voltage-clamp software (CED,

Cambridge, UK). Leak currents and residual capacitive artefacts were deducted off-

line, in software. Details of, and rationale for, pulse protocols are given in the results

text or associated figure legends. All electrophysiological experiments were

conducted at room temperature (20-24° C). Conductance (g) through Na⁺ channels

was calculated by dividing the peak Na⁺ current by the driving force (V-Er) where Er is the observed reversal potential in the clamped cell. Relative conductance was fitted to a Boltzmann function of the following form:-

g i g max l + exp[(Vϊ/2 - V)/ k]

where g _max is the peak conductance, Vι/₂ is the voltage at which half-maximal conductance occurs, k is the slope factor and V is the command voltage.

Statistical analysis In binding studies the IC₅₀ (inhibitor concentration which displaces 50% of specifically bound radioligand) of cOA was calculated using the probit method.

Effects of cOA on equilibrium binding parameters (Ki and B_max) of [³H]BTX-A were

determined. Least squares regression analysis was used to fit lines to data as

appropriate. Quantitative electrophysiological data were analysed using Microsoft

Excel 97 and GraphPad Prism 3 software (San Diego, CA, USA), and are cited as

mean ± S.E.M (n). P values were generated using Student's paired t test. Voltage-Clamp Electrophysiology RESULTS/DATA

All patch clamped cells were found to express fast inward currents in response to

step depolarisation. From a holding potential of -80 or -lOOmV, 0.1-1 μM TTX

produced complete block of evoked whole cell inward currents (n = 6) as previously

reported for this cell line in the ionic environment used. Preliminary experiments

showed that oleamide exerted a reversible block of peak Na⁺ currents (evoked at

0.1Hz) but that the onset and recovery kinetics were relatively slow, confirming

earlier observations in neural membranes (Fig. 7). Subsequently, 12-15 minute

incubations were used, following collection of control data, to ensure equilibration of

the lipid amide with cell membranes and its presumptive receptor site. Under these

conditions, cOA caused tonic inhibition of Na⁺ currents in response to a voltage step to 0 mV (0.5 Hz) in a voltage-dependent manner. 10 μM cOA resulted in a 40 ± 9%>

reduction in peak current at V = -60 mV, compared with 16 ± 5%> at V_h = -100 mV (n = 4; P < 0.05, Fig 7). The fractional block was concentration-dependent at the two

holding potentials used (Fig. 7c) and curves appeared to saturate at 64 μM. At negative holding potentials the curve was displaced to the right (log of concentration

required to block 20% of the peak current was raised significantly, P <0.01, n = 4)

raising the EC₂o from circa 2 μM at -60mV to circa 10.5 μM at -lOOmV. The maximal cOA fractional block was significantly enhanced at depolarised holding

potentials (maximal block at -100 mV, 40.8 ± 0.02 %: at -60 mV, 81.1 ± 0.02 %, n =

7, P < 0.0001).

3.2-64 μM cOA inhibited Na⁺ currents in a manner which was largely

independent of activation voltage (Fig. 8). No significant shifts in E_rev for the peak evoked currents were detected even at high cOA concentrations (P > 0.5: n = 4 at 10 μM and n= 6 at 32 μM). Conductance-voltage curves were virtually

superimposable before and after application of cOA (order of administration was randomised in these experiments) as shown in Fig 8. Compounding data from

replicated experiments confirmed that V50 values were not significantly altered by the

lipid amide even at the relatively high concentration of 32 μM (P > 0.05, n = 1 1).

Steady-state inactivation curves (prepulse of 90 ms between -120 mV and -20 mV prior to a test pulse to +10 mV) were fit to a single Boltzmann and showed a

concentration-dependent hyperpolarising shift in the presence of cOA (Fig. 9). The

maximum value of this shift in the V50 for inactivation was -15.4 ± 0.9 mV at 32 μM

cOA (n = 6;). At concentrations > 3.2 μM the negative shifts were significant (P <

0.05) and concentration-dependent. The magnitude of this shift was fitted to the Hill equation yielding an apparent EC₅0 of 11.6 μM (± log SEM of 0.057), a slope of 2.8 ± 1.0 (17 degrees of freedom) and a tendency to saturate at 64 μM which was

the highest concentration we could formulate at room temperature (Fig. 9). Recovery

from inactivation was studied by eliciting current with a 10 ms test pulse to 0 mV

from V_h = -100 mV with increasing time intervals following a 100 ms prepulse

between -100 and 0 mV (Fig 10). Normalised current data were plotted against recovery interval and fit to a single exponential for each cell examined, confirming

that 10 μM cOA slowed recovery from inactivation, with mean τ increasing from 3.7

± 0.4 ms to 6.4 ± 0.5 ms (n - 6; P < 0.001). Compounded data for recovery from inactivation are shown in Fig. 10b. In control physiological salines the peak Na⁺ current in these cells did not decrement significantly during a train of 50 ms stimulus

pulses to evoke maximal currents at 0.5-10Hz (not shown). Under these conditions, 10-32 μM cOA did not exhibit detectable frequency-dependent facilitation of block

(Fig 1 1) at frequencies up to 10 Hz (P> 0.1, n = 5 at 10 μM). Visual inspection of data at other concentrations revealed no frequency dependent fade but this data was

not subjected to statistical analysis.

Figure Legends

Figure 1 GABAA currents and spontaneous synaptic traffic are selectively modulated by the cis-isomcr of oleamide. a. Superfusion of 20μM cw-oleamide (black bar) to a neurone reversibly enhanced the effects of 4s pulses of exogenous GABA at 3.2 μM (indicated by black dots and shown at higher resolution in the traces below). Note the slow onset kinetics and the concurrent reduction in the incidence (and graded reduction in amplitude) of spontaneous synaptic currents. b. Identical experiment with trα -oleamide giving only a marginal response in contrast to the initial phases of cw-oleamide superfusion. c. Histogram (mean + s.e. mean) depicting compounded data from replicated experiments with exogenous GABA (ns, not significant; **P < 0.01). d. Histograms (mean + s.e. mean) depicting steady-state effects of oleamide isomers on the incidence of spontaneous synaptic currents (event detection by Spike

2 software with a bin width of 2 mins). Ns, not significant; * P < 0.05.

Figure 2 -oleamide, but not oleic acid, modulated responses to exogenous GABA. (a) Responses to 10 μM GABA expelled from a Y-tube for 5 s onto a neurone whole-cell clamped at -45mV. 20 μM cw-oleamide (horizontal bar) reversibly enhanced peak outward current. Note that the modulatory action took 10-12 min to attain steady-state: all subsequent data reported at equilibrium, (b) Compounded data from 5 cells (exposed to 500 ms-5 s pulses of 10 μM GABA, V_h -45mV) showing the peak GABA evoked-response in control saline or in the presence of 20 μM c s-oleamide. Data were analysed by repeated measures ANOVA: each column was compared with control data using Dunnett's po t-test (see Motulsky, 1995; **P<0.01. (c) Responses from a single cell, clamped at -45 mV, to 3.2 μM GABA (200 ms pulses) in control saline (left) and after 10 min exposure to 32 μM oleic acid (centre); and 32 μM cw-oleamide (right), (d) Data on peak responses from 5 identical experiments. The results were analysed by repeated measures ANOVA with Dunnett's post test (*P<0.05). Oleic acid did not significantly alter the amplitude of the peak GABA-evoked current.

Figure 3 The actions of cw-oleamide were concentration-dependent and saturable: the affinity of GABA for its receptor was enhanced by the brain lipid. (a) Discontinuous chart records of responses to repeated challenge with 400 ms pulses of 3.2 μM GABA in the presence of the indicated concentration of cw-oleamide (steady- state response). The cell was clamped at -45mV throughout. Note the different time bases and the marked reversible depressant effect of the higher concentrations on spontaneous multiquantal synaptic responses. Expanded data on left of each sweep: compressed data on right, (b) Data on peak currents (mean ± s.e. of mean) from the concentration-response experiments on cells clamped at -30 to -45 mV (numbers in parentheses denote the number of cells examined), (c) Normalised concentration- response curves to GABA in the absence and presence of 20 μM cw-oleamide (superfused for 10-12 min) in cells clamped at -30 mV. Data points represent mean and vertical lines ± s.e. mean (n ≥ 5 per point). The inset shows a response to a high concentration of GABA before, during and after equilibration with ct.y-oleam.ide: at 20 μM cw-oleamide had no significant effect on the maximal response to GABA.

Figure 4 cw-oleamide modulated spontaneous inhibitory synaptic currents. Inhibitory currents (upward deflections): excitatory currents (downward), (b) cis- oleamide 20 μM (bar) marginally reduced the frequency of synaptic currents. The apparent increase in i.p.s.c. amplitude can be attributed to the limited frequency response of the chart recorder and prolongation of the i.p.s.c. by cw-oleamide (below), (c) Average of 100 inhibitory synaptic currents (aligned at the mid-point of the rising phase) before and during (*) exposure to 20 μM cw-oleamide superimposed on bi-exponential fits to the decaying currents. Inset: traces superimposed with the response after washout demonstrated reversibility. In this and one other cell cw-oleamide selectively prolonged the slow component of decay (time constants were 5.8 ± 0.4 ms and 27.0 ± 0.7 ms pre-treatment: 4.4 ± 0.3 ms and 56.4 ± 1.2 ms). In the other replicates both τfast and τ_sι_ow were enhanced. The cells were exposed to cw-oleamide for at least 10 min before sampling data for kinetic analysis, (d) Compounded data from 8 replicated experiments; means ± s.w. mean are shown.

Figure 5 The actions of cis-oleamide on recombinant receptors in oocytes were subunit selective. All cells were clamped at -35 mV. Membrane conductance in response to brief hyperpolarizing steps (details in Methods) was used to quantify the evoked responses, (a) Representative effects of 10 μM cw-oleamide (left) and 333 nM diazepam (DZP, right) on the response to a fixed concentration of GABA (circa the EC ₀ for the indicated subunit combination). Note that both molecules markedly enhanced the evoked response with ιβ₂γ₂L receptors but that only DZP was antagonised by 1 μM FLUMAZENIL (Flum). (b) In identical experiments with oc]β receptors, DZP produced no significant effect. Both currents and conductance changes (difference between dense black bars at the top and bottom of each trace) evoked by GABA were marginally depressed by rø-oleamide. (c) Compounded data on conductance changes (normalised to pre-treatment responses) from a total of

5 cells for each modulator . Neither the positive- or negative-modulatory effects of -oleamide were significantly attenuated by the benzodiazepine antagonist.

Figure 6 cw-Oleamide is a specific state-dependent antagonist of the voltage-gated Na⁺ channel.

A. Spikes evoked by current passage through the patch micro-electrode (at 1 Hz, averages of 50 consecutive sweeps) were not altered by 64 μM czs-oleamide but were fully blocked by 50 nM TTX.

B. Compounded data from 7 replicated experiments on unitary action potentials. Bars represent mean + s.e. mean. C. In contrast cw-oleamide (10-64 μM) markedly suppressed SRF in salines containing Co⁺⁺ to suppress voltage-gated Ca^+_ currents.

D. Block of SRF was concentration-and voltage-dependent (the blocking action could be partially reversed by hyperpolarisation of the impaled cell, not shown). Each data point represents mean ± s.e. mean for the indicated number of replicates. Note the very steep slope (Hill coefficient of approximately 3).

E. The profound high affinity block produced by cz^'s-oleamide was not seen with high concentrations of hydrophobic congeners.

Figure 7 (A) Currents evoked brief voltage steps to OmV from the indicated holding potentials were differentially sensitive to the blocking action of 10 μM cOA (upper trace on superimposed sweep) under equilibrium conditions. Each trace is the average of 5 sweeps applied at 0.5Hz. (B) The onset of action and reversal of the cOA block exhibited slow kinetics. cOA application is indicated by the horizontal bar. Data depict the mean responses (± SEM) derived from 3 replicated experiments. (C) The effects of cOA were concentration dependent and appeared to approach saturation at 64 μM. Tonic block was clearly influenced by holding potential: the substantial shift in EC₂o for the two curves and the difference in maximal fractional block for the two holding potentials were both highly significant as indicated in text. Figures in brackets indicate number of cells examined.

Figure 8. (A) Families of peak current responses to a 10 ms depolarising step of increasing magnitude, from the same cell, before and after equilibration with 32 μM cOA. Details of voltage protocols are given below. (B) Cells were clamped at - 75mV then hyperpolarised to -115 mV to completely remove inactivation prior to the 10 ms test pulse. Test pulses were applied between -75 and + 120 mV using 15 mV increments. Twin pulses were repeated at 0.33Hz. (C) Current-voltage plot for the data depicted above demonstrates that the currents reverse close to the Nernstian Na⁺ equilibrium potential under these conditions (+ 68mV in our salines), that both inward and outward currents were antagonised and that cOA does not alter the measured reversal potential. No linear trend was observed when comparing extent of block to test potential. (D) Normalised conductance- voltage plots (derived from a different cell) were virtually superimposable even at these relatively high concentrations suggesting the block did not reflect a shift in voltage-dependence of activation gating (the mid point of the curves, V₅₀ ,was not significantly altered in replicated experiments).

Figure 9. (A) Steady-state inactivation curves before and after equilibration with 32 μM cOA from cells exposed to the indicated voltage protocol (right). Prepulse duration was 90 ms (15 mV increments) and the 10 ms test pulse was to +10 mV. The incrementing protocol was repeated every 2s. After cOA treatment for 15 min, the mid point for the curve was shifted by circa 15 mV in the hyperpolarising direction for this group of cells. (B) The size of the shift in V₅₀ was concentration dependent and saturated at 32 μM. Data were fitted to the Hill equation with two variable parameters (zero and observed maximum response were designated as constant minimal and maximal values, respectively). Derived parameters for EC₅₀ , Hillslope and their associated errors are given in text.

Figure 10. (A). Twin pulse protocol (applied every 4s) used to study recovery from inactivation. As the interpulse interval t was increased (1ms increments), evoked current amplitudes were proportionately increased (superimposed current sweeps from a control cell are depicted lower right). (B) The recovery from inactivation was well fitted by a single exponential and cOA prolonged tau for this process (values given in text).

Figure 11. (A) lOOmV step depolarisations were applied as a train with the characteristics described in the top component of the figure. In the lower traces the first and last events in the train are virtually superimposable even in the presence of 10 μM cO A. (B) Compounded data from 5 replicated experiments confirm that no frequency-dependent cumulative block is apparent in the presence of cOA.

Claims

1. A method of treating a convulsant disorder which comprises altering the levels of oleamide in the brain of a patient suffering from such a disorder.

2. A method according to claim 1 characterised in that the level of oleamide is altered by administering a therapeutically effective amount of an amido hydrolase inhibitor.

3. A method according to claim 1 characterised in that the level of oleamide is altered by administering a therapeutically effective amount of oleamide.

4. A method of treatment according to claim 2 which comprises administering an amount of an amido hydrolase inhibitor of from 13 μm to INm.

5. A method according to claim 2 characterised in that the amido hydrolase inhibitor is selected from the group; l-chloro-10Z-nonadecen-2-one, 8Z- heptadecenal, 2-oxo-9Z-octadecenamide, 2-oxo-10Z-nonadecenamide, ethyl 2- oxo-9Z-octadecenoate, ethyl 2-oxo-9Z-octadecenoate, ethyl 2-oxo-10Z- nonadecenoate, ethyl 2-oxo-nonadecanoate, tert-butyl 3-oxo-2, 2- dihydroxyoctadecanoate, 1,1,1-trifluoro-lOZ nonadecon-2-one, l,l,l-trifluoro-9Z- octadecen-2-one, 1,1,1 -trifluoro- 1 OE-nonadecen-2-one, 2-hydroxy-9Z- octadecenamide, 2-chloro-9Z-octadecenamide, l-diazo-10Z-nonadecen-2-one, N- amino-9Z-octadecenamide, methyl 1 OZ-nonadecenoate, 1 OZ-nonadecenoic acid, 2- hydroxy-1 OZ-nonadecenoic acid, 2-hydroxy-10Z-nonadecenamide, 8Z- heptadecenoic, 3-Oxo-2-(triphenylphosphoranylidene) octadecanoate.

6. A method of treatment according to claim 3 which comprises administering an amount of oleamide sufficient to provide a cellular concentration of oleamide of from 3.2 μm to 20 μm.

7. A method according to claim 1 characterised in that the convulsant disorder is epilepsy.

8. The use of an amido hydrolase inhibitor in the manufacture of an anticonvulsant medicament.

9. The use according to claim 8 characterised in that the amido hydrolase inhibitor is a fatty acid amide.

10. The use according to claim 8 characterised in that the amido hydrolase inhibitor is selected from those described in US Patent No 5,856,537.

11. The use according to claim 10 characterised in that the amido hydrolase inhibitor is selected from the group; l-chloro-10Z-nonadecen-2-one, 8Z- heptadecenal, 2-oxo-9Z-octadecenamide, 2-oxo-10Z-nonadecenamide, ethyl 2- oxo-9Z-octadecenoate, ethyl 2-oxo-9Z-octadecenoate, ethyl 2-oxo-10Z- nonadecenoate, ethyl 2-oxo-nonadecanoate, tert-butyl 3-oxo-2, 2- dihydroxyoctadecanoate, 1,1,1-trifluoro-lOZ nonadecon-2-one, l,l,l-trifluoro-9Z- octadecen-2-one, 1,1,1 -trifluoro- 1 OE-nonadecen-2-one, 2-hydroxy-9Z- octadecenamide, 2-chloro-9Z-octadecenamide, l-diazo-10Z-nonadecen-2-one, N- amino-9Z-octadecenamide, methyl lOZ-nonadecenoate, 1 OZ-nonadecenoic acid, 2- hydroxy-1 OZ-nonadecenoic acid, 2-hydroxy-10Z-nonadecenamide, 8Z- heptadecenoic, 3-Oxo-2-(triphenylphosphoranylidene) octadecanoate.

12. The use of oleamide in the manufacture of an anticonvulsant medicament.

13. The use according to claim 11 characterised in that the oleamide is cis- oleamide.

14. A formulation of an anticonvulsant medicament which comprises a therapeutically effective amount of an amido hydrolase inhibitor in combination with a pharmaceutically acceptable adjuvant, diluent or carrier.

15. A formulation according to claim 14 characterised in that the amido hydrolase inhibitor is selected from those described in US Patent No 5,856,537.

16. A formulation of an anticonvulsant medicament which comprises a therapeutically effective amount of oleamide in combination with a pharmaceutically acceptable adjuvant, diluent or carrier.

17. A method of treatment or alleviation of a CNS disorder, pain and/or ischaemia which comprises altering the levels of oleamide in the brain of a patient suffering from such a disorder.

18. The use or method substantially as described with reference to the accompanying examples.