BACKGROUND

About 30% of epilepsy patients do not gain satisfactory seizure control with currently available antiepileptic drugs (AEDs). Temporal lobe epilepsy (TLE) is the most common focal epilepsy syndrome and up to 80 % of TLE patients suffer from pharmacoresistance. Only a small percentage of TLE patients and patients with other forms of d iff icu It-to-treat focal epilepsies are eligible for surgical resection of epileptogenic tissue to achieve seizure control. Patients suffering status epilepticus present with similarly high rates of resistance to first-line treatment with benzodiazepines. Therefore, alternative anticonvulsant substances are required and interfering with purinergic signaling may be a viable option.

Adenosine triphosphate (ATP) is (co-)released in an activity-dependent manner from astrocytes and neurons and is subject to extracellular enzymatic degradation to adenosine. Also direct release of adenosine has been described. ATP/ADP and adenosine have been shown to modulate synaptic transmission and neuronal network activity in models of physiological and pathological network oscillations by activating P2 and A-i and A _2a receptors, respectively. Purinergic receptors have also been implicated in epileptogenic processes. While only few studies describe acute effects of P2 receptor activation on network activity adenosine receptor-mediated effects have been extensively studied. Anticonvulsant properties of adenosine, mediated by the G-protein coupled A-i receptor, have been known for some time and are mediated by reduced presynaptic transmitter release probability and postsynaptic hyperpolarization. Additionally, adenosine acts proconvulsantly via A _2a receptors. However, due to the lower affinity of adenosine to A _2a receptors, its acute net effect is a reduction or suppression of epileptiform activity in different model systems presumably serving as an intrinsic anticonvulsant. However, there remains a need for further means and modalities for treatment of status epilepticus and in particular for treatment of status epilepticus refractory to treatment with known anticonvulsive drugs like e.g. benzodiazepines, carbamazepine (CBZ), valproate (VPA) and/or phenytoin (PHT).

DESCRIPTION OF THE INVENTION

It is an object of the present invention to provide further means for use in the treatment of status epilepticus, preferably in the treatment of status epilepticus without prior epileptogenesis and being refractory to treatment with known anticonvulsive drugs like e.g. benzodiazepines, carbamazepine (CBZ), valproate (VPA) and/or phenytoin (PHT).

The present invention relates to adenosine A1 receptor agonists or pharmaceutically acceptable salts thereof for use in the treatment of status epilepticus.

It has surprisingly been found that direct targeting of adenosine A1 receptor with selective agonists is effective in treatment of status epilepticus, wherein it has been shown that use of adenosine A1 receptor agonists is particularly effective in treatment of status epilepticus being refractory to treatment with known anticonvulsive drugs like e.g. benzodiazepines, carbamazepine (CBZ), valproate (VPA) and/or phenytoin (PHT). Activating A-i receptors with a specific agonist completely suppressed seizure-like events (SLEs) in 73 % of human temporal cortex slices. In the remaining slices incidence of SLEs was markedly reduced. Also in slices insensitive to a high dose of carbamazepine (50 μΜ) the adenosine A-i agonist was equally efficient.

The adenosine A-i receptor is one member of the adenosine receptor group of G protein- coupled receptors with adenosine as endogenous ligand.

Activation of the adenosine A-i receptor by an agonist causes binding of Gn/2/3 or G ₀ protein. Binding of Gn/2/3 causes an inhibition of adenylate cyclase and, therefore, a decrease in the cAMP concentration. An increase of the inositol triphosphate/diacylglycerol concentration is caused by an activation of phospholipase C, whereas the elevated levels of arachidonic acid are mediated by DAG lipase, which cleaves DAG to form arachidonic acid. Several types of potassium channels are activated but N-, P-, and Q-type calcium channels are inhibited. In sum, activation of A1 receptors leads to lower presynapric transmitter release and postsynaptic hyperpolarization. Therefore A1 -receptor activation generally attenuates synaptic signaling. The present invention is directed to the use of adenosine A1 receptor agonists for treatment of status epilepticus. For the purpose of the present invention, the term "adenosine A1 receptor agonist" is used in its art recognized meaning and refers to small molecules which are capable of activating the adenosine A1 receptor in vivo. As used herein, the term "adenosine A1 receptor agonist" refers to the active compound as such and to a pharmaceutically acceptable salt thereof. Preferably, the adenosine A1 receptor agonist of the present invention exhibits an IC ₅₀ value of 1 μΜ or less with regard to the activation of human adenosine A1 receptor in vitro. The present invention is not limited to the use of a particular adenosine A1 receptor agonist but is independent from the actual choice of adenosine A1 receptor agonist provided said adenosine A1 receptor agonist is capable of activating the adenosine A1 receptor. Preferably, the adenosine A1 receptor agonist is selective for adenosine A1 receptor compared to other types of adenosine receptors like e.g. adenosine A2 receptor. For the present invention, an adenosine A1 receptor agonist is selective for adenosine A1 receptor if said agonist exhibits an IC ₅₀ value for activation of adenosine A1 receptor which is 2x, preferably 5x, more preferably 10x lower than the IC ₅₀ value for activation of adenosine A2 receptor.

In the prior art, a huge number of different adenosine A1 receptor agonists is known. Exemplary embodiments of suitable adenosine A1 receptor agonists of the present invention are disclosed and derivable from US 4,843,066, US 4,985,409, US 2014/0241990, WO 2001/045715, US 9,040,566 and WO 2008/130520. 9. The adenosine A1 receptor agonist for use of the present invention can be selected e.g. from alkinyl-substituted purine derivatives, exemplary embodiments are disclosed in WO 2008/130520, and substituted 3,5- dicyano-4-phenylpyridines, exemplary embodiments of which are disclosed in WO 03/53441 , WO 2009/015776, WO 2009/01581 1 , WO 2009/015812, WO 2010/072314, WO 2010/072315 and WO 2010/086101.

Preferably, the adenosine A1 receptor agonist for use according to the present invention is:

N ⁶ -cyclopentyladenosine (CPA);

- 2-chloro-N(6)-cyclopentyladenosine (CCPA);

- (2S)-N ⁶ -(2-endo-norbornyl)adenosine (S(-)-ENBA);

N ⁶ -[4-[[[4-[[[(2-aminoethyl)amino]carbonyl]methyl]-anili no]carbonyl]methyl]phenyl] adenosine (ADAC);

- 1 S-[1 a,23,33,4a(S ^* )]-4-[7-[[1 -[(3-chloro-2-thienyl)methyl]propylamino]-3H- imidazo[4,5-b]pyridin-3-yl]-N-ethyl-2,3-dihydroxy cyclopentanecarboxamide

(AMP579);

- 2-chloro-N-[(R)-[(2-benzothiazoly)thio]-2-propyl]adenosine (NNC-2I-0I36);

- /V-[(1 S,2S)-2-Hydroxycyclopentyl]adenosine (GR79236); - (2R _! 3S _! 4R,5R)-2-(hydroxymethyl)-5-[6-[[(3S)-oxolan-3-yl]amino ]purin-9-yl]oxolane- 3,4-diol (CVT-510, also known as tecadenoson);

- 5'-0-(N-methyl-carbamoyl)-N6 - [tetrahydrofuran-3(R)-yl] adenosine (CVT-2759); N- cyclohexyl-2'-0-methyladenosine (SDZ WAG 994);

2-amino-6-[[2-(4-chlorophenyl)-1 ,3-thiazol-4-yl]methylsulfanyl]-4-[4-(2-hydroxyethoxy) phenyl]pyridine-3,5-dicarbonitril (capadenoson);

N6-cyclohexyl-adenosine (CHA); R(-)-N6-(2-phenylisopropyl)adenosine (R-PIA);

- (2S,3S,4R,5R)-5-[6-(cyclopentylamino)purin-9-yl] -N-ethyl-3,4-dihydroxyoxolane-2- carboxamide (selodenoson, also known as RG 14202);

or a combination thereof.

In a particularly preferred embodiment, the adenosine A1 receptor agonist for use of the present invention is N-cyclohexyl-2'-0-methyladenosine (SDZ WAG 994).

The adenosine A1 receptor agonist for use of the present invention can comprise one particular type of adenosine A1 receptor agonist or may comprise a combination of two or more different adenosine A1 receptor agonists.

The adenosine A1 receptor agonists of the present invention may be presented in form of a pharmaceutically acceptable salt thereof. As used herein, the term "pharmaceutically acceptable salt" includes acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of an adenosine A1 receptor agonist with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo or by freeze-drying). When the adenosine A1 receptor agonist possesses a free base form, the compounds can be prepared as a pharmaceutically acceptable acid addition salt by reacting the free base form of the adenosine A1 receptor agonist with a pharmaceutically acceptable inorganic or organic acid, e.g. hydrohalides such as hydrochloride, hydrobromide, hydroiodide; other mineral acids and their corresponding salts such as sulfate, nitrate, phosphate, etc.; and alkyl- and monoarylsulfonates such as ethanesulfonate, toluenesulfonate and benzenesulfonate; and other organic acids and their corresponding salts such as acetate, tartrate, maleate, succinate, citrate, benzoate, salicylate and ascorbate. Further acid addition salts include adipate, alginate, arginate, aspartate, benxenesulfonate (hesylate), bisulfate, bisulfite, bromide, butyrate, camphorate, camphorsulfonate, caprylate, chloride, chlorobenzoate, cyclopentanepropionate, digluconate, dihydrogenphosphate, dini- trobenzoate, dodecylsulfate, ethanesulfonate, fumarate, galacterate (from mucic acid), galacturonate, glucoheptaoate, glucorrate, glutamate, glycerophosplrate, hemisueci- nate, hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, iodide, isethionate, iso-butyrate, lactate, lactobionate, malate, malonate, mandelate, metaphosphate, methanesulfonate, methylbenzoate, monohydro- genphosphate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, oleate, pamoate, pectinate, persulfate, phenylac- etate, 3- phenylpropionate, phosphate, phosphonate and phthalate. When the adenosine A1 receptor agonist of the present invention possesses a free acid form, a pharmaceutically acceptable base addition salt can be prepared by reacting the free acid form of the adenosine A1 receptor agonist with a pharmaceutically acceptable inorganic or organic base. Examples of such bases are alkali metal hydroxides including potassium, sodium and lithium hydroxides; alkaline earth metal hydroxides such as barium and calcium hydroxides; alkali metal alkoxides, e.g. potassium ethanolate and sodium propanolate; and various organic bases such as ammonium hydroxide, piperidine, diethanolamine and N-methylglutamine. Also included are the aluminum salts of the adenosine A1 receptor agonists of the present invention. Further base salts include copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium and zinc salts. Organic base salts include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, e.g., arginine, betaine, caffeine, chloroprocaine, choline, Ν,Ν'-dibenzylethylenediamine (benzathine), dicyclohexylamine, diethanolamine, diethylamine, 2-diethylaminoethanol, Z-dimethylami- noethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, iso-propylamine, lidocaine, lysine, meglumine, N -methyl- D-glucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethanolamine, triethylamirre, trimethylamine, tripropylamine and tris- (hydroxymethyl)-methylamine (tromethamine).

Salts may also be prepared by exchanging a counter-ion of an adenosine A1 receptor agonist of the invention in the form of a salt with another counter-ion, for example using a suitable ion exchange resin. The salt form may confer improved pharmacokinetic properties on the adenosine A1 receptor agonist as compared to the free form of the compound. The pharmaceutically acceptable salt form may also positively affect the pharmacodynamics of the compound with respect to its therapeutic activity in the body. An example of a pharmacodynamic property that may be favorably affected is the manner in which the compound is transported across cell membranes, which in turn may directly and positively affect the adsorption, distribution, biotransformation and excretion of the compound.

According to the invention, the adenosine A1 receptor agonist or a pharmaceutically acceptable salt thereof is used in the treatment of status epilepticus. The present invention also relates to the use of an adenosine A1 receptor agonist or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of status epilepticus.

The present invention also relates to a method of treating status epilepticus, wherein a patient in need of such therapy is administered a therapeutically effective dose of an adenosine A1 receptor agonist or a pharmaceutically acceptable salt thereof.

Status epilepticus (SE) is a medical condition defined by the presence of an exacerbated seizure or a series of seizures without recovering to baseline conditions lasting longer than 5 mins, preferably longer than 10 mins, more preferably more than 20 mins. For the purpose of the present invention, the term "seizure" is used in its art recognized meaning and refers to a highly coherent/synchronous network activity (HCSNA) which is excessive and overrides normal synchronous network activity and as a result interrupts normal brain function locally or over several brain regions.

SE is a separate seizure/HCSNA disorder and is classified in ICD-10 as G41 . Drug resistance rates in SE are around 30-40 %. Convulsive and non-convulsive SE exists, whereas non-convulsive SE tends to be more often resistant to pharmacological treatment.

SE requires ictogenesis, but not necessarily epileptogenesis.

The term "ictogenesis" describes an acute process that, in a short time scale, directly leads to a seizure/HCSNA (the ictus). For example, bathing brain tissue with ictogenic substances (like high potassium artificial cerebrospinal fluid combined with a GABA receptor antagonist) constitutes ictogenesis and acutely causes a seizure. A healthy brain can be pushed to have a seizure/ictus/HCSNA and a brain suffering from epilepsy can be pushed more "easily" to have a seizure/ictus/HCSNA. In both cases ictogenesis describes the acute process causing a seizure.

The term "epileptogenesis" describes a chronic process, witnessed across species, where in a healthy brain an initiating event occurs and the subject ultimately develops epilepsy later on. During the days/weeks (rodents, cats, dogs) and years (human) following the initial event no seizures/HCSNA are apparent. However, during this latent period network changes occur. After the latent period comes the chronic period which is characterized by repeated and "gradually" occurring clinically relevant seizures/HCSNAs. During the chronic period and after repeated seizures patients are usually diagnosed with epilepsy. Epileptogenesis, thus, describes the processes needed to transform a healthy brain into an epileptic brain and is a long-term process characterized by reproducible anatomical changes in multiple brain regions. Epileptogenesis describes a chronic process, comprising occurrence of reproducible anatomical changes in multiple brain regions, that ultimately leads to spontaneously recurring seizures/HCSNAs.

Preferably, the status epilepticus to be treated according to the present invention is a status epilepticus without prior epileptogenesis.

SE can occur without prior epileptogenesis e.g. as a spontaneous result of an acute insult like e.g. traumatic brain injury, stroke, metabolic abnormalities, hypoxia, systemic infection, anoxia, drug overdose, CNS infection, CNS hemorrhage and/or intoxication. Such SE patients do have an acute or unknown event (ictogenesis) that leads to a long lasting seizure or a series of seizures without recovery to baseline conditions which cannot be stopped through endogenous brain mechanisms. Moreover, SE in epilepsy patients tends to respond very well to treatment with anticonvulsive drugs whereas (acute) SE without prior epileptogenesis tends to be often difficult to treat.

Preferably, the adenosine A1 receptor agonist or a pharmaceutically acceptable salt thereof is used for treatment of status epilepticus, wherein the status epilepticus is refractory to treatment with one or more anticonvulsive drugs.

Anticonvulsant drugs (also commonly known as anticonvulsants or as antiseizure drugs) are a diverse group of pharmacological agents used in the treatment of seizures. Anticonvulsant drugs suppress the rapid and excessive firing of neurons during seizures. Anticonvulsant drugs may also prevent the spread of a seizure within the brain. Conventional anticonvulsive drugs may block sodium channels or enhance γ-aminobutyric acid (GABA) function. Next to the voltage-gated sodium channels and components of the GABA system, their targets include but are not restricted to GABA _A receptors, the GAT-1 GABA transporter, and GABA transaminase. Additional targets include voltage-gated calcium channels, its subunit α2δ, and the synaptic vesicle protein SV2A. Another potential target of antiepileptic drugs is the peroxisome proliferator-activated receptor alpha.

A huge number of anticonvulsant drugs is known in the art. Exemplary embodiments are compounds of the classes: aldehydes (e.g. paraldehyde);

aromatic allylic alcohols (e.g. stiripentol);

barbiturates (e.g. phenobarbital, methylphenobarbital, barbexaclone);

benzodiazepines (e.g. diazepam, midazolam, lorazepam, clobazam, clonazepam, clorazepate);

carbamates (e.g. felbamate);

carboxamides (e.g. carbamazepine, oxcarbamazepine); - fatty acids (e.g. valproates, valpryolamides, vigabatrin);

- fructose derivatives (e.g. topiramate);

GABA analogs (e.g. gabapentin, pregabalin);

hydantoins (e.g. ethotoin, phenytoin, mephenytoin, fosphenytoin);

oxazolidinediones (e.g. paramethadione, trimethadione, ethadione);

propionates (e.g. beclamide);

pyrimidinediones (e.g. primidone);

pyrrolidines (e.g. brivaracetam, levetiracetam, seletracetam);

succinimides (e.g. ethosuximide, phensuximide, mesuximide);

sulfonamides (e.g. acetazolamide, sultiame, methazolamide, zonisamide); and triazines (e.g. lamotrigine).

Preferably, the status epilepticus is refractory to treatment with one or more of carbamazepine (CBZ), valproate (VPA) and/or phenytoin (PHT).

According to another aspect of the invention, the status epilepticus to be treated is refractory to treatment with one or more modulators of GABA-A receptor signaling, preferably to treatment with an active agent of the class of benzodiazepines or dibenzazepines.

According to another aspect of the present invention, the adenosine A1 receptor agonist can be used in acute treatment of status epilepticus, which optionally is refractory to treatment with one or more known anticonvulsive drugs. An acute treatment regime is characterized by administration of the pharmaceutical in a scheme that occurs at irregular, not predetermined time intervals, wherein one particular administration event is triggered and dependent from the presence or occurrence of an individual seizure event.

In a preferred embodiment, the term "acute" as used herein defines that each treatment or administration of the adenosine A1 receptor agonist is directly triggered by the presence or reasoned expectance of the onset of a particular seizure event or status epilepticus. Even more preferably, the term "acute treatment" refers to a self-administration by the patient of the adenosine A1 receptor agonist of the invention or a pharmaceutically acceptable salt thereof, wherein each self-administration is directly triggered upon self-perception by the patient of presence or reasoned expectance of the onset of a particular seizure event or status epilepticus. In particular, each acute treatment or self-administration may be triggered by self-perception of signs of onset of a seizure event or status epilepticus by the patient, or by self-perception of an ongoing seizure event or status epilepticus. Preferably the term "acute" encompasses a time window starting 2 hours before the expected onset of a seizure event or status epilepticus until any time point during the persistence of the seizure event or status epilepticus of a particular patient. Even more preferred the term "acute" refers to a time window that begins not more than 1 hour before the expected onset of a particular seizure event or status epilepticus, most preferably not more than 30 min, 10 min or 5 min before the expected onset of a particular epileptic seizure event or status epilepticus.

Alternatively, the adenosine A1 receptor agonist can be used in chronic treatment of status epilepticus, preferably in chronic treatment of status epilepticus, which is refractory to treatment with one or more known anticonvulsive drugs. A chronic treatment regime is characterized by administration of the pharmaceutical in a scheme that occurs at regular, predetermined time intervals, wherein a particular administration event is independent from the presence or absence of an individual seizure or status epilepticus.

The adenosine A1 receptor agonist or a pharmaceutically acceptable salt thereof is preferably administered in combination with another pharmaceutically active agent. This other pharmaceutically active agent may be an anticonvulsive drug, an active agent for treatment of a concomitant disease or medical state or an active agent for treatment or prevention of side effects associated with the administration of the adenosine A1 receptor agonist of the invention. Preferably, the adenosine A1 receptor agonist is administered in combination with a non blood-brain-barrier permeable A1 antagonist. Exemplary embodiments of such non blood-brain-barrier permeable A1 receptor antagonists are 8- Sulphophenyltheophylline (8-SPT) und 8-Sulphophenylxanthine (DPCPX/8-SPX). Since the expression of adenosine A1 receptor is not specific to the brain, systemic treatment with an adenosine A1 receptor agonist may lead to side effects due to specific interaction with adenosine A1 receptor in the body periphery. In order to prevent or treat such side effects, it is beneficial to use an adenosine A1 receptor antagonist which does not cross the blood- brain barrier. By doing so, the activating effect of the adenosine A1 receptor agonist is counterbalanced in the body periphery by co-administration of a non blood-brain-barrier permeable A1 antagonist, while still allowing for specific activation of adenosine A1 receptor in the brain.

As used herein, the term "treating" or "treatment" encompasses to reversing, alleviating or inhibiting the progress of a disease, disorder or condition, or improvement of one or more symptoms of such disease, disorder or condition, to which such term applies. As used herein, "treating" or "treatment" may also refer to decreasing the probability or incidence of the occurrence of a disease, disorder or condition in a mammal as compared to an untreated control population, or as compared to the same mammal prior to treatment. For example, as used herein, "treating" or "treatment" may refer to preventing a disease, disorder or condition, and may include delaying or preventing the onset of a disease, disorder or condition, or delaying or preventing the symptoms associated with a disease, disorder or condition. As used herein, "treating" or "treatment" may also refer to reducing the severity of a disease, disorder or condition or symptoms associated with such disease, disorder or condition prior to a mammal's affliction with the disease, disorder or condition. Such prevention or reduction of the severity of a disease, disorder or condition prior to affliction relates to the administration of the adenosine A1 receptor agonist of the present invention, as described herein, to a subject that is not at the time of administration afflicted with the disease, disorder or condition. As used herein the term "treating" or "treatment" may also refer to preventing the recurrence of a disease, disorder or condition or of one or more symptoms associated with such disease, disorder or condition.

As used herein, the term "treating" or "treatment" refers to treatment of an individual, preferably of an animal, more preferably of a mammal, even more preferably of a human. The individual to be treated is preferably in need of such treatment and may suffer from status epilepticus, preferably from status epilepticus refractory to treatment with an anticonvulsive drug.

The present invention also relates to a pharmaceutical composition comprising as an active ingredient an adenosine A1 receptor agonist or a pharmaceutically acceptable salt thereof for use according to the present invention. Such pharmaceutical compositions may comprise, in addition to the adenosine A1 receptor agonist of the invention or their pharmaceutically acceptable salts, one or more pharmaceutically acceptable excipients. The term "excipient" is used herein to describe any ingredient other than the adenosine A1 receptor agonist of the invention. The choice of excipient will to a large extent depend on the particular mode of administration. Excipients can e.g. be suitable carriers, retardants, boosters, prolonging substances, adjuvants, stabilizers, binders, emulsifiers, surface active agents, penetration enhancers suspending agents, disintegrants, buffers, salts, dilutents, solvents, dispersion media, fillers, lubricants, propellants, preservatives, flavours or mixtures thereof.

The adenosine A1 receptor agonists are administered preferably at an effective dose. An "effective dose" is the dose of an adenosine A1 receptor agonist that upon administration to a patient yields a measurable therapeutic effect with regard to the disease of interest. In the present invention an effective dose is the dose of an adenosine A1 receptor agonist that upon administration to a patient yields a therapeutic effect with regard to at least one disease related symptom in a patient or patients suffering from a disease as specified above. Preferably, the adenosine A1 receptor agonist of the invention is administered at a dose of not more than 500 mg/kg/d. In particular, the adenosine A1 receptor agonist can be administered at a dose of 1 Mg/kg/d to 400 mg/kg/d, preferably of 20 Mg/kg/d to 150 mg/kg/d. In any event, the physician or the skilled person will be able to determine the actual dose which will be suitable for an individual patient, which is likely to vary with the age, weight, sex, and concomitant illnesses such as e.g. renal or hepatic dysfunction and response of the particular patient to be treated. The above mentioned dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are appropriate, and such are within the scope of the invention.

The adenosine A1 receptor agonists of the present invention are preferably administered orally, intravenously, subcutaneously, bucally, rectally, dermally, nasally, tracheally, bronchially or by any other parenteral route or via inhalation in a pharmaceutically acceptable dosage form.

The adenosine A1 receptor agonist of the present invention may be administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, or buccal or sublingual administration may be employed by which the compound enters the blood stream directly from the mouth.

Formulations suitable for oral administration include: solid formulations such as tablets; capsules containing particulates, liquids, or powders; lozenges (including liquid-filled); and chews; multi- and nano-particulates; gels; solid solutions; liposomes; films, ovules, sprays and liquid formulations. Liquid formulations include suspensions, solutions, syrups and elixirs. Such formulations may be employed as fillers in soft or hard capsules and typically comprise a carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and/or suspending agents. Liquid formulations may also be prepared by the reconstitution of a solid, for example, from a sachet.

For tablet dosage forms, depending on dose, the adenosine A1 receptor agonist may make up from 0.1 weight % to 80 weight % of the dosage form, more typically from 5 weight % to 60 weight % of the dosage form. In addition to the compound, tablets generally contain a disintegrant. Examples of disintegrants include sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methyl cellulose, microcrystalline cellulose, lower alkyl- substituted hydroxypropyl cellulose, starch, pregelatinised starch and sodium alginate. Generally, the disintegrant will comprise from 1 weight % to 25 weight %, preferably from 5 weight % to 20 weight % of the dosage form.

Binders are generally used to impart cohesive qualities to a tablet formulation. Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinised starch, hydroxypropyl cellulose and hydroxypropyl methylcellulose. Tablets may also contain diluents, such as lactose (monohydrate, spray-dried monohydrate, anhydrous and the like), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch and dibasic calcium phosphate dihydrate.

Tablets may also optionally comprise surface active agents, such as sodium lauryl sulfate and polysorbate 80, and glidants such as silicon dioxide and talc. When present, surface active agents may comprise from 0.2 weight % to 5 weight % of the tablet, and glidants may comprise from 0.2 weight % to 1 weight % of the tablet.

Tablets also generally contain lubricants such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulphate. Lubricants generally comprise from 0.25 weight % to 10 weight %, preferably from 0.5 weight % to 3 weight % of the tablet.

Other possible ingredients include anti-oxidants, colourants, flavouring agents, preservatives and taste-masking agents.

Exemplary tablets contain up to about 80% compound, from about 10 weight % to about 90 weight % binder, from about 0 weight % to about 85 weight % diluent, from about 2 weight % to about 10 weight % disintegrant, and from about 0.25 weight % to about 10 weight % lubricant.

The adenosine A1 receptor agonist of the present invention may also be administered directly into the blood stream, into muscle, or into an internal organ. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intracerebroventricular, intraurethral, intrasternal, intracranial, intramuscular and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques.

Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water. The preparation of parenteral formulations under sterile conditions, for example, by lyophilisation, may readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art.

The adenosine A1 receptor agonist of the invention may also be administered topically to the skin or mucosa, that is, dermally or transdermally. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibres, bandages and microemulsions. Liposomes may also be used. Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol and propylene glycol.

The adenosine A1 receptor agonist of the present invention can also be administered intranasally or by inhalation, typically in the form of a dry powder (either alone, as a mixture, for example, in a dry blend with lactose, or as a mixed component particle, for example, mixed with phospholipids, such as phosphatidylcholine) from a dry powder inhaler or as an aerosol spray from a pressurized container, pump, spray, atomizer (preferably an atomizer using electrohydrodynamics to produce a fine mist), or nebulizer, with or without the use of a suitable propellant, such as 1 ,1 ,1 ,2-tetrafluoroethane or 1 ,1 ,1 ,2,3,3, 3-heptafluoropropane. For intranasal use, the powder may comprise a bioadhesive agent, for example, chitosan or cyclodextrin. The pressurized container, pump, spray, atomizer, or nebulizer contains a solution or suspension of the adenosine A1 receptor agonist of the invention comprising, for example, ethanol, aqueous ethanol, or a suitable alternative agent for dispersing, solubilizing, or extending release of the active, a propellant(s) as solvent and an optional surfactant, such as sorbitan trioleate, oleic acid, or an oligolactic acid.

The use of adenosine A1 receptor agonist in the treatment of status epilepticus may have the advantage that such compounds may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, be more easily absorbable than, have better pharmacokinetic profile (e.g. higher oral bioavailability and/or lower clearance) than, and/or have other useful pharmacological, physical, or chemical properties over compounds known in the prior art for treatment of said diseases.

FIGURES

Fig. 1 : Seizure like event (SLE) parameters and time course in human cortical slices. A Representative field potential recording of SLEs (left) and SLE parameter analysis in the right panel. B Time course of SLE parameters in a group of control recordings (n = 13, six patients, ^* : p < 0.05, Friedman test and pairwise comparisons). Dotted wide lines in boxplots represent mean.

Fig. 2: Exogenous and endogenous ATP signaling and SLEs. A Sample field potential trace of exogenous activation of ATP and adenosine receptors. B SLE parameter box plots, wide dotted line represents mean. Two slices showed full suppression of SLEs during washin of ATP and were excluded for statistical analysis of remaining event parameters. C Specific P2 antagonists during SLEs. Neither incidence (time course depicted) nor other SLE parameters were relevantly altered by MRS 2179 (a) or by TNP-ATP (b) or by A740003 and A804598 (c). Experiments with P2 antagonists were discontinued due to scarcity of tissue and lack of SLE suppression/attenuation.

Fig. 3: ATP in presence of adenosine receptor block and exogenous adenosine A Original traces during broadband adenosine receptor block (upper trace) and specific A1 receptor antagonism (lower trace) which prevents SLE attenuation by ATP. B Quantification of SLE parameters. Wide dotted lines in box plots represent mean. (Wilcoxon for all four parameters; Inc: n = 8, p = 0.06; Dur, SFP, xAMP: n = 7 each, p = 0.61 , p = 0.37, p = 0.1 1 , respectively)

Fig. 4: Specific A1 agonist during SLEs and comparison of purinergic SLE block efficacy. A Sample trace of SLE suppression with A1 agonist. B SLE parameter quantification. 8 of 1 1 slices presented complete block of SLEs with 1 μΜ SDZ WAG 994. 3 slices were analyzed for the duration and amplitude parameters. Individual experiments in right three panels. C Proportional differences in efficacy to suppress SLEs with ATP, adenosine or the specific A1 agonist. ( ^* : p < 0.05; ^** : p < 0.01 )

Fig. 5: SLE suppression in CBZ resistant human cortical slices. A Upper grey trace represents SLEs sensitive to perfusion of CBZ (no subsequent A1 agonist) while lower trace is an example for CBZ resistance of SLEs in human cortical slices. B Quantification and comparison of SLE parameters in time-matched control recordings and a subset of slices resistant to CBZ. No statistically significant difference was found for SLE parameters of control and CBZ-resistant group (control: n = 13, CBZ: n = 17; Mann-Whitney U for all parameters; Inc: p = 0.62, Dur: p = 0.77, SFP: p = 0.26, xAmp: p = 0.3). Wide dotted line represents mean in box plots. C Sample recording of experiment testing CBZ response first, allowing 30 minutes of washout and subsequent application of the A1 agonist to CBZ-resistant SLEs. D Quantification of A1 agonist effects in CBZ-resistant SLEs. 71 % of 17 slices showed SLE suppression in spite of resistance to 50 μΜ CBZ. Dur, SFP and xAmp only analyzed for slices without suppression. Wide dotted lines represent mean in boxplots. ( ^*** : p < 0.001 )

EXAMPLES

About 30% of epilepsy patients do not gain satisfactory seizure control with currently available antiepileptic drugs (AEDs). Temporal lobe epilepsy (TLE) is the most common focal epilepsy syndrome and up to 80 % of TLE patients suffer from pharmacoresistance. Only a small percentage of TLE patients and patients with other forms of d iff icu It-to-treat focal epilepsies are eligible for surgical resection of epileptogenic tissue to achieve seizure control. Therefore alternative anticonvulsant substances are required and interfering with purinergic signaling may be a viable option. Studies from our and other labs have previously shown that slice preparations from resected human tissue are suitable to study pharmacological properties of induced epileptiform activities. Generally, resistance of seizure-like events (SLEs) to common AEDs like carbamazepine (CBZ), valproate (VPA) and phenytoin (PHT) is preserved in slice preparations of hippocampal and neocortical tissue from pharmacoresistant patients thereby providing a model of pharmacoresistant epilepsy and/or status epilepticus.

Here we study acute effects of purinergic substances on epileptiform activity induced by elevated [K ⁺ ] and bicuculline. In contrast to the low Mg ^2+" model, our induction protocol regularly presents with prolonged SLEs in human temporal neocortex slices from patients with pharmacoresistant epilepsy. Recurring, prolonged SLEs with short interictal time spans in vitro (seconds to minutes versus days to months in human in vivo condition) can be regarded to model a status epilepticus-like condition in vivo.

Materials & Methods

Human tissue acquisition and handling

Human temporal neocortex specimens were obtained as previously described. In brief, patients suffering from mesial temporal lobe epilepsy (MTLE) received pre-surgical evaluation in the Epilepsy-Center Berlin-Brandenburg. Seventeen eligible patients gave written informed consent before being included into this study (Tab. 1 ). The study was approved by the Ethics Committee of Charite-Universitatsmedizin Berlin (EA1/042/04) and was conducted in agreement with the Declaration of Helsinki.

Immediately after resection, temporal neocortex specimen were incubated in cold (1 ° - 4° C) carbogenated (95 % 0 ₂ and 5 % C0 ₂ ) transport solution containing (in mM) KCI 3, NaH ₂ P0 ₄ 1 .25, glucose 10, sucrose 200, MgS0 ₄ 2, MgCI ₂ 1 .6, CaCI ₂ 1 .6 and a-tocopherol 0.1 (pH 7.4, osmolality 304 mosmol/kg). a-Tocopherol was dissolved in ethanol yielding a final ethanol concentration of 0.005 v%. Tissue was rapidly transported to the laboratory and dissected into 500 μΜ thick slices in transport solution. Slices were immediately transferred to interface chambers and perfused with artificial cerebrospinal fluid (ACSF) at a flow rate of 1 .6 - 1 .8 ml/min (in mM: NaCI 129, KCI 3, NaH ₂ P0 ₄ 1 .25, glucose 10, MgS0 ₄ 1 .8, CaCI ₂ 1 .6, NaHC0 ₃ 21 , tocopherol in ethanol 0.1 , final ethanol v% 0.005, pH 7.4, osmolality 300 mosmol/kg) and allowed to recover for 3 hours before starting recordings. Electrophysiology and induction of SLEs

Field potential recordings were obtained in DC mode using electrodes filled with ACSF (3 - 10 mOhm) positioned in layer VA I of neocortex. Signals were digitized and saved on a computer hard drive at a sampling rate of 10 kHz with a 3 kHz low pass filter. SLEs were induced by perfusing slices with ACSF containing 8 mM K ⁺ and 50 μΜ bicuculline-methiodide (high-K ⁺ -Bic-ACSF). Pharmacological experiments began earliest 30 minutes after SLEs had appeared to allow for stabilization and registration of a sufficient prewash control time-period.

Data analysis and statistics

As described previously, SLEs from the last 10 min of drug application or corresponding control phases were analyzed with regard to incidence (events per minute, Fig 1 A, right panel), duration (s) and two amplitude values, namely, slow field potential (SFP; in mV; slow DC shift without superimposed faster activity) and maximum amplitude (xAmp; in mV, maximum amplitude of SLE including fast transients). Duration of SLEs is defined as the time between the initial fast deflection from baseline and the time point where the signal has recovered 2/3 towards baseline of the slow field potential. Data are reported as mean ± SD. Data set distribution was tested for normality (Kolmogorov-Smirnoff and Shapiro-Wilk) and subsequently non-parametric tests (Wilcoxon signed-rank test for dependent and Mann- Whitney U test for independent samples) were employed. For time-course analysis of the control group, Friedman test was used with subsequent pairwise comparisons. Fisher's Exact Test was used for proportional differences. All statistical calculations were performed using SPSS 23 (IBM, Armonk, NY, USA).

Drugs

ATP disodium salt, adenosine, the A-i receptor antagonist DPCPX, the A-i receptor agonist SDZ WAG 994, the P2Y-, receptor antagonist MRS2179, the Ρ2Χ·,, P2X ₃ and P2X _2/3 receptor antagonist TNP-ATP, the P2X ₇ receptor antagonists A740003 and A804598 were purchased from Tocris (Bristol, United Kingdom) and the broadband adenosine receptor antagonist CGS-15943, the GABA-A receptor antagonist bicuculline-methiodide, otocopherol and CBZ were purchased from Sigma (St. Louis, MO, USA). All drugs were added to the bath solution (high-K ⁺ -Bic-ACSF). A740003, DPCPX, SDZ WAG 994 and CGS15943 were pre-dissolved in dimethyl sulfoxide (DMSO) before adding to ACSF with a final DMSO concentration of < 0.02 v%. Results

Induced ictaform activity is stable over time

In total, we induced SLEs in 77 neocortical slices from 17 patients who underwent surgical treatment of pharmacoresistant TLE. SLEs were induced with 8 mM K-ACSF and 50 μΜ bicuculline-methiodide. Epileptiform events with durations longer than 5 s were classified as SLEs and typically were biphasic with a tonic-like and subsequent clonic-like activity superimposed on a slow negative DC shift (Fig 1A). SLEs in the control group were stable for at least 90 minutes regarding incidence and event duration (n = 13 slices, six patients; Fig. 1 B, two left panels), reminiscent of a status epilepticus situation in vivo. A slight, but significant, reduction of average slow field potential amplitude (SFP) and maximum amplitude (xAmp) after 60 and 90 minutes was observed when each time point was compared to the amplitude values of the control period (see table 2, Fig. 1 B, two right panels).

Effects on SLEs by exogenous activation of adenosine and P2 receptors with ATP

Initially, to test for purinergic modulation of SLEs, 1 1 slices from five patients were treated with 300 μΜ ATP for 20 min, a concentration known to rapidly attenuate peak frequency power in gamma oscillations. In two out of these 1 1 slices SLEs were suppressed upon ATP application (Fig. 2A and B, 4C). The remaining nine slices showed variable effects of ATP that may relate to differential activation of purinergic receptors after degradation due to variable presence of ATP degrading activity. Average values for incidence of SLEs in all slices was 0.7 ± 0.3 ev/min (control) and 0.5 ± 0.6 ev/min during ATP application (Wilcoxon, p = 0.15, n = 1 1 ). The two slices with SLE suppression were excluded from statistical analysis of the following SLE parameters. Average duration of non-suppressed slices was 20.9 ± 4.8 s during control and 17.4 ± 10.6 s during ATP application, SFP was 1 ± 0.5 mV and 0.9 ± 0.7 mV, xAmp 1 .6 ± 0.6 mV and 1 .4 ± 0.8 mV, respectively (n = 9 and Wilcoxon each; Dur: p = 0.17, SFP: p = 0.37, xAmp: p = 0.1 1 ).

We also tested for effects of endogenous activation of P2 receptors on SLEs by application of the P2Y R antagonist MRS2179 (30 μΜ), the P2X^, P2X ₃ and P2X _2/3 antagonist TNP-ATP (10 μΜ) or the P2X ₇ -R antagonists A740003 and A804598 (10 μΜ each) without adding ATP to the ACSF. None of these selective P2 antagonists relevantly influenced SLE incidence (Fig. 2C) and other parameters of SLEs compared to control phase (exemplified for Inc, see Fig. 2C). Since human tissue slices are a scarce resource, we stopped further testing of P2 antagonists after three to five slices with each specific antagonist. ATP effect is mediated by adenosine receptors

In order to differentiate whether the effect of exogenous ATP application on SLEs is mediated by adenosine receptors or P2 receptors, we next applied 300 μΜ ATP in presence of adenosine receptor blockers (total n = 5, 10 μΜ CGS15943 (n = 4) or 1 μ Μ DPCPX (n = 1 ), three patients; Fig. 3A). Under these conditions, one slice displayed SLE suppression. However, ATP application was insufficient to significantly alter SLE incidence (0.6 ± 0.4 ev/min during control and 0.5 ± 0.5 ev/min with ATP; n = 5, Wilcoxon, p = 0.41 ). In presence of adenosine receptor antagonists, mean duration and amplitudes values ± SD of SLEs during ATP application were also not relevantly altered. Control SLE duration was 32.2 ± 16.1 s and ATP: 35.3 ± 18.9 s. Amplitude values remained stable during ATP application (control SFP 1 .68 ± 0.4 mV and ATP: 1 .72 ± 0.3 mV; xAmp 2.09 ± 0.4 mV and 2.19 ± 0.4 mV, respectively). Therefore, the suppressing effect of ATP on SLEs is mainly mediated by adenosine receptors after extracellular degradation of ATP, presumably by the A-i receptor subtype.

We then studied the effect of 300 μΜ adenosine on SLEs without activating P2 receptors in eight slices from two patients (Fig 3B). In one slice adenosine suppressed SLEs (Fig 4C). Average SLE incidence for all eight slices decreased from 0.8 ± 0.3 ev/min during control period to 0.5 ± 0.4 ev/min during adenosine application (n = 8, Wilcoxon, p = 0.06). In the seven slices without SLE block, the remaining parameter values did not significantly differ (Fig. 3B). i receptor agonist suppresses SLEs in large majority of slices

Direct activation of the A-i receptor with 1 μΜ SDZ WAG 994 completely suppressed SLEs in 73 % of 1 1 slices (five patients, Fig. 4A and B). SLE incidence of all 1 1 slices decreased significantly from 0.5 ± 0.3 ev/min to 0.1 ± 0.1 ev/min (p = 0.003, Wilcoxon). Average SLE duration of three slices from two patients with remaining SLEs during drug application decreased slightly with 27.3 ± 15.2 s during control and 24.5 ± 19.8 s during the Ai agonist. Average SFP and xAmp values of SLEs were lower during Ai agonist application. Amplitude values were: SFP: 1 .2 ± 0.8 mV and 0.9 ± 0.5 mV and xAmp: 1 .9 ± 1 .1 mV and 1 .6 ± 0.8 mV during control and SDZ WAG 994, respectively (n = 3 each).

Additionally, we compared the efficacy of exogenous application of ATP, adenosine and the A-i agonist to block SLEs. With 1 μΜ of the A-i agonist, full blockade was observed in 73 % of slices vs. 18 % with 300 μΜ ATP vs. 13 % with 300 μΜ adenosine (Fig. 4C). The differences in efficacy of the A agonist compared to ATP and to adenosine were significant (p = 0.03, Fisher Exact Test for A agonist (n = 1 1 ) vs. ATP (n = 1 1 ); p = 0.02 for A agonist vs. adenosine (n = 8)) while no significant difference was observed between ATP and adenosine (p = 1 , Fisher Exact Test) rendering a relevant role of endogenous P2 signaling unlikely.

Ai receptor agonist efficacy is maintained in slices with proven resistance to CBZ

In a subset of slices, we determined SLE sensitivity to a very high concentration of CBZ with 50 μΜ exceeding maximal plasma levels and being about five times higher than in CSF in patients, but being at the upper end of tissue concentrations in resected human material. Slices, which upon CBZ administration did not show large reductions in SLE incidence, duration and/or amplitudes or a change in type of epileptiform activity from SLEs to recurrent epileptiform transients, were kept for subsequent testing with the A-i receptor agonist (n = 17, four patients, Fig. 5A). In these slices, SLE parameters during CBZ application were analyzed and compared to a set of time-matched control recordings (n = 13, six patients) where SLEs were induced without further pharmacological manipulation. All four parameter values characterizing SLE in presence of CBZ did not significantly differ from time-matched control recordings (Fig 5B). We therefore conclude that this subset of slices was indeed CBZ-resistant.

SDZ WAG 994 (1 μΜ) was applied to CBZ-resistant slices after CBZ was washed out for at least 30 minutes in order to minimize CBZ-mediated effects (Fig 5C). The A-i agonist completely suppressed CBZ-resistant SLE activity in 71 % of 17 slices. Incidence of SLEs was consistently and significantly reduced in all slices during application of the Ai agonist from an average control value of 0.7 ± 0.3 ev/min to 0.05 ± 0.1 ev/min (Wilcoxon, n = 17, p < 0.001 , Fig. 5D). In the five slices without suppression, alterations of average SLE parameters Dur, SFP and xAmp were not statistically significant. Dur declined from 30.1 ± 5.4 s to 23.3 ± 15.3 s (Wilcoxon, n = 5, p = 0.5, Fig. 5D). Average SFP and xAmp values were slightly lower with the Ai agonist (SFP: 1 ± 0.4 mV to 0.8 ± 0.6 mV, Wilcoxon, n = 5, p = 0.23; xAmp: 1 .4 ± 0.5 mV to 1 .3 ± 0.6 mV, Wilcoxon, n = 5, p = 0.23). We conclude that the Ai agonist suppressed SLE activity in a great majority of slices in spite of resistance to CBZ.

Discussion

The principal finding of our study is that adenosine A-i agonists like e.g. SDZ WAG 994 are sufficient to suppress seizure-like activity in human neocortical slices and, in addition, are equally effective in blocking SLEs that are CBZ-resistant.

Except for one slice, we found that all effects of ATP could be explained by activation of adenosine A-i receptors. This observation compares well to the studies in which the anticonvulsant activity of adenosine or modulation of adenosine levels was examined, including a mouse model of CBZ resistant temporal lobe epilepsy. Moreover, our data did not indicate intrinsic ATP effects on ATP-activated ionotropic P2X or metabotropic P2Y receptors. Importantly, we demonstrated that induced SLEs in human neocortical slices of pharmacoresistant patients are sufficiently stable to conduct extended pharmacological experiments. It is important to note that in our model epileptiform activity is unlikely to be pharmacoresistant a priori by virtue of induction since CBZ (and VPA and PHT) block or reduce induced SLEs in a minority of slices (Fig. 5A upper trace). Moreover, pharmacoresistance of SLEs was independent of induction protocol in temporal cortex, dentate gyrus and subiculum. As exemplified here with CBZ, reproducing previous findings from our lab, we suggest that pharmacoresistance is preserved in most of our slices.

Effects of i receptor activation and efficacy of agonist compared to CBZ

The attenuating effects of adenosine on network activity have been known for decades. The main inhibitory mechanism of A-i receptors on synaptic transmission is a reduced release probability of presynaptic glutamate at least in part due to reduced calcium influx. Also postsynaptic G protein-coupled inwardly rectifying potassium (GIRK) channels are activated by G-protein coupled A-i receptors, which leads to hyperpolarization. Our results suggest that A-i receptor mediated modulation of presynaptic transmitter release and postsynaptic hyperpolarization is sufficient to control CBZ-resistant seizure-like activity in a large proportion of slices form patients with pharmacoresistant epilepsy.

CBZ concentrations in our slice experiments were on the upper end of the maximal CBZ concentrations found in brain tissue of TLE patients. This condition may point to a superior efficacy of the A-i agonist inhibiting epileptiform activity in slices compared to CBZ.

Albeit lower affinity, adenosine still activates A _2a receptors which are known to mediate proconvulsant effects by facilitating glutamate release and NMDA-receptor activation. Although here we used a relatively high concentration of SDZ WAG 994 (1 μΜ) it is still tenfold below the Ki value for the A _2a receptor and high rates of SLE suppression, especially compared to adenosine application, render a relevant activation of A _2a receptors unlikely. In addition, intraslice drug concentrations are expected to be considerably lower than bath concentrations due to slow diffusional equilibration and possible metabolic or uptake processes. Fast metabolism and activation of proconvulsant adenosine receptors may be responsible for less efficient SLE control by adenosine compared to selective A-i receptor agonists. Our comparison of adenosine and SDZ WAG 994 suggests higher efficacy of the A-i agonist. Given that inflammatory processes may play a major role in epileptogenesis and adenosine differentially modulates immune responses via A-i and A _2a receptors, it seems preferable to directly target the A-i receptor for its anticonvulsant and antiepileptogenic functions.

In summary, it is conceivable that direct targeting of the A-i receptor with specific agonists is effective in preventing ictogenesis. Moreover, fewer adverse effects might be expected when applying specific A- _\ agonists instead of adenosine.

Bullet points:

Specific A1 -receptor activation by use of adenosine A1 receptor agonists suppresses seizure-like events in 73 % of human neocortical slices and significantly reduces incidence in remainder.

Adenosine A1 agonists are very efficient in blocking seizure-like events shown to be insensitive in vitro to a high dose of carbamazepine.

Efficacy in suppressing seizure-like events is significantly higher with the specific A1 agonist compared to adenosine or ATP application.

Human neocortical slices from patients with pharmacoresistant epilepsy allow extended pharmacological experiments on induced seizure-like events in vitro.

SDZ WAG 994 is expected to be efficient in stopping status epilepticus with tolerable side effects in patients where first and second line treatments are not efficient.