HK1166613A - Treating seizures using ice inhibitors - Google Patents

Description

Treatment of seizures using ICE inhibitors

The application is a divisional application of Chinese patent application with the application number of 2005800228318(PCT/US2005/017177), the application date of 5/16/2005 and the invention name of 'treating epileptic seizure by using ICE inhibitor'.

Technical Field

The present invention relates to methods and compositions for treating or preventing seizures using ICE inhibitors.

Background

Cytokines (especially IL-1. beta. and TNF-. alpha.) are optimal therapeutic targets because they are capable of initiating and sustaining a number of diseases. Various strategies are used to block cytokines, such as soluble receptors, antibodies, receptor antagonists or inhibitors. These specific anti-cytokine-based therapies have been shown to reduce inflammation in many chronic inflammatory or autoimmune diseases and are FDA approved for use in humans (Bresinahan et al, 1998; Mohler et al, 1993; Nuki et al, 2002; vanDeven, 1999).

Interleukin-1 β converting enzyme (ICE, also known as caspase-1) is an intracellular protease that cleaves precursors of IL-1 β and IL-18 into active cytokines (Akita et al, 1997; Kuida et al, 1995). Although other proteases (including bacterial and host proteases) can possess pro-IL-1. beta. properties, ICE-deficiency (ICE)^-/-) Mice have been shown to be unable to release mature IL-1 β in response to endotoxin [ Fantuzzi et al, 1997; li et al, 1995]。

Expression of pro-and anti-inflammatory cytokines in the brain is linked to seizures [ a. vezzani et al, "Interleukin-1 β; immunoactive and Microgliaare Enhanced in the Rat hipppocampus by Focal Kainate Application: functional evaluation for Enhancement of electronic devices "J.Neurosci., 19, pp.5054-5065 (1999); desimoni et al, "infectious cells and related genes and are induced in the rate of the pathological status epilayers" Eur.J.Neurosci., 12, pp.2623-2633 (2000); vezzani et al, "Power full anticionvulsant Action of IL-Receptor Antagonist one anticentropic temporal Injection and astrological Overexpression in Mice" PNAS, 97, pp.11534-11539(2000) ]. However, there is currently no acceptable anti-cytokine or anti-inflammatory drug for use as an anti-convulsant or anti-epileptic therapy.

Disclosure of Invention

The present invention relates to methods of treating or preventing seizures, convulsions, epilepsy, and related conditions by administering an ICE inhibitor.

The invention also relates to compositions for treating or preventing seizures, convulsions, epilepsy, or related disorders.

The invention also relates to methods of identifying components useful for treating or preventing such disorders.

The invention also relates to a method for preparing the composition and a kit for carrying out the method of the invention.

Drawings

FIG. 1 depicts the effect of Compound 1 (containing 25 μ g of 4 μ L icv) on levels of aspartate-specific cysteine protease (caspase) -1 (assessed by Western blotting) in the hippocampus of kainic acid-treated rats. Rats were sacrificed 90min after onset of EEG seizures induced by intrahippocampal microinjection of 40ng kainic acid (see also fig. 2A and 2B).

FIGS. 2A and 2B represent the results of Western blot analysis of ICE/caspase-1 and IL-1 β levels 90min after kainic acid-induced seizures in Pseudohippocampus, with or without treatment with Compound 1. Fig. 2A and 2B are histogram representations of western blot data, which are the mean ± SEM of 4 rats. Compound 1(25 μ g/4 μ L) or vehicle was injected intracerebroventricularly 45 and 10min before injection of kainic acid (40ng) in the hippocampus. Compound 1 blocks seizure-induced production of the mature form of caspase-1 (see also FIG. 1) and the mature form of IL-1 β. P < 0.05; p < 0.001, according to Tukey test. See example 1 and example 6.

Detailed description of the invention

The present invention provides methods of treating or preventing seizures by administering an amount of an ICE inhibitor effective to treat or prevent seizures.

Applicants have demonstrated that the use of ICE inhibitors is effective in treating rodent seizures. In particular, applicants have demonstrated that treatment with ICE inhibitors extends the time of onset of seizures, shortening the time spent on seizures. ICE inhibitor compound 1 is equally effective with high doses of phenytoin or carbamazepine, which are known anticonvulsant compounds.

Accordingly, one embodiment of the present invention provides a therapeutic strategy for inhibiting seizures. These methods may be used to modulate, ameliorate, treat or prevent seizures. These methods can also be used to ameliorate, treat or prevent the progression or worsening of seizures. Such methods would involve administering an ICE inhibitor, for example, after traumatic brain injury, infection, or febrile seizures, to prevent or reduce the severity of permanent seizure disorders.

Other embodiments of the present invention provide therapeutic strategies for modulating, ameliorating, treating or preventing epilepsy, convulsions, and related disorders.

Applicants have also shown that compound 1 and compound 2 inhibit seizures when administered by the intraperitoneal route (table 3).

ICE inhibitor compounds are known to have anti-inflammatory activity in animal models of rheumatoid Arthritis, skin inflammatory diseases, inflammatory bowel diseases, and the like [ G.Ku et al, "selected Interlukin-1 Converting Enzyme (ICE/Caspase-1) inhibition with Pralnacasan (HMR 3480/VX-740) reduction infection and Joint deletion in Murine Type-II Collagen-induced Arthritis (CIA)" American College of Rheumatology, San Francisco, November 12-15, 2001; ku et al, "Interleukin-1 β converting enzyme (ICE, Caspase-1) Inhibition with VX-765Reduces Inhibition and Cytokine Levels in Murine Dermatitis and deratthritis Models" International consistency of Immunology, Stockholm, Sweden, July 22-27, 2001; ku et al, "interference-1 β Converting Enzyme (ICE, Caspase-1) Inhibition with VX-765 recovery and Cytokine Levels in MurinOxazolone-induced Dermatitis" The Society for investigative Dermatology, May 9-12, 2001 Abstract # 856; see also the ICE inhibitor literature cited therein ]. Compound 1 has also been shown to have anti-inflammatory activity in Rheumatoid arthritis patients [ K.Pavelka et al, "Clinical Effects of Pralnacasan (PRAL), an organic-active interference in-1 β Converting Enzyme (ICE) Inhibitor, in a 285 Patient PHII Trial in Rheumatoid Arthritis (RA)" American College of Rheumatology 2002 Conference Late-Breaking extract, New Orelans, October 25-29, 2002 ]. ICE inhibitors have not been used to treat seizures or seizure disorders.

The pharmacokinetics underlying the anti-inflammatory activity of these compounds in animals and humans are well understood. Furthermore, the applicant has observed that these compounds penetrate into the brain, although at significantly lower concentrations than blood and certain peripheral tissues. This latter feature is postulated to be essential for the activity of any anticonvulsant or antiepileptic agent, and it is not clear whether these compounds achieve brain concentrations sufficient to inhibit ICE/caspase-1 production and IL-1 β production in the brain and its contribution to seizure formation. Applicants have demonstrated that compound 1 and compound 2 nonetheless have anticonvulsant activity when administered peripherally.

The beneficial effects of ICE inhibitors on seizures are not directly related to the anti-inflammatory activity of ICE inhibitors. A known anti-inflammatory agent, ibuprofen, was tested in applicants' seizure model and administered by the intraperitoneal route. Ibuprofen increases seizure activity compared to vehicle (see table 4). Ibuprofen prolongs the duration of status epilepticus relative to the vehicle, indicating that ibuprofen increases or induces seizure activity.

The examples provided herein relate to rodent models of seizures, which are considered good models of epilepsy and convulsive disorders in humans. For example, antiepileptic drugs (e.g., carbamazepine and phenytoin) are known to exhibit anticonvulsant activity in this model, as are ICE inhibitors.

Although applicants have investigated the anticonvulsant activity of these compounds after intracerebroventricular and intraperitoneal administration, previous experience with compound 1 and compound 2 administered by a variety of peripheral routes, including intraperitoneal, oral, and intravenous, has shown that these compounds will also have anticonvulsant activity when administered by these alternative routes. In a preferred embodiment, the ICE inhibitor is administered peripherally (i.e., orally or parenterally, not intracranially).

The present invention relates to the use of ICE inhibitor compounds. Such compounds may be ICE selective. Or such compounds may be active against ICE and active against another caspase or some other caspase (e.g., 2-14). As demonstrated herein, inhibiting ICE and inhibiting IL-1 β production will delay the time to onset of seizures, shorten the amount of time spent on seizures, or reduce the frequency of seizures, including any one or more or all of the foregoing. The data generated in examples 1 and 6 demonstrate that anticonvulsant doses of compound 1 have the expected mechanism-related effects on ICE/caspase-1 activation and IL-1 β production.

In the methods of the invention, the compound will be administered in an amount effective to inhibit ICE, thus treating seizures (or other related conditions). Treating a seizure (or other related condition) includes reducing the duration of the seizure, reducing the severity of the seizure, reducing the sensitivity to the onset of the seizure, delaying the onset of the seizure, and eliminating the occurrence of the seizure. Accordingly, the present invention also provides methods of preventing seizures (or other related conditions) by administering an amount of an ICE inhibitor effective to prevent seizures.

The methods of the invention may be used to treat animals, preferably mammals, including humans and non-human mammals. Any compound that inhibits ICE may be used in the methods and compositions of the present invention. Such compounds include those that selectively inhibit ICE and those that inhibit caspases or one or more enzymes of the ICE/CED-3 family. The compounds used in the present invention inhibit the catalytic activity of ICE in a reversible or irreversible manner.

The compounds of the invention inhibit ICE and/or reduce the levels of IL-1, particularly IL-1 β and IL-18. These compounds can be assayed for their ability to inhibit ICE, IL-1 β and/or IL-18 production, modulate IL-1 and/or IL-18 levels, and/or affect IL-1 β and/or IL-18 activity, for example. Assays for testing each of these activities are known in the art (see examples herein, WO 95/35308, WO97/22619, WO 99/47545, or WO 01/90063). Thus, these compounds are capable of targeting and inhibiting events in ICE and/or IL-1 mediated diseases as described herein.

Compounds that may be used in the present invention include, but are not limited to, the following: WO04/058718, WO04/002961, WO 03/088917, WO 03/068242, WO03/042169, WO98/16505, WO 93/09135, WO 03/106460, WO 03/103677, WO 03/104231, WO 02/085899, WO 00/55114, WO 00/55127, WO00/61542, WO 01/05772, WO 01/10383, WO 01/16093, WO 01/42216, WO 01/72707, WO 01/90070, WO 01/94351, WO 02/094263, WO 02/42278, US 6,184,210, US 6,184,244, US 6,187,771, US 6,197,750, US 6,242,422, April 2001 American Chemical Society (ACS) meeting in San Diego, California, USA, WO 02/22611, US 2002/0058630, WO 02/12638, WO 95/35308, 5,716,929, WO97/22619, U.S. Pat. No. 6,204,261, WO 99/47545, WO01/90063, U.S. Pat. No. 2004/0014753, U.S. Pat. No. 2004/0009966, U.S. Pat. No. 2003/0236296, U.S. Pat. No. 6,693,096, U.S. Pat. No. 6,610,683, U.S. Pat. No. 6,531,467, U.S. Pat. No. 6,528,506, U.S. Pat. No. 6,200,969, WO 2003/072528, WO 2003/032918, WO 01/00658, WO 98/10778, U.S. Pat. No. 6,716,818, U.S. Pat. No. 6,620,782, U.S. Pat. No. 6,566,338, U.S. Pat. No. 6,495,522, U.S. Pat. No. 6,355,618,6,153,591, WO 2005/003100, WO 2004/002401, WO00/61542, WO 00/55114, WO99/47154, U.S. Pat. No. 6,083,981, U., U.S. Pat. No. 6,316,415, U.S. Pat. No. 5,932,549, U.S. Pat. No. 5,919,790, U.S. Pat. No. 5,744,451, EP1082127, EP 1049703, EP 0932600, EP 0932598, WO 99/56765, WO93/05071, EP 0600880, and EP 1378573 (all of which are incorporated herein by reference as if set forth herein). Preferred compounds for use in the present invention include those of WO04/058718, WO04/002961, WO 95/35308, WO97/22619, WO 99/47545 and WO 01/90063. Other preferred compounds for use in the present invention include those of WO 95/35308, WO97/22619, WO 99/47545 and WO 01/90063. More preferred compounds are those cited in the claims herein. These compounds can be obtained by methods known to the skilled worker and disclosed in the literature cited herein.

The invention also provides assays for testing the compounds for anti-seizure, anti-epileptic, or anti-convulsive activity according to the methods herein. Such methods involve, for example, identifying a compound that is useful for treating seizures, convulsions, epilepsy, or a related disorder, including determining the ability of the compound to inhibit ICE and/or inhibit seizures, convulsions, epilepsy, or a related disorder. Other methods of the invention relate to determining the anticonvulsant activity of an ICE inhibitor. Such methods and assays are useful for identifying compounds for treating seizures, convulsions, epilepsy, or related disorders. In preferred embodiments, these assays can be performed by methods substantially as described herein (see, e.g., examples 1, 2 or 3).

The pharmaceutical compositions and methods of the invention will therefore be useful for controlling IL-1 levels and/or activity in vitro or in vivo. The compositions and methods of the invention will thus be useful for controlling IL-1 levels in vivo, treating certain disorders or reducing their progression, severity or outcome, including the diseases, disorders or outcomes described herein.

According to another embodiment, the present invention provides a composition comprising a compound of the present invention, or a pharmaceutically acceptable derivative (e.g., salt) thereof, as described above, and a pharmaceutically acceptable carrier.

According to another embodiment, the compositions and methods of the present invention may further comprise another therapeutic agent. Such ingredients include, but are not limited to, compounds that treat or inhibit seizures, convulsions, or epilepsy, such as barbiturates (e.g., mebendazole, pentobarbital), benzodiazepinesClasses (e.g. lorazepam, clonazepam, chlordiazepoxideDiazepam), GABA analogs (e.g., tiagabine, gabapentin, pregabalin, vigabatrin), hydantoins (e.g., phenytoin), phenyl triazines (e.g., lamotrigine), succinimides (e.g., methylsuccinamide, ethosuximide) or others, compounds (e.g., carbamazepine, riluzole, valproate, divalproex, felbamate, praminone or topiramate), anti-inflammatory agents, matrix metalloproteinase inhibitors, lipoxygenase inhibitors, cytokine antagonists, immunosuppressive agents, anticancer agents, antiviral agents, cytokines, growth factors, immunomodulatory agents (e.g., bromocriptine, anti-human interferon-alpha antibodies, IL-2, GM-CSF, methionine enkephalin, interferon-alpha, diethyl dithiocarbamate, tumor necrosis factor, naltrexone, and rEPO), prostaglandins, or anti-hyperproliferative vascular compounds.

The term "pharmaceutically acceptable carrier" means a non-toxic carrier which can be administered to a patient with a compound of the present invention without destroying its pharmacological activity.

Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances (e.g., phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (e.g., protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polypropylene-block polymers, polyethylene glycol, and wool fat.

In pharmaceutical compositions comprising only the compounds of the present invention as active ingredients, the methods of administering these compositions may additionally comprise the step of administering to the subject an additional ingredient. Such ingredients include, but are not limited to, compounds that treat or inhibit seizures, convulsions, or epilepsy, such as barbiturates (e.g., mebendazole, pentobarbital), benzodiazepinesClasses (e.g. lorazepam, clonazepam, chlordiazepoxideDiazepam), GABA analogs (e.g., tiagabine, gabapentin, pregabalin, vigabatrin), hydantoins (e.g., phenytoin), phenyl triazines (e.g., lamotrigine), succinimides (e.g., methylsuccinamide, ethosuximide) or others, compounds (e.g., carbamazepine, riluzole, valproate, divalproex, felbamate, praminone or topiramate), anti-inflammatory agents, matrix metalloproteinase inhibitors, lipoxygenase inhibitors, cytokine antagonists, immunosuppressive agents, anticancer agents, antiviral agents, cytokines, growth factors, immunomodulatory agents (e.g., bromocriptine, anti-human interferon-alpha antibodies, IL-2, GM-CSF, methionine enkephalin, interferon-alpha, diethyl dithiocarbamate, tumor necrosis factor, naltrexone, and rEPO), prostaglandins, or anti-hyperproliferative vascular compounds. When a second component is used, it may be administered in a separate dosage form or as part of a single dosage form with a compound or composition of the invention.

The amount of compound in the above compositions should be sufficient to result in a detectable decrease in the severity of the disease or in ICE inhibition, IL-1 levels, or IL-1 activity.

If pharmaceutically acceptable salts of the compounds of the present invention are employed in these compositions, these salts are preferably derived from inorganic or organic acids and bases. Such acid salts include the following: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate. Base salts include ammonium salts, alkali metal salts (e.g., sodium and potassium salts), alkaline earth metal salts (e.g., calcium and magnesium salts), salts with organic bases (e.g., dicyclohexylamine salts, N-methyl-D-glucamine salts), and salts with amino acids (e.g., arginine, lysine), and the like.

Further, the basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates, such as the dimethyl, diethyl, dibutyl, and diamyl esters of sulfuric acid; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; aralkyl halides such as benzyl and phenethyl bromides and others. Thereby obtaining a water-or oil-soluble or dispersible product.

The compounds used in the compositions and methods of the present invention may also be modified by the addition of suitable functional groups to enhance selective biological properties. Such modifications are known in the art and include increasing the biological permeability into a given biological system (e.g., blood, lymphatic system, central nervous system), increasing oral availability, increasing solubility for administration by injection, altering metabolism, and/or altering excretion rates.

According to a preferred embodiment, the compositions of the present invention may be formulated for administration to a subject, such as a mammal, preferably a human.

Such pharmaceutical compositions of the invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the composition is administered orally.

Sterile injectable forms of the compositions of the present invention may be aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Among the acceptable carriers and solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oily solutions or suspensions may also contain a long chain alcohol diluent or dispersant, for example, carboxymethyl cellulose or similar dispersing agents, which are commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. For formulation purposes, other commonly used surfactants such as tweens, spans, and other emulsifying agents or bioavailability enhancers, which are commonly used in the preparation of pharmaceutically acceptable solid, liquid, or other dosage forms, may also be used.

If a solid carrier is used, the preparation may be compressed, placed in a hard gelatin capsule, in the form of a powder or granules, or in the form of tablets or lozenges. The amount of solid carrier will vary, for example, from about 25mg to 400 mg. When a liquid carrier is used, the preparation may be in the form of, for example, a syrup, an emulsion, a soft gelatin capsule, a sterile injectable liquid such as an ampoule or a non-aqueous liquid suspension. Where the composition is in the form of a capsule, any conventional encapsulation method is suitable, for example using the above-mentioned carriers in a hard gelatin capsule shell.

Syrup formulations may consist of a suspension or solution of the compound in a liquid carrier, such as ethanol, glycerol or water, containing flavoring or coloring agents. Aerosol formulations may consist of a solution or suspension of the compound in a liquid carrier, for example, water, ethanol or glycerol; in dry powder aerosols, the formulation may include, for example, a humectant.

The formulations of the invention comprise the active ingredient together with one or more acceptable carriers and optionally any other therapeutic ingredients. A carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

The pharmaceutical compositions of the present invention may be administered orally in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, commonly used carriers include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in capsule dosage forms, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral administration, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.

Alternatively, the pharmaceutical compositions of the present invention may be administered rectally in the form of suppositories. They may be prepared by mixing the drug with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of the present invention may also be administered topically, particularly when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, skin or lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.

Topical administration to the lower intestinal tract may be carried out as a rectal suppository (see above) or as a suitable enema. Topical transdermal patches may also be used.

For topical administration, the pharmaceutical compositions may be formulated in a suitable ointment containing the active ingredient suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions may be formulated in a suitable lotion or cream containing the active ingredient suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

For ophthalmic purposes, the pharmaceutical compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or preferably as solutions in isotonic, pH adjusted sterile saline, with or without preservatives such as benzalkonium chloride. Alternatively, for ophthalmic use, the pharmaceutical compositions may be formulated as ointments, such as petrolatum. In one embodiment, the composition is formulated, for example, as in U.S. patent 6,645,994 and/or U.S. patent 6,630,473.

The pharmaceutical compositions of the present invention may also be administered by means of nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.

One skilled in the art will recognize that the form and characteristics of the pharmaceutically acceptable carrier or diluent will depend on the amount of active ingredient combined therewith, the route of administration, and other well known variables.

The above compounds and compositions are also useful in therapeutic applications involving certain diseases associated with seizures or convulsions.

The compounds of the invention are capable of inhibiting the release of IL-1 β and/or IL-18 and thus can be used to inhibit or block several of the pathophysiological consequences of certain diseases described herein.

The present invention also relates to therapeutic methods for treating certain diseases by (1) inhibiting the release of IL-1 from cells, and/or (2) preventing the undesirable, toxic or fatal consequences of excessive tissue levels of IL-1 in mammals, including humans. Such methods comprise administering to the mammal an effective ICE inhibiting amount of one or more ICE/CED-3 inhibitors. Such methods can also be used for prophylactic treatment or prevention of certain diseases for which they are indicated, including seizures, convulsions, epilepsy, or related conditions. The present invention provides methods of treating these conditions by administering to a mammal (including a human being) in need thereof an effective amount of such compounds.

These compounds directly promote the arrest or resolution of certain diseases and the restoration of normal function by inhibiting ICE and retarding the release of IL-1 or reducing IL-1 levels and activity, as well as the pathophysiological effects of excess IL-1 levels in each of these environments. Together, these effects relate to their novel use in the treatment of seizures and related conditions.

ICE inhibition may be measured by methods known in the art and described more fully herein.

These compounds can be used to inhibit the release of IL-1 by monocytes, macrophages, neuronal cells, endothelial cells, epidermal cells, mesenchymal cells (e.g., fibroblasts, skeletal muscle cells, smooth muscle cells, cardiac muscle cells) and many other cell types.

The term "disorder" or "condition" means any disease, disorder or effect that produces a detrimental biological outcome in a subject.

The term "seizure" as used herein generally refers to sudden and involuntary contractions of muscles throughout all or part of the body, which contractions result from abnormal excitation of a group of neurons of the central nervous system. Seizures are a symptom of epilepsy. The motor manifestations of epileptic seizures are accompanied by changes in the electroencephalogram (EEG). These changes can also occur in the absence of significant motor performance.

The level of IL-1 protein in the patient's blood or cells or cell culture (i.e., within the cells or cell culture medium) can be determined, for example, by measuring immunospecific binding to IL-1 or other proteins known to be produced as a result of the presence of active IL-1. Such methods are known in the art. For example, immunoassays that may be used include, but are not limited to, competitive and non-competitive assay systems, western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein a immunoassays, and FACS analyses with labeled antibodies. Such assays are well known in the art (see, e.g., Ausubel et al, eds., 1994, Current Protocols in molecular biology, Vol.1, John Wiley & Sons, Inc., New York, incorporated herein by reference in its entirety).

The level of IL-1 may also be determined using a competitive binding assay. An example of a competitive binding assay is a radioimmunoassay, includingIncubation of tagged proteins from IL-1 expressing cells in the presence of increasing amounts of unlabeled IL-1 (e.g.³H or¹²⁵I) And IL-1 antibody, and detecting the IL-1 antibody bound to the labeled IL-1. From this data, the affinity and binding rate of the relevant antibody to a particular antigen can be determined by means of Scatchard plot analysis. The competition of the second antibody can also be determined using radioimmunoassay. In this case, the antigen is incubated with the relevant antibody in the presence of increasing amounts of unlabeled secondary antibody, which is incubated with the labeled compound (e.g., with a labeled compound)³H or¹²⁵I) And (6) conjugation.

IL-1 levels can also be determined by activity, e.g., IL-1 levels can be determined by cell lines capable of detecting levels of biological activity of cytokines, such as IL-1 or growth factors. According to one embodiment, the level of biologically active IL-1 in a biological sample is detected by incubating a cell line genetically processed with isopropyl-b-D-thiogalactopyranoside. The cell lines are incubated with test samples and cell death in the cell lines is monitored by measuring the intensity of the blue color, which is indicative of a biologically active cytokine or growth factor in the sample being tested. See, for example, Burns (1994)20 (1): 40-44 assays for IL-1 activity in patient sera.

Dosage levels of the active ingredient compounds useful in the monotherapy are between about 0.01 and about 100mg/kg body weight per day, preferably between about 0.5 and about 75mg/kg body weight per day, most preferably between about 1 and about 50mg/kg body weight per day. Doses of about 50mg/kg to about 200mg/kg have been tested and found to be effective (see examples herein). For intracranial administration, useful dosage levels of the active ingredient compound are between 1ng and 1g, preferably between 100ng and 100 mg.

Typically, the pharmaceutical compositions of the present invention will be administered from about 1 to 5 times per day, or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that may be combined with the carrier materials to form a single dosage form will vary depending upon the host treated and the particular mode of administration. Typical formulations will contain from about 5% to about 95% active compound (w/w). Preferably, such preparations contain from about 20% to about 80% active compound.

When the compositions of the present invention comprise a compound of the present invention in combination with one or more additional therapeutic agents, both the compound and the additional ingredient should be present in about 10% to about 80% of the normally administered dose in a monotherapy regimen.

Once the patient's condition has improved, maintenance doses of the compounds, compositions or combinations of the present invention can be administered, if necessary. Subsequently, the dose or frequency of administration, or both, can be reduced as a function of the symptoms to a level that maintains the improved condition, and the treatment should be discontinued when the symptoms have been alleviated to the desired level. However, once any recurrence of disease symptoms occurs, the patient may require intermittent treatment on a long-term basis.

It will also be understood that the specific dose and regimen of treatment for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the particular disease, the patient's predisposition to the disease being treated, and the judgment of the attending physician. The amount of active ingredient will also depend on the particular compound and other therapeutic agents in the composition, if any.

Thus, a method of treating or preventing a disease of the invention in a subject comprises the step of administering to the subject any of the compounds, pharmaceutical compositions, or combinations described herein.

In a preferred embodiment, the present invention provides a method of treating a mammal suffering from one of the above-mentioned diseases, comprising the step of administering to said mammal a pharmaceutically acceptable composition as described above. In such embodiments, if the patient is also administered another therapeutic agent, it may be delivered in a single dosage form with the compound of the invention, or as a separate dosage form. When administered as a separate dosage form, the additional therapeutic agent may be administered prior to, simultaneously with, or subsequent to the administration of the pharmaceutically acceptable composition comprising the compound of the present invention.

Methods of identifying compounds or compositions for treating diseases according to the invention include screening a plurality of compounds or compositions for the ability to improve the outcome of certain diseases and to improve the condition of patients suffering from certain diseases of the invention. According to one embodiment of the invention, high throughput screening can be achieved by placing cell cultures in a large number of wells of a microtiter plate, adding different compounds or compositions to each well, and comparing the ICE inhibition and/or IL-1 levels and/or activity in each cell culture to the levels or activity in the cell culture medium of control wells. Controls may be used in the comparison step according to the present invention, including cells or test material that have not been treated with a compound or composition and cells or test material that have been treated with a compound or composition known to have no effect on ICE inhibition or activity. According to one embodiment of the invention, high throughput screening is automated so that the data collection and analysis steps, including the addition of cells to the plate until after the compound or composition is added, are both performed by the machine. Instruments useful for the comparison step of the present invention, for example, instruments capable of detecting objects with a label (e.g., a radiolabel, a fluorescent or colored object) or objects that are themselves detectable, are commercially available and/or known in the art. Thus, compounds and compositions according to the present invention can be rapidly and efficiently screened for use in treating certain diseases disclosed herein.

Detailed Description

All applications, patents, and references disclosed herein are incorporated by reference. In order that the invention may be more fully understood, the following preparation and test examples are set forth. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention in any way.

Example 1

One-sided microinjection of kainic acid (0.5 μ L containing 40ng) into the dorsal hippocampus of free-moving rats induced seizure experimental models in male adult Sprague-Dawley rats using chronically implanted cannulas and electrodes. Briefly, animals were deeply anesthetized with Equisthesin (1% phenobarbital and 4% chloral hydrate; 3ml/kg, i.p.). Bipolar nichrome wire insulated electrodes (60 μm) were implanted bilaterally into the dentate gyrus (septal foramen) of the dorsal hippocampus, and a guide cannula (22 gauge) was positioned unilaterally on top of the dura mater, bonded to one of the deep electrodes, for intra-hippocampal injection of kainic acid. The coordinates of the hippocampal electrode implant from bregma were (in mm): nasal bridge-2.5, AP-3.5, L + -2.4, subdural 3.

Another guide cannula was positioned unilateral on top of the dura for intracerebroventricular injection of the compound (in mm, bridge of the nose-2.5, AP-1, L + 1.5). The ground wire was positioned over the sinus and two helical electrodes placed bilaterally over the apical cortex. The electrodes were attached to a multi-tap socket (March Electronics, NY) and fixed to the skull with acrylic dental cement along with an injection cannula.

Compound 1 (25. mu.g/4. mu.L) or an equal volume of vehicle was administered by intraventricular injection. Seizure was recorded and quantified by EEG analysis based on the following parameters: 1) the start time of the first episode, 2) the number of episodes during the 3 hour recording, and 3) the duration of each episode event were added together to estimate the time taken for episode activity. Compound 1 treatment significantly extended the latency to onset of convulsions, reducing the number of seizures and the total time spent in seizure activity (table 1).

The effect of compound 1 on ICE/caspase-1 activation was evaluated based on the amount of the active 20kD subunit detected by Western blotting of these rat samples. FIG. 1 shows that compound 1 treatment not only abolished the increase in caspase-120 kD subunit induced by kainate seizures, but also reduced this subunit to very low levels. The level of the inactive 45kD subunit of pro-caspase-1 is not altered by either kainate or Compound 1.

Table 1: rats received compound 1 (25. mu.g/4. mu.L) icv 45 and 10min before injection of 40ng of 0.5. mu.L kainic acid into the left hippocampus. The control (vehicle) received a 20% Cremophor saline solution.

P < 0.01, relative to the vector, according to student's t-test

Example 2

Rat seizure experimental models were induced by unilateral microinjection of kainic acid (0.5 μ L containing 40ng) into the dorsal hippocampus of free-moving rats using chronically implanted cannulae. Compound 1(30mg/kg) or vehicle was administered by intraperitoneal injection 45 and 10min prior to kainic acid. EEG seizures were recorded using chronically implanted hippocampal electrodes. Seizure and inter-seizure epileptic activity was quantified by EEG analysis based on the following parameters: 1) the start time of the first episode, 2) the number of episodes during the 3 hour recording, and 3) the duration of each episode event were added together to estimate the time taken for episode activity. Compound 1 treatment significantly extended the latency to onset of convulsions, reducing the total time taken to attack activity by-30%, although this difference did not reach statistical significance (table 2). These data suggest that higher doses will be effective to produce greater and statistically significant effects. See example 4, where higher doses of compound 2 produced statistically significant effects.

Table 2: rats received compound 1 intraperitoneally (30mg/kg) 45 and 10min before application of 0.5 μ L kainic acid containing 40ng in the left hippocampus. Control animals (vehicle) received 20% Cremophor saline solution.

P < 0.01, relative to the vector, according to student's t-test

Example 3

Inhibition of ICE

Compounds can be tested for their ability to inhibit ICE by methods known in the art (see, e.g., the references cited in figures 2-4).

Example 4

EEG seizures were induced in adult male Sprague-Dawley rats using a long-term implanted cannulated intra-hippocampal injection of 40ng Kainic Acid (KA). EEG seizures were recorded using chronically implanted hippocampal electrodes. Seizure and inter-seizure epileptic activity was quantified by EEG analysis based on the following parameters: 1) the start time of the first episode, 2) the number of episodes during the 3 hour recording, and 3) the duration of each episode event were added together to estimate the time spent by the episode activity. Intraperitoneal injection of Compound 2 or its vehicle (50-200mg/kg) was performed for 3 consecutive days. On day 4, rats received compound 2 45 and 10min prior to injection of 40ng of 0.5 μ L kainic acid into the hippocampus.

Table 3: effect of Compound 2 on Halinate-induced seizures in rats

Data are mean + -SE (7-15 rats)

P < 0.01, relative to the vector, according to one-way ANOVA followed by Dunnett's test

Example 5

The effect of ibuprofen on seizures was also examined using the method described in example 4. Rats received ibuprofen (50mg/kg, i.p.) 60min prior to injection of 40 μ g of 0.5 μ L kainic acid in the hippocampus on one side. Controls (vehicle) received saline, p < 0.05 vs vehicle, according to student's t-test. Seizures were analyzed and quantified using EEG. Status epilepticus represents continuous seizure activity lasting more than 30min continuously.

Table 4: carrier

Rat	Start (min)	Number of epileptic seizures	Seizure time (min)	Status epilepticus
					1	11.6	13.0	16.0	-
2	7.5	16.0	18.5	-
					3	21.0	20.0	21.0	-
4	10.0	15.0	23.0	-
					5	21.0	20.0	21.0	-
6	10.0	15.0	23.0	-
					7	11.6	17.0	25.0	-
Mean. + -. SE	13.2±2.1	16.6±1.0	21.1±1.1	-

Ibuprofen

Rat	Start (min)	Number of epileptic seizures	Seizure time (min)	Status epilepticus
					1	14.4	13	13.0	75
2	7.9	10	8.4	-
					3	11.0	13	11.0	66.6
4	12.3	12	12.5	80
					5	13.3	16	11.2	-
6	21.4	8	9.8	70
					7	10.0	10	9.4	80
Mean. + -. SE	13.0±1.7	11.7±1.0	10.8±0.6	74.4±2.6(5)

Example 6

The effect of compound 1 on kainate-induced IL-1 β production was also investigated as described in example 1. Hippocampus homogenate products were obtained from rats 90 minutes after intra-hippocampal kainate (40ng) microinjection and IL-1 β production, an ICE/caspase-1 activation, was assessed by Western blot analysis. Total protein (170. mu.g) was isolated from hippocampal homogenate products using SDS PAGE 10% acrylamide and transferred to Hybond nitrocellulose membrane by electron blotting. ICE/caspase-1 and IL-1. beta. immunoreactivity was assessed using selective antibodies, with an enhanced chemiluminescence assay. Hippocampus kainate injection induces the production of the active 20kD subunit of ICE/caspase-1 and the production of the active 17kD IL-1 β. Intraventricular injection (25. mu.g/4. mu.L) of Compound 1 inhibited ICE/caspase-1 activation, as evidenced by the inhibition of production of the active 20kD subunit of ICE/caspase-1, and by a reduction in production of the mature active 17kD IL-1 β (see FIGS. 1 and 2A for caspase-1 and 2B for IL-1 β).

Example 7

Tablet preparation

Compound 2 can be formulated for oral administration as described below and in table 6. The drug product was formulated to give 300mg of compound 2 per tablet.

Table 6: compound 2 composition, 300mg tablet

Components	Amount (mg/tablet)	Function(s)
			Compound A	300	Active ingredient
Microcrystalline cellulose (NF)	277.50	Filler
			Pregelatinized starch (NF)	131.25	Disintegrating agent
Sodium starch glycolate (NF)	15.00	Disintegrating agent
			Colloidal silicon dioxide (NF)	11.25	Glidants
Talc (USP)	7.50	Glidants
			Magnesium stearate (NF)	7.50	Lubricant agent
Total of	750

Reference to the literature

Vezzani et al, "Power ful anticionvsannt Action of IL-Receptor Antagonist on Intra temporal Injection and astrological overexpression in Mice" PNAS, 97, pp.11534-11539(2000).

Viviani et al, "Interleukin-1. beta. Enhances NMDA Receptor-Mediated Intracellular Calcium Increase Activation soft-he Src Family of KinasesJ.Neurosci.，23，pp.8692-8700(2003).

Rizzi et al, "glia activation and Cytokine inclusion in RatHippocampus by Kainic Acid-induced Status DuringPostnat Development" 14, pp.494-503(2003).

De Simoni et al, "inflammation genes and Related genetic Induced in the Rat Hippocampus by Limbic Status" 12, pp.2623-2633(2000).

Vezzani et al, "Interleukin-1 β Immunoreactivity and micro 1ia are Enhanced in the Rat Hippocampus by Focal KainateApp1 action: functional event for Enhancement of E1 electronic therapeutic SeizesJ.Neurosci.19，pp.5054-5065(1999).

All documents cited herein are incorporated herein by reference.

While a number of embodiments of the present invention have been described, it is apparent that the basic example may be altered to provide other embodiments in which the compounds and methods of the present invention may be employed. It is, therefore, to be understood that the scope of the invention is defined by the claims rather than the specific embodiments, which are presented for purposes of illustration only.

Claims (22)

1. A method of treating a seizure in a patient, comprising administering to the patient a compound that inhibits ICE/caspase-1.

2. A method of treating convulsions in a patient comprising administering to the patient a compound that inhibits ICE/caspase-1.

3. A method of treating epilepsy in a patient, comprising administering to the patient a compound that inhibits ICE/caspase-1.

4. A method for preventing epilepsy in a patient, comprising administering to the patient a compound that inhibits ICE/caspase-1.

5. A method according to any one of claims 1-4, wherein said compound inhibits ICE/caspase-1 and one or more other caspases.

6. The method according to any one of claims 1-5, wherein said compound is a selective ICE/caspase-1 inhibitor.

7. The method according to any one of claims 1 to 6, wherein the compound is a compound according to any of WO 95/35308, WO97/22619, WO 99/47545 and WO 01/90063.

8. The method according to any one of claims 1-6, wherein said compound is:

or any stereoisomer thereof, including:

(Compound 1).

9. The method according to any one of claims 1-6, wherein said compound is:

10. the method according to any one of claims 1-6, wherein said compound is:

or any stereoisomer thereof, including:

compound 2.

11. The method according to any one of claims 1-6, wherein said compound is:

or any stereoisomer thereof, including:

12. the method according to any one of claims 1-11, wherein said compound is administered peripherally (i.e., orally or parenterally, not intracranially).

13. The method according to any one of claims 1-12, further comprising administering an additional compound, wherein the additional compound is an anticonvulsant compound.

14. The method according to claim 13, wherein said additional compound is meprobitum, pentobarbital, lorazepam, clonazepamDiazepam, tiagabine, gabapentin, pregabalin, vigabatrin, hydantoins, phenytoin, lamotrigine, mesunamine, ethosuximide, carbamazepine, riluzole, valproate, divalproex, feurette, pramipexone or topiramate.

15. A pharmaceutical composition for ameliorating, treating or preventing seizures, convulsions or epilepsy in a patient, comprising a compound that inhibits ICE/caspase-1 and a pharmaceutically acceptable carrier.

16. The pharmaceutical composition according to claim 15, wherein the composition further comprises another anticonvulsant compound.

17. The pharmaceutical composition according to claim 16, wherein the additional compound is meprobitum, pentobarbital, lorazepam, clonazepamDiazepam, tiagabine, gabapentin, pregabalin, vigabatrin, hydantoins, phenytoin, lamotrigine-methyl succinate, ethosuximide, carbamazepine, riluzole, valproate, divalproex, feurette, pramipexole or topiramate.

18. A kit comprising a compound that inhibits ICE and instructions for using the compound to treat seizures, convulsions, or epilepsy.

19. A pharmaceutical composition according to any one of claims 15 to 17 or a kit according to claim 18 wherein the compound is as disclosed in any one of WO 95/35308, WO97/22619, WO 99/47545 or WO01/90063 or as referred to in claims 8 to 11.

20. An assay for identifying a compound for treating seizures, convulsions or epilepsy comprising determining the ability of said compound to inhibit ICE/caspase-1.

21. An assay for identifying an ICE/caspase-1 inhibitor having anti-seizure, anticonvulsant, or anti-epileptic activity, comprising determining the ability of the ICE/caspase-1 inhibitor to inhibit seizures, convulsions, or epilepsy.

22. An assay according to claim 20 or claim 21, wherein the assay is carried out by means of a method substantially as described herein.