Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
  • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
  • A61K31/404—Indoles, e.g. pindolol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/08—Antiepileptics; Anticonvulsants

Definitions

• the present invention relates to the anticonvulsant activity of GSK-3 inhibitors and the use of GSK-3 inhibitors as agents for the treatment of neurological disorders characterized by seizures such as epilepsy.
• a common and often debilitating symptom of many neurological disorders is abnormal neuronal activity in the brain.
• epilepsy describes a diverse set of neurological disorders that are characterized by seizures, convulsions, and/or other involuntary changes in body movement or function. Approximately 65 million people worldwide are estimated to suffer from epilepsy, but effective treatment options are limited.
• AEDs anti-epileptic drugs
• target neuronal receptors such as GABA receptors and sodium, glutamate, or calcium channels, in order to reduce neuronal excitability.
• traditional AEDs based on these targets have failed to control seizures in about 25-30% of all epilepsy patients, and further, some patients who initially respond to AEDs later experience drug-resistant (pharmacoresistant) seizures.
• GSK-3 inhibitors comprise heterocyclic compounds such as indoles, pyrrolo-pyrazines, benzofurans, as well as GSK-3 antisense molecules.
• the present invention relates to inhibitors of GSK-3 phosphorylation for use in the treatment of neurological disorders characterized by seizures, such as epilepsy.
• the present invention concerns an inhibitor of phosphorylation by
• the indole is an indole dimer.
• the indole dimer may be, for example, an indirubin compound.
• the indirubin compound is a compou e salt or prodrug thereof:
• R 2 is selected from O, NOH, and NO-CO-R 4 ;
• each R 3 is independently selected from -H and halo
• R 4 is Ci_6 alkyl
• n 0-5.
• the indole is a compound of formula (VI), or a pharmaceutically acceptable salt or prodrug thereof:
• a further aspect of the present invention relates to an inhibitor of phosphorylation by GSK3 for use as an anticonvulsant agent in treating neurological disorders characterized by seizures, wherein the inhibitor is a benzofuran or a pyrrolo-pyrazine.
• the GSK-3 inhibitor is a GSK-3 antisense molecule.
• the GSK-3 antisense molecule is a morpholino.
• GSK-3 is inhibited by NAi.
• the present invention concerns the use of an inhibitor of phosphorylation by GSK3 for the manufacture of a medicament for treating a neurological disorder characterized by seizures.
• the present invention concerns a method for treating a neurological disorder characterized by seizures in a subject in need of such treatment, said method comprising the administration of a therapeutically effective amount of an inhibitor of phosphorylation by GSK3 .
• the inhibitor of phosphorylation by GSK3 in said use for the manufacture of a medicament, or for use in said method for treating in particular is a compound or molecule as described herein such as e.g. a compound of formula (I), a salt or prodrug thereof.
• the neurological disorder characterized by seizures is epilepsy.
• Epilepsy may be a form of pharmacoresistant epilepsy and/or a form of genetic epilepsy.
• FIG. 1 shows a schematic timeline of the experimental procedures. Electrographic recordings (EEG) described in the upper part, locomotor tracking - in the lower part. Treatment order and timing are indicated by arrows.
• EEG Electrographic recordings
• FIG. 3 shows that indirubin suppresses PTZ induced epileptiform discharges.
• Fig. 3A shows the number of interictal-like spikes
• Fig. 3B shows ictal-like spikes
• Fig. 3C shows the total cumulative duration of each type of epileptiform activity (ictal and inter-ictal activity) measured. All values are expressed as mean ⁇ SD.
• Statistical significance relative to PTZ control was determined using the unpaired Student's t-test with *, * * and * * * * denoting p ⁇ 0.05, p ⁇ 0.01 and p ⁇ 0.001 respectively.
• Figure 5 shows the quantitative analysis of the electrographic activity in response to BIO- acetoxime and TCS2002.
• Fig. 5A shows the number of interictal-like spikes
• Fig. 5B shows the number of ictal-like spikes
• Fig. 5C shows the total cumulative duration of each type of epileptiform activity (ictal and inter-ictal activity) measured. All values are expressed as mean ⁇ SD.
• Statistical significance relative to PTZ control was determined using the unpaired Student's t-test with *, * * and * * * * denoting p ⁇ 0.05, p ⁇ 0.01 and p ⁇ 0.001 respectively.
• FIG. 6 shows that GSK-3 knockdown protects against PTZ-induced convulsions in zebrafish larvae.
• Figure 7 shows that indirubin protects against pilocarpine-induced focal seizures in rats and against seizures in the 6 Hz assay.
• Fig. 7B shows results of the 6 Hz assay.
• FIG. 8 shows that BlO-acetoxime protects against pilocarpine-induced focal seizures in rats and against seizures in the 6 Hz assay.
• Fig. 8B shows results of the 6 Hz assay. For each dose the percentage of protected is shown as white segments and unprotected mice is shown as the segments completing 100% .
• mice Values that were significantly different from VHC treated mice were determined using Fisher's exact test with * * * denoting p ⁇ 0.001.
• FIG. 9 shows that TCS2002 protects against pilocarpine-induced focal seizures in rats and against seizures in the 6 Hz assay.
• Fig. 9B shows results of the 6 Hz assay. For each dose the percentage of protected and unprotected mice are shown as white and black columns respectively.
• mice Values that were significantly different from VHC treated mice were determined using Fisher's exact test with * * * denoting p ⁇ 0.001.
• Figure 11 shows that indirubin does not alter motor coordination in the beamwalking test. It shows the performance of mice in the beamwalking test after treatment with diazepam (Fig. 11A) or indirubin (Fig. 11B).
• the y-axis denotes (left panel) time on beam (in seconds), (middle panel) number of footslips or (right panel) number of falls.
• the x-axis denotes the treatment dose (1 mg/kg diazepam; 10 mg/kg indirubin).
• VHC used was phosphate buffered saline (PBS) and PEG400/H2O for diazepam and indiru bin respectively.
• Figure 12 shows the effects of various indirubin analogues on PTZ-induced convulsions in zebrafish larvae.
• Fig. 12A shows the structure of indirubin and the effects of this compound when administered at 30 ⁇ , 100 ⁇ , and 300 ⁇ .
• Fig. 12B shows the structure of indirubin- oxime and the effects of this compound when administered at dosages of 30 ⁇ , 100 ⁇ , and 300 ⁇ .
• Fig. 12C shows the structure of (Z)-5'chloro-[2,3'-biinolinylidene]-2',3-dione and the effects of this compound when administered at dosages of 30 ⁇ and 100 ⁇ .
• Fig. 12A shows the structure of indirubin and the effects of this compound when administered at 30 ⁇ , 100 ⁇ , and 300 ⁇ .
• Fig. 12B shows the structure of indirubin- oxime and the effects of this compound when administered at dosages of 30 ⁇ , 100 ⁇ , and 300
• FIG. 12D shows the structure of (2Z,3E)-5'-chloro-3-(hydroxyimino)-[2,3'-biindolinylidene]-2'-one and the effects of this compound when administered at dosages of 30 ⁇ , 100 ⁇ , and 300 ⁇ .
• Fig. 12E shows the structure of an indirubin analogue and the effects of this compound when administered at dosages of 30 ⁇ , 100 ⁇ , and 300 ⁇ .
• Fig. 12F shows the structure of an indirubin analogue and the effects of this compound when administered at dosages of 30 ⁇ , 100 ⁇ , and 300 ⁇ .
• Fig. 12D shows the structure of (2Z,3E)-5'-chloro-3-(hydroxyimino)-[2,3'-biindolinylidene]-2'-one and the effects of this compound when administered at dosages of 30 ⁇ , 100 ⁇ , and 300 ⁇ .
• Fig. 12E shows the structure of an indirubin an
• FIG. 12G shows the structure of 7-bromo-indirubin-3'-monoxime and the effects of this compound when administered at dosages of 100 ⁇ and 300 ⁇ .
• Fig. 12H shows the structure of 5-iodo-indirubin-3'-monoxime and the effects of this compound when administered at dosages of 100 ⁇ and 300 ⁇ .
• Fig. 121 shows the structure of indigo and the effects of this compound when administered at dosages of 100 ⁇ , and 300 ⁇ .
• halogen or halo means any atom selected from the group consisting of fluorine, chlorine, bromine and iodine.
• Ci_ 6 alkyl defines straight or branched chain saturated hydrocarbon radicals having from 1 to 6 carbon atoms such as methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2- butyl, 2-methyl-propyl, t.butyl, pentyl, 2-methyl butyl, hexyl, 2-methylhexyl, and the like.
• Ci_ 4 alkyl or C 1-2 alkyl which define straight or branched chain saturated hydrocarbon radicals having from 1 to 4 respectively 1 or 2 carbon atoms.
• a pharmaceutically acceptable salt or prodrug thereof is meant to include a pharmaceutically acceptable salt, a prodrug, or a pharmaceutically acceptable salt of a prodrug.
• the pharmaceutically acceptable salt forms of the compounds for use in the present invention include acid addition salts of said compounds with acids such as inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric, nitric, phosphoric, and the like acids; or organic acids such as acetic, aspartic, dodecyl-sulfuric, heptanoic, hexanoic, nicotinic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic, malonic, succinic, maleic, fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, salicylic, p-amino-salicylic acid.
• acids such as inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric,
• the compounds for use in the present invention containing acidic protons can be used as pharmaceutically acceptable metal or amine addition salt forms with appropriate organic and inorganic bases such as ammonium salts, alkali and earth alkaline metal salts, e.g. lithium, sodium, potassium, magnesium, or calcium salts, salts with organic bases, e.g.
• amines such as methylamine, ethylamine, propylamine, isopropylamine, the four butylamine isomers, dimethylamine, diethylamine, diethanolamine, dipropylamine, diisopropylamine, di-n-butylamine, pyrrolidine, piperidine, morpholine, trimethylamine, triethylamine, tripropylamine, quinuclidine, pyridine, quinolone, isoquinoline, and the like.
• prodrug as used herein means the pharmacologically acceptable derivatives such as esters, amides and phosphates, such that the resulting in vivo biotransformation product of the derivative is the active drug as defined herein.
• Prodrugs preferably have excellent aqueous solubility, increased bioavailability and are readily metabolized into the active inhibitors in vivo.
• Prodrugs of a compound of the present invention may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either by routine manipulation or in vivo, to the parent compound.
• pharmaceutically acceptable ester prodrugs that are hydrolysable in vivo and are derived from those compounds having a hydroxyl or a carboxyl group.
• the present invention relates to inhibitors of GSK-3 phosphorylation for use in the treatment of neurological disorders characterized by seizures, such as epilepsy. Accordingly, anticonvulsant agents may be effective in reducing seizures, convulsions, and/or other involuntary changes in body movement or function.
• anticonvulsant agents agents useful in the treatment of neurological disorders characterized by seizures
• anticonvulsant agents agents useful in the treatment of neurological disorders characterized by seizures
• antiepileptic agents agents useful in the treatment of neurological disorders characterized by seizures
• AEDs anti-epileptic drugs
• seizure medications agents useful in the treatment of neurological disorders characterized by seizures. These terms are used herein interchangeably.
• Glycogen synthase kinase 3 (GSK-3) is a serine/threonine protein kinase whose phosphorylation activity has been implicated in diverse cellular processes including cell differentiation and proliferation, cell migration, inflammation and immune responses, glucose regulation, and apoptosis. Given its prominent role in these processes, GSK-3 and other proteins in the signaling pathway are also associated with disorders such as Type I I diabetes, cancer, and autoimmune disease.
• GSK-3 GSK-3
• AD Alzheimer's Disease
• GSK-3 function has not been directly correlated with seizures, although patients diagnosed with AD and/or neurofibrillary tangles (N FTs) containing hyperphosphorylated tau (p-tau) have been known to suffer from seizures.
• N FTs neurofibrillary tangles
• p-tau and NFTs are also found in a mouse model of Lafora Disease, an autosomal recessive form of progressive myoclonus epilepsy (Puri et al., The Journal of Biological
• the GSK-3 inhibitor may be selected from an indole, a pyrrole-pyrazine, and a benzofuran.
• the GSK-3 inhibitor is a GSK-3 antisense molecule.
• the GSK-3 inhibitor is a heterocyclic compound comprising at least one of an indole, a pyrrole, a pyrazine, a pyrrolo-pyrazine, a quinoline, a thiazole, a pyridine, a pyrimidine, an imidazole, an azepine, an oxadiazole, a piperazine, and a substituted phenol.
• the GSK-3 inhibitor is an indole.
• the indole is an indole dimer, for example, a molecule made up of two indole subunits.
• the indole dimer is an indirubin compound.
• the indirubin compound may be indirubin or a chemical analogue of indirubin.
• a chemical analogue of indirubin may be a structural analogue, in which one or more atoms, functional groups, and/or substructures have been replaced with another atom, functional group, and/or substructure, or may be a functional analog which has similar physical, chemical, biochemical and/or pharmacological properties to indirubin.
• An analogue may show increased solubility as compared to indirubin.
• R 2 is O.
• R 2 is NOH.
• R 2 is NOAc.
• R 3 may be -H, or R 3 may be halo.
• n is 0, or n is 1-4, or n is 1-3, or n is 1-2.
• n is 1 and in a further embodiment each R 3 independently is halo.
• each R 3 independently is Br, CI, or I, in particular bromo.
• each R 3 has the same meaning.
• R 3 may be Ci_ 4 alkyl, or Ci_ 2 alkyl, or methyl.
• the indirubin compound is (Z)-[2,3'-biindolinylidene]-2',3-dione (which i ich can be represented by formula (II):
• Indirubin has been previously characterized as a bioactive compound with anti-leukemic activity and anti-proliferative effects, as well as inhibitor of cyclin dependent kinase 5 (CDK5), the aryl hydrocarbon receptor (AhR), and GSK-3 .
• CDK5 cyclin dependent kinase 5
• AhR aryl hydrocarbon receptor
• GSK-3 the anticonvulsant activity of indirubin is mediated by its inhibition of GSK-3 .
• Chemical analogues of indirubin, such as those described herein, may also selectively inhibit GSK-3 , and these analogues may be particularly useful in the treatment of seizures or as anticonvulsants, specifically in the treatment of epilepsy, as described herein.
• the indirubin compound is (2'Z, 3'E)-6-bromoindirubin-3'- acetoxime, which may also referred to as BlO-acetoxime, and can be represented by formula (III), or a pharmaceutically acceptable salt or prodrug thereof:
• BlO-acetoxime is a selective inhibitor of the GSK-3a and GSK-3 isomers of GSK-3, and has low selectivity for CDK molecules.
• the indirubin compound is (2'Z,3'E)-6-bromoindirubin-3'-oxime (which is also referred to as BIO or 6BIO), which can be represented by formula (IV), or a pharmaceutically acceptable salt or prodrug thereof:
• the indirubin compound is 3-[l,3-dihydro-3-(hydroxyimino)-2H- indol-2-ylidene]-l,3-dihydro-2H-indol-2-one, which may also be referred to as indirubin-3'- oxime, and which can be represented (V), or a pharmaceutically acceptable salt or prodrug
• the indirubin compound is a compound having the formula:
• the indole having the chemical structure of formula (VI) as specified above, or a pharmaceutically acceptable salt or prodrug thereof is 9-bromo-7,12- dihydro-pyrido[3',2':2,3]azepino[4,5-b]indol-6(5H)-one, which is also known as 1- azakenpaullone.
• the indole is 9-bromo-7,12-dihydro-indolo[3,2- d][l]benzazepin-6(5H)-one, which is also known as Kenpaullone.
• the GSK-3 inhibitor is a benzofuran.
• a benzofuran as mentioned herein is a heterocyclic compound comprising a benzofuran moiety.
• the GSK-3 inhibitor is a benzofuran.
• the benzofuran may be a compound having the chemical structure of formula (VII), or a pharmaceutically acceptable salt or prodrug thereof:
• the GSK-3 inhibitor is a pyrrolo-pyrazine compound, in particular a pyrrolo[2,3-b]pyrazine.
• a pyrrolo-pyrazine as mentioned herein is a heterocyclic compound comprising a fused ring in which a pyrrole is fused to a pyrazine.
• An exemplary pyrrolo-pyrazine is a chemical compound of formula (VIII), or a pharmaceutically acceptable salt or prodrug thereof:
• the pyrrole-pyrazine is 7-n-butyl-6-(4-hydroxyphenyl)[5H]- pyrrolo[2,3-b]pyrazine, which may also be referred to as aloisine.
• GSK-3 inhibitors as specified herein, in particular those of formula (II), (III), (IV), (V, (VI), (VII), and (VIII), and more specifically the compounds of formula (I), all as specified herein, that are selective towards GSK-3 .
• compounds with selectivity for GSK-3 i.e. compounds that selectively inhibit GSK-3 .
• the selectivity may be towards CDK molecules, or towards all of CDK1, CDK2, and CDK5; and/or towards Ah .
• selectivity means an IC 50 of one order of magnitude difference, or in another embodiment two orders of magnitude.
• Other GSK-3 inhibitors that may be used in the present invention are the compounds listed in Table 2, including the pharmaceutically acceptable salts or prodrugs thereof and if a compound is listed as a salt, including the base-form and other pharmaceutically acceptable salts thereof.
• One or more functional groups may be protected in the compounds for use in the present invention.
• a particular protecting group for hydroxyl is the acetyl group.
• Such protected compounds may also exhibit altered, and in some cases, optimized properties in vitro and in vivo, such as passage through cellular membranes and resistance to enzymatic degradation or sequestration. In this role, protected compounds with intended therapeutic effects may be referred to as prodrugs.
• Another function of a protecting group is to convert the parental drug into a prodrug, whereby the parental drug is released upon conversion of the prodrug in vivo.
• prodrugs may possess greater potency in vivo than the parental drug.
• Protecting groups are removed either in vitro, in the instance of chemical intermediates, or in vivo, in the case of prodrugs. With chemical intermediates, it is not particularly important that the resulting products after deprotection, e.g. alcohols, be physiologically acceptable, although in general it is more desirable if the products are pharmacologically innocuous.
• prodrug may also relate to an inactive or significantly less active derivative of a compound such as represented by the structural formulae herein described, which undergoes spontaneous or enzymatic transformation within the body in order to release the pharmacologically active form of the compound.
• the GSK-3 inhibitors for use in the invention are known compounds or can be made from these known compounds by appropriate derivatization reactions or from known intermediates using standard synthesis procedures.
• the GSK-3 inhibitor is a GSK-3 antisense oligonucleotide.
• An antisense oligonucleotide having a complementary or substantially complementary base sequence to the base sequence of an oligonucleotide encoding GSK-3 or a fragment thereof may be any antisense oligonucleotide, so long as it possesses a base sequence complementary or substantially complementary to the base sequence of the oligonucleotide (e.g., DNA) of
• the base sequence substantially complementary to the DNA of GSK-3 may include, for example, a base sequence having at least about 70% homology, preferably at least about 80% homology, more preferably at least about 90% homology and most preferably at least about 95% homology, to the entire base sequence or to its partial base sequence (i.e., complementary strand to the DNA of GSK-3 ), and the like.
• Antisense oligonucleotides may comprise nucleic acid (DNA, RNA, or a chemical analogue) that binds to GSK-3 mRNA and inhibits expression of GSK-3 .
• Antisense molecules may target any part of the GSK-3 RNA, such as the 5' untranslated region of RNA, splicing sites on the pre-mRNA, and/or exons in the mRNA.
• the GSK-3 antisense molecules are morpholinos.
• the morpholinos may be nucleic acid analogues which block regions of the GSK-3 RNA.
• the GSK-3 inhibitor may be a short interfering nucleic acid (siNA), such as an siRNA, which mediates RNA interference (RNAi) of GSK-3 .
• RNAi refers to the process of sequence specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (U.S. Patent No. 7989612).
• RNAi can also involve small RNA (e.g., micro-RNA or miRNA) mediated gene silencing, presumably though cellular mechanisms that regulate chromatin structure and thereby prevent transcription of target gene.
• siNA molecules may be used to mediated gene silencing via interaction with RNA transcripts, for example, RNA transcripts of GSK-3 , or alternately by interaction with particular gene sequences, wherein such interaction results in gene silencing either at the transcriptional level or post-transcriptional level.
• RNA transcripts for example, RNA transcripts of GSK-3
• siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. 21 nucleotide siRNA duplexes are most active when containing two 2-nucleotide 3'-terminal nucleotide overhangs.
• siRNA molecules lacking a 5'-phosphate are active when introduced exogenously, suggesting that 5'-phosphorylation of siRNA constructs may occur in vivo.
• siNA molecules may be synthesized.
• the siNAs are no more than 100 nucleotides in length, preferably no more than 80 nucleotides in length, and most preferably no more than 50 nucleotides in length (e.g., individual siNA oligonucleotide sequences or siNA sequences synthesized in tandem) are preferably used for exogenous delivery.
• the simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of protein and/or RNA structure.
• Exemplary molecules of the instant invention are chemically synthesized, and others can similarly be synthesized.
• a siNA molecule may also be assembled from two distinct nucleic acid strands or fragments wherein one fragment includes the sense region and the second fragment includes the antisense region of the RNA molecule.
• Exemplary siNA molecules may be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-0-methyl, 2'-H.
• siNA constructs can be purified by gel electrophoresis using general methods or can be purified by high pressure liquid
• siNA molecules are expressed from transcription units inserted into DNA or RNA vectors.
• the recombinant vectors can be DNA plasmids or viral vectors.
• siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus.
• the recombinant vectors capable of expressing the siNA molecules can be delivered as described herein, and persist in target cells.
• viral vectors can be used that provide for transient expression of siNA molecules.
• chemically synthesizing nucleic acid molecules with modifications prevents their degradation by serum ribonucleases, which increases their potency.
• GKEAPPAPPQSP SEQ ID NO:3 (which may include myristolation on Glyl,
• the inhibitors of GSK-3 phosphorylation in accordance with the present invention are used in the treatment of neurological disorders characterized by seizures, such as epilepsy.
• Epileptic seizures may result from any abnormal, excessive, or hypersynchronous neuronal activity in the brain.
• epileptic seizures which require treatment with anticonvulsants are caused by infection, stroke, trauma, fever, tumors, drug use, damage to the blood-brain barrier, and/or neurodegenerative disease.
• epileptic seizures are triggered by emotional state, by response to light and/or sound, sleep, sleep deprivation, hormones, metabolic disorders, and/or congenital defects.
• Epileptic seizures for which the anticonvulsants disclosed herein provide treatment may be classified as partial seizures, such as simple partial seizures and/or complex partial seizures, or they may be classified as generalized seizures, such as absence seizures, myoclonic seizures, clonic seizures, tonic seizures, tonic-clonic seizures, and/or atonic seizures, or a mixed seizure.
• the seizures may be symptoms of temporal lobe epilepsy, whose features may include epileptic foci in the limbic system, an initial precipitating injury, a latent period, and the presence of hippocampal sclerosis leading to reorganization of neuronal networks (Curia et al., J. Neurosci Methods, 172(2-4): 143-157 (2008)).
• the anticonvulsants described herein may also provide treatment for therapy-resistant forms of seizure.
• the 6 Hz psychomotor seizure model of partial epilepsy has been used as a model of therapy-resistant forms of seizures, including limbic seizures (Barton et al., Epilepsy research, 47(3), 217-227 (2001)).
• Patients suffering from epileptic seizures may be infants aged 0-6 months, 6-12 months, 12-18 months, 18-24 months. In certain embodiments, patients suffering from epileptic seizures are individuals aged 65-70, 75-80, 85-90, 95-100, 100-105, and older. Patients may also be children aged 2-12, adolescents aged 13-19, or adults aged 20-64.
• Anticonvulsants may be used for the treatment of epileptic seizures, including treatment of symptoms associated with epileptic seizures and/or epilepsy. Anticonvulsants may also be used to treat epileptic seizures that result from central nervous system disorders such as cerebrovascular diseases and/or neurodegenerative diseases.
• One goal of an anticonvulsant agent i.e., an "anticonvulsant” is to suppress the rapid and excessive firing of neurons that start a seizure.
• Another goal of an anticonvulsant is to prevent the spread of the seizure within the brain and offer protection against possible excitotoxic effects that may result in brain damage.
• Anticonvulsants may also be referred to as anti-seizure drugs.
• epilepsy an area of the brain and/or nervous system is typically hyper-irritable. Antiepileptic drugs function to help reduce this area of irritability and thus prevent epileptic seizures.
• GSK-3 inhibitors mentioned herein and specifically the compounds of formulae (I I), (II I), (IV), (V, (VI), (VII), and (VII I), and more specifical ly the compounds of formula (I), all as specified herein, show superior results in the treatment of neurological disorders characterized by seizures, such as epilepsy, in particular the seizure types mentioned above.
• compositions typically are administered formulated as pharmaceutical compositions, which comprise an effective amount of the active ingredient mixed with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of preparation desired for administration.
• pharmaceutical compositions are desirably in unitary dosage form suitable, for example, for oral, rectal, or percutaneous administration.
• any of the usual pharmaceutical media may be employed such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid preparations such as suspensions, syrups, elixirs, emulsions, and solutions; or solid carriers such as starches, sugars, kaolin, diluents, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules, and tablets.
• the carrier optionally comprises a penetration enhancing agent and/or a suitable wetting agent.
• compositions may be administered in various ways, e.g., as a transdermal patch, as a spot-on, or as an ointment.
• the pharmaceutical compositions are preferably formulated in unit dosage form for ease of administration and uniformity of dosage.
• Unit dosage form as used herein refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of such unit dosage forms are tablets, capsules and suppositories.
• an effective daily amount would be from 0.01 mg/kg to 50 mg/kg body weight, more preferably from 0.1 mg/kg to 10 mg/kg body weight. It may be appropriate to administer the required dose as two, three, four or more sub-doses at appropriate intervals throughout the day. Said sub-doses may be formulated as unit dosage forms, for example, containing 1 to 1000 mg, and in particular 5 to 200 mg of active ingredient per unit dosage form.
• the exact dosage and frequency of administration depends on the particular compound used, the particular condition being treated, the severity of the condition being treated, the age, weight and general physical condition of the particular patient as well as other medication the individual may be taking, as is well known to those skilled in the art. Furthermore, it is evident that the effective amount may be lowered or increased depending on the response of the treated subject and/or depending on the evaluation of the physician prescribing the compounds of the instant invention. The effective amount ranges mentioned above are therefore only guidelines and are not intended to limit the scope or use of the invention to any extent.
• Example 1 Identification of indirubin as active anticonvulsant principle in Indigofera arrecta
• MO concentration was titered so as not to cause any observable gross developmental dysmorphologies that could potentially impair locomotor ability (data not shown).
• GSK-3 knockdown embryos and corresponding controls were also video-tracked at 3 dpf, as MO-mediated translational inhibition of target proteins is transient, and presumably would no longer reduce the levels of GSK-3 at 7 dpf sufficiently.
• the concentration of PTZ was also reduced to 10 mM as 3 dpf embryos displayed the highest level of locomotor acitvity at this concentration (data not shown).
• GSK-3 knockdown was able to counter PTZ-induced seizure behavior in a
• indirubin analogues were tested in the larval zebrafish assay, using concentrations ranging from 30 ⁇ - 300 mM.
• BlO-acetoxime and TCS2002 were tested in the same panel of rodent seizure assays used for indirubin. As in the case of indirubin, the two compounds were ineffective in suppressing any of the seizure behaviors measured in both the acute mouse PTZ and pilocarpine i.v.
• roscovitine was the only none GSK-3 inhibitor that also showed anticonvulsant activity in the behavioral PTZ assay in zebrafish. Nevertheless, roscovitine did not show any activity in the 6 Hz model in mice up to a dose of 50 mg/kg (Fig. 10). Additionally intrahippocampal administration of roscovitine (10 ⁇ and 100 ⁇ ) did not significantly change the TSSS in the rat intrahippocampal pilocarpine limbic seizure model (data not shown).
• Exemplary GSK-3 inhibitors (Table 2) were selected from commercially available compounds and were tested in zebrafish larvae to determine the MTC and effective concentration range in the PTZ assay. PTZ was administered to the zebrafish larvae, as described hereinafter and each GSK-3 inhibitor was assayed for efficacy in reducing larval movement induced by PTZ. To confirm the seizure suppressing activity of the GSK-3 inhibitors, tectal field recordings were performed on zebrafish larvae.
• Each GSK-3 inhibitor was then tested in the mouse PTZ and pilocarpine intravenous infusion assays. Doses of 0.05 mg/kg, 0.5 mg/kg , 2 mg/kg, 5 mg/kg, 10 mg/kg, and other doses were used. The seizure inhibitory activity of each inhibitor was also measured in both the rat intrahippocampal pilocarpine limbic seizure and mouse 6 Hz models.
• larvae of 7 dpf of the Fli strain were used. In this experimental setup, it was found that larvae displayed the most consistent basal activity levels when raised under constant light conditions.
• morpholino (MO) injection embryos of the AB line were used.
• pilocarpine and PTZ tail vein infusion test male C57BI/6 mice (20-30 g) (Charles River Laboratories) were used.
• 6 Hz test and the beamwalking assay male N MRI mice (20-30 g) (Charles River Laboratories) were used.
• Indirubin, BlO-acetoxime and TCS2002 were dissolved in propylene glycol : saline (50 : 50) for intraperitoneal (i.p.) injections.
• Pilocarpine (Sigma-Aldrich) was dissolved in Ringer's solution (147 mM NaCI, 2.3 mM CaCI 2 and 4 mM KCI) for intrahippocampal application and in saline for intravenous tail infusion.
• PTZ was purchased from Sigma-Aldrich and was dissolved in embryo medium for zebrafish experiments and in purified water for mice experiments.
• the dried plant material was extracted multiple times with acetone. The extracts were combined, evaporated to dryness under reduced pressure on kieselguhr as a sorbent, and the resulting powder applied to the top of a silica gel 60 column. The fractionation was performed by liquid chromatography with ethyl acetate as the mobile phase. Fractions containing the bio-active constituent were pooled and dried. The residue was dissolved in dichloromethane by heating and stored at 4°C overnight. The precipitate formed contained the active constituent and was collected for further purity and N MR analysis.
• Toxicological evaluation in zebrafish MTC determination Zebrafish larvae were incubated with compound or VHC in the dark. After an overnight incubation (18 hours), each larva was individually checked under the microscope for death and for the following signs of acute toxicity: hypoactivity, decreased or no touch/escape response upon a light touch of the tail with a fine needle (Fetcho et al., Brain research reviews, 57(1), 86- 93 (2008) Pietri et al., Developmental neurobiology, 69(12), 780-795 (2009)), loss of posture, body deformation, exophthalmos (bulging of the eyes out of their sockets), and slow or absent heartbeat. Larvae were considered normal if they could cover a distance twice its body length.
• MTC maximum tolerated concentration
• the larva After exposure for 15 minutes (time interval selected corresponds to the peak of locomotor activity) the larva was embedded in 2% low-melting- point agarose, a glass electrode filled with artificial cerebrospinal fluid composed of (mM): 124 NaCI, 2 KCI, 2 MgS04, 2 CaCI2, 1.25 KH2P04, 26 NaHC03 and 10 glucose (resistance 1-5 ⁇ ) was placed into its optic tectum and recording was performed in current clamp mode, low-pass filtered at 1 kHz, high-pass filtered 0.1 Hz, digital gain 10, sampling interval 10 ⁇ (MultiClamp 700B amplifier, Digidata 1440A digitizer, both Axon instruments, USA). The recordings started each time 5 minutes after proconvulsant exposure and were continued for 10 minutes.
• mM artificial cerebrospinal fluid composed of (mM): 124 NaCI, 2 KCI, 2 MgS04, 2 CaCI2, 1.25 KH2P04, 26 NaHC03 and 10 glucose (resistance 1-5
• the threshold for different phases of PTZ-induced seizure activity was determined by an i.v. (intravenous) infusion of PTZ (7.5 mg/ml) in the lateral tail vein at a constant rate of 150 ⁇ /min. During the experiment, the animal was able to move freely in a Plexiglas cage. Vehicle or compound was delivered via i.p. injection 30 min before PTZ tail infusion.
• the following endpoints were used to determine the seizure threshold for PTZ: ear, tail and myoclonic twitch, forelimb clonus, falling, tonic hindlimb extension and death. Time was measured from the start of the infusion until the onset of these stages.
• the seizure thresholds were determined for each animal according to the following equation: dose (mg kg)
• Tables A and B show that indirubin does not inhibit seizure behaviors in mouse PTZ and pilocarpine i.v. infusion assays.
• Table A shows the results of the PTZ-infusion assay;
• Table B shows the results of pilocarpine-infusion assay.
• a higher value means that a higher dose of PTZ or pilocarpine is needed to evoke the same end point.
• mice were injected i.p. with either VHC or compound 30 minutes prior to testing.
• Seizures were induced via corneal stimulation using the Ugo-Basil device (6 Hz, 0.2 ms rectangular pulse width, 3 s duration). Prior to the placement of corneal electrodes, a drop of 0,5% tetracaine was applied to the eyes of the animal. Animals were restrained manually and released immediately in a cage of plexi glass following the stimulation. Then, the animal was observed. The seizure was characterized by stun, forelimb clonus, twitching of vibrissae, straub-tail for at least 45 s. Protection was defined as the absence of a seizure.
• Rats were anesthetized with an i.p. injection of ketamine HCkdiazepam (94.5:4.5 mg/kg).
• ketamine HCkdiazepam 94.5:4.5 mg/kg.
• a cannula with a replaceable guide (CMA Microdialysis) was implanted randomly into the left or right hippocampus. The exact coordinates were 4.6 mm lateral and 5.6 mm anterior to bregma, and 4.6 mm ventral starting from the dura (Paxinos and Watson, 1986). The inner guide of the cannula was then replaced by a 3 mm CMA 12 microdialysis probe (CMA Microdialysis).
• Ketoprofen (4 mg/kg i.p.) was administered to assure post-operative analgesia.
• This scale consists of 6 stages, which correspond to the successive developmental stages of motor seizures: (0) normal non- epileptic activity, (1) mouth and facial movements, hyperactivity, grooming, sniffing, scratching, wet dog shakes, (2) head nodding, staring, tremor, (3) forelimb clonus, forelimb extension, (4) rearing, slavering, tonic-clonic activity, (5) falling.
• Total seizure severity was determined by summation of the SSS's of each collection period, resulting in a Total Seizure Severity Score (TSSS) for each individual animal. 7.
• TSSS Total Seizure Severity Score
• the beamwalking assay was used to discover possible adverse effects such as problems with motor coordination or sedation, induced by the administration of indirubin. Mice were trained to walk from a start platform along a ruler towards a closed box. The trained mice were administered compound or VHC and were again tested on the beam. Mice that fell were returned to the position they fell from, with a maximum time of 60 s allowed on the beam. Measurements that were taken are time on beam, the number of foot slips (one or both hind limbs) and the number of falls.
• a GSK-3 morpholino (MO) targeting the translation start site of zebrafish gsk-3 (3' lissamine red-emitting fluorescent tag - excitation peak: 575 nm, emission peak: 593 nm; 5'- GTTCTGGGCCGACCGGACATTTTTC-3'; Gene Tools, LLC) were injected into zebrafish embryo's at the 1- to 2-cell stages. Injected embryos were raised at 28.5° C until 3dpf.
• MO GSK-3 morpholino
• the injected embryos were used in the behavioral PTZ assay to score for locomotor activity in the presence of 10 mM PTZ. Uninjected 3-dpf embryos were used as controls.
• the intrahippocampal pilocarpine limbic seizure model data were analyzed using one-way ANOVA followed by the Bonferroni post hoc test.
• the results of the 6 Hz test in mice were analysed using the chi-square test. If a significant effect was shown, the Fisher's exact test was used to compare each dose with the sham-treated group.
• the results of the beamwalking test were analyzed using one-way ANOVA followed by Dunnett post hoc test. All statistical analyses were performed using Graph Pad Prism.
• Table 1 MTC and reported IC 50 values for different enzymes.

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