WO2014053580A1 - Anticonvulsant activity of gsk-3beta inhibitors - Google Patents

Description

ANTICONVULSANT ACTIVITY OF GSK-3P INHIBITORS

Field of the Invention

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.

Background of the Invention

A common and often debilitating symptom of many neurological disorders is abnormal neuronal activity in the brain. The term 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. To date, almost all currently available anti-epileptic drugs (AEDs) target neuronal receptors such as GABA receptors and sodium, glutamate, or calcium channels, in order to reduce neuronal excitability. However, 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. Surgery can be an option for epilepsy patients, but neurosurgery carries significant risks and an estimated 10% of patients continue to experience seizures even after surgery. For these reasons, the identification of new anticonvulsant compounds and the development of new, effective AEDs remain important areas of research.

To identify new anticonvulsant compounds, research has turned to the medicinal plants used in traditional medicine. Moshi et al. in Journal of Ethnopharmacology, 97(2), 327- 336 (2005) describe plants that have been used in Tanzania to treat epilepsy, and Schachter et al. in Neurotherapeutics, 6, 415-420 (2009) review preclinical studies in which anticonvulsant activity has been attributed to various plant extracts. In spite of this knowledge, the active ingredients in plants and decoctions are often unknown, as are the targets of the active ingredients. Similarly, the precise compounds that can be used for treating specific types of seizures, such as drug-resistant seizures, are also unknown. Thus remains a need in the art for identifying and characterizing compounds and/or classes of compounds that may be used to treat different aspects of epilepsy. Summary of the Invention

It now has been found that certain compounds and molecules can be used as agents to reduce seizures and symptoms of epilepsy. These compounds and molecules target the glycogen synthase kinase 3 (GSK-3) signaling pathway and belong to the family of inhibitors that block phosphorylation by the isoform GSK-3 . Exemplary GSK-3 inhibitors comprise heterocyclic compounds such as indoles, pyrrolo-pyrazines, benzofurans, as well as GSK-3 antisense molecules.

In one aspect, the present invention relates to inhibitors of GSK-3 phosphorylation for use in the treatment of neurological disorders characterized by seizures, such as epilepsy.

In a further aspect, the present invention concerns an inhibitor of phosphorylation by

GSK3 for use in the treatment of neurological disorders characterized by seizures, wherein the inhibitor is an indole.

In some embodiments, the indole is an indole dimer. The indole dimer may be, for example, an indirubin compound. In certain embodiments, the indirubin compound is a compou e salt or prodrug thereof:

I)

wherein

1 is O;

R² is selected from O, NOH, and NO-CO-R⁴;

each R³ is independently selected from -H and halo;

R⁴ is Ci_6 alkyl; and

n is 0-5.

In certain embodiments, 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.

In certain embodiments, the GSK-3 inhibitor is a GSK-3 antisense molecule. For example, the GSK-3 antisense molecule is a morpholino. In some embodiments, GSK-3 is inhibited by NAi.

In a further aspect, 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.

In still a further aspect, 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.

In some embodiments, the neurological disorder characterized by seizures is epilepsy. Epilepsy may be a form of pharmacoresistant epilepsy and/or a form of genetic epilepsy.

Description of the Figures

Figure 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.

Figure 2 shows the behavioral response of zebrafish larvae to indirubin in conjunction with PTZ (pentylenetetrazole). Indirubin was tested at three different concentrations: 30 μΜ, 100 μΜ and 300 μΜ. All time interval points were compared to the corresponding PTZ control using the unpaired Student's t-test. Values that were significantly different from PTZ control are marked with *(p<0.05). Total locomotor activity is expressed in actinteg units and averaged for 5-min intervals. Standard deviations are only shown for vehicle control (VHC) + PTZ for visual clarity. The number of larvae tested for each concentration is n=24.

Figure 3 shows that indirubin suppresses PTZ induced epileptiform discharges. Fig. 3A shows the number of interictal-like spikes, Fig. 3B shows ictal-like spikes, and 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. The number of recordings analyzed was: VHC (n=10), VHC + PTZ (n=14), 300 μΜ indirubin + PTZ (n=8).

Figure 4 shows the behavioral response of zebrafish larvae to compounds with different affinities for GSK-3 , CDK and Ah in conjunction with PTZ. All compounds were tested at their MTC (maximum tolerated concentration) values. All time interval points were compared to the corresponding PTZ control using the unpaired Student's t-test. Values that were significantly different from PTZ control are marked with * (p<0.05), * * (p<0.01) or * * * (p<0.001). Total locomotor activity is expressed in actinteg units and averaged for 10-min intervals. Standard deviations are only shown for VHC + PTZ for visual clarity. The number of larvae tested for each concentration was n=12.

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, and 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. The number of recordings analyzed was: VHC (n=10), VHC + PTZ (n=14), 200 μΜ BlO-acetoxime + PTZ (n=14), 30 μΜ TCS2002 + PTZ (n=6).

Figure 6 shows that GSK-3 knockdown protects against PTZ-induced convulsions in zebrafish larvae. The graph denotes total larval activity (expressed in actinteg units), averaged for 10-min intervals over a period of 60 minutes. All time intervals were compared to the corresponding PTZ control using the unpaired Student's t-test. Values that were significantly different from PTZ control are marked with *(p<0.05) or * * * (p<0.001). Standard deviations are only shown for uninjected embryos + PTZ for visual clarity. The number of larvae tested for each concentration is: n=52 for uninjected, uninjected + PTZ, 200 μΜ MO, 200 μΜ MO + PTZ and n = 48 for 300 μΜ MO, 300 μΜ MO + PTZ. Figure 7 shows that indirubin protects against pilocarpine-induced focal seizures in rats and against seizures in the 6 Hz assay. Fig. 7A show results from the intrahippocampal pilocarpine limbic seizure model. Mean behavioral total seizure severity score (mean ± SD) is shown in graph. Significance was determined using the two-tailed Mann Whitney test and marked with * and * * * for p<0.05 and p<0.001 respectively. The number of recordings analyzed was: VHC (n=ll), 2 mg/kg indirubin (n=8), 10 mg/kg indiru bin (n=10). Fig. 7B 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% . Values that were significantly different from VHC treated mice were determined using the Fisher's exact test with * and * * denoting p<0.05 and p<0.01 respectively. For each dose 6 mice were used (n=6).

Figure 8 shows that BlO-acetoxime protects against pilocarpine-induced focal seizures in rats and against seizures in the 6 Hz assay. Fig. 8A shows results of the intrahippocampal pilocarpine limbic seizure model. The gaph denotes the mean behavioral total seizure severity score (mean ± SD). Significance was determined using the two-tailed Mann Whitney test and marked with * ** for p<0.001. The number of rats used was: VHC (n=10) and 0.5 mg/kg (n=5). 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% . Values that were significantly different from VHC treated mice were determined using Fisher's exact test with * * * denoting p<0.001. The number of mice used for every dose was: VHC (n=7), 0.05 mg/kg (n=8), 0.5 mg/kg (n=7) and 5 mg/kg (n=8).

Figure 9 shows that TCS2002 protects against pilocarpine-induced focal seizures in rats and against seizures in the 6 Hz assay. Fig. 9A shows results of the intrahippocampal pilocarpine limbic seizure model. The graph denotes the mean behavioral total seizure severity score (mean ± SD). Significance was determined using the two-tailed Mann Whitney test and marked with * ** for p<0.001. The number of rats used was: VHC (n=10) and 0.5 mg/kg (n=6). 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. Values that were significantly different from VHC treated mice were determined using Fisher's exact test with * * * denoting p<0.001. The number of mice used for every dose was: VHC (n=7), 0.05 mg/kg (n=8), 0.5 mg/kg (n=8) and 5 mg/kg (n=7).

Figure 10 shows that roscovitine is inactive in the 6 Hz assay. For each dose the percentage of protected and unprotected mice is shown as white and black columns, respectively. For each dose 6 mice were used (n=6). 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. The number of animals used was n=3 for VHC and diazepam and n=6 for indirubin. All results were evaluated using one-way ANOVA followed by Dunnett's multiple comparison test. The level of statistical significance is indicated as * for p<0.05 and * * for p<0.01.

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 μΜ, 100 μΜ, and 300 μΜ. Fig. 12B shows the structure of indirubin- oxime and the effects of this compound when administered at dosages of 30 μΜ, 100 μΜ, and 300 μΜ. 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 μΜ and 100 μΜ. 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 μΜ, 100 μΜ, and 300 μΜ. Fig. 12E shows the structure of an indirubin analogue and the effects of this compound when administered at dosages of 30 μΜ, 100 μΜ, and 300 μΜ. Fig. 12F shows the structure of an indirubin analogue and the effects of this compound when administered at dosages of 30 μΜ, 100 μΜ, and 300 μΜ. Fig. 12G shows the structure of 7-bromo-indirubin-3'-monoxime and the effects of this compound when administered at dosages of 100 μΜ and 300 μΜ. Fig. 12H shows the structure of 5-iodo-indirubin-3'-monoxime and the effects of this compound when administered at dosages of 100 μΜ and 300 μΜ. Fig. 121 shows the structure of indigo and the effects of this compound when administered at dosages of 100 μΜ, and 300 μΜ.

Detailed Description of the Invention

As used herein, the singular is meant to include the plural and vice versa, the plural is meant to include the singular. For example the term "a" includes "one or more" and the term "one or more" includes "a". The term "about" when used in relation to a numerical value has the meaning generally known in the relevant art. In certain embodiments the term "about" may be left out or it may be interpreted to mean the numerical value +10%; or +5%; or +2%; or +1%. Any feature described in relation to a particular aspect or embodiment is meant to be applicable to one or more of the other aspects or embodiments described herein. All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

As used herein and unless otherwise stated, the term "halogen" or "halo" means any atom selected from the group consisting of fluorine, chlorine, bromine and iodine.

As used herein, "Ci_₆alkyl" 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. Of interest among Ci_₆alkyl is Ci_₄alkyl or C_1-2alkyl, which define straight or branched chain saturated hydrocarbon radicals having from 1 to 4 respectively 1 or 2 carbon atoms.

"Ac" represents acetyl.

As used herein the term "or 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. 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.

The term "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. Of interest are 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.

Agents useful in the treatment of neurological disorders characterized by seizures can be referred to as anticonvulsant agents, or as antiepileptic agents, anti-epileptic drugs (AEDs), anti-seizure agents, or seizure medications. 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. In the nervous system, the β isoform of GSK-3 (GSK-3 ) has been linked with hyperphosphorylation of tau , regulation of the dopamine D2 receptor (Tripathi et al., Neuroreport, 21(12), 846-850 (2010)), and neurological disorders such as schizophrenia, autism spectrum disorders, and Alzheimer's Disease (AD), (Hur & Zhou, Nature reviews, Neuroscience, 11(8), 539-551. (2010)). To date, 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. Notably, 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

Chemistry, 284(34), 22657-22663 (2009)), and in human patients with intractable epilepsy (Xi et al., Med Hypotheses, 76(6):897-900 (2011)). However, no seizures have been specifically attributed to GSK-3 , and GSK-3 inhibitors have not been previously used to treat seizures.

The GSK-3 inhibitor may be selected from an indole, a pyrrole-pyrazine, and a benzofuran. In certain embodiments, the GSK-3 inhibitor is a GSK-3 antisense molecule. In some embodiments, 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. In certain embodiments, the GSK-3 inhibitor is an indole. In some embodiments, the indole is an indole dimer, for example, a molecule made up of two indole subunits. In certain embodiments, the indole dimer is an indirubin compound. For example, 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.

In some embodiments, in the indirubin compounds having a structure of formula (I), as specified above, or a pharmaceutically acceptable salt or prodrug thereof, R² is O. In some embodiments, R² is NOH. In some embodiments, R² is NOAc. R³ may be -H, or R³ may be halo. In some embodiments, n is 0, or n is 1-4, or n is 1-3, or n is 1-2. In one embodiment, n is 1 and in a further embodiment each R³ independently is halo. In some embodiments, each R³ independently is Br, CI, or I, in particular bromo. In one embodiment each R³ has the same meaning. In some embodiments, R³ may be Ci_₄alkyl, or Ci_₂alkyl, or methyl.

In certain embodiments, the indirubin compound is (Z)-[2,3'-biindolinylidene]-2',3-dione (which i ich can be represented by formula (II):

or a pharmaceutically acceptable salt or prodrug thereof.

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 . In certain embodiments, 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.

In certain embodiments, 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:

(III)

BlO-acetoxime is a selective inhibitor of the GSK-3a and GSK-3 isomers of GSK-3, and has low selectivity for CDK molecules.

In certain embodiments, 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:

(IV)

In other embodiments, 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

(v)

In certain embodiments, the indirubin compound is a compound having the formula:

or a pharmaceutically acceptable salt or prodrug thereof.

In certain embodiments, 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. In certain embodiments, the indole is 9-bromo-7,12-dihydro-indolo[3,2- d][l]benzazepin-6(5H)-one, which is also known as Kenpaullone.

In certain embodiments, the GSK-3 inhibitor is a benzofuran. A benzofuran as mentioned herein is a heterocyclic compound comprising a benzofuran moiety. In certain embodiments, 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:

(VII),

which can also be referred to as 2-methyl-5-[3-[4-(methylsulfinyl)phenyl]-5-benzofuranyl]- 1,3,4-oxadiazole, or as TCS2002.

In some embodiments, 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:

In one embodiment the pyrrole-pyrazine is 7-n-butyl-6-(4-hydroxyphenyl)[5H]- pyrrolo[2,3-b]pyrazine, which may also be referred to as aloisine.

Of interest for use in the present invention are those 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 . Of specific interest are 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 . In one embodiment, selectivity means an IC₅₀ 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. Such functional groups may be hydroxyl groups, including hydroxyimino groups (=N-OH), or amino groups. 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. Because active prodrugs may be absorbed more effectively than the parental drug, 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.

More specifically the term "prodrug", as used herein, 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.

In certain embodiments, 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

GSK-3 and capable of suppressing the expression of said DNA. For example, antisense DNA or antisense NA may be used. 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. Especially in the entire base sequence of the complementary strand to the DNA of GSK-3 , exemplary antisense oligonucleotides are (a) an antisense oligonucleotide 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 complementary strand of the base sequence which encodes the N-terminal region of the protein of the present invention (e.g., the base sequence around the initiation codon) in the case of antisense oligonucleotide directed to translation inhibition and (b) an antisense oligonucleotide 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 complementary strand of the entire base sequence of the DNA of GSK-3 having intron, in the case of antisense oligonucleotide directed to RNA degradation by RNaseH, respectively. Exemplary antisense oligonucleotides generally comprise about 10 to about 40 bases, for example, about 15 to about 30 bases.

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. In some embodiments, the GSK-3 antisense molecules are morpholinos. For example, the morpholinos may be nucleic acid analogues which block regions of the GSK-3 RNA.

In some embodiments, 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.

Accordingly, 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. There are specific requirements for 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. Furthermore, substitution of one or both siRNA strands with 2'-deoxy or 2'-0-methyl nucleotides abolishes NAi activity, whereas substitution of 3'- terminal siRNA nucleotides with deoxy nucleotides was shown to be tolerated. Mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5'-end of the siRNA guide sequence rather than the 3'-end. Other studies have indicated that a 5'-phosphate on the target-complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5'-phosphate moiety on the siRNA; however, siRNA molecules lacking a 5'-phosphate are active when introduced exogenously, suggesting that 5'-phosphorylation of siRNA constructs may occur in vivo.

After a target, for example a region of the GSK-3 gene has been selected, siNA molecules may be synthesized. In some embodiments, 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

chromatography (HPLC) and re-suspended in water.

In some embodiments, 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. Alternatively, viral vectors can be used that provide for transient expression of siNA molecules. In certain embodiments, chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) prevents their degradation by serum ribonucleases, which increases their potency. Various chemical modifications can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules described herein. Such modifications can be made to enhance their efficacy in cells, and/or remove of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements.

In some embodiments, inhibitors of GSK-3 may be peptide inhibitors. Exemplary peptide inhibitors include but are not limited to fragments derived from F AT1, the mammalian version of a GSK-3-binding protein, and peptide inhibtors derived from GSK-3 recognition motifs:

SQPETRTGDDDPHRLLQQLVLSGN LIKEAVRRLHSRRLQ (SEQ ID NO: l)

KEAPPAPPQSP (SEQ I D NO:2)

GKEAPPAPPQSP (SEQ ID NO:3) (which may include myristolation on Glyl,

phosphorylation on Serll, and Pro-12 = C-terminal amide).

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. In some embodiments, 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. In certain embodiments, 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, such as GSK-3 inhibitors, may also provide treatment for therapy-resistant forms of seizure. Notably, 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. In 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.

The particular 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.

The compounds for use in the present invention 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. These pharmaceutical compositions are desirably in unitary dosage form suitable, for example, for oral, rectal, or percutaneous administration. For example, in preparing the compositions in oral dosage form, 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. In compositions suitable for percutaneous administration, the carrier optionally comprises a penetration enhancing agent and/or a suitable wetting agent. These 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.

Those of skill in the treatment of the treatment of neurological disorders characterized by seizures, in particular of epilepsy, will be able to determine the effective amount from the test results presented hereinafter. In general it is contemplated that 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.

Examples

Having provided a general disclosure, the following examples help to illustrate the general disclosure. These specific examples are included merely to illustrate certain aspects and embodiments of the disclosure, and they are not intended to be limiting in any respect. Certain general principles described in the examples, however, may be generally applicable to other aspects or embodiments of the disclosure.

Example 1: Identification of indirubin as active anticonvulsant principle in Indigofera arrecta

Ten plants comprising part of a decoction used by traditional healers in Eastern Congo for the treatment of epilepsy, were collected and prepared as methanolic extracts for primary screening in a larval zebrafish PTZ seizure assay (Baraban et al., Neuroscience, 131(3), 759-768 (2005); Berghmans et al., Epilepsy research, 75(1), 18-28 (2007); Orellana-Paucar et al., Epilepsy & Behavior, 24(1), 14-22 (2012)). An extract of the leaves of Indigofera arrecta, a large shrub belonging to the family of Fabaceae and locally called "Kasholoza", particularly showed potent anticonvulsant activity. Hence, the secondary bio-active metabolites of the plant were isolated and identified. Bio-guided fractionation and structural elucidation using high- resolution mass spectrometry and N M resulted in the isolation of indiru bin. Purity was determined by H PLC (diol column, mobile phase: ethyl acetate) and was 85% (as monitored at UV289). The spectral data were in agreement with those previously reported. Commercially available indirubin (95% pure) was retested in zebrafish larvae to determine the MTC and effective concentration range in the PTZ assay. No signs of toxicity were observed up to 300 μΜ. Higher concentrations could not be tested as indirubin precipitated out of solution. 300 μΜ significantly decreased larval locomotor activity induced by PTZ (Fig. 2). Treatment with 30 μΜ and 100 μΜ did not result in a significant reduction of larval movement.

To confirm the seizure suppressing activity of indirubin tectal field recordings were performed on zebrafish larvae. Electrographic recordings showed that 300 μΜ indirubin was able to decrease the number of interictal and ictal-like spikes (Fig. 3A, 3B) as well as the total cumulative duration of epileptiform activity (Fig. 3C).

Example 2: Mechanism of action studies in zebrafish

In order to determine the mechanism by which indirubin exerts its anticonvulsant activity, a number of compounds were tested at their MTC values in the zebrafish acute PTZ assay with different affinities for its primary targets GSK-3 , CDK or AhR (Table 1; Fig. 4).

Compounds with greater selectivity towards GSK-3 (BlO-acetoxime, TCS2002, azakenpaullone, and aloisine were active in the larval zebrafish seizure assay, while those more selective towards CDK (olomoucine) and AhR (MeBIO) were inactive. Roscovitine, a compound selective towards CDK2 and CDK5 was modestly active.

For instance, analysis of the 30 minutes tracking period revealed a significant decrease in the total locomotor activity induced by PTZ when co-administered with 200 μΜ BlO-acetoxime. This effect was seen over the complete tracking period (Fig. 4). For TCS2002 a significant decrease in larval movement was seen when administering 30 μΜ from 5 minutes onwards. (Fig- 4).

Analysis of electrographic recordings in larval zebrafish showed that 200 μΜ BlO- acetoxime did not decrease the number of interictal-like spikes or ictal-like spikes (Fig. 5A, 5B), but was capable of decreasing the total cumulative duration of epileptiform activity (Fig. 5C). Similarly, TCS2002 (30 μΜ) also did not decrease the number of interictal-like and ictal-like spikes (Fig. 5A, 5B). However, in contrast to BlO-acetoxime and indirubin, TCS2002 caused an increase in the total cumulative duration of epileptiform activity (Fig. 5C).

Targeted inhibition of GSK-3 was achieved using antisense-mediated morpholino (MO) knockdown in zebrafish embryos (Summerton & Weller, Antisense and Nucleic Acid Drug Development, 7(3), 187-195 (1997); Nasevicius & Ekker, Nature Genetics, 26(2), 216-220

(2000); Bill et al., Zebrafish, 6(1), 69-77 (2009)). Importantly, the MO concentration was titered so as not to cause any observable gross developmental dysmorphologies that could potentially impair locomotor ability (data not shown). In addition, 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

concentration dependent manner (Fig. 6).

An additional selection of indirubin analogues (Fig. 12) was tested in the larval zebrafish assay, using concentrations ranging from 30 μΜ - 300 mM.

Example 3: Evaluation of anticonvulsant activity of a selection of compounds in rodent models

Indiru bin was tested in the mouse PTZ and pilocarpine intravenous infusion assays at a dose of 10 mg/kg but did not appear to suppress any of the seizure parameters scored for (Tables A and B). Although indirubin was ineffective in the acute PTZ and pilocarpine rodent assays, it showed significant seizure inhibitory activity in both the rat intrahippocampal pilocarpine limbic seizure and mouse 6 Hz models. Both 2 mg/kg and 10 mg/kg reduced the total seizure severity score in the rat intrahippocampal pilocarpine test in a dose-dependent manner (Fig. 7A). In the 6 Hz model a dose of 2 mg/kg was only mildly seizure protective (one mouse out of six did not undergo seizures), while 10 mg/kg and 30 mg/kg protected each four and five out of six mice respectively (Fig. 7B).

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.

infusion assays (data not shown). However, BlO-acetoxime and TCS2002 were found to be active in both the rat pilocarpine limbic seizure model and in the mouse 6 Hz model. At a dose of 0.5 mg/kg, both compounds caused a significant reduction in limbic seizures (Fig. 8A and Fig. 9A) and protected at least 50% and 37.5% of mice after corneal stimulation at doses of 0.05 mg/kg, 0.5 mg/kg and 5 mg/kg for BlO-acetoxime and TCS2002 respectively (Fig. 8B and Fig. 9B).

Interestingly, 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 μΜ and 100 μΜ) did not significantly change the TSSS in the rat intrahippocampal pilocarpine limbic seizure model (data not shown).

Example 4: Evaluation of anticonvulsant activity of GSK-3 inhibitors

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.

Animals

Zebrafish (Danio rerio) stocks of the Tg(flila:EGFP)yl line or the AB line were maintained at 28.5°C, on a 14/10 hour light/dark cycle under standard aquaculture conditions, and fertilized eggs were collected via natural spawning. Embryos were reared under constant light conditions in 0.3x Danieau's buffer (also referred to in text simply as 'embryo medium' - 1.5 mM H EPES, pH 7.6, 17.4 mM NaCI, 0.21 mM KCI, 0.12 mM MgS04, and 0.18 mM Ca(N03)2) in an incubator at 28.5°C. For the behavioral and electrographic assays, 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. For the morpholino (MO) injection embryos of the AB line were used. For the pilocarpine and PTZ tail vein infusion test male C57BI/6 mice (20-30 g) (Charles River Laboratories) were used. For the 6 Hz test and the beamwalking assay male N MRI mice (20-30 g) (Charles River Laboratories) were used.

For the intrahippocampal pilocarpine limbic seizure model male al bino Wistar rats (Charles River Laboratories), weighing 260-320 g, were used.

Chemicals and Reagents

The leaves of Indigofera arrecta were collected on Idjwi Island, Lake Kivu (Congo) and dried. For the testing in zebrafish, dried plant material was extracted with methanol. After centrifugation the clear supernatants were collected and concentrated using a rotavapor (Buchi). The extract was suspended in DMSO before use (stock solution: 20 mg/ml).

Indiru bin (purity: 95%) was obtained from Apin Chemicals Ltd; BlO-acetoxime (purity >98%) and TCS2002 (purity >99%) were obtained from Tocris Bioscience; aloisine RP106 (purity >95%), 1-azakenpaullone (purity >95%) and MeBIO (purity >97%) were obtained from Merck; and olomoucine (purity >98%) and roscovitine (purity >98%) were obtained from Sigma. For zebrafish experiments, all compounds were dissolved in DMSO and diluted in embryo medium to achieve a final DMSO concentration of 1% w/v. Embryo medium prepared with DMSO to a final concentration of 1% w/v served as a vehicle control (VHC).

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 CaCI₂ 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.

For isolation of the active constituent of Indigofera arrecta 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. A shorter distance travelled or movement in the same place was scored as a decreased or impaired touch response. The MTC (maximum tolerated concentration) was thus defined as the maximum concentration that did not cause death and where not more than two out of 12 larvae exhibited any sign of acute toxicity.

Evaluation of anticonvulsant activity

1. Zebrafish

Six-dpf larvae were placed in a 96-well plate with one larva per well. Twenty-four larvae were used per treatment group. The larvae were pre-incubated with compound or VHC for 18 hours in a volume of 100 μΙ (for the schematic timeline, see Fig. 1). After pre-incubation with each compound, normal embryo medium was added to twelve of the twenty-fourhalf of the larvae per treatment group. To the other twelve larvaehalf, 100 μΙ of PTZ solution was added to obtain a final concentration of 20 mM (Berghmans et al., Epilepsy research, 75(1), 18-28 (2007); Orellana-Paucar et al., Epilepsy & Behavior, 24(1), 14-22 (2012)). Larvae were allowed to habituate for 5 min in a darkened chamber of an automated tracking device (ZebraBox, Viewpoint, France). The total locomotor activity was then measured using an automated tracking system (VideoTrack, Viewpoint, France). Total movement or activity was expressed in "actinteg" units. The actinteg value of Viewpoint software is defined as the sum of all image pixel changes detected during the time slice defined for the experiment.

Each larva (6 dpf) was pre-incubated with either compound or VHC for 18 hours in a volume of 400 μΙ in individual wells of a 24-well plate (for the schematic timeline, see Figure 1). After the pre-incubation time, an equal volume of PTZ solution was added to the well to obtain a final concentration of 20 mM. 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 ΜΩ) 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 10μ≤ (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.

A spike was defined as an interictal-like if its amplitude exceeded three times the background noise and it lasted for less than 3 seconds. Longer discharges were counted as ictal-like ones (D'Antuono et al., Epilepsia, 51(3), 423-431 (2010)). The changes in the numbers and average duration of either type of electrographic activity were analyzed and also total cumulative duration of all forms of epileptiform discharges during recording time using Clampfit 10.2 software.

2. Mouse PTZ tail infusion model

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 μΙ/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)

duration of infusion (s) * rate of infusion (rol/sj ^« drug concentration (mg/ml)

weight of mouse (kg)

3. Mouse pilocarpine tail infusion model

The threshold for different phases of pilocarpine-induced seizure activity was determined by an i.v. infusion of pilocarpine (24 mg/ml) in the lateral tail vein at a constant rate of 150 μΙ/min. Methylscopolamine (1 mg/kg, s.c.) was injected 30 min before administration of pilocarpine to prevent peripheral cholinergic symptoms. 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 pilocarpine tail infusion.

The following endpoints were used to determine the seizure threshold for pilocarpine: shivering, tail twitch, rearing with bilateral myoclonus, clonic seizures with loss of righting reflexes, tonic hind limb 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 equation as described under 2.1.

The data (mean ± SD) listed in the following 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.

Ta ble A:

Table B:

Pilocarpine dose required to elicit

individual seizure parameters (mg/kg)

VHC Indirubin (10 mg/kg)

Seizure parameters

n=5 n=7

Shivering 364.4 ± 96.0 406.5 ± 48.6

Tail twitch 500.2 ± 74.7 533.3 ± 52.1

Rearing 532.3 ± 110.9 599.9 ± 62.9

Falling 612.1 ± 49.6 656.3 ± 97.3

Tonic hindlimb

635.4 ± 52.3 676.3 ± 98.5

extension

4. 6 Hz test in rats

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.

5. Intrahippocampal pilocarpine limbic seizure model in rats

Rats were anesthetized with an i.p. injection of ketamine HCkdiazepam (94.5:4.5 mg/kg). Three mm above the final probe position (CA1-CA3 region), 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.

The rats received either vehicle (50% propyleneglycol in saline; i.e. control group rats) or compound. 30 min after vehicle or compound injection, 12 mM pilocarpine was perfused intrahippocampally for 40 minutes to evoke limbic seizures. Rats were observed carefully during the following 100 minutes (15 periods in total). Within each rat receiving the chemoconvulsant pilocarpine, the behavioral manifestations were scored during each period after the start of pilocarpine administration in order to assess seizure severity. The Seizure Severity Score (SSS) was adapted from Racine's scale to take into account the typical behavioral changes associated with pilocarpine-induced motor seizures. 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. Beamwalking assay in mice

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.

8. Morpholino treatment

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.

At 3 dpf 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.

Statistical analysis

Data were analyzed using the unpaired Student's t-test for the locomotor behavioral tests, tectal field recordings, and the PTZ and pilocarpine i.v. infusion tests in mice. For the rat, 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 IC₅₀ values for different enzymes.

Compound MTC GSK-3 CDK1 CDK2 CDK5 Ah

references

Indirubin 300 μΜ 0.6 μΜ 10 μΜ 7.5 μΜ 5.5 μΜ 2 ηΜ

(1) (2) (3) (4)

BlO-acetoxime 200 μΜ 10 nM 63 μΜ 4.3 μΜ 2.4 μΜ

(5) (6) TCS2002 (7) 30 μΜ 35 nM >10 μΜ >10 μΜ >10 μΜ

Olomoucine (8) 1 mM 7 μΜ 3 μΜ

1-Azakenpaullone 10 μΜ 18 nM 2 μΜ 4.2 μΜ

(9)

Aloisine P106 30 μΜ 0.92 μΜ 0.7 μΜ 1.5 μΜ

(10)

Roscovitine (11) 100 μΜ 0.7 μΜ 0.16 μΜ

MeBIO (6) (12) 1 mM >100 μΜ >92 μΜ >100 μΜ 20 ηΜ

(1) Hoessel et al., Nature cell biology, 1(1), 60-67 (1999).

(2) Leclerc et al., The Journal of biological chemistry, 276(1), 251-260 (2001).

(3) Marko et al., British journal of cancer, 84(2), 283-289 (2001).

(4) Guengerich et al., Archives of biochemistry and biophysics, 423(2), 309-316 (2004).

(5) Meijer et al., Chemistry and Biology, 10, 1255-1266 (2003).

(6) Polychronopoulos et al. Journal of medicinal chemistry, 47(4), 935-946 (2004).

(7) Saitoh et al., Journal of medicinal chemistry, 52(20), 6270-6286 (2009).

(8) Vesely et al., European journal of biochemistry, 224(2), 771-786 (1994)

(9) Kunick et al., Bioorganic & Medicinal Chemistry Letters, 14(2), 413-416 (2004).

(10) Mettey et al., Journal of medicinal chemistry, 46(2), 222-236 (2003).

(11) Meijer et al., European journal of biochemistry, 243(1-2), 527-536 (1997).

(12) Knockaert et al. (2004), Oncogene, 23(25), 4400-4412 (2004). Table 2: Exem lary GSK-3 inhibitors (selected from commercially available compounds)

Claims

CLAI MS

1. An inhibitor of phosphorylation by GSK3 for use in the treatment of neurological disorders characterized by seizures, wherein the inhibitor is an indole.

2. The inhibitor of claim 1, wherein the indole is an indirubin compound.

3. The inhibitor of claim 2, wherein the indirubin compound is a compound of formula (I), or a pharmaceutically acceptable salt or prodrug thereof:

1 is O; R² is selected from O, NOH, and NO-CO-R⁴; R³ is selected from -H and halo; R⁴ is Ci-C₆ alkyl; and n is 0-5.

4. The inhibitor of claim 3, wherein R² is O.

5. The inhibitor of claim 3 or claim 4, wherein R² is NOH.

6. The inhibitor of claim 3 or claim 4, wherein R² is NOAc.

7. The inhibitor of any one of claims 3-6, wherein R³ is -H.

8. The inhibitor of any one of claims 3-6, wherein R³ is selected from Br, CI, and I.

9. The inhibitor of any one of claims 3-8, wherein n is 1-4.

10. The inhibitor of any one of claims 3-8, wherein n is 0 or 1.

11. The inhibitor of any one of claims 3-8, wherein n is 1 and the R³ substituent is in 6- position.

12. The inhibitor of claim 2, wherein the indirubin compound is a compound of formula (I I), or a pharmaceutically acceptable salt or prodrug thereof:

13. The inhibitor of claim 2, wherein the indirubin compound is a compound of formula (I I I), or a pharmaceutically acceptable salt or prodrug thereof:

(III)

14. The inhibitor of 2, wherein the indirubin compound is a compound of formula (IV), or a pharmaceutically acceptable salt or prodrug thereof:

15. The inhibitor of claim 2, wherein the indirubin compound is a compound of formula (V) or a pharmaceutically acceptable salt or prodrug thereof:

(V)

16. The inhibitor of claim 2, wherein the indirubin compound is a compound is a

compound of formula :

or a pharmaceutically acceptable salt or prodrug thereof.

17. The inhibitor of any of claims 1-16, wherein the neurological disorder is epilepsy.