Glucose oxidase compositions as neonatal anticonvulsants

Abstract

Neonatal epileptic seizures differ from adult epileptic seizures, and many antiepileptic drugs that are effective in adults often fail to treat neonatal epilepsy. Here we show that gluconic acid, a natural organic acid abundant in fruit and honey, and the glucose oxidase enzyme effectively suppress neonatal epilepsy both in vitro and in vivo. Sodium gluconate is demonstrated to suppress epileptiform burst activity in cell culture and protect neurons from kainic acid-induced cell death. Sodium gluconate also suppresses epileptiform burst activity in brain slices in a manner far more effective in neonatal animals than in aged animals. Sodium gluconate suppresses seizure activity in neonatal animals in a manner far more effective than in adult animals. Consistently, in vivo EEG recordings also revealed that sodium gluconate suppresses seizure activity in neonatal animals in a manner far more effective than in adult animals. Mechanistically, sodium gluconate suppresses voltage-dependent CLC‑3 C1 in neuronal cultures and hippocampal slices ‑ Together, these data suggest a new class of antiepileptic drugs, gluconate, that blocks CLC‑3 C1 ‑ The channel effectively suppresses neonatal epileptic seizures.

Description

[0001] Government Support Statement

[0002] This invention was made with government grants Nos. MH083911 and AG045656 awarded by the National Institutes of Health. The government has certain rights in this invention. Field of the Invention

[0003] The present invention relates to the field of neurological disorders. In particular, the prevention and treatment of convulsive disorders, including but not limited to myotonic / clonic convulsions, epilepsy, Jackson's epilepsy disorder and / or involuntary tremors. For example, some embodiments relate to the treatment and prevention of convulsive disorders in newborns and / or infants. It has been found that the gluconate base and glucose oxidase composition have selective effectiveness in the treatment and / or prevention of convulsive disorders in newborns and / or infants.

[0004] background

[0005] Epilepsy incidence is highest in the first year of life, with reported rates in the United States of approximately 1.8-3.5 / 1,000 live births. Silverstein et al., "Neonatal seizures" Annals Of Neurology 62:112-120 (2007); and Jensen F., "Neonatal seizures: An update on mechanisms and management" Clinics In Perinatology 36:881-900 (2009). Although many anti-epileptic drugs (AEDs) have been developed over the past few decades, neonatal epileptic seizures are often refractory to current AEDs, which are more effective for older children or adults. Painter et al., “Phenobarbital compared with phenytoin for the treatment of neonatal seizures” The New England Journal Of Medicine 341:485-489 (1999); van Rooij et al., “Treatment of neonatal seizures” Seminars In Fetal&NeonatalMedicine 18:209-215 (2013); and Dzhala et al., “NKCC1 transporter facilitates seizures in the developing brain” Nature Medicine 11:1205-1213(2005).

[0006] In some cases, even after AED application, electroencephalogram (EEG) recordings show persistent cortical epileptic activity in neonates, which may impair cognitive development and later lead to epilepsy. Connell et al., "Clinical and EEG response to anticonvulsants in neonatal seizures" Archives Of Disease In Childhood 64:459-464 (1989); Glykys et al., "Differences in cortical versus subcortical GABAergic signaling: a candidate mechanism of electroclinical uncoupling of neonatal seizures" Neuron 63:657-672 (2009); and Puskarjov et al., "Pharmacotherapeutic targeting of cation-chloride cotransporters in neonatal seizures" Epilepsia 55:806-818 (2014).

[0007] Seizures are often triggered by overactivation of brain circuits, which can be induced by enhancing GABA, the major inhibitory receptor in the adult brain. A GABA receptor A -R). Therefore, AEDs are often developed to increase GABA A -R function, such as benzodiazepines and barbiturates. Bialer, M. & White, HS Key factors in the discovery and development of new antiepileptic drugs. Nature reviews. Drug discovery 9, 68-82 (2010). However, although GABA A-Rs are mainly inhibitory in the adult brain, but they are excitatory in the developing brain. Chen, G., Trombley, PQ & van den Pol, AN Excitatory actions of GABA in developing rat hypothalamic neurones. The Journal of physiology 494 (Pt 2), 451-464 (1996); and Ben-Ari, Y. Excitatory actions of gaba during development: then nature of the nurture. Nature reviews. Neuroscience 3, 728-739 (2002).

[0008] Excitatory GABAergic transmission in the developing brain also explains why GABA agonists are often ineffective in controlling neonatal seizures and may sometimes even exacerbate neonatal seizure activity. Farwell, JR, et al. Phenobarbital for febrile seizures--effects on intelligence and on seizure recurrence. The New England journal of medicine 322, 364-369 (1990).

[0009] Classically, GABA excitatory and inhibitory functions have been attributed to Cl - Regulation of cotransporters NKCC1 and KCC2. 10. Kaila, K., Price, TJ, Payne, JA, Puskarjov, M. & Voipio, J. Cation-chloride cotransporters in neuronal development, plasticity and disease. Nature reviews. Neuroscience 15, 637-654 (2014). Blaesse, P., Airaksinen, MS, Rivera, C. & Kaila, K. Cation-chloride cotransporters and neuronal function. Neuron 61, 820-838 (2009). Previous studies have found that NKCC1 may promote neonatal epileptic seizures in rodents. 12However, recent clinical trials in infants and young children have found that the NKCC1 blocker bumetanide has serious side effects and is very limited in its effectiveness in treating neonatal epileptic seizures. 13 . Dzhala, VI, et al. NKCC1 transporter facilitates seizures in the developing brain. Nature medicine 11, 1205-1213 (2005); and Pressler, RM, et al. Bumetanide for the treatment of seizures in newborn babies with hypoxicischaemic encephalopathy (NEMO): an open-label, dose finding, and feasibilityphase 1 / 2 trial. Lancet Neurol 14,469-477(2015).

[0010] Unfortunately, to date, there is no effective drug that can successfully treat neonatal epileptic seizures, thereby urgently searching for new drugs to treat neonatal epilepsy. What is needed in the art is a simple, effective and safe anti-epileptic drug to treat the unique characteristics of neonatal epileptic seizures. SUMMARY OF THE INVENTION

[0012] The present invention relates to the field of neurological disorders. In particular, the prevention and treatment of convulsive disorders, including but not limited to myotonic / clonic convulsions, epilepsy, Jackson syndrome and / or involuntary tremors. For example, some embodiments relate to the treatment and prevention of convulsive disorders in newborns and / or infants. Gluconate-based and glucose oxidase compositions have been found to be selectively effective in treating and / or preventing convulsive disorders in newborns and / or infants.

[0013] In one embodiment, the present invention contemplates a method comprising: a) providing; i) a neonatal patient comprising glucose and experiencing convulsions; and ii) a composition comprising glucose oxidase; and b) administering the composition to the patient under conditions where the convulsions are reduced. In one embodiment, the conditions include conversion of the glucose to gluconate. In one embodiment, administration further includes supplemented glucose. In one embodiment, the composition further includes supplemented glucose. In one embodiment, the composition and the supplemented glucose are administered continuously. In one embodiment, the composition and the supplemented glucose are administered simultaneously. In one embodiment, the conditions include conversion of the supplemented glucose to gluconate. In one embodiment, the composition is a pharmaceutically acceptable composition. In one embodiment, the administration further includes an effective amount of the composition.

[0014] In one embodiment, the present invention contemplates a method comprising: a) providing: i) a neonatal patient experiencing convulsions, wherein the patient does not require a divalent cation-based therapy; and ii) a composition comprising a gluconate complex; and b) administering the composition to the patient under conditions such that the convulsions are reduced. In one embodiment, the gluconate complex does not contain divalent cations. In one embodiment, the gluconate complex is sodium gluconate. In one embodiment, the divalent cation-based therapy is a calcium-based therapy. In one embodiment, the divalent cation-based therapy is a magnesium-based therapy. In one embodiment, the divalent cation-based therapy is a zinc-based therapy. In one embodiment, the gluconate complex comprises a gluconic acid derivative.

[0015] In one embodiment, the present invention contemplates a method comprising:

[0016] a) providing: i) a neonatal patient experiencing convulsions, wherein the patient does not require a divalent cation-based therapy; ii) a composition consisting of a gluconate complex, wherein the complex does not contain divalent cations; and b) administering the composition to the patient under conditions such that the convulsions are reduced. In one embodiment, the gluconate complex is sodium gluconate. In one embodiment, the divalent cation-based therapy is a calcium-based therapy. In one embodiment, the divalent cation-based therapy is a magnesium-based therapy. In one embodiment, the divalent cation-based therapy is a zinc-based therapy. In one embodiment, the gluconate complex comprises a gluconic acid derivative.

[0017] In one embodiment, the present invention contemplates a composition comprising a gluconate derivative wherein at least one hydroxyl moiety or carboxylic acid moiety is substituted with a group including, but not limited to, substituted or unsubstituted aryl or heteroaryl, unsubstituted or substituted C1-C6-alkyl, substituted or unsubstituted 5-6 membered saturated or unsaturated fused ring, substituted or unsubstituted 5-6 membered saturated or unsaturated ring, natural or synthetic amino acid residue, trihalomethyl, substituted or unsubstituted C1-C6-alkoxy, NH2, SH, thioalkyl, aminoacyl, aminocarbonyl, substituted or unsubstituted C1-C6-alkoxycarbonyl, aryl, heteroaryl, substituted or unsubstituted 4-8 membered cycloalkyl, optionally containing 1-3 heteroatoms, carboxyl, cyano, halogen, hydroxyl, nitro, acetoxy, aminoacyl, sulfonyloxy, sulfonyl, C1-C6-thio R5 and R6 together can form a substituted or unsubstituted 4-8 membered saturated cycloalkyl or heteroalkyl.

[0018] In one embodiment, the present invention contemplates a method comprising: a) providing i) a neonatal patient exhibiting convulsions, wherein the patient comprises at least one chloride ion channel; ii) a composition comprising a chloride ion channel blocker; and b) administering the composition to the patient under conditions that reduce the convulsions. In one embodiment, the chloride ion channel blocker is a gluconate complex that does not contain divalent cations. In one embodiment, chloride ion channel blockers include but are not limited to niflumic acid (NFA), flufenamic acid, 4,4'-diisothiocyanate-2,2'-stilbene disulfonic acid disodium salt (DIDS) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). In one embodiment, the gluconate complex is sodium gluconate. In one embodiment, the patient does not require divalent cation-based treatment. In one embodiment, the divalent cation-based treatment is calcium-based treatment. In one embodiment, the divalent cation-based treatment is magnesium-based treatment. In one embodiment, the divalent cation-based treatment is zinc-based treatment. In one embodiment, the gluconate complex comprises a gluconic acid derivative.

[0019] definition

[0020] To facilitate understanding of the present invention, a number of terms are defined below. The terms defined herein have the meanings commonly understood by those of ordinary skill in the art in the field relevant to the present invention. Terms such as "a", "an", and "the" are not intended to refer only to a single entity but also to multiple entities, and also include general classes that can be used to illustrate specific examples. The terms herein are used to describe specific embodiments of the present invention, but their usage does not limit the present invention except as outlined in the claims.

[0021] The term "gluconic acid" or "gluconate" as used herein refers to a six α-carbon chain comprising five hydroxyl moieties and one carboxylic acid moiety. As disclosed herein, each of the hydroxyl and carboxylic acid moieties can be substituted and / or derivatized to provide anticonvulsant gluconate derivatives with improved clinical effectiveness compared to traditional divalent cation therapy.

[0022] The term "convulsion" or "seizure" refers to any abnormal violent and involuntary contraction or series of contractions of muscles and / or body manifestations, including but not limited to convulsions, sensory disturbances and / or loss of consciousness, caused by abnormal electrical discharges from the brain.

[0023] The term "neonate" or "neonatal" as used herein refers to a newborn baby that is usually less than one month old. In general, reference to the age of a newborn is characterized by "P#", where "P" means after birth, "#" means age in days, and P0 means day of birth.

[0024] The term "divalent cation" refers to any ion with a positive divalent charge (eg, 2+). For example, the ion Ca 2+ ,Mg 2 + and / or Zn 2+ It is considered a divalent cation.

[0025] As used herein, the term "about" in any context of any assay measurement refers to + / - 5% of the given measurement.

[0026] The term "effective amount" as used herein refers to a specific amount of a pharmaceutical composition comprising a therapeutic agent that achieves a clinically beneficial result (e.g., symptom relief). The toxicity and therapeutic efficacy of these compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as for determining LD 50 (a dose lethal to 50% of the population) and ED 50(the dose that is therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as LD 50 / ED 50 Compounds that exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and additional animal studies can be used to formulate a range of dosages for use in humans. Doses of such compounds are preferably within the range that includes the ED with little or no toxicity. 50 The dosage varies within this range depending on the dosage form used, the sensitivity of the patient, and the route of administration.

[0027] The term "symptom" as used herein refers to any subjective or objective evidence of a disease or physical disorder observed by a patient. For example, subjective evidence is typically based on patient self-report and may include, but is not limited to, pain, headache, visual disturbances, nausea, and / or vomiting. Alternatively, objective evidence is typically the result of a medical test, including, but not limited to, temperature, complete blood count, lipid panel, thyroid panel, blood pressure, heart rate, electrocardiogram, tissue, and / or body imaging scans.

[0028] As used herein, the term "disease" or "medical condition" refers to any impairment of the normal state of the body of a living animal or plant, or one of its parts, which interferes with or alters the manifestation of vital functions. It is usually manifested by noticeable signs and symptoms, usually in response to: i) environmental factors (malnutrition, industrial hazards, or climate); ii) specific infectious agents (such as worms, bacteria, or viruses); iii) an inherent defect of the organism (as a genetic abnormality); and / or iv) a combination of these factors.

[0029] The terms "reduce," "inhibit," "reduce," "suppress," "lower," "prevent," and grammatical equivalents (including "lower," "smaller," etc.) when referring to any symptom in an untreated subject relative to a treated subject mean that the amount and / or magnitude of the symptoms in the treated subject is lower than in the untreated subject by any amount that any medically trained person would consider to be a clinically relevant amount. In one embodiment, the amount and / or magnitude of the symptoms in the treated subject is at least 10%, at least 25%, at least 50%, at least 75%, and / or at least 90% lower than the amount and / or magnitude of the symptoms in the untreated subject.

[0030] The term "attachment" as used herein refers to any interaction between a medium (or carrier) and a drug. Attachment may be reversible or irreversible. Such attachment includes, but is not limited to, covalent bonds, ionic bonds, van der Waals forces, or frictional forces, etc. If the drug is impregnated, incorporated, coated, suspended, dissolved, mixed, etc., the drug is attached to the medium (or carrier).

[0031] The term "medium" as used herein refers to any material or combination of materials that serves as a carrier or vehicle for delivering a drug to a treatment site (e.g., a wound, a surgical site, etc.). Therefore, for all practical purposes, the term "medium" is considered to be a synonym of the term "carrier". It will be appreciated by those skilled in the art that a medium comprises a carrier, wherein the carrier is attached to a drug or drug, and the medium facilitates the delivery of the carrier to the treatment site. In addition, the carrier may comprise an attached drug, wherein the carrier facilitates the delivery of the drug to the treatment site. Preferably, the medium is selected from, but is not limited to, foams, gels (including but not limited to hydrogels), xerogels, microparticles (i.e., microspheres, liposomes, microcapsules, etc.), bioadhesives, or liquids. The present invention particularly contemplates a medium comprising a combination of microparticles and hydrogels, bioadhesives, foams, or liquids. Preferably, hydrogels, bioadhesives, and foams include any one or combination of polymers contemplated herein. Any medium contemplated by the present invention may include a controlled release formulation. For example, in some cases, the medium constitutes a drug delivery system that provides controlled and sustained drug release for a period of about 1 day to 6 months.

[0032] The term "drug" or "compound" as used herein refers to any pharmacologically active substance that can be administered to obtain a desired effect. The drug or compound can be a synthetic or naturally occurring non-peptide, protein or peptide, oligonucleotide or nucleotide, polysaccharide or sugar.

[0033] As used herein, the term "administered" or "administering" refers to any method of providing a composition to a patient so that the composition has its intended effect on the patient. Exemplary methods of administration are by direct mechanisms such as local tissue administration (e.g., extravascular placement), oral ingestion, intravenous injection, transdermal patch, topical, inhalation, suppository, etc.

[0034] The term "patient" or "subject" as used herein is a human or animal, not necessarily an inpatient. For example, an outpatient, a person in a nursing home is a "patient". A patient can include any age of human or non-human animal, thus including adults and juveniles (i.e., newborns, infants, and / or children). The term "patient" is not intended to imply a need for medical treatment, and thus, a patient may voluntarily or involuntarily become part of a clinical or experimental trial to support basic scientific research.

[0035] The term "effective amount" as used herein refers to a specific amount of a pharmaceutical composition comprising a therapeutic agent that achieves a clinically beneficial result (e.g., symptom relief). The toxicity and therapeutic efficacy of these compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as for determining LD 50 (a dose lethal to 50% of the population) and ED 50 (the dose that is therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as LD 50 / ED 50 Compounds that exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and additional animal studies can be used to formulate a range of dosages for use in humans. Doses of such compounds are preferably within the range that includes the ED with little or no toxicity. 50 The dosage varies within this range depending on the dosage form used, the sensitivity of the patient, and the route of administration.

[0036] As used herein, the terms "pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered to animals or humans.

[0037] The term "pharmaceutically acceptable carrier" as used herein includes any and all solvents or dispersion media, including, but not limited to, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, as well as vegetable oils, coatings, isotonic and absorption delaying agents, liposomes, commercially available detergents, etc. Supplementary biologically active ingredients may also be incorporated into such carriers.

[0038] As used herein, the term "small organic molecule" refers to any molecule of a size comparable to those organic molecules commonly used in pharmaceuticals. The term does not include biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size from about 10 Da to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

[0039] The term "biologically active" refers to any molecule that has a structural, regulatory or biochemical function. For example, biological activity can be determined, for example, by restoring wild-type growth in cells lacking protein activity. Cells lacking protein activity can be generated by a number of methods (e.g., point mutations and frameshift mutations). Complementation is achieved by transfecting cells lacking protein activity with an expression vector expressing the protein, its derivatives or portions thereof.

[0040] The term "transfection" or "transfected" refers to the introduction of foreign DNA into a cell.

[0041] The term "binding" as used herein includes any physical attachment or intimate association that may be permanent or temporary. Typically, interactions such as hydrogen bonding, hydrophobic forces, van der Waals forces, covalent bonds, and ionic bonds facilitate physical attachment between the target molecule and the analyte being measured. In the case where the binding results in a chemical reaction, the "binding" interaction may be brief. This is typical when the binding component is an enzyme and the analyte is a substrate for the enzyme. For purposes of the present invention, the reaction resulting from contact between the binding agent and the analyte is also within the definition of binding.

[0042] As used herein, the term "supplemented glucose" refers to the intravenous administration of glucose to a pediatric patient in combination with glucose oxidase. For example, a preferred glucose infusion rate (GIR) is 7.3 mg / kg / minute.

[0043] Brief description of the attached figure

[0044] The patent file contains at least one drawing executed in color. Copies of patents with color drawings will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.

[0045] Figure 1 shows exemplary data showing the anti-burst activity of gluconate on cultured neurons. Data are presented as mean±sem, *P<0.05, **P<0.01, ***P<0.001.

[0046] Figure 1A : Spontaneous burst action potentials in cultured neurons were immediately arrested by application of 20 mM NaGcA (n=10 from 3 batches), extension graphs before, during and after NaGcA application are shown at the bottom.

[0047] Figure 1B : CTZ induced robust burst activity in cultured neurons (upper panel) and was completely blocked by 10 mM NaGcA (bottom panel).

[0048] Figure 1C: Dose-dependent inhibition of CTZ-induced burst activity by NaGcA. Black bars indicate burst frequency, and gray bars indicate burst duration (n=11, from 4 batches, paired Student t-test).

[0049] Figure 1D : Representative traces of kainic acid (1 μM, 2 hours) induced a strong burst activity (upper panel), and it was also significantly inhibited by 10 mM NaGcA (bottom panel).

[0050] Figure 1E : Dose-dependent response of KA-induced burst activity to NaGcA (n=12, from 3 batches, paired Student's t-test).

[0051] Figure 1F : Representative traces of robust activity induced by 4-AP (50 μM, 2 hours) (upper panel) that was significantly inhibited by 10 mM NaGcA (bottom panel).

[0052] Figure 1G : Dose response of 4-AP-induced burst activity to NaGcA (n=9, from 3 batches, paired Student's t-test).

[0053] Figure 1H : Representative traces showing recurrent epileptiform bursts induced by 10 μM CTZ (upper panel) and no burst activity in co-administration of CTZ and NaGcA (10 mM).

[0054] Figure 1I (ac): Quantitative data of panel h show that chronic co-treatment with 5 and 10 mM NaGcA significantly reduced the proportion of neurons showing burst activity (Figure 1I (a), χ2 test), burst frequency (Figure 1I (b), one-way ANOVA) and burst duration (Figure 1I (c) and Figure 1I (b)) one-way ANOVA. n = 22 for CTZ, n = 16 for CTZ + 5 mM NaGcA, n = 17 for CTZ + 10 mM NaGcA, all data from 5 batches, paired Student t test.

[0055] Figure 1J : Live / Death assay images showing calcein-AM (green for live) and ethidium homodimer-1 (red for dead) in different groups. Scale bar, 100 μm.

[0056] Figure 1K: Quantification of cell survival / death assay. The results indicate that NaGcA has neuroprotective function against kainic acid-induced neuronal excitotoxicity without any side effects on neuronal survival itself.

[0057] Figure 2 shows exemplary data that gluconate inhibits chloride currents in cultured cortical neurons. Data are shown as mean ± sem.

[0058] (af) NaGcA (10 mM) to Na + Current (a, b), K + Current (c, d) or Ca 2+ The current (e, f) has no effect.

[0059] (g) Typical Cl recorded before (black) and after (green) 10 mM NaGcA administration. - Electric current.

[0060] (h) IV curve shows the effect of Cl - Significant NaGcA inhibition of currents (n=7 from 3 batches of cultures, paired Student's t-test).

[0061] (i, j) Classic Cl - The channel blocker NPPB (100 μM) inhibited the Cl - Electric current.

[0062] (k, l) Another common Cl - Effect of DIDS (100 μM), a channel blocker, on the Cl - The inhibitory effect of DIDS on Cl - Sustained inhibition of current. (m, n) NPPB (m) and DIDS (n) both effectively inhibited CTZ-induced epileptiform activity, supporting the Cl - Channels involved in epileptogenesis.

[0063] Figure 3 shows exemplary data showing the age-dependent effects of gluconate on epileptic activity in hippocampal slices. Data are shown as mean ± sem.

[0064] (a) Extracellular field potential recordings in the CA3 pyramidal cell layer of hippocampal slices from neonatal (P7) mice. Epileptic events are induced by Mg-free 2+ aCSF induced with Mg-free 2+ The expanded traces before, during and after NaGcA administration are shown at the bottom.

[0065] (b) Power spectra of epileptic activity in a 5-minute time window before, during and after NaGcA administration. The power amplitude (integrated area under the spectral trace) was significantly suppressed after NaGcA administration.

[0066] (c, d) Comparable efficacy of NaGcA against epileptiform activity was examined in P12 hippocampal slices.

[0067] (e) NaGcA has a moderate antiepileptic discharge effect in P26 hippocampal slices compared to neonatal slices. The bottom panel shows the extended traces before, before, and after NaGcA perfusion. (f) The power amplitude was slightly reduced during NaGcA application and almost recovered after washing out NaGcA.

[0068] (g) In a continuous 5-min window before, during, and after NaGcA administration, 0Mg 2+ Normalized power of aCSF-induced epileptic activity.

[0069] NaGcA significantly reduced epileptic activity in the P6-8 and P10-12 groups, but had little effect on the P21-33 group. The data from the control group showed that the addition of 20 mM NaCl to 0 Mg 2+ There were no significant changes in extracellular field potential recordings during the 80 min period after aCSF.

[0070] Figure 4 provides exemplary data showing the anti-epileptic activity of NaGcA tested in different neonatal slice seizure models. Data are shown as mean ± sem.

[0071] (a) Extracellular field potential recordings in the CA3 pyramidal cell layer, 50 μM 4-AP-induced epileptic activity. NaGcA attenuated epileptic discharges in response to 50 μM 4-AP. The respective traces are expanded at the bottom.

[0072] (b) Power spectra of epileptic activity in a 5-minute window before, during, and after NaGcA administration. The amplitude of the power spectrum was significantly suppressed after NaGcA administration.

[0073] (c) The mean power of 4-AP-induced epileptic activity was significantly reduced by perfusion of NaGcA.

[0074] (d) NaGcA significantly inhibited neonatal high K + Model of burst activity. Bottom panel shows enlarged view of the field before, during, and after NaGcA perfusion.

[0075] (e) During NaGcA administration, the power spectrum amplitude was significantly reduced.

[0076] (f) In a continuous 5-min window before, during, and after NaGcA administration, the high K + Normalized power of aCSF-induced epileptic activity. NaGcA significantly reduced high K + Efficacy of induced epileptic activity.

[0077] Figure 5 shows the Cl mediated by CLC-3 channels in CA3 pyramidal neurons - Example data for inhibition of current by gluconate. Values ​​are mean ± s.e.m.

[0078] (a) Representative Cl in CA3 pyramidal neurons - Flow. By applying 20 mM NaGcA (green), Cl - The current is significantly suppressed.

[0079] (b) The IV curve shows a significant outward rectification with a reversal potential close to 0 mV. The presence of NaGcA significantly reduces Cl - Current (green).

[0080] (c) Example Cl - Current traces are from CA3 pyramidal neurons following intracellular dialysis (10 min) with normal CsCl titration solution (left red), anti-CsCl-3 antibody (center orange), or control rabbit IgG (right black).

[0081] (d) Panel IV shows a significant decrease after dialysis of anti-ClC-3 antibody (orange), but no significant change after dialysis of control IgG (black).

[0082] (e) Immunostaining images of HEK293T cells transfected with eGFP (left row) or ClC-3-eGFP fusion protein plasmid (right row). ClC-3 immunoreactivity (red) was strongly detected in ClC-3-eGFP transfected cells (arrows), whereas no immunoreactivity was detected in non-transfected or eGFP transfected cells. Scale bar, 40 μm.

[0083] (f) Typical Cl in HEK cells transfected with eGFP (upper panel) or ClC-3-eGFP plasmid (lower panel). - Current and response to 20 mM NaGcA (middle green).

[0084] (g) IV relationships before, during and after NaGcA removal for cells infected with ClC-3-eGFP (right, n=7 from two batches), ClC-3-mediated currents were significantly inhibited by NaGcA.

[0085] Figure 6 presents exemplary data showing that CLC-3 is upregulated in neonatal epileptic slices. Data are presented as mean ± sem, ***P < 0.001.

[0086] (a) Comparable expression levels of ClC-3 in hippocampal CA3 at 0Mg 2+ CLC-3 immunoreactivity increased after 1 hour incubation of slices in aCSF. Asterisks indicate magnified typical neurons with overlapping CLC-3 immunosignals (red) in the CA3 region. Scale bar = 20 μm (5 μm in magnified images).

[0087] (b) at 0Mg 2+ Slices were incubated in aCSF for 1 hour (n=10 slices from 4 pups for control, n=10 slices from 4 pups for 0 Mg 2+ : n=11 slides from 4 puppies, Student's t-test) after which the quantitative data of ClC-3 immunoreactivity intensity showed a significant increase.

[0088] (c) from control and 0Mg 2+ Western blot of hippocampal ClC-3 protein in slices treated with aCSF (1 hour).

[0089] (d) Use 0Mg 2+ Quantitative data showed a significant increase in CLC-3 protein in hippocampal slices after aCSF treatment (n=6 puppies per group, Student's t test).

[0090] (e) Control, Mg 2+ Incubate for 1 hour, Mg 2+ Representative Cl recordings were made after 1 hour of incubation in 20 mM NaGcA. - Current traces.

[0091] (f)Cl - The average IV graph of the current shows 0Mg 2+ group was significantly increased, and it was also significantly inhibited by NaGcA.

[0092] Fig. 7A -G shows exemplary data showing that gluconate is an effective treatment for KA-induced de novo seizures.

[0093] FIG. 7( a ) Representative EEG traces of P12 rats injected with saline (0.1 ml / 10 g, ip) 10 minutes after KA (2 mg / kg, intraperitoneal (ip)) show recurrent epileptic burst discharges.

[0094] FIG7( b ) is an expanded view of a single clustered epileptic discharge in frame b from FIG7 a , showing typical interictal, tonic, and clonic seizure traces.

[0095] Figure 7(c) Example EEG of P12 rats injected with NaGcA (2 g / kg, ip) 10 minutes after KA injection (2 mg / kg, ip), showing increased spike activity but no development of strong burst discharges after NaGcA administration. Scale bar is the same as Figure 7a.

[0096] Fig. 7(d) Enlarged area in box d in Fig. 7c. The scale bar is the same as that in Fig. 7b.

[0097] Compared to the two side baseline traces, a single spike discharge was observed in the extended trace. Scale bar is the same as in Fig. 7c.

[0098] Figure 7(e) Individual EEG power in the 1-minute time window of panels a (black) and d (green).

[0099] Figure 7(f) Group average EEG power in the 1-minute time window of saline (black, P10-12, n=11) and NaGcA administration (green, P10-12, n=11). Black arrows indicate KA injection points, and red arrows indicate saline or NaGcA injection points. In the presence of NaGcA, the average power of epileptic activity induced by KA injection was reduced by 70%.

[0100] Figure 7(g) The working mode of our study. Cl through the chloride channel - Flux promotes synchronized network activity (left), but this is inhibited by gluconate (GA) due to GA's inhibition of chloride channels (right).

[0101] FIG8 shows exemplary data demonstrating the effects of gluconate on adult epilepsy.

[0102] (ac) Representative EEG recordings showing epileptiform activity induced by injection of pentylenetetrazol (PTZ) (50 mg / kg, ip) followed by injection of saline (a), NaGcA, 1 g / kg (b), or NaGcA, 2 g / kg (c). The open triangle indicates the first spike, and the small black arrow indicates the first burst.

[0103] (d, e) Bar graphs showing the dose-dependent effect of NaGcA in prolonging the latency of epileptiform spikes (d) or epileptiform bursts (e) induced by PTZ (50 mg / kg, ip).

[0104] (f) Bar graph showing the mild inhibitory effect of NaGcA on behavioral seizure scores induced by PTZ (50 mg / kg, ip).

[0105] (g) The bar graph shows that NaGcA dose-dependently prolonged the latency to epileptic seizure behavior (Racine score above 3).

[0106] FIG9 shows exemplary data demonstrating the effects of various chloride ion channel inhibitors:

[0107] (a) Niflumic acid (NFA) (100 μM)

[0108] (b) Flufenamic acid (100 μM)

[0109] (c) DIDS (100 μM)

[0110] (d) NPPB (100 μM)

[0111] FIG10 shows exemplary data showing the effects of various chloride ion channel inhibitors on epileptic activity in newborn hippocampal slices:

[0112] (a) Sodium gluconate (20 mM) (NaGcA)

[0113] (b) NFA (100 μm)

[0114] (c) NPPB (100 μm)

[0115] Figure 11 shows the Cl - Exemplary data on the evolution of current.

[0116] Fig.11A : Representative Cl recordings from CA3 pyramidal neurons in mouse hippocampal slices obtained from animals of different ages - Current (top row, black trace). The green trace in the middle row shows Cl after inhibition by 20 mM NaGcA. - The figure below shows the Cl current of mice of different ages after control and NaGcA treatment. - IV curve of the current.

[0117] Fig. 11B : Quantitative Cl after control (black) or NaGcA treatment (green) (HP = +90 mV) - Current density. Note that the Cl - The current density was significantly reduced. ***P<0.001.

[0118] Fig. 11C :NaGcA inhibits Cl -Dose-response curves of current.

[0119] Fig.11D : Typical Cl recorded under control (black), anti-CLC-3 antibody (orange) or pre-absorbed control antibody (grey). - Current trace. IV curve shows Cl after anti-CLC-3 antibody dialysis (orange) - The current is significantly reduced.

[0120] Fig.11E : Immunostaining confirms lack of CLC-3 signaling in CLC-3 knockout mice. Scale bar, 10 μm.

[0121] Fig.11F :Voltage-dependent outward rectifier Cl in CLC-3 KO mice - The current was mostly absent, supporting that CLC-3 channels mediate Cl - Electric current.

[0122] Fig.11G : Effect of NaGcA on the remaining small Cl in CLC-3 KO mice - The IV curves showed little inhibition of the current, supporting that NaGcA is an inhibitor of CLC-3 channels.

[0123] Fig.11H :CLC-3 Cl in HEK293T cells - Expression of channel. Scale bar, 40 μm.

[0124] Fig.11I : Large Cl recorded from HEK293T cells expressing CLC-3 channels (bottom row) - The application of 20 mM NaGcA significantly inhibited the CLC-3 channel-mediated Cl - Current (green trace).

[0125] Fig.11J : NaGcA was shown to inhibit CLC-3 channel-mediated Cl - IV curves of current (CLC-3, 925±124 pA, n=7; CLC-3+NaGcA, 544±59 pA, n=7; P<0.004, paired t-test; HP=+90 mV). Values ​​are mean±sem.

[0126] FIG. 12 shows exemplary data demonstrating the relationship between the CLC-3 chloride channel and neonatal epileptogenesis.

[0127] Fig. 12A : In 0Mg 2+CLC-3 channel expression is upregulated (red) in hippocampal CA3 neurons after induction of epileptiform activity (1 hour) in artificial cerebrospinal fluid (aCSF). Scale bar = 5 μm.

[0128] Fig. 12B : In the control and 0Mg 2+ Quantitative CLC-3 immunointensity in aCSF (control, n = 10 slices from 4 pups; 0 mg 2+ , n = 11 slices from 4 pups; p < 0.05, Student's t test).

[0129] Fig. 12C and 12D Western blot analysis also showed that 2+ The CLC-3 protein level in the hippocampus was significantly increased after aCSF treatment (n=6 pups in both groups, p<0.001, Student's t test).

[0130] Fig.12E : In the control 0Mg 2+ (1 hour) and 0Mg 2+ Representative voltage-dependent Cl under +20 mM NaGcA (1 hour) conditions - Current traces.

[0131] Fig.12F : Display 0Mg 2+ Outwardly rectified Cl in the aCSF group (red) - IV curves where the current was significantly increased and inhibited by NaGcA (green).

[0132] Figure 12G : Showing 0Mg in the CA3 pyramidal layer in hippocampal slices of newborn mice (postnatal day 7, P7) 2+ aCSF-induced epileptiform activity and its strong inhibition by 20 mM NaGcA were recorded from extracellular field potentials.

[0133] Fig.12H : Power spectra of epileptiform activity (5-minute time window) before (red), during (green), and after (black) NaGcA administration. The power amplitude (integrated area under the power spectrum trace) was significantly reduced after NaGcA administration.

[0134] Fig.12I and 12J : NaGcA also showed a strong efficacy against epileptiform activity in P12 hippocampal slices.

[0135] Figure 12K and 12L : However, in P26 hippocampal slices, NaGcA showed only moderate effects on epileptiform activity.

[0136] Figure 12M :Show that NaGcA inhibits 0Mg 2+ Normalized power of the time course of aCSF-induced epileptiform activity. NaGcA significantly reduced the power of epileptiform activity in the P6-8 and P10-12 groups, but the inhibitory effect was much smaller in the P21-33 group. As a control, the gray line represents the effect of 20 mM NaCl on epileptiform activity in newborns (P8-12).

[0137] Fig.12N and 12O : Hippocampal slices from WT, WT+20mM NaGcA, and CLC-3 KO mice (all at P8-12) were treated with 0Mg 2+ Representative traces of aCSF-induced epileptiform activity. Note that the epileptiform activity does not last long in CLC-3 KO mice.

[0138] Figure 12P : Shows the expression of 0Mg in hippocampal slices from WT, WT+20mM NaGcA and CLC-3 KO mice 2+ Summary data of aCSF-induced burst latency (left panel) and burst frequency (right panel). Data are mean ± sem, *P < 0.05, **P < 0.01, ***P < 0.001.

[0139] FIG. 13 shows exemplary data demonstrating the broad-spectrum anti-epileptic effects of NaGcA in various epilepsy models in neonatal hippocampal slices.

[0140] Fig.13A : Extracellular field potential recordings showing 50 μM 4-AP-induced epileptic activity in the CA3 pyramidal cell layer of newborn hippocampal slices and its inhibition by NaGcA (20 mM).

[0141] Fig. 13B : Power spectra of epileptic activity in a 5-minute time window before (red), during (green), and after (black) NaGcA administration. Note that NaGcA (green) significantly reduced the power amplitude.

[0142] Fig. 13C : Shows the normalized power of NaGcA to inhibit the time course of epileptic activity induced by 4-AP.

[0143] Fig.13D :NaGcA (20 mM) significantly inhibited the high K + Epileptiform activity in epilepsy models.

[0144] Fig.13E: Power spectra showing a significant reduction in epileptic activity during NaGcA application.

[0145] Fig.13F :It shows that NaGcA is sensitive to high K + Normalized power of the time course of aCSF-induced suppression of epileptic activity.

[0146] Fig. 13C and 13F The grey line in Figure 2 represents the control effect of 20 mM NaCl on epileptiform activity in neonates (P8-12). Data are shown as mean ± sem.

[0147] FIG. 14 shows exemplary data that the CLC-3 channel blocker gluconate effectively inhibits de novo seizure activity in vivo.

[0148] Fig.14A : Representative EEG traces showing recurrent epileptic burst discharges in P12 rats after KA injection (2 mg / kg, ip) followed by saline injection (0.1 ml / 10 g, ip) 10 min apart.

[0149] Fig. 14B : Fig.14A Expanded view of epileptic burst discharge in box.

[0150] Fig. 14C and 14D : Representative EEG traces (P12 rats) showing that NaGcA injection (2 g / kg, ip) significantly suppressed epileptic burst discharges 10 min after KA injection (2 mg / kg, ip).

[0151] Fig.14E and 14F : Representative EEG traces showing the moderate effect of phenobarbital (25 mg / kg, ip) on epileptic seizure activity in neonatal rats (P12).

[0152] Figure 14G and 14H : Representative EEG traces showing the effects of bumetanide (2 mg / kg, ip) on epileptic burst activity in neonatal rats (P12).

[0153] Fig.14I ,14J, 14K and 14L: In adult mice, KA injection (10 mg / kg, ip) also induced strong epileptic burst activity as shown by EEG recordings (14I and 14J), but NaGcA (2 g / kg, ip) showed only moderate effects on KA-induced epileptic burst activity (14K and 14L).

[0154] Fig.14M: Data summarized in neonatal animals showing the average EEG power (5 min time window) before and after KA injection followed by injection of saline (black), NaGcA (green), phenobarbital (magenta), or bumetanide (blue). Note that NaGcA effectively inhibits the KA-induced increase in EEG power.

[0155] Fig.14N : In adult animals, NaGcA (green) moderately inhibits the power of the EEG induced by KA.

[0156] exist Fig.14M and 14N In both, black arrows indicate KA injections, and red arrows indicate drug injections. Data are mean ± sem.

[0157] FIG. 15 shows exemplary data demonstrating the acute effects of NaGcA on KA-induced epileptic activity in neonatal rats.

[0158] Fig.15A : Typical epileptic EEG activity induced by ip injection of KA. After recording stable epileptic activity (~1 hour), NaGcA (2g / kg, ip) was administered to the rats (green). Note the gradual decrease in epileptic activity after NaGcA injection.

[0159] Fig. 15B : From Fig.15A Extended trace of the middle segment, showing strong epileptic burst activity.

[0160] Fig. 15C : Fig.15A Expanded view of the EEG recording after NaGcA injection.

[0161] Fig.15D : Fig. 15B A further expanded view of the middle clip shows repeated bursts of activity.

[0162] Fig.15E : Fig. 15C A further expanded view of the EEG after NaGcA injection, showing the significant suppression of epileptic activity by NaGcA.

[0163] Fig.15F : Summary data showing that NaGcA administration (2 g / kg ip, n=6, P10-12 rats, green) significantly reduced the power of epileptic burst activity. Data are mean ± sem.

[0164] FIG16 shows a graph showing that activation of CLC-3 channels alters intracellular Cl in developing neurons. - Exemplary data for homeostasis.

[0165] Fig.16A :0Mg in neonatal CA3 pyramidal neurons 2+ aCSF-induced epileptiform burst activity showed long-lasting membrane depolarization.

[0166] Fig. 16B :Long-duration membrane depolarization pulses (40 mV, 10 s) that mimic epileptic burst activity significantly enhanced GABAA-R currents induced by the GABAA-R agonist isotetrahydronicotinic acid (100 μM) in whole-cell recordings perforated with gramicidin. This depolarization-induced enhancement was absent in CLC-3KO mice and was strongly inhibited by the CLC-3 channel blocker NaGcA (20 mM).

[0167] Fig. 16C : Summary data of the effects of membrane depolarization on GABAA-R currents in WT, CLC-3 KO, and WT+NaGcA groups.

[0168] Fig.16D : In 0Mg 2+ After aCSF-induced epileptiform activity, gramicidin perforation recordings revealed a positive shift in the GABAA-R reversal potential (EGABA) in CA3 neurons.

[0169] Fig.16E Bath application of the NKCC1 inhibitor bumetanide induces a negative shift of EGABA in normal aCSF but does not eliminate the negative localization of EGABA in aCSF induced by 0Mg 2+ aCSF-induced positive shift of EGABA.

[0170] Fig.16F :KCC2 inhibitor VU0240551 had no effect on EGABA in normal aCSF and had no effect on 0Mg 2+ There was no effect on the positive displacement induced by aCSF.

[0171] Figure 16G : NaGcA is completely eliminated by 0Mg 2+ aCSF-induced positive EGABA shift. Gluconate itself does not affect EGABA at all in normal aCSF.

[0172] Fig.16H : In CLC-3 KO mice, when treated with 0Mg 2+ EGABA was also unchanged when treated with aCSF.

[0173] Fig.16I : Quantitative data showing changes in EGABA under various conditions in neonatal CA3 pyramidal neurons (eg, P8-9).

[0174] Fig.16J: Bar graph showing changes in EGABA in adult CA3 pyramidal neurons. Note that NaGcA (20 mM) did not alter EGABA displacement in adult animals.

[0175] Figure 16K : Typical traces of cell-attached recordings showing spiking activity induced by isoguvacine (10 μM, 30 s) in different groups in newborn animals (eg, P8-9).

[0176] Figure 16L : Summary data showing the percentage of neurons excited by isothiahydronicotinic acid. Note that NaGcA essentially eliminated 0Mg 2+ GABA excitatory activity in aCSF. Data are shown as mean ± sem, *P < 0.05, **P < 0.01, ***P < 0.001.

[0177] FIG17 shows a graph showing that gluconic acid complexed with various counterions inhibits Cl in P8-12 neonatal mice. - Exemplary data of current. Data are presented as mean ± sem.

[0178] Fig.17A : Out-rectifying Cl in neonatal CA3 pyramidal neurons (black, n=12) - Representative trace of current flow.

[0179] Fig. 17B : Cl in the presence of 20 mM NaGcA (green, n=7) - Typical trace of current flow.

[0180] Fig. 17C : Cl in the presence of 10 mM Mg(GcA)2 - Typical trace of current flow.

[0181] Fig.17D : Cl in the presence of 20 mM gluconic acid (blue, n=6) - Typical trace of current flow.

[0182] Fig.17E : IV diagram showing Cl after bath application of gluconic acid and its salts - The current intensity is significantly reduced.

[0183] FIG. 18 presents exemplary data showing that epileptiform activity in newborn hippocampal slices is strongly inhibited by glucose oxidase.

[0184] FIG. 18A : Glucose oxidase (GOx) on 0Mg 2+Dose-dependent acute effects of aCSF (artificial cerebrospinal fluid with 20 mM glucose)-induced epileptiform activity. Note that acute application of >1 U / ml GOx effectively suppresses epileptic activity.

[0185] Figure 18B: By high K + The induced epileptiform activity was also inhibited by 1U / ml GOx.

[0186] FIG. 19 presents exemplary data showing that the inhibitory effect of glucose oxidase on epileptiform activity is enhanced after prolonged incubation in aCSF.

[0187] FIG. 19A : Epileptiform activity is induced by 0Mg containing 4 mM glucose 2+ aCSF induction. 1U / ml GOx was added to 0Mg 2+ After preincubation in aCSF (4 mM glucose) at room temperature for 90 min, epileptic activity was significantly inhibited.

[0188] Figure 19B. Even used in 0Mg 2+ The epileptiform activity was also inhibited by pre-incubation of 0.1 U / ml GOx in aCSF (20 mM glucose) for 90 min.

[0189] FIG. 20 presents exemplary data showing that glucose oxidase inhibits epileptiform activity.

[0190] Fig. 20A : Experimental setup for testing the acute effects of GOx on epileptiform activity in hippocampal slices. Field potential recordings were placed in the CA3 pyramidal cell layer with 0Mg 2+ aCSF induced epileptiform activity. After inducing stable epileptiform activity, GOx was added to 0Mg 2+ aCSF.

[0191] Fig. 20B :0.1U / ml Gox to 0Mg 2+ Acute effects of aCSF-induced epileptiform activity.

[0192] Fig. 20C :0.3U / ml GOx to 0Mg 2+ Acute effects of aCSF-induced epileptiform activity.

[0193] Fig.20D :1U / ml GOx to 0Mg 2+ Acute effects of aCSF-induced epileptiform activity.

[0194] Fig.20E :1U / ml GOx to 0 glucose; 0Mg 2+Acute effects of aCSF-induced seizure-like activity. Glucose was replaced with 5 mM lactate and 3 mM pyruvate.

[0195] Fig.20F : Add 0.1U / ml GOx to 0Mg 2+ aCSF and incubated for more than 1 h at room temperature before evaluating induced epileptiform activity.

[0196] Figure 20G : Relative changes in field potential power under different conditions. Note that the inhibitory effect of 1 U / ml GOx on epileptiform activity was almost abolished when glucose was removed from the bath. Data are presented as mean ± sem.

[0197] FIG21 presents exemplary data showing that glucose oxidase inhibits epileptiform activity induced by: i) high K + aCSF; or ii) 4-aminopyridine (4-AP) plus Mg-free 2+ of aCSF.

[0198] Fig.21A :1U / ml GOx inhibition by high K + (8.5 mM) aCSF-induced epileptiform activity.

[0199] Fig. 21B :1U / ml GOx inhibits 4-AP(50μM)+0Mg 2+ aCSF-induced epileptiform activity.

[0200] Fig. 21C : Before, during and after administration of 1 U / ml GOx, the + aCSF (purple) or 4-AP (50 μM) + 0Mg 2 + Normalized epileptiform activity power induced by aCSF (blue). DETAILED DESCRIPTION

[0201] The present invention relates to the field of neurological disorders. In particular, the prevention and treatment of convulsive disorders, including but not limited to myotonic / clonic convulsions, epilepsy, Jackson syndrome and / or involuntary tremors. For example, some embodiments relate to the treatment and prevention of convulsive disorders in newborns and / or infants. Gluconate compositions have been found to be selectively effective in treating and / or preventing convulsive disorders in newborns and / or infants.

[0202] Neonatal epilepsy is difficult to treat compared to adult epilepsy. In one embodiment, the present invention contemplates the use of the anti-epileptic drug gluconate, which targets CLC-3Cl - The channel effectively inhibits neonatal epilepsy.

[0203] Gluconic acid is a large organic anion that is often used as a food or pharmaceutical additive in salt form, such as magnesium gluconate, calcium gluconate, or potassium gluconate, where the gluconate salt is used to transport ions. However, the functional role of gluconate itself has been largely overlooked. The data presented here show that gluconic acid itself acts as a Cl - "Gluconate is a channel blocker and directly inhibits epileptiform activity. In addition, gluconate was shown to be particularly effective for inhibiting neonatal epileptic seizures, with a potency superior to other currently available anti-epileptic drugs. Because gluconate is safe for human consumption, this compound represents a new platform for treating neonatal epilepsy."

[0204] Gluconate has been widely used as an anion in many pharmaceutical drugs, but is usually labeled as an inactive ingredient. Gluconate itself has never been directly linked to epilepsy in the literature, as the specific targets of these studies have focused on factors such as Ca. 2+ and Mg 2+ For example, one case report described a patient with epilepsy who was treated with calcium gluconate and subsequently had a gradual disappearance of epileptic spasticity. Boulenguez et al., “Down-regulation of the potassium-chloride coransporter KCC2 contributes to spasticity after spinal cord injury” Nature Medicine 16:302-307 (2010). This analysis attributed the improvement to Ca 2+ , without mentioning a possible role for gluconate. Other observations that magnesium gluconate may have an antiepileptic effect have been suggested to be due to Mg 2+ function.

[0205] The data presented herein suggest that the active ingredient in these anti-epileptic compositions is more likely to be gluconate rather than a divalent cation. Gluconate can be produced from the oxidation of glucose and is therefore abundant in natural foods such as fruit and honey. Anastassiadis et al., "Gluconic acid production" Recent Patents On Biotechnology 1: 167-180 (2007). In one embodiment, the present invention contemplates that gluconate has anti-epileptic effects and can support certain traditional dietary treatments for epilepsy.

[0206] CLC-3 channels are voltage-gated outward-rectifying Cl channels belonging to the CLC superfamily. -Previous studies have shown that CLC-3 channels are essential for regulating endosomal acidification, presynaptic neurotransmitter accumulation, insulin secretion, and glioma cell proliferation.Hara-Chikuma et al., "ClC-3 chloride channels facilitate endosomal acidification and chloride accumulation", The Journal Of Biological Chemistry 280:1241-1247 (2005); Riazanski et al., "Presynaptic CLC-3 determines quantal size of inhibitory transmission in the hippocampus", Nature Neuroscience 14:487-494 (2011); Deriy et al., "The granular chloride channel ClC-3 is permissive for insulin secretion", Cell Metabolism 10:316-323 (2009): Li et al., "Suppression of sulfonylurea- and glucose-induced insulin secretion in vitro and in vivo in mice lacking the chloride transport protein ClC-3", Cell Metabolism 10:309-315 (2009); Cuddapah et al., "Bradykinin-induced chemotaxis of human gliomas requires the activation of KCa3.1 and ClC-3", The Journal Of Neuroscience: The official journal of the Society for Neuroscience 33:1427-1440 (2013); and Habela et al., "ClC3 is a critical regulator of the cell cycle in normal and malignant glial cells", The Journal Of Neuroscience: the official journal of the Society for Neuroscience 28:9205-9217 (2008).

[0207] Although it is not necessary to understand the mechanism of the invention, it is believed that CLC-3 Cl - Channel-mediated large outward rectification of Cl - Currents, which are prominent in the early postnatal brain but largely disappear in the adult brain. Therefore, gluconate is a more effective anti-epileptic drug for the treatment of neonatal epilepsy. It is further believed that CLC-3 Cl - Activation of the channel changes the intracellular Cl - The internal environment is stable and GABA function is more exciting. In addition, the data presented in this paper show that CLC-3 Cl - Cl channels may play a greater role in neonatal epilepsy than previously thought - Transport proteins play a greater role. In fact, due to the Cl - Channel and Cl - Differences in voltage dependence of transporters, Cl - Channel and Cl - Transporters may control intracellular Cl - For example, under resting conditions, the voltage-sensitive CLC-3 Cl - The channel is inactive, therefore, Cl - Transport proteins are important in maintaining intracellular Cl - A major player in homeostasis in developing neurons. Ben-Ari, Y., “Excitatory actions of gaba during development: the nature of development” Nature Reviews. Neuroscience 3: 728-739 (2002); Kaila et al., “Cation-chloride cotransporters in neuronal development, plasticity and disease” Nature Reviews. Neuroscience 15: 637-654 (2014); and Blaesse et al., “Cation-chloride cotransporters and neuronal function” Neuron 61: 820-838 (2009).

[0208] However, during epilepsy, CLC-3 Cl - The channel is activated, and the large Cl - The inflow increased significantly [Cl - ] i, leading to enhanced excitatory GABAergic transmission and aggravated epileptic activity. In some embodiments, the present invention contemplates that blocking CLC-3 channels with gluconate disrupts this positive circuit and inhibits neonatal epilepsy in the developing brain. In addition, CLC-3 knockout mice do not have large outwardly rectifying Cl - Current, so reciprocal burst activity is significantly reduced.

[0209] By comparing gluconate with other potential antiepileptic drugs (AEDs), the data presented here suggest that gluconate is more effective than phenobarbital or bumetanide in suppressing new seizures. Gluconate acts differently from bumetanide in that it blocks NKCC1 and affects EGABA under resting conditions, whereas gluconate does not affect normal EGABA under physiological conditions but blocks CLC-3 channel activation during epileptogenesis.

[0210] Since gluconate is a natural organic acid and has been used as a food and drug additive with minimal side effects, in one embodiment, the present invention contemplates that gluconate compositions are therapeutic agents useful for treating neonatal epilepsy that is resistant to many current anticonvulsants in the absence of divalent cations.

[0211] I. Convulsive disorders

[0212] The term convulsion is often used interchangeably with epileptic seizure. A convulsion occurs when a person's body shakes rapidly and uncontrollably. During a convulsion, the person's muscles contract and relax repeatedly. There are many different types of epileptic seizures. Some symptoms are mild with no shaking. Some seizures simply cause a person to stare for a spell. These may go unnoticed. The specific symptoms of a convulsion may depend on which part of the brain is affected. Symptoms usually occur suddenly and may include, but are not limited to: brief amnesia followed by a period of confusion (the person cannot remember for a short period of time); behavioral changes such as picking at clothes; drooling or foaming at the mouth; eye movements; grunting and sniffing; loss of bladder or bowel control; mood changes such as sudden anger, unexplained fear, panic, joy, or laughter; shaking of the entire body; sudden collapse, tasting a bitter or metallic taste; clenching of teeth; temporary cessation of breathing, and / or uncontrollable muscle spasms with twitching and jerking of the limbs. Symptoms may stop after a few seconds or minutes, or last up to 15 minutes, but rarely last longer.

[0213] Seizures are often triggered by overexcitation of brain circuits, which can be activated by enhancing the major inhibitory receptor family in the adult brain - GABA A GABA receptor A-R) to inhibit. Therefore, antiepileptic drugs (AEDs) are usually developed to increase GABAA-R function, such as benzodiazepines and barbiturates, which are usually targeted at adult patients. Bialer et al., "Key factors in the discovery and development of new antiepileptic drugs. Nature reviews. Drug discovery 9, 68-82 (2010). However, although GABA A -R is primarily inhibitory in the adult brain, but in the developing brain of a newborn and / or infant, GABA A -R is mainly excitatory. Chen et al., "Excitatory actions of GABA in developing rat hypothalamic neurones" The Journal Of Physiology 494 (Pt 2): 451-464 (1996); and Ben-Ari Y., "Excitatory actions of gaba during development: the nature of the nurture" Nature Reviews Neuroscience 3: 728-739 (2002). The excitatory GABAergic transmission in the developing brain also explains why GABA agonists are generally ineffective in controlling neonatal epileptic seizures and may sometimes even exacerbate neonatal epileptic seizure activity. Farwell et al., "Phenobarbital for febrile seizures--effects on intelligence and on seizure recurrence" The New England Journal Of Medicine 322: 364-369 (1990). Therefore, some AEDs may enhance the excitatory GABA in the developing brain. A -R function to intensify neonatal epileptic seizures.

[0214] Classically, GABA A The excitatory function of -R in developing neurons has been attributed to chloride ions (Cl -) regulation of the transporters NKCC1 and KCC2. Ben-Ari et al., "GABA: a pioneer transmitter that excites mature neurons and generates primitive oscillations" Physiol Rev 87: 1215-1284 (2007); Kahle et al., "Roles of the cation-chloride cotransporters in neurological disease" Nat Clin Pract Neurol 4: 490-503 (2008); and Kaila et al., "Cation-chloride cotransporters in neuronal development, plasticity and disease" Nature Reviews. Neuroscience 15: 637-654 (2014). Previous studies have found that NKCC1 may promote neonatal epileptic seizures in rodents. Dzhala et al., "NKCC1 transporter facilitates seizures in the developing brain" Nature Medicine 11: 1205-1213 (2005). However, recent infant clinical trials have found serious side effects of the NKCC1 blocker bumetanide and very limited efficacy in treating neonatal epileptic seizures. Pressler et al., "Bumetanide for the treatment of seizures in newborn babies with hypoxic ischaemic encephalopathy (NEMO): an open-label, dose finding, and feasibility phase 1 / 2 trial" Lancet Neurol 14: 469-477 (2015). Because excitatory GABAergic transmission plays a key role in many neurodevelopmental processes, blocking NKCC1 may significantly alter GABA function under physiological conditions and increase the potential risk of disrupting normal brain development.Ben-Ari, Y., "Excitatory actions of gaba during development: the nature of the nurture" Naturereviews.Neuroscience 3:728-739 (2002); Kaila et al., "Cation-chloridecotransporters in neuronal development, plasticity and disease" Naturereviews.Neuroscience 15:637-654 (2014); Wang et al., "Blocking early GABA depolarization with bumetanide results in permanent alterations in corticalcircuits and sensorimotor gating deficits" Cerebral cortex 21:574-587(2011); and Deidda et al., "Early depolarizing GABA controls critical-period plasticity in the rat visual cortex" Nature neuroscience 18:87-96(2015). Recent studies have shown that, in addition to Cl. - Factors other than cotransporters may also contribute to Cl -The internal environment is stable, such as the anion-impermeable, voltage-gated inwardly rectifying chloride channel CLC-2 and the voltage-gated outwardly rectifying chloride channel CLC-3. Glykys et al., "Local impermeantanions establish the neuronal chloride concentration" Science 343:670-675 (2014); Foldy et al., "Regulation of fast-spiking basket cell synapses by the chloride channel ClC-2" Nature neuroscience 13:1047-1049 (2010); Rinke et al., "ClC-2 voltage-gated channels and Wang et al., “CLC-3channels modulate excitatory synaptic transmission in hippocampalneurons” Neuron 52:321-333 (2006).

[0215] Recent studies have shown that Cl - Channels, such as the CLC-3 channel, may also contribute to intracellular Cl - Concentration ([Cl - ] i ) changes. Zhou et al., “Regulation of intracellular Cl -concentration through volume-regulated ClC-3chloride channels in A10 vascular smooth muscle cells” The Journal Of Biological Chemistry 280:7301-7308 (2005). CLC-3 channels are voltage-dependent outwardly rectifying Cl - Jentsch TJ, "Chloride and the endosomal-lysosomal pathway: emerging roles of CLC chloride transporters" The Journal of Physiology 578: 633-640 (2007); Graves et al., "The Cl - / H+ antiporter ClC-7 is the primary chloride permeation pathway in lysosomes" Nature 453:788-792 (2008); Picollo et al., "Chloride / proton antiporter activity of mammalian CLC proteins ClC-4andClC-5" Nature 436:420-423 (2005); Scheel et al., "Voltage-dependent electrogenic chloride / proton exchange by endosomal CLC proteins" Nature 436:424-427 (2005); and Hara-Chikuma et al., "ClC-3chloride channels facilitate endosomalacidification and chloride accumulation" The Journal Of Biological Chemistry 280:1241-1247 (2005). CLC-3 channels are ubiquitously expressed throughout the brain, with high expression levels in the hippocampus and cerebellum. Duran et al., "Chloride channels: often enigmatic, rarely predictable” Annual Review Of Physiology 72:95-121 (2010); Verkman et al., “Chloride channels as drug targets” Nature Reviews. Drug discovery 8:153-171 (2009); and Kawasaki et al., “Cloning and expression of a protein kinase C-regulated chloride channel abundantly expressed in rat brain neuronal cells” Neuron 12:597-604 (1994). For example, CLC-3 knockout mice show selective postnatal neurodegeneration in the hippocampus.Stobrawa et al., "Disruption of ClC-3, a chloride channel expressed on synaptic vesicles, leads to a loss of the hippocampus" Neuron 29:185-196 (2001); and Dickerson et al., "Altered GABAergic function accompanies hippocampal degeneration in mice lacking ClC-3 voltage-gated chloride channels" Brain Research 958:227-250 (2002). However, voltage-dependent Cl is involved in epileptogenesis. - The exact role of the channel is largely unknown.

[0216] Compared with adult epilepsy, neonatal epilepsy is difficult to treat. Neonatal epilepsy is different from adult epilepsy, and many anti-epileptic drugs (AEDs) that are effective in adults are generally not able to treat neonatal epilepsy. For example, the data presented in this article show that CLC-3 Cl is present in the early developing brain. - The large voltage-dependent outward rectification of Cl - Although it is not necessary to understand the mechanism of the invention, it is believed that gluconate, a naturally occurring organic acid, effectively inhibits neonatal epileptic seizures by blocking the CLC-3 chloride channel.

[0217] Interestingly, CLC-3 Cl - The channel may be upregulated during epileptic seizures, and gluconate is required for CLC-3 Cl - Inhibition of the channel essentially eliminated neonatal seizure activity, with significantly greater potency compared to other AEDs. This result is believed to be supported by the observation that CLC-3 knockout mice have no outwardly rectifying Cl channels in the developing brain. - Although it is not necessary to understand the mechanism of the present invention, it is believed that during epileptic seizures, CLC-3Cl - Activation of the channel significantly changes the intracellular Cl - The internal environment is stable and the excitatory function of GABA is enhanced. Other Cl - The data presented herein identify gluconate as a regulator of CLC-3 Cl -Channels Effective Anti-Epileptic Drugs for Treating Neonatal Seizures In some embodiments described herein, gluconic acid is identified as an anti-epileptic drug that is particularly effective in inhibiting neonatal seizures.

[0218] II. Divalent cation-based seizure therapy

[0219] Administration of calcium gluconate, magnesium sulfate, or phenobarbital to infants 4-7 days of age with convulsions associated with hypocalcemia has been reported. Although all three compositions appear to reduce the rate of convulsions, an intra-treatment comparison showed that magnesium sulfate was more effective than calcium gluconate or phenobarbital. However, the references do not provide any disclosure of the effectiveness of gluconate compositions in infants who do not require a cation-based treatment. Turner, T., "Comparisons of phenobarbitone, magnesium sulphate, and calcium gluconate in treatment of neonatal hypocalcaemic convulsions" Paediatric Research Society Abstracts, pg244.

[0220] It has been reported that calcium gluconate was used for seizure control in infants less than 20 days old. After this treatment, swelling, inflammation, extraosseous calcification of the left leg occurred, and serum calcium levels were determined to be within the normal range. After stopping the calcium gluconate treatment, these symptoms were relieved and it was diagnosed as iatrogenic cutaneous calcinosis. However, the references do not provide any disclosure about the effectiveness of gluconate compositions in infants who do not require divalent cation-based treatment (e.g., calcium). Arora et al., "Latrogenic calcinosis cutis—a rare differential diagnosis of soft-tissue infection in a neonate: A case report" Journal of Orthopaedic Surgery 13(2): 195-198 (2005).

[0221] Administration of plant extracts for the treatment of seizure disorders and / or epilepsy has been reported. For example, plant extracts have been combined with various preparations of gluconates, such as potassium gluconate, zinc gluconate, and / or copper gluconate, and screened for anticonvulsant activity using a known mouse electroshock clone model. In addition, the commercially available product NutriiVeda TMadministered to human patients between the ages of four and a half (4.5) and thirty-seven (37) years old to reduce seizures and / or epilepsy. However, it should be noted that NutriiVeda TM The detailed composition analysis of the invention has not been disclosed, therefore, the exact amount of gluconate in the product and its relationship to divalent cations is unknown. However, the reference does not provide any disclosure on the effectiveness of gluconate compositions in infants who do not require divalent cation-based treatments such as calcium or zinc. Geng L., "Methods for Treating Neurological Disorders Using Nutrient Compositions" U.S. Pat. No. 8,962,042 (incorporated herein by reference).

[0222] Cases of neonates with early onset seizures associated with hypocalcemia, hyperphosphatemia, and elevated parathyroid hormone are presented. The infant did not have any features of pseudohypoparathyroidism. The hypocalcemia was initially resistant to calcium therapy but responded to vitamin D analog therapy. The diagnosis was "neonatal pseudohypoparathyroidism"; during follow-up at 3 and 8 months, the infant remained stable and seizure-free with normal serum biochemistry. Narang et al., "Neonatal pseudohypoparathyroidism" Indian J Pediatr. 73(1):97-98(2006); and Manzar et al., "Transient pseudohypoparathyroidism and neonatal seizure" J Trop Pediatr. 47(2):113-114(2001).

[0223] Failure of calcium-based seizure therapy has been reported, with the patient developing generalized tonic-clonic seizures when she was 9 years old, and these were associated with hypocalcemia. Despite treatment with calcium, seizures continued and the patient required antiepileptic drugs. She was eventually controlled with oxcarbazepine. Head MRI was normal. EEG showed independent spike and wave discharges from the left temporal and right frontal regions. The presence of lesions found on the EEG, the lack of a complete response to calcium treatment, and the need for antiepileptic drug treatment suggest that some of these patients may be inherently predisposed to developing epilepsy. Gonzalez et al., "Seizures and EEG findings in an adult patient with DiGeorge syndrome: a case report and review of the literature" Seizure 18(9): 648-651(2009).

[0224] Hypocalcemia is a relatively rare but reversible cause of left ventricular dysfunction in infants and children. A 30-day-old boy with idiopathic hypocalcemia presented with congestive heart failure and convulsive seizures. He had no evidence of underlying heart disease. The heart failure responded to calcium therapy. It is suggested that hypocalcemia should be considered as a possible cause of left ventricular dysfunction in infants. Karademir et al., "Left ventricular dysfunction due to hypocalcemia in a neonate" JPN Heart J. 34(3): 355-359 (1993).

[0225] Such reports as above teach those skilled in the art that the purpose of administering divalent cation gluconate treatment is to provide divalent cations (e.g., calcium, magnesium, zinc, etc.) as active ingredients to control convulsive disorders. For example, the condition of "hypocalcemia" is described as a pre-existing condition of convulsions. On the other hand, calcium excess leads to serious side effects during the treatment of convulsions experienced after calcium gluconate treatment. Therefore, these observations teach that the purpose of administering divalent cation gluconate complexes to neonates is because: it is believed that divalent cation supplementation is required for convulsion control, and gluconate is only a suitable counter ion. Therefore, the present invention shows that divalent cation gluconate compositions are surprisingly more effective in treating convulsive disorders.

[0226] III. Gluconate-based seizure therapy

[0227] In one embodiment, the present invention contemplates voltage-dependent CLC-3 Cl - The channel may control intracellular Cl - Concentration ([Cl - ] i ), particularly during neonatal epilepsy. For example, CLC-3 channels can mediate large voltage-dependent outward rectifiers that can be inhibited by gluconate in the neonatal but not in the adult brain. - For example, the data showed that gluconate effectively inhibited neonatal epileptic activity with less effect in adult animals. In addition, CLC-3 knockout mice showed an absence of voltage-dependent outwardly rectifying Cl currents in neonatal brains. - Currents and reduced recurrent epileptic activity. EEG recordings also confirmed that gluconate was more effective in suppressing seizure burst activity in newborn animals but less effective in adult animals. Finally, the significant increase in CLC-3 channel activation observed during neonatal epilepsy [Cl - ] i , whereas blocking CLC-3 channels with gluconate inhibited [Cl- ] accumulation. In one embodiment, the present invention contemplates that gluconate (a natural organic chemical) is an effective CLC-3 Cl - channel blocker and may be effective as an anticonvulsant in the treatment of neonatal epilepsy.

[0228] Gluconic acid is a large organic anion that is commonly used as a food or drug additive in salt form, such as magnesium gluconate, calcium gluconate, or potassium gluconate, where gluconate is used to help deliver calcium, magnesium, or potassium. However, the functional role of gluconate itself has been largely overlooked in the past. The data presented here show that gluconic acid itself acts as a Cl - Channel blockers and directly inhibit epileptiform activity. In some embodiments, the present invention provides that gluconate is particularly effective in inhibiting neonatal epileptic seizures, thereby providing a selective treatment for neonatal epilepsy.

[0229] Gluconate is an alkanoyl organic compound with the following structure:

[0230]

[0231] It should be noted that various hydroxyl and carboxylic acid groups provide active species that can be functionalized and / or derivatized. Such reactive groups can be partially substituted by groups, including but not limited to substituted or unsubstituted aryl or heteroaryl, unsubstituted or substituted C1-C6-alkyl, substituted or unsubstituted 5-6-membered saturated or unsaturated fused ring, substituted or unsubstituted 5-6-membered saturated or unsaturated ring, natural amino acid residue or synthetic amino acid residue, trihalomethyl, substituted or unsubstituted C1-C6-alkoxy, NH2, SH, thioalkyl, aminoacyl, aminocarbonyl, substituted or unsubstituted C1-C6 alkoxycarbonyl, aryl, heteroaryl, substituted or unsubstituted 4-8-membered cycloalkyl, optionally containing 1-3 heteroatoms, carboxyl, cyano, halogen, hydroxyl, nitro, acetoxy, aminoacyl, sulfonyloxy, sulfonyl, C1-C6-thioalkoxy, C1-C6-aliphatic alkyl, optionally containing A substituted or unsubstituted saturated cyclic C4-C8-alkyl group having 1-3 heteroatoms and optionally fused to an aryl or heteroaryl group; a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, wherein the aryl or heteroaryl group is optionally substituted by: a substituted or unsubstituted C1-C6-alkyl group, such as a trihalomethyl group, a substituted or unsubstituted C1-C6-alkoxy group, a substituted or unsubstituted C2-C6-alkenyl group, a substituted or unsubstituted C2-C6 alkynyl group, an amino group, an aminoacyl group, an aminocarbonyl group, a substituted or unsubstituted C1-C6-alkoxycarbonyl group, an aryl group, a carboxyl group, a cyano group, a halogen group, a hydroxyl group, a nitro group, an acetoxy group, an aminoacyl group, a sulfonyloxy group, a sulfonyl group, a C1-C6-thioalkoxy group; or R5 and R6 together can form a substituted or unsubstituted 4-8-membered saturated cycloalkyl group or a heteroalkyl group. It is expected that at least one or more of these gluconate derivatives of interest have an anticonvulsant effect that is superior to conventionally used divalent cation therapy.

[0232] Gluconate has been widely used as an anion for many pharmaceutical purposes, usually labeled as an inactive ingredient. Those skilled in the art who reported anticonvulsant drugs with gluconate as a counterion never suggested that gluconate was associated with any antiepileptic effect. Instead, as discussed above, previous studies have focused on divalent cations such as Ca 2+ and Mg 2+ , as a mediator of anticonvulsant effects. For example, one case study reported that an epileptic patient was treated with calcium gluconate and his epileptic spasms gradually disappeared. Boulenguez et al., "Down-regulation of the potassium chloride cotransporter kCC2 contributes to spasticity after spinal cord injury" Nature Medicine 16: 302-307 (2010). The authors attributed this effect to Ca 2+, completely ignoring the question of whether gluconate may play any role. Similarly, it is generally accepted in the art that magnesium-related anticonvulsant products, such as magnesium gluconate, may have antiepileptic effects, but the anticonvulsant effects are also believed to be due to Mg 2+ Contrary to these commonly held beliefs, the data presented here suggest that gluconate may be as effective or more effective than divalent cations alone.

[0233] Gluconate can be produced by the oxidation of glucose and is therefore present in large quantities in natural foods such as fruit and honey. Anastassiadis et al., "Gluconic acid production" Recent Patents On Biotechnology 1: 167-180 (2007). Therefore, dietary sources of gluconate may also be useful for anti-epileptic treatment. Since gluconate is a natural organic acid that has been used as a food and drug additive with minimal side effects, gluconate derivatives may lead to a new generation of therapeutic drugs for the treatment of neonatal epilepsy.

[0234] In one embodiment, the present invention contemplates that gluconic acid (e.g., gluconate) effectively inhibits epileptic burst activity in neuronal cultures and hippocampal slices. In addition, field potential recordings in hippocampal slices showed that sodium gluconate effectively inhibited neonatal epileptic activity, with less effect on aged animals. Electroencephalogram (EEG) recordings confirmed that sodium gluconate was more effective in inhibiting epileptic seizures in neonatal animals, but less effective in adult animals. Whole-cell patch clamp recordings showed that sodium gluconate significantly inhibited voltage-dependent Cl in hippocampal pyramidal neurons, which is primarily mediated by CLC-3 channels. - Data presented herein identify the natural organic chemical gluconic acid and its derivatives, as previously unknown, as potent anticonvulsant drugs for neonatal epilepsy.

[0235] A. Inhibition of epileptiform activity

[0236] During epileptogenesis, extracellular Cl in the bath solution is replaced by large anions such as gluconic acid. - (i.e., for example, 137 mM Cl - ) to study Cl - Surprisingly, concentrations between about 5-20 mM sodium gluconate (NaGcA) in the bath solution inhibit spontaneous epileptiform activity in cultured cortical neurons. Figure 1A This potent suppression of epileptiform activity by low concentrations of NaGcA cannot be purely due to Cl - concentration change, since there is still >117 mM Cl in the bath solution. -. To determine whether NaGcA might directly inhibit epileptiform activity, cyclothiazide (CTZ) was used to induce robust epileptiform burst activity in neuronal cultures. Qi et al., "Cyclothiazide induces robust epileptiform activity in rat hippocampal neurons both in vitro and in vivo" The Journal of Physiology 571: 605-618 (2006). The data showed that CTZ-induced epileptiform activity was completely blocked by 10 mM NaGcA, and the inhibition was dose-dependent. See respectively Figure 1B and Figure 1C The effects of NaGcA are reversible, as burst activity returns to control levels after removal of NaGcA. Figure 1A and Figure 1C These data suggest that NaGcA has a potent inhibitory effect on epileptiform burst activity in cultured neurons.

[0237] To ensure that this inhibitory effect of NaGcA was not limited to the CTZ epilepsy model, NaGcA was further tested in two additional epilepsy models. First, cortical neurons were treated with 1 μM kainic acid (KA), a potent neurotoxin, for 2 h to induce epileptic burst activity. Whole-cell patch clamp recordings showed that 10 mM NaGcA greatly inhibited the epileptic burst activity induced by KA. Figure 1D and Figure 1E . Second, cortical neurons were incubated in 50 μM 4-aminopyridine (4-AP) for 2 h, which also induced long-lasting epileptiform burst activity. Similarly, application of 10 mM NaGcA completely blocked 4-AP-induced epileptiform activity. See Figure 1F and Figure 1G Together, these results indicate that NaGcA is a potent and general inhibitor of epileptiform activity in cultured neurons.

[0238] It was then determined whether NaGcA could inhibit the induction of epileptic activity. For this purpose, a combination of NaGcA and CTZ was added during the induction period. Neurons treated with CTZ alone showed strong epileptic-like activity; however, neurons treated with 10 μM CTZ and 10 mM NaGcA showed a significant reduction in epileptic-like activity. Figure 1H, refer to the upper part to the lower part. Almost 90% of the control neurons showed epileptiform activity after CTZ treatment (e.g., about 19 out of 22 neurons), while only 18% of the neurons co-treated with 10mM NaGcA (e.g., about 3 out of 17 neurons, p<0.001, χ2 test) showed epileptiform activity. See Figure 1I (a). The average frequency and duration of epileptiform bursts were also significantly reduced in neurons co-treated with NaGcA. See Figure 1I (b) and Figure 1I (c). In summary, these data indicate that acute and chronic NaGcA administration can inhibit epileptiform activity in cultured neurons.

[0239] B. Kainic acid-induced protection against cell death

[0240] Neuronal death is a serious side effect of epilepsy. Sagar et al., "Hippocampal neuron loss intemporal lobe epilepsy: correlation with early childhood convulsions" Annals of Neurology 22:334-340 (1987); and Sass et al., "Verbal memory impairment resulting from hippocampal neuron loss among epileptic patients with structural lesions" Neurology 45:2154-2158 (1995). The data provided herein show that gluconate not only has anti-epileptic effects, but also exerts a neuroprotective effect on KA-induced neuronal death.

[0241] Epileptic activity may induce cell death in epilepsy patients and animal models. Chen et al., "Differential roles of NR2A- and NR2B-containing NMDA receptors in activity-dependent brain-derived neurotrophic factor gene regulation and limbic epileptogenesis" The Journal Of Neuroscience: the official journal of the Society for Neuroscience 27: 542-552 (2007); and Naseer et al., "Maternal epileptic seizure induced by pentylenetetrazol: apoptotic neurodegeneration and decreased GABAB1 receptor expression in prenatal rat brain" Molecular Brain 2: 20 (2009). To evaluate whether the antiepileptic effect of NaGcA may be neuroprotective, the kainic acid (KA)-induced cell death model was compared with the LIVE / The activity / cytotoxicity assay kit (L3224, Life Technologies, Inc.) was used in combination to analyze neuronal survival. Wu et al., "Protective effect of resveratrol against kainate-induced temporal lobe epilepsy in rats" Neurochemical Research 34:1393-1400 (2009). In the control group, most neurons appeared morphologically healthy and stained with calcein (green, alive), but did not stain with ethidium homodimer-1 (EthD-, red, dead). See Figure 1J left. After exposure to 10 μM KA for 24 hours, most neurons died, as shown by EthD-1 staining. See Figure 1J , middle left). Interestingly, KA-induced neuronal death was abolished by co-application of 20 mM NaGcA. Figure 1J , middle right. Exposure of neurons to 20 mM NaGcA alone had no adverse effect on their survival rate. Figure 1J , right. Quantitative data showed that the neuroprotective effect of NaGcA was also dose-dependent. Figure 1K These experiments suggest that gluconate not only suppresses epileptiform activity but also exerts a neuroprotective effect.

[0242] C. Chloride ion current inhibition

[0243] The results presented in this paper show that gluconic acid blocks Cl - Although it is not necessary to understand the mechanism of the present invention, it is believed that gluconic acid blocks these channels because it is a large organic compound with a negative charge. For example, as a large anion, it is believed that gluconic acid may not be as strong as Cl - ions pass through the Cl - Ch. Cl -Channels are associated with human epilepsy patients. Mutations in CLC-1 channels have been identified in many patients with idiopathic epilepsy. Chen et al., "Novel brain expression of ClC-1 chloride channels and enrichment of CLCN1 variants inepilepsy" Neurology 80: 1078-1085 (2013). Mutations in CLC-2 channels have also been found in human patients, but some studies suggest that the mutations may not contribute to epilepsy. Haug et al., "Mutations in CLCN2 encoding a voltage-gated chloride channel are associated with idiopathic generalized epilepsies" Nat Genet 33:527-532 (2003); Kleefuss-Lie et al., "CLCN2 variants in idiopathic generalized epilepsy" Nat Genet 41:954-955 (2009); and Niemeyer et al., "No evidence for a role of CLCN2 variants in idiopathic generalized epilepsy" Nat Genet 42:3 (2010). CLC-3 channels are widely expressed in different brain regions, with the hippocampus being one of the highest expression areas. Duran et al., "Chloride channels: often enigmatic, rarely predictable" Annual Review Of Physiology 72:95-121 (2010); Verkman et al., "Chloride channels as drug targets" Nature Reviews. Drug Discovery 8: 153-171 (2009); and Kawasaki et al., "Cloning and expression of a protein kinase C-regulated chloride channel abundantly expressed in rat brain neuronal cells" Neuron 12: 597-604 (1994).

[0244] The experiments were designed to answer the question of what the molecular target of gluconate is. We first investigated the ion channel regulation of NaGcA comparing sodium, potassium or calcium channels. The data showed that after NaGcA treatment, Na + , K + and Ca 2+ There is no significant change in current. Figures 2A-2F Although it is not necessary to understand the mechanism of the invention, it is believed that because gluconic acid is an organic anion, it may affect neuronal anion channels.

[0245] The data provided by the present invention show that Cl in cultured cortical neurons - The current was outwardly rectifying and was significantly reduced in the presence of 10 mM NaGcA. Figure 2G , H; control, 913 ± 171 pA; NaGcA, 499 ± 89 pA; n = 7; P < 0.007, paired t test; recorded at +90 mV). Therefore, one target of NaGcA may be Cl - In order to consolidate Cl - channels and epileptogenesis, we examined two classic Cl - Effects of channel blockers; 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and 4,4'-diisothiocyanate 2,2'-stilbene disulfonic acid disodium salt (DIDS). Administration of NPPB (100 μM) or DIDS (100 μM) significantly inhibited Cl - Current. Fig.2I , L. This also demonstrated that both NPPB (100 μM) and DIDS (100 μM) significantly inhibited CTZ-induced epileptiform activity (10 μM, 24 hours). Figure 2M , N; n = 9. In conclusion, these results suggest that NaGcA can inhibit the Cl - aisle.

[0246] D. Inhibition of epileptiform activity in newborn hippocampal slices

[0247] Given the possibility that the aforementioned effects of gluconate on cultured neurons are artifacts, the antiepileptic activity of NaGcA was examined in hippocampal slices with relatively intact neuronal circuits. Field potentials were recorded in the CA3 pyramidal layer and in the absence of Mg. 2+ After induction of stable epileptic seizure activity (~30 min), 20 mM NaGcA was applied to the Mg-free 2+ The data showed that NaGcA exerted anti-epileptic effects in brain slices of P6-P12 newborn animals ( Figure 2Aand 2c), but exerted a moderate effect in older animals around one month old. See Figures 3A and 3C, see Figure 3E. The power spectrum of the field potential was then analyzed before, during, and after NaGcA administration. The amplitude of the power was significantly reduced after administration of NaGcA in newborn animals, but only slightly reduced in older animals (e.g., P26). See Figures 3B and 3D, see Figure 3F. Quantitatively, NaGcA inhibited 60% of the average power of epileptic activity in newborn animals (P6-8, 59.6±4.3%, n=10 slices from 5 puppies; P10-12, 62.1±3.8%, n=10 slices from 5 puppies; P<0.001, paired t-test), but only reduced it by 20% in older animals (P21-33, 23.8±4.1%, n=7 slices from 4 puppies; p<0.005, paired t-test). See Figure 3G. These results suggest that NaGcA may have potent anti-epileptic activity in the neonatal brain.

[0248] We further determined whether the effects of NaGcA on epileptic activity were generally applicable to the 0Mg-free period in P6-P12 neonatal animals. 2+ Other epilepsy models other than the 4-AP model. For example, using the 4-AP model, where K + Bath application of the channel blocker 4-AP (50 μM) induced strong epileptic burst activity in the CA3 pyramidal layer. Further addition of 20 mM NaGcA to the 4-AP-containing bath solution significantly reduced epileptic activity, which was reversible after NaGcA removal. See Figure 4A. The amplitude of power also showed a significant decrease in the presence of NaGcA, and the summarized data showed that NaGcA caused an average decrease in power amplitude of 70.2 ± 5.3% (n = 9 slices from 4 pups; P < 0.001, paired t-test). See Figures 4B and 4C, respectively. High K can also be applied + model, in which high extracellular K in the CA3 pyramidal layer + (+8.5mM) induced epileptic burst activity. See Figure 4D. Similarly, at high K + Addition of 20 mM NaGcA to aCSF also blocked epileptic burst activity and reduced power amplitude (see Figure 4D, E). Statistical analysis showed that in high K + In the model, NaGcA significantly reduced the power amplitude by 70.8±4.9% (n=5 slices from 2 pups; P<0.001, paired t test). See Figure 4F. In summary, these results indicate that NaGcA can effectively inhibit neonatal epilepsy in various epilepsy models.

[0249] E. CLC-3 chloride channel inhibition

[0250] As mentioned above, NaGcA inhibits Cl in cultured neurons -See Figure 2. Previous studies have reported that cultured hippocampal neurons have voltage-dependent CLC-3 Cl - Channels. Wang et al., "CLC-3 channels modulate excitatory synaptic transmission in hippocampal neurons" Neuron 52: 321-333 (2006). As shown herein, gluconate modulates the outwardly rectified Cl ion mediated by CLC-3 channels. - The current has a strong inhibitory effect. Postsynaptic Cl - Influx can enhance NMDA receptor-mediated excitatory currents, and overactivation of NMDA receptors is involved in seizure-induced neuronal cell death. Therefore, the neuroprotective effect of gluconate may be indirectly inhibiting the function of NMDA receptors.

[0251] Although the hippocampus is known to be one of the most common epileptogenic structures in the rodent brain, to date, there have been few studies directly examining the relationship between CLC-3 channels and epilepsy, except for one report that CLC-3 knockout mice showed significant resistance to PTZ-induced epilepsy. Dickerson et al., "Altered GABAergic function accompanies hippocampal degeneration in mice lacking ClC-3 voltage-gated chloride channels" Brain Research 958:227-250 (2002). The electrophysiological studies presented here found that CLC-3 channels mediate Cl in hippocampal CA3 pyramidal neurons. - The data also showed that CLC-3 channels were upregulated in newborn hippocampal slices after induction of epileptic activity and that sodium gluconate abolished the increased chloride current.

[0252] Therefore, CLC-3 channels are clearly involved in neonatal epilepsy. Supporting these observations, CLC-3 channels have been found to be highly expressed in human glioma cells and can be blocked by gluconate. Olsen et al., "Expression of voltage-gated chloride channels in human glioma cells" The Journal of Neuroscience: the official journal of the Society for Neuroscience 23: 5572-5582 (2003). In addition, it has been reported that the high incidence between glioma and epilepsy is evident, and ... - Disturbances in homeostasis are associated with glioma-related epilepsy. Buckingham et al., “Glutamate release by primary brain tumors induces epileptic activity” Nature Medicine 17: 1269-1274 (2011); and Pallud et al., “Cortical GABAergic excitation contributes to epileptic activities around human glioma” Sci Transl Med 6: 244ra289 (2014). In summary, these findings suggest that Cl - There is a potential link between the channel and neonatal epileptogenesis. As a potent inhibitor of CLC-3 channels, gluconate may be a previously unknown anti-epileptic drug for the treatment of neonatal epilepsy.

[0253] Consistently, the data presented here show that voltage-dependent outwardly rectifying Cl in CA3 pyramidal neurons in hippocampal slices - Current. See Figure 5A , left. For example, after applying NaGcA (20 mM), Cl - The current is significantly reduced. Figure 5A , right. NaGcA before and after Cl - Quantitative data analysis of current IV curves. Figure 5B To directly test the voltage sensitivity of Cl - Is the current from CLC-3 Cl -Channel-mediated, CLC-3 specific antibodies are introduced into the titration solution to block CLC-3 channels. Wang et al., "Functional effects of novel anti-ClC-3 antibodies on native volume-sensitive osmolyte and anion channels in cardiac and smooth muscle cells" American Journal Of Physiology. Heart and circulatory physiology 285: H1453-1463 (2003); and Duan et al., "Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3antibody" The Journal Of Physiology 531: 437-444 (2001).

[0254] In fact, after 10 min of dialysis with CLC-3 antibody (1:100), the outwardly rectified Cl - The current was greatly reduced, but there was no significant change in control IgG dialyzed neurons. Figure 5C Quantitatively, under control conditions Cl - The current density at 90 mV was 36.2 ± 1.4 pA / pF (n = 10 cells from 4 pups), 33.5 ± 2.3 pA / pF (n = 10 cells from 3 pups) in the IgG group, but decreased to 13.7 ± 2.3 pA / pF in the CLC-3 antibody group (n = 11 cells from 4 pups; P < 0.001, one-way ANOVA with Tukey post hoc tests). See Figure 5D Therefore, Cl in CA3 pyramidal neurons - The currents were mainly mediated by CLC-3 channels, consistent with previous findings in CLC-3- / - hippocampal neurons.

[0255] NaGcA was also tested to determine whether this compound could specifically inhibit CLC-3 channels. HEK293T cells were transfected with CLC-3-EGFP plasmid and expression of CLC-3 channels was confirmed using a CLC-3-specific antibody. Figure 5E, and Matsuda et al., "Overexpression of CLC-3 in HEK293T cells yields novel currents that are pH-dependent" American Journal Of Physiology. Cell physiology 294: C251-262 (2008). Whole-cell recordings also showed large externally rectified currents in CLC-3 transfected HEK 293T cells (control, 1012 ± 123 pA at +90 mV, n = 7 cells from 2 batches of culture), but no currents were observed in EGFP-transfected control cells. See Fig. 5F In addition, the administration of 20 mM NaGcA significantly reduced the CLC-3-mediated Cl - Current (NaGcA, 544 ± 59 pA at +90 mV, from 2 batches of n = 7 cells; P < 0.004 compared with control, paired t test), which was also reversible after NaGcA removal (925 ± 124 pA at +90 mV, n = 7 cells, two batches; P > 0.4 compared with control, paired t test). Fig. 5F , G. Taken together, these data suggest that NaGcA is a CLC-3 Cl - A potent inhibitor of the channel, which mediates the major outward rectifying Cl - Electric current.

[0256] F. Upregulation of epilepsy genes by CLC-3 channels

[0257] To determine whether CLC-3 channels are involved in epileptogenesis, CLC-3 expression levels were measured in P8-P12 neonatal brain slices before and after induction of epileptic activity. 2+ The slices were incubated in aCSF for 1 hour to induce epileptic activity, and then CLC-3 immunostaining was performed to detect CLC-3 expression levels. Control slices were incubated in normal aCSF for 1 hour accordingly. Interestingly, CLC-3 immunoreactivity in the CA3 pyramidal layer of the hippocampus was shown to be higher in 0Mg compared with the control. 2+ 6A, B; control, 11.2±1.5 au, n=10 slices from 4 puppies; 0Mg 2+ , 20.4±2.8 au, n=11 slices from 4 pups; P<0.02, Student t test. This elevated expression level of CLC-3 was further confirmed by Western blot. See Figure 6C, D. Therefore, CLC-3 channels are upregulated in neonatal epileptic slices.

[0258] In addition, patch clamp recordings directly measured control and 0Mg 2+ Cl of CA3 pyramidal neurons in treated slices - Consistent with the results of immunostaining and western blotting, whole-cell recordings also showed 0Mg 2+ Cl in treated neurons - The current was significantly increased. See Figure 6E, F; control, 31.2 ± 1.8 pA / pF at +90 mV, n = 15 cells from 3 pups; 0 Mg 2+ , 42.2±2.4 pA / pF, n=15 cells from 3 pups; P<0.001, one-way ANOVA with Tukey post hoc test. In addition, NaGcA significantly inhibited 0Mg 2+ After treatment, most of the Cl - Current, see Fig. 6E, F; NaGcA, 7.3 ± 0.6 pA / pF, n = 11 cells from 2 pups; P < 0.001, one-way ANOVA with Tukey post hoc test. These results suggest that CLC-3 channels may play a role in neonatal epileptogenesis.

[0259] G. Neonatal epileptic seizure suppression

[0260] To induce neonatal epileptic seizures, the commonly used KA model was used. KA (2 mg / kg, ip) was injected into P10-P12 newborn mice, which induced strong epileptic seizures as shown by in vivo EEG recordings. Fig. 7A , B. When NaGcA (2 g / kg, ip) was injected 10 min after KA injection, seizure activity was essentially abolished. Figure 7C , D. Power analysis confirmed that NaGcA injection significantly inhibited the amplitude of power. Fig. 7E , F. These data show that NaGcA is an effective anti-neonatal epilepsy drug in vivo.

[0261] H. Seizure suppression in adults

[0262] It was determined whether NaGcA could also be used to treat adult epilepsy. Preliminary data showed that the KA seizure model had a high mortality rate. However, a similar PTZ model resulted in reliable seizures in adult mice. See Figure 8A. The data showed that NaGcA prolonged the latency of PTZ-induced seizure bursts but did not suppress seizure burst activity as effectively as in neonatal animals. See Figure 8. Therefore, NaGcA may be a unique anti-epileptic drug that is more effective in treating neonatal seizures than adult epilepsy.

[0263] I. Whole-cell Inhibition with Chloride Channel Blockers

[0264] A related question is whether other ClA inhibitors that inhibit neonatal epileptic seizures - Channel blockers, such as NPPB and NFA, also block Cl - Ion channel related activities. See Figures 9 and 10. In one embodiment, the present invention contemplates that compounds including but not limited to gluconate, NPPB, niflumic acid, DIDS, flufenamic acid may play a similar role in inhibiting neonatal epileptic seizures. Although it is not necessary to understand the mechanism of the invention, it is believed that neonatal epileptic seizure inhibition may occur through chloride ion channel inhibition.

[0265] IV. Glucose oxidase-based seizure therapy

[0266] In one embodiment, the present invention contemplates that glucose oxidase can be used as an anticonvulsant to inhibit seizure activity. Although it is not necessary to understand the mechanism of the present invention, it is believed that glucose oxidase converts glucose to gluconate (supra), which has been shown herein to reduce epileptic activity. The data provided herein suggest that gluconate may be involved in the inhibition of CLC-3 Cl - The data also showed that glucose oxidase (GOx), an enzyme that oxidizes glucose to gluconic acid, also had anti-epileptic-like activity in hippocampal slices from newborn mice.

[0267] About 1% of the world's population suffers from epilepsy. Although many seizures in epilepsy patients can be partially controlled by drugs, about one-third of epilepsy patients are resistant to antiepileptic drugs. Sada et al., "Epilepsy treatment. Targeting LDH enzymes with a stiripentol analog to treat epilepsy" Science 347: 1362-1367 (2015); and Juge et al., "Metabolic control of vesicular glutamate transport and release" Neuron 68: 99-112 (2010). Fortunately, several new antiepileptic drugs have been developed in the past few years that show some other effects on those resistant patients. Bialer et al., "Key factors in the discovery and development of new antiepileptic drugs" Nature Reviews: Drug Discovery 9: 68-82 (2010). Unfortunately, there are no new drugs specifically developed to suppress neonatal epileptic seizures.

[0268] For example, phenobarbital, a GABA receptor agonist, was discovered in 1912 and is the oldest but still the drug of choice for treating neonatal epilepsy, but is limited to only 50% efficacy and exhibits multiple side effects. Chamberlain et al., "Lorazepam vs diazepam for pediatric status epilepticus: arandomized clinical trial" JAMA 311: 1652-1660 (2014); Painter et al., "Phenobarbital compared with phenytoin for the treatment of neonatal seizures" The New England Journal Of Medicine 341: 485-489 (1999); and Khanna et al., "Limitations of Current GABA Agonists in Neonatal Seizures: Toward GABA Modulation Via the Targeting of Neuronal Cl (-)Transport” Front Neurol 4:78 (2013). Video-EEG studies have shown that phenobarbital is less effective in suppressing electroencephalographic epileptiform activity than clinically apparent seizures. The discrepancy between clinical symptoms and electrographic recordings is called “electroclinical dissociation” and can reach 80% in neonates after anticonvulsant treatment. Glykys et al., “Differences in cortical versus subcortical GABAergic signaling: a candidate mechanism of electroclinical uncoupling of neonatal seizures” Neuron 63:657-672 (2009).

[0269] Therefore, some studies have shown that phenobarbital can resolve the clinical symptoms of epileptic seizures caused by sedation without correcting the underlying abnormal epileptiform activity in the developing brain, which may lead to an overestimation of the true efficacy of phenobarbital. Boylan et al., "Phenobarbitone, neonatal seizures, and video-EEG" Arch Dis Child Fetal Neonatal Ed 86: F165-170 (2002). In addition, fasting and ketogenic diet therapy have long been used to treat epileptic seizures and are still mainly used to treat refractory epilepsy in children. However, the mechanism of the fasting / ketogenic diet effect is not yet fully understood. Zupec-Kania et al., "An overview of the ketogenic diet for pediatric epilepsy" Nutr Clin Pract 23: 589-596 (2008).

[0270] It is generally believed that fasting or ketogenic diet therapy forces the human body to burn fat instead of carbohydrates. Generally, carbohydrates (such as glucose) are used to produce the main energy source of human adenosine triphosphate (ATP), especially the main energy substance for the brain. However, when lacking carbohydrates, it is believed that fat will be converted into ketone bodies in the liver, wherein ketone bodies will enter the brain and replace glucose as an energy source. It is reported that these ketone bodies can also inhibit brain epileptiform activity. Lima et al., "Neurobiochemical mechanisms of a ketogenic diet in refractory epilepsy" Clinics (Sao Paulo) 69: 699-705 (2014). For most epileptic patients, it has been proposed that the use of a ketogenic diet will cause a variety of adverse reactions. Wheless JW, "The ketogenic diet: an effective medical therapy with side effects" Journal Of Child Neurology 16: 633-635 (2001): and Neal et al., "The ketogenic diet for the treatment of childhood epilepsy: a randomised controlled trial" Lancet Neurol 7: 500-506 (2008). Therefore, there is a need to develop alternative therapeutic approaches to treat pediatric epilepsy.

[0271] Because the ketogenic diet apparently achieves the effect of controlling epilepsy by forcibly changing the patient's dietary composition, and the data in this article show that gluconate has a strong inhibitory effect on neonatal epileptiform activity both in vitro and in vivo, a possible mechanism is that gluconate can be locally produced by oxidizing glucose through glucose oxidase. Glucose oxidase is an enzyme commonly found in several fungi and insects and is widely used as a food additive. Glucose oxidase (GOx) catalyzes a reaction in which glucose produces hydrogen peroxide and gluconolactone, which can be hydrolyzed to gluconic acid. The data in this article show that gluconate (e.g., gluconate) inhibits epileptic activity.

[0272] To test the effects of GOx on neonatal epileptiform activity, baseline field potentials were recorded in CA3 in horizontal sections of hippocampal slices (P10-12) and then stimulated with 0Mg2+. 2+ GOx was added to the bath after induction of stable epileptiform activity lasting at least 20 min. Fig. 20AThe data showed that epileptiform activity was significantly inhibited by 0.3 U / ml GOx and 1 U / ml GOx, but not by 0.1 U / ml Gox (n=8, p<0.001). Figures 20B-20D .

[0273] To confirm the involvement of glucose in the GOx-mediated inhibition, glucose was replaced by lactate (5 mM) and pyruvate (3 mM) and then in 0 Mg 2+ Incubation in aCSF, GOx was observed to have no effect on neonatal epileptiform activity. Fig.20E .

[0274] It is generally believed that the available concentration of gluconic acid produced from glucose in the extracellular solution depends on enzyme activity and reaction time. Therefore, increasing the reaction time in the presence of a low dose of GOx (e.g., 0.1 U / ml) may produce a sufficient dose of gluconic acid to suppress epileptic activity in the developing brain. For example, 0.1 U / ml GOx in 0 Mg 2+ Incubate in aCSF for at least one (1) hour. Figure 2F ; Bubble with 95% O2 / 5% CO2. These data indicate that 0.1U / ml GOx exhibits a strong inhibitory effect on epileptiform activity in neonatal mouse brain slices after one (1) hour of incubation. Fig.20F ;refer to Fig. 20B It should be noted that these various GOx concentrations reduced the mean power by 64.3 ± 8.2% (0.3 U / ml, n = 9, p < 0.001), 83.3 ± 2.2% (1 U / ml, n = 6, p < 0.001), and 81.4 ± 2.0% (0.1 U / ml incubated for 1 hour, n = 5, p < 0.001). Figure 20G .

[0275] In addition to evaluating the effects of GOx on 0Mg 2+ In addition to the effects of aCSF-induced epileptiform activity, we also determined whether GOx (1 U / ml) could inhibit i) high K + or ii) 4-aminopyridine (4-AP) + 0Mg 2+ aCSF-induced neonatal epileptiform activity. The data confirmed that 1U / ml GOx also showed effective inhibition of K in developing brain hippocampal slices (P10-12). + aCSF or 4-AP+0Mg 2+ aCSF-induced epileptiform activity. Figures 21A-21C Therefore, these data demonstrate that GOx has strong activity in suppressing neonatal epileptiform activity.

[0276] V.CLC-3 Cl -Channels regulate large outward rectifying currents in developing neurons

[0277] Changes in GABA function are closely related to epilepsy. GABA function can be regulated by intracellular Cl - Concentration ([Cl - ] i )) to determine, because GABAA receptor (GABAA-R) is considered to be a ligand-gated Cl - For example, previous studies have extensively investigated Cl - Transporters such as KCC2 and NKCC1 play a role in regulating [Cl - ] i and thus the role of GABA in function. However, other Cl - How might channels affect [Cl - ] i And GABA function is not well understood.

[0278] The data presented here examine the voltage dependence of Cl - Does the channel contribute to [Cl - ] i Homeostasis and / or regulation of GABA function. Previously, recording voltage-dependent Cl - Currents showing large outwardly rectifying Cl in CA3 pyramidal neurons of hippocampal slices from newborn mice (e.g., P8-12) - Current (3.3±0.3nA). Fig.11A , upper left panel. Surprisingly, this voltage-dependent outward rectification Cl - The current is significantly reduced in the adult brain (eg, P60-62). Fig.11A and 11B These data suggest that Cl - We recorded Cl from CA3 pyramidal neurons - The currents were similar to those previously reported in cultured hippocampal neurons for voltage-dependent CLC-3 Cl -current. Wang et al., "CLC-3channels modulate excitatory synaptic transmission in hippocampal neurons" Neuron 52: 321-333 (2006); Huang et al., "Calcium-activated chloride channels (CaCCs) regulate action potential and synaptic response in hippocampal neurons" Neuron 74: 179-192 (2012).

[0279] To test CLC-3 Cl - Does the channel mediate Cl - Current, CLC-3 specific antibodies were tested for blocking CLC-3 channels. Wang et al., "Functional effects of novel anti-ClC-3 antibodies on native volume-sensitive osmolyte and anion channels in cardiacand smooth muscle cells" American Journal Of Physiology. Heart and Circulatory Physiology 285: H1453-1463 (2003); and Duan et al., "Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody" The Journal Of Physiology 531: 437-444 (2001). In fact, after 10 minutes of dialysis with CLC-3 antibody (1:100), outwardly rectifying Cl - The current was greatly reduced but did not change significantly upon dialysis with preabsorbed control antibody. Fig.11D .

[0280] Furthermore, direct examination of Cl in CLC-3 knockout mice -Current. Huang et al., "ClC-3 deficiency protects preadipocytes against apoptosis induced by palmitate in vitro and intype 2 diabetes mice" Apoptosis: An International Journal On Programmed Cell Death 19: 1559-1570 (2014); Liu et al., "ClC-3 deficiency prevents apoptosis induced by angiotensin II in endothelial progenitor cells via inhibition of NADPHoxidase" Apoptosis: An International Journal on Programmed Cell Death 18: 1262-1273 (2013); and Zheng et al., "Deficiency of volume-regulated ClC-3 chloride channelattenuates cerebrovascular remodeling in DOCA-salt hypertension" Cardiovascular Research 100: 134-142 (2013). Immunostaining confirmed the absence of CLC-3 signals in the CA3 region of CLC-3 KO mice. Fig.11E Therefore, in CA3 neurons of CLC-3 KO mice, outwardly rectifying Cl - Current (WT, 33.1±1.3 pA / pF, n=10; CLC-3KO, 9.0±1.6 pA / pF, n=6). Fig.11F and 11G These results together suggest that CLC-3 Cl - Channels mediate large outward rectifiers in the newborn brain - Current, but in the adult brain this Cl - The current is significantly reduced.

[0281] Further testing found that sodium gluconate (NaGcA) was a promising candidate for blocking Cl in the brain of newborns. - Effective inhibitor of electric current. Fig.11A -C; Green trace. NaGcA response to Cl in adult brain or CLC-3 KO mice - The current has no effect. Fig.11A , 11B and 11G. These data suggest that NaGcA may be an inhibitor of CLC-3 channels. Olsen et al., "Expression of voltage-gated chloride channels in human glioma cells" The Journal of Neuroscience: The Official Journal Of The Society For Neuroscience 23: 5572-5582 (2003).

[0282] To directly test this idea, CLC-3 channels were overexpressed in HEK293T cells and expression was confirmed by binding with a CLC-3-specific antibody. Fig.11H and Matsuda et al., "CLC-3 overexpression in HEK293T cells generates a novel pH-dependent current." American Journal of Physiology. Cell Physiology 294: C251-262 (2008). Whole-cell recordings revealed large outwardly rectifying Cl in CLC-3-transfected HEK293T cells (1012±123 pA, n=7). - current, but not in EGFP-transfected control cells. Fig.11I In addition, the administration of 20 mM NaGcA significantly reduced the CLC-3 channel-mediated Cl - Electric current. Fig.11I and 11J Therefore, the present data identified NaGcA as CLC-3 Cl - Potent inhibitors of the channel.

[0283] In the presence or absence of divalent cations (e.g., calcium or magnesium), gluconate can inhibit Cl - The data presented in this article compare the Cl between sodium gluconate, magnesium gluconate, and gluconic acid. - The data show that equivalent Cl - Current suppression. Fig.17A -E. Therefore, these data suggest that it is gluconic acid, rather than the cation it carries, that is the active ingredient. Previous studies using gluconate often attributed the clinical effects to the cation it carries, with gluconic acid being considered a mere food / drug additive rather than a primary effector. The data suggest that such an interpretation is incorrect in the art, and more specifically, that divalent cations are not a necessary component of gluconate for its anti-epileptic efficacy.

[0284] VI. CLC-3 channels and neonatal epilepsy

[0285] To understand the functional role of CLC-3 channels in neonatal epilepsy, we used 0Mg 2+ Neonatal brain slices were treated with artificial cerebrospinal fluid (aCSF), and the expression levels of CLC-3 channels were measured after induction of epileptiform activity.

[0286] Mg-free 2+ aCSF-induced epileptic activity significantly upregulates CLC-3 channel expression in neonatal brain slices (eg, P8-P12). Fig. 12A and 12B ; Control: 11.2±1.5 artificial units (n=10); 0Mg 2+ : 20.4±2.8 artificial units (n=11); P<0.02, Student's t test. Western blotting further confirmed the upregulation of CLC-3 channel expression after epileptic activity. Fig. 12C and 12D In addition, patch clamp recordings also showed that outward rectification of Cl after induction of epileptiform activity - The current increased significantly, and the administration of NaGcA significantly blocked the Cl - Electric current. Fig.12E and 12F ; Control: 31.2±1.8pA / pF (n=15); 0Mg 2+ : 42.2±2.4 pA / pF (n=15); NaGcA: 7.3±0.6 pA / pF (n=11); P<0.001, one-way ANOVA with Tukey post hoc test. These results suggest that CLC-3 channels may play a role in neonatal epilepsy.

[0287] To directly test this hypothesis, CLC-3 channels were inhibited with NaGcA and the effects on epileptic activity were examined. Significantly, the data showed that NaGcA produced potent anti-epileptic effects in brain slices from postnatal animals (P6-12). Figure 12G , 12H, 12I and 12J. However, the antiepileptic effect of NaGcA becomes much smaller in aged animals, such as animals of about one month of age (eg, P26). Figure 12K and 12L Quantitatively, NaGcA suppressed the mean power of epileptiform activity in early postnatal animals by 60% (P6-8: 59.6±4.3% (n=10); P10-12: 62.1±3.8% (n=10); P<0.001, paired t-test). However, in aged animals, the power was reduced by only 20% after weaning (P21-33: 23.8±4.1% (n=7); p<0.005, paired t-test). Figure 12M This significant difference between the inhibition of epileptic activity by NaGcA in neonates and adults is consistent with the effect of NaGcA on CLC-3 Cl in animals of different ages. - The relative inhibition of currents was consistent, suggesting that CLC-3 channels may play a role in neonatal epilepsy.

[0288] To further investigate the functional role of CLC-3 channels in epileptiform activity, epileptiform burst activity was detected by Mg-free culture in hippocampal slices of CLC-3 KO mice. 2+ In addition, the epileptiform activity was not induced by aCSF, but was maintained for a shorter period of time when compared with WT mice. Interestingly, when hippocampal slices from WT mice were pretreated with NaGcA to block CLC-3 channels, the epileptiform activity also did not persist for a long time, mimicking that observed in CLC-3 KO mice. Fig.12N and 12O Quantitative data showed that burst latency was significantly prolonged and total burst activity was significantly reduced when CLC-3 channels were inhibited or knocked down. Figure 12P Taken together, these data suggest that CLC-3 Cl - The channel may play a role in epileptogenesis in the early developing brain.

[0289] We also investigated whether the effects of NaGcA on neonatal epileptic activity can be generally applicable to epileptic animals other than 0Mg 2+ Other epilepsy models. + The channel blocker 4-AP (50 μM) was added to the bathing solution to induce strong epileptic burst activity in immersed hippocampal slices. Fig.13A The addition of 20 mM NaGcA to 4-AP solution significantly reduced the epileptic activity, which was reversible after NaGcA removal. Fig.13A , 13B and 13C. NaGcA reduced the power amplitude by 70.2±5.3% (n=9). (P<0.001, paired t-test). + (8.5mM) to induce epileptic burst activity. Fig.13D Similarly, in high K + The addition of 20 mM NaGcA to aCSF also significantly reduced epileptic burst activity. Fig.13D , 13E and 13F. NaGcA reduced the power amplitude by 70.8±4.9% (n=5). (P<0.001, paired t test). In conclusion, these results indicate that NaGcA can effectively suppress neonatal epilepsy in various epilepsy models.

[0290] VII. Gluconate inhibits neonatal epilepsy

[0291] In one embodiment, the present invention contemplates a method of treating neonatal epilepsy comprising using a gluconate compound. In one embodiment, the gluconate compound is administered systemically.

[0292] For example, gluconate was administered to neonatal rats to determine the effect on epileptic seizures in neonatal and adult animals. To induce epileptic seizures, the neurotoxin kainic acid (KA) (2 mg / kg, ip) was injected into neonatal rats (P10-12) to induce strong seizure activity as revealed by in vivo EEG recordings. Fig.14A and 14B ; Dzhala et al., "NKCC1 transporter facilitates seizures in the developing brain" Nature Medicine 11: 1205-1213 (2005). In addition, when NaGcA (2 g / kg, intraperitoneal) was injected 10 minutes after KA injection, epileptic seizure activity was essentially eliminated in newborn animals. Fig. 14C and 14D .

[0293] The antiepileptic effects of gluconate have also been compared with previously reported anticonvulsant drugs such as phenobarbital and bumetanide in neonatal animals. Phenobarbital is currently the drug of choice for the treatment of neonatal seizures, despite only approximately 50% efficacy and potential negative neurodevelopmental consequences. Slaughter et al., “Pharmacological treatment of neonatal seizures: a systematic review” Journal of Child Neurology 28:351-364 (2013). Bumetanide is a potent loop diuretic that is currently being evaluated as a prospective antiepileptic drug. Loscher et al., “Cation-chloride cotransporters NKCC1 and KCC2 as potential targets for novel antiepileptic and antiepileptogenic treatments” Neuropharmacology 69:62-74 (2013). Although both phenobarbital and bumetanide can suppress epileptic activity to some extent in neonatal animals, their suppression is not as effective as that of gluconate. See also Figures 14E-14H and Fig. 14C and 14DQuantitatively, when EEG power was calculated during the last 30 min of the 2-h recording period after KA injection, the relative power was reduced by 72.3% in the NaGcA group, 35.5% in the phenobarbital group, and 54.3% in the bumetanide group, respectively. Fig.14M Gluconate therefore appears to be an effective antiepileptic drug for the treatment of neonatal seizures.

[0294] The antiepileptic effects of gluconate were also compared in adult animals. Unlike the strong suppression of seizures in neonatal animals, gluconate had less inhibitory effects on seizure activity in adults. Figures 14I-14L and 14N. These data are consistent with the results of brain slice recordings. Figure 14G-14M In addition, newborn animals were tested for the effect of NaGcA on KA-induced stable seizure activity and it was found that NaGcA still suppressed seizure activity 1 hour after KA injection (Figure 15). Therefore, these data allow the conclusion that NaGcA is an effective drug for treating neonatal epileptic seizures.

[0295] VIII. CLC-3 channels regulate Cl during epileptogenesis - Internal environment is stable

[0296] Although it is not necessary to understand the mechanism of the invention, it is believed that [Cl - ] i The changes in Cl may represent at least one molecular mechanism of CLC-3 channels in neonatal epilepsy, because CLC-3 channels are voltage-dependent, outwardly rectifying Cl - For example, epileptic bursts often last more than 10 seconds. Fig.16A .

[0297] To investigate the effects of this sustained epileptiform burst on GABA function, whole-cell recordings were performed using gramicidin-perforated wells to maintain intracellular Cl in newborn animals. - Intact. GABAA receptor (GABAA-R) currents induced by the receptor agonist isoguvacine (100 μM, 50 ms) before and after a membrane depolarization shift (40 mV, 10 s) that mimics epileptiform burst activity. Interestingly, GABAA-R currents increased significantly after a membrane depolarization shift in WT but not in CLC-3 KO neurons, nor in the presence of the CLC-3 channel blocker NaGcA. Fig. 16B and 16C These data suggest that CLC-3 channels may modulate GABA function during epileptogenesis in neonatal animals.

[0298] To further test this idea, we directly measured the GABAA-R reversal potential (EGABA), which controls the excitatory and inhibitory functions of GABA, using whole-cell recordings from gramicidin-perforated neonatal mouse brain slices (e.g., P8-P9). 2+ Hippocampal slices were treated with aCSF for 1 hour (aCSF: -59.2±2.0 mV, n=13; 0Mg 2+ : -48.2±1.1 mV, n=10), epileptiform activity induced a large depolarizing shift of EGABA. Fig.16D Compared with the control EGABA (dashed line), the use of bumetanide (Bum, 10 μM), a specific blocker of NKCC1 at low concentrations, induced a hyperpolarizing shift in EGABA (blue line). Figure 6E. These results are consistent with the role of NKCC1 in the regulation of Cl - The reports of introduction into neuronal cells are consistent. Kaila et al., "Cation-chloride cotransporters in neuronal development, plasticity and disease" Nature Reviews. Neuroscience 15: 637-654 (2014); Dzhala et al., "NKCC1 transporter facilitates seizures in the developing brain" Nature Medicine 11: 1205-1213 (2005); and Loscher et al., "Cation-chloride cotransporters NKCC1 and KCC2 as potential targets for novel antiepileptic and antiepileptogenic treatments" Neuropharmacology 69: 62-74 (2013). However, in the presence of bumetanide, epileptic activity still causes depolarization shift of EGABA, which indicates that factors other than NKCC1 regulate EGABA during epilepsy. Fig.16E , yellow line; Bum, -68.0±1.7mV(n=9); 0Mg 2+ +Bum, -50.2±1.4mV (n=9); (p<0.002).

[0299] Treatment with the KCC2 blocker VU0240551 (10 μM) did not affect EGABA in newborn animals. Fig.16F, purple line; -60.8±1.9 mV (n=6). These data may be explained by the low expression level of KCC2 in the early stage. Epileptic activity also triggered a positive shift of EGABA. Fig.16F , orange line; -48.9 ± 1.3 mV (n = 8). Thus, epileptiform activity-induced EGABA shifts in neonatal animals are not controlled by NKCC1 or KCC2.

[0300] On the other hand, when NaGcA (20 mM) was used to inhibit CLC-3 Cl - channel, when exposed to 0Mg 2+ When aCSF was added, EGABA was not further altered. Figure 16G ;0Mg 2+ +NaGcA: -59.3±1.7 mV (n=6). In addition, in CLC-3 KO mice, EGABA 2+ There were also no changes in aCSF. Fig.16H ; CLC-3 KO+0Mg 2+ :-55.6±2.1mV(n=10). In addition, the administration of NaGcA to CLC-3KO mice had an effect on the 2+ EGABA in aCSF had no effect (-56.6±2.4 mV, n=5). Inhibition of CLC-3 channels with NaGcA or knockout of CLC-3 did not change the normal EGABA under normal conditions. Figure 16G ; NaGcA: -60.3±0.9mV (n=5); CLC-3 KO: -55.4±2.9mV (n=9).

[0301] Therefore, these results indicate that CLC-3 Cl - The channel plays a role in controlling EGABA during epilepsy in newborn animals. However, in old animals (e.g., P30-90), in the presence of NaGcA, 0Mg 2+ aCSF still induced a large depolarizing shift of EGABA. Fig.16J These data are consistent with the outwardly rectified Cl ions mediated by CLC-3 channels in adult animals. - This is consistent with the observation that the current is substantially reduced. Fig.16A .

[0302] In addition to EGABA, the effects of different drugs on GABA-induced neuronal activity during epileptogenesis were investigated in newborn animals (e.g., P8-9). In order to accurately measure GABA-induced neuronal activity, spike firing monitored by cell-attached recordings was initiated by local application of the GABAA-R agonist isotetrahydronicotinic acid (10 μM, 30 s). Tyzio et al., "Maternaloxytocin triggers a transient inhibitory switch in GABA signaling in the fetal brain during delivery" Science 314: 1788-1792 (2006). Most resting CA3 pyramidal neurons do not respond to isotetrahydronicotinic acid. Figure 16K , control, (n=32). However, in 0Mg 2+ After treatment, 68% of neurons showed spiking activity upon isothiazolinone administration. Figure 16K ,0Mg 2+ (n=19) Blockade of NKCC1 with bumetanide or KCC2 with VU0240551 did not alter the stimulatory activity elicited by isothiahydronicotinic acid. Figure 16K ;0Mg 2+ +Bum (n=19); and 0Mg 2+ +VU (n=21). In contrast, administration of NaGcA inhibited CLC-3 Cl - The channel is essentially eliminated at 0Mg 2+ Stimulatory activity induced by isotetrahydronicotinic acid after treatment. Figure 16K ;0Mg 2+ +NaGcA (n=18) Quantification of these observations is shown in Figure 4L.

[0303] These data suggest that CLC-3 Cl - Activation of the channel increases GABA excitatory activity during epileptogenesis, and blocking CLC-3 channels is an effective way to inhibit neonatal epileptic seizures by reducing excitatory GABA activity.

[0304] IX. Pharmaceutical Preparations and / or Compositions

[0305] The present invention also provides pharmaceutical compositions (e.g., comprising the above compounds). The pharmaceutical compositions of the present invention can be administered in a variety of ways, depending on whether local or systemic treatment is needed and the area to be treated. Administration can be local (including eyes and mucous membranes, including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including sprayers, intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, such as intrathecal or intraventricular administration. In particular, intramuscular and / or intravenous injections of sodium gluconate can be delivered in a sterile saline solution at an approximate concentration range of 1-100 mM gluconate. Although it is not necessary to understand the mechanism of the present invention, it is believed that these concentration ranges are achievable because sodium gluconate is highly water-soluble.

[0306] Pharmaceutical compositions and preparations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0307] Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

[0308] Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0309] The pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be produced from a variety of components, including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids.

[0310] The pharmaceutical preparation of the present invention which can be conveniently present in unit dosage form can be prepared according to conventional techniques known in the pharmaceutical industry. These techniques include the step of combining the active ingredient with a pharmaceutical carrier or excipient. Usually, the preparation is prepared by uniformly and directly combining the active ingredient with a liquid carrier or a finely dispersed solid carrier or both, and then, if necessary, shaping the product.

[0311] The compositions of the present invention can be formulated into any of a number of possible dosage forms, such as but not limited to tablets, capsules, liquid syrups, soft gels, suppositories and enemas. The compositions of the present invention can also be formulated into suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions can further contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension can also contain a stabilizer.

[0312] In one embodiment of the invention, the pharmaceutical composition can be formulated and used as a foam. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies, and liposomes. Although similar in nature, these formulations differ in the composition and consistency of the final product.

[0313] The reagent that strengthens the oligonucleotide absorption of cellular level can also be added in medicine and other compositions of the present invention.For example, cationic lipid, for example cationic liposome (lipofectin) (U.S. Patent number 5,705,188), cationic glycerol derivative and polycationic molecule such as polylysine (WO 97 / 30731) also strengthen the cellular uptake of oligonucleotide.

[0314] The composition of the present invention can contain other auxiliary components usually found in pharmaceutical compositions in addition.Therefore, for example, the composition can contain additional, compatible pharmaceutically active substances, such as antipruritic drugs, astringents, local anesthetics or anti-inflammatory agents, or can contain other substances for the various dosage forms of the physical preparation of the composition of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, sunscreens, thickeners and stabilizers.However, when added, these materials should not interfere with the biological activity of the components of the composition of the present invention inappropriately.Preparation can be sterilized, and if necessary, mixed with auxiliary agents (such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for affecting osmotic pressure, buffers, coloring agents, flavoring agents and / or aromatic substances, etc.), which are different from the nucleic acid of the preparation and interact harmfully.

[0315] The dosage depends on the severity and responsiveness of the disease state to be treated, and the course of treatment lasts from a few days to a few months, or until a cure is achieved or a reduction in the disease state is achieved. The optimal dosage schedule can be calculated by measuring the accumulation of the drug in the patient's body. The administering physician can readily determine the optimal dosage, method of administration, and repetition rate. Depending on the relative potency of the individual oligonucleotides, the optimal dosage may vary and can generally be based on the EC values ​​found to be effective in in vitro and in vivo animal models. 50SOr estimated based on the embodiments described herein. Typically, the dosage per kg body weight is 0.01 μg to 100 g, and may be administered once or more daily, weekly, monthly or annually. The treating physician may estimate the repetition rate of administration based on the drug residence time and concentration measured in body fluids or tissues. After successful treatment, it may be necessary to subject the subject to maintenance therapy to prevent the recurrence of the disease state, wherein the compound is administered at a maintenance dose of 0.01 μg to 100 g per kg body weight, once or more daily, to once every 20 years.

[0316] X. Drug Delivery Systems

[0317] The present invention contemplates several drug delivery systems that provide a generally uniform distribution with a controlled release rate. Various different media useful in preparing drug delivery systems are described below. Any one medium or carrier is not intended to limit the present invention. Note that any medium or carrier can be combined with another medium or carrier; for example, in one embodiment, a polymer microparticle carrier attached to a compound can be combined with a gel medium.

[0318] Carriers or vehicles contemplated by the present invention include those selected from the group consisting of gelatin, collagen, cellulose esters, dextran sulfate, pentose polysulfate, chitin, sugars, albumin, fibrin sealants, synthetic polyvinyl pyrrolidone, polyethylene oxide, polypropylene oxide, block polymers of polyethylene oxide and polypropylene oxide, polyethylene glycol, acrylates, acrylamides, methacrylates, including but not limited to 2-hydroxyethyl methacrylate, poly(orthoesters), cyanoacrylates, gelatin-resorcinol-aldehyde type bioadhesives, polyacrylic acid and copolymers and block copolymers thereof.

[0319] One embodiment of the present invention contemplates a drug delivery system comprising a therapeutic agent described herein.

[0320] particle

[0321] One embodiment of the present invention contemplates a medium comprising microparticles. Preferably, microparticles include liposomes, nanoparticles, microspheres, nanospheres, microcapsules and nanocapsules. Preferably, some microparticles contemplated by the present invention include poly(lactide-co-glycolide), aliphatic polyesters, including but not limited to polyglycolic acid and polylactic acid, hyaluronic acid, modified polysaccharides, chitosan, cellulose, dextran, polyurethane, polyacrylic acid, pseudo-poly(amino acid), polyhydroxybutyrate related copolymers, polyanhydrides, polymethyl methacrylate, poly(ethylene oxide), lecithin and phospholipids.

[0322] Liposomes

[0323] One embodiment of the present invention contemplates liposomes capable of attaching and releasing therapeutic agents described herein. Liposomes are microscopic spherical lipid bilayers made of amphiphilic molecules such as phospholipids surrounding an aqueous core. For example, liposomes can capture therapeutic agents between the hydrophobic tails of phospholipid micelles. Water-soluble agents can be captured in the core, and fat-soluble agents can be dissolved in the shell-like bilayer. Liposomes have special characteristics because they enable water-soluble and water-insoluble chemicals to be used together in a medium without the use of surfactants or other emulsifiers. Liposomes can be formed spontaneously by vigorously mixing phospholipids in an aqueous medium. Water-soluble compounds are dissolved in an aqueous solution that can hydrate phospholipids. Therefore, when liposomes are formed, these compounds are captured in the center of the liposome aqueous solution. The liposome wall, which is a phospholipid membrane, retains fat-soluble substances such as oils. Liposomes provide controlled release of incorporated compounds. In addition, liposomes can be coated with water-soluble polymers such as polyethylene glycol to increase the pharmacokinetic half-life. One embodiment of the present invention takes into account ultra-high shear technology to improve liposome production, thereby producing stable unilamellar (single layer) liposomes with specific designed structural features. These unique liposome properties allow for the simultaneous storage of normally immiscible compounds and their ability to control release.

[0324] In some embodiments, the present invention contemplates cationic and anionic liposomes and liposomes with neutral lipids. Preferably, cationic liposomes contain negatively charged materials by mixing materials and fatty acid liposome components and allowing them to undergo charge association. Obviously, the choice of cationic or anionic liposomes depends on the desired pH of the final liposome mixture. Examples of cationic liposomes include lipofectin, lipofectamine and lipofectace.

[0325] One embodiment of the present invention contemplates a medium comprising liposomes that provide controlled release of at least one therapeutic agent. Preferably, the liposomes capable of controlled release: i) are biodegradable and non-toxic; ii) carry water- and oil-soluble compounds; iii) dissolve recalcitrant compounds; iv) prevent compound oxidation; v) promote protein stability; vi) control hydration; vii) control compound release by changes in bilayer composition, such as but not limited to fatty acid chain length, fatty acid lipid composition, relative amounts of saturated and unsaturated fatty acids, and physical form; viii) have solvent dependence; iv) have pH dependence, v) have temperature dependence.

[0326] The composition of liposomes can be roughly divided into two categories. Conventional liposomes are usually a mixture of stable natural phosphatidylcholine (PC), which may contain synthetic homologous chain phospholipids, which may or may not contain glycolipids. Special liposomes may include: i) bipolar fatty acids; ii) the ability to attach antibodies for tissue-targeted therapy; iii) coating with materials such as, but not limited to, lipoproteins and carbohydrates; iv) multiple encapsulation and v) emulsion compatibility.

[0327] Liposomes can be readily prepared in the laboratory by methods such as, but not limited to, ultrasound and vibration. Alternatively, compound-delivery liposomes are commercially available. For example, Collaborative Laboratories, Inc. is known to manufacture custom-designed liposomes for specific delivery requirements.

[0328] Microspheres, microparticles and microcapsules

[0329] Microspheres and microcapsules are useful because they have the ability to maintain a generally uniform distribution, provide a steady controlled release of the compound, and are economical to manufacture and distribute. Preferably, the associated delivery gel or compound-impregnated gel is transparent or, alternatively, is colored for easy viewing by medical personnel.

[0330] Microspheres are commercially available ( , Alkerme's: Cambridge, Mass.). For example, a freeze-dried medium containing at least one therapeutic agent is homogenized in a suitable solvent and sprayed to prepare microspheres ranging from 20 to 90 microns. The technique is then followed to maintain sustained release integrity during the purification, encapsulation and storage stages. Scott et al., Improving Protein Therapeutics With Sustained Release Formulations, Nature Biotechnology, Vol. 16: 153-157 (1998).

[0331] The ability to control the release rate of therapeutic agents can be provided by using biodegradable polymer-modified microsphere compositions. Miller et al., Degradation Rates of Oral Resorbable Implants {Polylactates and Polyglycolates: Rate Modification and Changes in PLA / PGA Copolymer Ratios, J. Biomed. Mater. Res., Vol. II: 711-719 (1977).

[0332] Alternatively, a sustained or controlled release microsphere preparation is prepared using a water drying method, wherein an organic solvent solution of a biodegradable polymer metal salt is first prepared. Subsequently, a medium of a dissolved or dispersed therapeutic agent is added to the biodegradable polymer metal salt solution. The weight ratio of the therapeutic agent to the biodegradable polymer metal salt can be, for example, about 1:100000 to about 1:1, preferably about 1:20000 to about 1:500, more preferably about 1:10000 to about 1:500. Next, an organic solvent solution containing a biodegradable polymer metal salt and a therapeutic agent is poured into an aqueous phase to prepare an oil / water emulsion. The solvent in the oil phase is then evaporated to provide microspheres. Finally, these microspheres are recovered, washed and freeze-dried. Thereafter, the microspheres can be heated under reduced pressure to remove residual water and organic solvent.

[0333] Other methods for preparing microspheres compatible with the biodegradable polymer metal salt and therapeutic agent mixture are: i) phase separation during gradual addition of a coagulant; ii) an in water drying process or a phase separation process wherein a deflocculant is added to prevent particle aggregation, and iii) by a spray drying process.

[0334] In one embodiment, the present invention contemplates a medium comprising microspheres or microcapsules capable of delivering a controlled release therapeutic agent over a duration of about 1 day to 6 months. In one embodiment, the microspheres or microparticles can be colored to allow the physician to clearly see the ability of the medium when dispensing. In another embodiment, the microspheres or microcapsules can be transparent. In another embodiment, the microspheres or microparticles are impregnated with a radiopaque fluorescent dye.

[0335] Controlled release microcapsules can be prepared by using known encapsulation techniques such as centrifugal extrusion, pan coating and air suspension. Such microspheres and / or microcapsules can be designed to achieve a desired release rate. For example, (Macromed) is a controlled release microsphere system. These specific microspheres can be obtained with uniform sizes between 5-500 microns and are composed of biocompatible and biodegradable polymers. The specific polymer composition of the microspheres can control the therapeutic agent release rate, making customized microspheres possible, including effective management of burst effects. (Epic Therapeutics, Inc.) is a protein-matrix delivery system. The system is aqueous in nature and is suitable for standard drug delivery models. In particular, Biodegradable protein microspheres that can deliver both small and large molecule drugs and can be customized for microsphere size and desired release characteristics.

[0336] In one embodiment, the microsphere or microparticle is included in a pH-sensitive encapsulation material that is stable at a pH value less than the internal mesenteric pH. The typical range of the internal mesenteric is pH 7.6 to pH 7.2. Therefore, the microcapsule should be maintained at a pH value less than 7. However, if the pH change is expected, the pH-sensitive material can be selected based on the different pH standards required for the dissolution of the microcapsules. Therefore, the encapsulated compound will be selected for the pH environment in which the pH is required to be dissolved and stored in a preselected pH to maintain stability. Examples of pH-sensitive materials that can be used as capsule materials are Eudragit L-100 or S-100 (Rohm GMBH), hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, cellulose acetate phthalate and cellulose acetate trimellitate. In one embodiment, lipids include the inner coating of the microcapsule. In these compositions, these lipids can be, but are not limited to, partial esters of fatty acids and adipic anhydride (hexitiolanhydride), and edible fats such as triglycerides. Lew CW, Controlled-Release pH Sensitive Capsule And Adhesive System And Method. U.S. Patent No. 5,364,634 (incorporated herein by reference).

[0337] In one embodiment, the present invention contemplates microparticles comprising gelatin or other polymeric cations having a similar charge density to gelatin (i.e., poly-L-lysine) and used as a complex to form primary microparticles. Primary microparticles are produced as a mixture of the following ingredients: i) gelatin (60 bloom, type A from porcine skin), ii) chondroitin sulfate 4-sulfate (0.005%-0.1%), iii) glutaraldehyde (25%, grade 1), and iv) 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC hydrochloride) and ultrapure sucrose (Sigma Chemical Co., St. Louis, Mo.). The source of the gelatin is not considered critical; it can be from bovine, porcine, human or other animal sources. Typically, the polymeric cation is 19,000-30,000 Daltons. Chondroitin sulfate is then added to the complex with sodium sulfate or ethanol as a coagulant.

[0338] After the microparticles are formed, the therapeutic agent is directly bound to the microparticle surface or indirectly attached using a "bridge" or "spacer". The amino groups of the lysine groups of gelatin are easily derivatized, thereby providing sites for direct coupling of the compound. Alternatively, a spacer (i.e., a derivatized portion connecting the molecule and the targeting ligand) such as avidin-biotin can also be used to indirectly bind the targeting ligand to the microparticles. The stability of the microparticles is controlled by the amount of glutaraldehyde spacer crosslinks induced by EDC hydrochloride. The controlled release medium is also empirically determined by the final density of the glutaraldehyde-spacer crosslinks.

[0339] In one embodiment, the present invention contemplates microparticles formed by spray drying a composition comprising fibrinogen or thrombin and a therapeutic agent. Preferably, the microparticles are soluble and the selected protein (i.e., fibrinogen or thrombin) produces the wall of the microparticles. Thus, the therapeutic agent is incorporated into and between the protein walls of the microparticles. Heath et al., Microparticles And Their Use In Wound Therapy. U.S. Pat. No. 6,113,948 (incorporated herein by reference).

[0340] After application of the microparticles to living tissue, the subsequent reaction between fibrinogen and thrombin produces a tissue sealant, thereby releasing the introduced compounds into the immediate surrounding area.

[0341] Those skilled in the art will appreciate that the shape of the microspheres need not be a precise sphere; only as very small particles that can be sprayed or diffused into a surgical site (either open or closed). In one embodiment, the microparticles are composed of a biocompatible and / or biodegradable material selected from polylactide, polyglycolide and copolymers of lactide / glycolide (PLGA), hyaluronic acid, modified polysaccharides and any other known materials.

[0342] test

[0343] Animal protocols for cell culture and brain slices were approved by the Pennsylvania State University IACUC in accordance with the National Institutes of Health Guide for the Care and use of Laboratory Animals. For in vivo experiments with adult mice or neonatal rats, all procedures were in accordance with the Animal Research Ethics Guidelines and approved by the Animal Use Research and Education Committee of Fudan University or South China Normal University, respectively. The animal room was automatically controlled on a 12-h light / dark cycle, and water and food were available ad libitum.

[0344] Embodiment 1

[0345] Cell culture and transfection

[0346] Mouse cortical neurons were prepared from newborn C57BL / 6 mice as previously described. Qi et al., "Cyclothiazide induces robust epileptiform activity in rat hippocampal neurons both in vitro and in vivo" The Journal Of Physiology 571:605-618 (2006). Briefly, newborn mouse cerebral cortex was dissected in ice-cold HEPES buffered saline solution, washed and digested with 0.05% trypsin-EDTA at 37°C for 20 minutes. After inactivating trypsin with serum-containing medium, cells were centrifuged, resuspended, and plated at 10,000 cells / cm 2 The density of 24-well plates was seeded on a monolayer of cortical astrocytes. Neuronal culture medium contained MEM (500ml, Invitrogen), 5% fetal bovine serum (Atlanta Biologicals), 10ml B-27 supplement (Invitrogen), 100mg NaHCO3, 2mM Glutamax (Invitrogen) and 25 units / ml penicillin & streptomycin. AraC (4μM, Sigma) was added to inhibit excessive proliferation of astrocytes. Cell cultures were maintained in a humidified incubator with 5% CO2 at 37°C for 14-21 days.

[0347] Human embryonic kidney (HEK) 293T cells were maintained in DMEM supplemented with 10% FBS and 25 units / ml penicillin / streptomycin. PEI kit (molecular weight 25,000, Polysciences, Inc.) was used for HEK cell transfection. Briefly, 1 μg DNA was diluted into 50 μl of OptiMEM (Invitrogen), then mixed with 4 μl PEI (1 μg / μl), incubated for 5 minutes, and added dropwise to a culture well containing 500 μl of culture medium. After incubation for 5 hours, the transfection reagent was washed off with fresh culture medium. Two days after transfection, HEK293T cells were used for electrophysiological studies. Rat CLC-3 short transcripts fused to eGFP plasmid (pCLC3sGFP) were purchased from Addgene (plasmid #52423). Li et al., "The ClC-3 chloride channel promotes acidification of lysosomes in CHO-K1 and Huh-7 cells," American Jjournal of Physiology, Cell Physiology 282: C1483-1491 (2002).

[0348] Example II

[0349] Cell viability assay

[0350] Using LIVE / Viability / Cytotoxicity Assay Kit (L3224, Life Technologies) was used to detect cell viability. Ethidin homodimer-1 binds to cellular DNA and usually marks dead cells with red fluorescence, while calcein-AM can be cleaved by esterases in living cells to produce strong green fluorescence. After drug treatment, neurons were incubated in a bath solution containing 1 μM calcein-AM and 4 μM ethidium homodimer-1 at room temperature for 40 minutes. Cell survival and mortality were measured by quantifying the percentage of green and red fluorescent cells, respectively. For each group, at least 5 areas of each coverslip were imaged for data analysis.

[0351] Example III

[0352] Mouse brain slice preparation

[0353] Brain slices were prepared from C57BL / 6 mice (male and female). The animals were anesthetized with Avertin (tribromoethanol, 250 mg / kg) and decapitated. Horizontal slices (400 μm) of the hippocampus were prepared by a Leica VT1200S vibrating slicer in ice-cold artificial cerebrospinal fluid (aCSF) ((mM): 125 NaCl, 26 NaHCO3, 10 glucose, 2.5 KCl, 2.5 CaCl2, 1.25 NaH2PO4 and 1.3 MgSO4, osmotic pressure of 290-300 mOsm, gassed with 95% O2 / 5% CO2). The slices were then transferred to an incubator containing normal aCSF saturated with a mixture of oxygen and carbon dioxide (carbogen) (95% O2 / 5% CO2) at 33°C for 30 minutes and then recovered at room temperature for 1 hour before use. Individual slices were transferred to a submerged recording chamber where they were continuously perfused (2-3 ml / min) with aCSF saturated with 95% O2 / 5% CO2 at 31-33°C (TC-324B, Warner Instruments Inc). Slices were visualized with infrared optics using an Olympus microscope equipped with DIG optics.

[0354] Example IV

[0355] Electrophysiology

[0356] Cell culture

[0357] Cultured neurons were placed in a recording chamber and continuously perfused with a bath solution consisting of (mM): 128 NaCl, 10 glucose, 25 HEPES, 5 KCl, 2 CaCl2, 1 MgSO4, pH 7.3 adjusted with NaOH and an osmotic pressure of approximately 300 mOsm. To record spontaneous firing in current clamp mode, pipettes were filled with an internal standard solution containing (mM): 125 K gluconate, 5 Na-phosphocreatine, 5 EGTA, 10 KCl, 10 HEPES, 4 Mg-ATP, 0.3 Na-GTP, 280-290 mOsm, pH 7.3 adjusted with KOH. Epileptiform activity in cultured neurons was induced by 10 μM CTZ for 24 h, or 1 μM KA for 2 h or 50 μM 4-AP for 2 h. Burst activity was defined as previously described. Qi et al., "Cyclothiazide induces robust epipileptiform activity in rat hippocampal neurons both in vitro and in vivo" The Journal of Physiology 571: 605-618 (2006). Briefly, at least five consecutive action potentials are superimposed on a large depolarizing shift (≥10 mV depolarization and duration ≥300 ms).

[0358] For Cl - Current recording, 0Ca 2+ The titration solution contained (mM): 125 CsCl, 5 Na-creatine phosphate, 10 HEPES, 5 EGTA, 5 TEACl, 4 MgATP (pH 7.3, adjusted with CsOH, 280-290 mOsm).

[0359] To separate Cl - Current: (1) Extracellular Na + By NMDG + Instead, the voltage-dependent Na + The channel is blocked by tetrodotoxin (TTX); (2) K + and Ca 2+ Remove from the bath solution; (3) Use Cs in the titration solution + and tetraethylammonium (TEA) to block K + channels and blocked them with 4-AP in the bath solution; (4) CdCl2 was added to the bath solution to block the Ca 2+channel; (5) also included picrotoxin (50 μM) to block GABAA receptors. Therefore, the external solution contained the following (in mM): 135 NMDG-Cl, 20 HEPES, 20 glucose, 5 4-AP and 2 MgSO4, supplemented with 1 μM TTX, 200 μM CdCl2 and 50 μM picrotoxin, osmotic pressure ~300 mOsm, pH 7.3-7.4 after aeration with 95% O2 / 5% CO2. From 0 mV (ECl - Voltage steps from -80 to +90 mV (10 mV increments) were applied with a holding potential of -0 mV. Data were collected using a MultiClamp 700A amplifier and pCLAMP9 software (Molecular Devices).

[0360] Brain slice recordings

[0361] Field potential recordings were performed with glass electrodes (2-4 MΩ tip resistance) filled with external solution. The glass electrodes were placed into the CA3 pyramidal layer. For current clamp (I=0) recordings, the amplifier was set to 100x with a bandpass filter of 0.1-5 KHz. All recordings were performed at 31-33°C. Epileptic activity was detected by Mg-free 2+ or by adding 50 μM 4-AP or 8.5 mM K + .

[0362] The power spectrum analysis was preceded by a Hamming window function. The power was calculated by integrating the RMS value of the signal in a frequency band from 0.1 to 1000 Hz in consecutive 5-min time windows before, during, and after drug administration. To avoid slice-to-slice and electrode contact variations, power values ​​were normalized to control conditions before drug administration in each slice and then averaged across different slices for statistical analysis.

[0363] Example V

[0364] Immunostaining and Western Blot

[0365] Brain slices were prepared according to the electrophysiology protocol described above at 200 μm thickness for immunostaining and 400 μm thickness for western blot. Typically, the slices were recovered at room temperature for 1 hour and then randomly divided into two groups. One group of slices was incubated in normal aCSF at 33 °C for 1 hour. The other group of slices was incubated at 0 Mg 2+ The sections were incubated in aCSF for 1 hour at 33°C and then fixed with 4% PFA at 4°C overnight.

[0366] Immunostaining

[0367] For CLC-3 staining, the sections were pretreated with blocking solution (0.3% Triton-X and 5% normal donkey and goat serum in 0.1M PBS) for 2 hours and then incubated with CLC-3 primary antibody for 72 hours (rabbit, 1:200, Alomone, ACL-001). Some sections were incubated with rabbit IgG as a control. After washing three times in PBS containing 0.01% triton-X, brain sections were incubated with goat anti-rabbit secondary antibody conjugated to Cy3 (1:500, Jackson ImmunoResearch) at room temperature for 2 hours. Brain sections were mounted on slides with anti-fading mounting solution (Invitrogen). Fluorescence images were obtained on an Olympus confocal microscope system (FV1000). For each section, at least 2-3 regions of CA3 were imaged. To quantify the CLC3 fluorescence intensity in the CA3 pyramidal layer, confocal images were analyzed using NIH Image J software.

[0368] Western blotting

[0369] After 1 hour of recovery at room temperature, 400 μm thick hippocampal slices were randomly divided into two groups, which were used for immunostaining as described above. The hippocampus was dissected in ice-cold aCSF. Proteins were extracted with RIPA lysis buffer (containing 1:100 phenylmethylsulfonyl fluoride and 1:200 protease inhibitors). Then, lx loading dye buffer (NuPAGE LDS sample buffer (4x)) and 1% β-ME were added to the protein samples and mixed thoroughly. The protein extract (2μg / μ1, 15μl per lane) was boiled at 55°C for 20 minutes, loaded on a 10% gel, and separated at room temperature using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (70V, 50 minutes, then switched to 90V for 2 hours). The gel was then blotted onto a PVDF membrane. The blot was blocked for 1 hour at room temperature using a solution of 5% milk in Tris buffer solution (1xTBS) containing 1% Tween-20. Primary antibodies to CLC3 (rabbit, 1:300, Alomone, ACL-001) and GAPDH (rabbit, 1:20000, Sigma, G9545) were applied overnight in a blocking solution containing 5% milk and 1% Tween-20 in 1xTBS at 4°C on a rotator. The blot was then incubated with anti-rabbit secondary antibody at a dilution of 1:20000 for 1.5 hours at room temperature, washed three times with 1xTBS, and then the signal was imaged using an Odyssey scanner system (Li-Cor Biosciences).

[0370] Example VI

[0371] Electroencephalogram (EEG) recording

[0372] To test the antiepileptic effects of gluconate in vivo in neonates and adult rodents, P8-12 Sprague-Dawley rats or 2-month-old male C57BL / 6 mice were deeply anesthetized with sodium pentobarbital (50 mg / kg for neonates and 100 mg / kg for adult mice, intraperitoneally). Two stainless steel screws (1 mm in diameter) were inserted into the skull above the cerebral cortex as EEG recording electrodes, a ground electrode and a reference electrode located at +1.8 mm anterior margin, ±0.5 mm lateral to the midline, and 1 mm below the cortical surface. All electrodes were connected to microconnectors and fixed to the skull with dental adhesive. After surgery, neonatal rats were returned to their mothers and allowed to recover for 2 days before subsequent EEG recordings. Adult mice were housed individually to prevent damage to the implanted electrodes and allowed to recover for at least 5 days before EEG recordings.

[0373] Baseline EEG was recorded for 0.5-1 hour to acclimate the animals to the environment. Kainic acid (2 mg / kg) was administered intraperitoneally (ip) for neonatal epilepsy induction. D-gluconate sodium salt (2 g / kg) or 0.9% saline was injected ip 10 minutes after KA administration. Intraperitoneal injection of pentylenetetrazol (PTZ) was used to induce stable epileptic burst activity in adult mice, and D-gluconate sodium salt (1 g / kg and 2 g / kg) or 0.9% saline was injected ip 10 minutes before PTZ administration. Epileptic activity was monitored for 1 hour after PTZ injection. Qian et al., "Epileptiform response of CA1 neurones to convulsantstimulation by cyclothiazide, kainic acid and pentylenetetrazol in anaesthetized rats" Seizure 20:312-319 (2011). After the experiment, the animals were injected with diazepam to protect the animals from recurrent epilepsy.

[0374] Electrophysiological signals were amplified (1000×) and filtered (0–500 Hz) using the NeuroLog System (Digitimer Ltd, Hearts, UK), visualized via a DA converter, CED 1401 micro (Cambridge Electronic Design, Cambridge, UK), and stored in a PC.

[0375] EEG analysis

[0376] Power spectrum analysis was used to reveal the power levels of different frequency components in the neonatal EEG signal. The power was calculated in a 1-minute time window by integrating the power of the main frequency band from 1 to 100 Hz (EEG frequency band).

[0377] Adult EEG signals were analyzed by spike 2 software (Analysis Program of CED 1401, Cambridge, UK). EEG spikes were defined as more than twice the baseline amplitude, and EEG bursts were defined as multiple spikes with high amplitude (>1 Hz) and lasting for more than 10 s. The latency of a spike or burst was defined as the time from PTZ injection to the first spike or first burst.

[0378] Example VII

[0379] Seizure Behavioral Testing

[0380] Male mice were divided into three groups: vehicle control group (saline, n=9), 1 g / kg sodium gluconate group (n=10), and D-gluconate sodium 2 g / kg group (n=11). PTZ (50 mg / kg) was administered intraperitoneally 1 hour after drug treatment. After PTZ injection, behavior was monitored and observed for 1 hour. Seizure behavior was classified according to the Racine score: stage 0, no response; stage 1, facial convulsions; stage 2, nodding; stage 3, forelimb clonus, tail vertical; stage 4, standing with forelimb clonus; stage 5, standing and falling, jumping, general clonic seizures, general clonic and tonic seizures or death. Racine et al., "Modification of seizure activity by electrical stimulation 3, Mechanisms" Electroencephalogr Clin Neurophysiol 32: 295-299 (1972).

[0381] Example VIII

[0382] Data analysis

[0383] Data are shown as mean ± sem. Student t test (paired or unpaired) was performed for comparison of two groups, and the χ2 test was used to compare the percentage differences between the two groups. For comparison of multiple groups, post hoc one-way ANOVA was used. Statistical significance was set at P < 0.05.

[0384] Example IX

[0385] Decreased epileptiform activity produced by glucose oxidase

[0386] This example presents preliminary data indicating that glucose oxidase can effectively inhibit epileptiform activity.

[0387] Hippocampal slices were prepared from newborn mice. Seizure-like activity was induced by Mg-free 2+ aCSF (artificial cerebrospinal fluid) or high K + Glucose oxidase (GGx) was either applied to the slices acutely or pre-incubated in aCSF for 90 min before application to the slices.

[0388] The data showed that glucose oxidase dose-dependently inhibited the epileptiform activity induced by 0Mg2+ aCSF. Figure 18A. Glucose oxidase was also found to inhibit the epileptiform activity induced by high K+. Figure 18B.

[0389] Different glucose concentrations from 4 mM to 20 mM were also tested. When 1 unit of glucose oxidase / ml was provided, suppression of epileptiform activity was observed. Figure 19A. In contrast, acute application of 0.1 unit of glucose oxidase / ml had no significant effect. Figure 18A, top row. However, after ninety (90) minutes of pre-incubation, 0.1 U / ml of glucose oxidase also suppressed epileptiform activity. Figure 19B.

[0390] These results suggest that glucose oxidase may be used as an anti-epileptic drug.

Claims

1. Use of glucose oxidase for preparing a medicament for treating epilepsy in neonatal patients.

2. The use according to claim 1, wherein the medicament is configured to convert glucose to gluconate in the neonatal patient.

3. The use according to claim 2, wherein the gluconate inhibits CLC-3 channel-mediated Cl in the neonatal patient. - Electric current.

4. The use according to claim 1, wherein the glucose oxidase is in a pharmaceutically acceptable composition.

5. The use according to claim 4, wherein the pharmaceutically acceptable composition is formulated to be administered to the neonatal patient under conditions such that the epilepsy is reduced. The use according to claim 5 , wherein the pharmaceutically acceptable composition comprises an effective amount of the glucose oxidase.

7. The use according to claim 4, wherein the pharmaceutically acceptable composition further comprises supplemented glucose.

8. The use according to claim 1, wherein the medicament is administered to the neonatal patient in combination with supplemental glucose.

9. The use according to claim 8, wherein the supplemented glucose is administered by intravenous injection at a fixed dose with a glucose infusion rate of 7.3 mg / kg / min.

10. The use according to any one of claims 1-9, wherein the neonatal patient has been diagnosed with neonatal epilepsy.

11. The use according to any one of claims 4 to 7, wherein the pharmaceutically acceptable composition further comprises a pharmaceutically acceptable carrier.

12. The use according to any one of claims 4 to 7, wherein the pharmaceutically acceptable composition comprises a fixed dose of the glucose oxidase and an inactive carrier.

13. The use according to claim 12, wherein the inactive carrier is saline.

14. The use according to any one of claims 1 to 9, wherein the medicament is administered via a transdermal patch.

15. The use according to any one of claims 1-9, wherein the neonatal patient is less than 1 month old.

Images