Scorpion toxin mutant ChTX-Q18W and application of scorpion toxin mutant ChTX-Q18W in preparation of anti-epileptic drugs

Abstract

The invention relates to a scorpion toxin mutant ChTX-Q18W and an application of the scorpion toxin mutant ChTX-Q18W in preparation of an anti-epileptic medicine. Specifically, the invention provides a scorpion toxin mutant ChTX-Q18W and an active fragment thereof, or a pharmaceutically acceptable salt thereof, and the scorpion toxin mutant ChTX-Q18W and the active fragment thereof are used for preparing a preparation or a composition for treating and / or preventing epilepsy. The scorpion toxin mutant comprises an amino acid sequence as shown in SEQ ID NO: 2; or the scorpion toxin mutant has at least 50% identity with the amino acid sequence as shown in SEQ ID NO: 2 on the basis of keeping the 18th amino acid residue as W. It is proved for the first time that ChTX-Q18W can effectively prevent and / or treat epilepsy symptoms by measuring the influence of ChTX-Q18W on recurrent convulsion behaviors of a mouse PTZ convulsion model, and the ChTX-Q18W has a good clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the field of polypeptide drugs, and in particular to a scorpion toxin mutant ChTX-Q18W and an application thereof in the preparation of anti-epileptic drugs. Background Art

[0002] Epilepsy is a chronic disease that causes transient brain dysfunction, characterized by repeated seizures caused by sudden abnormal discharges of brain neurons. It is estimated that there are about 400,000 new epilepsy patients each year. In China, epilepsy has become the second most common disease in neurology after headache.

[0003] The pathogenesis of epilepsy is very complex, but it is generally believed that the imbalance between excitation and inhibition in the central nervous system will lead to epileptic seizures. Based on the different starting sites and transmission methods of abnormal discharges, the causes of epilepsy are divided into various factors such as abnormal ion channel function, abnormal neurotransmitters, and abnormal glial cells. Ion channels are one of the foundations of regulating tissue excitability in the body. Studies have shown that channels such as sodium ion channels, potassium ion channels, and calcium ion channels have a certain correlation with epilepsy.

[0004] In response to the diverse pathogenesis of epilepsy, researchers have developed various anti-epileptic drugs with different mechanisms of action and different targets. Small molecule drugs such as phenytoin sodium and carbamazepine can selectively act on voltage-dependent sodium ion channels, blocking the rapid release of sodium ion-dependent action potentials to achieve anticonvulsant effects. Trimethadione is a selective T-type calcium channel blocker that inhibits excessive neuronal excitation. Perampanel is an AMPA-type glutamate receptor (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, mediating rapid excitatory synaptic transmission in the central nervous system) antagonist that inhibits postsynaptic AMPA receptor glutamate activity and reduces excessive neuronal excitation, thereby preventing and treating epilepsy.

[0005] However, the effects of current epilepsy treatment drugs are far from satisfactory. For example, most small molecule drugs have serious side effects when taken for a long time. In addition, the causes of epilepsy are diverse, so it is difficult for current small molecule drugs to prescribe the right medicine for the right disease.

[0006] Therefore, there is an urgent need in the art to develop new drugs with high specificity and / or small side effects for preventing and / or treating epilepsy. Summary of the invention

[0007] In order to solve the technical problem of lack of drugs with high specificity and / or small side effects for preventing and / or treating epilepsy in the art, the present invention provides a scorpion toxin mutant ChTX-Q18W and its use in the preparation of anti-epileptic drugs.

[0008] A first aspect of the present invention provides a scorpion toxin mutant or an active fragment thereof, wherein the scorpion toxin mutant comprises the amino acid sequence shown in SEQ ID NO:2; or the scorpion toxin mutant has at least 50% identity with the amino acid sequence shown in SEQ ID NO:2 on the basis of keeping the amino acid residue at position 18 as W.

[0009] In some preferred embodiments, the scorpion toxin mutant has 1-7 amino acid differences from the amino acid sequence shown in SEQ ID NO: 2;

[0010] Preferably, the amino acid sequence of the scorpion toxin mutant is as shown in SEQ ID NO: 2 or 3.

[0011] The second aspect of the present invention provides a scorpion toxin mutant combination, which includes the scorpion toxin mutant or an active fragment thereof as described in the first aspect;

[0012] Preferably, it further comprises one or more scorpion toxin mutants selected from the group consisting of Q18F, Q18Y, Q18H, Q18V and Q18L.

[0013] The third aspect of the present invention provides a pharmaceutical composition or a pharmaceutically acceptable salt thereof, which comprises the scorpion toxin mutant or an active fragment thereof as described in the first aspect, or the scorpion toxin mutant combination as described in the second aspect.

[0014] Preferably, it further comprises a pharmaceutically acceptable carrier or excipient or buffer.

[0015] The fourth aspect of the present invention provides a use of a scorpion toxin mutant or an active fragment thereof as described in the first aspect, or a scorpion toxin mutant combination as described in the second aspect, or a pharmaceutical composition or a pharmaceutically acceptable salt thereof as described in the third aspect in the preparation of a drug for treating and / or preventing epilepsy.

[0016] Preferably, the epilepsy includes secondary epilepsy, primary epilepsy and cryptogenic epilepsy.

[0017] The fifth aspect of the present invention provides a polynucleotide, wherein the polynucleotide encodes the scorpion toxin mutant or an active fragment thereof as described in the first aspect.

[0018] Preferably, the nucleotide sequence of the polynucleotide is as shown in SEQ ID NO:4.

[0019] The sixth aspect of the present invention provides a recombinant expression vector comprising the polynucleotide as described in the fifth aspect.

[0020] In some preferred embodiments, the expression vector includes a eukaryotic expression vector, a prokaryotic expression vector or a viral vector.

[0021] The seventh aspect of the present invention provides a host cell comprising the recombinant expression vector described in the sixth aspect, or the polynucleotide described in the fifth aspect.

[0022] In some preferred embodiments, the starting cell of the host cell is a prokaryotic cell or a eukaryotic cell, and the prokaryotic cell is preferably Escherichia coli such as E. coli BL21 (DE3).

[0023] The eighth aspect of the present invention provides a pharmaceutical composition comprising:

[0024] (i) a first pharmaceutical composition, which is the scorpion toxin mutant or an active fragment thereof as described in the first aspect, or the scorpion toxin mutant combination as described in the second aspect, or the pharmaceutical composition as described in the third aspect or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier;

[0025] (ii) a second pharmaceutical composition, wherein the second pharmaceutical composition contains a second active ingredient other than the first pharmaceutical composition, and a pharmaceutically acceptable carrier; the second active ingredient is selected from carbamazepine, fluazifop, gabapentin, lamotrigine, oxcarbazepine, phenytoin, phenytoin sodium, retigabine, topiramate, cefotaxime, ethosuximide, sodium valproate, or a combination thereof.

[0026] A ninth aspect of the present invention provides a medicine kit, wherein the medicine kit comprises:

[0027] (1) a first container, and a first pharmaceutical composition in the first container, wherein the first pharmaceutical composition is the scorpion toxin mutant or an active fragment thereof as described in the first aspect, or the scorpion toxin mutant combination as described in the second aspect, or the pharmaceutical composition as described in the third aspect or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier; and

[0028] (2) a second container, and a second pharmaceutical composition in the second container, wherein the second pharmaceutical composition contains a second active ingredient other than the first pharmaceutical composition, and a pharmaceutically acceptable carrier; the second active ingredient is selected from carbamazepine, fluazifop, gabapentin, lamotrigine, oxcarbazepine, phenytoin, phenytoin sodium, retigabine, topiramate, cefotaxime, ethosuximide, sodium valproate, or a combination thereof.

[0029] In some preferred embodiments, the medicine kit further includes (3) instructions.

[0030] In some preferred embodiments, the first container and the second container are the same or different containers.

[0031] In some preferred embodiments, the first pharmaceutical composition in the first container is a single-ingredient preparation containing ChTX-Q18W or an active fragment thereof.

[0032] In some preferred embodiments, the pharmaceutical composition in the second container is a single preparation containing other or additional active pharmaceutical ingredients for treating and / or preventing epilepsy.

[0033] In some preferred embodiments, the instructions record instructions for administering the first active ingredient and the optional second active ingredient to treat and / or prevent epilepsy.

[0034] In some preferred embodiments, the instructions state that the dosage form of the first pharmaceutical composition and optionally the second pharmaceutical composition is an injection.

[0035] In some preferred embodiments, the injection is administered into the subject via intracerebroventricular (ICV).

[0036] The eighth aspect of the present invention provides a method for treating and / or preventing epilepsy, comprising administering the scorpion toxin mutant polypeptide described in the second aspect of the present invention or the pharmaceutical composition described in the sixth aspect of the present invention to a subject in need thereof.

[0037] In another preferred embodiment, the subject is a human or a non-human mammal.

[0038] In another preferred embodiment, the subject suffers from or is suspected of suffering from epilepsy.

[0039] In another preferred embodiment, the epilepsy includes epilepsy caused by increased excitability of the cerebral cortex (or abnormal neuronal excitability type epilepsy).

[0040] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0041] The reagents and raw materials used in the present invention are commercially available.

[0042] The positive and progressive effects of the present invention are:

[0043] (1) ChTX-Q18W has the characteristics of strong effect and small dosage (the dosage can be as low as 0.32 μg / kg in the mouse PTZ model). It not only significantly prolongs the latency period of convulsive seizures, but also significantly relieves the symptoms of epilepsy (especially significantly reduces the incidence of grade 3, 4 and 5 epilepsy). Therefore, it can be used as a new polypeptide drug for the treatment of epilepsy.

[0044] (2) ChTX-Q18W is one of the first peptide substances that has been experimentally confirmed to have the effect of alleviating epilepsy. It can be expressed in prokaryotes and prepared by recombinant methods, and is expected to provide a safe, effective, and inexpensive anti-epileptic drug for epilepsy patients.

[0045] (3) ChTX-Q18W is a neurotoxin with a very small molecular weight (only about 4KDa) that can effectively penetrate the blood-brain barrier and act on the brain. It is selective for the α+β4 subtype of BK potassium channels with large conductance calcium ions and voltage-activated potassium channels, but has no effect on the cardiovascular, endocrine, and reproductive systems, so it has few side effects.

[0046] (4) In addition, the present invention also found that the π-π stacking interaction between ChTX-Q18W and the amino acid H101 of the target BK channel β4 subunit is stronger than that of the wild-type scorpion toxin, and is stronger than that of other mutants such as ChTX-QF18 and H101. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 A map of the mouse brain.

[0048] Figure 2 The modulation of the number of Nissl bodies in the hippocampus of mice with PTZ epilepsy model by ChTX-Q18 mutant peptide. a is the expression of Nissl bodies in the hippocampus of mice in different groups on the ipsilateral side of the drug administration; b is the statistical diagram of the number of Nissl bodies in different subregions of the hippocampus on the ipsilateral side of the drug administration; c is the expression of Nissl bodies in the hippocampus of mice in different groups on the contralateral side of the drug administration; d is the statistical diagram of the number of Nissl bodies in different subregions of the hippocampus on the contralateral side of the drug administration (n=3, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001: drug administration group vs. blank group; #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001: fusion peptide group, VPA group vs. model group).

[0049] Figure 3 The inhibitory effect of ChTX-Q18 mutant on BK(α+β4) channel. ae: Whole cell current of HEK293T expressing BK(α+β4) channel, the clamping potential is -80mV, the current is induced by +80mV voltage stimulation and 400nM internal calcium; f: I f It represents the percentage of residual current under the action of 10 μM ChTX-Q18 mutant, n=5, *P<0.05, **P<0.01: after administration of ChTX-Q18 mutant compared with before administration. DETAILED DESCRIPTION

[0050] After extensive and in-depth research, through the screening of a large number of different compounds (including a large number of screenings for compounds such as sodium ion channels), the inventor unexpectedly discovered for the first time that a scorpion toxin mutant (i.e., ChTX-Q18W) can be used to extremely effectively treat and / or prevent epilepsy. Experiments have shown that in the mouse PTZ (pentetrazol) convulsion model, by measuring the effect of ChTX-Q18W on recurrent convulsion behavior, it was discovered for the first time that the scorpion toxin mutant ChTX-Q18W can effectively alleviate epilepsy symptoms. On this basis, the present invention was completed.

[0051] The patent of this invention uses molecular cloning and other technologies to prepare the toxin peptide ChTX and its mutant ChTX-Q18W. As control samples, physiological saline, ChTX and mutants ChTX-Q18H, Q18V, Q18Y, and Q18L are used to examine their effects on the recurrent convulsive behavior of mice PTZ, whether they cause damage to hippocampal neurons after convulsive attacks in mice, and current blocking electrophysiological experiments targeting BK (a+b4) channels. It is proved that ChTX-Q18W toxin has little damage to hippocampal neurons, can inhibit convulsive attacks in mice, and has anti-epileptic efficacy. The following is an explanation of the terms.

[0052] Scorpion toxin (charybdotoxin) mutant

[0053] As used herein, the terms "charybdotoxin Q18W protein", "Charybdotoxin polypeptide mutant Q18W", "ChTX toxin mutant Q18W" and "ChTX polypeptide mutant Q18W", "recombinant ChTX toxin Q18W", "scorpion toxin mutant Q18W", "ChTX-Q18W protein", "polypeptide ChTX-Q18W", "toxin peptide ChTX-Q18W" and the like are used interchangeably, all referring to scorpion toxin mutant Q18W. Scorpion toxin mutant mutant Q18W is composed of 37 amino acids, has three pairs of disulfide bonds, and is a short-chain polypeptide toxin. In the present invention, the term includes not only chemically synthesized charybdotoxin protein mutant Q18W (ChTX-Q18W), but also recombinant ChTX-Q18W, such as recombinantly expressed ChTX-Q18W with or without an initiating Met, and recombinantly expressed ChTX-Q18W with or without an expression tag or 1-3 amino acids of enzyme cleavage residues.

[0054] Natural wild-type charybdotoxin can be isolated and purified from the venom of the North African scorpion (Leiurus quinquestratus), and is the first blocker of the BK channel, and does not have anti-epileptic activity targeting the BK (α+β4) channel. The toxin peptide ChTX-Q18W of the present invention is difficult to obtain by natural separation, but can be obtained by chemical synthesis (including solid phase cooperation) or recombinant technology.

[0055] For recombination, the gene can be expressed in host cells such as Escherichia coli through conventional recombination techniques and obtained through separation and purification.

[0056] The amino acid sequence of wild-type charybdotoxin is shown in SEQ ID NO: 1:

[0057] EFTNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS

[0058] The amino acid sequence of the toxin peptide ChTX-Q18W is shown in SEQ ID NO: 2:

[0059] EFTNVSCTTSKECWSVCWRLHNTSRGKCMNKKCRCYS

[0060] A recombinant ChTX-Q18W amino acid sequence is shown in SEQ ID NO: 3:

[0061] GS EFTNVSCTTSKECWSVCWRL HNTSRGKCMNKKCRCYS

[0062] The nucleic acid sequence encoding the amino acid sequence of ChTX-Q18W protein SEQ ID NO:2 is shown in SEQ ID NO:4.

[0063] GAGTTCACCAACGTTAGCTGCACCACGAGTAAAGAATGCTGGAGCGTGTGCTGGCGTTTGCATAATACTTCTCGCGGTAAGTGCATGAACAAGAAGTGCCGCTGCTATAGC

[0064] The present invention experimentally confirms for the first time that in a mouse PTZ convulsion model, the scorpion toxin mutant ChTX-Q18W can extremely significantly preventively and therapeutically alleviate abnormal neuronal excitation and epilepsy symptoms, and therefore serves as a new polypeptide drug for treating epilepsy symptoms.

[0065] It should be understood that although the wild type of the polypeptide ChTX-Q18W provided in the examples of the present invention is derived from the North African scorpion, ChTX-Q18W polypeptide mutants derived from other similar species (especially scorpions belonging to the same family or genus as the North African scorpion) and having a certain identity (or conservation) with the sequence of the present invention (preferably, the sequence is shown in SEQ ID NO: 2)) can also be used in the present invention.

[0066] It should be understood that although the gene provided in the examples of the present invention is derived from the North African scorpion, the nucleotide sequence of the gene encoding ChTX-Q18W derived from other similar species (especially scorpions belonging to the same family or genus as the North African scorpion) and having a certain identity (or conservation) with the sequence of the present invention (for example, as shown in SEQ ID NO: 4) is also included in the scope of the present invention, as long as a person skilled in the art can easily isolate the sequence from other species (especially scorpions) based on the information provided in the present application after reading the present application.

[0067] The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA forms include: DNA, genomic DNA or artificially synthesized DNA, and the DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The coding region nucleotide sequence encoding the mature polypeptide can be the same as the coding region sequence shown in SEQ ID NO:4 or a degenerate variant thereof.

[0068] The polynucleotide encoding a mature polypeptide includes: a coding sequence encoding only a mature polypeptide; a coding sequence for a mature polypeptide and various additional coding sequences; a coding sequence for a mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0069] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may include a polynucleotide further including additional coding and / or non-coding sequences. The present invention also relates to variants of the above-mentioned polynucleotides, which encode fragments, analogs and derivatives of polyglycosides or polypeptides having the same amino acid sequence as the present invention. The variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the polypeptide encoded by it.

[0070] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) addition of denaturing agents during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Wicoll, 42°C, etc.; or (3) hybridization occurs only when the identity of one sequence to the complementary sequence of another sequence is at least 90%, preferably 95%.

[0071] It should be understood that, although the wild type of the ChTX-Q18W gene of the present invention is preferably derived from a North African scorpion and then subjected to a single point mutation, other genes from other species (especially scorpions) that are highly homologous to the North African scorpion ChTX gene (e.g., having more than 80%, such as 85%, 90%, 95% or even 98% sequence identity) are also within the scope of the present invention. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0072] The full-length ChTX-Q18W nucleotide sequence or fragments thereof of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and a commercially available DNA library or a cDNA library prepared by conventional methods known to those skilled in the art is used as a template to amplify the relevant sequence.

[0073] In addition, artificial synthesis methods can also be used to synthesize relevant sequences, especially when the fragment length is shorter. Usually, by synthesizing multiple small fragments first and then connecting them, very long fragments of the sequence can be obtained. At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0074] The present invention relates to a polypeptide ChTX-Q18W for treating epilepsy, and the amino acid sequence of the polypeptide is shown in SEQ ID NO: 2. The polypeptide of the present invention can effectively treat and / or prevent epilepsy.

[0075] The present invention also includes polypeptides or proteins with the same or similar functions that have 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more, such as 99%) identity with the sequence shown in SEQ ID NO: 2 of the present invention.

[0076] The “same or similar functions” mainly refer to: “relieving the symptoms of epilepsy”.

[0077] The polypeptide of the present invention can be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide. The polypeptide of the present invention can be a naturally purified product, or a chemically synthesized product, or produced using recombinant technology from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, plants, insects, and mammalian cells). Depending on the host used in the recombinant production scheme, the polypeptide of the present invention can be glycosylated, or can be non-glycosylated. The polypeptide of the present invention may also include or not include an initial methionine residue.

[0078] The present invention also includes ChTX-Q18W polypeptide fragments and analogs having ChTX-Q18W polypeptide activity. As used herein, the terms "fragment" and "analog" refer to polypeptides that substantially retain the same biological function or activity of the ChTX-Q18W polypeptide of the present invention.

[0079] The polypeptide fragments, derivatives or analogs of the present invention may be: (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having a substitution group in one or more amino acid residues; or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein). According to the definition herein, these fragments, derivatives and analogs belong to the scope known to those skilled in the art.

[0080] In the present invention, the polypeptide variant is an amino acid sequence as shown in SEQ ID NO: 2, on the basis of maintaining the amino acid residue at position 18 as W, after several (usually 1-10, preferably 1-7, more preferably 1-4, and most preferably 1-2) substitutions, deletions or additions of at least one amino acid residue, and one or several (usually within 10, preferably within 5, and more preferably within 3) amino acid residues are added to the C-terminus and / or N-terminus (for example, the amino acid sequence shown in SEQ ID NO: 3). For example, in the protein, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed, and adding one or several (such as 1-3) amino acid residues to the C-terminus and / or N-terminus usually does not change the function of the protein. These conservative variations are preferably generated by substitution according to Table 1.

[0081] Table 1

[0082]

[0083]

[0084] The present invention also includes analogs of the claimed proteins. The difference between these analogs and SEQ ID NO: 2 may be a difference in the amino acid sequence, or a difference in the form of modification that does not affect the sequence, or both. Analogs of these proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, or by site-directed mutagenesis or other known molecular biological techniques. Analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (such as β, γ-amino acids). It should be understood that the proteins of the present invention are not limited to the representative proteins listed above.

[0085] Modifications (usually without altering the primary structure) include: chemical derivatization of proteins in vivo or in vitro such as acetylating or carboxylating. Modifications also include glycosylation, such as those carried out during protein synthesis and processing. Such modifications can be accomplished by exposing the protein to glycosylation enzymes (such as mammalian glycosylases or deglycosylases). Modifications also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine).

[0086] Pharmaceutical composition and method of administration thereof

[0087] The present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of the following active ingredients: a scorpion toxin mutant ChTX-Q18W or an active fragment thereof and a pharmaceutical active ingredient thereof for treating and / or preventing epilepsy.

[0088] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals.

[0089] As used herein, "pharmaceutically acceptable" ingredients are suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), i.e., substances with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

[0090] The pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Usually, the pharmaceutical preparation should match the mode of administration, and the dosage form of the pharmaceutical composition of the present invention is an injection. For example, it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions.

[0091] The effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. For example, several divided doses may be administered daily, or the dose may be reduced proportionally, depending on the urgency of the treatment situation.

[0092] The pharmaceutically acceptable carrier of the present invention includes, but is not limited to, water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should match the mode of administration, which is well known to those of ordinary skill in the art.

[0093] The first active ingredient (a) ChTX-Q18W or its active fragment provided by the present invention can be used in combination with the second active ingredient (b) other drugs for treating and / or preventing epilepsy. The second active ingredient (b) is an anti-epileptic drug that is available in the prior art, including but not limited to: carbamazepine, flupirtine, gabapentin, lamotrigine, oxcarbazepine, phenytoin, phenytoin sodium, retigabine, topiramate, cephalosporin, ethosuximide, sodium valproate, or a combination thereof.

[0094] PTZ (pentylenetetrazole)

[0095] Pentylenetetrazol (PTZ), also known as pentylenetetrazol, pentamethylenetetrazolyl, and cardiazolyl, is a white crystalline powder chemical. Its chemical name is 1,5-pentamethylene-1H-tetrazole, its molecular formula is C6H10N4, and its molecular weight is 138.1704. Pentylenetetrazol is a central nervous system stimulant, and is mainly used clinically to rescue central respiratory failure caused by severe barbiturate and anesthetic poisoning; it can also be used for respiratory depression and acute circulatory failure caused by acute infectious diseases, anesthetics, and barbiturate poisoning.

[0096] Pentylenetetrazol can excite the respiratory center and cardiovascular motor center. Its effect is rapid and strong, deepening and accelerating breathing and slightly increasing blood pressure. If the dose is slightly larger, the excitement can extend to the cerebral cortex and spinal cord, causing convulsions.

[0097] PTZ mainly acts on the chloride ion channel of GABAA receptors (γ-aminobutyric acid type A receptors), inhibiting the activity of GABA neurons, thereby over-enhancing the excitability of the nervous system and inducing clonic or generalized tonic epileptic seizures in animals and humans. The PTZ-induced rodent seizure model has been widely used in the study of epilepsy mechanisms and new anti-epileptic drugs.

[0098] In the present invention, PTZ is injected intraperitoneally into animals to induce convulsions / epilepsy, so as to conduct subsequent experimental studies on the effect of the ChTX-Q18W toxin peptide of the present invention on epilepsy.

[0099] The purpose of the present invention is to provide a drug with high specificity and / or small side effects for preventing and / or treating epilepsy and its application.

[0100] The present invention provides a new scorpion toxin (charybdotoxin) mutant ChTX-Q18W, which is different from the wild-type scorpion toxin in that position 18 is replaced by W from Q. It can be a mutant ChTX-Q18 W formed by mutating the glutamic acid at position 18 of the wild-type scorpion toxin to tryptophan; it can also be a mutant ChTX-Q18 W formed by amino acid substitution of other scorpion toxin mutants.

[0101] In another preferred embodiment, the ChTX-Q18W comprises a recombinant or artificially synthesized ChTX-Q18 W polypeptide.

[0102] In another preferred embodiment, the ChTX-Q18W comprises the amino acid sequence shown in SEQ ID NO:2.

[0103] In another preferred embodiment, the ChTX-Q18W includes an amino acid sequence obtained by replacing, deleting, changing, inserting or adding one or more amino acids based on the sequence SEQ ID NO: 2 while maintaining the activity of the protein.

[0104] In another preferred embodiment, the ChTX-Q18W includes inserting one or more amino acids at the N-terminus or C-terminus of the sequence SEQ ID NO: 1 while maintaining protein activity, and the number of inserted amino acid residues includes 1 to 10, preferably 1 to 7, more preferably 1 to 3, for example 1, 2 or 3.

[0105] In another preferred embodiment, the ChTX-Q18W comprises retaining the active site amino acid Trp18 on the basis of the sequence SEQ ID NO: 2 and further changing the resulting amino acid sequence.

[0106] In another preferred embodiment, the ChTX-Q18W includes a recombinant protein with one or more protein tags at the N-terminus or C-terminus of the sequence SEQ ID NO: 2 within the range of maintaining protein activity.

[0107] In another preferred embodiment, the protein tag is selected from the following group: MBP tag, His tag, GST tag, SUMO tag, TRX tag, HA tag, Wlag tag, NusA tag, or a combination thereof.

[0108] In another preferred embodiment, the ChTX-Q18W comprises the amino acid sequence shown in SEQ ID NO:2.

[0109] In another preferred embodiment, the ChTX-Q18W polypeptide includes one or more amino acids inserted at the N-terminus or C-terminus of the sequence SEQ ID NO: 2 within the range of maintaining protein activity, and the number of the inserted amino acid residues includes 1 to 10, preferably 1 to 5, and more preferably 1 to 3.

[0110] In another preferred embodiment, the ChTX-Q18W is recombinant.

[0111] In another preferred embodiment, the ChTX-Q18W is recombinantly expressed in Escherichia coli.

[0112] In another preferred embodiment, the amino acid sequence of the recombinant ChTX-Q18W is shown in SEQ ID NO:3.

[0113] In another preferred embodiment, the preparation or pharmaceutical composition comprises: component (a) scorpion toxin mutant ChTX-Q18W and its active fragment, or a pharmaceutically acceptable salt thereof, and component (b) a pharmaceutically acceptable carrier,

[0114] In another preferred embodiment, the component (a) accounts for 0.1-99.9wt%, preferably 10-99.9wt%, and more preferably 70%-99.9wt% of the total weight of the preparation or pharmaceutical composition.

[0115] In another preferred embodiment, the pharmaceutically acceptable carrier is selected from the following group: an infusion carrier and / or an injection carrier. Preferably, the carrier is one or more carriers selected from the following group: physiological saline, glucose saline, or a combination thereof.

[0116] In another preferred embodiment, the pharmaceutical composition is liquid, solid, or semisolid.

[0117] In another preferred embodiment, the pharmaceutical composition is in the form of an injection or an external-use pharmaceutical dosage form.

[0118] In another preferred embodiment, the dosage form of the pharmaceutical composition includes an injection or a lyophilized preparation.

[0119] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0120] In another preferred embodiment, the pharmaceutical composition is administered via intravenous, subcutaneous, intramuscular or intracranial routes.

[0121] In another preferred embodiment, the pharmaceutical composition is administered via microinfusion pumps.

[0122] In another preferred embodiment, the pharmaceutical composition is administered via intracranial administration, preferably via intracerebroventricular (ICV) delivery into the subject.

[0123] In another preferred embodiment, the subject includes: a mammal.

[0124] In another preferred embodiment, the mammal includes a human or a non-human mammal.

[0125] In another preferred embodiment, the non-human mammals include: rodents (such as mice, rats), primates (such as monkeys).

[0126] In another preferred embodiment, the preparation or pharmaceutical composition can be administered alone or in combination.

[0127] In another preferred embodiment, the combined administration includes: combined administration with other therapeutic drugs for treating and / or preventing epilepsy.

[0128] In another preferred embodiment, the other therapeutic drugs for treating and / or preventing epilepsy are selected from the following group: carbamazepine, fluopyridine, gabapentin, lamotrigine, oxcarbazepine, phenytoin, phenytoin sodium, retigabine, topiramate, cephalosporin, ethosuximide, sodium valproate, or a combination thereof.

[0129] In another preferred embodiment, the epilepsy includes epilepsy caused by increased excitability of the cerebral cortex (or abnormal neuronal excitability type epilepsy).

[0130] In another preferred embodiment, the epilepsy has the following characteristics: the activity of large-conductance calcium ion and voltage-activated potassium ion channels is enhanced, thereby leading to increased excitability of the cerebral cortex.

[0131] In another preferred embodiment, the epilepsy includes epilepsy in humans and non-human mammals (such as rodents).

[0132] In another preferred embodiment, the epilepsy includes PTZ-induced epilepsy, especially PTZ-induced epilepsy in mice.

[0133] In another preferred embodiment, the epilepsy includes refractory epilepsy.

[0134] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0135] Experimental Materials and Methods

[0136] Experimental animals and drugs

[0137] Experimental animals: Adult male C57BL / 6J mice (Spef Biotech Co., Ltd.), weighing 20-22 g, 5 mice per cage, were raised under routine laboratory conditions, with room temperature maintained at 22 ± 1 °C and a natural circadian rhythm.

[0138] Related drugs: The expression and purification of the recombinant North African scorpion toxin ChTX-Q18W is shown in Preparation Example 1. Pentylenetetrazol (PTZ, CAS: 54-95-5) was purchased from Sigma, USA.

[0139] Animal surgery

[0140] After the mice were anesthetized with ether, they were fixed in a prone position on a stereotaxic instrument. The hair on the top of the head was removed, the skin was disinfected with alcohol, and the scalp was cut to fully expose the midline of the mouse skull and the anterior fontanel (Bregma, the intersection of the sagittal and coronal planes), the posterior fontanel (Lambda, the intersection of the sagittal and lambdoid sutures) and the target position. The vertical distance between the anterior fontanel and the posterior fontanel was measured by the stereotaxic instrument to ensure that the vertical distance between the two was less than 0.5 mm. According to the mouse brain positioning map, the drug administration site was determined on the skull 0.3 mm behind the anterior fontanel; 1.0 mm to the side; 2.0 mm deep under the dura mater. With the positioning point as the center, a micro drill was used to drill a skull bone window of about 1×1 mm above the skull. After the dura mater was gently opened with a sterile needle, a small hole (1 mm) was drilled with a dental drill, the inner plate was peeled off, the dura mater was opened with the needle tip, and injection was performed with a micro syringe.

[0141] Preparation Example 1

[0142] The ChTX stored in the laboratory on pET32a was synthesized by artificial synthesis, and primers containing the Gln18 site were designed for full plasmid PCR mutation to Q18W, Q18H, Q18Y, Q18L, and Q18V. The plasmids were transformed into E. coli BL21 (DE3) expression strains for prokaryotic expression.

[0143] After recombinant expression, a fusion protein was obtained, and the toxin peptide ChTX mutant peptides Q18W, Q18H, Q18Y, Q18L and Q18V were prepared by the following method. The specific purification steps are as follows:

[0144] (1) The fusion protein in Buffer A first binds to the nickel column, and then is gradient eluted with Buffer B imidazole salt solution to remove most of the impurities, completing the first affinity column purification and performing SDS-PAGE electrophoresis detection.

[0145] (2) The target fusion protein was collected and dialyzed in 2 L Buffer C at 18°C. Thrombin enzyme was added at 6 U / mL and the digestion was stirred in a 1.0 kDa dialysis bag overnight.

[0146] (3) The enzyme cleavage mixture was then loaded onto an Amylose Resin chromatography column using a constant flow pump, and the flow-through fraction (WL) and the elution fraction from Buffer D were collected. The His-MBP-tag was removed by elution from Buffer E, and the second affinity column was used for purification and SDS-PAGE electrophoresis was performed.

[0147] (4) The FL component and Buffer D washing solution were then concentrated to 2 mL for the next step of gel chromatography column purification.

[0148] (5) The gel chromatography column Superdex 30 was equilibrated with Buffer F in advance, and then the sample was loaded onto the column and eluted with Buffer F. The elution peak with a retention volume of about 110 mL was collected, which was the ChTX mutant peptide Q18W, Q18H, Q18Y, Q18L or Q18V sample, and the previous absorption peak was the MBP-tag or the uncut fusion protein. Thus, high-purity ChTX mutant peptides Q18W, Q18H, Q18Y, Q18L and Q18V were obtained.

[0149] The relevant buffer components during the purification process are shown in Table 2.

[0150] Table 2 Relevant buffers during purification of recombinant ChTX-Q18W toxin

[0151]

[0152] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0153] Unless otherwise specified, the materials and reagents used in the examples are commercially available products.

[0154] Example 1 Effect of ChTX-Q18W on PTZ-induced recurrent seizures in mice

[0155] 1.1 Experimental procedures

[0156] Adult male C57BL / 6J mice were placed in a 40×30×50cm transparent glass box to observe the convulsive reaction after drug administration. Before drug injection, mice were placed in the box 1 hour in advance and moved freely to adapt to the environment, and then PTZ (60mg / kg) was injected intraperitoneally to induce convulsions in mice. The experiment was divided into toxin injection group, saline blank control group and VPA group.

[0157] ChTX mutant peptide Q18W, Q18H, Q18Y, Q18L or Q18V injection group: After a single intraperitoneal injection of PTZ to induce seizures, the next day, the CA1 area of ​​the hippocampus ( Figure 1) were injected with ChTX-Q18W toxin (dissolved in 1 μL saline) and then PTZ was injected. The toxin dose was 0.08 μg, n=6 (n is the number of experimental mice; the weight of mice is about 20-22 g / mouse);

[0158] Normal saline blank control group: After intraperitoneal injection of PTZ once to induce epileptic seizures, an equal amount of normal saline was injected 1 day later, and then PTZ was injected again, n=6.

[0159] VPA group: After PTZ was injected intraperitoneally once to induce epileptic seizures, VPA solution was injected intraperitoneally, and then PTZ was injected again, n=6.

[0160] During the experiment, the "double-blind method" of drug administration and behavioral observation was used to reduce human errors. The latency period of convulsions within 2 hours after PTZ injection, mortality, duration of convulsions of different severity and number of seizures were measured as statistical indicators. The severity of convulsions in mice was rated according to the following standards [Neurophysiol. 1972, 32, 281-294; Brain Res. 1997, 758, 92-98]:

[0161] Level 0: No reaction;

[0162] Grade 1: Rhythmic twitching of the mouth and face;

[0163] Grade 2: body wave-like migratory spasms;

[0164] Grade 3: generalized myoclonus, buttocks tilted upward;

[0165] Level 4: The body turns to one side;

[0166] Grade 5: Supine position, whole body tonic spasm.

[0167] A complete seizure was defined as the period from the onset of seizure to recovery after seizure. When the interval between seizures was more than 5 seconds, it was defined as another independent seizure. The latent period was defined as the time from the injection of PTZ to the onset of the first grade 2 seizure.

[0168] 1.2 Experimental Results

[0169] Compared with the saline blank control group, the regulatory effects of toxin ChTX mutant peptides Q18W, Q18H, Q18Y, Q18L or Q18V on PTZ-induced recurrent convulsive behavior in mice were investigated. The experimental results are shown in Tables 3, 4 and 5.

[0170] Table 3 The inhibitory effect of ChTX-Q18W on PTZ-induced recurrent convulsive behavior within 2 hours

[0171]

[0172] (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001: ChTX Q18W, VPA and model groups.)

[0173] Table 4 Effects of ChTX Q18Y and ChTX Q18H on PTZ-recurrent convulsive behavior

[0174]

[0175] (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001: ChTX Q18Y, ChTX Q18H, VPA and model groups.)

[0176] Table 5 Effects of ChTX Q18 L and ChTX Q18 V on PTZ-recurrent convulsive behavior

[0177]

[0178] (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001: ChTX Q18L, ChTX Q18V, VPA and model groups.)

[0179] From Table 3, we can see that:

[0180] Compared with the saline group, the ChTX Q18W treatment group can significantly shorten the duration of convulsion recurrence (P<0.05), with a decrease of 51% (100%-49%=51%). In terms of the number of epileptic seizures, compared with the saline group, ChTX Q18W can significantly reduce the number of grade 3 convulsions by 50% (100%-50%=50%), and at the same time, it can significantly reduce the duration of grade 4 and 5 major convulsions by 38% (100%-62%=38%). ChTX Q18W can reduce the total duration of seizures and the duration of major epileptic seizures, and can reduce the number of grade 3 epileptic seizures. It can be concluded that ChTX Q18W can effectively improve the epileptic seizures of PTZ-induced mice.

[0181] Therefore, the ChTX-Q18W of the present invention exhibits a significant inhibitory effect on the latency, duration and number of seizures at different severities of seizures, indicating that ChTX-Q18W effectively alleviates abnormal neuronal excitation and epileptic symptoms.

[0182] Example 2 Investigation of hippocampal neuron damage after seizures in mice

[0183] 2.1 Experimental procedures

[0184] After the PTZ-induced status epilepticus behavior experiment, the animals were anesthetized by intraperitoneal injection of sodium pentobarbital (60 mg / kg). The blood vessels were cleaned by perfusion of 50 mL of normal saline through the left ventricle and ascending aorta, and then perfused with 40 mL of fixative (0.1 M PBS containing 40% paraformaldehyde, pH 7.4, 4°C). The brain tissue was removed and placed in the same fixative overnight, then moved to a 20% sucrose solution and immersed until it sank to the bottom of the container, and then immersed in a 30% sucrose solution until it sank to the bottom of the container.

[0185] The mouse brain tissue was sliced ​​into hippocampus with a cryostat slicer (Leica 1900, Germany) with a thickness of 25 μm, attached to a glass slide, and stored at -20°C for later use.

[0186] (1) Take the slices out of the -20°C freezer, return to the temperature for 30 min, draw a frame around the slices with a tissue pen, and let them dry;

[0187] (2) Soak the slices in distilled water for 2 min;

[0188] (3) Nissl staining solution (purchased from Biyuntian) was dropped onto the brain slices and stained in a 37°C water bath for 10 min;

[0189] (4) Wash twice with distilled water, 10 seconds each time;

[0190] (5) The sections were dehydrated with 70%, 80%, 95%, and 100% alcohol for 2 min each time, and then transparentized with xylene twice for 5 min each time;

[0191] (6) Seal the slides with neutral gum and observe Nisslera body staining under a microscope.

[0192] From the above two groups of animals (each group had 6 animals) in the behavioral study, 6 to 8 slices were randomly taken to count the neuronal cells in different hippocampal regions (CA1, CA3 and DG regions), and the average value was finally taken, and the rate of increase of the number of neurons was calculated.

[0193] 2.2 Experimental Results

[0194] The damage or death of hippocampal neurons in mice after PTZ-induced epilepsy was determined by Nissl staining. The experimental results are shown in Figure 2 ad.

[0195] The analysis results are as follows:

[0196] (1) The ChTX-Q18W group retained a relatively complete hippocampal structure, with the highest density of hippocampal neurons, especially in the DG region, and the most densely arranged cells, and the darkest Nissler body staining. In contrast, the arrangement of neurons in the saline group became loose, the cell density decreased, and the staining was the lightest, indicating that PTZ induced epilepsy and caused the greatest damage to the neurons in the saline control group, while the ChTX-Q18W toxin of the present invention protected the hippocampal neurons after epilepsy with minimal damage;

[0197] (2) The increase rates of the CA1, CA3 and DG neurons in the ipsilateral hippocampus of the ChTX-Q18W group and the saline group were 47.6%, 63.2% and 59.8%, respectively; the increase rates of the CA1, CA3 and DG neurons in the contralateral hippocampus were 38.7%, 43.6% and 50.2%, respectively. Figure 2 ad, indicating that the protective effect of ChTX-Q18W on the ipsilateral hippocampal neurons is stronger than that on the contralateral hippocampus.

[0198] Example 3 Current Inhibitory Effect of ChTX Q18 Mutant on BK (α+β4) Channels

[0199] 3.1 Experimental procedures

[0200] (1) Human embryonic kidney cell line (HEK293T) was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences and cultured in a cell culture incubator at 37°C and 5% CO2 using DMEM medium containing 10% FBS.

[0201] (2) Observe under an optical microscope. When the cells in the culture dish grow to about 80% confluence, subculture. Take a clean 60mm culture dish, add 3ml DMEM culture medium containing 10% FBS, aspirate 200μl of cell suspension and add it to the culture dish. After shaking and mixing, place it in a 37℃, 5% CO2 cell culture incubator for culture. The next subculture will be performed in about 2-3 days. The day before transfection, plate cells on a 24-well plate (using culture medium without antibiotics), and transfect when the cell density is about 90-95%. Replace the cell culture medium in advance and place it in the incubator for standby use. Prepare two clean 1.5ml EP tubes to prepare plasmid DNA-liposome complexes. Take one EP tube and add 25μL DMEM, 4μg plasmid, 1μL P3000, and 0.4μL GFP to dilute the plasmid; take another EP tube and add 25μL DMEM and 1μL Lipofectamin 3000. After mixing, add Lipofectamin 3000 dilution to plasmid DNA dilution, mix gently, let stand at room temperature for 5-20 minutes, then gently drop DNA-liposome complex into cell culture medium, shake gently and place in 37℃, 5% CO2 cell culture incubator for culture. Perform whole-cell patch clamp experiment 24-48 hours after subculture

[0202] (3) Whole-cell recording was performed within 24-48 hours after cell transfection and passage. Whole-cell voltage clamp experiments were performed using an Axon Multiclamp700B microelectrode amplifier. The resistance was kept between 2 and 4 MΩ using a PC-10 glass electrode puller. The amplifier was preheated for half an hour in advance, and the cells were attached to the coverslip in the culture dish. Data acquisition and stimulation methods were controlled by pCLAMP10.3. During the recording process, the cells were kept at a fixed voltage of -80 mV, and a fixed increasing positive current was injected for 1000 ms. The leakage current was removed during the recording using the P / 4 mode. Cells with a series resistance higher than 15% were not used. The error was compensated by the system by 60%-70%. The drug was added using a homemade gravity dosing system (ALA Scientific Instruments). The dosing tube had a diameter of about 100 μm and was about 100 μm away from the cells. The drug delivery rate was 200-300 μl / min. The cells were clamped at -80 mV using a glass microelectrode, and the BK (α+β4) channel was activated by a step stimulus with a duration of 200 ms and a step of 5 mV. The current under the same voltage stimulation was recorded, and then 10 μM ChTX Q18 mutant was applied to record the current. The difference between the two currents is the inhibitory effect of ChTX Q18 mutant on the macroscopic current of BK (α+β4) channel.

[0203] 3.2 Experimental Results

[0204] like Figure 3 As shown in af, compared with the saline group and other mutant groups, at a concentration of 10 μM, the ChTX mutant Q18W has a significant inhibitory effect on the BK (α+β4) ion channel current blocking effect, and the final current flow rate If value is 0.15±0.07; the If values ​​of other mutants Q18V, Q18Y and Q18H are 0.56±0.12, 0.20±0.14, 0.26±0.22 respectively; while the ChTX mutant Q18L unexpectedly shows an effect of promoting current flow. Therefore, ChTX-Q18W has the best (α+β4) ion channel current blocking effect.

[0205] In summary, PTZ-induced status epilepticus will lead to damage or death of rat hippocampal neurons. The results showed that the damage degree of neurons in the ChTX-Q18W injection experimental group was greatly reduced, and the number of neurons stained by Nissl was the largest and the arrangement was the most compact.

[0206] The above experiments collectively show that the ChTX-Q18W of the present invention can inhibit the convulsions of mice behaviorally and reduce the degree of damage to hippocampal neurons, indicating that the ChTX-Q18W of the present invention has an anti-epileptic effect.

[0207] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A scorpion toxin mutant or an active fragment thereof, characterized in that: The scorpion toxin mutant comprises the amino acid sequence shown in SEQ ID NO: 2; or the scorpion toxin mutant has at least 50% identity with the amino acid sequence shown in SEQ ID NO: 2 while keeping the 18th amino acid residue as W.

2. The scorpion toxin mutant or active fragment thereof according to claim 1, characterized in that: The scorpion toxin mutant has 1-7 amino acid differences from the amino acid sequence shown in SEQ ID NO: 2; Preferably, the amino acid sequence of the scorpion toxin mutant is as shown in SEQ ID NO: 2 or 3.

3. A scorpion toxin mutant combination, characterized in that: It includes the scorpion toxin mutant or active fragment thereof as described in claim 1 or 2; Preferably, it further comprises one or more scorpion toxin mutants selected from the group consisting of Q18F, Q18Y, Q18H, Q18V and Q18L.

4. A pharmaceutical composition or a pharmaceutically acceptable salt thereof, characterized in that: It comprises the scorpion toxin mutant or active fragment thereof as claimed in claim 1 or 2, or the scorpion toxin mutant combination as claimed in claim 3; Preferably, it further comprises a pharmaceutically acceptable carrier or excipient or buffer.

5. Use of the scorpion toxin mutant or active fragment thereof as claimed in claim 1 or 2, or the scorpion toxin mutant combination as claimed in claim 3, or the pharmaceutical composition as claimed in claim 4 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating and / or preventing epilepsy; Preferably, the epilepsy includes secondary epilepsy, primary epilepsy and cryptogenic epilepsy.

6. A polynucleotide, characterized in that The polynucleotide encodes the scorpion toxin mutant or active fragment thereof according to claim 1 or 2; Preferably, the nucleotide sequence of the polynucleotide is as shown in SEQ ID NO:

4.

7. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the polynucleotide according to claim 6.

8. A host cell, characterized in that The host cell comprises the recombinant expression vector of claim 7, or the polynucleotide of claim 6; Preferably, the starting cell of the host cell is a prokaryotic cell or a eukaryotic cell, and the prokaryotic cell is preferably Escherichia coli such as E. coli BL21 (DE3).

9. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises: (i) a first pharmaceutical composition, which is the scorpion toxin mutant or active fragment thereof according to claim 1 or 2, or the scorpion toxin mutant combination according to claim 3, or the pharmaceutical composition or pharmaceutically acceptable salt thereof according to claim 4; and a pharmaceutically acceptable carrier; (ii) a second pharmaceutical composition, wherein the second pharmaceutical composition contains a second active ingredient other than the first pharmaceutical composition, and a pharmaceutically acceptable carrier; the second active ingredient is selected from carbamazepine, fluazifop, gabapentin, lamotrigine, oxcarbazepine, phenytoin, phenytoin sodium, retigabine, topiramate, cefotaxime, ethosuximide, sodium valproate, or a combination thereof.

10. A medicine box, characterized in that: The medicine kit comprises: (1) a first container, and a first pharmaceutical composition in the first container, wherein the first pharmaceutical composition is the scorpion toxin mutant or active fragment thereof according to claim 1 or 2, or the scorpion toxin mutant combination according to claim 3, or the pharmaceutical composition or pharmaceutically acceptable salt thereof according to claim 4, and a pharmaceutically acceptable carrier; and (2) a second container, and a second pharmaceutical composition in the second container, wherein the second pharmaceutical composition contains a second active ingredient other than the first pharmaceutical composition, and a pharmaceutically acceptable carrier; the second active ingredient is selected from carbamazepine, fluazifop, gabapentin, lamotrigine, oxcarbazepine, phenytoin, phenytoin sodium, retigabine, topiramate, cefotaxime, ethosuximide, sodium valproate, or a combination thereof.

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