CN115710307A - Application of scorpion toxin and mutant thereof in resisting epilepsy - Google Patents

Abstract

The invention relates to scorpion toxin (chrybdotoxin) (ChTX toxin) and application of a mutant Q18F (ChTX-Q18F) thereof. Specifically, the invention provides application of the polypeptide ChTX-Q18F and active fragments thereof, or pharmaceutically acceptable salts thereof, which are used for preparing preparations or compositions for treating and/or preventing epilepsy. The ChTX-Q18F is proved to be effective in preventing and/or treating epileptic symptoms for the first time by measuring the influence of the ChTX-Q18F on recurrent convulsion behaviors of a rat PTZ convulsion model, the influence on c-Fos expression of hippocampus after the convulsion of the rat and the investigation on the damage condition of the hippocampus neurons, and the investigation on action potential and local field potential of the hippocampus neurons of the rat.

Description

Application of scorpion toxin and mutant thereof in anti-epilepsy

Technical Field

The invention relates to the field of polypeptide drugs, in particular to an anti-epileptic toxin peptide scorpion toxin (Charybdotoxin) and application of a mutant Q18F (ChTX-Q18F) thereof in anti-epilepsia.

Background

Epilepsy (epilepsy) is a chronic disease that causes transient cerebral dysfunction and is characterized by recurrent seizures resulting from sudden abnormal firing of cerebral neurons. It is estimated that about 40 million new epileptic patients are added each year, and epilepsy has become the second most common disease after headache in neurology in China.

The pathogenesis of epilepsy is very complex, however it is generally believed that an imbalance between excitation and inhibition of the central nervous system will lead to seizures.

Based on the difference in the initiation site and transmission mode of abnormal discharge, the factors of epilepsy are classified into various factors such as ion channel dysfunction, neurotransmitter dysfunction, and glial cell dysfunction. Ion channels are one of the bases for the regulation of tissue excitability in vivo. Research shows that channels such as sodium ion channels, potassium ion channels, calcium ion channels and the like have certain relevance with epilepsy.

In response to the diverse pathogenesis of epilepsy, researchers have developed various antiepileptic drugs with different mechanisms of action and different targets. The micromolecular drugs such as phenytoin sodium, carbamazepine and the like can selectively act on the voltage-dependent sodium ion channel to block the rapid release of the sodium ion-dependent action potential, thereby achieving the anticonvulsant effect. The trimethadione is a selective T-type calcium ion channel blocker and inhibits the hyperexcitation of neurons. Perampanel is an AMPA type glutamic acid receptor (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor, mediating the rapid excitatory synaptic transmission of the central nervous system) antagonist, and can reduce the excessive excitation of neurons by inhibiting the activity of postsynaptic AMPA receptor glutamic acid so as to prevent and treat epileptic diseases.

However, the effect of the current therapeutic agents for epilepsy is still unsatisfactory. For example, most small molecule drugs have significant side effects when taken for a long period of time. In addition, the causes of epilepsy are various, so that the current small-molecule drugs are difficult to be applied to the symptoms.

Therefore, there is an urgent need in the art to develop a novel drug for preventing and/or treating epilepsy, which has high specificity and/or low side effects.

Disclosure of Invention

The invention aims to provide a medicament which has high specificity and/or small side effect and is used for preventing and/or treating epilepsy and application thereof.

In a first aspect of the invention, there is provided a use of scorpion toxin (charybdotoxin) and active fragments thereof, or pharmaceutically acceptable salts thereof, for preparing a preparation or a pharmaceutical composition for treating and/or preventing epilepsy,

the scorpion toxin comprises wild type scorpion toxin or a mutant thereof with an amino acid sequence shown in SEQ ID NO. 1, and the mutant comprises a mutant formed by mutating the 18 th glutamic acid point of the wild type scorpion toxin.

In another preferred embodiment, the mutant is selected from the group consisting of: chTX-Q18F, chTX-Q18Y, chTX-Q18H, chTX-Q18V, or ChTX-Q18L.

In another preferred embodiment, the mutant comprises a mutant ChTX-Q18F formed by mutating glutamic acid at position 18 of wild type scorpion toxin into phenylalanine.

In another preferred embodiment, the ChTX-Q18F comprises a recombinant or synthetic ChTX-Q18F polypeptide.

In another preferred embodiment, chTX-Q18F comprises the amino acid sequence shown as SEQ ID NO. 2.

In another preferred embodiment, the ChTX-Q18F comprises an amino acid sequence obtained by performing one or more amino acid substitutions, deletions, alterations, insertions or additions on the basis of the sequence SEQ ID NO. 2 within the range of keeping the activity of the protein.

In another preferred embodiment, the ChTX-Q18F comprises one or more amino acid insertions ranging from 1 to 10, preferably from 1 to 5, more preferably from 1 to 3, at the N-or C-terminus of the sequence SEQ ID NO. 1, while maintaining the activity of the protein.

In another preferred embodiment, the ChTX-Q18F comprises an amino acid sequence which is obtained by further changing the active site amino acid Phe18 on the basis of the sequence SEQ ID NO. 2.

In another preferred embodiment, chTX-Q18F comprises a recombinant protein with one or more protein tags at the N-terminal or C-terminal of the sequence SEQ ID NO. 2, within the range of maintaining protein activity.

In another preferred embodiment, the protein tag is selected from the group consisting of: MBP tag, his tag, GST tag, SUMO tag, TRX tag, HA tag, flag tag, nusA tag, or a combination thereof.

In another preferred embodiment, the ChTX-Q18F comprises the amino acid sequence shown as SEQ ID NO. 2.

In another preferred embodiment, the ChTX-Q18F polypeptide comprises one or more amino acid insertions ranging from 1 to 10, preferably from 1 to 5, more preferably from 1 to 3, at the N-or C-terminus of the sequence SEQ ID NO. 2, while maintaining the activity of the protein.

In another preferred embodiment, the ChTX-Q18F is recombinant.

In another preferred embodiment, the ChTX-Q18F is recombinantly expressed in E.coli.

In another preferred embodiment, the amino acid sequence of the recombinant ChTX-Q18F is shown as SEQ ID NO. 3.

In another preferred embodiment, the formulation or pharmaceutical composition comprises: a component (a) scorpion toxin (charybdotoxin) and active fragments thereof, or pharmaceutically acceptable salts thereof, and a component (b) pharmaceutically acceptable carriers,

the scorpion toxin comprises wild type scorpion toxin or a mutant thereof with an amino acid sequence shown in SEQ ID NO. 1, and the mutant comprises a mutant formed by mutating the 18 th glutamic acid point of the wild type scorpion toxin.

In another preferred embodiment, the mutant is selected from the group consisting of: chTX-Q18F, chTX-Q18Y, chTX-Q18H, chTX-Q18V, or ChTX-Q18L.

In another preferred embodiment, the formulation or pharmaceutical composition comprises: chTX-Q18F, and a pharmaceutically acceptable carrier.

In another preferred embodiment, the component (a) is 0.1-99.9wt%, preferably 10-99.9wt%, more preferably 70-99.9 wt% of the total weight of the formulation or pharmaceutical composition.

In another preferred embodiment, the pharmaceutically acceptable carrier is selected from the group consisting of: an infusion solution carrier and/or an injection carrier, preferably, the carrier is one or more selected from the following group: normal saline, dextrose saline, or combinations thereof.

In another preferred embodiment, the pharmaceutical composition is a liquid, solid, or semi-solid.

In another preferred embodiment, the dosage form of the pharmaceutical composition is an injection, or a topical pharmaceutical dosage form.

In another preferred embodiment, the dosage form of the pharmaceutical composition comprises an injection or a lyophilized preparation.

In another preferred embodiment, the dosage form of the pharmaceutical composition is an injection.

In another preferred embodiment, the pharmaceutical composition is administered by intravenous, subcutaneous, intramuscular or intracranial routes.

In another preferred example, the pharmaceutical composition is administered by micro infusion pumps (microinfusion pumps).

In another preferred embodiment, the pharmaceutical composition is administered by intracranial administration, preferably by intracerebroventricular Injection (ICV) delivery into the subject.

In another preferred embodiment, the subject comprises: a mammal.

In another preferred embodiment, the mammal comprises a human or non-human mammal.

In another preferred embodiment, the non-human mammal comprises: rodents (e.g., rats, mice), primates (e.g., monkeys).

In another preferred embodiment, the formulations or pharmaceutical compositions may be administered alone, or in combination.

In another preferred embodiment, said co-administration comprises: in combination with other therapeutic agents for the treatment and/or prevention of epilepsy.

In another preferred embodiment, said other therapeutic agent for the treatment and/or prevention of epilepsy is selected from the group consisting of: carbamazepine, fluoropyridine, gabapentin, lamotrigine, oxcarbazepine, phenytoin sodium, retigabine, topiramate, atropizin, ethosuximide, sodium valproate, or combinations thereof.

In another preferred embodiment, the epilepsy comprises epilepsy caused by cerebral cortex excitability enhancement (or neuron excitation type epilepsy).

In another preferred embodiment, the epilepsy has the following characteristics: the activity of large conductance calcium ions and voltage activated potassium channels is enhanced, resulting in enhanced excitability of the cerebral cortex.

In another preferred embodiment, the epilepsy comprises epilepsy in both human and non-human mammals (e.g., rodents).

In another preferred embodiment, the epilepsy comprises PTZ-induced epilepsy, especially PTZ-induced epilepsy in rats.

In another preferred embodiment, the epilepsy comprises refractory epilepsy.

In a second aspect, the invention provides a scorpion toxin mutant polypeptide, wherein the mutant comprises a mutant formed by mutating the 18 th glutamic acid point of wild type scorpion toxin with an amino acid sequence shown as SEQ ID NO. 1.

In another preferred embodiment, the mutant is selected from the group consisting of: chTX-Q18F, chTX-Q18Y, chTX-Q18H, chTX-Q18V, or ChTX-Q18L.

In another preferred embodiment, the mutant comprises a mutant ChTX-Q18F formed by mutating glutamic acid at position 18 of wild scorpion toxin into phenylalanine.

In another preferred embodiment, the ChTX-Q18F comprises the amino acid sequence shown as SEQ ID NO. 2.

In another preferred embodiment, the ChTX-Q18F comprises an amino acid sequence obtained by performing one or more amino acid substitutions, deletions, alterations, insertions or additions based on the sequence SEQ ID NO. 2 within the range of keeping the activity of the protein.

In another preferred embodiment, the ChTX-Q18F comprises one or more amino acid insertions ranging from 1 to 10, preferably from 1 to 5, more preferably from 1 to 3, at the N-or C-terminus of the sequence SEQ ID NO. 1, while maintaining the activity of the protein.

In another preferred embodiment, the ChTX-Q18F includes the active site amino acid Phe18 retained on the basis of the sequence SEQ ID NO. 2 to further alter the resulting amino acid sequence.

In another preferred embodiment, the ChTX-Q18F comprises a recombinant protein with one or more protein tags at the N-terminal or C-terminal of the sequence SEQ ID NO. 2 within the scope of maintaining the activity of the protein.

In another preferred embodiment, the protein tag is selected from the group consisting of: an MBP tag, a His tag, a GST tag, a SUMO tag, a TRX tag, an HA tag, a Flag tag, a NusA tag, or a combination thereof.

In another preferred embodiment, chTX-Q18F comprises the amino acid sequence shown as SEQ ID NO. 2.

In another preferred embodiment, the ChTX-Q18F polypeptide comprises one or more amino acid insertions ranging from 1 to 10, preferably from 1 to 5, more preferably from 1 to 3, at the N-or C-terminus of the sequence SEQ ID NO. 2, while maintaining the activity of the protein.

In another preferred embodiment, the ChTX-Q18F is recombinant.

In another preferred embodiment, the ChTX-Q18F is recombinantly expressed in E.coli.

In another preferred embodiment, the amino acid sequence of the recombinant ChTX-Q18F is shown as SEQ ID NO. 3.

In a third aspect of the invention, there is provided a polynucleotide encoding a scorpion toxin mutant polypeptide according to the second aspect of the invention.

In another preferred embodiment, the sequence of the polynucleotide is shown in SEQ ID NO. 4.

In a fourth aspect of the invention, there is provided an expression vector comprising a polynucleotide according to the third aspect of the invention.

In another preferred embodiment, the expression vector comprises a eukaryotic expression vector, a prokaryotic expression vector, or a viral vector.

In a fifth aspect of the invention, there is provided a host cell comprising an expression vector according to the fourth aspect of the invention, or having integrated into its genome a polynucleotide according to the third aspect of the invention.

In another preferred embodiment, the host cell comprises a eukaryotic cell or a prokaryotic cell.

In a sixth aspect of the present invention, there is provided a pharmaceutical composition comprising:

(i) A first pharmaceutical composition comprising a first active ingredient (Z1) a scorpion toxin mutant polypeptide or an active fragment thereof according to the second aspect of the invention, and a pharmaceutically acceptable carrier;

(ii) A second pharmaceutical composition comprising the second active ingredient (Z2) in addition or alternatively a pharmaceutically active ingredient for the treatment and/or prophylaxis of epilepsy, and a pharmaceutically acceptable carrier.

In another preferred embodiment, the first pharmaceutical composition and the second pharmaceutical composition are the same composition or different compositions.

In another preferred embodiment, the pharmaceutical composition comprises:

(a) A first active ingredient that is ChTX-Q18F or an active fragment thereof;

(b) A second active ingredient which is a further or additional pharmaceutically active ingredient for the treatment and/or prophylaxis of epilepsy; and

(c) A pharmaceutically acceptable carrier.

In another preferred embodiment, the other or additional pharmaceutically active ingredients for treating and/or preventing epilepsy comprise: carbamazepine, fluoropyridine, gabapentin, lamotrigine, oxcarbazepine, phenytoin sodium, retigabine, topiramate, atropizin, ethosuximide, sodium valproate, or combinations thereof.

In a seventh aspect of the invention, there is provided a kit comprising:

(C1) A first container, and a first pharmaceutical composition comprising a first active ingredient (Z1) a scorpion toxin mutant polypeptide or active fragment thereof according to the second aspect of the invention, and a pharmaceutically acceptable carrier; and

(C2) A second container, and a second pharmaceutical composition in said second container, said second pharmaceutical composition comprising a second active ingredient (Z2) in addition or in addition to a pharmaceutically active ingredient for the treatment and/or prevention of epilepsy, and a pharmaceutically acceptable carrier.

In another preferred embodiment, the kit further comprises (iii) instructions.

In another preferred embodiment, the first active ingredient (Z1) is ChTX-Q18F or an active fragment thereof, and a pharmaceutically acceptable carrier.

In another preferred example, the second active ingredient (Z2) comprises: carbamazepine, fluoropyridine, gabapentin, lamotrigine, oxcarbazepine, phenytoin sodium, retigabine, topiramate, atropizin, ethosuximide, sodium valproate, or combinations thereof.

In another preferred embodiment, the first container and the second container are the same or different containers.

In another preferred embodiment, the first pharmaceutical composition in the first container is a single formulation comprising ChTX-Q18F or active fragment thereof.

In another preferred embodiment, the pharmaceutical composition in the second container is a single formulation containing other or additional pharmaceutically active ingredients for treating and/or preventing epilepsy.

In another preferred embodiment, the description describes instructions for administering the first active ingredient (Z1) and optionally the second active ingredient (Z2) in order to treat and/or prevent epilepsy.

In another preferred embodiment, the specification states that the dosage form of the first pharmaceutical composition and optionally the second pharmaceutical composition is an injection.

In another preferred embodiment, the injection is injected into the subject via the lateral ventricle of the brain (ICV).

In an eighth aspect of the invention, there is provided a method for treating and/or preventing epilepsy, comprising administering to a subject in need thereof a scorpion toxin mutant polypeptide according to the second aspect of the invention or a pharmaceutical composition according to the sixth aspect of the invention.

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

In another preferred example, the subject has or is suspected of having epilepsy.

In another preferred embodiment, the epilepsy comprises epilepsy caused by cerebral cortex excitability enhancement (or neuron excitation type epilepsy).

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be repeated herein, depending on the space.

Drawings

Figure 1 shows a rat brain map.

FIG. 2 shows the inhibitory effect of toxin peptides ChTX, chTX-Q18F and ChTX-T23F on PTZ-induced c-Fos expression in the ipsilateral (A) and ipsilateral (B) hippocampus of injection. (a-d) represent the saline control group and experimental group of ChTX, chTX-Q18F and ChTX-T23F injected into the ipsilateral hippocampus, respectively. (a 1-d 1) represents the CA1 region, (a 2-d 2) represents the CA3 region, and (a 3-d 3) represents the DG region. (C) Histogram of c-Fos from ipsilateral hippocampus injected with toxin peptide (n = 3). (F-i) represents a normal saline control group and experimental groups of ChTX, chTX-Q18F and ChTX-T23F injected into the hippocampus heteropleura, respectively. (f 1-i 1) represents the CA1 region, (f 2-i 2) represents the CA3 region, and (f 3-i 3) represents the DG region. (D) Histogram of c-Fos from the hippocampus heteropolaris injected with toxin peptide (n = 3). P compared to saline group<0.05，**p<0.01，***p<0.001 (One-way ANOVA). In comparison to the ChTX group, ^# p<0.05， ^## p<0.01， ^### p<0.001(One-way ANOVA)。

FIG. 3 shows that the hippocampus, following ipsilateral (A) and ipsilateral (B) injection of toxin peptides ChTX, chTX-Q18F and ChTX-T23F, developsMicronaire staining showed ptz-induced integrity of hippocampal tissue in rats with epilepsy. (a-d) represent the saline control group and experimental group of ChTX, chTX-Q18F and ChTX-T23F injected into the ipsilateral hippocampus, respectively. (a 1-d 1) represents the CA1 region, (a 2-d 2) represents the CA3 region, and (a 3-d 3) represents the DG region. (C) histogram showing C-Fos of injected hippocampus heteropolaris. (F-i) represents a normal saline control group and experimental groups of ChTX, chTX-Q18F and ChTX-T23F injected into the hippocampus heteropleura, respectively. (f 1-i 1) represents the CA1 region, (f 2-i 2) represents the CA3 region, and (f 3-i 3) represents the DG region. (D) Histogram of c-Fos from the hippocampus of the xenogeneic hippocampus injected with toxin peptide (n = 3). P compared to saline group<0.05，**p<0.01，***p<0.001 (one-way ANOVA). In comparison to the ChTX group, ^# p<0.05， ^## p<0.01， ^### p<0.001(One-way ANOVA)。

FIG. 4 shows the effect of toxin peptides ChTX, chTX-Q18F and ChTX-T23F on firing characteristics of hippocampal pyramidal neurons. (a) The PTZ pre-treated neurons were induced to produce action potentials using current clamps, saline (black), chTX (red), chTX-Q18F (blue), chTX-T23F (magenta). (b) the number of action potentials under different current injections. (c) an action potential width under 300pA current injection. (d) Fast post-hyperpolarization (fAHP) amplitude from pre-spike voltage to peak after post-hyperpolarization at 300pA current injection. (e) Post hyperpolarization deactivation time constant under 300pA current injection. (f) 6 th-7 th spike interval under 300pA current injection. P compared to saline group<0.05，**p<0.01，***p<0.001; in comparison to the ChTX group, ^# p<0.05， ^## p<0.01， ^### p<0.001(One-way ANOVA)。

FIG. 5 shows the regulation of PTZ-induced systemic tonic clonic seizures LFP profile and PSD by the toxin peptides ChTX, chTX-Q18F and ChTX-T23F. (A) Representative seizure treatment efficacy of LFP signal and spectrothermograph in saline-injected control, chTX-Q18F and ChTX-T23F toxins and VPA experimental groups, respectively. (B) Spectral plots and cumulative profiles of PTZ-induced seizure mice from saline-injected control groups, chTX-Q18F and ChTX-T23F toxins and VPA experimental groups, respectively, are shown. (C) The results represent the spectrum analysis of ptz-induced epilepsy group and PSD values of δ, θ, α, β, γ waves of each drug group (n = 4) after saline injection control, chTX-Q18F, and ChTX-T23F toxins and VPA, respectively.

Detailed Description

The present inventors have conducted extensive and intensive studies and, as a result of screening a large number of different compounds, including a large number of compounds such as sodium ion channels, have for the first time unexpectedly found that a polypeptide substance scorpion toxin, and in particular a mutant thereof (i.e., chTX-Q18F), can be used for the extremely effective treatment and/or prevention of epilepsy. Experiments show that in a rat PTZ (pentetrazol) convulsion model, the inventor finds that the scorpion toxin and the mutant ChTX-Q18F thereof can effectively relieve epileptic symptoms for the first time by measuring the influence of the polypeptide ChTX-Q18F on recurrent convulsion behaviors, the influence on hippocampal c-Fos expression after rat convulsion and the investigation on damage conditions of hippocampal neurons. The present invention has been completed on the basis of this finding.

Scorpion toxin (charybdotoxin) and mutant thereof

As used herein, the terms "Charybdotoxin Q18F protein," "Charybdotoxin polypeptide mutant Q18F," "ChTX toxin mutant Q18F," and "ChTX polypeptide mutant Q18F," "recombinant ChTX toxin Q18F," "scorpion toxin mutant Q18F," "ChTX-Q18F protein," "polypeptide ChTX-Q18F," "toxin peptide ChTX-Q18F," and the like, are used interchangeably and all refer to Charybdotoxin protein mutant Q18F. The Charybdotoxin protein mutant Q18F 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 encompasses not only chemically synthesized mutant charybdotoxin protein Q18F (ChTX-Q18F), but also recombinant ChTX-Q18F, such as recombinantly expressed charybdotoxin Q18F protein with or without the initiating Met, and recombinantly expressed mutant charybdotoxin protein Q18F with or without the expression tag or enzyme cleavage residue of 1-3 amino acids.

The natural wild charybdotoxin can be obtained by separating and purifying venom of a northern Africa scorpion (Leiurus quinquestratus), is a first blocking agent of a BK channel, and does not have the anti-epileptic activity of a target BK (alpha + beta 4) channel. The toxin peptide ChTX-Q18F of the invention is difficult to be obtained by natural separation, but can be obtained by chemical synthesis (including solid phase cooperation) or recombinant technology.

For recombination, the recombinant DNA can be obtained by expression in host cells such as Escherichia coli by conventional recombinant techniques, and isolation and purification.

The amino acid sequence of the wild type charybdotoxin is shown in SEQ ID NO. 1:

EFTNVSCTTSKECWSVCQRL HNTSRGKCMNKKCRCYS(SEQ ID NO:1)。

the amino acid sequence of the toxin peptide ChTX-Q18F is shown as SEQ ID NO. 2:

EFTNVSCTTSKECWSVCFRL HNTSRGKCMNKKCRCYS(SEQ ID NO:2)。

a recombinant ChTX-Q18F amino acid sequence is shown as SEQ ID NO. 3:

GSEFTNVSCTTSKECWSVCFRLHNTSRGKCMNKKCRCYS(SEQ ID NO:3)。

the nucleic acid sequence of the amino acid sequence SEQ ID NO. 1 of the coding ChTX-Q18F protein is shown as SEQ ID NO. 4.

GAGTTCACCAACGTTAGCTGCACCACGAGTAAAGAATGCTGGAGCGTGTGCTTTCGTTTGCATAATACTTCTCGCGGTAAGTGCATGAACAAGAAGTGCCGCTGCTATAGC(SEQ ID NO:4)。

Experiments prove that in a rat PTZ convulsion model, the scorpion toxin mutant ChTX-Q18F can remarkably and preventively relieve abnormal excitation of neurons and epileptic symptoms, so that the scorpion toxin mutant ChTX-Q18F can be used as a novel polypeptide medicine for treating epileptic symptoms.

It is to be understood that although the wild-type of the polypeptide ChTX-Q18F provided in the examples of the invention is derived from the North Africa scorpion, mutants of ChTX-Q18F polypeptides derived from other similar species, particularly scorpions belonging to the same family or genus as the North Africa scorpion, having some homology (conservation) to the sequences of the invention (preferably, the sequences are shown in SEQ ID NO: 2) may also be used in the invention.

It is to be understood that although the genes provided in the examples of the invention are derived from the Arctia andraenus Kirschaum, the gene sequences of ChTX-Q18F derived from other similar species (particularly, from the Scorpion of the same family or genus as the Arctia andraena), which have some homology (conservation) to the sequences of the invention (preferably, the sequences are shown in SEQ ID NO: 2), are included within the scope of the invention, as long as the sequences can be readily isolated from other species (particularly, scorpions) by one skilled in the art after reading the present application, based on the information provided herein.

The polynucleotide of the present invention may be in the form of DNA or RNA. The DNA forms include: DNA, genomic DNA or artificially synthesized DNA, the DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The nucleotide sequence of the coding region encoding the mature polypeptide may be identical to the sequence of the coding region shown in SEQ ID NO. 4 or may be a degenerate variant.

Polynucleotides encoding a mature polypeptide include coding sequences that encode only a mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences. The invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of the polyglycosides or polypeptides having the same amino acid sequence as the invention. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the polypeptide encoded thereby.

The present invention also relates to polynucleotides which hybridize to the above-described sequences and which 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 which hybridize under stringent conditions to the polynucleotides of the present invention. In the present invention, "stringent conditions" mean: (1) Hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) methylphthalamide, 0.1% calf serum/0.1% Ficoll,42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 90% or more, preferably 95% or more.

It is to be understood that although the wild type ChTX-Q18F gene of the invention is preferably derived from the andropa pterocarpus, by single point mutation, other genes from other species (particularly scorpions) that are highly homologous (e.g., have greater than 80%, such as 85%, 90%, 95%, or even 98% sequence identity) to the andropa pterocarpus ChTX gene are also within the contemplation of the invention. Methods and means for aligning sequence identity are also well known in the art, for example BLAST.

The full-length ChTX-Q18F nucleotide sequence or the fragment thereof can be obtained by a PCR amplification method, a recombination method or an artificial synthesis method. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the sequences can be amplified using a commercially available DNA library or a cDNA library prepared by conventional methods known to those skilled in the art as a template. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order. Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. Usually, it is cloned into a vector, transferred into a cell, and then isolated from the propagated host cell by a conventional method to obtain the relevant sequence.

In addition, the sequence of interest can be synthesized by artificial synthesis, especially when the fragment length is short. Typically, long fragments are obtained by first synthesizing a plurality of small fragments and then ligating them together. 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 may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The invention relates to a polypeptide ChTX-Q18F for treating epilepsy, and in a preferred embodiment of the invention, the amino acid sequence of the polypeptide is shown as SEQ ID NO. 2. The polypeptide of the invention can effectively treat and/or prevent epilepsy.

The present invention also includes polypeptides or proteins having 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, e.g., 99%) homology to the sequence of SEQ ID NO. 2 of the present invention, and having the same or similar functions.

The "same or similar functions" mainly refer to: "alleviating the symptoms of epilepsy".

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide. The polypeptides of the invention can be naturally purified products, or chemical synthesis products, or using recombinant technology from prokaryotic or eukaryotic host (for example, bacteria, yeast, plant, insect and mammalian cells) in the production of. Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated or may be non-glycosylated. The polypeptides of the invention may or may not also include the initial methionine residue.

The invention also includes fragments and analogs of ChTX-Q18F polypeptides having ChTX-Q18F polypeptide activity. As used herein, the terms "fragment" and "analog" refer to a polypeptide that retains substantially the same biological function or activity as a ChTX-Q18F polypeptide of the invention.

The polypeptide fragment, derivative or analogue of the invention may be: (i) Polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) a polypeptide having a substituent group in one or more amino acid residues; or (iii) a polypeptide formed by fusing the mature polypeptide with another compound, such as a compound that increases the half-life of the polypeptide, e.g., polyethylene glycol; or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (e.g., a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein). Such fragments, derivatives and analogs are within the scope of those skilled in the art as defined herein.

In the present invention, the polypeptide variant is an amino acid sequence shown in SEQ ID NO. 2, a derivative sequence obtained by substituting, deleting or adding at least one amino acid by several (usually 1-10, preferably 1-8, more preferably 1-4, most preferably 1-2), and one or several (usually less than 10, preferably less than 5, more preferably less than 3) amino acids (such as the amino acid sequence shown in SEQ ID NO. 3) added at the C-terminal and/or N-terminal. For example, substitutions in the protein with amino acids of similar or analogous properties will not generally alter the function of the protein, nor will the addition of one or more (e.g., 1-3) amino acids at the C-and/or N-terminus. These conservative variations are best generated by making substitutions according to table 1.

TABLE 1

Initial residue(s)	Representative substitutions	Preferred substitutions
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

The invention also includes analogs of the claimed proteins. These analogs may differ from SEQ ID NO. 2 by amino acid sequence differences, by modifications that do not affect the sequence, or by both. Analogs of these proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, site-directed mutagenesis, or other known molecular biological techniques. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the proteins of the present invention are not limited to the representative proteins listed above.

Modified (generally without altering primary structure) forms include: chemically derivatized forms of the protein such as acetoxylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those that are modified during protein synthesis and processing. Such modification may be accomplished by exposing the protein to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine).

Pharmaceutical compositions and methods of administration thereof

The invention provides a pharmaceutical composition, which comprises a pharmaceutically acceptable carrier and effective amounts of the following active ingredients: the toxin peptide ChTX-Q18F or an active fragment thereof and a pharmaceutical active ingredient thereof for treating and/or preventing epilepsy.

As used herein, the term "effective amount" or "effective dose" refers to an amount that produces a function or activity in, and is acceptable to, a human and/or an animal.

As used herein, a "pharmaceutically acceptable" ingredient is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at 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.

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 vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Usually, the pharmaceutical preparation should match the administration mode, and the pharmaceutical composition of the present invention is in the form of injection. For example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions.

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, etc. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such 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 weight of the patient, the immune status of the patient, the route of administration, and the like. For example, divided doses may be administered several times per day, or the dose may be proportionally reduced, as may be required by the urgency of the condition being treated.

The pharmaceutically acceptable carrier of the present invention includes (but is not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptidic substances, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched with the mode of administration, and these are well known to those skilled in the art.

The first active ingredient (a) ChTX-Q18F or the active fragment thereof provided by the invention can be combined with the second active ingredient (b) other drugs for treating and/or preventing epilepsy. Wherein said second active ingredient (b) is an antiepileptic drug already available in the art, including but not limited to: carbamazepine, fluoropyridine, gabapentin, lamotrigine, oxcarbazepine, phenytoin sodium, retigabine, topiramate, atropizin, ethosuximide, sodium valproate, or combinations thereof.

c-Fos gene

The c-Fos gene is one of the immediate early genes [ Trends neurosci.1995,18,66-67] (IEGs, protooncogenes that can be induced by second messengers), and is closely related to various pathophysiological processes after the onset of epilepsy. Under physiological conditions, the c-Fos gene is expressed at low levels in the central nervous system; when neurons are physically or chemically stimulated, IEGs such as c-Fos are activated by excitation of the neurons, and mRNA transcribed from the IEGs is translated to produce a c-Fos protein [ Annu. Rev. Neurosci.1991,14,421-451], and the like. The protein can regulate the expression of various Late Response Genes (LRGs), and simultaneously cause epileptic seizure through various mechanisms, and finally cause the formation of epileptic focus [ Neuron1990,4,477-485]. The c-Fos protein is rapidly expressed in a large quantity during the epileptic seizure, and if the epileptic seizure is controlled by using a medicament or other therapeutic means, the expression of the protein can be obviously inhibited, so the expression determination of the c-Fos can be used as an effective index for evaluating the action mechanism and the curative effect of the antiepileptic medicament.

Nile body

Nissl is a specific infectious agent found in the cytoplasm of neuronal dendrites. Usually, the nissl body has a fixed shape, the neuron cell bodies are large, the cytoplasm is pale and uniformly stained, the cell nucleus is large and round, and all the cell stains are deeply stained. The pyramidal cells are tightly packed in the hippocampal Dentate Gyrus (DG) region, with 4 or 5 layers. However, when the brain is damaged, the shape of the brain changes, the cell edges are blurred, the integrity of the cells is reduced, the staining of the cell bodies is shallow, the distribution of neurons is disturbed, and the number of cells or the number of cell layers is reduced. The presence, distribution and pathological changes of neurons can therefore be identified by the morphology and number of nisseurs.

PTZ (Pentylenetetrazole)

Pentylenetetrazol (PTZ), also known as pentamtrazol (petetrazol), pentamethylenetetrazole, and cadiazole, are chemicals that are white crystalline powders. The chemical name is 1, 5-pentamethylene-1H-tetrazole and the molecular formula is C ₆ H ₁₀ N ₄ And the molecular weight is 138.1704. Pentaerythrine is a central stimulant and is mainly used for clinically rescuing central respiratory failure caused by serious barbiturates and narcotic poisoning; it can also be used for respiratory depression and acute circulatory failure caused by acute infectious diseases, anesthetic and barbiturate poisoning.

Pentaerythrine can excite respiratory center and cardiovascular movement center, and its action is rapid and strong, so that it can make respiration deepen and accelerate, and its blood pressure is microliter; at a slightly larger dose, excitation may extend to the cerebral cortex and spinal cord, causing convulsions.

PTZ acts primarily on GABA _A The chloride channel of the receptor (gamma-aminobutyric acid A type receptor) inhibits the activity of GABA neurons, thereby enhancing the excitability of the nervous system and inducing clonic or systemic tonic epileptic seizures of animals and human beings. The PTZ-induced rodent convulsive seizure model has been widely used in the study of epileptogenic mechanisms and novel antiepileptic drugs.

In the present invention, animals were induced to develop convulsions/epilepsy by intraperitoneal PTZ injection for subsequent experimental studies of the effect of the ChTX-Q18F toxin peptides of the present invention on epilepsy.

The main advantages of the present invention include:

(1) The ChTX-Q18F has the characteristics of strong effect and small dosage (the dosage can be as low as 0.32 mu g/kg in a rat PTZ model), not only obviously prolongs the latent period of seizure, but also obviously relieves the symptoms of epilepsy (especially can obviously reduce the morbidity of 3-grade, 4-grade and 5-grade epilepsy), thereby being used as a novel polypeptide medicament for treating epilepsy.

(2) The recombinant ChTX-Q18F can be prepared by prokaryotic expression and recombination, and is expected to provide a safe, effective and cheap antiepileptic drug for epileptics.

(3) ChTX-Q18F is a neurotoxin with a very small molecular weight (only about 4 KDa) and can effectively penetrate the blood brain barrier and act on the brain.

(4) ChTX-Q18F is a high-specificity neurotoxin, has selectivity on large-conductance calcium ions of alpha + beta 4 subtype BK potassium ion channels and voltage-activated potassium ion channels, and has no influence on cardiovascular, endocrine, reproductive system and the like, so that side effects are low.

(5) ChTX-Q18F is one of polypeptide substances which are proved to have the efficacy of relieving epilepsy through experiments for the first time.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: conditions described in a Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

The materials and reagents used in the examples were all commercially available products unless otherwise specified.

Experimental materials and methods

Laboratory animals and drugs

Experimental animals: adult male SD rats (provided by Shanghai laboratory animal center of Chinese academy of sciences) with weight of 250-300g, 5 rats per cage, are raised under conventional laboratory conditions, and are maintained at room temperature of 22 + -1 deg.C and natural circadian rhythm.

Related drugs: the expression and purification of recombinant North Africa scorpion toxin ChTX-Q18F are described in preparation example 1.

Penetraazan (PTZ, CAS: 54-95-5), available from Sigma, USA.

Animal surgery

After anesthesia by intraperitoneal injection of 10% chloral hydrate (300 mg/kg), the rats were fixed in a stereotaxic apparatus (NS-2, narishige, japan), the hair at the vertex was shaved off and the scalp was disinfected, the skin was cut off and the subcutaneous tissue was cauterized with 10% hydrogen peroxide, the Bregma point of the skull was exposed, and the implantation site of the drug delivery base (AP-4.3 mm, L2.2 mm) was determined on the skull according to the rat brain localization map. After positioning, a small hole (1 mm) was drilled with a dental drill, the inner plate was peeled off, the dura mater was opened by the tip of the needle, and the drug delivery base cannula was implanted 2.5mm below the skull in the CA1 region of the hippocampus. The base is fixed on the surface of the rat skull by dental cement.

Preparation of example 1

DNA sequences of His-MBP-Thrombin site ChTX, chTX-Q18F and ChTX-T23F fusion proteins are respectively synthesized and expressed by an artificial synthesis method, are cut by Nco I and Not I and then are connected with pETDuet-1 plasmid cut by the same enzyme, so that recombinant plasmids pETDuet-1-ChTX, pETDuet-1-Q18F and pETDuet-1-T23F are obtained and are transformed into an E.coli Origami B (DE 3) expression strain for prokaryotic expression.

The fusion protein is obtained through recombinant expression, and the toxin peptide ChTX and mutant ChTX-Q18F and ChTX-T23F polypeptides thereof are prepared by the following method.

The specific purification method comprises the following steps:

(1) The fusion protein in Buffer A environment is firstly combined with a nickel column, then Buffer B imidazole salt solution is used for gradient elution to remove most of hybrid protein, the first affinity column purification is completed, and SDS-PAGE electrophoresis detection is carried out.

(2) The target fusion protein was collected and dialyzed at 18 ℃ in 2L Buffer C, thrombin enzyme was added at 6U/mL, and the mixture was stirred overnight in a 3.5kDa dialysis bag.

(3) The cleavage mixture was then loaded onto an Amylose Resin column using a constant flow pump, and the flow-through Fraction (FL) and the Buffer D eluted fraction were collected. His-MBP-tag is eluted and removed by Buffer E, and the second affinity column purification is completed and SDS-PAGE electrophoresis detection is carried out.

(4) The FL content and Buffer D washes (usually containing small amounts of recombinant ChTX, chTX-Q18F, or ChTX-T23F toxin) were then concentrated to 2mL for further gel chromatography column purification.

(5) The gel chromatography column, superdex 75, was equilibrated with Buffer F in advance, and then the sample was loaded on the column and eluted with Buffer F. And collecting an elution peak with the retention volume of about 110mL, namely a recombinant ChTX, chTX-Q18F or ChTX-T23F sample, wherein the previous absorption peak is MBP-tag or uncut fusion protein. Thus obtaining high-purity ChTX, chTX-Q18F or ChTX-T23F.

The relevant buffer components during purification are shown in table 2.

TABLE 2 relevant buffer solutions during purification of recombinant ChTX-Q18F toxin

Example 1: effect of ChTX-Q18F on rat PTZ recurrent convulsive behavior

1.1 Experimental procedures

Adult male SD rats were placed in a 40X 30X 50cm clear glass box and observed for convulsive responses following administration. Before drug injection, rats were placed in the box 1h in advance, allowed to move freely to adapt to the environment, and then injected with PTZ (60 mg/kg) intraperitoneally to induce seizure in rats. The experiment was divided into toxin injection group and physiological saline blank control group.

ChTX toxin and mutants thereof, namely ChTX-Q18F and ChTX-T23F injection groups: following a single PTZ-induced episode by intraperitoneal injection, the next day the ChTX-Q18F toxin (dissolved in 2 μ L of saline) was injected into the hippocampus of the brain, followed by another PTZ injection. Toxin dose 0.08 μ g, n =7-8 (n is the number of experimental rats);

saline blank control group: after one intraperitoneal PTZ injection to induce seizures, equal amounts of saline were injected in the brain hippocampus 1 day later, followed by another PTZ injection, n =6.

In the experimental process, a double-blind method is adopted for dosing and behavioral observation so as to reduce human errors. The latency of convulsive episodes within 2h after PTZ injection, mortality, duration and number of episodes at different convulsive episode severity were used as statistical measures. Severity of convulsive episodes in rats was determined according to the following criteria [ neurophysiol.1972,32,281-294; brain Res.1997,758,92-98] rating:

level 0: no reaction is carried out;

level 1: rhythmic twitching of the mouth and face;

stage 2: somatic wave-like migratory spasms;

and 3, level: myoclonus of the whole body and upwarping of the buttocks;

and 4, stage 4: the body is turned to one side;

stage 5: supine position, the attack of general spasmodic rigidity.

One complete seizure was defined as the seizure from the beginning of convulsion to the return to normality after convulsion, and another independent seizure was defined when the interval between convulsions reached more than 5 s. Latency is defined as the time after PTZ injection until the onset of the first grade 2 convulsive episode.

1.2 results of the experiment

Compared with a physiological saline blank control group, the control effect of the toxins ChTX, chTX-Q18F and ChTX-T23F on the rat PTZ recurrent convulsion behaviors is examined, and the experimental result is shown in table 2.

TABLE 2 inhibitory Effect of ChTX-Q18F on PTZ recurrent convulsive behavior over 2h

By one-way ANOVA, the ChTX-Q18F group showed significant differences in latency, duration and number of seizures compared to the saline control group, p <0.05, p <0.01, p <0.001.

As can be seen from Table 2:

(1) The latency of recurrence of convulsions in the ChTX-Q18F group was significantly prolonged compared to the blank control group (the latency of the ChTX-Q18F group was 340.73 ± 49.77s, n =11, × <0.001, > the saline group was 103.56 ± 7.90s, n =9), which was 229% (329% -1 =229%).

(2) ChTX-Q18F also significantly reduced seizure duration (duration 223.27 ± 35.99s, n =11, × p <0.01; 429.80 ± 65.19s, n = 9) by 48% (1-52% = 48%).

(3) ChTX-Q18F significantly reduced the degree of convulsive seizures at various levels, particularly at levels 3, 4 and 5, in terms of seizure number. 0.08 μ g of Q18F toxin significantly reduced the number of seizures (4 &5 grades) (1.18 ± 0.26 in the ChTX-Q18F group, n =11, <0.05 for p; 2.17 ± 0.40 in the saline group, n = 9) by 46% (100% -54% = 46%). In terms of the number of 3 episodes, the number of ChTX-Q18F groups was also significantly reduced compared to the saline group (. P < 0.05), by 46% (100% -54% = 46%).

Therefore, chTX-Q18F of the invention shows obvious inhibiting effect in the latent period and duration of the convulsive attack and the times of the seizures under different convulsive attack severity degrees, which indicates that ChTX-Q18F effectively relieves the abnormal excitation and epileptic symptoms of neurons.

Example 2: effect of ChTX-Q18F on hippocampal c-Fos expression following seizure in rats

2.1 Experimental procedures

After the PTZ-induced status epilepticus behavior experiment was completed, animals were anesthetized by intraperitoneal injection of sodium pentobarbital (60 mg/kg). The blood vessels were washed by 200mL of physiological saline through left ventricular ascending aorta perfusion, then perfused with 400mL of a fixative (0.1M PBS containing 40% paraformaldehyde, pH7.4,4 ℃) for 1-2h, the brain tissue was taken out and placed in the same fixative overnight, and then was transferred to a 20% sucrose solution to be soaked until it settled to the bottom of the vessel, and then was soaked in a 30% sucrose solution until it settled to the bottom of the vessel.

Rat brain tissue was sectioned into slices of 20 μm thick in the hippocampal region using a cryostat microtome (Leica 1900, germany), attached to gelatin-potassium chromium sulfate-treated slides, and stored frozen at-20 ℃ for future use. The c-Fos immunohistochemistry was as follows:

(1) Taking out the slices from a refrigerator at-20 deg.C, heating for 30min, drawing with a combined pen frame around the slices, and air drying;

(2) Adding 1% of H to 1% ₂ O ₂ Soaking the slices in water for 30min;

(3) Washing in 0.01M PBS (pH7.4) buffer for 5min 3 times;

(4) Blocking with 5% Goat serum at 37 ℃ for 1h;

(5) The sera were blotted dry with filter paper, rabbit anti-c-Fos antibody (1, 400, sc-52, santa Cruz, USA) was added, the antibody was diluted with 0.01M PBS, approximately 100. Mu.L of each section, and incubated in a wet box for 48h at 4 ℃;

(6) Washing with 0.01M PBS (pH7.4) for 10min, and repeating for 3 times;

(7) Adding biotin-labeled goat anti-rabbit IgG (1: 200) diluted with 0.01M PBS, and incubating for 2h;

(8) 0.01M PBS wash for 10min, repeat 3 times. Adding an ABC compound (the preparation ratio is A: B: PBS = 1) at normal temperature for 2h;

(9) Rinsing with 0.01M PBS (pH 7.4) for 10min, repeating 3 times;

(10) Performing light-shielding dyeing for 10min by using a DAB-nickel ammonium sulfate-glucose oxidase method (DAB, a living organism);

(11) Dehydrating with 70%, 80%, 95%, and 100% (. Times.2) ethanol for 5min each time, and soaking in xylene for 5min each time for 2 times;

(12) The gel was preserved with neutral gum seals and the expression of c-Fos was observed under a microscope.

Counting the number of C-Fos-immunoreactive (FLI) neurons on different partitions (CA 1 and CA 3) of the hippocampus and Dentate Gyrus (DG), randomly taking 6-8 slices for carrying out FLI counting (respectively counting the same side and the different side of toxin injection or physiological saline) of different partitions on two animal groups (each group is 6 animals) for behavior investigation, and finally taking the average value of the FLI counting. The inhibition rate of c-Fos expression was calculated according to the following equation.

Inhibition ratio (inhibition ratio) = (A-B)/A × 100%

Wherein A represents the number of FLI neurons in a normal saline control group; b represents the number of FLI neurons in the ChTX-Q18F toxin-injected group in the corresponding area of the hippocampus.

2.2 results of the experiment

The hippocampus of a rat is divided into a hippocampus and a Dentate Gyrus (DG) according to the morphology of the cell. The hippocampus mainly comprises CA1 (cornu amonis), CA2, CA3 and a portal area, and mainly comprises some pyramidal neurons, wherein the CA1 area is connected with the inferior support, and the portal area is adjacent to the dentate gyrus. The dentate gyrus is the cortex layer between the hippocampus cleft and the hippocampus umbrella shaped like a tooth, and is C-shaped. The structure is divided into three layers: a molecular layer, a granular cell layer, and a multi-layer, consisting essentially of granular cells. See fig. 1 for specific locations.

The c-Fos expression in hippocampus after seizures in rats was determined according to the experimental procedures and data processing described above and the results are shown in FIG. 2.

The analytical results were as follows:

(1) After PTZ-induced status epilepticus behavioral experiments, all animals, including the saline control group and the toxin injection group, showed c-Fos protein expression in the ipsilateral and ipsilateral hippocampus at the injection site, see fig. 2;

(2) In the normal saline control group, c-Fos positive neurons were mainly concentrated in the granular cell layer of the DG region of the hippocampus, with fewer positive neurons in the CA1 and CA3 regions;

(3) ChTX-Q18F has obvious inhibition effect on PTZ-induced hippocampal c-Fos expression. The inhibition rate of ChTX-Q18F on CA1, CA3 and DG areas of ipsilateral hippocampus injection compared to saline control group was 82.68%, 69.41%, 53.49%, respectively, see fig. 2; and the inhibitory effect on c-Fos expression of ipsilateral hippocampus was stronger than that of the heteropolaral hippocampus (the inhibition rates of ChTX-Q18F on CA1, CA3 and DG regions of the heteropolaral hippocampus were 78.76%, 74.27% and 39.80% respectively compared with the saline control group), see FIG. 2.

Therefore, chTX-Q18F has a remarkable inhibiting effect on c-Fos protein expression of rat hippocampus after PTZ induces epilepsy, and the injection ipsilateral effect is stronger than that of heteropolaral hippocampus, which shows that the ChTX-Q18F toxin of the invention may have antiepileptic effect.

Example 3: investigation of damage condition of hippocampal neurons after convulsion attack of rat

3.1 Experimental procedures

(1) Taking out the slices from a refrigerator at-20 deg.C, heating for 20min, drawing with a combined pen frame around the slices, and air drying;

(2) Soaking the slices in distilled water for 2min;

(3) Dripping Nile dyeing liquor (purchased from Biyuntian) on brain slice, and dyeing in water bath at 37 deg.C for 10min;

(4) Washing twice with distilled water for 10s each time;

(5) Dehydrating the slices with 70%, 80%, 95% and 100% ethanol for 2min each time, and soaking in xylene for 5min each time for 2 times;

(6) The slides were preserved with neutral gum and examined under a microscope for staining with ninx.

And randomly taking 6-8 slices for the two animal groups (each group is 6 animals) for counting the neuron cells of different hippocampal subareas (CA 1, CA3 and DG areas), and finally taking the average value of the neuron cells and calculating the rising rate of the neuron number.

3.2 results of the experiment

The damage or death of hippocampal neurons following PTZ-induced epilepsy in rats was measured by nissl staining and the results are shown in figure 3.

The analytical results were as follows:

(1) The ChTX-Q18F group retains a relatively intact hippocampal structure, with highest cell density and close arrangement in hippocampal neurons, particularly in DG, and with deepest Netzerlian staining. Compared with the normal saline group, the neurons become loose in arrangement, the cell density is reduced, the staining is the lightest, the damage to the neurons of the normal saline group is the largest after PTZ induces epilepsy, and the ChTX-Q18F toxin protects the hippocampal neurons after epilepsy, and the damage is the smallest;

(2) The ascent rates of neurons in CA1, CA3 and DG regions of the hippocampus ipsilateral to injection in the ChTX-Q18F group and the saline group were respectively: 213.90%, 220.22%, 273.16%, see fig. 3; the rate of increase of neurons in the CA1, CA3 and DG regions of the hippocampus heteropleura are respectively as follows: 114.29%, 113.66%, 136.41%, see fig. 3, demonstrating that ChTX-Q18F protects the ipsilateral hippocampal neurons more strongly than the ipsilateral hippocampus.

Taken together, PTZ induced status epilepticus resulted in damage or death of rat hippocampal neurons. According to the determination, the damage degree of the neurons in the ChTX-Q18F injection experimental group is greatly reduced, the number of the neurons dyed in the Nib type is the largest, and the arrangement is the most compact.

The experiments jointly show that the ChTX-Q18F can inhibit the convulsion attack of a rat in behavior, simultaneously inhibit the expression of c-Fos in the hippocampus, reduce the damage degree of hippocampal neurons, and prove that the ChTX-Q18F has the antiepileptic effect.

Example 4: examination of action potential of rat hippocampal neurons

4.1 Experimental procedure

(1) Slides were placed in 24-well plates, coated with polylysine (0.01 mg/ml, PDL), the PDL was aspirated the next day and washed twice with sterile PBS before air drying on a clean bench.

(2) Anaesthetizing pregnant mouse, taking out fetal mouse, fixing head at eye position with ophthalmological elbow forceps, tearing off skin with another elbow forceps, and exposing skull. Cut off along the midline of the skull by a spring scissors, take a picture by an elbow and tear off the skull to expose the brain. The whole brain was picked up with forceps into pre-cooled DMEM medium and hippocampal tissue was carefully isolated.

(3) Collecting tissue, cutting into tissue blocks of about 1mm with ophthalmic scissors, carefully aspirating DMEM, addingAdding 0.25% pancreatin, digesting in 37 deg.C incubator for 10min, and mixing up and down once every 3 min. After completion of digestion, the tubes were removed and the digestion was terminated by adding pre-cooled DMEM medium containing 10% FBS. Gently blow 20 times with a pasteur tube, leave on ice for 2min, and centrifuge the single cell suspension at 1000rpm for 5min. After centrifugation, the cell debris in the supernatant was removed and resuspended in 2ml of pre-cooled DMEM medium containing 10% of the f12 nutrient mixture (F12, invitrogen, usa), 10% of fbs (Invitrogen, usa), 1% of streptomycin (Invitrogen, usa). At 5X 10 ⁴ The density of (2) was inoculated into a well plate and cultured in an incubator at 37 ℃.

(4) Half-changes were performed 6h later in serum-free Neurobasal medium (containing 2mM Glutamax-I and 2% B27supplement), followed by half-changes every three days later. Meanwhile, 2. Mu.M cytarabine was added to each well to inhibit the growth of glial cells, and patch clamp current clamp recordings were performed after two weeks of continuous culture.

(5) The neuron selects-70 mV resting potential and 350M omega or more output resistance to record the current clamp. Cells were held at a fixed voltage of-80 mV during recording, and different frequencies of spiking were modulated between PTZ-treated groups (recorded after 24h of PTZ treatment) and epileptic cell groups (recorded after 10min of drug addition) to which ChTX or its mutants, chTX-Q18F, chTX-T23F, was applied after PTZ treatment by injecting a fixed increasing positive current for 1000 ms. The width of the spike is measured at half the spike threshold, the magnitude of the AHP is measured as the difference between the minimum voltage after the peak of the action potential and the spike threshold, the decay of the AHP is measured by an exponential function from the highest point of the AHP to the next 5-10ms, and the time interval between peaks is the time between the peaks of the action potential.

4.2 results of the experiment

Results referring to fig. 4, PTZ-pretreated pyramidal neurons (saline group) showed lower action potential frequency adaptation, resulting in shorter intervals between current peaks. Compared with the cells of the PTZ pretreatment group, the interval between two action potentials of the epileptic cells treated by the ChTX-Q18F is obviously prolonged (p <0.001, n = 11), and even the cells treated by the ChTX-Q18F cannot continuously generate the action potentials at the time of high current injection and simultaneously have more action potentials to be deleted, which is completely different from the results of the PTZ pretreatment group. The ChTX-Q18F treatment group showed longer action potential intervals, wider action potentials and decreased action potential firing frequency compared to PTZ pretreatment (p <0.001 at 200 and 300pA, p <0.05pA curve at 100p, n = 4). At 300pA current, hyperpolarization (AHP) decay rate was slower after the action potential was wider for the ChTX-Q18F group compared to the saline group (p <0.001, n = 11). Meanwhile, after ChTX-Q18F treatment, the AHP size was significantly increased (p <0.001,n =11 at the 300pA curve), and the AHP decay time was significantly extended and the AHP was inhibited from generating the neuron firing threshold. In addition, the number of action potentials and the interval of action potentials were smaller in the epileptic cells treated with ChTX-Q18F than in the ChTX and ChTX-T23F group. These results suggest that the time to reach the neuronal firing threshold for seizure cells treated with ChTX-Q18F is significantly extended.

Example 5: examination of local field potential of hippocampus of rat PTZ (Pantograph-derived hypothalamus) epilepsy model

5.1 Experimental procedure

The local field potential of hippocampus of a rat PTZ epilepsy model is measured by a multi-electrode electroencephalogram technology, and the experimental result is shown in figure 5.

Local field potential data are collected through an OmniPlex in-vivo multichannel recording system (Plexon, hong Kong), a preamplifier amplifies 1000 times, the wave amplitude range is-2- +2V, the filtering range is 1.6-100Hz, the recording frequency of the local field potential is 1kHz, 50Hz high-pass filtering and 300Hz low-pass filtering are set, and the single recording time is not less than 30min.

Before the experiment, 30min of the signals are collected in a waking state as a basic brain electrical level, and then PTZ is injected into the abdominal cavity to induce epileptic seizure and continuously record the epileptic seizure for 1h. During the epileptic seizure, the rat should be observed constantly when the rat moves violently, and if the electrode loosens or falls off, the recording should be stopped in time so as to avoid larger errors or data loss.

The local field potential is exported in a pi 2 format, the Offline Sorter V4 software is used for visual preview, and electroencephalogram data is exported through MATLAB R2015b software. Defining the time from PTZ injection to the first epileptiform discharge as a latent period, and decomposing the wave to obtain four physiological rhythms with different frequencies of delta, theta, alpha, beta and gamma (delta is 0-4Hz, theta is 4-8Hz, alpha is 8-13Hz, beta is 13-30Hz, and gamma is 30-100 Hz); power Spectral Density (PSD) analysis is performed by using pwelch commands, and local field potentials are decomposed into waves of different frequencies according to frequency ranges of delta, theta, alpha, beta and gamma. Welch method, hamming window) and fast Fourier transform are adopted to calculate the frequency domain information of the local field potential of the power spectrum analysis.

The local field potential Power Spectral Density (PSD) is calculated as follows:

5.2 results of the experiment

To determine whether ChTX, and its ChTX-Q18F, chTX-T23F mutants, were able to alleviate seizures, and the effect on Power Spectral Density (PSD) after PTZ-induced convulsions, the Field Potential (FP) signals after PTZ (60 mg/kg) induced convulsions were compared for ChTX and ChTX-Q18F, chTX-T23F, and sodium Valproate (VPA) groups, respectively, and the change in field potential after PTZ induction was shown using a power spectral density heatmap generated by OmniPlex software (Plexon, USA).

The results were analyzed as follows:

the Field Potential (FP) activity of PTZ-induced epileptic rats was rapidly inhibited by the application of ChTX-Q18F compared to the saline group (fig. 5A), while it can be seen from the power spectral density plot that ChTX had no significant effect on the field potential activity of PTZ-induced epileptic rats (fig. 5B), the saline group and ChTX group showed a peak at low frequency (δ) band, chTX-T23F showed a peak at medium and low frequency (θ) band, while the ChTh-Q18F group did not show similar peaks at the same band, indicating that the application of ChTX-Q18F fundamentally changed the neural connection network of neurons responsible for generating low frequency waves.

Statistically, the PSD of the delta wave (p <0.001, n = 3), the theta wave (p <0.001, n = 3) and the alpha wave (p <0.01, n = 3) of ChTX-Q18F is significantly lower than that of the saline group (fig. 5C). Whereas the α, β, γ waves of the ChTX-T23F group (fig. 5c, p <0.05, n = 3) were not significantly different from the saline group. The PSD of the delta wave (p <0.01, n = 3) of ChTX-Q18F was significantly lower than that of the wild-type ChTX group. The low frequency waves after ChTX-T23F administration (delta waves: p <0.05n =3; theta waves: p <0.05, n = 3) were significantly lower than in the saline group.

The experiments jointly show that the ChTX-Q18F toxin can inhibit rat convulsion, and the ChTX-Q18F toxin peptide has antiepileptic effect.

Conclusion

The invention discloses a method for preparing toxin peptide ChTX and mutants ChTX-Q18F and ChTX-T23F thereof by using technologies such as molecular cloning. As a control sample, physiological saline, chTX and ChTX-T23F are used, and the influence of the physiological saline, chTX-Q18F on the rat PTZ recurrent convulsion behavior, the influence on the hippocampal c-Fos expression after the rat convulsion, whether the injury on hippocampal neurons after the rat convulsion, the change on the action potential of the rat hippocampal neurons, the change on the local field potential of the rat PTZ epilepsy model hippocampal is caused or not and the like are considered, so that the ChTX-Q18F toxin has small damage on the hippocampal neurons, can inhibit the rat convulsion and has the antiepileptic effect.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Shanghai institute of organic chemistry, national academy of sciences

<120> scorpion toxin and application of mutant thereof in anti-epilepsy

<130> P2021-1852

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 37

<212> PRT

<213> Leiurus quinquestriatus

<400> 1

Glu Phe Thr Asn Val Ser Cys Thr Thr Ser Lys Glu Cys Trp Ser Val

1 5 10 15

Cys Gln Arg Leu His Asn Thr Ser Arg Gly Lys Cys Met Asn Lys Lys

20 25 30

Cys Arg Cys Tyr Ser

35

<210> 2

<211> 37

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> ChTX-Q18F

<400> 2

Glu Phe Thr Asn Val Ser Cys Thr Thr Ser Lys Glu Cys Trp Ser Val

1 5 10 15

Cys Phe Arg Leu His Asn Thr Ser Arg Gly Lys Cys Met Asn Lys Lys

20 25 30

Cys Arg Cys Tyr Ser

35

<210> 3

<211> 39

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> recombinant ChTX-Q18F polypeptide

<400> 3

Gly Ser Glu Phe Thr Asn Val Ser Cys Thr Thr Ser Lys Glu Cys Trp

1 5 10 15

Ser Val Cys Phe Arg Leu His Asn Thr Ser Arg Gly Lys Cys Met Asn

20 25 30

Lys Lys Cys Arg Cys Tyr Ser

35

<210> 4

<211> 111

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotides ChTX-Q18F

<400> 4

gagttcacca acgttagctg caccacgagt aaagaatgct ggagcgtgtg ctttcgtttg 60

cataatactt ctcgcggtaa gtgcatgaac aagaagtgcc gctgctatag c 111

Claims (10)

1. Use of a scorpion toxin (charybdotoxin) and active fragments thereof, or pharmaceutically acceptable salts thereof, for preparing a preparation or a pharmaceutical composition for treating and/or preventing epilepsy,

the scorpion toxin comprises wild type scorpion toxin or a mutant thereof with an amino acid sequence shown in SEQ ID NO. 1, wherein the mutant comprises a mutant formed by mutating the 18 th glutamic acid point of the wild type scorpion toxin.

2. The use according to claim 1, wherein the mutant is selected from the group consisting of: chTX-Q18F, chTX-Q18Y, chTX-Q18H, chTX-Q18V, or ChTX-Q18L.

3. The use of claim 1, wherein the mutant comprises a mutant ChTX-Q18F in which glutamic acid at position 18 of wild-type scorpion toxin is mutated to phenylalanine.

4. The use according to claim 1, wherein the epilepsy comprises epilepsy resulting from enhanced excitability of the cerebral cortex (or neuronal excitability type epilepsy).

5. A scorpion toxin mutant polypeptide, which is characterized in that the mutant comprises a mutant formed by mutating the 18 th glutamic acid point of wild scorpion toxin with the amino acid sequence shown as SEQ ID NO. 1.

6. A polynucleotide encoding the scorpion toxin mutant polypeptide of claim 5.

7. An expression vector comprising the polynucleotide of claim 6.

8. A host cell comprising the expression vector of claim 7, or having the polynucleotide of claim 6 integrated into its genome.

9. A pharmaceutical composition, comprising:

(i) A first pharmaceutical composition comprising a first active ingredient (Z1) the scorpion toxin mutant polypeptide or active fragment thereof according to claim 5 and a pharmaceutically acceptable carrier;

(ii) A second pharmaceutical composition comprising the second active ingredient (Z2) in addition or alternatively a pharmaceutically active ingredient for the treatment and/or prophylaxis of epilepsy, and a pharmaceutically acceptable carrier.

10. A kit, comprising:

(C1) A first container, and a first pharmaceutical composition comprising a first active ingredient (Z1) the scorpion toxin mutant polypeptide or active fragment thereof of claim 5 and a pharmaceutically acceptable carrier; and

(C2) A second container, and a second pharmaceutical composition in said second container, said second pharmaceutical composition comprising a second active ingredient (Z2) in addition or in addition to a pharmaceutically active ingredient for the treatment and/or prevention of epilepsy, and a pharmaceutically acceptable carrier.

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