WO2020094002A1 - 抗癫痫的毒素Martentoxin及其应用 - Google Patents

抗癫痫的毒素Martentoxin及其应用 Download PDF

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WO2020094002A1
WO2020094002A1 PCT/CN2019/115704 CN2019115704W WO2020094002A1 WO 2020094002 A1 WO2020094002 A1 WO 2020094002A1 CN 2019115704 W CN2019115704 W CN 2019115704W WO 2020094002 A1 WO2020094002 A1 WO 2020094002A1
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epilepsy
martx
pharmaceutical composition
toxin
use according
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PCT/CN2019/115704
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English (en)
French (fr)
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曹春阳
吉永华
刘新莲
陶杰
张淑章
王春喜
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中国科学院上海有机化学研究所
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Priority to US17/291,016 priority Critical patent/US20220040260A1/en
Publication of WO2020094002A1 publication Critical patent/WO2020094002A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1767—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants

Definitions

  • the invention relates to the field of polypeptide medicines, in particular, to the antiepileptic toxin Martentoxin and its application.
  • Epilepsy is a chronic disease that causes transient brain dysfunction and is characterized by repeated episodes caused by sudden abnormal discharge of brain neurons. It is estimated that about 400,000 new patients with epilepsy are added each year. In China, epilepsy has become the second most common disease in neurology after headache.
  • the factors of epilepsy are divided into various factors such as abnormal ion channel function, abnormal neurotransmitter and abnormal glial cell.
  • Ion channels are one of the foundations of excitability regulation in the body. Studies have shown that sodium ion channels, potassium ion channels, calcium ion channels and other channels have a certain correlation with epilepsy.
  • Phenytoin sodium and carbamazepine small molecule drugs can selectively act on voltage-dependent sodium ion channels, block the rapid release of sodium ion-dependent action potentials, and achieve anticonvulsant effects.
  • Metformin is a selective T-type calcium channel blocker that inhibits excessive neuronal excitation.
  • Pirimpanaide is an AMPA-type glutamate receptor ( ⁇ -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor, which mediates rapid excitatory synaptic transmission in the central nervous system) Antagonist, by inhibiting the post-synaptic AMPA receptor glutamate activity, reduce neuronal hyperexcitability, to achieve the prevention and treatment of epilepsy diseases.
  • AMPA-type glutamate receptor ⁇ -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor, which mediates rapid excitatory synaptic transmission in the central nervous system
  • Antagonist by inhibiting the post-synaptic AMPA receptor glutamate activity, reduce neuronal hyperexcitability, to achieve the prevention and treatment of epilepsy diseases.
  • the object of the present invention is to provide a medicine and its application for preventing and / or treating epilepsy with high specificity and / or side effects.
  • MarTX toxin or an active fragment thereof, or a pharmaceutically acceptable salt thereof for preparing a formulation or composition, the formulation or composition is used for treatment and / Or prevent epilepsy.
  • the epilepsy includes epilepsy (or neuronal abnormally excited epilepsy) caused by increased excitability of the cerebral cortex.
  • the epilepsy has the following characteristics: the activity of large-conductance calcium ions and voltage-activated potassium ion channels is enhanced, resulting in increased excitability of the cerebral cortex.
  • the epilepsy includes epilepsy in human and non-human mammals (such as rodents).
  • the epilepsy includes PTZ-induced epilepsy, especially PTZ-induced epilepsy in rats.
  • the MarTX toxin includes recombinant, synthetic or natural MarTX polypeptide.
  • the MarTX toxins include wild-type and mutant MarTX toxins.
  • the MarTX toxin includes the amino acid sequence SEQ ID NO: 2.
  • the MarTX toxin 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.
  • the protein tag is selected from the group consisting of MBP tag, His tag, GST tag, SUMO tag, TRX tag, HA tag, Flag tag, or a combination thereof.
  • the MarTX toxin includes amino acid substitutions, deletions, changes, insertions, or additions of one or more amino acids based on the sequence SEQ ID NO: 2 within the range of maintaining protein activity. sequence.
  • the MarTX toxin includes inserting one or more amino acids at the N-terminus or C-terminus of the sequence SEQ ID NO: 2 within the range of maintaining protein activity, the number of inserted amino acid residues It includes 1 to 10, preferably 1 to 5, more preferably 1 to 3.
  • the MarTX toxin is recombinant.
  • the MarTX toxin is recombinantly expressed in E. coli.
  • amino acid sequence of the MarTX toxin is shown in SEQ ID No .: 3.
  • the composition is a pharmaceutical composition.
  • the pharmaceutical composition contains a pharmaceutically acceptable carrier and (a) MarTX toxin or active fragment thereof.
  • the component (a) accounts for 0.1-99.9% by weight of the total weight of the pharmaceutical composition, preferably 10-99.9% by weight, more preferably 70% -99.9% by weight.
  • the pharmaceutical composition is liquid, solid, or semi-solid.
  • the dosage form of the pharmaceutical composition is an injection, or an external pharmaceutical dosage form.
  • the dosage form of the pharmaceutical composition includes an injection or a lyophilized preparation.
  • the composition is a liquid composition.
  • the carrier is selected from the group consisting of infusion carrier and / or injection carrier.
  • the carrier is one or more carriers selected from the group consisting of physiological saline and glucose Salt water, or a combination thereof.
  • composition or formulation can be used alone or in combination.
  • the combined use includes: in combination with other therapeutic drugs for treating and / or preventing epilepsy.
  • the other therapeutic drugs are selected from the following group:
  • Carbamazepine fluopyridine, gabapentin, lamotrigine, oxcarbazepine, phenytoin, phenytoin sodium, retigabine, topiramate, indochlor, ethosuximide, sodium valproate, or a combination thereof.
  • the dosage form of the pharmaceutical composition is an injection.
  • the pharmaceutical composition is administered intravenously, subcutaneously, intramuscularly or intracranially.
  • the injection is administered by microinfusion pumps.
  • the injection is administered by intracranial administration, preferably by intracerebroventricular (ICV) delivery into the subject.
  • ICV intracerebroventricular
  • the injection is administered to the ipsilateral hippocampus of the subject.
  • the subject includes: a mammal.
  • the mammal includes a human or non-human mammal.
  • the non-human mammal includes rodents (such as rats and mice) and primates (such as monkeys).
  • composition product comprising:
  • a first pharmaceutical composition containing the first active ingredient (a) MarTX toxin or its active fragment and a pharmaceutically acceptable carrier;
  • a second pharmaceutical composition containing the second active ingredient (b) other drugs for treating and / or preventing epilepsy and a pharmaceutically acceptable carrier.
  • the first pharmaceutical composition and the second pharmaceutical composition are the same composition.
  • the pharmaceutical composition product contains:
  • a first active ingredient is MarTX toxin or an active fragment thereof;
  • the first pharmaceutical composition and the second pharmaceutical composition are different compositions.
  • the content of the component (a) is 0.1-99.9wt%, preferably 10-99.9wt%, more preferably 70% -99.9wt%.
  • the other or additional pharmacologically active ingredients for treating and / or epilepsy include:
  • Carbamazepine fluopyridine, gabapentin, lamotrigine, oxcarbazepine, phenytoin, phenytoin sodium, retigabine, topiramate, indomethacin, ethosuximide, sodium valproate, or a combination thereof.
  • a kit in a third aspect of the present invention, includes:
  • the kit also contains (ii) instructions.
  • the active ingredient (b) includes: carbamazepine, fluopyridine, gabapentin, lamotrigine, oxcarbazepine, phenytoin, phenytoin sodium, retigabine, topiramate, indomethacin, Ethylsuccinamine, sodium valproate, or a combination thereof.
  • first container and the second container are the same or different containers.
  • the medicine in the first container is a unilateral preparation containing MarTX toxin or its active fragment.
  • the medicine in the second container is a unilateral preparation containing the medicine for treating and / or preventing epilepsy.
  • the description describes instructions for administering the active ingredient (a) and optionally (b) to treat and / or prevent epilepsy.
  • the description states that the dosage form of the active ingredient (a) and optionally (b) is an injection.
  • the injection is injected into the subject through the lateral ventricle (ICV).
  • IOV lateral ventricle
  • (A, B) represent the saline control group injected with ipsilateral hippocampus and the MarTX toxin experimental group, respectively.
  • (A1, B1) represents the CA1 area
  • (A2, B2) represents the CA3 area
  • (A3, B3) represents the DG area.
  • (C) shows the c-Fos histogram of ipsilateral hippocampus. Compared with saline group, * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001 (one-way ANOVA).
  • (A, B) represent the saline control group and the MarTX toxin experimental group injected with heterolateral hippocampus, respectively.
  • (A1, B1) represents the CA1 area
  • (A2, B2) represents the CA3 area
  • (A3, B3) represents the DG area.
  • (C) H-horse hippocampus c-Fos histogram. Compared with saline group, * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001 (one-way ANOVA).
  • Figure 4 The effect of MarTX toxin on PTZ-induced injection of ipsilateral hippocampal neurons.
  • (A, B) represent the saline control group injected with ipsilateral hippocampus and the MarTX toxin experimental group, respectively.
  • (A1, B1) represents the CA1 area
  • (A2, B2) represents the CA3 area
  • (A3, B3) represents the DG area.
  • (C) Statistical graph showing the number of neurons stained with ipsilateral hippocampus. Compared with saline group, * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001 (one-way ANOVA).
  • Figure 5 The effect of MarTX toxin on PTZ-induced injection of heterolateral hippocampal neurons.
  • (A, B) represent the saline control group and the MarTX toxin experimental group injected with heterolateral hippocampus, respectively.
  • (A1, B1) represents the CA1 area
  • (A2, B2) represents the CA3 area
  • (A3, B3) represents the DG area.
  • (C) Histogram of the number of neurons stained neurons injected into the lateral hippocampus. Compared with saline group, * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001 (one-way ANOVA).
  • Figure 6 The inhibitory effect of MarTX toxin on PTZ-induced field potential power spectral density in rat hippocampus.
  • (A) shows the field potential signal and the spectral heat map of the rat PTZ epilepsy model in normal saline group (black) and MarTX toxin group (red), respectively.
  • (B) shows the relationship between the power spectral density of the PTZ epilepsy model of the normal saline group (black) and the MarTX toxin group (red) rats and the brain wave frequency.
  • (C) The power spectral density values of the brain waves of ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ waves at different frequencies in the rat PTZ epilepsy model in the saline group (black) and MarTX toxin group (red) respectively.
  • * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001 one-way ANOVA.
  • Figure 7 The inhibitory effect of MarTX toxin on the action potential frequency of rat hippocampal neurons induced by PTZ.
  • A Modulation of normal saline (black) and MarTX toxin (red) on the action potential of hippocampal neurons pretreated with PTZ.
  • B The number of action potentials caused by each current injection.
  • C The width of the action potential calculated at half the height of the action potential when the 300pA current is injected.
  • D Post-hyperpolarization potential amplitude calculated from the pre-spike potential of the action potential to the post-hyperpolarization peak at 300 pA current injection.
  • E During the 300pA current injection period, the action potential interval before the ninth action potential. Compared with saline group, * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001 (one-way ANOVA).
  • MarTX toxin a polypeptide substance
  • the present inventors unexpectedly discovered for the first time that a polypeptide substance (ie, MarTX toxin) can be used for extremely effective treatment and / Or prevent epilepsy.
  • a polypeptide substance ie, MarTX toxin
  • the inventors measured the effect of MarTX toxin on the behavior of recurrent convulsions, the effect on the expression of c-Fos in the hippocampus after convulsive seizures in rats, and the investigation of neuronal damage in the hippocampus
  • MarTX toxin can effectively relieve epilepsy symptoms.
  • the present invention has been completed.
  • Martentoxin protein As used herein, "Martentoxin protein”, “Martentoxin polypeptide”, “MarTX toxin” and “MarTX polypeptide” “recombinant MarTX toxin”, “Marten toxin” are used interchangeably and refer to the Martentoxin protein.
  • the wild-type Martentoxin protein is composed of 37 amino acids, has three pairs of disulfide bonds, and is a short-chain polypeptide toxin.
  • the term includes not only wild type but also mutant type; not only includes naturally isolated Martentoxin protein, but also recombinant Martentoxin protein, such as recombinantly expressed Martentoxin protein with or without initial Met, And recombinantly expressed Martentoxin protein with or without expression tags or 1-3 amino acid residues.
  • Martentoxin can be separated and purified from the venom of Buthus martensi Karsch (BmK).
  • MarTX polypeptides provided in the examples of the present invention are derived from Scorpio scorpioides, they are derived from other similar species (especially scorpions belonging to the same family or genus as Scorpio scorpioides), and the sequences of the present invention ( Preferably, MarTX polypeptides with a certain homology (conservation) whose sequence is shown in SEQ ID NO: 2 can also be used in the present invention.
  • genes provided in the examples of the present invention are derived from Scorpio scorpioides, they are derived from other similar species (especially scorpions belonging to the same family or genus as Scorpio scorpioides). Ground, the sequence is shown in SEQ ID NO: 1)
  • the gene sequence of MarTX with certain homology (conservative) is also included in the scope of the present invention, as long as a person skilled in the art reads this application The information provided can easily isolate this sequence from other species (especially scorpions).
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • the form of DNA includes: DNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • the coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • Polynucleotides encoding mature polypeptides include: coding sequences encoding only mature polypeptides; mature polypeptide coding sequences and various additional coding sequences; mature polypeptide coding sequences (and optional additional coding sequences) and non-coding sequences.
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or a polynucleotide further including additional coding and / or non-coding sequences.
  • the present invention also relates to variants of the aforementioned polynucleotides, which encode fragments, analogs and derivatives of polyglycosides or polypeptides having the same amino acid sequence as the present invention.
  • This polynucleotide variant may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants.
  • allelic variant is a form of substitution of a polynucleotide. It may be a substitution, deletion, or insertion of one or more nucleotides, but it will not substantially change the function of the polypeptide it encodes. .
  • the 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.
  • stringent conditions means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60 ° C; or (2) There are denaturants, such as 50% (v / v) methylphthalamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc .; or (3) Only the identity between the two sequences is at least 90%, More preferably, the hybridization occurs only when it exceeds 95%.
  • MarTX gene of the present invention is preferably derived from Scorpion scorpion, other species (especially scorpions) are highly homologous (e.g., have 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 aligning sequence identity are also well known in the art, such as BLAST.
  • the full-length MarTX nucleotide sequence of the present invention or a fragment thereof can generally be obtained by PCR amplification method, recombination method or artificial synthesis method.
  • primers can be designed according to the relevant nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available DNA library or cDNA prepared according to conventional methods known to those skilled in the art
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
  • the relevant sequence can be obtained in large quantities by the recombination method. It is usually cloned into a vector, and then transferred into cells, and then the relevant sequences are isolated from the proliferated host cells by conventional methods.
  • synthetic methods can be used to synthesize the relevant sequences, especially when the length of the fragments is short.
  • a long sequence can be obtained by synthesizing multiple small fragments and then connecting them.
  • the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequence of the invention by chemical synthesis.
  • the present invention relates to a MarTX polypeptide and its variants for treating epilepsy.
  • 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.
  • the present invention also includes 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, and most preferably 98 % Or more, such as 99%) homologous polypeptides or proteins with the same or similar functions.
  • the "same or similar function” mainly refers to: “alleviates the symptoms of epilepsy”.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide.
  • the polypeptide of the present invention may be a naturally purified product, or a chemically synthesized product, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated or may be non-glycosylated.
  • the polypeptide of the present invention may or may not include the starting methionine residue.
  • the present invention also includes MarTX polypeptide fragments and analogs having MarTX polypeptide activity.
  • fragment and “analog” refer to a polypeptide that substantially retains the same biological function or activity of the natural MarTX polypeptide of the present invention.
  • the polypeptide fragment, derivative or analog of the present invention may be: (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues
  • the group may or may not be encoded by the genetic code; or (ii) a polypeptide having a substitution group in one or more amino acid residues; or (iii) a mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, For example, a polypeptide formed by fusion of polyethylene glycol; or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or a secretion sequence or a sequence or proprotein sequence used to purify the polypeptide, or Fusion protein).
  • These fragments, derivatives and analogs are within the scope of those skilled in the art according to the definitions herein.
  • the polypeptide variant is the amino acid sequence shown in SEQ ID NO .: 2, after several (usually 1-10, preferably 1-8, more preferably 1-4) , Optimally 1-2) derived, substituted or deleted at least one amino acid derived derivative sequence, and the C-terminal and / or N-terminal to add one or several (usually within 10, preferably within 5 , More preferably within 3) amino acids.
  • SEQ ID NO .: 2 amino acid sequence shown in SEQ ID NO .: 2
  • Optimally 1-2) derived, substituted or deleted at least one amino acid derived derivative sequence and the C-terminal and / or N-terminal to add one or several (usually within 10, preferably within 5 , More preferably within 3) amino acids.
  • the function of the protein is usually not changed.
  • Adding one or several (such as 1-3) amino acids at the C-terminus and / or N-terminus usually also Does not change the function of the protein.
  • substitution Ala 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; Thr
  • the invention also includes analogs of the claimed proteins.
  • the difference between these analogues and the natural SEQ ID NO: 2 can be the difference in amino acid sequence, the difference in the modification form that does not affect the sequence, or both.
  • Analogues 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 mutagen, or by site-directed mutagenesis or other known molecular biology techniques. Analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), as well as analogs with non-naturally occurring or synthetic amino acids (such as ⁇ , ⁇ -amino acids). It should be understood that the protein of the present invention is not limited to the representative proteins listed above.
  • Modified (usually without changing the primary structure) forms include: in vivo or in vitro chemically derived forms of proteins such as acetate or carboxylation. Modifications also include glycosylation, such as those that are modified during protein synthesis and processing. This modification can be accomplished by exposing the protein to an enzyme that performs glycosylation (such as mammalian glycosylation or deglycosylation enzymes). Modified forms also include sequences with phosphorylated amino acid residues (eg phosphotyrosine, phosphoserine, phosphothreonine).
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of the following active ingredients: MarTX toxin or its active fragments and its active ingredients for treating and / or preventing epilepsy.
  • the term "effective amount” or “effective dose” refers to an amount that can produce a function or activity in humans and / or animals and is acceptable to humans and / or animals.
  • pharmaceutically acceptable ingredients are suitable for humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergies), that is, substances that have a reasonable benefit / risk ratio.
  • 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 carriers include (but are not limited to): saline, buffer, glucose, water, glycerin, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched with the mode of administration, and the dosage form of the pharmaceutical composition of the present invention is an injection.
  • it can be prepared by normal methods 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 according to the present invention may vary with the mode of administration and the severity of the disease to be treated.
  • the selection of a preferred effective amount can be determined by those of ordinary skill in the art based on various factors (eg, through clinical trials).
  • the factors include but are not limited to: the pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc .; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, administration Way, etc. For example, due to the urgent requirements of the treatment situation, separate doses can be given several times a day, or the dose can be reduced proportionally.
  • the pharmaceutically acceptable carriers of the present invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or Its combination.
  • the choice of carrier should match the mode of administration, which is well known to those of ordinary skill in the art.
  • the first active active ingredient (a) MarTX toxin or active fragment thereof 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) described therein is an antiepileptic drug available in the prior art, which includes but is not limited to: carbamazepine, fluopyridine, gabapentin, lamotrigine, oxcarbazepine, phenytoin, phenytoin Sodium, retigabine, topiramate, indomethacin, ethosuximide, sodium valproate, or a combination thereof.
  • the c-Fos gene is one of the immediate early genes [Trends Neurosci. 1995, 18, 66-67] (IEGs, proto-oncogenes that can be induced by the second messenger), and it is associated with various pathophysiological processes after the onset of epilepsy Closely related. Under physiological conditions, the c-Fos gene is expressed at low levels in the central nervous system; when neurons are stimulated by physical or chemical stimulation, neuron excitement activates c-Fos and other IEGs, and the mRNA generated by the transcription of IEGs is translated into c-Fos Protein [Annu. Rev. Neurosci. 1991, 14, 421-451], etc.
  • This protein can regulate the expression of various late-response genes (LRGs), and at the same time lead to seizures through various mechanisms, and eventually lead to the formation of epileptic lesions [Neuron1990, 4,477-485].
  • LRGs late-response genes
  • c-Fos protein is rapidly expressed in large quantities during seizures. If drugs or other treatments are used to control seizures, the expression of this protein can be significantly inhibited. Therefore, c-Fos expression measurement can be used as an antiepileptic drug mechanism of action and efficacy evaluation Of effective indicators.
  • Nissl body is a special susceptible substance found in the cytoplasm of neuronal dendrites. Nissl body usually has a fixed shape, the neuron cell body is large, the cytoplasm is pale and stained uniformly, the nucleus is large and round, and all cells are stained deep. In the hippocampal dentate gyrus (DG) area, the nerve pyramidal cells are closely packed, and the cells are 4 or 5 layers. However, when the brain is damaged, their morphology changes, and the blurring of the cell edges reduces the integrity of the cell. At the same time, the cell body staining becomes lighter, the distribution of neurons becomes disordered, and the number of cells or cell layers becomes less. Therefore, the existence, distribution and pathological changes of neurons can be recognized by the morphology and number of Nissl bodies.
  • DG hippocampal dentate gyrus
  • Pentylenetetrazole also known as Pentetrazol, pentatetrazole, and caradiazole, is a white crystalline powder chemical.
  • the chemical name is 1,5-pentamethylene-1H-tetrazole, the molecular formula is C 6 H 10 N 4 , and the molecular weight is 138.1704.
  • Pentylenetetrazole is a central stimulant. It is mainly used clinically to rescue central respiratory failure caused by severe barbiturates and narcotic poisoning; it can also be used for acute infectious diseases, narcotic and barbiturate poisoning Respiratory depression and acute circulatory failure caused by time.
  • Pentylenetetrazole can excite the respiratory center and cardiovascular sports center, and its effect is rapid and strong, which accelerates the deepening of breathing and the blood pressure rises slightly; at a slightly larger dose, the excitement can extend to the cerebral cortex and spinal cord, causing convulsions.
  • PTZ mainly acts on the chloride channel of GABA A receptor ( ⁇ -aminobutyric acid type A receptor), inhibits the activity of GABA neurons, so that the excitability of the nervous system is excessively enhanced, inducing clonic or whole body rigidity in animals and human Seizures.
  • GABA A receptor ⁇ -aminobutyric acid type A receptor
  • animals are induced by intraperitoneal injection of PTZ to induce convulsions / epilepsy, so as to conduct subsequent experimental studies on the effect of the MarTX toxin of the present invention on epilepsy.
  • MarTX toxin has the characteristics of strong effect and small dosage (the dosage can be as low as 0.32 ⁇ g / kg in the rat PTZ model), which not only significantly prolongs the incubation period of seizures, but also significantly relieves the symptoms of epilepsy (especially can Reduce the incidence of grade 3, 4 and 5 epilepsy), so it can be used as a new peptide drug for epilepsy treatment.
  • the recombinant MarTX toxin of the present invention can be prokaryotically expressed and recombinantly prepared, and is expected to provide a safe, effective, and inexpensive antiepileptic drug for epilepsy patients.
  • MarTX toxin is a neurotoxin with a very small molecular weight (only about 4KDa), which can effectively pass through the blood-brain barrier and act on the brain.
  • MarTX toxin is a highly specific neurotoxin, selective for ⁇ + ⁇ 4 subtype ion channels, large conductance calcium ions, and voltage-activated potassium ion channels, but not for cardiovascular, endocrine, and reproductive systems. Impact, so side effects are small.
  • MarTX toxin is the first peptide substance that has been proven to have epilepsy-relieving effects by experiments.
  • Experimental animals Adult male SD rats (provided by Shanghai Experimental Animal Center, Chinese Academy of Sciences), weighing 250-300g, 5 per cage, reared under routine laboratory conditions, room temperature maintained at 22 ⁇ 1 °C, natural circadian rhythm.
  • Pentylenetetrazole (PTZ, CAS: 54-95-5), purchased from Sigma Corporation of the United States.
  • the rats were fixed on a stereotaxic instrument (NS-2, Narishige, Japan), the hair on the top of the head was shaved and the scalp was disinfected, the skin was cut and treated with 10% hydrogen peroxide
  • the subcutaneous tissue was burned to expose the Bregma point of the skull, and the implantation point of the drug delivery base (AP-4.3mm, L2.2mm) was determined on the skull according to the localization map of the rat brain.
  • a small hole (1mm) was drilled with a dental drill, the inner plate was peeled off, the dura mater was removed by the needle tip, and the drug-based base sleeve was implanted into the hippocampal CA1 area 2.5mm below the skull.
  • the base is fixed to the surface of the rat skull by dental cement.
  • fusion protein (sequence such as SEQ ID No .: 5) was obtained, and MarTX polypeptide was prepared by the following method.
  • the fusion protein in the Buffer A environment is first combined with the nickel column, and then the gradient elution is performed with the Buffer B imidazole salt solution to remove most of the contaminated proteins, the first affinity column is purified and subjected to SDS-PAGE electrophoresis detection.
  • the FL component and Buffer D washing solution (usually containing a small amount of recombinant MarTX toxin) are concentrated to 2 mL for the next gel chromatography column purification.
  • Example 1 The effect of MarTX toxin on the behavior of PTZ relapse and convulsion in rats
  • Level 1 rhythmic twitching of the mouth and face
  • Level 3 Full-body myoclonus, buttocks upturn
  • Level 4 Turn the body to one side
  • Level 5 Suspended position, seizures of tonic spasticity.
  • a complete convulsive seizure is defined as the time from the onset of the seizure to the return to normal after the seizure. When the interval between seizures reaches more than 5s, it is defined as another independent seizure.
  • the incubation period is defined as the time after the injection of PTZ to the beginning of the first grade 2 seizure.
  • the MarTX toxin of the present invention exhibits a significant inhibitory effect during the incubation period, duration of seizures, and the number of seizures of different convulsive seizure severity, indicating that the MarTX toxin effectively relieves neuronal abnormal excitement and epilepsy symptoms.
  • Example 2 Effect of MarTX toxin on the expression of c-Fos in hippocampus after seizures in rats
  • the animals were anesthetized by intraperitoneal injection of pentobarbital sodium (60 mg / kg). Wash the blood vessels with 200 mL of normal saline through the ascending aorta of the left ventricle, then perfuse with 400 mL of fixative solution (0.1 M PBS containing 40% paraformaldehyde, pH 7.4, 4 ° C) for 1-2 h, and remove the brain tissue in the same After overnight in the fixing solution, move to 20% sucrose solution and soak until it sinks to the bottom of the container, and then immerse in 30% sucrose solution until it sinks to the bottom of the container.
  • fixative solution 0.1 M PBS containing 40% paraformaldehyde, pH 7.4, 4 ° C
  • the brain tissue of the rat was sliced into the hippocampus with a constant cold box microtome (Leica 1900, Germany). The thickness of the slice was 20 ⁇ m. It was pasted on a glass slide treated with gelatin-chromium potassium sulfate, and stored at -20 ° C. .
  • the steps of c-Fos immunohistochemistry are as follows:
  • DAB-nickel amine sulfate-glucose oxidase method (10) DAB-nickel amine sulfate-glucose oxidase method (DAB, biotechnology) staining for 10 min in the dark;
  • A represents the number of FLI neurons in the saline control group
  • B represents the number of FLI neurons in the MarTX toxin injection group corresponding to the hippocampus.
  • Rat hippocampus can be divided into hippocampal gyrus and dentate gyrus (DG) according to different cell morphology.
  • the hippocampus mainly includes CA1 (cornuammonis), CA2, CA3 area and portal area, which is mainly composed of some pyramidal neurons, in which CA1 area is connected with the lower support, and the portal area is adjacent to the dentate gyrus.
  • the dentate gyrus is a C-shaped cortical layer between the hippocampus fissure and the hippocampus umbrella. Its structure is divided into three layers: molecular layer, granular cell layer and polymorphic layer, basically composed of granular cells. For the specific location, see Figure 1 [The Rat Brain in stereotaxic coordinates. 2007].
  • c-Fos positive neurons are mainly concentrated in the granular cell layer of the hippocampus DG area, and there are fewer positive neurons in the CA1 and CA3 areas;
  • MarTX toxin has a significant inhibitory effect on the expression of c-Fos protein in rat hippocampus after PTZ-induced epilepsy, and the ipsilateral injection effect is stronger than the heterolateral hippocampus, indicating that the MarTX toxin of the present invention may have antiepileptic drug effects.
  • the slices are dehydrated with 70%, 80%, 95%, and 100% alcohol for 2 minutes each time, and then soaked in xylene for 2 times for 5 minutes each time;
  • the analysis results are as follows: (1) The MarTX toxin group retains a relatively complete hippocampal structure. The neurons in the hippocampus, especially the DG area, have the highest cell density and tight arrangement, and the nicotine staining is the deepest. 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 caused the greatest damage to the neurons in the saline control group, and the MarTX toxin of the present invention protected the post-epileptic Hippocampal neurons with minimal damage;
  • PTZ-induced epilepsy will cause damage or death of rat hippocampal neurons. It was found that the degree of neuron damage in the experimental group injected with MarTX was greatly reduced, and the number of neurons stained by Nissan was the largest and arranged the most closely.
  • Implanted microarray electrodes with the sixth edition of the "Three-Dimensional Atlas of Rat Brain” written by George Paxinos and Charles Watson ( Figure 1) to determine the position of the electrodes: AP: 4.3 mm, MR: 2.2 mm, DV: 2.5 mm, Fix 3-4 screws in the blank area of the skull as reference electrodes.
  • the recording electrode and electrode base are fixed with dental cement.
  • the field potential (FP) was recorded after the rat was awake.
  • FP signals and synchronized video can be recorded through Omniplex (plexon, USA).
  • the rear end of the electrode is connected to a preamplifier and coupled to an analog-to-digital converter box.
  • the patch clamp current is considered acceptable.
  • the record was kept at -80mV as the holding current and induced by increasing the positive current injection (for 1000ms).
  • the judgment compares the difference in action potential between PTZ-treated cells (measured 24 hours after application) and PTZ-treated epilepsy cells treated with rMarTX (measured 10 minutes after application).
  • the peak width was measured at the peak amplitude of half of the action potential.
  • the difference between the peak threshold and the minimum voltage after the peak of the action potential is taken as the measured value of the post-supercharge potential.
  • the peak interval is the time between the peaks of the action potential.
  • the standard external solution consists of 1.2 mM MgCl 2 , 10 mM HEPES, 10 mM glucose, 1.5 mM CaCl 2 , 2.5 mM KCl, and 145 mM NaCl.
  • the pH of the solution was adjusted to 7.4 with NaOH.
  • the internal fluid consisted of 1 mM CaCl 2 , 4 mM MgCl 2 , 10 mM HEPES, 11 mM EGTA and 140 mM KCl.
  • the pH of the solution was adjusted to 7.2 with KOH.
  • Pentylenetetrazole induces BK channel function gain and high discharge rate of neocortical and hippocampal pyramidal neurons. Therefore, we compared the changes in action potential characteristics caused by current injection in PTZ-induced pre-epilepsy cells after application of saline and MarTX (Figure 7A-E). 7A shows an example of the action potential curve of neurons after the application of normal saline and MarTX pretreated with PTZ. Current injection into hippocampal pyramidal neurons can induce the generation of action potentials, and its trigger frequency may be limited by two mechanisms.

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Abstract

本发明提供了抗癫痫的毒素Martentoxin及其衍生物的应用。具体地，本发明提供了Martentoxin（简称MarTX毒素）或其活性片段、或其药学上可接受的盐的用途，其被用于制备治疗/或预防癫痫的制剂或组合物。

Description

抗癫痫的毒素Martentoxin及其应用 技术领域
本发明涉及多肽药物领域，具体地，涉及抗癫痫的毒素Martentoxin及其应用。
背景技术
癫痫(epilepsy)是一种导致短暂的大脑功能障碍的慢性疾病，以大脑神经元突发性异常放电引起反复发作为特征。据估计每年新增加癫痫患者约40万，在中国癫痫已成为神经科仅次于头痛的第二大常见病。
癫痫的发病机制非常复杂，然而一般认为，中枢神经系统兴奋与抑制之间的不平衡将导致癫痫发作。
基于异常放电的起始部位和传递方式的不同，癫痫的发病因素分为离子通道功能异常、神经递质异常、神经胶质细胞异常等各种不同的因素。
离子通道是体内组织兴奋性调节的基础之一。研究表明，钠离子通道、钾离子通道、钙离子通道等通道与癫痫有一定的相关性。
针对癫痫多样的发病机制，研究人员发展了各类不同作用机制和不同靶标的抗癫痫药物。苯妥英钠、卡马西平等小分子药物可选择性作用于电压依赖性钠离子通道，阻断钠离子依赖性动作电位的快速发放，达到抗惊厥作用。三甲双酮药物为选择性T型钙离子通道阻断剂，抑制神经元的过度兴奋。吡仑帕奈是一种AMPA型谷氨酸受体(α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体，介导中枢神经系统快速兴奋性突触传递)拮抗剂，通过抑制突触后AMPA受体谷氨酸活性，减少神经元过度兴奋，达到预防和治疗癫痫疾病。
然而，目前的癫痫治疗药物的效果还难以令人满意。例如，大多数小分子药物长期服用副作用很大。此外，引起癫痫的病因多种多样，因此目前的小分子药物难以对症下药。
因此，本领域迫切需要开发新的特异性高和/或副作用小的、用于预防和/或治疗癫痫的药物。
发明内容
本发明的目的就是提供了特异性高和/或副作用小的、用于预防和/或治疗癫痫的药物及其应用。
在本发明的第一方面，提供了一种MarTX毒素或其活性片段、或其药学上可接受的盐的用途，用于制备一制剂或组合物，所述制剂或组合物用于治疗和/或预防癫痫。
在另一优选例中，所述的癫痫包括大脑皮层兴奋性增强所导致的癫痫(或神经元异常兴奋型癫痫)。
在另一优选例中，所述癫痫具有以下特征：大电导钙离子和电压激活的钾离子通道的活性增强，从而导致大脑皮层的兴奋性增强。
在另一优选例中，所述的癫痫包括人和非人哺乳动物(如啮齿动物)的癫痫。
在另一优选例中，所述的癫痫包括PTZ诱导型癫痫，尤其是大鼠的PTZ诱导型癫痫。
在另一优选例中，所述的MarTX毒素包括重组的、人工合成的或天然的MarTX多肽。
在另一优选例中，所述的MarTX毒素包括野生型和突变型的MarTX毒素。
在另一优选例中，所述的MarTX毒素包括氨基酸序列SEQ ID NO:2。
在另一优选例中，所述MarTX毒素包括在保持蛋白活性范围内，在序列SEQ ID NO:2的N端或C端带有一个或多个蛋白标签的重组蛋白。
在另一优选例中，所述蛋白标签选自下组：MBP标签、His标签、GST标签、SUMO标签、TRX标签、HA标签、Flag标签，或其组合。
在另一优选例中，所述MarTX毒素包括在保持蛋白活性范围内，在序列SEQ ID NO:2的基础上进行一个或多个氨基酸的替换、缺失、改变、插入或增加，所得到的氨基酸序列。
在另一优选例中，所述MarTX毒素包括在保持蛋白活性范围内，在序列SEQ ID NO:2的N端或C端进行一个或多个氨基酸的插入，所述插入的氨基酸残基个数包括1个至10个，较佳地1个至5个，更佳地1个至3个。
在另一优选例中，所述的MarTX毒素是重组的。
在另一优选例中，所述的MarTX毒素是大肠杆菌中重组表达的。
在另一优选例中，所述的MarTX毒素的氨基酸序列如SEQ ID No.:3所示。
在另一优选例中，所述组合物为药物组合物。
在另一优选例中，所述的药物组合物含有药学上可接受的载体以及 (a)MarTX毒素或其活性片段。
在另一优选例中，所述组分(a)占所述药物组合物总重量的0.1-99.9wt％,较佳地10-99.9wt％，更佳地70％-99.9wt％。
在另一优选例中，所述药物组合物为液态、固体、或半固体。
在另一优选例中，所述的药物组合物的剂型为注射剂、或外用药物剂型。
在另一优选例中，所述药物组合物的剂型包括注射剂或冻干制剂。
在另一优选例中，所述的组合物为液态组合物。
在另一优选例中，所述的载体选自下组：输液剂载体和/或注射剂载体，较佳地，所述的载体是选自下组的一种或多种载体：生理盐水、葡萄糖盐水、或其组合。
在另一优选例中，所述组合物或制剂可单独使用，或联合使用。
在另一优选例中，所述的联合使用包括：与治疗和/或预防癫痫的其它治疗药物联合使用。
在另一优选例中，所述的其他治疗药物选自下组：
卡马西平、氟吡啶、加巴喷丁、拉莫三嗪、奥卡西平、苯妥英、苯妥英钠、瑞替加滨、托吡酯、颠扑净、乙琥胺、丙戊酸钠、或其组合。
在另一优选例中，所述药物组合物的剂型为注射剂。
在另一优选例中，所述的药物组合物通过静脉内、皮下、肌内或颅内方式给药。
在另一优选例中，所述注射剂通过微量输液泵(microinfusion pumps)进行施用。
在另一优选例中，所述的注射剂通过颅内给药进行施用，较佳地通过侧脑室注射(intracerebroventricular(ICV)delivery)入受试者体内。
在另一优选例中，所述注射剂被施用于受试者的同侧海马。
在另一优选例中，所述受试者包括：哺乳动物。
在另一优选例中，所述的哺乳动物包括人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括：啮齿动物(如大鼠、小鼠)、灵长动物(如猴)。
在本发明的第二方面，提供了一种组合物产品，所述组合物产品包括：
(i)第一药物组合物，所述第一药物组合物含有第一活性成分(a)MarTX毒素或其活性片段和药学上可接受的载体；
(ii)第二药物组合物，所述的第二药物组合物含有第二活性成分(b)其他治疗和/或预防癫痫的药物和药学上可接受的载体。
在另一优选例中，所述的第一药物组合物和第二药物组合物是同一组合物。
在另一优选例中，所述的药物组合物产品含有：
(a)第一活性成分，所述第一活性成分为MarTX毒素或其活性片段；
(b)第二活性成分，所述第二活性成分为其他的或额外的治疗和/或癫痫的药物活性成分；
(c)药学上可接受的载体。
在另一优选例中，所述的第一药物组合物和第二药物组合物是不同的组合物。
在另一优选例中，所述组分(a)的含量为0.1-99.9wt％,较佳地10-99.9wt％，更佳地70％-99.9wt％。
在另一优选例中，所述其他的或额外的治疗和/或癫痫的药物活性成分包括：
卡马西平、氟吡啶、加巴喷丁、拉莫三嗪、奥卡西平、苯妥英、苯妥英钠、瑞替加滨、托吡酯、颠扑净、乙琥胺、丙戊酸钠、或其组合。
在本发明的第三方面，提供了一种药盒，所述药盒包括：
(i)第一容器，以及位于所述第一容器中第一药物组合物，所述第一药物组合物含有第一活性成分(a)MarTX毒素或其活性片段和药学上可接受的载体；
(ii)第二容器，以及位于所述第二容器中第二药物组合物，所述的第二药物组合物含有第二活性成分(b)其他治疗和/或预防癫痫的药物和药学上可接受的载体。
在另一优选例中，所述的药盒还含有(ii)说明书。
在另一优选例中，所述活性成分(b)包括：卡马西平、氟吡啶、加巴喷丁、 拉莫三嗪、奥卡西平、苯妥英、苯妥英钠、瑞替加滨、托吡酯、颠扑净、乙琥胺、丙戊酸钠、或其组合。
在另一优选例中，所述的第一容器和第二容器是相同或不同的容器。
在另一优选例中，所述的第一容器的药物是含MarTX毒素或其活性片段的单方制剂。
在另一优选例中，所述的第二容器的药物是含其治疗和/或预防癫痫的药物的单方制剂。
在另一优选例中，所述说明书中记载了给予活性成分(a)和任选地(b)从而治疗和/或预防癫痫的说明。
在另一优选例中，所述说明书中记载了所述活性成分(a)和任选地(b)的剂型为注射剂。
在另一优选例中，所述注射剂通过(ICV)侧脑室注射入受试者体内。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1大鼠脑图谱[The rat brain in stereotaxic coordinates.2007]。
图2 MarTX毒素对PTZ诱导的注射同侧海马的c-Fos表达抑制效果。
(A，B)分别代表注射同侧海马的生理盐水对照组和MarTX毒素实验组。(A1、B1)代表CA1区，(A2、B2)代表CA3区，(A3、B3)代表DG区。(C)表示注射同侧海马的c-Fos柱状统计图。与生理盐水组相比*p<0.05，**p<0.01，***p<0.001(one-way ANOVA)。
图3 MarTX毒素对PTZ诱导的注射异侧海马的c-Fos表达抑制效果。
(A，B)分别代表注射异侧海马的生理盐水对照组和MarTX毒素实验组。(A1、B1)代表CA1区，(A2、B2)代表CA3区，(A3、B3)代表DG区。(C)表示注射异侧海马的c-Fos柱状统计图。与生理盐水组相比*p<0.05，**p<0.01，***p<0.001(one-way ANOVA)。
图4 MarTX毒素对PTZ诱导的注射同侧海马神经元的影响。
(A，B)分别代表注射同侧海马的生理盐水对照组和MarTX毒素实验组。 (A1、B1)代表CA1区，(A2、B2)代表CA3区，(A3、B3)代表DG区。(C)表示注射同侧海马的尼式体染色神经元数目统计图。与生理盐水组相比*p<0.05，**p<0.01，***p<0.001(one-way ANOVA)。
图5 MarTX毒素对PTZ诱导的注射异侧海马神经元的影响。
(A，B)分别代表注射异侧海马的生理盐水对照组和MarTX毒素实验组。(A1、B1)代表CA1区，(A2、B2)代表CA3区，(A3、B3)代表DG区。(C)表示注射异侧海马的尼式体染色神经元数目柱状统计图。与生理盐水组相比*p<0.05，**p<0.01，***p<0.001(one-way ANOVA)。
图6 MarTX毒素对PTZ诱导的大鼠海马区场电位功率谱密度的抑制效果。
(A)分别显示了生理盐水组(黑色)和MarTX毒素组(红色)大鼠PTZ癫痫模型的场电位信号和光谱热图。(B)分别显示了生理盐水组(黑色)和MarTX毒素组(红色)大鼠PTZ癫痫模型的功率谱密度与脑电波频率的关系曲线。(C)分别显示了生理盐水组(黑色)和MarTX毒素组(红色)大鼠PTZ癫痫模型的在不同频率δ、θ、α、β、γ波脑电波的功率谱密度值。与生理盐水组相比*p<0.05，**p<0.01，***p<0.001(one-way ANOVA)。
图7 MarTX毒素对PTZ诱导的大鼠海马神经元动作电位频率的抑制效果。
(A)生理盐水(黑色)、MarTX毒素(红色)对经PTZ预处理的海马神经元动作电位的调制作用。(B)每次电流注入时引发的动作电位数量。(C)300pA电流注入时，在动作电位一半高度处计算的动作电位宽度。(D)在300pA电流注入时，从动作电位尖峰前电位到超极化后峰值计算的后超极化电位振幅。(E)在300pA电流注入期间，第9个动作电位之前的动作电位间隔。与生理盐水组相比*p<0.05，**p<0.01，***p<0.001(one-way ANOVA)。
具体实施方式
本发明人经过广泛而深入的研究，通过对大量不同化合物的筛选(包括针对钠离子通道等化合物的大量筛选)，首次意外地发现一种多肽物质(即MarTX毒素)可用于极其有效地治疗和/或预防癫痫。实验表明，在大鼠PTZ(pentetrazol)惊厥模型中，本发明人通过测定MarTX毒素对复发惊厥行为的影响、对大鼠惊厥发作后海马c-Fos表达的影响以及对海马神经元损伤情况的考察，首次发现MarTX毒素可有效缓解癫痫症状。在此基础上完成了本发明。
Martentoxin蛋白及其编码序列
如本文所用，“Martentoxin蛋白”、“Martentoxin多肽”、“MarTX毒素”和“MarTX多肽”“重组MarTX毒素”、“马腾毒素”可互换使用，指Martentoxin蛋白。野生型的Martentoxin蛋白由37个氨基酸构成，具有三对二硫键，是一种短链多肽毒素。在本发明中，所述术语不仅包括野生型，还包括突变型；不仅包括天然分离的Martentoxin蛋白，也包括重组的Martentoxin蛋白，例如重组表达的带有或不带有起始Met的Martentoxin蛋白，以及重组表达的带有或不带有表达标签或酶切残留的1-3个氨基酸的Martentoxin蛋白。
对于分离而言，Martentoxin可从东亚钳蝎(Buthus martensi Karsch,BmK)毒液中分离纯化得到。
对于重组而言，可通过常规的重组技术，在大肠杆菌等宿主细胞中进行表达，并经分离纯化而获得。
野生型Martentoxin蛋白的氨基酸序列如SEQ ID No.:2所示：
Figure PCTCN2019115704-appb-000001
一种Martentoxin蛋白的编码序列如SEQ ID No.:1所示
Figure PCTCN2019115704-appb-000002
一种重组的Martentoxin蛋白的氨基酸序列如SEQ ID No.:3所示：
Figure PCTCN2019115704-appb-000003
本发明实验首次证实，在大鼠PTZ惊厥模型中，MarTX毒素可极其显著地预防性地和治疗性地缓解神经元的异常兴奋和癫痫症状，因此作为治疗癫痫症状的新型多肽药物。
应理解，尽管本发明的实例中提供的MarTX多肽来源于东亚钳蝎，但是来源于其它类似的物种(尤其是与东亚钳蝎属于同一科或属的蝎类)的、与本发明的序列(优选地，序列如SEQ ID NO:2所示)具有一定同源性(保守性)的MarTX多肽也可用于本发明。
应理解，尽管本发明的实例中提供的基因来源于东亚钳蝎，但是来源于其它类似的物种(尤其是与东亚钳蝎属于同一科或属的蝎类)的、与本发明的序列(优选地，序列如SEQ ID NO:1所示)具有一定同源性(保守性)的MarTX的基因 序列，也包括在本发明的范围内，只要本领域技术人员在阅读了本申请后根据本申请提供的信息可以方便地从其它物种(尤其是蝎类)中分离得到该序列。
本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括：DNA、基因组DNA或人工合成的DNA，DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。编码成熟多肽的编码区序列可以与SEQ ID NO:1所示的编码区序列相同或者是简并的变异体。
编码成熟多肽的多核苷酸包括:只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多苷或多肽的片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码的多肽的功能。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酞胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。
应理解，虽然本发明的MarTX基因优选来自东亚钳蝎，但是来自其它物种(尤其是蝎类)的与东亚钳蝎MarTX基因高度同源(如具有80％以上，如85％,90％,95％甚至98％序列相同性)的其它基因也在本发明考虑的范围之内。比对序列相同性的方法和工具也是本领域周知的，例如BLAST。
本发明的MarTX核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本发明所公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的DNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按 正确次序拼接在一起。一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明涉及一种用于治疗癫痫的MarTX多肽及其变体，在本发明的一个优选例中，所述多肽的氨基酸序列如SEQ ID NO:2所示。本发明的多肽能够有效治疗和/或预防癫痫。
本发明还包括与本发明的SEQ ID NO:2所示序列具有50％或以上(优选60％以上，70％以上，80％以上，更优选90％以上，更优选95％以上，最优选98％以上，如99％)同源性的具有相同或相似功能的多肽或蛋白。
所述“相同或相似功能”主要是指：“缓解癫痫的症状”。
本发明的多肽可以是重组多肽、天然多肽、合成多肽。本发明的多肽可以是天然纯化的产物，或是化学合成的产物，或使用重组技术从原核或真核宿主(例如，细菌、酵母、植物、昆虫和哺乳动物细胞)中产生。根据重组生产方案所用的宿主，本发明的多肽可以是糖基化的，或可以是非糖基化的。本发明的多肽还可包括或不包括起始的甲硫氨酸残基。
本发明还包括具有MarTX多肽活性的MarTX多肽片段和类似物。如本文所用，术语“片段”和“类似物”是指基本上保持本发明的天然MarTX多肽相同的生物学功能或活性的多肽。
本发明的多肽片段、衍生物或类似物可以是：(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的；或(ii)在一个或多个氨基酸残基中具有取代基团的多肽；或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽；或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或融合蛋白)。根据本文的定义这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明中，所述的多肽变体是如SEQ ID NO.:2所示的氨基酸序列，经过若干个(通常为1-10个，较佳地1-8个，更佳地1-4个，最佳地1-2个)取代、缺失或添加至少一个氨基酸所得的衍生序列，以及在C末端和/或N末端添加一个或数个(通常为10个以内，较佳地为5个以内，更佳地为3个以内)氨基酸。例如，在所述蛋白中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能，在C末端和/或N末端添加一个或数个(如1-3个)氨基酸通常也不会改变蛋白质的功能。这些保守性变异最好根据表1进行替换而产生。
表1
最初的残基 代表性的取代 优选的取代
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
本发明还包括所要求保护的蛋白的类似物。这些类似物与天然SEQ ID NO:2差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。这些蛋白的类似物包括天然或诱导的遗传变异体。诱导变异体可以通过各种技术得到，如通过辐射或暴露于诱变剂而产生随机诱变，还可通过定点诱变法或其他已知分子生物学的技术。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的蛋白并不限于上述列举的代表性的蛋白。
修饰(通常不改变一级结构)形式包括：体内或体外蛋白的化学衍生形式如乙酸化或羧基化。修饰还包括糖基化，如那些在蛋白质合成和加工中进行糖基化修饰。这种修饰可以通过将蛋白暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。
药物组合物及其施用方法
本发明提供了一种药物组合物，包括药学上可接受的载体和有效量的以下活性成分：MarTX毒素或其活性片段和其治疗和/或预防癫痫的药物活性成分。
如本文所用，术语“有效量”或“有效剂量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。
如本文所用，“药学上可接受的”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性、刺激和变态反应)的，即具有合理的效益/风险比的物质。术语“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。
本发明的药物组合物含有安全有效量的本发明的活性成分以及药学上可接受的载体。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂应与给药方式相匹配，本发明的药物组合物的剂型为注射剂。例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的药物组合物宜在无菌条件下制造。
本发明所述的活性成分的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的活性成分的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体 重、患者的免疫状况、给药的途径等。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。
本发明所述的药学上可接受的载体包括(但不限于)：水、盐水、脂质体、脂质、蛋白、蛋白-抗体缀合物、肽类物质、纤维素、纳米凝胶、或其组合。载体的选择应与给药方式相匹配，这些都是本领域的普通技术人员所熟知的。
本发明所提供的第一活性活性成分(a)MarTX毒素或其活性片段，可与第二活性成分(b)其他治疗和/或预防癫痫的药物联用。其中所述的第二活性成分(b)为现有技术中已经可用的抗癫痫药物，其包括但不限于：卡马西平、氟吡啶、加巴喷丁、拉莫三嗪、奥卡西平、苯妥英、苯妥英钠、瑞替加滨、托吡酯、颠扑净、乙琥胺、丙戊酸钠、或其组合。
c-Fos基因
c-Fos基因是即刻早期基因[Trends Neurosci.1995,18,66-67](IEGs，能被第二信使所诱导的原癌基因)的一种，它与癫痫发病后的多种病理生理过程息息相关。在生理情况下，c-Fos基因在中枢神经系统低水平表达；而当神经元受到物理或化学等刺激后，神经元兴奋激活了c-Fos等IEGs，IEGs转录生成的mRNA翻译生成c-Fos蛋白[Annu.Rev.Neurosci.1991,14,421-451]等。该蛋白可调节多种迟反应基因(LRGs)的表达，同时通过多种机制导致癫痫发作，最终导致癫痫病灶的形成[Neuron1990,4,477-485]。c-Fos蛋白在癫痫发作时迅速大量表达，若使用药物或其他治疗手段控制癫痫发作，则可明显抑制该蛋白的表达，因此c-Fos的表达测定可作为抗癫痫药物的作用机制以及疗效评价的有效指标。
尼氏体
尼氏体是在神经元树突的细胞质中发现的一种特殊的易染物质。通常尼氏体具有固定形状，神经元细胞体较大，胞浆颜色呈苍白色且染色均匀，细胞核大而圆，所有细胞染色呈深染。在海马齿状回(DG)区域神经锥体细胞排列紧密，细胞为4层或5层。但当脑部受到损伤时它们的形态会发生变化，细胞边缘发生模糊降低了细胞的完整性，同时细胞体染色变浅，神经元分布变紊乱，细胞数目或细胞层数变少。因此神经元的存在、分布以及病理变化可以通过尼氏体的形态和数目进行识别。
PTZ(pentylenetetrazole，即戊四唑)
戊四唑(PTZ)，别名戊四氮(Pentetrazol)、五甲烯四氮唑、卡地阿唑，是一种白色结晶粉末的化学品。化学名称为1,5-五亚甲基-1H-四唑，分子式为C 6H 10N 4，分子量为138.1704。戊四氮为中枢兴奋药，临床上主要用于解救严重的巴比土酸盐类及麻醉药中毒所引起的中枢性呼吸衰竭；也可用于急性传染病、麻醉药及巴比妥类药物中毒时引起的呼吸抑制、急性循环衰竭。
戊四氮能兴奋呼吸中枢和心血管运动中枢，其作用迅速而强烈，使呼吸加深加快，血压微升；剂量稍大，兴奋可扩展到大脑皮质和脊髓，引起惊厥。
PTZ主要作用于GABA A受体(γ-氨基丁酸A型受体)的氯离子通道，抑制GABA神经元活动，从而使神经系统兴奋性过度增强，诱发动物和人的阵挛性或全身强直性癫痫发作。PTZ诱导的啮齿类动物惊厥发作模型已被广泛应用于癫痫发生机制和新型抗癫痫药物的研究。
在本发明中，通过对动物进行腹腔注射PTZ来诱导其发生惊厥/癫痫，以进行后续的本发明MarTX毒素对癫痫的效应的实验研究。
本发明的主要优点包括：
(1)MarTX毒素具有作用强、用量小(在大鼠PTZ模型中用量可低至0.32μg/kg)的特点，不仅显著延长惊厥发作的潜伏期，还可显著缓解癫痫的症状(尤其是可显著降低3级、4级和5级癫痫的发病率)，因此可作为癫痫治疗的新型多肽药物。
(2)本发明的重组MarTX毒素可经原核表达、重组制备，有望为癫痫患者提供一种安全、有效、且价廉的抗癫痫药物。
(3)MarTX毒素是一种分子量很小(仅约4KDa)的神经毒素，可有效透过血脑屏障作用于脑部。
(4)MarTX毒素是一种高特异性的神经毒素，对α+β4亚型的离子通道大电导钙离子和电压激活的钾离子通道具有选择性，而对心血管、内分泌以及生殖系统等无影响，因此副作用小。
(5)MarTX毒素是首次经实验证实具有缓解癫痫功效的多肽类物质。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说 明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
如无特别说明，实施例所用的材料和试剂均为市售产品。
实验材料与方法
实验动物和药品
实验动物：成年雄性SD大鼠(中国科学院上海实验动物中心提供)，体重250-300g，每笼5只，实验室常规条件下饲养，室温维持在22±1℃，自然昼夜节律。
相关药品：重组的东亚钳蝎毒素Martentoxin(简称MarTX毒素)的表达纯化参见制备实施例1。
戊四氮(PTZ，CAS：54-95-5)，购于美国Sigma公司。
动物手术
经10％的水合氯醛(300mg/kg)腹腔注射麻醉后，大鼠固定于立体定位仪上(NS-2，Narishige,Japan)，剃去头顶毛发并消毒头皮，剪开皮肤并用10％双氧水烧灼皮下组织，暴露颅骨Bregma点，根据大鼠脑定位图谱在颅骨上确定给药基座植入点(AP-4.3mm、L 2.2mm)。位置确定后，用牙科钻钻一小孔(1mm)，剥净内板，针尖挑开硬脑膜，将给药基座套管植入至颅骨下2.5mm的海马CA1区。基座通过牙科水泥固定于大鼠颅骨表面。
制备实施例1
采用人工合成方法，合成一表达His-MBP-Thrombin位点-MarTX融合蛋白的DNA序列(SEQ ID No.:4)，将SEQ ID No.:4经Nco I和Not I酶切后，与相同酶切的pETDuet-1质粒进行连接，从而获得重组质粒pETDuet-1-MarTX，将其转化入E.coli Origami B(DE3)表达菌株中进行原核表达。
经重组表达，获得一融合蛋白(序列如SEQ ID No.:5)，用以下方法制备 MarTX多肽。
具体纯化方法步骤如下：
1)处于Buffer A环境中的融合蛋白先与镍柱结合，然后用Buffer B咪唑盐溶液进行梯度洗脱除去大部分杂蛋白，完成第一根亲和柱纯化并进行SDS-PAGE电泳检测。
2)收集目的融合蛋白(约46.8kDa)于18℃、2L Buffer C中进行透析酶切，按照6U/mL加入Thrombin酶，于3.5kDa透析袋中搅拌酶切过夜。
3)随后用恒流泵将酶切混合物加载至Amylose Resin层析柱上，收集穿流成分(FL)和Buffer D洗脱成分。His-MBP-tag经Buffer E洗脱除去，完成第二根亲和柱纯化并进行SDS-PAGE电泳检测。
4)然后将FL成分和Buffer D洗涤液(通常含有少量重组MarTX毒素)浓缩至2mL进行下一步凝胶色谱柱纯化。
5)凝胶色谱柱Superdex 75提前用Buffer F平衡，然后将样品加载到柱子上，用Buffer F进行洗脱。收集保留体积为110mL左右的洗脱峰即为重组MarTX毒素样品，之前的吸收峰为MBP-tag或未切开的融合蛋白。即得到高纯度的MarTX毒素，其序列如SEQ ID No.:3所示。
纯化过程中的相关缓冲液组分如表2所示。
表2重组MarTX毒素纯化过程中的相关缓冲液
Figure PCTCN2019115704-appb-000004
实施例1：MarTX毒素对大鼠PTZ复发惊厥行为的影响
1.1实验步骤
将成年雄性SD大鼠放置于一个40×30×50cm的透明玻璃盒内，观察给药后的惊厥反应。药物注射前，大鼠提前1h放置于盒中，自由活动以适应环境，然后于腹腔注射PTZ(60mg/kg)诱发大鼠惊厥发作。实验分为毒素注射组和生理盐水空白对照组。
(1)MarTX毒素注射组：腹腔注射一次PTZ诱导发作后，第二天在大脑海马内注射MarTX毒素(溶于2μL生理盐水中)，然后再进行PTZ注射。毒素剂量为0.08μg，n＝7-8(n为实验大鼠数目)；
(2)生理盐水空白对照组：腹腔注射一次PTZ诱导癫痫发作后，1天后于大脑海马内注射等量生理盐水，然后再进行PTZ注射，n＝6。
实验过程中采用“双盲法”加药和行为学观察，以减少人为误差。以大鼠在PTZ注射后2h内惊厥发作的潜伏期、死亡率、不同惊厥发作严重程度下的持续时间和发作次数为统计指标进行测定。大鼠的惊厥发作严重程度根据以下标准[Neurophysiol.1972,32,281-294；Brain Res.1997,758,92-98]进行评级：
0级：无反应；
1级：嘴部和面部节律性抽动；
2级：躯体波动样游走性痉挛；
3级：全身肌阵挛、臀部上翘；
4级：躯体向一侧翻转；
5级：仰翻位，全身强直痉挛发作。
一次完整的惊厥发作定义为从惊厥开始发作至惊厥后恢复正常，惊厥发作间隔时间达到5s以上时，定义为另一次独立的惊厥发作。潜伏期定义为注射PTZ后至首次2级惊厥发作开始的时间。
1.2实验结果
与生理盐水空白对照组相比，考察了MarTX毒素对大鼠PTZ复发惊厥行为的调控作用，实验结果如表2所示。
表2 MarTX毒素在2h内对PTZ复发惊厥行为的抑制效果
Figure PCTCN2019115704-appb-000005
通过单因子变异数分析(one-way ANOVA)，相比于生理盐水对照组，MarTX毒素组在潜伏期、发作持续时间和次数上有显著性差异，*p<0.05，**p<0.01。
由表2可知：(1)MarTX毒素组与空白对照组相比，能够显著延长惊厥复发的潜伏期(MarTX组的潜伏期为427.17±62.21s，n＝6，**p<0.01；生理盐水组为157.33±33.63s，n＝6)。
(2)MarTX毒素也显著降低了癫痫持续时间(MarTX组持续时间为160.00±38.47s，n＝6，*p<0.05；对照组为400.00±97.57s，n＝6)，下降了约60％。
(3)在癫痫发作次数方面，MarTX毒素可显著降低各种级别的惊厥，尤其是3级、4级和5级的惊厥。0.08μg的MarTX毒素显著降低了惊厥大发作(4级&5级)的次数(MarTX组为0.50±0.34次，n＝6，*p<0.05；对照组为2.17±0.40次，n＝6)，下降幅度达77％(100％-23％＝77％)。在3级发作次数方面，与空白对照组相比MarTX组的次数也更为显著降低(**p<0.01)，下降幅度达86％。
因此，本发明的MarTX毒素在惊厥发作的潜伏期、持续时间和不同惊厥发作严重程度下的发作次数都体现出显著的抑制效果，说明MarTX毒素有效缓解神经元的异常兴奋和癫痫症状。
实施例2：MarTX毒素对大鼠惊厥发作后海马c-Fos表达的影响
2.1实验步骤
在PTZ诱发的癫痫持续状态行为实验结束后，动物通过腹腔注射戊巴比妥钠(60mg/kg)进行麻醉。以200mL生理盐水经左心室升主动脉灌注冲洗净血管，然后用400mL固定液(含40％多聚甲醛的0.1M PBS，pH7.4，4℃)灌注1-2h，取出脑组织于同一固定液中过夜后，移至20％蔗糖溶液中浸泡直至沉于容器底部，再浸入30％的蔗糖溶液中直至沉于容器底部。
将大鼠的脑组织用恒冷箱切片机(Leica 1900，Germany)对海马区进行切片，片厚20μm，贴于经明胶-硫酸铬钾处理的载玻片上，置于-20℃冻存备用。c-Fos免疫组化按步骤如下：
(1)从-20℃冰箱中取出切片，回温30min，在切片四周用组化笔画框，晾干；
(2)在1％Triton-X溶液中加入1％的H 2O 2，将切片浸泡破膜30min；
(3)在0.01M PBS(pH7.4)缓冲液中冲洗3次，每次5min；
(4)用5％Goat serum 37℃封闭1h；
(5)用滤纸将血清吸干，加兔抗c-Fos抗体(1：400，Sc-52，Santa Cruz，USA)，抗体用0.01M PBS稀释，每张切片约100μL，置于湿盒中4℃下孵育48h；
(6)0.01M PBS(pH7.4)冲洗10min，重复3遍；
(7)加用0.01M PBS稀释的生物素标记羊抗兔IgG(1：200)，常温2h；
(8)0.01M PBS冲洗10min，重复3遍。加入ABC复合物(配置比例为A：B：PBS＝1:1:100)，常温2h；
(9)0.01M PBS(pH 7.4)冲洗10min，重复3遍；
(10)DAB-硫酸镍胺-葡萄糖氧化酶法(DAB,生工生物)避光染色10min；
(11)脱水，用70％、80％、95％、100％(×2)酒精脱水，每次5min，再用二甲苯浸泡2次，每次5min；
(12)用中性树胶封片保存，并在显微镜下观察c-Fos的表达。
统计海马不同分区(CA1、CA3)及齿状回(DG)上c-Fos免疫反应阳性(c-Fos-like immunoreactive,FLI)神经元的数目，对行为考察的两个动物分组(每组均为6只动物)随机取6～8张切片进行不同分区的FLI计数(注射毒素或生理盐水的同侧和异侧分别统计)，最后取其平均值。c-Fos表达的抑制率按以下公式进行计算。
抑制率(Innibitory ratio)＝(A-B)/A×100％
式中A表示生理盐水对照组的FLI神经元数目；B表示海马对应区域的MarTX毒素注射组的FLI神经元数。
2.2实验结果
大鼠的海马根据细胞形态的不同可分为海马回和齿状回(dentate gyrus,DG)两部分。海马回主要包括CA1(cornu ammonis)、CA2、CA3区和门区，主要由一些锥体神经元组成，其中CA1区与下托相连接，门区与齿状回相邻。齿状回是海马裂和海马伞之间形如齿状的皮质层，呈C形。其结构分为三层：分子层、颗粒细胞层和多形层，基本由颗粒细胞构成。具体位置参见图1[The rat brain in stereotaxic coordinates.2007]。
按照上述实验步骤和数据处理的方法进行了大鼠癫痫发作后海马内c-Fos表达的测定，实验结果参见图2和图3。
分析结果如下：(1)在PTZ诱发癫痫持续状态行为实验后，所有动物包括生理盐水对照组和毒素注射组均在注射点的同侧和异侧海马显示c-Fos蛋白表达，参见图2和图3；
(2)在生理盐水对照组，c-Fos阳性神经元主要集中于海马DG区的颗粒细胞层，其CA1和CA3区的阳性神经元较少；
(3)MarTX毒素对PTZ诱发的海马c-Fos的表达具有明显抑制效果。与生理盐水对照组相比，MarTX对注射同侧海马的CA1、CA3和DG区的抑制率分别为60.71％、51.18％、84.98％，参见图2；且对同侧海马c-Fos表达抑制效应比异侧海马更强(与生理盐水对照组相比，MarTX对异侧海马的CA1、CA3和DG区的抑制率分别为54.01％、55.25％、78.53％)，参见图3。
因此，MarTX毒素对PTZ诱发癫痫后大鼠海马的c-Fos蛋白表达具有显著的抑制效果，且注射同侧效果强于异侧海马，说明本发明的MarTX毒素可能具备抗癫痫药效。
实施例3：大鼠惊厥发作后对海马神经元损伤情况的考察
3.1实验步骤
(1)从-20℃冰箱中取出切片，回温20min，在切片四周用组化笔画框，晾干；
(2)将切片经蒸馏水浸泡2min；
(3)将尼式染色液(碧云天购买)滴在脑片上，于37℃水浴锅中染色10min；
(4)用蒸馏水洗涤两次，每次10s；
(5)切片经70％、80％、95％、100％的酒精脱水，每次2min，再用二甲苯浸泡透明2次，每次5min；
(6)用中性树胶封片保存，并在显微镜下观察尼式体染色。
对行为考察的以上两个动物分组(每组均为6只动物)随机取6～8张切片进行不同海马分区(CA1、CA3和DG区)的神经元细胞计数，最后取其平均值，并计算神经元数目的上升率。
3.2实验结果
通过尼氏染色实验对大鼠PTZ诱发癫痫后海马神经元的损伤或死亡情况进行了测定，实验结果参见图4和图5。
分析结果如下：(1)MarTX毒素组保留了较为完整的海马结构，海马区神经元尤其是DG区细胞密度最高且排列紧密，尼式体染色最深。与之对比的生理盐水组神经元排列变得疏松，细胞密度减小，染色最浅，说明PTZ诱发癫痫后对生理盐水对照组神经元的损伤最大，而本发明的MarTX毒素保护了癫痫后的海马神经元，损伤最小；
(2)MarTX毒素组与生理盐水组在注射同侧海马的CA1、CA3和DG区神经元的上升率分别为：95.73％、102.02％、93.43％，参见图4；对异侧海马CA1、CA3和DG区神经元的上升率分别为：79.50％、94.82％、79.62％，参见图5，说明MarTX对注射同侧海马神经元的保护作用强于异侧海马。
综上所述，PTZ诱发癫痫持续状态后将导致大鼠海马神经元的损伤或死亡。经测定发现注射MarTX实验组神经元的损伤程度大大降低，尼式染色的神经元数目最多，排列最紧密。
以上实验共同表明本发明的MarTX毒素在行为上可抑制大鼠惊厥发作，同时抑制了海马内c-Fos的表达，降低了海马神经元的损伤程度，说明本发明的 MarTX毒素具有抗癫痫药效。
实施例4：大鼠惊厥发作后对海马区场电位的考察
4.1实验步骤
用George Paxinos和Charles Watson编写的第六版《大鼠脑立体定位图谱》(图1)植入微阵列电极，以确定电极的位置：AP：4.3毫米，MR：2.2毫米，DV：2.5毫米，在颅骨的空白区域固定3-4个螺钉作为参考电极。记录电极和电极底座用牙科水泥固定。大鼠清醒后记录场电位(FP)。FP信号和同步视频可以通过omniplex(plexon，美国)录制。根据Nesquet的采样理论，将电极后端连接到一个前置放大器，并偶联在模拟数字转换器盒。取1Hz作为局部场电位记录的采样频率，并设置50Hz高通滤波器和300Hz低通滤波器，连续记录30分钟以上。将局部场电位记录结果导出为*.pl2文件格式，并使用离线sorter v4软件进行可视化预览。局部场电位分析选用同一分析通道，通过MATLAB(MathWorks，USA)程序导出数据，并利用小波变换对不同频率的局部场电位信号进行分解，得到不同频率的生理节律(δ：～0-4hz，θ：～4-8hz，α：～8-13hz，β：～13-30hz，γ：～30-100hz)。在功率谱分析中，采用韦尔奇法、汉明窗法和快速傅立叶变换法计算局部FP的功率谱密度变化。
4.2实验结果比较生理盐水、MarTX对PTZ癫痫模型场电位信号的调制作用。利用omniplex软件(美国plexon)生成的光谱密度热图，观察FP的功率谱密度变化。与生理盐水组相比，MarTX能快速抑制PTZ致痫大鼠的FP功率谱密度(图6A和B)。在功率谱密度曲线图(图6B)中，生理盐水组在低频(δ)波带出现一个尖峰，而在MarTX组则不能观察到这个尖峰，这表明施加MarTX抑制了产生低频波的神经网络。据统计，MarTX组的功率谱密度在脑电δ波(P<0.001，n＝3)、θ波(P<0.001，n＝3)、α波(P<0.01，n＝3)、β波(P<0.05，n＝3)和γ波(P<0.05，n＝3)上均显著低于生理盐水组(图6C)。
实施例5：大鼠海马神经元膜片钳电流记录和动作电位分析
5.1实验步骤
如果细胞的静息膜电位为-70mV，输入电阻为350MΩ或更大时，则认为膜片钳电流是可以接受的。将记录保持在-80mV作为保持电流，并通过增加正电流注入(持续1000ms)来诱发。评判比较PTZ处理的细胞(施加24小时后测量)与施加rMarTX的PTZ处理的癫痫细胞(施加10分钟后测量)之间的动作电位差异。在动作电位一半的峰值振幅处测量峰值宽度。以峰值阈值与动作电位峰值后的最小电压之差作为后超级化电位大小的测量值。峰值间隔是动作电位峰值之间的时间。
在当前膜片钳记录中，标准外液由1.2mM MgCl 2、10mM HEPES，10mM葡萄糖，1.5mM CaCl 2、2.5mM KCl和145mM NaCl组成。用NaOH将溶液的pH调节至7.4。内液由1mM CaCl 2、4mM MgCl 2、10mM HEPES，11mM EGTA和140mM KCl组成。溶液的pH用KOH调节至7.2。
5.2实验结果
戊四唑(PTZ)会诱导BK通道功能获得以及新皮质和海马锥体神经元的高放电率。因此，我们比较了施加生理盐水与MarTX后PTZ诱导的癫痫前细胞中电流注入引起的动作电位特征变化(图7A-E)。图7A中分别显示了经PTZ预处理的施加生理盐水和MarTX后的神经元动作电位曲线示例。电流注入海马锥体神经元可诱导动作电位的产生，其触发频率可能受到两种机制的限制。一个是电流注入期间的峰值间隔随时间增加，导致动作电位减少；另一个是在较高的电流注入，例如300pA，导致动作电位发生失败。我们发现经PTZ预处理的癫痫细胞(生理盐水组)的动作电位峰值间隔时间较短(图7E)。与经PTZ预处理的细胞相比(图7A)，用MarTX处理的癫痫细胞(MarTX组)的动作电位峰值间隔明显延长(图7E，P<0.001，n＝4)。此外，用MarTX处理的癫痫细胞在更高的电流注入(300pA)时，导致更多的动作电位发生失败(图7A)，这与PTZ预处理的癫痫细胞完全不同(生理盐水组)(图7A)。因此，与PTZ预处理的癫痫细胞(生理盐水组)相比，MarTX处理的癫痫细胞，其动作电位峰值间隔越长，动作电位宽度越大(图7C,P<0.01,n＝4)，动作电位发生失败的出现导致MarTX处理的癫痫神经元的放电率显著降低(图7B,P<0.001 at 200 and 300 pA,P<0.05 at 100 pA trace,n＝4)。我们还发现MarTX处理明显增加了后超级化电位 的幅值(图7D，在300pA曲线处P<0.01，n＝4)。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (13)

  1. 一种MarTX毒素或其活性片段、或其药学上可接受的盐的用途，其特征在于，用于制备一制剂或组合物，所述制剂或组合物用于治疗和/或预防癫痫。
  2. 如权利要求1所述的用途，其特征在于，所述的癫痫包括大脑皮层兴奋性增强所导致的癫痫(或神经元异常兴奋型癫痫)。
  3. 如权利要求1所述的用途，其特征在于，所述的MarTX毒素包括氨基酸序列SEQ ID No:2或3所示。
  4. 如权利要求1所述的用途，其特征在于，所述MarTX毒素包括在保持蛋白活性范围内，在序列SEQ ID NO:2的基础上进行一个或多个氨基酸的替换、缺失、改变、插入或增加，所得到的氨基酸序列。
  5. 如权利要求1所述的用途，其特征在于，所述的MarTX毒素包括在保持蛋白活性范围内，在序列SEQ ID NO:2的N端或C端进行一个或多个氨基酸的插入，所述插入的氨基酸残基个数包括1个至10个，较佳地1个至5个，更佳地1个至3个。
  6. 如权利要求1所述的用途，其特征在于，所述组合物为药物组合物。
  7. 如权利要求6所述的用途，其特征在于，所述药物组合物的剂型为注射剂。
  8. 如权利要求6或7所述的用途，其特征在于，所述的药物组合物通过静脉内、皮下、肌内或颅内方式给药。
  9. 如权利要求8所述的用途，其特征在于，所述注射剂通过微量输液泵(microinfusion pumps)进行施用。
  10. 如权利要求7所述的用途，其特征在于，所述的注射剂通过颅内给药进行施用，较佳地通过侧脑室注射(intracerebroventricular(ICV)delivery)入受试者体内。
  11. 如权利要求10所述的用途，其特征在于，所述注射剂被施用于受试者的同侧海马。
  12. 一种组合物产品，其特征在于，所述组合物产品包括：
    (i)第一药物组合物，所述第一药物组合物含有第一活性成分(a)MarTX毒素或其活性片段和药学上可接受的载体；
    (ii)第二药物组合物，所述的第二药物组合物含有第二活性成分(b)其他治疗和/或预防癫痫的药物和药学上可接受的载体。
  13. 一种药盒，其特征在于，所述药盒包括：
    (i)第一容器，以及位于所述第一容器中第一药物组合物，所述第一药物组合物含有第一活性成分(a)MarTX毒素或其活性片段和药学上可接受的载体；
    (ii)第二容器，以及位于所述第二容器中第二药物组合物，所述的第二药物组合物含有第二活性成分(b)其他治疗和/或预防癫痫的药物和药学上可接受的载体。
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