WO2023019846A1 - Utilisation d'une neurotoxine cobra et sa préparation dans la préparation d'un médicament pour la prévention et le traitement de la maladie de parkinson - Google Patents

Utilisation d'une neurotoxine cobra et sa préparation dans la préparation d'un médicament pour la prévention et le traitement de la maladie de parkinson Download PDF

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WO2023019846A1
WO2023019846A1 PCT/CN2021/140916 CN2021140916W WO2023019846A1 WO 2023019846 A1 WO2023019846 A1 WO 2023019846A1 CN 2021140916 W CN2021140916 W CN 2021140916W WO 2023019846 A1 WO2023019846 A1 WO 2023019846A1
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disease
preparation
parkinson
cobotide
neurotoxin
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Chinese (zh)
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秦正红
吴峰
朱路佳
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苏州人本药业有限公司
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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
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/58—Reptiles
    • A61K35/583—Snakes; Lizards, e.g. chameleons
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs

Definitions

  • the invention relates to the field of medicine, in particular to the application of a cobra neurotoxin and its preparation in the preparation of drugs for preventing and/or treating Parkinson's disease.
  • Ataxia is the state of being unable to perform complex movements in an orderly manner despite the absence of muscle abnormalities.
  • the maintenance of human body posture and the completion of voluntary movements are regulated by the deep sensation from muscles, joints, etc. and the effects of the cerebellum, brain, eyes, and vestibular organs. Damage to these systems will lead to poor coordination of movement, balance disorders, etc., including Parkinson's disease (Parkinson's disease, Parkinson's disease, PD), Parkinson's syndrome (Parkinsonism), Huntington's disease, ataxia, muscle tension Obstacles etc.
  • Parkinson's disease Parkinson's disease, Parkinson's disease, PD
  • Parkinson's syndrome Parkinson's syndrome
  • Huntington's disease ataxia
  • muscle tension Obstacles etc There are many types of movement disorders with different causes.
  • the treatment of different causes of movement disorders requires different drugs, and the curative effect varies greatly.
  • the etiology of ataxia includes cerebellar ataxia, deep sensory impairment ataxia, cerebral ataxia, vestibular ataxia, pa Treatment of tremor and bradykinesia in Kinson's disease varies widely.
  • the prominent pathological changes of Parkinson's disease are the degeneration and death of dopamine (DA) neurons in the substantia nigra of the midbrain, the significant reduction of DA content in the striatum, and the appearance of eosinophilic inclusion bodies in the cytoplasm of residual neurons in the substantia nigra.
  • the etiology is still unknown. Genetic factors, environmental factors, aging, oxidative stress, etc. may be involved in the degeneration and death process of PD dopaminergic neurons.
  • DA dopamine
  • Parkinson's disease also known as primary parkinsonism
  • Parkinson's disease is the second largest neurodegenerative disease with the characteristics of high incidence, progressive course, high disability rate, and irreversibility.
  • the number of patients with Parkinson's disease in my country has been increasing year by year.
  • Epidemiology shows that the prevalence rate is 15-328/100,000 population, about 1% of people >55 years old, and the incidence rate is 10-21/100,000 population/year.
  • Parkinson's disease has always been an important task in the medical field.
  • the main pathological basis of the disease is the defect of dopamine neurons in the substantia nigra of the midbrain; in theory, drugs (neuroprotective agents) targeting dopamine neuron damage can protect brain cells and improve the resistance to genetic and environmental pathogenic factors of Parkinson's disease. Tolerance: According to the current research results, most drugs that have curative effects in animal experiments often end in failure in clinical trials.
  • the most effective drug for symptomatic treatment is levodopa, but with the continuous loss of dopamine neurons after long-term use of levodopa, the ability to convert levodopa into dopamine is further lost, and the curative effect is gradually lost. And long-term use of levodopa will produce complications of tardive dyskinesia, which seriously affects the quality of life and self-care ability of PD patients.
  • the cobra family is widely distributed, with more than 10 species mainly distributed in Africa and Asia, including Australia and the ocean, and more than 50 other species.
  • my country's folk medicine and traditional Chinese medicine have a long history of using snake venom to treat diseases, mainly in the fields of cancer, arthritis and pain.
  • Neurotoxin is the main active ingredient of cobra snake venom, and also the main active ingredient of analgesia. It can be divided into ⁇ and ⁇ according to the site and mode of action, and there are more than 50 structural types in total.
  • the analgesic effect of ⁇ -neurotoxin (cobotide) of cobra venom has been discovered for many years and used clinically.
  • the research on the analgesic effect of cobra venom mainly focuses on Thai cobra and Chinese cobra.
  • the cobratoxin contained in Thai cobra is a long-chain ⁇ -neurotoxin consisting of 71 amino acids with 5 disulfide bonds.
  • Chinese cobra contains three short-chain ⁇ -neurotoxins, among which cobrotoxin (Chinese name: Kebotide) consists of 62 amino acids and has 4 disulfide bonds, which are considered necessary to maintain activity.
  • the other two short-chain neurotoxins have similar structures and pharmacological effects.
  • the homeopathic pharmacopoeia of the United States describes the preparation of Asian cobra venom for pain.
  • Cobra venom preparations approved by the US Pharmacopoeia include Nyloxin, cobraxin, and peperon. These preparations contain ⁇ -neurotoxin components and have similar pharmacological effects to short-chain ⁇ -neurotoxins.
  • the Kunming Institute of Zoology in my country isolated short-chain neurotoxins from Chinese cobras in the early years and made them into the analgesic drug Ketongning, and later developed a compound preparation - Compound Ketongning, which contains the cobra neurotoxins cobratoxin, tramadol and ibuprofen.
  • This cobra neurotoxin is the early named Ketongning and Cobotide (actually cobrotoxin, here the short-chain neurotoxin of Chinese cobra is mistakenly named as the long-chain neurotoxin cobratoxin of Thailand). The latter was once clinically used for pain caused by cancer and achieved good results, but it has been discontinued. Cobratide injection (cobratide, named Ketongning in the early years) is also used clinically for analgesia.
  • the object of the present invention is to provide a new application of cobra neurotoxin and its preparation in the preparation of drugs for preventing and/or treating Parkinson's disease.
  • the study found that the cobra neurotoxin cobotide plays a role in the treatment of Parkinson's disease by activating the nerve growth factor (NGF)-pTrkA pathway.
  • NGF nerve growth factor
  • This discovery clarifies the new mechanism of action of cobotide on Parkinson's disease, as well as the new application of cobotide and its preparations in the prevention and treatment of Parkinson's disease.
  • New targets and drugs for drug intervention provide new ideas for the study of new drugs for the prevention and treatment of Parkinson's disease and other neurodegenerative diseases.
  • the present invention provides the application of the cobra neurotoxin and its preparation in the preparation of medicaments for preventing and/or treating Parkinson's disease.
  • the drug has at least one application in the following (1)-(3);
  • the drug is used to improve motor coordination and/or improve bradykinesia
  • the drug is used to enhance muscle strength
  • the drug is used to improve the nigrostriatal system lesions of the brain tissue.
  • said improving muscle ability includes improving grip strength.
  • the improvement of brain tissue nigrostriatal system lesions includes increasing the number of dopamine neurons in the substantia nigra, increasing the content of dopamine and/or its metabolites in the substantia nigra and striatum, and increasing tyrosine hydroxylase at least one of the contents.
  • the invention also provides the application of the cobra neurotoxin and its preparation in the preparation of NGF-pTrkA pathway activator.
  • cobra neurotoxin is selected from cobrotoxin (cobotide).
  • cobra neurotoxin is selected from cobra long-chain ⁇ -neurotoxin and/or cobra short-chain ⁇ -neurotoxin.
  • cobra neurotoxin is selected from cobrotoxin and/or cobratoxin.
  • cobra neurotoxin preparation is selected from at least one of cobratide injection (cobratide, or clotine), Nyloxin, cobraxin and peperon.
  • the medicine includes a pharmaceutically effective dose of cobra neurotoxin and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is selected from pharmaceutically acceptable solvents, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators Agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrating agents, penetration enhancers, pH regulators, buffers, plasticizers , surfactants, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, polymer framework materials and film-forming materials .
  • the water can be distilled water, water for injection, etc.
  • the buffer can be phosphate buffer, Tris buffer, etc.
  • the medicament is in the form of oral and buccal administration, nasal cavity administration, transdermal administration or injection; preferably it is an injection dosage form.
  • the oral dosage form is selected from oral liquid, capsule, effervescent tablet, and oral drug film;
  • the nasal administration dosage form is selected from conventional dosage forms such as spray and nasal drops.
  • the injection form is selected from conventional dosage forms such as powder injection and water injection.
  • Conventional dosage forms such as microneedle patches, paints, and cataplasms can be used for transdermal dosage forms, and the microneedle patches include soluble microneedles and insoluble microneedles.
  • the drug administration methods include external administration and oral administration.
  • Oral administration includes buccal, sublingual.
  • External administration includes transdermal, subcutaneous or intramuscular injection, intravenous injection or intravenous drip.
  • the drug also includes another additional therapeutic agent for treating Parkinson's disease
  • the additional therapeutic agent is selected from synthetic small molecule drugs, genetic engineering drugs, biological agents or gene therapy drugs.
  • the additional therapeutic agent can be a therapeutic agent that also agonizes the NGF-pTrkA signaling pathway, or other drugs that treat Parkinson's disease, such as anticholinergics, amantadine, levodopa, and peripheral dopamine decarboxylase Compound preparations composed of inhibitors, etc.
  • Cobotide can be extracted from Chinese cobra or other species of snake venom (such as cobras in India, Thailand, Malaysia, etc.), or obtained through genetic engineering expression, or obtained through artificial synthesis.
  • Cobotide discovered by the inventor has the effect of regulating NGF-pTrkA signal transduction, reducing the loss of dopamine neurons, increasing the content of dopamine in the brain, and reducing the symptoms of movement disorders. It has important application value in the treatment of Parkinson's disease and can be used It is being developed as a drug for the treatment of PD.
  • the present inventors have discovered a drug cobotide that regulates a neurotrophic pathway and a drug that regulates this neurotrophic pathway by studying whether cobotide protects dopamine neurons in the substantia nigra of the midbrain from neurotoxin damage in the pathological process of Parkinson's disease.
  • the neuroprotective effect of the pathway in Parkinson's disease can be used as a therapeutic drug and target for the treatment of Parkinson's disease. Therefore, the present invention provides a new application of cobotide for treating Parkinson's disease. Specifically, the inventors of the present invention have found through research that cobotide protects dopamine neurons from neurotoxin MPTP damage by activating the NGF-pTrkA signal transduction pathway.
  • Intraperitoneal injection of cobotide before modeling with MPTP neurotoxin in Parkinson's disease mice can significantly improve the behavioral disorder of Parkinson's disease mice, prolong the running time of the wheel test, shorten the climbing time of mice, and increase the grip of forepaws and front and rear paws Strength, up-regulate the number of dopamine neurons in the substantia nigra, increase the content of dopamine neurotransmitters in the striatum, promote the synthesis of NGF by astrocytes in the substantia nigra of the mouse, and up-regulate the NGF-related receptor TrkA at position 490 of threonine
  • the phosphorylation level of dots indicated that cobotide had a protective effect on neurotoxin damage in Parkinson's disease.
  • This discovery will provide new ideas and strategies for the study of the pathological mechanism of Parkinson's disease, and will have a major impact on the discovery of new targets for neuroprotective agents.
  • Cobotide is found to be an effective drug for the
  • This target is that cobotide exerts neuroprotective effect by activating NGF-pTrkA signal transduction pathway, and can be used as an activator of NGF-pTrkA signal transduction pathway;
  • no obvious toxic and side effects are found in the therapeutic dose; it has the advantages of small dosage, safety, transdermal administration and injection administration, etc.
  • Figure 1 is the effect of cobotide on the motor coordination of Parkinson's disease mice, and the ordinate is the time the mice stay on the wheel;
  • Figure 2 shows the effect of cobotide on the slowness of movement in mice with Parkinson's disease.
  • the ordinate of the left figure is the time from the beginning of the movement to the time when the mouse is completely turned head down; time to the bottom of the pole;
  • Figure 3 is the effect of cobotide on the grip strength of front and rear limbs of Parkinson's disease mice
  • Figure 4 is the effect of cobotide on the number of dopamine neurons in the substantia nigra of the Parkinson's disease mice;
  • Figure 5 is the effect of cobotide on the content of striatum tyrosine hydroxylase in Parkinson's disease mice
  • Figure 6 is the effect of cobotide on the content of dopamine and its metabolites in the striatum of Parkinson's disease mice;
  • Figure 7 shows the effect of cobotide on the expression of NGF protein in the astrocytes in the substantia nigra region of the Parkinson's disease mice
  • Figure 8 is the effect of cobotide on NGF content in the ventral midbrain of Parkinson's disease mice
  • Figure 9 shows the effect of cobotide on the phosphorylation level of TrkA protein in the ventral midbrain of Parkinson's disease mice
  • Figure 10 is the effect of different concentrations of hydrogen peroxide treatment on the purity of cobotide.
  • Example Cobotide inhibits Parkinson's pathological process caused by neurotoxin MPTP by activating NGF-pTrkA signal transduction pathway
  • the source of Cobotide can be obtained through artificial synthesis, semi-synthesis, and biological extraction.
  • Cobotide used in this experiment was extracted from the venom of Cobra sinensis by Suzhou Renben Pharmaceutical Co., Ltd., with a purity of 98% and a protein content of 93%.
  • the amino acid sequence obtained by protein full-coverage amino acid sequencing is lechnqqssq tptttgcsgg etncykkrwr dhrgyrterg cgcpsvkngieinccttdrc nn (see SEQ ID No: 1). It is consistent with the amino acid sequence of cobrotoxin in the gene bank.
  • the molecular mass determined by mass spectrometry was 6944, which was consistent with the theoretically calculated molecular weight.
  • mice Sixty C57bl/6 mice were randomly divided into 4 groups (control group, Parkinson's disease model group, cobotide low-dose and high-dose treatment groups). Parkinson's disease model group, cobotide low-dose and high-dose treatment groups were continuously injected intraperitoneally with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP M0896, purchased from Sigma-Aldrich, St.Louis, MO, USA), injected once every 2 hours on the same day, a total of 4 times, each dose was 20mg ⁇ kg -1 , to establish an acute Parkinson's disease (PD) mouse model, and the control group received an equal volume of 0.9% normal saline was injected intraperitoneally.
  • MPTP M0896 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
  • Cobotide low-dose (3 ⁇ g ⁇ kg -1 ) treatment group started to inject 0.3 ⁇ g ⁇ ml -1 aqueous solution of cobotide 5 days before modeling (including the day of modeling), and high-dose cobotide (9 ⁇ g ⁇ kg -1 1 )
  • the treatment group started to inject 0.9 ⁇ g ⁇ ml -1 cobotide aqueous solution 5 days before modeling (including the day of modeling), and both groups were intraperitoneally injected once a day until the animals were sacrificed (12 days of drug intervention in total) ). Behavioral analysis was done the next day after MPTP modeling, and the animals were sacrificed 7 days later. During this period, no animals in each group died.
  • a Rotarod test (Rotarod test) This test requires animals to maintain balance on a rotating shaft and move continuously. It is a widely used test for evaluating animal movement coordination. The score of this item decreased in MPTP model mice, and was highly correlated with the content of striatal tyrosine hydroxylase. The test requires mice to balance on a rotating rod at a constant speed, and the time spent on the rod is recorded. Slow acceleration in the test limits inter-individual variability in performance.
  • the required props include: (1) Instrument: The diameter of the roller shaft is about 5cm, made of strong plastic and wrapped with gray rubber foam. Before the formal measurement, the animals were trained for 5 days, 3 times a day. The instrument is set to accelerate from 4 rpm to 40 rpm within 300 s. The instrument was operated at a constant speed of 4 revolutions/min before the start, and the dwell time of the animal on the wheel was recorded.
  • B Pole test (Pole test) Pole test was used to evaluate MPTP-induced motor retardation in mice.
  • the experiment adopts a wooden straight rod with a diameter of 1 cm and a length of 50 cm.
  • the mouse was placed facing the top of the pole about 10 cm away from the top of the pole, and the time from the start of the movement to the time when the mouse completely turned its head down and the time when the mouse reached the bottom of the pole were recorded respectively. Both were elongated in MPTP model mice, and were highly correlated with striatal dopamine content. Similarly, the mice were trained for 5 days, 3 times a day, before the formal measurement. The longest recording time is 120s.
  • C Grip strength test mouse grip instrument is used to evaluate the impact of disease on animal limb strength.
  • the mice were allowed to hold the matching metal grid with their forelimbs or front and rear limbs respectively, and the experimenter gently dragged the tail of the mouse until the mouse got out of the grid, and the instrument recorded its maximum grasping force.
  • MPTP model mice showed motor dysfunction, and the grip strength was significantly down-regulated, which was highly correlated with the concentration of dopamine in the striatum. Mice were trained for a total of 5 days, 3 times a day.
  • mice 1 C57bl/6 mice were anesthetized with chloral hydrate 7 days after the model was established, and the brain tissue was fixed by cardiac perfusion with 0.01M PBS and 4% paraformaldehyde solution containing 0.01M PBS in turn.
  • the brain was quickly removed, placed in 4% paraformaldehyde and fixed for 6 hours, then transferred to 20% sucrose solution and stored at 4°C. After settling to the bottom of the container, transfer to 30% sucrose solution. After settling to the bottom of the container, a coronal frozen section was performed, and the thickness of the brain slice was 40 ⁇ m. Brain slices were stored in antifreeze solution at -30°C for future use.
  • the lysate (125mM Tris–HCl, pH 6.8, 2% SDS, 10% glycerol, 10% ⁇ -mercaptoethanol, 0.004% bromophenol blue) was used to treat the homogenate of brain tissue, boiled for 5 minutes to denature, and the protein content was measured by lorry method. The lysate was used to adjust the concentration of the sample protein to be consistent.
  • 3SDS-PAGE electrophoresis Prepare the glass plate, wash it with ethanol, dry it, and install it. Prepare 8% separating gel, and slowly add double distilled water to the top layer as a covering solution to prevent the inhibition of air oxygen on the gel. After the gel has been polymerized, the stacking gel is prepared.
  • TrkA polyclonal antibody #2505, Cell Signaling Technology, Danvers, Massachusetts, USA, 1:1000
  • Phospho-TrkA polyclonal antibody Tyr490, #9141, Cell Signaling Technology, Danvers, Massachusetts, USA, 1:1000
  • ⁇ -actin mouse monoclonal antibody A5441, Sigma- Aldrich, St.Louis, MO, USA, 1:5000
  • the ventral midbrain of the mouse was homogenized at low temperature (50mM Tris–HCl, pH 7.4, 150mM NaCl, 2.0mM EDTA, 10mM ⁇ -mercaptoethanol, 1.0mM sodium vanadate, 50mM sodium fluoride, 1.0mM AEBSF, 10 ⁇ g/ml aprotinin enzyme, 10 ⁇ g/ml albumin and 10 ⁇ g/ml pepsin inhibitor), the mouse NGF ELISA kit (EM9RB, Thermo Fisher Scientific, Waltham, MA, USA) was used to quantitatively detect the NGF content according to the method described in the kit manual.
  • EM9RB Thermo Fisher Scientific, Waltham, MA, USA
  • tissue homogenate was centrifuged at 14,000 g for 30 min, and 100 ⁇ L of the supernatant was taken out, together with the standard and control, incubated in a 96-well plate for 2.5 h at room temperature on a low-speed shaker. After washing, add 100 ⁇ L biotin conjugate solution, incubate for 1 hour, wash, then add 100 ⁇ L Streptavidin-HRP solution and incubate for 45 minutes. Finally, 100 ⁇ L of chromogenic substrate was added, and the stop solution was added after 30 min of reaction. Detect with a microplate reader at a wavelength of 450nm, calculate the standard curve, and use the total amount of protein in each sample to standardize the final result.
  • mice After the decapitation of the mice, the whole brain was quickly taken out, and the striatum was quickly separated in an ice bath at 4°C, weighed and put into dry ice immediately. :10, w/v). Then centrifuge at 15,000g at 4°C for 30min, filter the supernatant with a 0.22 ⁇ m filter membrane, take 10 ⁇ l sample injection (717 autosampler, Waters, Milford, MA, USA), connect to high performance liquid chromatography (Waters 1525) , electrochemical detector (Waters 2465) to analyze the content of dopamine and its metabolites homovanillic acid HVA and 3,4-dihydroxybenzeneacetic acid DOPAC.
  • sample injection 717 autosampler, Waters, Milford, MA, USA
  • electrochemical detector Waters 2465
  • the mobile phase solution contains 50mM sodium dihydrogen phosphate, 0.25mM 1-octanesulfonic acid, 50mM sodium citrate, 2mM NaCl, 20mg disodium edetate and acetonitrile (1:3, v/v), pH Adjust to 4.3 with phosphoric acid, pump flow rate 1ml/min. After establishing a standard curve, the calculated values were standardized and quantified by striatum weight.
  • Figure 1 shows the effect of cobotide on the motor coordination of Parkinson's disease mice.
  • the results of the wheel test to test the balance exercise ability of the mice showed that compared with the exercise coordination of the mice in the MPTP model group, although there was no significant difference between the mice in the cobotide low-dose and high-dose groups (P>0.05), each dose of the treatment group
  • P>0.05 the number of dose of the treatment group
  • Figure 2 shows the effect of cobotide on the slow movement of Parkinson's disease mice.
  • Figure 3 shows the effect of cobotide on the grip strength of front and rear limbs of Parkinson's disease mice.
  • Figure 4 shows the effect of cobotide on the number of dopamine neurons in the substantia nigra region of the Parkinson's disease mice.
  • Figure 5 is the effect of cobotide on the content of tyrosine hydroxylase in the striatum of Parkinson's disease mice.
  • Figure 6 shows the effect of cobotide on dopamine and its metabolites in the striatum of Parkinson's disease mice.
  • Fig. 7 is the effect of cobotide on the expression of NGF protein in the astrocytes in the substantia nigra region of the Parkinson's disease mice.
  • Fig. 8 is the effect of cobotide on the NGF content in the ventral midbrain of Parkinson's disease mice.
  • Figure 9 shows the effect of cobotide on the phosphorylation level of threonine 490 site of TrkA protein in the ventral midbrain of Parkinson's disease mice.
  • Figure 10 is the effect of different concentrations of hydrogen peroxide on the purity of cobotide. As the concentration of hydrogen peroxide increases, the degradation ratio of the treated cobotide increases, and the purity decreases. The purity of Cobotide decreased to 69.49%, 50.09%, and 21.74% after being treated with 5%, 10%, and 20% hydrogen peroxide for 12 hours.

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Abstract

L'invention concerne l'utilisation d'une neurotoxine cobra et une préparation de celle-ci dans la préparation d'un médicament pour la prévention et le traitement de la maladie de Parkinson. L'invention concerne spécifiquement une nouvelle utilisation de cobratide pour traiter la maladie de Parkinson et une nouvelle cible de cobratide pour la neuroprotection. La cible de cobotide joue un rôle dans la neuroprotection au moyen de l'activation d'une voie de transduction de signal NGF-pTrkA, et la cobotide peut être utilisée en tant qu'activateur de la voie de transduction de signal NGF-pTrkA. La présente invention trouve que l'injection intrapéritonéale de cobotide présente un bon effet sur le traitement de la maladie de Parkinson, et en outre, aucun effet secondaire toxique évident n'est trouvé dans la dose de traitement ; et la cobotide présente les avantages d'une administration, d'une injection et d'une distribution de médicament transdermiques sûres à petite dose, etc.
PCT/CN2021/140916 2021-08-19 2021-12-23 Utilisation d'une neurotoxine cobra et sa préparation dans la préparation d'un médicament pour la prévention et le traitement de la maladie de parkinson WO2023019846A1 (fr)

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