WO2019141256A1 - 可利霉素或其活性成分的用途 - Google Patents

可利霉素或其活性成分的用途 Download PDF

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WO2019141256A1
WO2019141256A1 PCT/CN2019/072413 CN2019072413W WO2019141256A1 WO 2019141256 A1 WO2019141256 A1 WO 2019141256A1 CN 2019072413 W CN2019072413 W CN 2019072413W WO 2019141256 A1 WO2019141256 A1 WO 2019141256A1
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Prior art keywords
disease
medicament
drugs
isovalerylspiramycin
preventing
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PCT/CN2019/072413
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English (en)
French (fr)
Inventor
夏明钰
赵小峰
姜勋雷
姜勋东
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沈阳福洋医药科技有限公司
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Priority to MX2020007627A priority Critical patent/MX2020007627A/es
Priority to AU2019209740A priority patent/AU2019209740A1/en
Priority to US16/962,592 priority patent/US11413302B2/en
Priority to RU2020126396A priority patent/RU2771046C2/ru
Priority to JP2020539282A priority patent/JP7152052B2/ja
Priority to EP19741498.0A priority patent/EP3741373B1/en
Priority to KR1020207023822A priority patent/KR20200112898A/ko
Priority to CA3088823A priority patent/CA3088823A1/en
Publication of WO2019141256A1 publication Critical patent/WO2019141256A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • 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
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to the field of pharmaceuticals, and in particular to the use of colimycin or an active ingredient thereof.
  • Carrimycin clones the 4"-o-acyl-transferase of the carbomycin producing strain into the spiramycin producing strain by transgenic technology, and the oriented acylation helix 4'-OH, a novel antibiotic with a 4'-position isovalerylspiramycin as a main component formed by adding an isovaleryl side chain at the 4' position.
  • Colimycin is composed of a variety of spiramycin derivatives, the main active ingredient isopyryl spiromycin (I + II + III) total content of not less than 60%, is an acceptable pharmaceutical combination in pharmacy Things.
  • the central structure is a 16-membered lactone ring, which is formed by linking one molecule of felosamide, one molecule of carbamelamine sugar and one molecule of carbonaceous sugar. Its main components are isovalerylspiramycin I, II, III and spirochetes.
  • the difference in the structure of the prime is that the group attached to the 4' position of the carbonaceous sugar is isovaleryl rather than the hydroxyl group.
  • the drug is jointly declared by the Shenyang Tonglian and the like.
  • R H
  • R' COCH 2 CH (CH 3 ) 2 is isovaleryl spiromycin I;
  • R′ COCH 2 CH(CH 3 ) 2 is isovalerylspiramycin II;
  • R′ COCH 2 CH(CH 3 ) 2 is isovalerylspiramycin III;
  • the molecular structure contains two dimethylamine groups which are weakly basic and easily soluble in an acidic aqueous solution; and have a "negative solubility" property in which the solubility decreases with an increase in temperature. Since the main component of colimycin, isovalerylspiramycin, has a longer carbon chain at the 4' position and poor hydrophilicity, and its solubility in water is smaller than that of spiramycin and 4"-acetylspiramycin.
  • Colimycin is a white non-crystalline powder with slightly hygroscopicity, specific rotation of about -80.8 °, maximum ultraviolet absorption wavelength of 231 ⁇ 232 nm, itself with weak fluorescent chromophores, in case of concentrated sulfuric acid or hydrochloric acid It has a purple reaction and produces strong purple fluorescence with a maximum absorbance at 231 to 232 nm.
  • the drug has good lipophilicity, strong tissue penetration ability, rapid oral absorption, long body maintenance time, and sustained antibiotic effect. According to the relationship between pharmacodynamics and chemical conformation, the lipophilicity and in vivo activity of macrolide antibiotics after 4′ position acylation, the antibacterial activity and clinical therapeutic effect in vivo are significantly improved, and the stability of antibiotics in vivo. As the carbon chain of the 4" hydroxy ester grows, it is enhanced, that is, isovalerylspiramycin > butyrylspiramycin > propionylspiramycin > acetylspiramycin.
  • the preliminary in vitro and in vivo pharmacodynamic tests showed that the drug not only has good antibacterial activity against most G + bacteria, but also has certain effects on some G - bacteria.
  • the technical indicators are obviously superior to azithromycin, erythromycin, acetylspiramycin, Medicamycin, especially for Mycoplasma pneumoniae, has the strongest antibacterial activity against erythromycin-resistant bacteria, gonococcal bacteria, pneumococcus, Staphylococcus aureus, Pseudomonas aeruginosa, influenza bacillus, Haemophilus influenzae, Bacteroides fragilis Legionella, Pseudomonas aeruginosa and Clostridium perfringens also have certain antibacterial activity, and have little cross-resistance to clinical erythromycin-resistant Staphylococcus aureus.
  • Colimycin will be mainly used for the treatment of infectious diseases of Gram-positive bacteria, especially upper respiratory tract infections, and may be used for urinar
  • colimycin or its pharmaceutically active ingredient isopyryl spiromycin I, isovaleryl spiromycin II or isovaleryl spiromycin III or a combination thereof is better.
  • the anti-aging, anti-diabetic or anti-Alzheimer's disease provides a theoretical basis for the clinical promotion of the drug colimycin or its active ingredients in this aspect, and has important economic and social benefits.
  • the present invention adopts the following technical solutions:
  • a medicament for preventing and/or treating a disease wherein: the disease is Alzheimer's disease, diabetes or aging; the medicament comprises a first active ingredient, and the first active ingredient comprises a bacterium One of leucine, isovalerylspiramycin I, isovalerylspiramycin II and isovalerylspiramycin III;
  • the medicament further includes a second active ingredient
  • the second active ingredient is at least one of anti-Alzheimer's disease drugs
  • the second active ingredient is at least one of the anti-diabetic drugs
  • the second active ingredient is at least one of a drug that delays aging or prolongs life.
  • the medicament and the pharmaceutically acceptable carrier are formulated into a clinically acceptable preparation, preferably a tablet, a capsule, a pill, an injection, a sustained release agent, and various microparticle delivery systems.
  • the dose of the drug is 10 to 1500 mg/kg, preferably 50 to 1000 mg/kg, more preferably 100 to 500 mg/kg.
  • the present invention also provides a combination product for preventing and/or treating a disease, wherein the disease is Alzheimer's disease, diabetes or aging; the combined product comprises a first agent, the first
  • the active ingredient of the medicament comprises one of colimycin or isovaleryl spiromycin I or isovaleryl spiromycin II or isovaleryl spiromycin III;
  • the combined product further includes a second medicament
  • the second agent is at least one of for preventing and/or treating Alzheimer's disease-related drugs
  • the second agent is at least one of a drug for preventing and/or treating diabetes
  • the second agent is at least one of anti-aging or prolonging life-related drugs.
  • the ratio of the first agent to the second agent is from 1 to 99:99 to 1, preferably from 5 to 95:95 to 5, more preferably from 10 to 90:90 to 10, still more preferably from 20 to 80: 80 to 20.
  • the related drugs for preventing and/or treating Alzheimer's disease include a drug acting on a cholinergic system, a drug acting on an N-methyl-D-aspartate receptor, and an antioxidant drug.
  • anti-inflammatory drugs drugs that inhibit the formation of A ⁇ protein, estrogen, nerve growth factor, nimodipine, anti-apoptotic agents;
  • Related drugs for preventing and/or treating diabetes include biguanide hypoglycemic agents, sulfonylurea hypoglycemic agents, alpha glycosidase inhibitors, insulin sensitizers, non-sulfonylurea insulin secretagogues or insulin At least one of them.
  • the present invention also provides the use of the medicament or the combination thereof for the preparation of a medicament for preventing and/or treating Alzheimer's disease, puzzle, diabetes, delaying aging or prolonging life.
  • the use of the medicament for the preparation of a medicament for preventing and/or treating Alzheimer's disease and the like is for the preparation of a medicament for reducing acetylcholine hydrolyzate, for improving cognitive impairment and dyskinesia, Protects the application of nerve cell drugs in the brain, does not reduce body weight, improves immunity or enhances the application in leukocyte drugs;
  • the use in the preparation of a medicament for preventing and/or treating diabetes is for the preparation of a medicament for preventing and/or treating type I or type II diabetes or a particular type of diabetes, preferably for preparing for promoting insulin secretion or lowering blood sugar.
  • the use of the drug in delaying aging or prolonging life is to prepare a drug for delaying aging and/or prolonging life by altering the transcription factor DAF-16 activity; by increasing the expression level of the SIR2 homologous protein SIR2.1 , SIR2.1 by delaying aging and/or prolonged life by affecting DAF-16 activity; or by directly activating AMPK, enhancing FOXO/DAF-16 activity, delaying aging and/or prolonging life .
  • a first object of the present invention is to provide a medicament for preventing and/or treating Alzheimer's disease.
  • the present invention provides a medicament for preventing and/or treating Alzheimer's disease, the active ingredients of which include colimycin or isovalerylspiramycin III or isovalerylspiramycin II or different One of pivaloylspiramycin I; or a combination of two or three of isovalerylspiramycin I, isovalerylspiramycin II, isovalerylspiramycin III.
  • Colimycin is a mixture of various active ingredients, including the three active ingredients of isovalerylspiramycin I, isovalerylspiramycin II, isovalerylspiramycin III, and other impurities.
  • the medicament comprises a pharmaceutically acceptable carrier.
  • the medicament is formulated into a clinically acceptable tablet, capsule, pill, injection, sustained release agent, and various microparticle delivery systems.
  • the dose of the drug is 10 to 1500 mg/kg.
  • the dose of the drug is 50 to 1000 mg/kg.
  • the dose of the drug is 100 to 500 mg/kg.
  • the present invention also provides a combination product for preventing and/or treating Alzheimer's disease, the combination product comprising a first agent, and the active ingredient of the first agent comprises colimycin or isoprene One of acylomycin III or isovaleryl spiromycin II or isovaleryl spiromycin I; or isovaleryl spiromycin I, isovaleryl spiromycin II, isovaleryl spiromycin III A combination of two or three.
  • the combination product further includes a second medicament.
  • the second agent includes at least one of related drugs for preventing and/or treating Alzheimer's disease.
  • the related drugs for preventing and/or treating Alzheimer's disease include a drug acting on a cholinergic system, a drug acting on an N-methyl-D-aspartate receptor, and an antioxidant drug.
  • anti-inflammatory drugs drugs that inhibit the formation of A ⁇ protein, estrogen, nerve growth factor, nimodipine, anti-apoptotic agents.
  • the invention also provides the use of a medicament or combination according to any of the above, in the prevention and/or treatment of Alzheimer's disease, puzzle.
  • AD Alzheimer's disease
  • AD Alzheimer's disease
  • the hypoxia hypothesis is an earlier recognized Alzheimer's disease (AD) theory.
  • Cholinergic damage is Considered to be an important cause of AD, the cholinergic system is also considered to be an important target for AD drugs.
  • the drug of the present invention increases the acetylcholine content in the hippocampus and cortex of the brain by reducing the hydrolysis of acetylcholine, thereby improving cognitive function. Prevention and/or treatment of Alzheimer's disease is achieved.
  • the invention also provides the use of a medicament or combination of any of the above, for reducing acetylcholine hydrolysis.
  • the invention also provides the use of a medicament or combination according to any of the above, for improving cognitive impairment and dyskinesia.
  • the invention also provides the use of a medicament or combination as described above for the protection of nerve cells in the brain.
  • the present invention also provides the use of a medicament or a combination product according to any of the above, without lowering body weight, improving immunity, and increasing white blood cells.
  • a second object of the present invention is to provide a medicament for preventing and/or treating diabetes.
  • the present invention adopts the following technical solutions:
  • a medicament for preventing and/or treating diabetes the active ingredients of which include one of colimycin, isovalerylspiramycin I, isovalerylspiramycin II, isovalerylspiramycin III, or A combination of two or three of isovaleryl spiromycin I, isovaleryl spiromycin II, isovaleryl spiromycin III.
  • the medicament of the present invention comprises a pharmaceutically acceptable carrier.
  • the medicament of the present invention can be prepared as a tablet, capsule, pill, injection, sustained release or microparticle delivery system with a pharmaceutically acceptable carrier.
  • the dose of the effective drug component in the medicament of the present invention is 100 to 400 mg 1500 mg/kg, preferably 50 to 1000 mg/kg, more preferably 100 to 500 mg/kg.
  • the diabetes is type I or type II diabetes or a particular type of diabetes.
  • the drugs for preventing and/or treating diabetes in the present invention are mainly directed to diabetes caused by the following factors: imbalance of Th1 and Th2 cells and their expression factors, loss of ⁇ cells by virus invasion into the body, and overexpression of UCP2 gene. Or autosomal changes.
  • the invention also provides a combination product for preventing and/or treating diabetes, the combination product comprising a first medicament, the active ingredients of the first medicament comprising colimycin, isovalerylspiramycin I, isoprene One of acetylspiramycin II, one of isovalerylspiramycin III, or a combination of two or three of isovalerylspiramycin I, isovalerylspiramycin II, isovalerylspiramycin III.
  • the combination of the invention further comprises a second agent comprising at least one of the drugs useful for the treatment of glycosaminoglycans.
  • the second agent comprises at least one of a biguanide hypoglycemic agent, a sulfonylurea hypoglycemic agent, an alpha glycosidase inhibitor, an insulin sensitizer, a non-sulfonylurea insulin secretagogue or insulin.
  • the first agent and the second agent are used in an amount of from 1 to 99:99 to 1, preferably from 5 to 95:95 to 5, more preferably from 10 to 90:90 to 10, still more preferably from 20 to 80:80 to 20 .
  • the present invention also provides a medicament for preparing a medicament for preventing and/or treating diabetes by using at least one of colimycin or isovalerylspiramycin I or isovalerylspiramycin II or isovalerylspiramycin III. use;
  • the diabetes is type I or type II diabetes or a particular type of diabetes.
  • the present invention also provides for the preparation of at least one of colimycin, isovaleryl spiromycin I, isovaleryl spiromycin II and isovalerylspiramycin III for preparing insulin secretion or lowering blood sugar or protecting islets Use in the drug of beta cells.
  • the present invention also provides for the preparation of at least one of colimycin, isovalerylspiramycin I, isovalerylspiramycin II and isovalerylspiramycin III for the preparation of a prophylactic and/or therapeutic diabetes and maintaining body weight Use of the drug.
  • a third object of the present invention is to provide a medicament for delaying aging and/or prolonging life.
  • the present invention adopts the following technical solutions:
  • compositions for delaying aging and/or prolonging life wherein said composition comprises a first active ingredient, said first active ingredient being colimycin, isovaleryl spiromycin I, isovaleryl One of spiramycin II, one of isovalerylspiramycin III, or a combination of isovalerylspiramycin I, isovalerylspiramycin II, isovalerylspiramycin III or a combination of three.
  • composition further includes a second active ingredient.
  • the second active ingredient comprises at least one of a drug that delays aging or prolongs life.
  • At least one of the first active ingredients and at least one of the second active ingredients may be formulated as a compound.
  • the ratio of the first active ingredient to the second active ingredient is from 1 to 99:99 to 1, preferably from 5 to 95:95 to 5, more preferably from 10 to 90:90 to 10, More preferably, it is 20-80:80-20.
  • composition is prepared as a pharmaceutical, nutraceutical or food additive with an acceptable adjuvant.
  • the medicament is in a pharmaceutically acceptable dosage form.
  • the pharmaceutically acceptable dosage form is a tablet, a capsule, a pill, an injection, a sustained release agent, and a microparticle administration preparation.
  • the present invention also provides a combination product for delaying aging and/or prolonging life, wherein the combination product comprises a first agent, and the active ingredient of the first agent comprises colimycin, isovalerylspiramycin I, one of isovalerylspiramycin II, one of isovalerylspiramycin III, or two or three of isovalerylspiramycin I, isovalerylspiramycin II, isovalerylspiramycin III combination.
  • kalliktomycin is a mixture of a plurality of active ingredients, in addition to three active ingredients including isopyryl spiromycin I, isovaleryl spiromycin II, isovaleryl spiromycin III, and other Impurities.
  • the combined product further includes a second medicament.
  • the second medicament comprises at least one of anti-aging or anti-aging drugs.
  • the first agent and the second agent may be used in combination.
  • the order of administration of the first agent and the second agent may be indefinitely, and the first agent may be used first, or the second agent may be used first.
  • the medicament can also be used simultaneously with two medicaments.
  • the ratio of the first agent to the second agent is from 1 to 99:99 to 1, preferably from 5 to 95:95 to 5, more preferably from 10 to 90:90 to 10, still more preferably from 20 to 80:80. ⁇ 20.
  • the first agent is in a pharmaceutically acceptable dosage form.
  • the pharmaceutically acceptable dosage form is a tablet, a capsule, a pill, an injection, a sustained release agent, and a microparticle administration preparation.
  • the invention also provides for the use of the described compositions or combination products in the manufacture of products which delay aging and/or prolong life.
  • the combination product in the preparation of a medicament for delaying aging and/or prolonging life by altering the transcription factor DAF-16 activity; by increasing the expression level of the SIR2 homologous protein SIR2.1, SIR2.1 Use in drugs that affect DAF-16 activity to delay aging and/or prolong life; or activate AMPK to directly enhance FOXO/DAF-16 activity, delay aging and/or prolong life applications.
  • the product is a medicine, a health care product or a food additive.
  • the wild type C. elegans sub-administered group and the blank control group were selected, and the growth curves of different concentrations of nematodes were firstly administered.
  • the physiological indicators related to lifespan such as nematode spawning and behavioral activities were investigated.
  • the impact of changes in capabilities The survival rate of nematodes after high temperature stress and ultraviolet irradiation stimulation at 37 ° C after the administration of colimycin was also determined.
  • the present invention utilizes Caenorhabditis elegans as a model organism for screening anti-aging drugs.
  • the nematode-rich genetic resources can be used to select appropriate mutants according to the research purpose to study the mechanism of aging and anti-aging. In essence, several major theories of aging mechanisms are proved in nematodes. Therefore, drugs that have anti-aging effects on nematodes are also generally considered to have the same effect on humans.
  • Caenorhabditis elegans has been used for life analysis and has been in existence for 30 years. Because of its unique advantages, it has become the first model of aging research.
  • the nematode has a short cycle, usually about 3 days, and usually has a short life span of about 3 weeks. This makes it possible to repeat the stability of the experiment. In order to ensure the reliability of the experimental method and the accuracy of the experimental results, drug screening can obtain more accurate and credible information, repeated experiments are necessary.
  • Caenorhabditis elegans has the above unique advantages, it has become the first model of aging research. Therefore, nematodes can be used to evaluate the anti-aging effect of the composition, thereby judging that the composition is used to prepare an anti-aging drug.
  • the medicament can be prepared into various pharmaceutically acceptable dosage forms such as tablets, capsules and the like by a method conventional in the art.
  • the dose of the drug is 10 to 1500 mg/kg; preferably 50 to 1000 mg/kg; more preferably 100 to 500 mg/kg.
  • colimycin or its pharmaceutically active ingredient isopyryl spiromycin I, isovaleryl spiromycin II or isovaleryl spiromycin III or a combination thereof has better resistance to Alzheim
  • the role of silent disease, anti-diabetes or anti-aging provides a theoretical basis for the clinical promotion of the drug colimycin or its active ingredients in this aspect, and has important economic and social benefits.
  • Figure 2 is a total number of advance arms of each group of rats in the rat Y maze test of the present invention
  • Figure 3 is a prioritized index of new objects in each group of rats in the rat new object discrimination experiment of the present invention
  • Figure 4 is a priority index of new objects in each group of rats in the rat new object discrimination experiment of the present invention after 24 hours;
  • Figure 5 is a discrimination coefficient of a new object after 1 hour in each rat of the rat in the new object discrimination experiment of the present invention
  • Figure 6 is a discrimination coefficient of a new object after 24 hours in each rat of the rat new object discrimination experiment of the present invention
  • Figure 7 is an escape latency of each group of rats in the rat water maze test of the present invention.
  • Figure 8 is the number of times of rats in each group of rats in the rat water maze test of the present invention.
  • Figure 9 is a swimming speed of each group of rats in the rat water maze test of the present invention.
  • Figure 10 shows the effect on life cycle after administration of colimycin
  • Figure 11-a shows the measurement of exercise index after administration of 5 ⁇ g/ml of colimycin
  • Figure 11-b shows the measurement of exercise index after administration of 10 ⁇ g/ml of colimycin
  • Figure 12 is a graph showing the survival rate under heat stress after administration of colimycin
  • Figure 13 is a measurement of the survival rate under UV irradiation after administration of colimycin
  • Figure 14-a shows the fluorescence intensity after 10 days of administration of colimycin
  • Figure 14-b shows the fluorescence intensity after 15 days of administration of colimycin
  • Figure 15 is a measurement of fluorescence intensity after administration of colimycin
  • Figure 16-a shows the determination of DJ-16 entry into the nucleus of TJ356 nematode by colimycin for 6 days;
  • Figure 16-b shows the fluorescence intensity of TJ356 nematode on the 6-day administration of colimycin
  • KL represents roscomycin and RES represents resveratrol.
  • the drug colimycin involved in the following examples may also be one or more of isovaleryl spiromycin I or isovaleryl spiromycin II or isovaleryl spiromycin III. Compositions.
  • the main drug and the auxiliary material are respectively passed through a 100 mesh sieve, and the prescription amount of colimycin, microcrystalline cellulose and 1/2 prescription amount of sodium carboxymethyl starch are uniformly mixed, and then 5% povidone K 30 is added.
  • the soft material made of aqueous solution is granulated by 18 mesh sieve, the wet granules are dried under the condition of 60 ° C for 2 hours; after drying, the granules are sieved with 18 mesh, and then 1/2 prescription amount of sodium carboxymethyl starch and stearic acid are added.
  • the magnesium was uniformly mixed, it was tableted with a shallow concave die having a diameter of 11 mm to obtain a tablet core containing a tablet weight of 350 mg and a hardness of 6.5 kg.
  • Preparation of coating liquid Weigh the required Opadry II (white) into the liquid container, add the required amount of water, add in several portions, after all the addition, reduce the stirring speed, make the spiral disappear, continue to stir 30min, that's it.
  • Preparation of film coated tablets The core is placed in a coating pan to determine the coating conditions, the host speed is 20r/min, the inlet air temperature is 40 ° C, the outlet air temperature is 30 ° C, the spray pressure is 0.02 Mpa, and the spray flow rate is The coating was carried out at 1 ml/min, and the coating was continued for 1.5 hours after constant, until the surface of the granules was smooth and the color was uniform, which was in compliance with the film coating inspection standard. The coating gains about 5%.
  • Preparation process weigh the appropriate amount of starch, dilute to 15% concentration, heat to a paste, to make a binder; main material can be lignin, adjuvant starch, low-substituted hydroxypropyl cellulose, sodium carboxymethyl starch, hard The magnesium sulphate was passed through a 100 mesh sieve, and the required main ingredients and auxiliary materials were weighed according to the prescription amount; the drug A, starch, and low-substituted hydroxypropyl cellulose were well mixed, and then softened with a starch paste of 15% starch concentration.
  • main material can be lignin, adjuvant starch, low-substituted hydroxypropyl cellulose, sodium carboxymethyl starch, hard
  • the magnesium sulphate was passed through a 100 mesh sieve, and the required main ingredients and auxiliary materials were weighed according to the prescription amount; the drug A, starch, and low-substituted hydroxypropyl cellulose were well mixed, and then softened with a
  • Preparation process the main material of colamycin, auxiliary medicinal starch is weighed according to the process formula, and then mixed into the mixer for 1.5-2 hours; the data obtained by sampling and testing should be basically consistent with the theoretical data (each grain)
  • the weight of the capsule is about 0.105g), and the qualified medicinal No. 3 capsule and the mixed raw materials to be loaded are filled in the filling device according to the operation requirements of the automatic capsule machine, and the filled capsules are filled.
  • Difference test ( ⁇ 10% or less, ⁇ 0.3g), dissolution rate meets the requirements, the capsules that meet the requirements after inspection, put into the polishing machine, add liquid paraffin for 15-20 minutes of light, and then take out the finished packaging box test.
  • Preparation process the original powder of colimycin, citric acid and sucrose are respectively pulverized into 85% of the particles by 300 mesh, 15% through 180 mesh, and then the fine powder after crushing is weighed according to the prescription amount and fully mixed. 1-1.5 hours, measure the content, calculate the loading (theoretical capacity is 500mg per bag), then put the mixture into the bagging machine, install the aluminum foil paper, and pack according to the operation of the filling machine. The difference is ⁇ 5 Within %, after the installation, the outer packaging is carried out after passing the inspection.
  • Preparation process the original powder of colimycin, powdered sugar, dextrin passed through a 120 mesh sieve, and the colimycin, sugar powder and dextrin were uniformly mixed according to the prescription amount, and the above materials were uniformly mixed with 5% PVP-K. 30 glue is made into soft material, and the swinging granules are granulated at 70 ° C for drying and granulation.
  • Example 6 lysine lyophilized powder injection
  • Test Example 1 Using cell experiments to determine whether colicin has the effect of protecting islet cells
  • the purpose of the assay was to evaluate the effect of the test sample, available, on the protection of islet beta cells in vitro.
  • Rat insulinoma cell INS cells were purchased from the Cell Resource Center of the Institute of Basic Research, Chinese Academy of Medical Sciences.
  • RPMI1640 medium and fetal bovine serum were purchased from Gibco, USA, trypsin, glutamine, penicillin, streptomycin, dimethyl sulfoxide (DMSO), tetramethyl azozolium (MTT), alloxan (Alloxan). Monohydrate, purity ⁇ 98.0%) were purchased from Sigma, USA.
  • Carbon dioxide incubator (Sanyo, Japan), enzyme-linked immunosorbent analyzer (Tecan, Austria), 96-well culture plate (Corning, USA), inverted microscope (Motic, China).
  • INS-1 is an adherent cell, taking INS-1 cells in logarithmic growth phase
  • the cells in the drug-administered group were given 24mmol/L alloxan injury, and different concentrations (0.2, 0.4, 0.8mM) of the drug to be tested were given to the drug, and 4 duplicate holes were set for each concentration.
  • the culture was continued at 37 ° C for 24 h.
  • a normal control group without any drug treatment
  • a model group only with alloxan injury
  • a positive control group with metformin 0.5 mmol/L added to the alloxan treatment
  • wash carefully 3 times with PBS add 100 ⁇ L of freshly prepared medium containing 0.5 mg/ml MTT to each well, and continue to incubate for 4 h at 37 °C.
  • the supernatant was carefully discarded, and 150 mL of DMSO was added thereto, and after mixing for 10 minutes with a micro-oscillator, the optical density value was measured at 492 nm with a microplate reader.
  • the purpose of the assay was to evaluate the effect of the test sample, available, on the blood glucose of diabetic mice.
  • Alloxan monohydrate purity ⁇ 98.0%) and metformin (Metformin, purity 97%) were supplied by Sigma-Aldrich.
  • the insulin assay kit was purchased from Shanghai Rongsheng Biological Pharmaceutical Co., Ltd. (batch number: E02I0006).
  • One-touch blood glucose tester and blood glucose test strip (American Johnson & Johnson), electronic analytical balance.
  • mice with stable blood glucose levels at the model level (10-25 mmol/L) were randomly divided into model controls.
  • the group, the positive drug control group (metformin 200mg/kg), the drug-administered group (the drug is coli-mycin, 25, 50, 100mg/kg), and the normal control group was also established, and the normal saline was administered.
  • mice in each group were given a glucose solution by intragastric administration of 2 g/kg, blood was collected from the tail tip, and blood glucose values of 0, 0.5, and 2 h after glucose administration were determined (Bg0, Bg0). .5, Bg2), calculate the area under the blood glucose curve (AUC) (reactive glucose tolerance) as follows:
  • the rats were fasted for 16 hours, and blood was taken from the fundus venous plexus. Before the blood was taken, the capillary was moistened with heparin, and 0.5 ml of blood was taken from the fundus venous plexus, 3500 r/min, and centrifuged for 10 min, and the plasma was separated for testing. The supernatant was aspirated and insulin was measured by ELISA. The specific operation is carried out according to the kit instructions.
  • the insulin resistance index (Insulin Resistance): After measuring the concentration of fasting blood glucose and insulin, the insulin resistance index was calculated by the formula.
  • Insulin resistance index fasting blood glucose * fasting insulin / 22.5 (recently given >2.6 when insulin resistance was judged)
  • Colimycin has the symptoms of reducing body weight in diabetic mice, lowering fasting blood glucose in mice, increasing glucose tolerance, and not affecting fasting serum insulin, but it can reduce insulin resistance index, indicating that colimycin has good Hypoglycemic effect.
  • Test Example 3 Effect of colimycin on AD model induced by injection of A ⁇ 1-42 in bilateral hippocampal CA1 region of SD rats
  • a ⁇ 1-42 purchased from (Sigma, USA);
  • Resveratrol Aladdin (Lot #K1414052) (L.A., CA, USA);
  • Grouping male SD rats, weighing 220-260 g, adaptive feeding for 3 days, free drinking water, 12h day and night cycle. They were randomly divided into 6 groups, namely sham-operated control group, model group, coli-mycin 25, 50 and 100 mg/kg, and positive drug resveratrol 30 mg/kg.
  • a ⁇ 1-42 was dissolved in hexafluoroisopropanol/sterilized physiological saline solution to a concentration of 2 ⁇ g/ ⁇ L, and incubated at 4 ° C for 24 h to form A ⁇ oligomers, which were used.
  • the rats were anesthetized with 3.5% chloral hydrate (350 mg/kg) and fixed on a stereotaxic instrument.
  • the previous point was a dot, 3.6 mm backward, ⁇ 2.5 mm left, and a needle depth of 3.1 mm.
  • the bilateral hippocampal CA1 area was injected once with a micro-injector, and each side of the hippocampus was injected 2.5 ⁇ L (0.5 ⁇ L/) within 5 min.
  • the needle was left for 5 min, causing the rat dementia model;
  • the sham-operated control group was injected with the same volume of normal saline in the hippocampal CA1 area by the same operation method, ie animal model selection: using A ⁇ 1-42 lateral ventricle injection method
  • the sham-operated control rats were injected with an equal volume of normal saline in the bilateral hippocampal CA1 region by the same method.
  • the amyloid plaque of ⁇ -amyloid (A ⁇ ) is currently recognized as a pathological marker of AD.
  • the sham-operated control group and the model group were intragastrically administered with the corresponding solvent, and the other experimental groups were given glyphosine 25, 50, and 100 mg/kg, respectively, and the positive drug resveratrol 30 mg/kg.
  • Y-maze, new object discrimination and water maze experiments were performed on the 12th day after injection of A ⁇ 1-42 into the hippocampus. Dosing continued during the behavioral experiment, once a day until the end of behavior.
  • Rat Y maze test On the 12th day of administration, each group of rats was subjected to Y maze test.
  • the rat Y maze test was designed to investigate the effects of colimycin on spontaneous spontaneous activity and working memory in rats.
  • the device consists of three wooden arms with an angle of 120 degrees, which are three arms of A, B and C respectively.
  • the rats were placed at the end of the A-arm, and they were allowed to freely enter and exit the three arms.
  • the total number of times each rat entered the three arms and the sequence of the arm in 8 minutes were recorded to continuously enter three different arms for a correct alternation. Reaction, record the number of correct alternating reactions.
  • the spatial working memory ability is reflected by the spontaneous alternating reaction rate.
  • Rat new object discrimination experiment On the 14th-15th day of administration, each group of rats was subjected to a new object discrimination experiment.
  • the rat new object discrimination experiment was designed to investigate the effect of colimycin on the image recognition of rats.
  • the experimental device is a black plastic circular open field with a diameter of about 60 cm and a height of 20 cm. This experiment is divided into adaptation phase and testing phase. In the adaptation phase, 2 to 3 rats were placed in the open field each time, allowing them to freely explore for 3 minutes to adapt to the environment, 2 times a day for 2 days. On the third day, the test was carried out. Each time, one rat was placed in the open field and freely explored for 3 minutes.
  • the rats were taken out, and two identical objects (A1, A2) were placed in the center of the open field and placed in the rat. Record the time (tA1, tA2) of exploring two objects within 5 minutes. After 1 h, the A2 object was replaced with a new object B, and the rats were placed again, and the time (tA1, tB) at which the two objects were explored was recorded. After 24 hours, the B object was replaced with a C object, and the rats were placed again, and the time (tA1, tC) used to explore the two objects was recorded.
  • the criterion for the exploration is that the rat points the object with the nose and is within 1 cm of the object, or touches the nose, rubbing the object, or touching the object with the front paw. Calculate the Preferential index and Discrimination index for new objects.
  • Priority index (1h) tB / (tA1 + tB)
  • Priority index (24h) tC / (tA1 + tC)
  • Discrimination coefficient (24h) (tC-tA1) / (tA1 + tC)
  • Rat water maze test On the 16th to 20th day of administration, the rats in each group were subjected to the Morris water maze test.
  • the Morris water maze experiment was designed to investigate the effects of colimycin on spatial learning and memory impairment.
  • the water labyrinth consists of a black stainless steel circular pool 1.5 meters in diameter and 50 centimeters high and a circular metal platform with a diameter of 10 cm. The platform is free to move. Water was injected into the pool before the experiment (water temperature 24 ⁇ 1 ° C) so that the water surface was 1 cm above the platform.
  • each training session was conducted every day and afternoon for 6 days. The platform was placed in the fourth quadrant, and the rat was placed in the water facing the pool wall for 90 seconds.
  • the rat found the platform within 90 seconds, he was allowed to rest on the platform for 10 seconds. If the platform is not found in 90 seconds, the rats are guided to the platform for 10 seconds. After the training, the test was carried out, the platform was removed, and the rats were allowed to swim for 90 seconds. The maze system automatically records the time the rat stays in the original platform quadrant (target quadrant).
  • Y-maze test of working memory ability of rats The experimental results showed that compared with the sham-operated control group, the spontaneous alternation rate of Y-maze in the model group was significantly decreased; compared with the model group, colimycin (25, 50, 100 mg) In the control group of /kg) and resveratrol (30 mg/kg), the rate of spontaneous alternation of Y maze was significantly increased (see Figure 1). There was no significant difference in the total number of arms in each group of rats (see Figure 2). It can be concluded that colimycin can improve A ⁇ 1-42 induced working memory impairment in rats.
  • the new object discrimination experiment was used to test the image memory ability of rats: the experimental results showed that there was no significant difference in the discrimination coefficients between the two objects of A1 and A2 in each group (see Table 7).
  • the model group rats showed a significant decrease in the priority index and discrimination coefficient of new objects after 1h and 24h; compared with the model group, colimycin and white peony
  • the rosin group showed a significant increase in the priority index (see Figure 3, Figure 4) and the discrimination coefficient (see Figure 5 and Figure 6) for new objects. Therefore, colimycin has an improved A ⁇ 1-42- induced damage to rat image memory.
  • the medicament of the invention has a better therapeutic effect on the central nervous system disease Alzheimer, and not only provides a theoretical basis for the application and clinical promotion of the medicament of the invention in the treatment of central nervous system diseases, and has Important economic and social benefits.
  • Test Example 4 Using life test to determine whether colimycin has a prolonged lifespan of C. elegans
  • the hermaphroditic nematodes were reared on Nematode growth medium (NGM) coated with Escherichia coli OP50 (E. coli OP50) at a culture temperature of 20 ° C to observe the growth of nematodes, and the nematodes were periodically transferred to a new coated E. .coli OP50 in NGM medium.
  • NGM Nematode growth medium
  • E. coli OP50 Escherichia coli OP50
  • nematodes in the spawning period are picked up in the same plate (the specific number depends on the number of nematodes required, and usually one nematode can lay 8 eggs in one hour). After 30 min, the nematodes in the plate were picked out, and the eggs in the plates were in the same developmental period after hatching.
  • the liquid administration method is used to set the administration group and the blank group, the drug concentration of the administration group is selected, and the drug is diluted to the desired concentration with physiological saline, and a 24-well plate is used.
  • Only nematodes were conided, and the surviving nematodes were transferred to the next new wells of the same conditions every 24 h until death.
  • the nematodes that did not respond to mechanical stimuli and did not vomit or excrete were judged to be death.
  • the number of surviving days and the average number of days of survival of each group of nematodes were recorded every 24 h after transfer.
  • Test Example 5 Detection of exercise capacity index to determine whether colimycin has a prolonged life span of C. elegans
  • the survival rate of C. elegans was determined under heat shock conditions to determine whether colimycin has a prolonged effect on the lifespan of C. elegans.
  • the nematodes were transferred to 37 ° C for 10 h, and the number of nematodes surviving after the end of the culture was recorded as the survival rate of the experiment. The experiment should be repeated 2-3 times in parallel, and the average value was taken as the final survival result.
  • the survival rate of C. elegans under UV irradiation was determined to determine whether colimycin has a prolonged effect on the lifespan of C. elegans.
  • the nematode death standard is the same as the life test.
  • the ultraviolet wavelength was 254 nm, and the height of the ultraviolet lamp from the culture plate was 15 cm, and the power was 1 ⁇ 10 -3 W/cm 3 .
  • the experiments were grouped into a blank group and a drug administration group.
  • colimycin can reduce the accumulation of lipofuscin in wild-type C. elegans can be detected.
  • lipofuscin Due to the accumulation and aggregation of cross-linked proteins and the like, lipofuscin is deposited in various tissues and organs of the human body, resulting in a decrease in cell metabolism and a decrease in activity, thereby causing aging of human organ function and aging. Therefore, lipofuscin is considered to be a sign of aging. Lipofuscin has the characteristic of autofluorescence, and the nematode is observed under a fluorescence microscope, and its senescence degree can be judged according to its fluorescence intensity. The results of the measurement are shown in Figures 14-a and 14-b. The results showed that the amount of lipofuscin accumulated in the administration group was significantly lower than that in the control group.
  • sod-3 content measurement showed that the expression level of sod-3 was decreased in the nematode group after administration, and the lipofuscin fluorescence content showed that the lipofuscin content of the drug-administered group was decreased after administration.
  • the present invention investigates whether colicin has an effect on the dod-16 upstream genes daf-16 and daf-15.
  • Coriomycin can increase the expression of anti-aging gene sod-3 in nematodes
  • ROS is the main cause of aging and disease.
  • the present invention can enhance the heat-resistance of wild-type nematodes and enhance the intracellular stress resistance of nematodes by investigating the colimycin. Therefore, the inventors suspected that KL was anti-aging by affecting the antioxidant gene sod-3, and after continuously treating the CF1553 (sod-3::GFP) type nematode with colimycin for 6 days, the nematode was observed by fluorescence microscopy. The amount of green fluorescent protein expressed was significantly higher than that of the control group. The results are shown in Figure 15. It indicates that colimycin may prolong the lifespan by increasing the level of ROS in the nematode by increasing the antioxidant gene sod-3.
  • Coriomycin can affect the distribution of DAF-16 in cells.
  • TJ356 (DAF-16::GFP) nematodes in L4 were picked from NGM plates with good OP50 and laid for 3-4 hours. The nematode was completed simultaneously. Eggs were administered in groups of 30 after the eggs grew to adulthood. The TJ356 nematode DAF-16 was enrolled in the nucleus for 6 days, and the DIF-16 nucleus was observed using an inverted fluorescence microscope.
  • DAF-16 The accumulation of DAF-16 in the nucleus or in the cytoplasm is directly related to its activity. Under normal conditions, DAF-16 is localized in the cytoplasm. If it is under external stress, different proteins are activated, which promotes transcription into the nucleus to initiate downstream gene expression to prolong the lifespan of the nematode. Studies have shown that lack of insulin or oxidative stress can cause DAF-16 to aggregate in the nucleus, improve the ability of nematodes to resist oxidative stress and prolong the lifespan of nematodes.
  • the present invention utilizes DAF-16::GFP transgenic nematode TJ356 to investigate the distribution of DAF-16 in cells.
  • the nematode that had just entered the adult stage was treated with colimycin, and after 6 days of administration, it was observed with an inverted fluorescence microscope.

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Abstract

一种用于预防和/或治疗疾病的药物，所述的疾病为阿尔茨海默病、糖尿病或衰老；所述药物包括第一活性成分，所述的第一活性成分包括可利霉素、异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ和异戊酰螺旋霉素Ⅲ中的一种；或者异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ中的两种或三种的组合。

Description

可利霉素或其活性成分的用途 技术领域
本发明涉及药物领域，具体地说，涉及可利霉素或其活性成分的用途。
背景技术
可利霉素(Carrimycin)是通过转基因技术将碳霉素产生菌的4”异戊酰基转移酶基团(4”-o-acyl-transferase)克隆至螺旋霉素产生菌中，定向酰化螺旋霉素4”-OH，在4”位加入异戊酰基侧链所形成的以4”位异戊酰基螺旋霉素为主要组分的新型抗生素。
可利霉素是由多种螺旋霉素衍生物组成，主要活性成分异戊酰螺旋霉素(I+II+III)总含量不低于60％，于药学上是一种可接受的药物组合物。中心结构为16元内酯环，与一分子福洛胺糖、一分子碳霉胺糖和一分子碳霉糖连接而成，其主要成分异戊酰螺旋霉素I、II、III与螺旋霉素结构不同之处在于碳霉糖4”位上连接的基团为异戊酰基而不是羟基。该药由沈阳同联等共同申报1.1类新药。
可利霉素主成分的化学结构，如式(1)所示：
Figure PCTCN2019072413-appb-000001
其中，当R＝H，R′＝COCH 2CH(CH 3) 2时为异戊酰螺旋霉素Ⅰ；
当R＝COCH 3，R′＝COCH 2CH(CH 3) 2时为异戊酰螺旋霉素Ⅱ；
当R＝COCH 2CH 3，R′＝COCH 2CH(CH 3) 2时为异戊酰螺旋霉素Ⅲ；
可利霉素属于16元大环内酯类抗生素，具有活性基团羧基、烷氧基、环氧基、酮基和醛基以及一对共轭的C＝C，分子量约为884～982。由于具有相似的化学结构，可利霉素与大环内酯类抗生素具有很多共性：易溶于酯类、丙酮、氯仿、醇类等大多数有机溶剂，微溶于石油醚，难溶于水；分子结构中含有两个二甲胺基而呈弱碱性，易溶于酸性水溶液；具有溶解度随温度的升高而降低的“负溶解度”性质。由于可利霉素主要组分异戊酰螺旋霉素4”位碳链较长，亲水性差，其水中溶解度比螺旋霉素及4”-乙酰螺旋霉素小。
可利霉素是一种白色非结晶粉末，略有引湿性，比旋度约为-80.8°，紫外最大吸收波长为231～232nm，本身带有弱荧光发色基团，遇浓硫酸或盐酸呈紫色反应，产生强紫色荧光，在231～232nm处有最大吸光值。
该药具有亲脂性好，组织渗透能力强，口服吸收快，体内维持时间长，有持续的抗生素后效应。根据药效与化学构象的关系，大环内酯类抗生素4”位酰化后，其亲脂性和体内活性提高，体内抗菌活性与临床治疗效果均得到了显著提升，并且抗生素在体内的稳定性随着4”羟基酯的碳链增长而增强，即异戊酰螺旋霉素＞丁酰螺旋霉素＞丙酰螺旋霉素＞乙酰螺旋霉素。
初步体内外药效学试验表明，该药不仅对多数G +菌有较好抗菌活性，对部分G -菌也有一定作用，各项技术指标明显优于阿奇霉素、红霉素、乙酰螺旋霉素、麦迪霉素，尤其对肺炎支原体的抗菌活性最强，对红霉素耐药菌、淋球菌、肺炎球菌、金葡菌、绿脓假单胞菌、流感杆菌、流感嗜血杆菌、脆弱拟杆菌、军团菌、多行杆菌和产气荚膜梭菌也有一定抗菌活性，对临床耐红霉素的金葡球菌仅有极少交叉耐药性。 可利霉素将主要用于治疗革兰氏阳性菌感染性疾病，尤其是上呼吸道感染，并可能用于泌尿系统感染等。
本申请人在近期的一项研究中发现，可利霉素或其药物活性成分异戊酰螺旋霉素I、异戊酰螺旋霉素II或异戊酰螺旋霉素III或其组合具有较好的抗衰老、抗糖尿病或抗阿尔茨海默病的作用，为药物可利霉素或其药物活性成分在这方面的临床推广提供了理论依据，而且具有重要的经济效益和社会效益。
有鉴于此，特提出本发明。
发明内容
本发明的目的在于提供一种用于预防和/或治疗疾病的药物。
为实现上述目的，本发明采用如下技术方案：
一种用于预防和/或治疗疾病的药物，其中：所述的疾病为阿尔茨海默病、糖尿病或衰老；所述药物包括第一活性成分，所述的第一活性成分包括可利霉素、异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ和异戊酰螺旋霉素Ⅲ中的一种；
或者异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ中的两种或三种的组合。
进一步的，所述的药物还包括第二活性成分；
优选，当所述的疾病为阿尔茨海默病时，所述的第二活性成分为抗阿尔茨海默病药物中的至少一种；
当所述的疾病为糖尿病时，所述的第二活性成分为抗糖尿病药物中的至少一中；
当所述的疾病为衰老时，所述的第二活性成分为延缓衰老或延长寿命的药物中的至少一种。
进一步的，所述药物与药学上可接受的载体制成临床上可接受的制剂，优选片剂、胶囊、丸剂、注射剂、缓释剂及各种微粒给药系统。
进一步的，所述药物的剂量为10～1500mg/kg，优选50～1000mg/kg，更优选100～500mg/kg。
本发明还提供一种用于预防和/或治疗疾病的组合产品，其中，所述的疾病为阿尔茨海默病、糖尿病或衰老；所述的组合产品包括第一药剂，所述的第一药剂的活性成分包括可利霉素或异戊酰螺旋霉素Ⅰ或异戊酰螺旋霉素Ⅱ或异戊酰螺旋霉素Ⅲ中的一种；
或者异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ中的两种或三种的组合。
进一步的，所述的组合产品还包括第二药剂；
优选，当所述的疾病为阿尔茨海默病时，所述的第二药剂为用于预防和/或治疗阿尔茨海默病相关药物中的至少一种；
当所述的疾病为糖尿病时，所述的第二药剂为用于预防和/或治疗糖尿病相关药物中的至少一种；
当所述的疾病为衰老时，所述的第二药剂为延缓衰老或延长寿命相关药物中的至少一种。
进一步的，所述的第一药剂和第二药剂的用量比为1～99：99～1，优选5～95：95～5，更优选10～90：90～10，再优选20～80：80～20。
进一步的，所述的用于预防和/或治疗阿尔茨海默病的相关药物包括作用于胆碱能系统药物、作用于N-甲基-D-门冬氨酸受体药物、抗氧化药物、抗炎药物、抑制Aβ蛋白形成的药物、雌激素、神经生长因子、尼莫地平、抗细胞凋亡剂；
所述的用于预防和/或治疗糖尿病的相关药物包括双胍类降糖药、磺脲类降糖药、α糖苷酶抑制剂、胰岛素增敏剂、非磺酰脲类促胰岛素分泌剂或胰岛素中的至少一种。
本发明还提供所述的药物或所述的组合产品在制备预防和/或治疗阿尔茨海默病、益智、糖尿病、延缓衰老或延长寿命方面的药物中的应用。
具体地说，所述的在制备预防和/或治疗阿尔茨海默病、益智方面的药物中的应用为制备减少乙酰胆碱水解药物中的应用、改善认知障碍和运动障碍药物中的应用、保护脑内神经细胞药物中的应用、不降低体重、提高免疫力或提高白细胞药物中的应用；
所述的在制备预防和/或治疗糖尿病方面的药物中的应用为制备预防和/或治疗Ⅰ型或Ⅱ型糖尿病或特殊类型糖尿病药物方面的应用，优选在制备用于促进胰岛素分泌或降低血糖或保护胰岛β细胞药物中的应用或预防和/或治疗糖尿病并保持体重药物方面的应用；
所述的在延缓衰老或延长寿命方面的药物中的应用为制备通过改变转录因子DAF-16活性来延缓衰老 和/或延长寿命药物中的应用；通过提高SIR2同源蛋白SIR2.1的表达水平，SIR2.1再通过影响DAF-16活性来延缓衰老和/或延长寿命药物中的应用；或通过激活AMPK，直接增强FOXO/DAF-16的活性，延缓衰老和/或延长寿命药物中的应用。
以下详细描述本发明：
本发明的第一目的在于提供一种用于预防和/或治疗阿尔茨海默病的药物。
为实现上述发明，本发明采用如下技术方案：
本发明提供了一种用于预防和/或治疗阿尔茨海默病的药物，所述药物的有效成分包括可利霉素或异戊酰螺旋霉素III或异戊酰螺旋霉素II或异戊酰螺旋霉素I中的一种；或者异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ两种或三种的组合。
可利霉素为多种活性成分的混合物，除了包括异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ这三种活性成分，还含有其他的杂质。
进一步地，所述药物包括药学上可接受的载体。
进一步地，所述药物制成临床上可接受的片剂、胶囊、丸剂、注射剂、缓释剂及各种微粒给药系统。
进一步地，所述药物的剂量为10～1500mg/kg。
进一步地，所述药物的剂量为50～1000mg/kg。
进一步地，所述药物的剂量为100～500mg/kg。
本发明还提供了一种用于预防和/或治疗阿尔茨海默病的组合产品，所述的组合产品包括第一药剂，所述的第一药剂的活性成分包括可利霉素或异戊酰螺旋霉素III或异戊酰螺旋霉素II或异戊酰螺旋霉素I中的一种；或者异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ两种或三种的组合。
进一步地，所述的组合产品还包括第二药剂。
进一步地，所述的第二药剂包括用于预防和/或治疗阿尔茨海默病的相关药物中的至少一种。
进一步地，所述的用于预防和/或治疗阿尔茨海默病的相关药物包括作用于胆碱能系统药物、作用于N-甲基-D-门冬氨酸受体药物、抗氧化药物、抗炎药物、抑制Aβ蛋白形成的药物、雌激素、神经生长因子、尼莫地平、抗细胞凋亡剂。
本发明还提供了一种如上任一所述的药物或组合产品在预防和/或治疗阿尔茨海默病、益智中的应用。
阿尔茨海默病(AD)是一种以进行性痴呆为主要临床表现的神经退行性疾病，胆碱能损伤假说是较早公认的阿尔茨海默病(AD)学说，胆碱能损伤被认为是AD的重要病因，胆碱能系统也被认为是AD药物的重要靶点，实验表明，本发明的药物通过减少乙酰胆碱的水解而增加大脑海马和皮层的乙酰胆碱含量，从而改善认知功能，实现了对阿尔茨海默病的预防和/或治疗。
本发明还提供了一种如上任一所述的药物或组合产品在减少乙酰胆碱水解中的应用。
本发明还提供了一种如上任一所述的药物或组合产品在改善认知障碍和运动障碍中的应用。
本发明还提供了一种如上任一所述的药物或组合产品在保护脑内神经细胞中的应用。
本发明还提供了一种如上任一所述的药物或组合产品在不降低体重、提高免疫力、提高白细胞中的应用。
本发明的第二目的在于提供一种用于预防和/或治疗糖尿病的药物。
为实现本发明的第二目的，本发明采用如下技术方案：
一种预防和/或治疗糖尿病的药物，所述药物的有效成分包括可利霉素、异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ之一，或者异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ中的两种或三种的组合。
本发明所述的药物包括药学上可接受的载体。
本发明所述的药物可以与药学上可接受的载体制备成片剂、胶囊、丸剂、注射剂、缓释剂或微粒给药系统。
本发明所述药物中的有效药分成分的剂量为100～400mg1500mg/kg，优选为50～1000mg/kg，更优选为100～500mg/kg。
所述糖尿病为Ⅰ型或Ⅱ型糖尿病或特殊类型糖尿病。
糖尿病的致病原因有很多，本发明的预防和/或治疗糖尿病的药物主要是针对由下列因素导致的糖尿病：Th1和Th2细胞及其表达因子失衡，病毒入侵机体损失β细胞，UCP2基因过度表达或者常染色体发生异变。
本发明还提供一种预防和/或治疗糖尿病的组合产品，所述的组合产品包括第一药剂，所述第一药剂的有效成分包括可利霉素、异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ之一，或者异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ中的两种或三种的组合。
本发明所述的组合产品还包括第二药剂，所述第二药剂包括可用于治疗糖尿糖的药物中的至少一种。
优选地，所述第二药剂包括双胍类降糖药、磺脲类降糖药、α糖苷酶抑制剂、胰岛素增敏剂、非磺酰脲类促胰岛素分泌剂或胰岛素中的至少一种。
优选地，第一药剂和第二药剂的用量比为1～99：99～1，优选5～95：95～5，更优选10～90：90～10，再优选20～80：80～20。
本发明还提供将可利霉素或异戊酰螺旋霉素I或异戊酰螺旋霉素II或异戊酰螺旋霉素III中的至少一种用于制备预防和/或治疗糖尿病的药物的用途；
所述糖尿病为Ⅰ型或Ⅱ型糖尿病或特殊类型糖尿病。
本发明还提供将可利霉素、异戊酰螺旋霉素I、异戊酰螺旋霉素II和异戊酰螺旋霉素III中的至少一种用于制备促进胰岛素分泌或降低血糖或保护胰岛β细胞的药物中的用途。
本发明还提供将可利霉素、异戊酰螺旋霉素I、异戊酰螺旋霉素II和异戊酰螺旋霉素III中的至少一种用于制备预防和/或治疗糖尿病并保持体重的药物中的用途。
本发明的第三目的在于提供一种延缓衰老和/或延长寿命的药物。
为实现本发明的第三目的，本发明采用如下技术方案：
一种延缓衰老和/或延长寿命的组合物，其中，所述的组合物包括第一活性成分，所述的第一活性成分为可利霉素、异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ之一，或者异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ两种或三种的组合。
进一步的，所述的组合物还包括第二活性成分。
进一步的，所述的第二活性成分包括延缓衰老或延长寿命药物中的至少一种。
本发明中，可以将第一活性成分中的至少一种与第二活性成分中的至少一种做成复方制剂。
进一步的，在制成复方制剂时，第一活性成分与第二活性成分的用量比为1～99：99～1，优选5～95：95～5，更优选10～90：90～10，再优选20～80：80～20。
进一步的，所述的组合物与可接受的辅料制备成药物、保健品或食品添加剂。
进一步的，所述的药物为药学上可接受的剂型。
进一步的，所述的药学上可接受的剂型为片剂、胶囊、丸剂、注射剂、缓释剂及微粒给药制剂。
本发明还提供一种延缓衰老和/或延长寿命的组合产品，其中，所述的组合产品包括第一药剂，所述的第一药剂的活性成分包括可利霉素、异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ之一，或者异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ两种或三种的组合。
本发明中，可利霉素为多种活性成分的混合物，除了包括异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ这三种活性成分，还含有其他的杂质。
进一步的，所述的组合产品还包括第二药剂。
进一步的，所述的第二药剂包括抗衰老或延缓衰老药物中的至少一种。
本发明中，还可以将第一药剂与第二药剂进行联合用药，在联合用药时，第一药剂与第二药剂的用药顺序不分先后，可以先用第一药剂，也可以先用第二药剂，也可以两个药剂同时使用。
在联合用药时，第一药剂和第二药剂的用量比为1～99：99～1，优选5～95：95～5，更优选10～90：90～10，再优选20～80：80～20。
进一步的，所述的第一药剂为药学上可接受的剂型。
进一步的，所述的药学上可接受的剂型为片剂、胶囊、丸剂、注射剂、缓释剂及微粒给药制剂。
本发明还提供所述的组合物或组合产品在制备延缓衰老和/或延长寿命产品中的应用。
具体地说，涉及所述的组合产品在制备通过改变转录因子DAF-16活性来延缓衰老和/或延长寿命的药物中的应用；通过提高SIR2同源蛋白SIR2.1的表达水平，SIR2.1再通过影响DAF-16活性来延缓衰老和/或延长寿命的药物中的应用；或通过激活AMPK，直接增强FOXO/DAF-16的活性，延缓衰老和/或延长寿命药物中的应用。
进一步的，所述产品为药物、保健品或食品添加剂。
研究选用野生型秀丽隐杆线虫分给药组、空白对照组，首先给予不同浓度的测定线虫的生长曲线；考察了可利霉素对与寿命相关的生理指标如线虫的产卵量和行为活动能力变化的影响。还通过测定给予可利霉素之后线虫在37℃高温胁迫、紫外照射刺激后的存活率。
线虫用于抗衰老模型具有以下优点：
由于线虫60％-80％的基因与人类的相关基因高度保守，而且至今发现的条信号传导途径，线虫具有其中的12条，因此本发明利用秀丽隐杆线虫作为筛选抗衰老药物的模型生物。可利用线虫丰富的遗传资源，根据研究目的选择合适的突变体，研究衰老及抗衰老机制，实质上衰老机制的几大理论都是在线虫中被证明。因此，对线虫有抗衰老作用的药物通常也被认为对人类也具有同样功效。
秀丽隐杆线虫被用于寿命分析，至今已有30年的历程。因其独特的优势，成为衰老研究的首推模型。线虫世代周期短，通常为3天左右寿命短，通常为3周左右。这使实验的重复稳定性成为可能。为了确保实验方法的可靠性和实验结果的准确性、药物筛选能得到更多准确的、可信的信息，重复实验是必要的。由于秀丽隐杆线虫具有以上独特的优势，而成为衰老研究的首推模型。因此线虫可以被用于评价组合物的抗衰老作用，进而判断组合物被用于制备抗衰老药物。
研究结果表明，可利霉素对秀丽隐杆线虫有抗衰老方面作用。
本发明中，所述的药物可采用本领域常规的方法制备成药学上可接受的各种剂型，如片剂、胶囊等。
本发明中，所述的药物的剂量为10～1500mg/kg；优选50～1000mg/kg；更优选100～500mg/kg。
本申请人经过试验证明，可利霉素或其药物活性成分异戊酰螺旋霉素I、异戊酰螺旋霉素II或异戊酰螺旋霉素III或其组合具有较好的抗阿尔茨海默病、抗糖尿病或抗衰老的作用，为药物可利霉素或其药物活性成分在这方面的临床推广提供了理论依据，而且具有重要的经济效益和社会效益。
附图说明
图1是本发明的大鼠Y迷宫实验中各组大鼠Y迷宫自发交替反应率；
图2是本发明的大鼠Y迷宫实验中各组大鼠的进臂总次数；
图3是本发明的大鼠新物体辨别实验中各组大鼠在1h后对新物体的优先指数；
图4是本发明的大鼠新物体辨别实验中各组大鼠在24h后对新物体的优先指数；
图5是本发明的大鼠新物体辨别实验中各组大鼠在1h后对新物体的辨别系数；
图6是本发明的大鼠新物体辨别实验中各组大鼠在24h后对新物体的辨别系数；
图7是本发明的大鼠水迷宫实验中各组大鼠的逃避潜伏期；
图8是本发明的大鼠水迷宫实验中各组大鼠的穿台次数；
图9是本发明的大鼠水迷宫实验中各组大鼠的游泳速度；
图10为给予可利霉素后对寿命周期的影响；
图11-a为给予5μg/ml可利霉素后运动指标测定；
图11-b为给予10μg/ml可利霉素后运动指标测定；
图12为给予可利霉素后热应激反应下生存率测定；
图13为给予可利霉素后UV照射下生存率测定；
图14-a为给予可利霉素10天后荧光强度测定；
图14-b为给予可利霉素15天后荧光强度测定；
图15为给予可利霉素后荧光强度测定；
图16-a为给予可利霉素6天TJ356线虫DAF-16入核情况测定；
图16-b为给予可利霉素6天TJ356线虫荧光强度测定；
其中图中KL表示可利霉素，RES表示白藜芦醇。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例，对实施例中的技术方案进行清楚、完整地描述，以下实施例用于说明本发明，但不用来限制本发明的范围。
需要说明的是：以下实施例中涉及的药物可利霉素，也可以是异戊酰螺旋霉素I或异戊酰螺旋霉素II或异戊酰螺旋霉素III中的一种或几种的组合物。
实施例1、可利霉素片
规格：200mg/350mg
片芯处方：
Figure PCTCN2019072413-appb-000002
包衣液处方：
Figure PCTCN2019072413-appb-000003
制备工艺：
片芯的制备：主药和辅料分别过100目筛，将处方量可利霉素、微晶纤维素与1/2处方量的羧甲淀粉钠混合均匀，然后加入5％聚维酮K 30水溶液制软材，以18目筛制粒，湿颗粒在60℃通风条件下干燥2h；干燥后以18目筛整粒，再加入1/2处方量的处方量羧甲淀粉钠与硬脂酸镁混合均匀后，用直径11mm的浅凹冲模压片，制得片重350mg、硬度6.5kg的含药片芯。
包衣液的配制：称好所需的欧巴代II(白色)在配液容器中加入所需量的水，分次加入，待全部加入后，降低搅拌速度，使蜗旋消失，继续搅拌30min，即得。
薄膜包衣片的制备：将片芯置于包衣锅内，确定包衣条件，主机速度为20r/min，进风温度40℃，出风温度30℃，喷雾压力0.02Mpa，喷浆流量为1ml/min进行包衣，恒定后持续喷包1.5h，至片粒表面光滑、色泽均匀，符合薄膜衣检验标准为合格。包衣增重5％左右。
实施例2、可利霉素素片(按10000片计算)
处方：
Figure PCTCN2019072413-appb-000004
制备工艺：称取适量淀粉，稀释至15％浓度，加热至糊状，制成粘合剂；主料可利霉素、辅料淀粉、低取代羟丙基纤维素、羧甲基淀粉钠、硬脂酸镁分别过100目筛，按处方量，称取所需主料和辅料；药物A、淀粉、低取代羟丙基纤维素充分混合均匀后，用15％淀粉浓度的淀粉糊制成软材，14目筛制粒，50-60℃干燥，水份控制在3-5％，14目筛整粒，加羧甲基淀粉钠，硬脂酸镁混合，测定颗粒含量；根据颗粒含量，计算片重，压片(Φ9mm浅凹冲头)，检测片重差异；经检验合格后进行包装。
实施例3、可利霉素胶囊剂(按10000粒计算)
处方：
Figure PCTCN2019072413-appb-000005
制备工艺：将主料可利霉素、辅料药用淀粉按工艺配方量分别称取后，装入混合器充分混合后1.5-2小时；取样检测含量所得数据应和理论数据基本一致(每粒胶囊所装重量约为0.105g)，将经检验合格的药用3号胶囊及混合好的待装原料按全自动胶囊机操作要求，分别填入装料器进行填充，将填充好的胶囊进行差异检验(±10％以内，＜0.3g)，溶出度符合要求，将检验后符合要求的胶囊，放入打光机内加入液体石蜡进行15-20分钟的打光，然后取出进行成品包装盒检验。
实施例4、可利霉素干糖浆(按10000袋计算)
处方：
Figure PCTCN2019072413-appb-000006
制备工艺：可利霉素原粉，柠檬酸、蔗糖分别用高速气流粉碎机粉碎成颗粒85％通过300目，15％通过180目，然后将粉碎后的细粉按处方量称取后充分混合1-1.5小时，测其含量，计算装量(理论装量为每袋500mg)，然后将混合物装入袋装机中，装好铝箔纸，按分装机操作要求分装，装量差异在±5％以内，装好后进行检验合格后外包装。
实施例5、可利霉素颗粒剂(按10000袋计算)
处方：
Figure PCTCN2019072413-appb-000007
制备工艺：可利霉素原粉、糖粉、糊精过120目筛，按处方量称取可利霉素、糖粉、糊精混合均匀，将混合均匀的上述物料用5％PVP-K 30胶浆制成软材，摇摆式颗粒剂制粒70℃干燥、整粒，送检合格后分装。
实施例6、可利霉素冻干粉针剂
称取可利霉素原粉500mg与等摩尔的己二酸混合均匀后溶解于5ml水中，得到淡黄色澄明溶液，pH在4.6-5.6之间。再加入甘露醇40mg作为冻干支撑剂，低温快速冷冻9h后，冷冻干燥，获得淡黄色疏松块状物，使用前用10ml无菌水溶解。
试验例1、利用细胞实验确定可利霉素是否具有保护胰岛细胞作用
所测定的目的是评价被测试样品可利霉素体外保护胰岛β细胞的作用。
细胞株：
大鼠胰岛素瘤细胞INS细胞购自中国医学科学院基础研究所细胞资源中心。
试剂：
RPMI1640培养液、胎牛血清购于美国Gibco公司，胰蛋白酶、谷氨酰胺、青霉素、链霉素、二甲基亚砜(DMSO)、四甲基偶氮唑(MTT)、四氧嘧啶(Alloxan monohydrate，纯度≥98.0％)均购于美国Sigma公司。
仪器：
二氧化碳培养箱(Sanyo，Japan)、酶联免疫分析仪(Tecan，Austria)、96孔培养板(Corning，USA)、倒置显微镜(Motic，China)。
操作步骤如下：
贴壁细胞：
INS-1为贴壁细胞，取对数生长期的INS-1细胞
，胰蛋白酶消化，用完全培养基吹打，制备成单个细胞悬液，将细胞浓度调整为1*10 5个/ml，接种于96孔板中，100μl/孔，置于5％CO 2、37℃培养箱中培养24h。根据实验需求分组，给药组细胞给予24mmol/L的四氧嘧啶损伤，同时分别给与不同浓度(0.2、0.4、0.8mM)的待测药物可利霉素，每个浓度设置4个复孔，37℃继续培养24h。另外还设立正常对照组(不给予任何药物处理)、模型组(仅给予四氧嘧啶损伤)、阳性对照组(在四氧嘧啶处理的基础上加入二甲双胍0.5mmol/L)弃去上清液，用PBS小心洗3次，每孔加入100μL新鲜配制的含0.5mg/ml MTT的培养基，37℃继续培养4h。小心弃去上清，并加入150mL DMSO，用微型振荡器混匀10min后，用酶标仪在492nm处测定光密度值。
结果评定：
按下式计算药物对INS-1细胞的存活率：
INS细胞的存活率(％)＝A 492(给药组)/A 492(正常对照组)×100％
结果：待测药物保护胰岛细胞评价结果如下表1：
表1、可利霉素对INS-1的保护作用
Figure PCTCN2019072413-appb-000008
Figure PCTCN2019072413-appb-000009
*p<0.05与正常对照组相比， ##p<0.01与模型对照组相比， ###p<0.001与模型对照组相比，样品对INS-1细胞显示了良好的保护作用。
试验例2：
所测定的目的是评价待测试样品可利霉素对糖尿病小鼠血糖的作用。
试剂：
四氧嘧啶(Alloxan monohydrate，纯度≥98.0％)及二甲双胍(Metformin，纯度97％)，均由Sigma-Aldrich公司提供。
胰岛素测定试剂盒购于上海荣盛生物药业有限公司(批号：E02I0006)。
仪器：
One-touch型血糖检测仪及血糖试纸条(美国强生公司)，电子分析天平。
动物：
SPF级昆明小鼠，体重18～20g
方法：
可利霉素降血糖作用研究选用昆明小鼠，四氧嘧啶160mg/kg单次腹腔注射造模后，选取血糖值稳定在成模水平(10～25mmol/L)的小鼠随机分为模型对照组，阳性药对照组(二甲双胍200mg/kg)，给药组(药物为可利霉素，25、50、100mg/kg)同时另设正常对照组，灌胃生理盐水。
在造模后及灌胃给药30、45天时，禁食16h，称量小鼠体重。称重后，尾尖采血，血糖仪测定空腹血糖值。测定空腹血糖值后继续灌胃给药1次，2h后，各组小鼠按2g/kg灌胃给予葡萄糖溶液，尾尖采血，测定给予葡萄糖后0、0.5、2h的血糖值(Bg0，Bg0.5，Bg2)，按下式计算血糖曲线下面积(AUC)(反应糖耐量)：
AUC＝0.25*(Bg0+4*Bg0.5+3*Bg2)。
在造模后及灌胃给药45天时，禁食16h，进行眼底静脉丛取血。取血之前将毛细管用肝素润化，眼底静脉丛取血0.5ml，3500r/min，离心10min，分离血浆待测。吸取上清液，采用ELISA的方法测定胰岛素。具体操作按试剂盒说明书进行。
胰岛素抵抗指数(Insulin Resistance)的测定：在测得空腹血糖和胰岛素的浓度后，通过公式计算出胰岛素抵抗指数。
胰岛素抵抗指数＝空腹血糖*空腹胰岛素/22.5(最近提出>2.6时判有胰岛素抵抗)
所有数据用SPSS16.0软件进行统计分析，实验所得数据均以平均数加减标准差
Figure PCTCN2019072413-appb-000010
表示，各组均数之间均用单因素方差(one-way ANOVA)检验进行差异性比较，其P<0.05表示差异具有显著性。结果如表2～表6所示，在造模及灌胃给药30天及45天后，与正常对照组相比模型组动物体重显著降低，空腹血糖显著升高，糖耐量降低，对空腹血清胰岛素的影响不明显，但使胰岛素抵抗指数显著升高。
可利霉素具有改善糖尿病小鼠体重降低的症状、降低小鼠的空腹血糖，提高糖耐量，对空腹血清胰岛素的影响不明显，但可以降低胰岛素抵抗指数，表明可利霉素有很好的降血糖功效。
表2、动物实验各组体重变化(g)
Figure PCTCN2019072413-appb-000011
Figure PCTCN2019072413-appb-000012
*p<0.05与正常对照组相比， #p<0.05模型对照组相比，n＝8。
表3、动物试验各组血糖变化(mmol/L)
Figure PCTCN2019072413-appb-000013
Figure PCTCN2019072413-appb-000014
*p<0.05与正常对照组相比，**p<0.01与正常对照组相比，***p<0.001与正常对照组相比， #p<0.05与模型对照组相比， ##p<0.01与模型对照组相比， ###p<0.001与模型对照组相比，n＝8。
表4、可利霉素对糖尿病小鼠糖耐量的影响
Figure PCTCN2019072413-appb-000015
Figure PCTCN2019072413-appb-000016
*p<0.05与正常对照组相比，**p<0.01与正常对照组相比，***p<0.001与正常对照组相比， #p<0.05与模型对照组相比， ##p<0.01与模型对照组相比， ###p<0.001与模型对照组相比，n＝8。
表5、可利霉素对糖尿病小鼠糖耐量的影响
Figure PCTCN2019072413-appb-000017
Figure PCTCN2019072413-appb-000018
*p<0.05与正常对照组相比，**p<0.01与正常对照组相比，***p<0.001与正常对照组相比， #p<0.05与模型对照组相比， ##p<0.01与模型对照组相比， ###p<0.001与模型对照组相比，n＝8。
表6、动物试验各组空腹胰岛素(mmol/L)和胰岛素抵抗指数(IR)
*p<0.05与正常对照组相比，**p<0.01与正常对照组相比，***p<0.001与正常对照组相比， #p<0.05与模
Figure PCTCN2019072413-appb-000019
型对照组相比， ##p<0.01与模型对照组相比， ###p<0.001与模型对照组相比，n＝8。
试验例3：可利霉素对SD大鼠双侧海马CA1区注射Aβ 1-42致AD模型的影响
实验动物：
雄性健康Sprague Dawley大鼠(等级SPF)，体重220-260g，购于辽宁长生生物技术有限公司，许可证号：SCXK(辽)2015-0001。
实验药品及试剂：
可利霉素
Aβ 1-42：购自(Sigma，USA)；
白藜芦醇(Resveratrol)：阿拉丁公司(Lot#K1414052)(L.A.,CA,USA)；
实验仪器：
脑立体定位仪：美国Stoelting，型号51600(Kiel，WI，USA)；
Morris水迷宫及自动采集分析设备：北京硕林苑科技有限公司(Beijing，China)；
Y迷宫及自动打个采集分析设备：北京硕林苑科技有限公司(Beijing，China)；
操作步骤：
分组：雄性SD大鼠，体重220-260g，适应性喂养3d，自由饮水进食，12h昼夜循环。随机分为6组，即假手术对照组，模型组，可利霉素25、50及100mg/kg，阳性药白藜芦醇30mg/kg。
实验过程：Aβ 1-42用六氟异丙醇/无菌生理盐水溶液溶解成浓度为2μg/μL，4℃孵育老化24h形成Aβ寡聚体，备用。手术时，腹腔注射3.5％的水合氯醛(350mg/kg)麻醉大鼠后，固定于脑立体定位仪上。以前囟点为圆点，向后3.6mm、左右±2.5mm，进针深度3.1mm，用微量进样器一次性注入双侧海马CA1区，每侧海马在5min内注射2.5μL(0.5μL/min)，留针5min，造成大鼠痴呆模型；假手术对照组大鼠通过相同的操作方法双侧海马CA1区注射等体积的生理盐水，即动物模型选择：采用Aβ 1-42侧脑室注射方法造模；假手术对照组大鼠通过相同的操作方法双侧海马CA1区注射等体积的生理盐水，其中，β-淀粉样蛋白(Aβ)的淀粉样斑块是目前公认的AD的病理标志物，采用以上动物模型考察可利霉素治疗中枢神经系统疾病阿尔茨海默的作用有充分理论依据。
建立模型后第二天，假手术对照组和模型组灌胃给予相应溶剂，其他各实验组分别灌胃给予可利霉素25、50及100mg/kg和阳性药白藜芦醇30mg/kg。海马内注射Aβ 1-42后第12天开始依次进行Y迷宫、新物体辨别和水迷宫实验。行为学实验期间继续给药，每天1次直至行为学结束。
大鼠Y迷宫实验：给药第12天，各组大鼠进行Y迷宫实验。大鼠Y迷宫实验旨在考察可利霉素对大鼠自发交替活动和工作记忆的影响。装置由三个夹角为120度的木制支臂组成，分别为A、B、C三臂。实验时将大鼠放入A臂末端，让其自由出入三个臂，记录8min内每只大鼠进入三个臂的总次数及进臂顺序，以连续进入三个不同的臂为一次正确交替反应，记录正确交替反应次数。用自发交替反应率反映空间工作记忆能力。
大鼠新物体辨别实验：给药第14-15天，各组大鼠进行新物体辨别实验。大鼠新物体辨别实验旨在考察可利霉素对大鼠形象辨别记忆的影响。实验装置为一黑色塑料圆形开放场，直径约60cm，高20cm。本实验分为适应阶段和测试阶段。适应阶段每次将2～3只大鼠放入开放场中，让其自由探索3min以适应环境，每天2次，进行2天。第3天进行测试，每次将一只大鼠放入开放场中先自由探索3min，后取出大鼠，将2个相同的物体(A1，A2)置于开放场中央，放入大鼠，记录5min内探索两物体的时间(tA1，tA2)。1h后，将A2物体换成新物体B，将大鼠再次放入，记录探索两物体的时间(tA1，tB)。24h后，将B物体换成C物体，将大鼠再次放入，记录探索两物体所用的时间(tA1，tC)。探索的判定标准为大鼠用鼻子指向物体并距离物体1cm以内，或触及鼻子、舔物体、或用前爪触碰物体。计算对新物体的优先指数(Preferential index)及辨别系数(Discrimination index)。
优先指数计算公式如下：
优先指数(1h)＝tB/(tA1+tB)
优先指数(24h)＝tC/(tA1+tC)
辨别系数计算公式如下：
辨别系数(1h)＝(tB-tA1)/(tA1+tB)
辨别系数(24h)＝(tC-tA1)/(tA1+tC)
大鼠水迷宫实验：给药第16-20天，各组大鼠进行Morris水迷宫实验。Morris水迷宫实验旨在考察可 利霉素对空间学习记忆障碍的影响。水迷宫装置由直径为1.5米、高为50厘米的黑色不锈钢圆形水池和一个直径为10厘米的圆形金属平台组成，平台可自由移动位置。实验前向水池中注水(水温24±1℃)，使水面高于平台1厘米。训练阶段，每天上下午各进行1次训练，历时6天。平台置于第四象限，将大鼠面向池壁放入水中，记录90秒，90秒内如果大鼠找到平台则让其在平台上休息10秒。如果90秒未找到平台，将大鼠引导至平台上休息10秒。训练结束后进行测试，撤除平台，让大鼠自由游泳90秒。迷宫系统自动记录大鼠在原平台象限(目标象限)停留的时间。
结果：
Y迷宫测试大鼠工作记忆能力：实验结果表明，与假手术对照组相比，模型组大鼠Y迷宫自发交替反应率显著下降；与模型组相比，可利霉素(25，50，100mg/kg)组及白藜芦醇(30mg/kg)对照组大鼠Y迷宫自发交替反应率显著增加(见图1)。各组大鼠的进臂总次数未见显著性差异(见图2)。可得出，可利霉素能够改善Aβ 1-42诱导的大鼠工作记忆损伤。
新物体辨别实验测试大鼠形象记忆能力：实验结果表明，各组大鼠对A1和A2两个相同物体的辨别系数无明显差异(见表7)。在新物体辨别实验中，与假手术对照组相比，模型组大鼠在1h和24h后，对新物体的优先指数和辨别系数显著降低；与模型组相比，可利霉素及白藜芦醇组大鼠在1h和24h，对新物体的优先指数(见图3、图4)和辨别系数(见图5、图6)均显著提高。因此，可利霉素具有改善Aβ 1-42诱导的大鼠形象记忆损伤。
表7、在Aβ 1-42诱导的大鼠的新物体辨别实验测试中，关于对A1和A2的辨别系数的试验结果(N＝4-5，平均数±标准误)
Figure PCTCN2019072413-appb-000020
Morris水迷宫测试大鼠学习记忆能力：实验结果表明，实验结果表明，在定位航行实验中，随着训练次数增加，各组实验大鼠的逃避潜伏期均缩短，提示大鼠的空间探索学习能力均得到提高。实验第2天，与假手术对照组相比，模型组大鼠逃避潜伏期显著延长；与模型组相比，可利霉素及白藜芦醇组的逃避潜伏期显著下降，在第3天和第4天，这种趋势得以保持(见图7)。
空间探索实验结果显示，与假手术对照组相比，模型组大鼠穿越平台的次数显著降低；与模型组相比，可利霉素及白藜芦醇组的穿台次数显著提高(见图8)；各组大鼠的游泳速度没有显著性差异(见图9)，说明可利霉素及白藜芦醇能够提高大鼠穿越平台的次数。实验结果表明，可利霉素可以改善Aβ 1-42诱导的大鼠学习记忆以及空间探索能力的损伤。
上述试验证明了本发明的药物具有较好的治疗中枢神经系统疾病阿尔茨海默的作用，不仅为本发明的药物在治疗中枢神经系统疾病中的应用及其临床推广提供了理论依据，而且具有重要的经济效益和社会效益。
试验例4：利用寿命实验确定可利霉素是否对秀丽隐杆线虫寿命有延长作用
实验材料、试剂和仪器
1.1菌株、线虫
E.coli OP50                 沈阳药科大学实验室保存
C.elegans N2                沈阳药科大学实验室保存
1.2主要试剂
Figure PCTCN2019072413-appb-000021
1.3主要仪器
Figure PCTCN2019072413-appb-000022
1.1线虫的培养
将雌雄同体线虫饲养于涂布大肠杆菌OP50(E.coli OP50)的线虫标准培养基(Nematode growth medium，NGM)上，培养温度20℃，观察线虫生长情况，定期将线虫转至新的涂有E.coli OP50的NGM培养基中。
1.2线虫的同期化
首先挑取处于产卵期的线虫若干条在同一平板中(具体数量视所需要的线虫数量而定，一般一条产卵期线虫一小时可以产卵8个左右)。30min后，将平板中线虫挑出，平板中的卵孵化后即处于同一发育时期。
1.3寿命周期实验
为了系统而准确地测定线虫的寿命，使用液体培养的方法设定给药组和空白组，选定给药组药物浓度，用生理盐水将药物稀释到所需浓度，使用24孔板，每孔分别加420微升液体培养液，30微升菌液，50微升可利霉素药物(空白组使用S-medium液体培养基)使得每孔液体总体积为500微升，每孔放入25只同期化线虫，每24h后将存活的线虫转移到下一个新的相同条件的培养液孔中，直到死亡。对机械刺激没有反应及不咽食或排泄的线虫判断为死亡每24h转移后记录每组存活只数统计每组线虫最长存活天数和平均存活天数。
实验结果见表8和图10，表明可利霉素可延长秀丽隐杆线虫的平均寿命，且在浓度5μg/ml浓度下有明显效果。
表8
Concentration(μg/ml) Mean life span±SD(days) N
0 23.38±0.90 25
0.5 23.64±0.84 25
1 25.32±0.83 25
5 26.72±0.82* 25
10 26.97±0.87* 25
试验例5：检测运动能力指标确定可利霉素是否对秀丽隐杆线虫寿命有延长作用
试剂：同试验例4
仪器：同试验例4
实验对象：秀丽隐杆线虫
方法：分别将给药组线虫和对照组线虫进行同期化处理，每组得到一定数量的同期化线虫进行培养。当其进入成虫期后(一般为第三天)，给药可利霉素48小时后测量其运动速度。每隔24小时每组随机挑选10条线虫测定其在一段时间内运动的距离，并记录20S内线虫走过的峰数。
实验结果分别给予5μg/ml及10μg/ml可利霉素后能够显著提高线虫的行为运动能力，见图11-a和图11-b所示。
试验例6：
检测热激条件下秀丽隐杆线虫存活率确定可利霉素是否对秀丽隐杆线虫寿命有延长作用。
试剂：同试验例4
仪器：同试验例4
实验对象：秀丽隐杆线虫
方法：在正常培养条件下同期培养线虫至L4期，将一定数量的雌雄同体线虫挑取到加有2ml S-medium的30mm一次性培养皿中，于20℃继续培养。培养液含有可利霉素，E.coli OP50菌体(OD600＝0.2-0.3)，50μM FUdR(阻止子代线虫生长)，对照组为不含可利霉素的S-medium液体培养基培养。注意应每天将线虫挑取到含新鲜培养基的培养皿中以更换培养液。48h以后，将线虫转到37℃继续培养10h，记录培养结束后存活的线虫的数目做为该实验的存活率，实验应平行重复2-3次，取平均值作为最终存活率结果。
实验结果从图中可以看出与对照组相比，可利霉素能够显著增强线虫的耐热能力，延长线虫在37℃的平均生存时间，见图12所示。
试验例7：
检测紫外照射实验下秀丽隐杆线虫存活率确定可利霉素是否对秀丽隐杆线虫寿命有延长作用。
试剂：同试验例4
仪器：同试验例4
实验对象：秀丽隐杆线虫
方法：将同期化至I4期的线虫的实验培养板固定在紫外灯下进行预实验照射，分别240s，记录线虫死亡、存活数目。线虫死亡标准同寿命实验。紫外线波长为254nm，紫外灯距离培养板的高度为15cm，功率为1×10 -3W/cm 3。实验分组为空白组和给药组。
实验结果从图中可知，可利霉素对紫外照射氧化损伤有一定的保护作用，见图13所示。
结果
实验至少重复3次。结果以均值和标准差的形式给出。显著性检验以t检验为主。所有数据统计分析和制图使用Excel和SPSS16.0软件。
试验例8：
检测可利霉素是否可以减少野生型秀丽隐杆线虫体内脂褐素积累。
方法：按照寿命实验中的培养方法培养10d、15d、20d。NaN3麻醉后，倒置荧光显微镜下，激发光340-380nm,发射光430nm，观察线虫体内的脂褐素水平，拍摄荧光图片，利用Image J软件分析处理线虫体内脂褐素荧光水平。
由于交联蛋白等的累积和聚集，脂褐素沉积在人体各组织器官细胞中，导致细胞代谢减慢，活性下降，从而造成人体器官功能衰退产生衰老。因此脂褐素被认为是衰老的标志。脂褐素具有自发荧光的特点，在荧光显微镜下观察线虫，可以根据其荧光强度来判断其衰老程度。测定结果见图14-a和图14-b。结果表明，给药组的脂褐素积累量较对照组显著减少。
试验例9：
研究可利霉素对线虫寿命的影响机制。
由于sod-3含量测定表明，给药后给药组线虫体内sod-3表达量降低，且脂褐素荧光含量表明，给药后给药组的脂褐素含量降低。本发明考察可利霉素是否对sod-3上游基因daf-16和daf-15有影响。
1、可利霉素可提高线虫体内抗衰老基因sod-3表达
方法：本实验采用带有gfp标签的线虫株CF1553观察sod-3的表达量。用可利霉素持续处理进入成熟期的线虫6天，分别用10m M的叠氮化钠将两组线虫进行麻醉，固定于5％琼脂凝胶中，然后用激发波长488nm，发射波长500nm-530nm的荧光显微镜对线虫荧光强度进行观察，拍照。用ImageJ软件对荧光强度进行定量分析，每组线虫样本数为15条，实验重复两次。
根据自由基与衰老理论，ROS是造成机体衰老及疾病的主要原因。随着衰老的过程线虫体内的其体内的自由基逐渐增多，本发明通过考察可利霉素可以增强野生型线虫的耐热能力，增强线虫的胞内胁迫抵抗。因此本发明人猜想KL是否通过影响抗氧化基因sod-3从而抗衰老，用可利霉素连续处理6天CF1553(sod-3::GFP)型线虫后，用荧光显微镜观察发现给药组线虫的绿色荧光蛋白表达量较对照组线虫显著提高。结果见图15。表明可利霉素可能是通过提高抗氧化基因sod-3降低线虫体内ROS的水平从而延长寿命。
2、可利霉素可以影响DAF-16在细胞内的分布
方法：将处于L4期TJ356(DAF-16：：GFP)线虫每组30只挑取到OP50长势良好的NGM平板，产卵3-4小时，线虫的同期化完毕。卵长至成虫后每组30只分组给药。测给药6天TJ356线虫DAF-16入核情况，使用倒置荧光显微镜观察DAF-16入核情况。
DAF-16在细胞核内聚集还是在细胞质内弥散分布同其活性直接相关。正常情况下DAF-16在体内定位于细胞质中，如果在外界胁迫环境下，不同的蛋白被激活，进而促进进入细胞核进行转录功能来启动下游基因表达来延长线虫的寿命。有研究表明缺乏胰岛素或氧化应激均能使DAF-16在细胞核内聚集，提高线虫抵抗氧化应激的能力并延长线虫寿命。
由于可利霉素延长线虫的寿命和DAF-16有关，本发明人推测可利霉素可能会影响DAF-16在细胞内的分布。本发明利用DAF-16::GFP转基因线虫TJ356考察DAF-16在细胞内的分布的研究。用可利霉素处理刚进入成虫期的线虫，给药6天后，用倒置荧光显微镜观察。
结果发现可利霉素处理的TJ356线虫绿色荧光集中于细胞核中成点状聚集状态，而未经可利霉素处理的TJ356线虫绿色荧光弥散分布于整个细胞中。可利霉素5,10μg/ml处理6天时，DAF-16在细胞核内的聚集较未处理的线虫显著增加。这表明可利霉素可以促进DAF-16进入细胞核，见图16-a和图16-b。

Claims (10)

  1. 一种用于预防和/或治疗疾病的药物，其特征在于：所述的疾病为阿尔茨海默病、糖尿病或衰老；所述药物包括第一活性成分，所述的第一活性成分包括可利霉素、异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ和异戊酰螺旋霉素Ⅲ中的一种；
    或者异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ中的两种或三种的组合。
  2. 根据权利要求1所述的药物，其特征在于，所述的药物还包括第二活性成分；
    优选，当所述的疾病为阿尔茨海默病时，所述的第二活性成分为抗阿尔茨海默病药物中的至少一种；
    当所述的疾病为糖尿病时，所述的第二活性成分为抗糖尿病药物中的至少一中；
    当所述的疾病为衰老时，所述的第二活性成分为延缓衰老或延长寿命的药物中的至少一种。
  3. 根据权利要求1或2所述的一种用于预防和/或治疗疾病的药物，其特征在于：所述药物与药学上可接受的载体制成临床上可接受的制剂，优选片剂、胶囊、丸剂、注射剂、缓释剂及各种微粒给药系统。
  4. 根据权利要求3所述的一种用于预防和/或治疗疾病的药物，其特征在于：所述药物的剂量为10～1500mg/kg，优选50～1000mg/kg，更优选100～500mg/kg。
  5. 一种用于预防和/或治疗疾病的组合产品，其特征在于：所述的疾病为阿尔茨海默病、糖尿病或衰老；所述的组合产品包括第一药剂，所述的第一药剂的活性成分包括可利霉素或异戊酰螺旋霉素Ⅰ或异戊酰螺旋霉素Ⅱ或异戊酰螺旋霉素Ⅲ中的一种；
    或者异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ中的两种或三种的组合。
  6. 根据权利要求5所述的组合产品，其特征在于：所述的组合产品还包括第二药剂；
    优选，当所述的疾病为阿尔茨海默病时，所述的第二药剂为用于预防和/或治疗阿尔茨海默病相关药物中的至少一种；
    当所述的疾病为糖尿病时，所述的第二药剂为用于预防和/或治疗糖尿病相关药物中的至少一种；
    当所述的疾病为衰老时，所述的第二药剂为延缓衰老或延长寿命相关药物中的至少一种。
  7. 根据权利要求6所述的组合产品，其特征在于，所述的第一药剂和第二药剂的用量比为1～99：99～1，优选5～95：95～5，更优选10～90：90～10，再优选20～80：80～20。
  8. 根据权利要求6所述的组合产品，其特征在于，所述的用于预防和/或治疗阿尔茨海默病的相关药物包括作用于胆碱能系统药物、作用于N-甲基-D-门冬氨酸受体药物、抗氧化药物、抗炎药物、抑制Aβ蛋白形成的药物、雌激素、神经生长因子、尼莫地平、抗细胞凋亡剂；
    所述的用于预防和/或治疗糖尿病的相关药物包括双胍类降糖药、磺脲类降糖药、α糖苷酶抑制剂、胰岛素增敏剂、非磺酰脲类促胰岛素分泌剂或胰岛素中的至少一种。
  9. 一种权利要求1-4任一所述的药物或权利要求5-8任一所述的组合产品在制备预防和/或治疗阿尔茨海默病、益智、糖尿病、延缓衰老或延长寿命方面的药物中的应用。
  10. 根据权利要求9所述的应用，其特征在于，所述的在制备预防和/或治疗阿尔茨海默病、益智方面 的药物中的应用为制备减少乙酰胆碱水解药物中的应用、改善认知障碍和运动障碍药物中的应用、保护脑内神经细胞药物中的应用、不降低体重、提高免疫力或提高白细胞药物中的应用；
    所述的在制备预防和/或治疗糖尿病方面的药物中的应用为制备预防和/或治疗Ⅰ型或Ⅱ型糖尿病或特殊类型糖尿病药物方面的应用，优选在制备用于促进胰岛素分泌或降低血糖或保护胰岛β细胞药物中的应用或预防和/或治疗糖尿病并保持体重药物方面的应用；
    所述的在延缓衰老或延长寿命方面的药物中的应用为制备通过改变转录因子DAF-16活性来延缓衰老和/或延长寿命药物中的应用；通过提高SIR2同源蛋白SIR2.1的表达水平，SIR2.1再通过影响DAF-16活性来延缓衰老和/或延长寿命药物中的应用；或通过激活AMPK，直接增强FOXO/DAF-16的活性，延缓衰老和/或延长寿命药物中的应用。
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