WO2020133605A1 - 一种人工合成多肽h-473及其应用 - Google Patents

一种人工合成多肽h-473及其应用 Download PDF

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WO2020133605A1
WO2020133605A1 PCT/CN2019/072013 CN2019072013W WO2020133605A1 WO 2020133605 A1 WO2020133605 A1 WO 2020133605A1 CN 2019072013 W CN2019072013 W CN 2019072013W WO 2020133605 A1 WO2020133605 A1 WO 2020133605A1
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polypeptide
group
drugs
drug
psoriasis
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PCT/CN2019/072013
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French (fr)
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侯万儒
侯怡铃
丁祥
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西华师范大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • 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
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention belongs to the technical field of biomedicine, and relates to a synthetic peptide H-473 and its application, specifically in preparing anti-cancer, anti-senile dementia, anti-asthma, analgesic, anti-psoriatic and anti-age-related macular degeneration (AMD ) Application of medicines such as diseases.
  • omalizumab omalizumab
  • omalizumab significantly reduces the level of free IgE by binding to free IgE, preventing Off IgE is combined with mast cells and basophils to prevent the release of inflammatory mediators.
  • infliximab can significantly improve the symptoms, lung function and quality of life of asthma patients, reduce the number of exacerbations of asthma, reduce the amount of glucocorticoids, safe to use, and well tolerated.
  • Rituximab is rheumatoid and systemic Lupus erythematosus also has a good therapeutic effect.
  • Infliximab can reduce the clinical symptoms of pain, morning stiffness and joint swelling in patients with rheumatoid arthritis by up to 60%. Another study shows that infliximab infusion can quickly and significantly relieve joint and skin lesions in patients with refractory psoriasis and arthritis.
  • Infliximab is effective against Crohn's disease, ulcerative colitis, and alcohol Liver diseases and other digestive system diseases have good curative effect and are safe in the recommended treatment range.
  • Ranibizumab is used to treat wet (neovascular) age-related macular degeneration (AMD). These examples illustrate that certain drugs have multi-target therapeutic properties.
  • the present invention analyzes related receptors (FZDs, ITG, FGFR, ErBB2, PDGFR, IGFR, KIT, FGFR, HGFR and TRK, etc.); related receptors in the molecular pathways of bronchial asthma and chronic inflammation (TLR4, TLR1, TLR5, TLR6, etc.); opioid receptors (opioid receptors), acetylcholinesterase (acetylcholinesterase) related to Alzheimer's disease, Based on the analysis of the molecular structure and functional sites of proteins such as psoriasis-related factors and age-related macular degeneration (AMD)-related factors, small-molecule peptides were designed as multi-target therapeutic drugs with the purpose of interfering with these receptors , The function of cytokines and enzyme molecules to achieve the purpose of treating related diseases.
  • FZDs related receptors
  • the inventors have synthesized the polypeptide H-473, so far, and provided the polypeptide H-473 in anti-cancer, anti-asthma, anti-dementia, analgesia, treatment of psoriasis and treatment of psoriasis and age-related macular degeneration (AMD ) The biomedical efficacy of the disease and its application.
  • the purpose of the present invention is to provide a synthetic polypeptide H-473 and its application, and to reveal its application prospects in the anti-cancer, anti-asthma, anti-senile dementia and analgesic treatment psoriasis active effects.
  • polypeptide H-473 An artificially synthesized polypeptide H-473.
  • the amino acid sequence of the polypeptide H-473 RGLRGLR, with a molecular weight of 827 Da; PI of 12.30.
  • the peptide is single-chain and has no chemical bond modification.
  • the polypeptide H-473 of the present invention has almost no effect on the growth inhibition of human normal liver cells Chang Liver (only weak growth inhibitory effect or weak growth promotion effect), while it has effects on human gastric cancer cell MGC-803 and human pancreatic cancer Cell cfpac-1 cell growth has a significant inhibitory effect.
  • the acetylcholinesterase in the brain tissue of each experimental group was measured and the expression level of the acetylcholinesterase expression in the brain cells of each experimental group, the polypeptide of the present invention was calculated
  • the inhibition rate of H-473 on the expression of acetylcholinesterase in brain tissue was 55.6%.
  • the expression rate of acetylcholinesterase by peptide H-473 interfered with brain tissue was 43.8%.
  • the polypeptide H-473 drug of the present invention has significant anti-asthma activity.
  • the polypeptide H-473 of the present invention can significantly reduce the number of writhing reactions in mice caused by acetic acid, and each dose group has a significant analgesic effect on the pain caused by acetic acid.
  • the polypeptide H-473 drug of the present invention has related factors such as CARMA, VEGF, IL6, IL8, IL1-R, TNF-R1, IL17RA, IL12, IL12R and other proteins at the molecular, cellular and animal levels for the development of psoriasis
  • CARMA CARMA
  • VEGF vascular endothelial growth factor
  • IL6 IL6-IL8
  • IL1-R TNF-R1, IL17RA
  • IL12 IL12R
  • other proteins at the molecular, cellular and animal levels for the development of psoriasis
  • the expression of is significantly down-regulated and is expected to become a powerful drug against psoriasis.
  • the polypeptide H-473 of the present invention significantly down-regulates the VEGF expression of cells, can effectively inhibit the biological activity of VEGF, and effectively interfere with VEGF-related receptors, such as VEGFR, PDGFR, and the like. Eventually, it can inhibit the proliferation of blood vessels and achieve the purpose of resisting age-related macular degeneration (AMD) disease.
  • AMD age-related macular degeneration
  • the present invention provides the use of the polypeptide H-473 in the prevention and/or treatment of anti-cancer drugs.
  • the anti-cancer drugs include anti-human gastric cancer drugs and anti-human pancreatic cancer drugs.
  • the present invention provides the use of the polypeptide H-473 in the prevention and/or treatment of anti-senile dementia drugs.
  • the present invention provides the use of the polypeptide H-473 in the prevention and/or treatment of analgesic drugs, the application includes the prevention and/or treatment of burns, external injuries, frostbite, inflammation Sexual pain, neuropathic injury, and other causes of pain.
  • the present invention provides the use of the polypeptide H-473 in medicine for preventing and/or treating asthma.
  • the present invention provides the use of the polypeptide H-473 in the prevention and/or treatment of psoriasis drugs.
  • the present invention provides the use of the polypeptide H-473 in the prevention and/or treatment of macular degeneration (AMD).
  • AMD macular degeneration
  • excipients can also be added to the above-mentioned drugs, and the excipients include conventional diluents, stabilizers, fillers, excipients, binders, wetting agents, absorption accelerators, Surfactants and lubricants.
  • the medicines of the present invention can be made into injections, lyophilized preparations, implants, sustained release agents and other forms of medicines.
  • the drugs of the above various dosage forms can be prepared according to conventional methods in the pharmaceutical field.
  • the polypeptide H-473 provided by the present invention is a small-molecule polypeptide, which is easy to prepare, its preparation process and cost are low, and it is easy to prepare. H-473 has very high application value in anti-cancer, anti-asthma, anti-senile dementia, analgesia and anti-psoriasis. Studies have shown that the growth inhibition rate of human gastric cancer cell MGC-803 cell reached 51.9%. The growth inhibition rate of human pancreatic cancer cell cfpac-1 cells reached 48.8%.
  • Polypeptide H-473 inhibited human gastric cancer cell MGC-803 xenograft tumor in nude mice up to 64.1%, peptide H-473 inhibited human pancreatic cancer cell cfpac-1 xenograft tumor in nude mice up to 68.04%, polypeptide
  • the inhibitory rate of H-473 on the expression of acetylcholinesterase in brain tissue is 55.6%, the expression rate of H-473 interferes with the expression of acetylcholinesterase in brain tissue is 43.8%, and the inhibitory rate of H-473 peptide drugs on the pain and writhing of mice caused by acetic acid At 0.5 mg/kg and 1 mg/kg doses, they were 81.95% and 95.11%, respectively.
  • the present invention provides valuable resources and directions for studying novel anti-cancer, anti-asthma, anti-senile dementia, analgesic, anti-psoriatic and anti-age-related macular degeneration (AMD) diseases.
  • Figure 1 is a graph of the cytological activity effect data of H-473 of the present invention.
  • Example 2 is a graph showing the effect of polypeptide H-473 on the growth of human gastric cancer xenograft tumor in nude mice in Example 2 of the present invention
  • FIG. 3 is a graph showing the effect of polypeptide H-473 on the growth of human pancreatic cancer xenograft tumor in nude mice in Example 3 of the present invention
  • Figure 4 is a standard curve of acetylcholinesterase.
  • 1.1.1.1 Cell lines Human normal liver cells Chang Liver (human liver cells), human gastric cancer cells MGC-803, human pancreatic cancer cells cfpac-1, the above cell lines are all commercial biological materials.
  • RPMI1640 powder purchased from Gibco, USA
  • fetal bovine serum purchased from Sijiqing Biological Products Co., China
  • double antibody penicillin, streptomycin, purchased from Gibco
  • H-473 The amino acid sequence of H-473 is: RGLRGLR, molecular weight is 827Da; PI is 12.30. The purity is 98.12%.
  • Human normal liver cell Chang Liver, human gastric cancer cell MGC-803, human pancreatic cancer cell cfpac-1 cell line were used to contain 10% inactivated fetal bovine serum (FBS) 100U/ml penicillin, 100 ⁇ g/ml Streptomycin, RPMI-1640 complete culture solution at pH 7.4 was cultured in a 37°C, 5% CO 2 incubator.
  • FBS fetal bovine serum
  • Chang Liver a normal liver cell of the logarithmic growth phase, human gastric cancer cell MGC-803, and human pancreatic cancer cell cfpac-1 cells were digested with trypsin to adjust the cell number to l ⁇ 10 5 cells/mL and inoculated into 96 wells Plate, the amount of cell fluid per well is 200 ⁇ L, put it in a CO 2 incubator, and incubate under the conditions of 37°C, 5% CO 2 and saturated humidity until the cell monolayer is covered with the bottom of the well (96-well flat bottom plate), and then add H-473, make the final concentration of 0.05, 0.1, 0.2, 0.5, 0.8, 1.5, 3.0 and 6.0 ⁇ g/ml; set 6 replicate wells, the blank group is RPMI1640 culture medium without cells, put CO 2 After incubating for 24, 36, and 48 hours in the incubator, remove the 96-well plate, add 10 ⁇ L of MTT (5 mg/mL) solution, and continue culturing for 4 hours, then add 150
  • Cell growth inhibition rate (1—A experimental group/A control group) ⁇ 100%.
  • the detection results of the MTT method in this example showed that different concentrations of the peptide drug H-473 had almost no effect on the growth inhibition of human liver cells Chang Liver at 36h (only weak growth inhibition or weak growth promotion), and It can significantly inhibit the growth of human gastric cancer cell MGC-803 and human pancreatic cancer cell cfpac-1.
  • the polypeptide drug H-473 has a dose-dependent effect on the growth inhibition rate of human gastric cancer cell MGC-803 and human pancreatic cancer cell cfpac-1. It was found that the growth inhibition rate of human gastric cancer cell MGC-803 at a concentration of 3.0 ⁇ g/ml reached 51.9%. It was found that the cell growth inhibition rate on human pancreatic cancer cell cfpac-1 cells reached 48.8% at a concentration of 6.0 ⁇ g/ml.
  • Preparation method 1mg of peptide drug is dissolved in 1ml of physiological saline, and then diluted multiple times to obtain the desired concentration of peptide solution.
  • Preparation method Before administration, a solution with a concentration of 20,000 IU/ml is prepared with physiological saline.
  • Model control group normal saline, 0.1ml/10g, once a day for 10 times;
  • Interleukin-2 Intravenous injection of 20,000 IU/ml interleukin-2 solution, 0.1ml/10g, once a day for 10 times;
  • Peptide H-473-1ug/kg group 0.1ug/ml peptide H-473 solution, 0.1ml/10g, was injected into the tail vein once a day for 10 times.
  • Source, germline, strain BALB/c nude mice, provided by the Laboratory Animal Center of the Chinese Academy of Military Medical Sciences.
  • Number of animals 8 animals in each group, a total of 24 animals.
  • the cultured human gastric cancer cell MGC-803 cell suspension was collected at a concentration of 1 ⁇ 10 7 cells/ml, and 0.1 ml of each was inoculated subcutaneously in the right armpit of nude mice.
  • Transplanted tumors of nude mice were measured with vernier calipers. After 8 days of inoculation, when the tumors grew to 100-150 mm 3 , the animals were randomly divided into groups of 8 animals. At the same time, the nude mice of each group began to administer the drugs. For the dosing regimen, see the group and dosing regimen. Using the method of measuring the tumor diameter, the anti-tumor effect of the test sample was dynamically observed. After 10 days of administration, the observation was continued for 4 days, then the nude mice were sacrificed, and the tumor mass was surgically peeled off and weighed.
  • TV tumor volume
  • RTV relative tumor volume
  • Evaluation index of antitumor activity relative tumor proliferation rate T/C (%), the calculation formula is as follows:
  • T RTV treatment group RTV
  • C RTV model group RTV.
  • the mean value is expressed by X ⁇ SD, and the t-test is used for statistical processing between groups.
  • SPSS Staffstical Package for the Social Science 17.0 is used for statistical analysis of the results.
  • a human gastric cancer cell MGC-803 xenograft tumor model in nude mice was established, and the antitumor activity of polypeptide H-473 at a concentration of 1 ug/kg was evaluated using this model.
  • the experimental results are as follows: the tumor inhibition rate of polypeptide H-473 is 64.1%. Under the same conditions, the tumor suppression rate of the positive control IL2 group (200,000 U/kg) was 60.9%. The conclusion is: the peptide drug H-473 has a significant anti-tumor activity at a concentration of 1 ug/kg, with a tumor suppression rate of >40%, and is significantly different from the model group.
  • the experimental results of this example are shown in Table 1. The effect of the polypeptide H-473 on the growth of human gastric cancer cell MGC-803 xenograft tumor in nude mice is shown in FIG. 2.
  • Preparation method 1mg of peptide drug is dissolved in 1ml of physiological saline, and then diluted multiple times to obtain the desired concentration of peptide solution.
  • Preparation method Before administration, a solution of 20,000 IU/ml is prepared with physiological saline.
  • Model control group normal saline, 0.1ml/10g, once a day for 10 times.
  • Interleukin-2 Intravenous injection of 20,000 IU/ml interleukin-2 solution, 0.1ml/10g, once a day for 10 times.
  • Peptide H-473-1ug/kg group 0.1ug/ml peptide H-473 solution, 0.1ml/10g, was injected into the tail vein once a day for 10 times.
  • Source, germline, strain BALB/c nude mice, provided by the Laboratory Animal Center of the Chinese Academy of Military Medical Sciences.
  • Number of animals 8 animals in each group, a total of 24 animals.
  • the cultured cfpac-1 cell suspension was collected at a concentration of 1 ⁇ 10 7 cells/ml, and 0.1 ml of each was inoculated subcutaneously in the right armpit of nude mice.
  • Transplanted tumors of nude mice were measured with vernier calipers. After 17 days of inoculation, when the tumors grew to 100-150 mm 3 , the animals were randomly divided into groups of 8 animals. At the same time, the nude mice of each group began to administer the drugs. For the dosing regimen, see the group and dosing regimen. Using the method of measuring the tumor diameter, the anti-tumor effect of the test sample was dynamically observed. After 10 days of administration, the observation was continued for 3 days, then the nude mice were sacrificed, and the tumor mass was surgically peeled off and weighed.
  • TV tumor volume
  • RTV relative tumor volume
  • Evaluation index of antitumor activity relative tumor proliferation rate T/C (%), the calculation formula is as follows:
  • T RTV treatment group RTV
  • C RTV model group RTV.
  • the mean value is expressed by X ⁇ SD, and the t-test is used for statistical analysis between groups.
  • SPSS Staffstical Package for the Social Science 17.0 is used for statistical analysis of the results.
  • a human pancreatic cancer cell cfpac-1 xenograft tumor model in nude mice was established, and the antitumor activity of polypeptide H-473 at a concentration of 1 ug/kg was evaluated using this model.
  • the experimental result is: the tumor suppressive rate of polypeptide H-473 is 56.1%. Under the same conditions, the tumor suppression rate of the positive control IL2 group (200,000 U/kg) was 68.04%.
  • the peptide drug H-473 has a significant anti-tumor activity at a concentration of 1 ug/kg, with a tumor suppression rate of >40%, and is significantly different from the model group.
  • Table 2 The experimental results of this example are shown in Table 2; in this example, the effect of polypeptide H-473 on the growth of human gastric cancer cell MGC-803 xenograft in nude mice is shown in FIG. 3.
  • mice Thirty-two mice were randomly divided into 4 groups, 8 in each group.
  • Control group no treatment, normal feeding every day.
  • Expression-suppressed group each mouse was intramuscularly injected with 200 ⁇ g/kg of drug every day for 30 days.
  • Model group each mouse was injected intraperitoneally with 150 mg/kg D-galactose and 120 mg/kg sodium nitrite every day for 30 days.
  • Interference group On the basis of the model group, each mouse was injected intramuscularly with 200 ⁇ g/kg peptide drug H-473 every day for 30 days.
  • the collected brain tissue was immediately treated with liquid nitrogen. After treatment, it was evenly divided into 3 portions and stored at -80° ultra-low temperature.
  • Brain tissue protein was extracted with animal brain tissue protein extraction kit.
  • the expression level of acetylcholinesterase in brain cells of each experimental group was measured by ELISA kit.
  • Expression inhibition rate% (expression level of control group-expression level of suppression group)/expression level of control group x 100%.
  • Drug interference expression rate% (expression of model group-expression of interference group)/expression of model group ⁇ 100%.
  • a standard curve of acetylcholinesterase (Figure 4), measure the acetylcholinesterase of brain tissue of each group, and then use the standard curve to calculate the expression of acetylcholinesterase in brain cells of each experimental group (Table 3).
  • the rate of expression inhibition 55.6% (compared to the control group).
  • Drug interference expression rate 43.8% (compared with model group).
  • the results showed that the peptide drug H-473 significantly inhibited the expression of acetylcholinesterase in brain cells.
  • Source, germline, strain BABL/C mice, provided by Shanghai Xipuer-Bikai Experimental Animal Co., Ltd.
  • the drug was administered one hour before the OVA nebulization.
  • mice were injected intraperitoneally with 0.2ml of sensitizing fluid (oval protein OVA80ug + adjuvant liquid aluminum 0.1ml), and injected twice on days 0 and 14. From the 24th day after the first sensitization, 2.5% OVA was inhaled by inhalation for 45 minutes, once a day for 18 days.
  • sensitizing fluid oval protein OVA80ug + adjuvant liquid aluminum 0.1ml
  • H-473 polypeptide drug 15ug/kg group and H-473 polypeptide drug 30ug/kg group were injected intramuscularly.
  • the positive control group was intramuscularly injected with dexamethasone 1mg/kg. Normal control group and model control group were injected with saline intramuscularly.
  • BALF Bronchoalveolar lavage fluid
  • Mouse serum Blood was collected from mouse eyeballs 24h after the last challenge, centrifuged, and serum collected.
  • Lung tissue is half fixed and half frozen.
  • mice in each group had reactions such as restlessness, nodding breath, and scratching of the nose 32 days after the first sensitization, that is, 11 days after nebulization. At 18 days after nebulization, the model group experienced dyspnoea and restlessness. The positive group showed no abnormal reactions. The other medication groups had the same symptoms as at 8 days of nebulization, and there was no aggravating trend.
  • H-473 peptide drug has significant anti-asthma activity.
  • Preparation method Drug, store at -20°C, take appropriate amount of drug, add saline to dissolve to the required concentration.
  • Model group intramuscular injection of normal saline, 5ml/kg.
  • Group H-473-L Intramuscular injection of 0.5 mg/kg, administered once.
  • Group H-473-H intramuscular injection of drug, 1 mg/kg, administered once.
  • Source, germline, strain ICR mouse, provided by Shanghai Lingchang Biological Technology Co., Ltd.
  • mice were adaptively fed for 3 days, and then randomly divided into 3 groups according to the group, with 8 animals in each group.
  • the mice in each group were injected intramuscularly. After 1 hour of administration, the mice were intraperitoneally injected with 0.2ml of 2% glacial acetic acid, and observed the writhing reaction of the mice within 15 minutes (abdominal contraction showed "S" shape, body twisted, hind limb Stretching and creeping, etc.), and calculate the twisting inhibition rate.
  • Writhing inhibition rate% (average number of writhing in control group-average number of writhing in administration group)/average number of writhing in control group ⁇ 100%.
  • the mean value is expressed by X ⁇ SD, and the t-test is used for statistical processing between groups.
  • SPSS Staffstical Package for the Social Science 17.0 is used for statistical analysis of the results.
  • H-473 can significantly reduce the number of writhing reactions in mice caused by acetic acid.
  • Each dose group has a significant analgesic effect on acetic acid-induced pain, which is significantly different from the blank control group (p ⁇ 0.01 ).
  • the article reports the histopathological phenotype and inflammatory response type of Card14E138A/+ spontaneous psoriasis mice, and clarifies the molecular mechanism of the deletion or mutation of Card14 gene affecting the activation of IL-17A signal in keratinocytes, as well as the role of keratinocytes in An important role in the initiation of psoriasis.
  • CARMA2 is an important regulatory protein of the IL-17A signaling pathway in keratinocytes, and its continuous activation in keratinocytes can induce the occurrence of psoriasis. This provides a new direction for the treatment of psoriasis. More importantly, Card14E138A/+ mice can be used as an appropriate model for psoriasis research, especially in the initial stage of mechanism research, as well as a model for screening and validation of therapeutic drugs.
  • the design idea is as follows: the designed peptide small molecule drugs can effectively interfere with the binding of TCR, BCR, IL1-R, TNF-R1 and other receptors to pathogenic factors, and down-regulate the expression level of CARMA through related signaling cascade pathways to further inhibit the VEGF Expression, to achieve the purpose of inhibiting vascular proliferation. Control the occurrence and development of psoriasis.
  • the designed peptide small molecule drugs can effectively inhibit IL17RA, NOTCH1-3, CD4, IL4R, CD58 and CD2 and other receptors and effectively bind to related cytokines. On the one hand, it down-regulates the expression level of VEGF through cell-related signaling pathways; another Aspects down-regulate the expression of inflammatory factors such as IL12, IL12R, IL1, IL1R, IL6, IL8, and control the occurrence and development of psoriasis.
  • H-473 peptide small molecule drugs can efficiently bind to TCR, BCR, IL1-R, TNF-R1, IL17RA, NOTCH1-3, CD4, IL12, IL12R, CD58 and CD2 molecules.
  • H-473 peptide small molecule drugs act on the cultured T and B cells respectively, and the expression of cell-related proteins was detected by ELISA experiments.
  • CARMA, VEGF, IL6, IL8, IL1-R, TNF-R1, IL17RA, IL12, IL12R were found The expression of other proteins was significantly down-regulated.
  • VEGF inhibitors such as those caused by injection, including conjunctival hemorrhage, acute intraocular Increased blood pressure, traumatic cataract, uveitis, retinal detachment and other side effects; and VEGF inhibitor injection may cause infection and endophthalmitis.
  • bevacizumab antibody causes some patients to suffer from endophthalmitis and blindness. side effect.
  • the design idea is that the designed small-molecule peptide drugs can: 1. Make the expression of VEGF in cells significantly down-regulated. 2. Can effectively inhibit the biological activity of VEGF. 3. Effectively interfere with VEGF-related receptors, such as VEGFR, PDGFR, etc. Eventually, it can inhibit the proliferation of blood vessels and achieve the purpose of resisting age-related macular degeneration (AMD) disease.
  • AMD age-related macular degeneration
  • H-473 peptide small molecule drugs can fully bind with VEGF molecules in vitro, and also with VEGFR, PDGFR, and GF related receptor molecules. Theoretically, not only the VEGF molecule is deactivated, but also the VEGF molecule is lost the opportunity to bind to the related receptor.
  • the H-473 peptide small molecule drugs acted on the cultured lung cancer cell A549 and gastric cancer cell MGC-803 respectively.
  • the expression of cell-related proteins was detected by ELISA experiment, and it was found that the expression of VEGF protein was significantly down-regulated.
  • the experimental results show that the H-473 peptide small-molecule drug has achieved its intended design and is expected to be a powerful drug against age-related macular degeneration (AMD) disease.
  • AMD age-related macular degeneration

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Abstract

一种人工合成多肽H-473及其应用，所述的多肽H-473的氨基酸序列是：RGLRGLR,分子量为827Da；PI为12.30。多肽H-473在制备抗癌药物，抗老年痴呆，镇痛等方面的应用。多肽H-473药物为抗癌，抗哮喘，抗老年痴呆和镇痛的研发提供了科学依据和方向，也为研究抗癌，抗哮喘，抗老年痴呆，镇痛和抗银屑病机理提供了有价值的资源，为研究新型的抗癌，抗哮喘，抗老年痴呆，镇痛，抗银屑病和抗年龄相关性黄斑变性(AMD)病的手段提供的新的方向。

Description

一种人工合成多肽H-473及其应用 技术领域
本发明属于生物医药技术领域，涉及一种人工合成多肽H-473及其应用，具体在制备抗癌，抗老年痴呆，抗哮喘，镇痛，抗银屑病和抗年龄相关性黄斑变性(AMD)病等药物方面的应用。
背景技术
蛋白质为行使其生物学功能，通常与其它蛋白质发生相互作用来体现其功能效应，即：蛋白质相互作用是生命活动在分子水平上的基本事件。单克隆抗体和某些特定结构的小分子药物，包括小分子多肽可以抑制这些相互作用。在生物医学领域也被称之为靶向治疗。单克隆抗体药物不只在肿瘤的治疗中取得了很好疗效，在其他疾病的治疗中也取得了一些疗效，例如奥马珠单抗(omalizumab)通过与游离IgE结合而显著降低游离IgE的水平，阻断IgE与肥大细胞、嗜碱粒细胞结合，防止炎症介质的释放。可显著改善哮喘病人的症状、肺功能及生活质量，减少哮喘恶化的发作次数，减少糖皮质激素的用量，使用安全，具有很好的耐受性.利妥昔单抗对类风湿和系统性红斑狼疮也有很好的治疗作用。英夫利昔单抗对类风湿性关节炎患者疼痛、晨僵、关节肿胀等临床症状减轻程度就可达60％。另有研究显示，输注英夫利昔单抗可快速、显著缓解顽固性银屑病、关节炎病人的关节和皮损症状.英利昔单抗对克罗恩病、溃疡性结肠炎、酒精性肝病等消化系统疾病都有较好疗效，且在推荐的治疗范围内安全性良好。雷珠单抗 是用于治疗湿性(新生血管性)年龄相关性黄斑变性(AMD)。这些例子说明某些药物具有多靶点治疗性。
根据蛋白分子受到其他分子作用后，将改变原蛋白分子的空间结构和功能特征的原理，本发明在分析癌分子通路中的相关受体(FZDs,ITG,FGFR,ErBB2,PDGFR,IGFR,KIT,FGFR,HGFR和TRK等)；支气管哮喘和慢性炎症分子通路中的相关受体(TLR4，TLR1，TLR5，TLR6等)；阿片受体(opioid receptor)，老年痴呆相关的乙酰胆碱酯酶(acetylcholinesterase)，银屑病相关因子和年龄相关性黄斑变性(AMD)相关因子等蛋白的分子结构及其功能位点进行分析的基础上，设计了小分子多肽作为多靶点治疗药物，目的在于干扰这些受体，细胞因子和酶分子的功能，达到治疗相关疾病的目的。发明人合成了多肽H-473，迄今为止，并提供了多肽H-473在抗癌，抗哮喘，抗老年痴呆，镇痛，治疗银屑病和治疗银屑病和年龄相关性黄斑变性(AMD)病的生物医学功效及其应用。
发明内容
本发明的目的旨在提供一种人工合成多肽H-473及其应用，并揭示其在抗癌，抗哮喘，抗老年痴呆，镇痛治疗银屑病活性效应的应用前景。
为实现上述技术目的，本发明具体通过以下技术方案实现：
一种人工合成多肽H-473，所述的多肽H-473的氨基酸序列:RGLRGLR,，分子量为827Da；PI为12.30。该肽段为单链，不具有化学键的修饰。
本发明所述的多肽H-473对人正常肝细胞Chang Liver的生长抑制几乎无影响(仅有微弱的生长抑制作用或微弱的生长促进作用)，而对人胃癌细胞MGC-803和人胰腺癌细胞cfpac-1细胞生长具有显著的抑制作用。
通过构建小鼠乙酰胆碱酶动物模型结合构建的乙酰胆碱酯酶的标准曲线，对各实验组的脑组织的乙酰胆碱酯酶进行测定和计算各实验组脑细胞乙酰胆碱酯酶表达量，本发明所述的多肽H-473对脑组织的乙酰胆碱酯酶表达抑制率为55.6％。多肽H-473干扰脑组织的乙酰胆碱酯酶表达率为43.8％。
本发明所述的多肽H-473药物有显著抗哮喘活性。
通过构建小鼠扭体反应次数的镇痛模型，观察H-473对小鼠镇痛作用的影响。本发明多肽H-473可明显减少醋酸所致的小鼠扭体反应次数，各剂量组对醋酸所致疼痛有显著的镇痛作用。
同时本发明多肽H-473药物在分子、细胞和动物水平上对银屑病发生发展的相关因子如：CARMA，VEGF，IL6，IL8,IL1-R，TNF-R1，IL17RA，IL12，IL12R等蛋白的表达显著下调，可望成为抗银屑病的强效药物。
本发明多肽H-473使细胞的VEGF表达显著下调，能够有效抑制VEGF的生物活性，有效地干扰VEGF的相关受体，如VEGFR，PDGFR等。最终实现抑制血管增生，达到抗年龄相关性黄斑变性(AMD)病的目的。
在本发明的另一方面，本发明提供了所述的多肽H-473在预防和 /或治疗抗癌药物中的应用，所述的抗癌药物包括抗人类胃癌药物和抗人胰腺癌药物。
在本发明的另一方面，本发明提供了所述的多肽H-473在预防和/或治疗抗老年痴呆药物中的应用。
在本发明的另一方面，本发明提供了所述的多肽H-473在预防和/或治疗镇痛药物中的应用，所述应用包括预防和/或治疗烧烫伤、外力损伤、冻伤、炎症性疼痛、神经病理性损伤以及其他原因导致的疼痛。
在本发明的另一方面，本发明提供了所述的多肽H-473在预防和/或治疗哮喘药物中的应用。
在本发明的另一方面，本发明提供了所述的多肽H-473在预防和/或治疗银屑病药物中的应用。
在本发明的另一方面，本发明提供了所述的多肽H-473在预防和/或治疗黄斑变性(AMD)药物中的应用。
上述药物中还可以加入一种或多种药学上可接受的辅料，所述辅料包括药学领域常规的稀释剂、稳定剂、填充剂、赋形剂、粘合剂、湿润剂、吸收促进剂、表面活性剂和润滑剂等。
本发明所述的药物，可以根据小分子多肽的理化性质制成注射剂、冻干制剂、埋植剂、缓释剂等多种形式的药物。上述各种剂型的药物均可以按照药学领域的常规方法制备。
本发明的有益效果为：
本发明提供的多肽H-473属于小分子多肽，容易制备，其制备工 艺和成本较低，易于制得。H-473在抗癌、抗哮喘、抗老年痴呆、镇痛和抗银屑病中都具有非常高的应用价值，研究表明，对人胃癌细胞MGC-803细胞生长抑制率达到51.9％。对人胰腺癌细胞cfpac-1细胞细胞生长抑制率达到48.8％。多肽H-473对人胃癌细胞MGC-803裸鼠异种移植瘤的抑瘤率高达64.1％，多肽H-473对人胰腺癌细胞cfpac-1裸鼠异种移植瘤的抑瘤率高达68.04％，多肽H-473对脑组织的乙酰胆碱酯酶表达抑制率为55.6％，肽H-473干扰脑组织的乙酰胆碱酯酶表达率为43.8％，H-473多肽药物对醋酸所致小鼠疼痛扭体抑制率在0.5mg/kg和1mg/kg剂量时分别为81.95％和95.11％。本发明为研究新型的抗癌、抗哮喘、抗老年痴呆、镇痛、抗银屑病和抗年龄相关性黄斑变性(AMD)病提供了有价值的资源和方向。
附图说明
图1是本发明H-473的细胞学活性效应数据图；
图2是本发明实施例2中多肽H-473对人胃癌裸鼠异种移植瘤生长的影响效果数据图；
图3是本发明实施例3中多肽H-473对人胰腺癌裸鼠异种移植瘤生长影响效果数据图；
图4是乙酰胆碱酯酶的标准曲线。
具体实施方式
下面将结合本发明具体的实施例，对本发明技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在 没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1细胞模型的构建与抗癌效果的验证
1.细胞模型
1.1材料与方法
1.1.1材料
1.1.1.1细胞株：人正常肝细胞Chang Liver(张氏肝细胞)，人胃癌细胞MGC-803，人胰腺癌细胞cfpac-1，上述细胞株均为已经商品化的生物材料。
1.1.2试剂：RPMI1640粉末(购于Gibco公司，美国)，胎牛血清(购于四季青生物制品公司，中国)，双抗(青霉素，链霉素，购于Gibco公司)。
1.1.1.3 H-473的氨基酸序列为：RGLRGLR,分子量为827Da；PI为12.30。纯度为98.12％。
1.1.2方法
1.1.2.1细胞培养
将人正常肝细胞Chang Liver，人胃癌细胞MGC-803，人胰腺癌细胞cfpac-1细胞株分别用含有10％的灭活的胎牛血清(fetalbovine serum,FBS)100U/ml青霉素、100μg/ml链霉素、pH7.4的RPMI-1640完全培养液于37℃、5％CO 2培养箱中进行培养。
1.1.2.2 MTT法检测细胞活性
取对数生长期的人正常肝细胞Chang Liver，人胃癌细胞 MGC-803，人胰腺癌细胞cfpac-1细胞用胰蛋白酶消化后将细胞数调整到l×10 5个/mL，接种于96孔板，每孔细胞液量为200μL，放入CO 2培养箱内，于37℃、5％CO 2及饱和湿度的条件下孵育至细胞单层铺满孔底(96孔平底板)，然后加H-473，使其终浓度分别为0.05，0.1，0.2，0.5，0.8，1.5，3.0和6.0μg/ml；设6个重复孔，空白组为不含细胞的RPMI1640培养液，放入CO 2培养箱内培养24、36和48h后取出96孔板，加入MTT(5mg/mL)溶液10μL，继续培养4h后，然后加入150μL的二甲基亚砜(DMSO)振荡10min，待蓝紫色结晶完全溶解后，用酶标仪在在490nm的波长下测定各孔的吸光度(OD)值，计算不同浓度下H-473(本实施例采用直接加入培养生长良好的癌细胞中观察结果，临床中，蛋白质或者多肽类的生物药物多采用注射剂的剂型，肌注或者静脉滴注)作用各类型细胞的抑制率(％)。细胞生长抑制率公式为：
细胞生长抑制率＝(1—A实验组/A对照组)×100％。
在以上实验的基础上选择了H-473生长抑制效果最好的人胃癌细胞MGC-803，人胰腺癌细胞cfpac-1细胞和人正常肝细胞Chang Liver进行对比实验，培养时间为36h，获取统计数据，具体方法与步骤同上。
1.2结果
本实施例采用MTT法检测结果显示：不同浓度多肽药物H-473在36h下对人正常肝细胞Chang Liver的生长抑制几乎无影响(仅有微弱的生长抑制作用或微弱的生长促进作用)，而对人胃癌细胞 MGC-803和人胰腺癌细胞cfpac-1细胞生长具有显著的抑制作用。从图1可以看出，多肽药物H-473对人胃癌细胞MGC-803和人胰腺癌细胞cfpac-1细胞的生长抑制率具有剂量的依赖性。发现在3.0μg/ml浓度下对人胃癌细胞MGC-803细胞生长抑制率达到51.9％。发现在6.0μg/ml浓度下对人胰腺癌细胞cfpac-1细胞细胞生长抑制率达到48.8％。
实施例2对人胃癌细胞MGC-803裸鼠异种移植瘤的药效实验
2.1实验目的：
根据《抗肿瘤药物药效学指导原则》和《细胞毒类抗肿瘤药物非临床研究技术指导原则》的要求，测试多肽药物多肽药物H-473对人胃癌细胞MGC-803异种移植肿瘤生长有无抑制作用及作用强度。
2.2受试样品：
多肽药物H-473。
配制方法：多肽药物1mg溶于1ml的生理盐水中，然后倍比稀释，得到所需浓度的多肽溶液。
2.3对照药物：
白介素-2
厂家：北京双鹭药业股份有限公司
批号：20120824
配制方法：给药前用生理盐水配成浓度为2万IU/ml的溶液。
2.4组别及给药方案：
模型对照组：生理盐水，0.1ml/10g，每天1次，持续10次；
白介素-2：尾静脉注射2万IU/ml白介素-2溶液，0.1ml/10g，每天1次，持续10次；
多肽H-473-1ug/kg组：尾静脉注射0.1ug/ml的多肽H-473溶液，0.1ml/10g，每天1次，持续10次。
2.5受试动物：
来源、种系、品系：BALB/c裸小鼠，由中国人民解放军军事医学科学院实验动物中心提供。
实验动物生产许可证：SCXK(军)2012-004
合格证编号：0039642
实验动物使用许可证：SYXK(苏)2011-0036
日龄：4-5w
体重：18-22g
性别：雄性
动物数：每组8只，共24只。
2.6实验方法：
2.6.1模型的制备
收集培养的人胃癌细胞MGC-803细胞悬液，浓度为1×10 7个/ml，以每只0.1ml接种于裸小鼠右侧腋窝皮下。
2.6.2分组与给药
裸鼠移植瘤用游标卡尺测量移植瘤直径，接种8天后，肿瘤生长至100-150mm 3时将动物随机分组，每组8只。同时，各组裸鼠开始给药，给药方案见组别与给药方案，使用测量瘤径的方法，动态观察 受试样品的抗肿瘤效应。给药10天后继续观察4天，随即处死裸鼠，手术剥取瘤块称重。
2.6.3观测指标
肿瘤体积(tumor volume,TV)的计算公式为：TV＝1/2×a×b 2，其中a、b分别表示长宽。
根据测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为：RTV＝V t/V 0，其中V 0为分笼给药时(即d 0)测量所得肿瘤体积，V t为每一次测量时的肿瘤体积。
抗肿瘤活性的评价指标：相对肿瘤增殖率T/C(％)，计算公式如下：
Figure PCTCN2019072013-appb-000001
其中，T RTV：治疗组RTV；C RTV：模型组RTV。
抗肿瘤活性的评价指标：肿瘤生长抑制率(％)，计算公式如下：
Figure PCTCN2019072013-appb-000002
2.6.4统计处理
均值用X±SD表示，组间分析用t检验进行统计学处理，应用SPSS(Staffstical Package for the Social Science)17.0对结果进行统计分析。
2.7实施例结果与结论：
本实施例建立了人胃癌细胞MGC-803裸鼠异种移植瘤模型，利用该模型评价了多肽H-473在1ug/kg浓度时的抗肿瘤活性。实验结 果为：多肽H-473的抑瘤率为64.1％。而相同条件下，阳性对照IL2组(20万U/kg)的抑瘤率为60.9％。结论为：多肽药物H-473在1ug/kg的浓度下有显著的抗肿瘤活性，抑瘤率>40％，且和模型组比有显著性差异。本实施例的实验结果如表1所示；本实施例中多肽H-473对人胃癌细胞MGC-803裸鼠异种移植瘤生长的影响效果如图2所示。
表1多肽H‐473对人胃癌细胞MGC-803裸鼠异种移植瘤生长的抑制作用
(X±SD，n＝8)
Figure PCTCN2019072013-appb-000003
实施例3对人胰腺癌细胞cfpac-1裸鼠异种移植瘤的药效实验
3.1实验目的：
根据《抗肿瘤药物药效学指导原则》和《细胞毒类抗肿瘤药物非临床研究技术指导原则》的要求，测试多肽药物H-473对人胰腺癌细胞cfpac-1异种移植肿瘤生长有无抑制作用及作用强度。
3.2受试样品：
多肽药物H-473
配制方法：多肽药物1mg溶于1ml的生理盐水中，然后倍比稀释，得到所需浓度的多肽溶液。
3.3对照药物：
白介素-2
厂家：北京双鹭药业股份有限公司
批号：20131037
配制方法：给药前用生理盐水配成浓度为2万IU/ml的溶液。
3.4组别及给药方案：
模型对照组：生理盐水，0.1ml/10g，每天1次，持续10次。
白介素-2：尾静脉注射2万IU/ml白介素-2溶液，0.1ml/10g，每天1次，持续10次。
多肽H-473-1ug/kg组：尾静脉注射0.1ug/ml的多肽H-473溶液，0.1ml/10g，每天1次，持续10次。
3.5受试动物：
来源、种系、品系：BALB/c裸小鼠，由中国人民解放军军事医学科学院实验动物中心提供。
实验动物生产许可证：SCXK(军)2012-004
合格证编号：0018558
实验动物使用许可证：SYXK(苏)2011-0036
日龄：4-5w
体重：18-22g
性别：雄性
动物数：每组8只，共24只。
3.6实验方法：
3.6.1模型的制备
收集培养的cfpac-1细胞悬液，浓度为1×10 7个/ml，以每只0.1ml 接种于裸小鼠右侧腋窝皮下。
3.6.2分组与给药
裸鼠移植瘤用游标卡尺测量移植瘤直径，接种17天后，肿瘤生长至100-150mm 3时将动物随机分组，每组8只。同时，各组裸鼠开始给药，给药方案见组别与给药方案，使用测量瘤径的方法，动态观察受试样品的抗肿瘤效应。给药10天后继续观察3天，随即处死裸鼠，手术剥取瘤块称重。
3.6.3观测指标
肿瘤体积(tumor volume,TV)的计算公式为：TV＝1/2×a×b 2，其中a、b分别表示长宽。
根据测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为：RTV＝V t/V 0，其中V 0为分笼给药时(即d 0)测量所得肿瘤体积，V t为每一次测量时的肿瘤体积。
抗肿瘤活性的评价指标：相对肿瘤增殖率T/C(％)，计算公式如下：
Figure PCTCN2019072013-appb-000004
其中，T RTV：治疗组RTV；C RTV：模型组RTV。
抗肿瘤活性的评价指标：肿瘤生长抑制率(％)，计算公式如下：
Figure PCTCN2019072013-appb-000005
3.6.4统计处理
均值用X±SD表示，组间分析用t检验进行统计学处理，应用 SPSS(Staffstical Package for the Social Science)17.0对结果进行统计分析。
3.7实施例结果与结论：
本实施例建立了人胰腺癌细胞cfpac-1裸鼠异种移植瘤模型，利用该模型评价了多肽H-473在1ug/kg浓度时的抗肿瘤活性。实验结果为：多肽H-473的抑瘤率为56.1％。而相同条件下，阳性对照IL2组(20万U/kg)的抑瘤率为68.04％。结论为：多肽药物H-473在1ug/kg的浓度下有显著的抗肿瘤活性，抑瘤率>40％，且和模型组比有显著性差异。本实施例的实验结果如表2所示；本实施例中多肽H-473对人胃癌细胞MGC-803裸鼠异种移植瘤生长的影响效果如图3所示。
表2多肽H‐473对人胰腺癌细胞cfpac-1裸鼠异种移植瘤生长的影响效果
(X±SD，n＝8)
Figure PCTCN2019072013-appb-000006
实施例4小鼠乙酰胆碱酶动物模型实验
4.1实验目的：
评价多肽药物H-473对脑细胞乙酰胆碱酯酶表达的抑制效应和干扰效果。
4.2试剂：D-半乳糖、亚硝酸钠、动物脑组织蛋白提取试剂盒。
4.3模型构造：
32只小鼠随机分成4组，每组8只。
对照组：不做任何处理，每天正常喂食。
表达抑制组：每只小鼠每天肌肉注射200μg/kg药物处理30天。
模型组：每只小鼠每天腹腔注射150mg/kg D-半乳糖和120mg/kg亚硝酸钠，处理30天。
干扰组：在模型组的基础上，同时每只小鼠每天肌肉注射200μg/kg多肽药物H-473，处理30天。
4.4样本收集：
收集每组的脑组织，将每组中各只取相同重量的脑组织混在一起。
将收集的脑组织立即用液氮处理，处理后平均分为3份，-80°超低温保存。
用动物脑组织蛋白提取试剂盒提取脑组织蛋白。
用ELISA试剂盒测定每个实验组的脑细胞乙酰胆碱酯酶表达量。
4.5数据统计
表达抑制率的计算公式：
表达抑制率％＝(控制组的表达量－抑制组表达量)/控制组的表达量×100％。
药物干扰表达率的计算公式：
药物干扰表达率％＝(模型组的表达量－干扰组表达量)/模型组的表达量×100％。
4.6实验结果。
按照ELISA试剂盒说明书的方法，构建乙酰胆碱酯酶的标准曲线(图4)，对各组的脑组织的乙酰胆碱酯酶进行测定，然后利用标准曲线计算各实验组脑细胞乙酰胆碱酯酶表达量(表3)。表达抑制率为：55.6％(与控制组比较)。药物干扰表达率为：43.8％(与模型组比较)。结果显示：多肽药物H-473有显著抑制脑细胞乙酰胆碱酯酶的表达。
表3各实验组脑组织乙酰胆碱酯酶表达的测定结果(nmol/L)
Figure PCTCN2019072013-appb-000007
实施例5多肽药物H-473对小鼠哮喘药效作用
5.1受试样品：
多肽药物H-473。
阳性药物，地塞米松，浙江仙崌制药股份有限公司，批号：150921。
5.2主要试剂：
卵白蛋白，Sigma公司，Lot SLBK7542V。
佐剂液态铝，Thermo公司，Lot RB231380B。
5.3主要仪器：
压缩空气式雾化器，黄山市雅适医疗器械有限公司，YS-01。
血液分析仪，山东恒拓科技发展有限公司，BTX-1800。
5.4受试动物：
来源、种系、品系：BABL/C小鼠，由上海西普尔-必凯实验动物有限公司提供。
实验动物生产许可证：SCXK(沪)2013-0016
实验动物使用许可证：SYXK(苏)2012-0047
体重：18-22g。
性别：雌性。
动物数：40只。
5.3药物分组、给药方式及周期
药物分组、给药方式及周期见表4。实验35天时，在OVA雾化前一小时给予药物。
表4药物分组、给药方式及周期
Figure PCTCN2019072013-appb-000008
实验35天时，在OVA雾化前一小时给予药物。
5.6实验方法：
5.6.1哮喘模型：小鼠腹腔注射0.2ml致敏液(卵蛋白OVA80ug+佐剂液态铝0.1ml)，于0、14天注射2次。从第一次致敏后第24天开始，雾化吸入2.5％的OVA 45min，每天一次，持续18d。
5.6.2给药：自实验第35天起，在OVA溶液雾化攻击前1h给予相应药物。H-473多肽药物15ug/kg组和H-473多肽药物30ug/kg组分别肌肉注射。阳性对照组肌肉注射地塞米松1mg/kg。正常对照组、模型对照组肌肉注射生理盐水。
5.6.3指标检测及取材：
一般整体观察：呼吸频率，有无呼吸困难、发绀、躁动及抽搐。
支气管肺泡灌洗液(BALF)细胞计数、分类(EOS比率是否升高)。
小鼠血清：末次激发后24h小鼠眼球采血，离心，收集血清。
肺组织一半固定，一半冷冻。
5.7实验结果
5.7.1一般观察：
各组小鼠第一次致敏后32天，即雾化后11天出现躁动、点头样呼吸，抓鼻等反应。雾化后18天模型组出现呼吸困难、躁动等反应，阳性组未出现异常反应，其他给药组和雾化8天时出现症状一样，没有加重趋势。
5.7.2 BALF细胞计数、分类(表5)
表5 BALF细胞计数、分类(M±SD，N＝6，10 ^9/L)
Figure PCTCN2019072013-appb-000009
结论：H-473多肽药物有显著抗哮喘活性。
实施例6H-473镇痛药物药效的评价
6.1实验目的：
观察H-473对小鼠镇痛作用的影响。
6.2受试样品：
药物名称：H-473。
配制方法：药物，-20℃保存，取适量药品，加生理盐水溶解至所需浓度。
6.3组别及给药方案：
模型组：肌肉注射生理盐水，5ml/kg。
H-473-L组：肌肉注射药物，0.5mg/kg，给药一次。
H-473-H组：肌肉注射药物，1mg/kg，给药一次。
6.4受试动物：
来源、种系、品系：ICR小鼠，上海灵畅生物科技有限公司提供。
实验动物生产许可证：SCXK(沪)2013-0018，合格证编号：2013001834301。
实验动物使用许可证：SYXK(苏)2017-0015。
日龄：5-6W。
体重：180-220g。
性别：雌性。
动物数：24只。
6.5实验方法：
6.5.1模型的制备
根据药理实验方法学将小鼠适应性饲养3天后，按照组别随机分为3组，每组8只动物。按照给药方案，各组小鼠肌肉注射药物，给药1h后，小鼠腹腔注射0.2ml2％冰醋酸，观察15min内小鼠出现扭 体反应(腹部收缩呈“S”形，身体扭曲，后肢伸展及蠕行等)的次数，并计算扭体抑制率。扭体抑制率％＝(对照组平均扭体次数-给药组平均扭体次数)/对照组平均扭体次数×100％。
6.5.2统计处理
均值用X±SD表示，组间分析用t检验进行统计学处理，应用SPSS(Staffstical Package for the Social Science)17.0对结果进行统计分析。
6.6实验结果：
如表6所示，H-473可明显减少醋酸所致的小鼠扭体反应次数，各剂量组对醋酸所致疼痛有显著的镇痛作用，与空白对照组比较有显著差异(p<0.01)。
表6 H‐473对醋酸所致小鼠扭体反应的影响(X±SD，n＝8)
Figure PCTCN2019072013-appb-000010
实施例7抗银屑病药物实验
7.1银屑病(俗称牛皮癣)最新研究成果：2018年7月3日，清华大学林欣实验室在细胞杂志子刊《Immunity》上在线发表了题为“Gainof function mutation of Card14 leadsto spontaneous psoriasis-like skin inflammation through enhanced keratinocyteresponse to interleukin-17A”的文章。文章报道了Card14E138A/+自发性银屑病小鼠的组织病理学表型和炎症反应类型，并阐明了Card14基因的缺 失或突变影响角质细胞中IL-17A信号激活的分子机制，以及角质细胞在银屑病起始过程中的重要作用。
目前，对于银屑病的起因尚不清楚。近期的GWAS研究发现，银屑病患者中尚存在多种NF-κB信号相关基因突变，如皮肤中高表达的CARD14基因等。这一发现表明CARD14基因可能在诱发银屑病的过程中起到重要作用。CARD14基因编码细胞内一个信号转导相关的蛋白CARMA2，林欣实验室多年来研究CARD家族编码蛋白在免疫与炎症反应中的功能，因此在这项研究中通过构建Card14突变小鼠模型来探讨CARMA2的激活能否诱发银屑病，以及如何诱发银屑病的发生。
研究发现CARMA2是角质细胞中IL-17A信号通路的重要调控蛋白，其在角质细胞中的持续激活，即可诱导银屑病的发生。这为银屑病的治疗提供新的方向。更重要的是，Card14E138A/+小鼠可作为银屑病研究，特别是起始阶段机制研究的恰当模型，以及治疗药物筛选和验证的模型。
7.2创新性抗银屑病药物的设计
我们根据现有药物的作用机制，结合最新研究进展，从分子水平设计出创新性抗银屑病的多肽小分子药物。
设计思路如下：设计的多肽小分子药物能有效地干扰TCR，BCR，IL1-R，TNF-R1等受体与致病因子结合，通过相关信号联级通路下调CARMA的表达水平，进一步抑制VEGF的表达，达到抑制血管增生的目的。控制银屑病的发生与发展。
设计的多肽小分子药物同时能有效地抑制IL17RA，NOTCH1-3，CD4，IL4R，CD58和CD2等受体和与相关细胞因子有效结合，通过细胞相关信号通路一方面下调VEGF的表达水平；另一方面下调IL12,IL12R,IL1,IL1R,IL6，IL8等炎性因子的表达，控制银屑病的发生与发展。
7.3摸底实验结果如下：
7.3.1分子水平研究结果
H-473多肽小分子药物在分子水平上能与TCR，BCR，IL1-R，TNF-R1，IL17RA，NOTCH1-3，CD4，IL12，IL12R,CD58和CD2等分子高效结合。
7.3.2细胞水平研究结果
H-473多肽小分子药物分别作用于培养的T和B细胞，利用ELISA实验检测细胞相关蛋白的表达量，发现CARMA，VEGF，IL6，IL8,IL1-R，TNF-R1，IL17RA，IL12，IL12R等蛋白的表达显著下调。
7.3.3动物水平研究结果
将H-473多肽小分子药物通过肌肉给药和鼻粘膜给药实验小鼠12天后，分别收集模型组和实验组动物的相应组织构建基因池，然后进行基因转录谱分析。结果显示CARMA，VEGF,IL6，IL8IL1-R，TNF-R1，IL17RA，IL12，IL12R等基因转录量显著下调。
实验结果表明H-473多肽小分子药物达到了预期的设计目的，可望成为抗银屑病的强效药物。
实施例8抗年龄相关性黄斑变性(AMD)病小分子多肽药物实 验
8.1抗年龄相关性黄斑变性(AMD)病小分子多肽药物的设计
根据已有的抗年龄相关性黄斑变性(AMD)病的几个抗体的药理学原理，同时又要克服现有VEGF抑制剂的主要副作用，如因注射产生的，其中包括结膜出血、急性眼内压上升、创伤性白内障、葡萄膜炎、视网膜脱离等副作用；以及VEGF抑制剂注射可能导致感染和眼内炎，在没有适当无菌技术的贝伐单抗体使一些患者罹患眼内炎和失明等副作用。
设计思路是：所设计的小分子多肽药物能够：1.使细胞的VEGF表达显著下调。2.能够有效抑制VEGF的生物活性。3.有效地干扰VEGF的相关受体，如VEGFR，PDGFR等。最终实现抑制血管增生，达到抗年龄相关性黄斑变性(AMD)病的目的。
8.2.摸底实验结果如下：
8.2.1分子水平研究结果
H-473多肽小分子药物在体外能与VEGF分子充分结合，也能与VEGFR，PDGFR，以及GF的相关受体分子充分结合。从理论上讲，既使VEGF分子失去活性，又使VEGF分子失去与相关受体结合的机会。
8.2.2细胞水平研究结果
H-473多肽小分子药物分别作用于培养的肺癌细胞A549和胃癌细胞MGC-803，利用ELISA实验检测细胞相关蛋白的表达量，发现VEGF蛋白的表达显著下调。
8.2.3动物水平研究结果
将H-473多肽小分子药物通过鼻粘膜给药实验小鼠15天后，分别收集模型组和实验组动物的相应组织构建基因池，然后进行基因转录谱分析。结果显示VEGF，IL6，IL8，IL1-R，TNF-R1，IL17RA，IL12，IL12R等基因转录量显著下调。
实验结果表明：H-473多肽小分子药物达到了预期的设计目的，可望成为抗年龄相关性黄斑变性(AMD)病的强效药物。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。

Claims (8)

  1. 一种人工合成多肽H-473，其特征在于，所述的多肽H-473的氨基酸序列:RGLRGLR，分子量为827Da；PI为12.30。
  2. 权利要求1所述的多肽H-473在预防和/或治疗抗癌药物中的应用。
  3. 根据权利要求2所述的应用，其特征在于所述的抗癌药物包括抗人类胃癌药物和抗人胰腺癌药物。
  4. 权利要求1所述的多肽H-473在预防和/或治疗抗老年痴呆药物中的应用。
  5. 权利要求1所述的多肽H-473在预防和/或治疗镇痛药物中的应用，其特征在于，包括预防和/或治疗烧烫伤、外力损伤、冻伤、炎症性疼痛、神经病理性损伤以及其他原因导致的疼痛。
  6. 权利要求1所述的多肽H-473在预防和/或治疗哮喘药物中的应用。
  7. 权利要求1所述的多肽H-473在预防和/或治疗银屑病药物中的应用。
  8. 权利要求1所述的多肽H-473在预防和/或治疗黄斑变性药物中的应用。
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