WO2016127788A1 - 绿原酸在制备治疗红斑狼疮的药物中的用途 - Google Patents

绿原酸在制备治疗红斑狼疮的药物中的用途 Download PDF

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WO2016127788A1
WO2016127788A1 PCT/CN2016/072124 CN2016072124W WO2016127788A1 WO 2016127788 A1 WO2016127788 A1 WO 2016127788A1 CN 2016072124 W CN2016072124 W CN 2016072124W WO 2016127788 A1 WO2016127788 A1 WO 2016127788A1
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lupus erythematosus
chlorogenic acid
preparation
treating lupus
glomerular
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PCT/CN2016/072124
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French (fr)
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张洁
贾静
黄望
陈晓光
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四川九章生物科技有限公司
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Priority to US15/550,647 priority Critical patent/US10058525B2/en
Priority to EP16748589.5A priority patent/EP3257507A4/en
Publication of WO2016127788A1 publication Critical patent/WO2016127788A1/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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/429—Thiazoles condensed with heterocyclic ring systems
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4706—4-Aminoquinolines; 8-Aminoquinolines, e.g. chloroquine, primaquine
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/498—Pyrazines or piperazines ortho- and peri-condensed with carbocyclic ring systems, e.g. quinoxaline, phenazine
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/2292—Thymosin; Related peptides
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • the invention relates to the use of chlorogenic acid in the preparation of a medicament for treating lupus erythematosus, belonging to the field of medicine.
  • Chlorogenic acid also known as coffee tannin, chemically known as 3-0-caffeoylquinic acid, is derived from caffeic acid and quinic acid.
  • Quinic acid consists of a carboxylic acid.
  • Chlorogenic acid is a product of aerobic respiration metabolism in plants. It is the main active ingredient in many Chinese herbal medicines and fruits and vegetables. It has many biological activities, such as cardiovascular protection, anti-oxidation, anti-ultraviolet and anti-radiation effects. Anti-mutagenic and anti-cancer effects, antibacterial effects, antiviral effects, lipid-lowering and hypoglycemic effects, immunomodulatory effects, etc. It has a wide range of applications in the fields of pharmaceutical, chemical and food.
  • Lupus erythematosus is a typical autoimmune connective tissue disease, more common in women aged 15 to 40 years.
  • Lupus erythematosus is a disease spectrum disease, which can be divided into discoid lupus erythematosus (DLE), subacute cutaneous lupus erythematosus (SCLE), systemic lupus erythematosus (SLE), deep lupus erythematosus (LEP), newborn Subtypes such as lupus erythematosus (NLE) and drug-induced lupus erythematosus (DIL).
  • DLE discoid lupus erythematosus
  • SCLE subacute cutaneous lupus erythematosus
  • SLE systemic lupus erythematosus
  • LEP deep lupus erythematosus
  • NLE drug-induced lupus erythematos
  • lupus erythematosus mainly through antimalarial drugs (such as hydroxychloroquine), thalidomide or oral low-dose hormones.
  • antimalarial drugs such as hydroxychloroquine
  • thalidomide or oral low-dose hormones.
  • the traditional Chinese medicine used in traditional Chinese medicine for the treatment of lupus erythematosus includes compound Shengdi mixture, which consists of raw land, raw gypsum and Lonicera japonica. It has the functions of nourishing yin and clearing heat and cooling blood and collaterals. It is mainly used for the treatment of light and moderate activities.
  • Patients with systemic lupus erythematosus also include lupus pill, which includes more than ten kinds of medicinal materials such as honeysuckle, forsythia, dandelion, berberine, rehmannia, rhubarb, licorice, and medlar, which have the functions of clearing away heat and detoxifying, cooling blood, promoting blood circulation and removing blood stasis.
  • lupus pill which includes more than ten kinds of medicinal materials such as honeysuckle, forsythia, dandelion, berberine, rehmannia, rhubarb, licorice, and medlar, which have the functions of clearing away heat and detoxifying, cooling blood, promoting blood circulation and removing blood stasis.
  • Increase cellular immune function improve the body's disease resistance, reduce circulating immune complexes, and be used for the treatment of systemic lupus erythematosus.
  • the traditional Chinese medicine compound is complex,
  • the above object of the present invention is achieved by the following technical solution: a use of chlorogenic acid for the preparation of a medicament for treating lupus erythematosus.
  • the inventors unexpectedly found in the drug screening process of lupus erythematosus that the aqueous extract of Eucommia ulmoides leaves and the alcohol extract have obvious inhibitory effects on lupus erythematosus; further screening of the medicinal components of the extract revealed that green
  • the inhibition of lupus erythematosus by ortho-acid, genipin, and peach leaf peony is more obvious, especially chlorogenic acid, and the inhibition is particularly significant. This hair It is through the improvement of immune function by chlorogenic acid to achieve the purpose of treating lupus erythematosus.
  • the drug is a preparation prepared by adding chlorogenic acid as an active ingredient, adding a pharmaceutically acceptable adjuvant or an auxiliary component.
  • Another object of the present invention is to provide a formulation for treating lupus erythematosus comprising chlorogenic acid and a pharmaceutically acceptable excipient or adjuvant.
  • the excipients or excipients include, but are not limited to, mannitol, sodium bisulfite, starch, dextrin powder, absolute ethanol, water for injection, powdered sugar, lactose, hypromellose, magnesium stearate, sucrose, Povidone K30.
  • the preparation may be an oral preparation, an injection preparation or a transdermal preparation for external use.
  • the preparation of the present invention contains 1-10 mg of chlorogenic acid per unit of preparation, and the clinical use dose is 1-100 mg/kg.
  • Another object of the present invention is to provide a combination drug for treating lupus erythematosus, which comprises chlorogenic acid and a medicament for treating lupus erythematosus.
  • the medicament for treating lupus erythematosus includes, but is not limited to, hydroxychloroquine, methotrexate, azathioprine, chlorophenol azine, thymosin, levamisole, prednisone.
  • the test of the invention shows that chlorogenic acid can improve the pathological changes of the lupus erythematosus model, has immunomodulatory effects, improves immune function, and provides a theoretical basis for clinical treatment of lupus erythematosus.
  • the present invention has an advantageous effect of providing a medicament for the prevention and treatment of lupus erythematosus, which aims to prevent and treat by improving the body's immune function.
  • Figure 1 shows the results at 14d treatment, from top to bottom:
  • the ENA polypeptide antibody was anti-Sm, anti-ssA and ssB antibody positive at 14 days after treatment with chlorogenic acid in high dose group;
  • the ENA polypeptide antibody was positive for anti-SsA and ssB antibodies at 14 days after treatment with chlorogenic acid in a small dose group;
  • ENA peptide antibody was anti-Sm, anti-Rib, anti-ssA and ssB antibody positive at 14d after artesunate treatment;
  • ENA polypeptide antibody was anti-Sm, anti-Rib, anti-ssA and ssB antibodies positive at 14 days of prednisone treatment.
  • Figure 2 shows that ANA fluorescence intensity is reduced by ⁇ 200 when chlorogenic acid (large dose) is treated for 14 days.
  • Figure 3 shows that ANA fluorescence intensity is reduced by ⁇ 200 when chlorogenic acid (high dose) is treated for 21 days.
  • Figure 4 shows that ANA fluorescence intensity is reduced by ⁇ 200 when treated with chlorogenic acid (large dose) for 28 days.
  • Figure 5 shows that the ANA fluorescence intensity was not reduced by 200 when artesunate was treated for 28 days.
  • Figure 6 shows that normal mouse kidney sections show a small amount of silver staining of the renal tubular basement membrane in the renal medulla.
  • Figure 7 shows the thickening of the liver vascular basement membrane of the model mouse at ip LPS 14d ⁇ 50.
  • Figure 8 shows that the glomerular volume of the mouse is increased compared with normal, the number of cells is increased, and the kidney is increased at 28 days after administration of the high dose group of chlorogenic acid.
  • the thickness of the glomerular and tubular basement membrane was reduced by ⁇ 50 compared with normal mice.
  • Figure 9 shows that the glomerular basement membrane was significantly thicker than normal mice at 28 days after administration of the artesunate treatment group.
  • Figure 10 shows that the glomerular basement membrane was significantly thicker than normal mice at 28 days after administration of the prednisone treatment group.
  • Figure 11 shows that the glomerular wall epithelial cells are severely proliferated at 24 h in the model mice, and monocytes appear, with a large increase in mitochondria ⁇ 3000.
  • Figure 12 shows that the degree of hyperplasia of glomerular wall epithelial cells decreased in model mice at 28 days, with monocytes and a large number of mitochondria ⁇ 6600.
  • Figure 13 shows that the degree of hyperplasia of glomerular wall epithelial cells was relieved in the high-dose group of chlorogenic acid for 21 days ⁇ 4000.
  • Figure 14 shows that the degree of hyperplasia of glomerular wall epithelial cells was slightly relieved on the 21st day after administration of the artesunate treatment group, but the degree of remission was smaller than that of the Qingteng capsule treatment group, accompanied by thrombosis ⁇ 3000.
  • Figure 15 shows that the degree of glomerular wall epithelial cell proliferation was also relieved in the prednisone group compared with the model group, but the degree of remission was smaller than that in the Qingteng capsule group, and the number of mitochondria increased ⁇ 12,500.
  • Example 1 In vivo pharmacodynamic study of chlorogenic acid in the treatment of lupus erythematosus
  • mice weighing 17.0-21.0 g, 5-7 weeks old, male and female, were provided by the Experimental Animal Center of Sichuan University.
  • Chlorogenic acid prepared by Sichuan Jiuzhang Biotechnology Co., Ltd., has a content of 99.2%. Lot number: 131101.
  • physiological saline was used to prepare a solution of three concentrations of high, medium and low.
  • Prednisone produced by Zhejiang Xianyi Pharmaceutical Co., Ltd., 5mg/tablet, batch number: 110502; Escherichia coli lipopolysaccharide, produced by sigma company, Lot58kp8725, prepared with 0.25g/L with physiological saline before use.
  • HEP-2 cell sheets were produced by Scimedx.
  • Fluorescein-labeled goat anti-mouse sera FITC Goat Anti-Mouse IgG
  • ENA peptide immunoblot membrane strip produced by Shenzhen Hengjia Company. Biotin-conjugated goat anti-mouse IgG, Vector. Horseradish peroxidase-glycoprotein conjugate (HRP-avidin), Vector.
  • UV lamp produced by Shanghai Special Lamp Factory, 30W, maximum energy spectrum is 312nm, energy is 2.0Mj/cm 2 , distance is 30cm, medium-wave ultraviolet (UVB) part accounts for more than 60% of its radiant energy; Olympus BH - 2 million microscope, produced by OLYMPUS, Japan; Philip M800 transmission electron microscope, produced by PHILIPS, the Netherlands.
  • UVB medium-wave ultraviolet
  • the back of the mouse was shaved, and the ultraviolet light was irradiated for 2h/d*3d, the distance was 30cm. After the irradiation, the ip LPS was 2.5mg/kg, and the model was successful in 24h.
  • the normal control group was ip normal saline.
  • the model mice were administered intragastrically 24 h after ip LPS.
  • the chlorogenic acid experimental group was administered in the high, medium and low dose groups, respectively, and the doses were 10 mg/kg*d, 20 mg/kg*d, and 40 mg/kg*d, respectively.
  • the mouse serum was diluted 1:100 with Shenzhen Hengjia special buffer, then the diluted serum 200uL was separately added to the corresponding numbered reaction tank, incubated in a 37 ° C incubator for 30 min, and the buffer was washed 4 times for 2 min each time; Add 200 uL of 1:100 diluted biotin goat anti-mouse IgG, incubate in a 37 ° C incubator for 30 min, wash the buffer 4 times for 2 min each time; add 1:100 diluted HRP-avidin 200 uL, incubate in a 37 ° C incubator for 30 min, wash Ibid. The reaction solutions A and B were added until a positive band appeared, and the buffer was washed.
  • Alignment with the standard band of ENA polypeptide antibody in the same kit can be judged as Anti-Sm, anti-Rib, anti-ssA and anti-ssB antibodies were positive.
  • the serum was diluted 1:40 with PBS buffer, and then the diluted serum 20-30 uL was separately dropped into the pits of HEp-2 cell sheets, one cell slide per group. During the process of adding serum, care should be taken not to touch the indication of the cell sheet.
  • the serum-immobilized cell sheets were placed in a wet box and allowed to stand at room temperature (25 ° C) for 30 min. After 30 min, the cell pellet was removed, and the serum was carefully washed out with PBS, and then carefully washed twice with PBS for 5 min each time; after washing, excess PBS was absorbed by Scimedx-specific absorbent paper (this process should be avoided to avoid the bottom of the cell). The matter becomes dry and affects the result). Diluted (PBS 1:100 dilution) FITC goat anti-mouse IgG 20-30 uL into the wet box for 30 min at room temperature. Wash with PBS, the same procedure as above. Absorb the excess PBS on the absorbent paper, add 1-2 drops of fluorescent sealant to the cell sheet, cover the slide, and observe with Olympus BH-2 microscope.
  • the specimens under light microscopy were fixed with 10% formalin, then dehydrated, transparent, dipped in wax and embedded, and sectioned.
  • the slice is 3um thick and is dewaxed to water at the beginning of dyeing. After oxidation with 0.5% periodate, it is immersed in hexamine silver working solution for about 2h (constant temperature 58°C), washed with distilled water, dyed with hematoxylin for 1min, washed with Lichun red dye.
  • the droplets were stained for 5 min, 70% ethanol was differentiated, the gradient alcohol was dehydrated, the xylene was transparent, and the neutral gum was sealed.
  • Specimens observed by electron microscopy on 21d were fixed with 2% paraformaldehyde + 0.25% glutaraldehyde mixed fixative for 4h, rinsed with TBS buffer, fixed with 1% citrate for 1h, buffer rinsed, acetone dehydrated, uranyl acetate stained for 2h, Epon812 Soaked and embedded, LEICA Ultra-thin slicer sectioning, lead liquid staining, buffer washing, the specimen can be dried and then observed by transmission electron microscopy.
  • the basement membrane of the renal tubules was significantly thicker than that of normal mice; the basal membrane of the liver was thicker than that of normal mice.
  • Fig. 7 When the chlorogenic acid was administered for 21 days, the renal tissue structure of the mice still showed a larger glomerular volume, increased cell number, glomerular and interstitial chronic inflammatory cell infiltration, glomerular and tubular basement. The membrane was thicker than normal mice; however, the condition was better than the model group, especially in the high dose group.
  • the glomerular and tubular basement membranes of the artesunate treatment group were significantly thicker than those of normal mice on the 21st day;
  • Fig. 9 The glomerulus and kidney were treated on the 21st day of the 5 prednisone administration group.
  • the tubule basement membrane was also significantly thicker than normal mice. ( Figure 10).
  • Electron microscopy showed that there was no proliferation of glomerular wall epithelial cells in normal mice, and the number of mitochondria was normal.
  • Fig. 11 The glomerular wall epithelial cells were severely proliferated on the 21st day of the 2 model mice, and monocytes appeared, and mitochondria were abundant. increase.
  • Fig. 12 3 The degree of hyperplasia of glomerular wall epithelial cells was relieved compared with the model group when chlorogenic acid was administered for 21 days, and the degree of remission was most obvious in the high dose group; (Fig.
  • mice could see glomerular volume increase, cell number increase, glomerular and interstitial chronic inflammatory cell infiltration, glomerular and tubular basement membrane at 24h. Significantly thicker than normal mice; liver vascular basement membrane thicker than normal mice. On the 2nd, 3d, 7d, 14d, and 21d, the glomerular volume increased, the number of cells increased, the glomerular and interstitial chronic inflammatory cells infiltrated, and the glomerular and tubular basement membranes were significantly smaller than normal. The rats were thickened and the basement membrane of the liver was thickened.
  • chlorogenic acid has a good therapeutic effect on lupus erythematosus. Its mechanism of action may be to improve the immune function of mice with lupus erythematosus.

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Abstract

本发明提供了一种绿原酸在制备治疗红斑狼疮的药物中的应用，其中绿原酸能够改善免疫机能。本发明提供了一种用于治疗红斑狼疮的制剂，其中包含绿原酸和药学上可接受的辅料。本发明还提供了一种含有绿原酸和治疗红斑狼疮药物的联合药物。

Description

绿原酸在制备治疗红斑狼疮的药物中的用途 技术领域
本发明涉及绿原酸在制备治疗红斑狼疮的药物中的用途，属于药物领域。
背景技术
绿原酸(Chlorogenic acid,CGA)，又名咖啡单宁酸，化学名为3-0-咖啡酰奎尼酸(3-0-caffeoylquinic acid)，是由咖啡酸(Caffeic acid)与奎尼酸(Quinic acid)组成的羧酚酸。
绿原酸是植物体有氧呼吸代谢的产物，是许多中药材以及水果蔬菜中的主要有效成分，具有多种生物活性，如:心血管保护作用、抗氧化作用、抗紫外及抗辐射作用、抗诱变及抗癌作用、抗菌作用、抗病毒作用、降脂降糖作用、免疫调节作用等。在医药化工和食品等领域都具有广泛的应用。
红斑狼疮(LE)是一种典型的自身免疫性结缔组织病，多见于15～40岁女性。红斑狼疮是一种疾病谱性疾病，可分为盘状红斑狼疮(DLE)、亚急性皮肤型红斑狼疮(SCLE)、系统性红斑狼疮(SLE)、深在性红斑狼疮(LEP)、新生儿红斑狼疮(NLE)、药物性红斑狼疮(DIL)等亚型。
目前临床治疗红斑狼疮主要是通过抗疟药(如羟氯喹)、沙利度胺或者口服小剂量激素。我国传统中医目前用于治疗红斑狼疮的药物包括有复方生地合剂，其由生地、生石膏、忍冬藤三味药组成，具有养阴清热，凉血通络之功，主要用于治疗轻、中度活动期系统性红斑狼疮患者；还包括有狼疮丸，其处方包括金银花、连翘、蒲公英、黄连、地黄、大黄、甘草、蜈蚣等十几种药材，为具有清热解毒，凉血，活血化瘀，增加细胞免疫功能，提高机体抗病能力，降低循环免疫复合物，用于系统性红斑狼疮的治疗。然而，中药复方成分复杂，其药效成分不确切，作用机理不明确，存在一定的安全隐患。因此，对于单一药效组分用于治疗红斑狼疮的需求极为迫切。
发明内容
针对上述技术问题，本发明的目的在于提供一种绿原酸的新的用途。
本发明的上述目的是通过如下技术方案实现的：一种绿原酸在制备治疗红斑狼疮的药物中的应用。
在本发明中，发明人在对红斑狼疮的药物筛选过程中意外发现，杜仲叶水提物及醇提物对红斑狼疮都具有明显抑制作用；再对提取物进一步的药效成分筛选发现，绿原酸、京尼平苷酸、桃叶珊瑚甙对红斑狼疮的抑制较为明显，尤其绿原酸，抑制作用尤为显著。本发 明正是通过绿原酸改善免疫机能从而达到治疗红斑狼疮的目的的。
在本发明中，作为优选，所述的药物是由绿原酸为有效成分，加入药学上可接受的辅料或辅助性成分制备而成的制剂。
本发明的另一目的在于，提供一种用于治疗红斑狼疮的制剂，所述制剂包含绿原酸和药物上可接受的赋形剂或辅料。所述辅料或赋形剂包括但不限于甘露醇、亚硫酸氢钠、淀粉、糊精粉、无水乙醇、注射用水、糖粉、乳糖、羟丙甲纤维素、硬脂酸镁、蔗糖、聚维酮K30。
在本发明中，所述的制剂可以是口服制剂、注射制剂或外用透皮给药制剂。作为优选，本发明所述的制剂每制剂单位含绿原酸1-1000mg，临床使用剂量为：1-100mg/kg。
本发明的另一目的在于，提供一种用于治疗红斑狼疮的联合用药物，所述联合用药物包括绿原酸和治疗红斑狼疮的药物。作为优选，所述治疗红斑狼疮的药物包括但不限于羟基氯喹、甲氨喋呤、硫唑嘌呤、氯苯酚嗪、胸腺素、左旋咪唑、强的松。
本发明试验表明，绿原酸能改善红斑狼疮模型的病理变化，具有免疫调节作用，改善免疫机能，为其临床治疗红斑狼疮提供了理论依据。
本发明的有益效果在于，提供一种红斑狼疮的预防和治疗的药物，通过改善机体免疫机能达到预防和治疗的目的。
附图说明
图1为治疗14d时结果，其中由上往下分别为：
1)：绿原酸大剂量组治疗14d时ENA多肽抗体抗Sm，抗ssA和ssB抗体阳性；
2)：绿原酸中剂量组治疗14d时ENA多肽抗体抗Sm，抗Rib，抗ssA和ssB抗体阳性：
3)：绿原酸小剂量组治疗14d时ENA多肽抗体抗Sm.抗ssA和ssB抗体阳性；
4)：青蒿琥酯治疗14d时ENA多肽抗体抗Sm，抗Rib，抗ssA和ssB抗体阳性；
5)：强的松治疗14d时ENA多肽抗体抗Sm，抗Rib，抗ssA和ssB抗体阳性。
图2为绿原酸(大剂量)治疗14d时，ANA荧光强度减弱×200。
图3为绿原酸(大剂量)治疗21d时，ANA荧光强度减弱×200。
图4为绿原酸(大剂量)治疗28d时，ANA荧光强度减弱×200。
图5为青蒿琥酯治疗28d时，ANA荧光强度未减弱×200。
图6为正常小鼠肾脏切片显示肾髓质中肾小管基底膜有少量银染×5。
图7为ip LPS 14d时模型小鼠肝脏血管基底膜增厚×50。
图8为绿原酸大剂量组给药28d时小鼠肾小球体积较正常增大，细胞数目增多，肾 小球和肾小管基底膜较正常小鼠增厚程度减轻×50。
图9为青蒿琥酯治疗组给药28d时肾小球基底膜明显较正常小鼠增厚×50。
图10为强的松治疗组给药28d时肾小球基底膜明显较正常小鼠增厚×50。
图11为模型小鼠24h时肾小球囊壁上皮细胞严重增生，出现单核细胞，线粒体大量增加×3000。
图12为模型小鼠28d时肾小球囊壁上皮细胞增生程度降低，有单核细胞，线粒体数目多×6600。
图13为绿原酸大剂量组给药21d肾小球囊壁上皮细胞增生程度有所缓解×4000。
图14为青蒿琥酯治疗组给药21d时肾小球囊壁上皮细胞增生程度稍有缓解，但缓解程度较青藤胶囊治疗组小，并伴有血栓×3000。
图15为强的松治疗组给药21d时肾小球囊壁上皮细胞增生程度较模型组也有所缓解，但缓解程度较青藤胶囊治疗组小，线粒体数目增多×12500。
具体实施方式
实例1：绿原酸治疗红斑狼疮的体内药效学试验研究
1、材料和方法
1.1动物
BALB/c小鼠，体重17.0-21.0g，5-7周龄，雌雄各半，由四川大学实验动物中心提供。
药物
绿原酸，由四川九章生物科技有限公司所制备，含量99.2％。批号：131101。实验时用生理盐水配制成高、中、低三个浓度的溶液备用。强的松，浙江仙琚制药股份有限公司生产，5mg/片，批号：110502；大肠杆菌脂多糖，sigma公司生产，Lot58kp8725，使用前用生理盐水分别配制成0.25g/L备用。HEP-2细胞片Scimedx公司生产。荧光素标记的山羊抗小鼠血清(FITC Goat Anti-Mouse IgG)，Vector公司。ENA多肽免疫印迹膜条，深圳恒佳公司生产。生物素结合山羊抗小鼠血清(biotin goat anti-mouse IgG)，Vector公司。辣根过氧化物酶-卵白素结合物(HRP-avidin)，Vector公司。
仪器
紫外灯：上海特种灯具厂生产，30W，最大能量光谱为312nm，能量为2.0Mj/cm2，距离为30cm，中波紫外线(UVB)部分占其辐射能量的60％以上；奥林巴斯BH-2万能显微镜，日本OLYMPUS公司生产；菲利普M800透射电镜，荷兰PHILIPS公司生产。
方法
小鼠背部剃毛，紫外线急性照射2h/d*3d，距离30cm，照射后一次性ip LPS 2.5mg/kg，24h造模成功；正常对照组小鼠ip生理盐水。模型小鼠ip LPS 24h后开始灌胃给药。绿原酸实验组分别以高、中、低剂量组给药，剂量分别为10mg/kg*d、20mg/kg*d、40mg/kg*d灌胃。阳性对照药A组青蒿琥酯(Artesunate)2mg/kg*d灌胃，B组强的松(Prednisone)1mg/kg*d灌胃。模型和正常对照组小鼠均0.4ml/只生理盐水灌胃。连续给药15d，各组动物分别于14d、21d、28d时去眼球采血，37℃水浴1h后离心，2000r/min，5min，抽取血清备用。21d时采过血的动物脱臼致死，立即取肝脏和肾脏，分别入电镜固定液和光镜固定液。
血清学检测
将小鼠血清用深圳恒佳专用缓冲液以1：100稀释，然后将稀释好的血清200uL分别加入相应编号的反应槽中，37℃温箱孵育30min，缓冲液洗涤4次，每次2min；加入200uL1:100稀释的biotin goat anti-mouse IgG，37℃温箱孵育30min，缓冲液洗涤4次，每次2min；再加入1:100稀释的HRP-avidin 200uL，37℃温箱孵育30min，洗涤同上。加入反应液A和B至阳性条带出现，缓冲液洗涤。与同一试剂盒内的ENA多肽抗体谱标准带比对，如阳性条带出现于28/29,13.5ku多肽；15,16.5,38ku多肽；52ku多肽及47/48,45ku多肽上，可判定为抗Sm，抗Rib，抗ssA和抗ssB抗体阳性。将血清用PBS缓冲液以1:40稀释，然后将稀释好的血清20-30uL分别滴入HEp-2细胞片小窝中，每组一张细胞片。在滴加血清的过程中应注意不能触及细胞片的表明。将加好血清的细胞片置于湿盒中，室温(25℃)下放置30min。30min后将细胞片取出，用PBS小心洗出血清，然后用PBS小心洗涤2次，每次5min；洗好后用Scimedx公司专用的吸水纸吸去多余的PBS(这一过程应注意避免细胞底物变干而影响结果)。滴入稀释好的(PBS1:100稀释)FITC goat anti-mouse IgG 20-30uL，湿盒内室温下30min。PBS洗涤，步骤同上。吸水纸吸去多余PBS，加1-2滴荧光封片剂于细胞片上，盖玻片封片，Olympus BH-2万能显微镜观察，照相。
三重染色标本的制备
21d时用于三重染色，光镜观察的标本用10％福尔马林固定，然后脱水、透明、浸蜡和包埋，进行切片。切片厚3um，染色开始时经常规脱蜡至水，0.5％高碘酸氧化后，浸入六胺银工作液中约2h(恒温58℃)，蒸馏水洗，苏木素染1min，水洗后丽春红染液滴染5min，70％乙醇分化，梯度酒精脱水，二甲苯透明，中性树胶封固。
电镜标本的制备
21d时电镜观察的标本用2％多聚甲醛+0.25％戊二醛混合固定液固定4h，TBS缓冲液漂洗，1％锇酸后固定1h，缓冲液漂洗，丙酮脱水，醋酸铀染色2h，Epon812浸透并包埋，LEICA 超薄切片机切片，铅液染色，缓冲液冲洗，标本变干后即可进行透射电镜观察。
结果
2.1动物死亡情况
60只动物ip LPS 24h时共有6只动物死亡，雌雄各3只，此后至给药第28d各给药组及各对照组无动物死亡。
血清学检测结果
治疗14d、21d、28d时所采动物血清ENA多肽抗体检测及ANA检测结果见表1、2、3，图1-5；各组结果均无性别差异。
表1  14d时ENA多肽抗体和ANA检测结果
Figure PCTCN2016072124-appb-000001
(+)：阳性(-)：阴性(++++)：荧光强(+++)：荧光较强。
表2  21d时ENA多肽抗体和ANA检测结果
Figure PCTCN2016072124-appb-000002
(+)：阳性(-)：阴性(++++)：荧光强(+++)：荧光较强(++)：荧光较弱。
表3  28d时ENA多肽抗体和ANA检测结果
Figure PCTCN2016072124-appb-000003
(+)：阳性(-)：阴性(++++)：荧光强(+++)：荧光较强(++)：荧光较弱。
三重染色标本观察结果
病理切片显示21d时①正常对照小鼠的肾脏其皮质结构中的肾小球基底膜有少量银染，肾小管基底膜也同样有少量银染，髓质结构中的肾小管有少量银染；肝脏血管基底膜未见增厚。(图6)②模型小鼠在第21d时银染结果可见肾小球体积增大，细胞数目增多，肾小球旁有单核细胞，肾小球及间质慢性炎细胞浸润，肾小球和肾小管基底膜明显较正常小鼠增厚；肝脏血管基底膜较正常小鼠增厚。(图7)③绿原酸给药21d时小鼠肾脏组织结构依然可见肾小球体积较正常增大，细胞数目增多，肾小球及间质慢性炎细胞浸润，肾小球和肾小管基底膜较正常小鼠增厚；但情况较模型组好，尤以高剂量组为甚。(图8)④青蒿琥酯治疗组给药21d时肾小球和肾小管基底膜明显较正常小鼠增厚；(图9)⑤强的松给药组治疗21d时肾小球和肾小管基底膜也明显较正常小鼠增厚。(图10)。
2.4电镜标本观察结果
电镜观察结果显示①正常小鼠的肾小球囊壁上皮细胞无增生，线粒体数目正常；(图11)②模型小鼠21d时肾小球囊壁上皮细胞严重增生，出现单核细胞，线粒体大量增加。(图12)③绿原酸给药21d时肾小球囊壁上皮细胞增生程度较模型组有所缓解，以大剂量组缓解程度最明显；(图13)④青蒿琥酯治疗组给药21d时肾小球囊壁上皮细胞增生程度较模型组有所缓解，但缓解程度较绿原酸治疗组小，并伴有血栓；(图14)⑤强的松给药组治疗21d时肾小球囊壁上皮细胞增生程度较模型组有所缓解，但缓解程度较绿原酸治疗组小，线粒体数目增多的同时伴有细胞器的丢失；(图15)。
3.结论
使用此模型所进行的绿原酸药效学研究表明，①从血清学检测结果可看出，造模成功。②三重染色标本观察结果表明：模型小鼠在第24h时银染结果即可见肾小球体积增大，细胞数目增多，肾小球及间质慢性炎细胞浸润，肾小球和肾小管基底膜明显较正常小鼠增厚；肝脏血管基底膜较正常小鼠增厚。第2d、3d、7d、14d、21d时同样显示肾小球体积增大，细胞数目增多，肾小球及间质慢性炎细胞浸润，并伴有肾小球和肾小管基底膜明显较正常小鼠增厚，肝脏血管基底膜增厚。绿原酸组28d时肾小球体积同样增大，细胞数目增多，但肾小球和肾小管基底膜较正常小鼠增厚的程度降低，银染色变浅，肝脏血管基底膜染色也有所恢复。③电镜标本观察结果也表明，正常小鼠的肾脏细胞肾小球囊壁上皮细胞无增生，线粒体数目正常；模型小鼠24h时肾脏细胞肾小球囊壁上皮细胞严重增生，出现单核细胞，线粒体大量增加。绿原酸给药21d时肾小球囊壁上皮细胞增生程度较模型组有所缓解，以大剂量组缓解程度最明显。
上述实验结果表明，绿原酸对红斑狼疮有良好的治疗作用。其作用机理可能在于改善红斑狼疮小鼠的免疫功能。

Claims (7)

  1. 一种绿原酸在制备治疗红斑狼疮的药物中的应用。
  2. 如权利要求1所述的应用，其中所述药物为改善免疫机能。
  3. 一种用于治疗红斑狼疮的制剂，所述制剂包含绿原酸和药物上可接受的赋形剂或辅料。
  4. 如权利要求3所述的制剂，所述的制剂为口服制剂、注射制剂或外用透皮给药制剂。
  5. 如权利要求3或4所述的制剂，所述制剂的每制剂单位含绿原酸1-1000mg，临床使用剂量为1-100mg/kg。
  6. 一种用于治疗红斑狼疮的联合用药物，所述联合用药物包括绿原酸和治疗红斑狼疮的药物。
  7. 如权利要求6所述的联合用药物，所述治疗红斑狼疮的药物选自羟基氯喹、甲氨喋呤、硫唑嘌呤、氯苯酚嗪、胸腺素、左旋咪唑、强的松中的一种或几种。
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