WO2014121492A1 - Sp肽或其衍生物在制备降低il-13水平的药物中的应用 - Google Patents

Sp肽或其衍生物在制备降低il-13水平的药物中的应用 Download PDF

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WO2014121492A1
WO2014121492A1 PCT/CN2013/071525 CN2013071525W WO2014121492A1 WO 2014121492 A1 WO2014121492 A1 WO 2014121492A1 CN 2013071525 W CN2013071525 W CN 2013071525W WO 2014121492 A1 WO2014121492 A1 WO 2014121492A1
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Prior art keywords
peptide
derivative
group
patient
gly
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PCT/CN2013/071525
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English (en)
French (fr)
Inventor
程云
虞瑞鹤
赵万洲
李红
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Cheng Yun
Yu Ruihe
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Priority to PCT/CN2013/071525 priority Critical patent/WO2014121492A1/zh
Priority to CN201380070423.4A priority patent/CN104918629B/zh
Publication of WO2014121492A1 publication Critical patent/WO2014121492A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides 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
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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

Definitions

  • the present invention relates to the field of medical technology, in particular, the present invention relates to the use of SP peptide or a derivative thereof for preparing a drug for lowering IL-13 level, in particular to the preparation of a patient with a liver disease or a patient with pulmonary diseases. Or the use of drugs with IL-13 levels in patients with kidney disease. Background technique
  • the present inventors have disclosed in Chinese patents CN1194986C and CN1216075C a 7P peptide or a derivative thereof (abbreviated as 7P peptide or a derivative thereof, referred to as SP peptide or a derivative thereof in the present invention), which is initially based on hepatitis C.
  • the 7P peptide or its derivative has the function of preventing and/or treating hepatitis C. .
  • the inventors disclosed in the patent application CN101822816A the use of the 7P peptide or a derivative thereof for preventing and treating pneumonia, and specifically describing the treatment of pneumonia by reducing the TNF- ⁇ level of the 7P peptide and its derivative. . Therefore, it has been reported that 7P peptide has the effect of lowering the level of TNF-a in patients with pneumonia and increasing the level of cytokine IL-4 in patients with hepatitis C.
  • IL-13 is known to be a cytokine that induces asthma in allergic asthma, which is highly expressed in lung tissue and induces inflammation, mucus hypersecretion, epithelial fibrosis, eosinophil activation/chemokine The production and airway hyperresponsiveness, and the relationship between the above 7P peptide or its derivative and IL-13 level has not been reported yet. Summary of the invention
  • the present invention provides the use of SP peptides or derivatives thereof for the preparation of a medicament for lowering IL-13 levels, broadening the potential pharmaceutical field of the SP peptide or its derivatives.
  • the present invention also provides a method for reducing the level of IL-13 in a patient by administering to a patient A therapeutically effective amount of a drug of the peptide or a derivative thereof is effective for improving a disease state of a patient.
  • a peptide (SP peptide) of the formula I or a derivative thereof according to the invention for the preparation of a medicament for reducing IL-13 levels:
  • Xaal is missing, Ala, Gly, Val, Leu or lie,
  • Xaa2 is Thr or Ser
  • Xaa3 is Tyr, Phe or Trp
  • Xaa4 is missing, Ala, Gly, Val, Leu, lie or Pro;
  • the derivative includes a pharmaceutically acceptable salt or ester of the peptide.
  • the drug may be a drug for treating liver disease, lung disease or kidney disease, for example, a liver disease patient who is clinically diagnosed with liver fibrosis disease, a lung disease patient whose clinical diagnosis is pulmonary fibrosis, or a clinical diagnosis of kidney disease.
  • Therapeutic medication for patients with interstitial fibrosis in kidney disease for example, a liver disease patient who is clinically diagnosed with liver fibrosis disease, a lung disease patient whose clinical diagnosis is pulmonary fibrosis, or a clinical diagnosis of kidney disease.
  • the basic structure and composition of the peptide represented by the above formula I or a derivative thereof are the 7P peptide or a derivative thereof obtained by the inventors in the previous studies, and are also referred to as SP peptide or a derivative thereof in the present invention.
  • the SP peptide or a derivative thereof can be synthesized by a solid phase synthesis method or a liquid phase synthesis method which has been known or known to those skilled in the art, and can also be obtained by genetic engineering fusion expression and purification.
  • ester refers to an ester that is suitable for contact with the tissues of a human or animal without excessive toxicity, irritation or allergic reaction, and the like.
  • esterification can reduce the hydrolysis of peptides by proteases in the body.
  • Modification of the terminal amino, carboxyl or side chain groups of the peptides of the invention can form pharmaceutically acceptable esters. Modifications to amino acid side chain groups include, but are not limited to, threonine, esterification of a serine side chain hydroxyl group with a carboxylic acid.
  • the amino acid terminal group is protected with a protecting group known to those skilled in the art of protein chemistry, such as acetyl, trifluoroacetyl, Fmoc (9-fluorenyl-fluorenyloxycarbonyl), Boc (tert-butoxycarbonyl) , Alloc (allyloxycarbonyl), C 1-3 alkyl, C 12 aralkyl, and the like.
  • a protecting group known to those skilled in the art of protein chemistry, such as acetyl, trifluoroacetyl, Fmoc (9-fluorenyl-fluorenyloxycarbonyl), Boc (tert-butoxycarbonyl) , Alloc (allyloxycarbonyl), C 1-3 alkyl, C 12 aralkyl, and the like.
  • the inventors have found that the peptides of the invention are not modified to be sufficient for the treatment or prevention of asthma under physiological conditions, and thus the polypeptide of formula I may be a peptide without any modification, for example,
  • the chemical group at the end is still the ⁇ -amino group (- ⁇ 2 ⁇ ) on the first amino acid, and the chemical group at the C-terminus is 1 ⁇ 2 (-COOH) of the C-terminal amino acid.
  • pharmaceutically acceptable salt refers to a salt suitable for contact with the tissues of a human or animal without excessive toxicity, irritation or allergies, etc., i.e., a pharmaceutically acceptable salt form well known in the art. Such salts may be prepared during the final isolation and purification of the polypeptides of the invention, or may be prepared separately by reacting the peptides with a suitable organic or inorganic acid or base.
  • Representative acid addition salts include, but are not limited to, acetate, dihexanoate, alginate, citrate, aspartate, benzoate, benzoate, hydrogen sulfate, butyrate , camphorate, camphorate, glycerol phosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate Acid salt, lactate, maleate, sulfonate, nicotinate, 2-naphthylate, oxalate, 3-phenylpropionate, propionate, succinate, tartrate , phosphate, glutamate, bicarbonate, p-toluene hydrochloride and undecanoate.
  • Preferred acids which can be used to form pharmaceutically acceptable salts are hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, oxalic acid, maleic acid, succinic acid and citric acid.
  • the cations in the pharmaceutically acceptable base addition salts include, but are not limited to, alkali metal or alkaline earth metal ions such as lithium, sodium, potassium, calcium and magnesium, and quaternary ammonium cations such as tetradecylammonium, tetraethylammonium, and the like.
  • Preferred base addition salts include phosphates, trishydroxyalkylaminos (tris) and acetates. These salts are generally capable of increasing the solubility of the polypeptide, and the salt formed does not substantially alter the activity of the polypeptide.
  • the drug for lowering the IL-13 level may be a pharmaceutical preparation in which the peptide (SP peptide) is directly used, or a pharmaceutically acceptable salt or a pharmaceutically acceptable ester form of the SP peptide.
  • peptide or a derivative thereof is a peptide of the formula II or a pharmaceutically acceptable salt or ester thereof:
  • the peptide represented by the formula ⁇ can also be abbreviated as GQTYTSG.
  • the peptide or a derivative thereof may be in a suitable preparation form depending on the subject to be administered and the route of administration, for example: injection, (injection) lyophilized powder, spray, oral solution, oral Suspensions, tablets, capsules, enteric-coated tablets, pills, powders, granules, sustained release agents (a dosage form that can control the slow release of the active ingredient of the agent) or a controlled release agent (a dosage form that can control the active ingredient of the drug)
  • a formulation which may comprise a conventional pharmaceutically acceptable carrier or excipient
  • said "pharmaceutically acceptable carrier” means a non-toxic solid, semi-solid or liquid filler, diluent, adjuvant , wrapping materials or other preparation excipients, such as: physiological saline, isotonic glucose solution, buffered saline, glycerin, ethanol and a combination of the above solutions.
  • a drug made of the peptide or a derivative thereof by injection that is, it is preferred to use an injection or a lyophilized powder injection, and to dissolve it with physiological saline as a carrier.
  • the present inventors have found that an increase in the level of IL-13 is also one of the causes of fibrotic symptoms in organs such as the liver, kidneys, and lungs. Therefore, the above-mentioned drugs containing a therapeutically effective amount of SP peptide or a derivative thereof are used.
  • the peptide or its derivative as an active ingredient reduces the level of IL-13 in patients, and can be used in clinical treatment to relieve symptoms and treat symptoms in patients with liver diseases, lung diseases or kidney diseases.
  • the present invention provides a method for reducing IL-13 levels in a patient comprising administering to a patient a therapeutically effective amount of a drug of the above formula I or a derivative thereof, said derivative
  • the pharmaceutically acceptable salt or ester of the peptide is included.
  • the present invention further provides a method of treating liver fibrosis, pulmonary fibrosis or renal interstitial fibrosis by reducing IL-13 levels in a patient.
  • the peptide or the derivative thereof as an active ingredient in the therapeutic drug may be the peptide represented by the above formula II or a pharmaceutically acceptable salt or ester thereof.
  • a drug containing a therapeutically effective amount of 200 to 3000 ⁇ s of the peptide or a derivative thereof, and further a drug of 300 to 2000 ⁇ of the peptide or a derivative thereof may be administered to a patient.
  • the therapeutically effective amount described herein is the amount of the active ingredient described in the general adult body weight, single administration.
  • administering the therapeutically effective amount of the said injection A drug of a peptide or a derivative thereof. More preferably, the patient is administered in a dose of a unit preparation which is a preparation which satisfies the active ingredient required for one administration, and a common unit preparation such as one unit (tablet) tablet, one unit (needle) injection or powder Injection, etc.
  • a unit preparation which is a preparation which satisfies the active ingredient required for one administration
  • a common unit preparation such as one unit (tablet) tablet, one unit (needle) injection or powder Injection, etc.
  • the amount of drug required for a single administration of a patient can be conveniently obtained by calculating the product of the patient's body weight and the unit weight dose required for the patient to take the drug once.
  • the adult body weight is 50-90 kg
  • the dose can be determined initially by an equivalent dose conversion relationship between the experimental animal and the human body weight dose.
  • it can be based on the guidance provided by the FDA, SFDA and other drug regulatory agencies, or refer to (Huang Jihan et al., "Equivalent dose conversion between animals and animals and humans in pharmacological tests", “Chinese Clinical Pharmacology and Therapeutics” , 2004 Sep ; 9(9) : 1069 - 1072 ) to determine.
  • the doses of human and rat according to the body surface area conversion coefficient of human and rat of 0.018 can be used.
  • the SP peptide or a derivative thereof in the unit preparation has a better therapeutic effect when administered to a rat at a dose of 20-30 (g/kg rat), and is 40-20 ( ⁇ g/kg large).
  • the dose of the mouse for example, 18 ( ⁇ g/kg, 9 ( ⁇ g/kg, and 45 g/kg of the rat dose is more effective when administered to the rat.
  • the pharmaceutical manufacturer can obtain the unit preparation for human according to the above conversion method).
  • the active ingredient content is used in the pharmaceutical process thereof.
  • the The unit preparation contains the peptide or a derivative thereof in an amount of from 200 to 3000 ⁇ , more preferably the peptide or a derivative thereof in a dose of from 300 to 2000 ⁇ .
  • SP peptide or its derivatives can effectively reduce IL-13 levels in patients with liver disease, lung disease or kidney disease, especially in patients with liver fibrosis, pulmonary fibrosis, or renal interstitial fibrosis. Levels, clinical manifestations are effective in improving the symptoms of these patients.
  • liver fibrosis may be a BSA-immunized adjuvant-induced liver
  • the pulmonary fibrosis may be bleomycin-induced pulmonary fibrosis, which may be renal interstitial fibrosis induced by unilateral ureteral obstruction), as can be seen from the data, the application site
  • the group of peptides or derivatives thereof showed significantly reduced IL-13 levels, as well as significantly improved tissue lesions, compared to the model group.
  • Figure 1 shows the effect of SP peptide on IL-13 levels in the liver of each group of rats.
  • Figure 2 shows the comprehensive score of liver tissue fibrosis in each group of rats.
  • Figure 3 shows the effect of SP peptide on IL-13 levels in the kidney serum of each group of rats.
  • Figure 4 shows a comprehensive score of the degree of renal interstitial fibrosis in each group of rats.
  • Figure 5 shows the effect of SP peptide on IL-13 levels in the lung serum of each group of rats.
  • Figure 6 shows the effect of SP peptide on hydroxyproline levels in lung tissue homogenates of each group of rats.
  • Figure 7 shows a comprehensive score of the degree of pulmonary fibrosis in each group of rats.
  • Example 1 SP peptide reduces IL-13 levels in liver disease animals and improves liver fibrosis
  • the description of Example 1 was dissolved in physiological saline at the time of use.
  • Positive control drug hydrocortisone injection, commercially available, nominal daily dose 5mg / kg-d.
  • BSA immunoadjuvant was purchased from Sigma.
  • Rat IL-13 ELISA Kit purchased from Shanghai Lanji Biotechnology Co., Ltd.
  • Microplate reader American Thermoelectric Reb MK3. 1.3 grouping and drug dosage
  • mice were randomly divided into 6 groups, and 10 rats in each group were:
  • model group (CC1 4 peanut oil and administered using saline formulated immunoadjuvant BSA);
  • SP peptide high dose group 5) SP peptide medium dose group, 6) SP peptide low dose group (administer CC1 4 peanut oil and BSA immunoadjuvant prepared with physiological saline, and the dose is 18 ( ⁇ g/kg- respectively) d, 9 ( ⁇ g/kg d, 45 g/kg d of SP peptide, configured with physiological saline to a desired concentration of SP peptide solution).
  • the BSA immunoadjuvant was formulated to a concentration of 0.8% by weight with physiological saline, and is now ready for use. At the same time, 40% (v/v) CCl 4 peanut oil was placed.
  • Each group of rats was administered as follows, and during the administration, rats of each group were normally fed daily.
  • rats in the other groups except the blank control group were intraperitoneally injected with 40% CC1 4 peanut oil solution 0.3 ml/100 g rat body weight, and subcutaneously injected with 0.8% BSA immunoadjuvant 0.3 ml/100 g large. Rat weight.
  • the blank control group was intraperitoneally injected with an equal volume of physiological saline for 5 weeks. Then, one group of animals was randomly selected, and the body weight was weighed by intraperitoneal injection of 1% sodium pentobarbital. The liver was taken and the morphology and pathological changes were observed. The rats in the other groups showed the same except the blank control group. Significant liver fibrosis indicates successful modeling.
  • the rats in the blank control group and the model group were injected with normal saline (0.1 ml normal saline per 100 g body weight), and administered once every other day; subcutaneous injection of SP peptide high, medium and low dose groups The corresponding dose of SP peptide, rats were administered 0.1 ml of SP peptide solution per 100 g body weight, and administered once every other day; rats in the positive drug group were intraperitoneally injected with hydrocortisone, and rats were administered 0.1 ml per 100 g body weight. Once a day. Each group of animals was co-administered 15 times in the manner described above.
  • the blank control group, the model group, the SP peptide high, medium and low dose groups, and the positive drug group rats were intraperitoneally injected with 10% chloral hydrate for anesthesia.
  • Blood was collected from the blood, and the IL-13 content in the serum was measured according to the operation requirements of the kit.
  • the IL-13 standard was diluted with the dilution in the kit, and then operated according to the kit instructions. Calculate the standard curve, read the value of the sample measured from the standard curve, and multiply by the dilution factor to obtain the level of IL-13 in the sample serum.
  • the rat liver was washed with ice physiological saline, blotted with filter paper, fixed in 10% neutral formalin solution for 24 hours, embedded in paraffin, 5 ⁇ slice, HE stained, optical Histopathological examination under the microscope includes: checking whether the structure of the hepatocytes is intact, whether the hepatic lobules are normal, and whether the connections between the hepatocytes are tight, and whether the arrangement of hepatic cells is regular.
  • the scores were 1 point, 2 points, 3 points, 4 points, no obvious lesions were 0 points, extremely mild lesions were 0.5 points, all scores were accumulated, and each group was calculated.
  • the average score of animals ( ⁇ SD) the higher the score, the more severe the lesion.
  • the rank sum test of the two sample comparisons of the lesion score results was compared with the model group.
  • Figure 1 shows the effect of SP peptide on serum IL-13 levels in each group of rats, expressed as a mean value, wherein the amount administered is as described in 1.3 above. It can be seen from Fig. 1 that the level of IL-13 in the serum of the rat model group of liver injury induced by BSA immunoadjuvant is significantly higher than that of the blank control group. According to the pathological examination below, IL is known.
  • the level of -13 increased the liver fibrosis lesions in rats; while the serum levels of IL-13 in the high, medium and low dose groups of SP peptide were significantly lower than those in the model group (* ⁇ 0.05 ** ⁇ 0.01) And the SP peptide high-dose group had better effect than the positive drug group, and the SP peptide medium- and low-dose groups had the same effect as the positive drug group.
  • the hepatocytes were structurally intact, the hepatic lobules were normal, the hepatic cell cords were arranged regularly, and the hepatocytes were closely connected.
  • the hepatocytes of the rats were severely damaged, the cytoplasm was empty, the nucleus was condensed or dissolved, the structure of the hepatic lobule was destroyed, and the pseudolobules were formed.
  • the number of fibroblasts increased from the portal area to the central hepatic vein, and a large number of hepatocytes were visible. Vacuolar structure, as well as cell turbidity, watery changes, etc. Dead performance.
  • hepatocyte destruction was alleviated, and the number of fibroblasts was reduced from the portal area to the central hepatic vein.
  • the hepatocyte destruction in the high, medium and low dose groups of SP peptide was significantly reduced, and the structural damage of the hepatic lobule was significantly reduced.
  • the scores of liver tissue lesions of each group were obtained by rank sum test.
  • the results of comprehensive scoring of pathological examination indexes were obtained as shown in Fig. 2. Histopathological examination showed that SP peptide was administered. Rats in each group showed significant reduction in hepatic fibrotic lesions, with statistically significant differences compared with the model group (* P ⁇ 0.05 or ** ⁇ 0.01).
  • Example 2 SP peptide reduces IL-13 levels in renal nephropathy and improves renal interstitial fibrosis
  • mice were randomly divided into 6 groups, and 10 rats in each group were:
  • SP peptide high-dose group 5) SP peptide middle dose group, 6) SP peptide low-dose group (surgery unilateral ureteral obstruction, the dose was 18 ( ⁇ g/kg.d, 9 ( ⁇ g) /kg.d, 45 g/kg-d of SP peptide, configured with physiological saline to a desired concentration of SP peptide solution).
  • Surgical treatment of unilateral ureteral obstruction in rats included: Inhalation of rats with diethyl ether, incision in the right mid-abdomen, dissection of the right ureter, in a free state, respectively in the renal pelvis and one third of the ureter The wires were ligated separately, and the ureter at the ligation site was cut, and the skin was sutured layer by layer. Each group of rats was administered as follows, and during the administration, rats of each group were normally fed daily.
  • rats in the other groups except the blank control group (model group, SP peptide high, medium, low dose group, positive drug group) were treated with unilateral ureteral obstruction. Then regular feeding
  • the rats in the blank control group and the model group were injected with normal saline (0.1 ml normal saline per 100 g body weight of rats), administered once every other day; subcutaneous injection of SP peptide high, medium and low dose groups
  • the corresponding dose of SP peptide, rats were administered 0.1 ml of SP peptide solution per 100 g body weight, once every other day; the rats in the positive drug group were intraperitoneally injected with hydrocortisone, and the rats were given 0.1 ml positive per 100 g body weight.
  • Drug once every other day.
  • Each group of animals was co-administered 15 times in the manner described above.
  • the blank control group, the model group, the SP peptide high, medium and low dose groups, and the positive drug group rats were intraperitoneally injected with 10% chloral hydrate for anesthesia, blood collection.
  • Serum, the serum IL-13 content is determined according to the operation requirements of the kit, and the IL-13 standard is diluted with the dilution in the kit, and then operated according to the kit instructions. Calculate the standard curve, read the value of the sample from the standard curve, and multiply by the dilution factor to obtain the level of IL-13 in the sample serum.
  • the rat kidney was washed with ice physiological saline, the filter paper was blotted, placed in a 10% neutral formalin solution for 24 hours, routine paraffin embedding, 5 ⁇ section, HE staining, optics Renal histopathological examination under the microscope, including: Checking the normal structure of the renal pelvis and renal tubules, whether the volume of the renal corpuscle changes.
  • the scores were 1 point, 2 points, 3 points, 4 points, no obvious lesions were 0 points, extremely mild lesions were 0.5 points, all scores were accumulated, and each group was calculated.
  • the average score of animals ( ⁇ SD) the higher the score, the more severe the lesion.
  • the rank sum test of the two sample comparisons of the lesion score results was compared with the model group.
  • Fig. 3 shows the effect of SP peptide on the serum IL-13 level in each group of rats, expressed as an average value, wherein the administration amount is as described in 1.3 above.
  • the level of IL-13 in the serum of the rat model of right renal interstitial fibrosis caused by unilateral ureteral obstruction was significantly higher than that of the blank control group, according to the histopathological examination below. Elevated levels of IL-13 induce renal right interstitial fibrosis in rats.
  • the structure of renal pelvis and renal tubules was normal, and the volume of renal corpuscles did not change significantly.
  • the transitional epithelium of the renal pelvis on the inner surface of the kidney was dilated, showing a very thin and flat, single row arrangement, and the smooth muscle and connective tissue on the inner side were significantly increased; the renal corpuscle was smaller in size, and it was oblate, vascular The number is significantly reduced and distributed in a single layer in the iliac wall; the normal structure of the renal tubules is rare, and most of them are replaced by a large number of interstitial components.
  • the smooth muscle and connective tissue on the medial side of the renal pelvis were reduced compared to the model group.
  • the renal tissues of the SP peptide high, medium and low dose groups showed mild fibrosis and tubular thickening compared with the model group, and there was a certain increase in infiltrating cells.
  • Rat IL-13 ELISA Kit purchased from Shanghai Lanji Biotechnology Co., Ltd.
  • Microplate reader American Thermoelectric Reb MK3.
  • mice were randomly divided into 6 groups, and 10 rats in each group were:
  • model group (administering physiological saline containing bleomycin);
  • SP peptide high dose group 5) SP peptide middle dose group, 6) SP peptide low dose group (administer saline containing bleomycin, and the dose is 18 ( ⁇ g/kg.d, 9 ( ⁇ , respectively) g/kg.d, 45 g/kg-d of SP peptide, configured with physiological saline to a desired concentration of SP peptide solution).
  • the dose of 5 mg of bleomycin was administered at a body weight of 1 kg of animal, and bleomycin was used in physiological saline.
  • Each group of rats was administered as follows, and during the administration, rats of each group were normally fed daily.
  • the rats in the other groups except the blank control group were fasted for 12 hours, and the rats were treated with 10% chlorinated chlorine.
  • the aldehyde was intraperitoneally injected (0.35ml/100g), it was placed on the fixed table and exposed to the trachea.
  • Each rat was injected with bleomycin physiological saline solution into the trachea with a lmL syringe. Rats were given 0.25 per 100 g of body weight. mL.
  • the blank control group was intraperitoneally injected with an equal volume of physiological saline. During the experiment, the rats in each group were fed normally.
  • rats in the blank control group and the model group were injected with normal saline (0.1 ml normal saline per 100 g body weight of rats), administered once every other day; high, medium and low doses of SP peptide
  • the group was injected subcutaneously with the corresponding dose of SP peptide.
  • Rats were administered 0.1 ml of SP peptide solution per 100 g body weight once every other day.
  • rats were intraperitoneally injected with hydrocortisone, and rats were administered 0.1 mg per 100 g body weight. Ml, administered once every other day.
  • Each group of animals was co-administered 15 times in the manner described above.
  • the blank control group, the model group, the SP peptide high, medium and low dose groups, and the positive drug group rats were intraperitoneally injected with 10% chloral hydrate for anesthesia, blood collection.
  • Serum, the serum IL-13 content is determined according to the operation requirements of the kit, and the IL-13 standard is diluted with the dilution in the kit, and then operated according to the kit instructions. Calculate the standard curve, read the value of the sample from the standard curve, and multiply by the dilution factor to obtain the level of IL-13 in the sample serum.
  • the whole lung is opened by opening the chest cavity, the surface of the tissue is washed with ice physiological saline, the filter paper is blotted, and the right lung tissue is fixed in a 10% neutral formalin solution for 24 hours, embedded in paraffin, 5 ⁇ sectioning, HE staining, pathological histological examination of lung tissue under light microscope, including: Checking whether the structure of the lung is clear, whether there is edema, no inflammation and fibrosis, and whether the alveolar septum is thickened.
  • Preparation of lung tissue homogenate Take 10% homogenate of left lung tissue, centrifuge at 3000 rpm for 10 minutes, take the supernatant, and detect the level of hydroxyproline (HyP) in the lung tissue according to the operation requirements of the kit, according to the kit. Operation requires detection of hydroxyproline (HyP) levels in lung tissue.
  • HyP hydroxyproline
  • the scores were 1 point, 2 points, 3 points, 4 points, no obvious lesions were 0 points, extremely mild lesions were 0.5 points, all scores were accumulated, and each group was calculated.
  • the mean ( ⁇ SD) of each animal the higher the score, the more severe the lesion.
  • the rank sum test of the two sample comparisons of the lesion score results was compared with the model group.
  • Figure 5 shows the effect of SP peptide on serum IL-13 levels in each group of mice, expressed as a mean value, wherein the amount administered is as described in 1.3 above.
  • serum IL-13 levels in the rat model group of pulmonary fibrosis induced by bleomycin were significantly higher than those in the blank control group.
  • IL-13 levels were elevated. Pulmonary fibrosis lesions were induced in rats.
  • the SP peptide is high, The serum levels of IL-13 in the middle and low dose groups were significantly lower than those in the model group (* P ⁇ 0.05 P ⁇ 0.01 ), indicating that the SP peptide can be treated by reducing IL-13 for pulmonary fibrosis in rats.
  • the high-dose SP peptide group had a better effect than the positive drug group, and the SP peptide medium-dose group was equivalent to the positive drug group.
  • Figure 6 shows the effect of SP peptide on hydroxyproline levels in the lung tissue of each group of mice, expressed as a mean value, wherein the amount administered is as described in 1.3 above.
  • the hydroxyproline level in the lung tissue homogenate of the rat model group of pulmonary fibrosis induced by bleomycin was significantly higher than that of the blank control group, and the hydroxyproline level was pulmonary fibrosis lesion.
  • the hydroxyproline level in the lung tissue homogenate of the SP peptide high, medium and low dose groups was significantly lower than that of the model group ( * P ⁇ 0.05 ** ⁇ 0.01); and the SP peptide high dose group was positive.
  • the drug group had a better effect, and the SP peptide middle dose group was equivalent to the positive drug group.
  • the lungs of the blank control group had clear structure, no edema, no inflammation and fibrosis, and no alveolar septum thickening.
  • the degree of alveolitis and fibrosis in the positive drug group was lighter than that in the model group.
  • the degree of alveolar septal thickening was lighter than that in the model group, and the inflammatory cell infiltration was less, and the lesion range was small.
  • the alveolitis symptoms and fibrosis degree of the SP peptide high, medium and low dose groups were lighter than the model group, the lung structure was clear, the alveolar septum thickening was lighter, and the inflammatory cell infiltration range was small.

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Abstract

本发明提供了一种SP肽或其衍生物在制备降低患者IL-13水平的药物中的应用，所述肽或其衍生物为如式I所示的SP肽或其药学上可接受的盐或酯。本发明的研究证实，所述肽或其衍生物可有效降低患者IL-13水平。Xaal-Gln-Xaa2-Xaa3-Thr-Ser-Gly-Xaa4（式I），其中，Xaa1为缺失、Ala、Gly、Val、Leu或Ile，Xaa2为Thr或Ser，Xaa3为Tyr、Phe或Trp，而且Xaa4为缺失、Ala、Gly、Val、Leu，Ile或Pro。

Description

SP肽或其衍生物在制备降低 IL-13水平的药物中的应用 技术领域
本发明属于医药技术领域， 具体而言， 本发明涉及 SP肽或其衍生物在 制备降低 IL-13水平的药物中的应用， 尤其涉及该 SP肽或其衍生物在制备 降低肝病患者、 肺病患者或肾病患者 IL-13水平的药物中的应用。 背景技术
本发明人在中国专利 CN1194986C和 CN1216075C中披露了一种 7P肽 或其衍生物（简称 7P肽或其衍生物， 本发明中称为 SP肽或其衍生物）， 是 一种最初根据丙型肝炎病毒而设计的免疫原性肽， 并证明所述 7P肽或其衍 生物具有诱导细胞因子 r-IFN, IL-4JL-10水平升高和抗体产生的功能， r-IFN 是 Thl分泌的细胞因子， 是人体免疫系统抵抗病毒感染的主要细胞因子之 一， 针对 HCV (丙型肝炎病毒)的清除具有相当重要的意义， 因此该 7P肽或 其衍生物具有预防和 /或治疗丙型肝炎的作用。 进一步的， 本发明人在专利 申请 CN101822816A中公开了所述 7P肽或其衍生物在预防和治疗肺炎中的 用途，并且具体记载该 7P肽及其衍生物通过降低 TNF-α水平对肺炎的治疗。 所以， 已经有的研究报道可以知道， 7P肽具有降低肺炎患者的 TNF-a水平， 以及具有提高丙型肝炎患者细胞因子 IL-4水平的作用。
IL-13已知是一种在过敏性哮喘中诱发哮喘发生的细胞因子， 其可在肺 组织高表达， 并诱导炎症、 粘液高分泌、 上下皮纤维化、 嗜酸粒细胞活化 / 趋化因子的产生和气道高反应， 而上述 7P肽或其衍生物与 IL-13水平之间 关系目前尚没有报道。 发明内容
本发明提供了 SP肽或其衍生物在制备降低 IL-13水平的药物中的应用， 拓宽了该 SP肽或其衍生物的潜在药用领域。
本发明还提供了一种降低患者 IL- 13水平的方法， 通过向患者施用含有 治疗有效量的所述肽或其衍生物的药物， 能有效改善患者的疾病状况。 本发明提供的如式 I所示的肽（ SP肽）或其衍生物在制备降低 IL-13水平 的药物中的应用：
Xaal-Gln-Xaa2-Xaa3-Thr-Ser-Gly-Xaa4 (式 I )
其中，
Xaal为缺失、 Ala、 Gly、 Val、 Leu或 lie,
Xaa2为 Thr或 Ser,
Xaa3为 Tyr、 Phe或 Trp, 而且
Xaa4为缺失、 Ala、 Gly、 Val、 Leu, lie或 Pro;
所述衍生物包括所述肽在药学上可接受的盐或酯。
发明人的研究证明， 施用有效剂量的所述肽或其衍生物能够有效降低 患者 IL-13水平， 尤其能降低肝病患者、 肺病患者或肾病患者 IL-13水平。 所 以， 在本发明的方案中， 所述药物可以为肝病、 肺病或肾病治疗用药， 例 如， 针对临床诊断为肝纤维化疾病的肝病患者、 临床诊断为肺纤维化的肺 病患者或临床诊断为肾间质纤维化的肾病患者的治疗用药。
上述式 I所示的肽或其衍生物的基本结构和组成即为发明人在之前的研 究中所得到的 7P肽或其衍生物， 在本发明中也称为 SP肽或其衍生物。 根据 在先专利公开的记载， 所述 SP肽或其衍生物可通过本领域技术人员已经了 解或熟知的固相合成法或液相合成法合成， 也可通过基因工程融合表达并 提纯获得。
在本文中，所述"药学上可接受的酯"指适于与人或动物的组织接触而且 无过多的毒性、 刺激或变态反应等的酯。 通常， 酯化修饰后能降低机体中 的蛋白酶对肽的水解。 对本发明的肽的末端氨基、 羧基或侧链基团进行修 饰可以形成药学上可接受的酯。 对氨基酸侧链基团的修饰包括但不限于苏 氨酸、 丝氨酸侧链羟基与羧酸发生的酯化反应。 优选氨基酸末端基团用蛋 白质化学领域的技术人员已知的保护性基团保护起来， 如乙酰基、 三氟乙 酰基、 Fmoc ( 9-芴基 -曱氧羰基） 、 Boc (叔丁氧羰基） 、 Alloc (烯丙氧羰 基）、 C1-3烷基、 C 12芳烷基等。有关该 SP肽的药用酯在 PCT/CN2006/001176 中有详细说明， 因此将该在先公开申请文件中的相关内容并入本案作为参 考。 在本发明的具体实施方式中， 发明人发现， 本发明的肽不经修饰也足 以在生理条件下用于治疗或预防哮喘，因此式 I 多肽可以是不经任何修饰的 肽段，例如， Ν末端的化学基团仍旧为第一个氨基酸上的 α-氨基（-ΝΗΒ2Β ) , C末端的化学基团是 C末端氨基酸的 ½ ( -COOH ) 。
在本文中，所述"药学上可接受的盐"指适于与人或动物的组织接触而且 无过多的毒性、 刺激或变态反应等的盐， 即， 本领域熟知的药用盐形式。 这种盐可以在本发明多肽的最终分离和纯化的过程中制备， 也可以将所述 的肽与适当的有机或无机酸或碱反应单独制备。 代表性酸加成盐包括但不 限于乙酸盐、 二己酸盐、 藻酸盐、 柠檬酸盐、 天冬氨酸盐、 苯曱酸盐、 苯 横酸盐、 硫酸氢盐、 丁酸盐、 樟脑酸盐、 樟脑横酸盐、 甘油磷酸盐、 半硫 酸盐、 庚酸盐、 己酸盐、 富马酸盐、 盐酸盐、 氢溴酸盐、 氢碘酸盐、 2-羟基 乙磺酸盐、 乳酸盐、 马来酸盐、 曱磺酸盐、 烟酸盐、 2-萘横酸盐、 草酸盐、 3-苯基丙酸盐、 丙酸盐、 琥珀酸盐、 酒石酸盐、 磷酸盐、 谷氨酸盐、 碳酸氢 盐、 对曱苯横酸盐和十一烷酸盐。 能用于形成药学上可接受盐的优选的酸 是盐酸、 氢溴酸、 硫酸、 磷酸、 草酸、 马来酸、 琥珀酸和柠檬酸。 药学上 可接受的碱加成盐中的阳离子包括但不限于碱金属或碱土金属离子如锂、 钠、 钾、 钙和镁等， 季铵阳离子 （如四曱基铵、 四乙基铵等） 、 以及铵、 曱基胺、 二曱基胺、 三曱基胺、 三乙基胺、 二乙基胺、 乙基胺、 二乙胺、 乙醇胺、 二乙醇胺、 哌啶、 哌嗪等的阳离子。 优选的碱加成盐包括磷酸盐、 三羟曱基氨基曱烷（tris )和乙酸盐。 这些盐一般能够增加多肽的溶解性， 而且所形成的盐基本上不改变多肽的活性。
总之， 根据本发明的方案， 所述降低 IL-13水平的药物可以是直接采用 所述肽（SP肽） ， 也可以是 SP肽的药用盐或药用酯形式的药物制剂。
进一步的， 所述肽或其衍生物为式 II所示的肽或其药学上可接受的盐 或酯：
Gly-Gln-Thr-Tyr-Thr-Ser-Gly (式 II )
根据本领域公知的氨基酸表示方式， 式 Π所示的肽还可以缩写为 GQTYTSG。
在本发明的方案中， 所述肽或其衍生物可根据施用对象的不同以及施 用途径的不同采用适当的制剂形式， 例如： 注射剂、 （注射用） 冻干粉、 喷雾剂、 口服溶液、 口服混悬液、 片剂、 胶嚢、 肠溶片、 丸剂、 粉剂、 颗 粒剂、 緩释剂 （可控制药剂有效成分緩慢译放的剂型）或控释剂 （可控制 药剂有效成分译放的剂型）等制剂， 所述制剂中可包含常规的药学上可接 受的载体或赋形剂，所述"药学上可接受的载体"指无毒固态、半固态或液态 填充剂、 稀释剂、 佐剂、 包裹材料或其他制剂辅料， 例如： 生理盐水、 等 渗葡萄糖溶液、 緩冲盐水、 甘油、 乙醇及上述溶液的组合。 本发明的方案 中优选以注射方式施用由所述肽或其衍生物制成的药物， 即， 使用注射针 剂或冻干粉针剂是优选的， 以生理盐水作为载体溶解即可。
本发明的研究发现， IL-13水平的升高也是导致肝脏、 肾脏以及肺等器 官出现纤维化症状的诱因之一， 所以， 利用上述含有治疗有效量的 SP肽或 其衍生物的药物（所述肽或其衍生物作为有效成分） 降低患者 IL-13水平， 引用在临床治疗中可以起到对肝病患者、 肺病患者或肾病患者的症状緩解 及治疗的效果。
在此研究结果的基础上，本发明提供的一种降低患者 IL-13水平的方法， 包括， 向患者施用含有治疗有效量的上述式 I所示的肽或其衍生物的药物， 所述衍生物包括所述肽在药学上可接受的盐或酯。
本发明进一步提供了一种通过降低患者 IL-13水平实现对肝纤维化疾 病、 肺纤维化疾病或肾间质纤维化疾病治疗的方法。
根据本发明的优选方案， 所述治疗用药中作为有效成分的肽或其衍生 物可以为上述式 II所示的肽或其药学上可接受的盐或酯。
本发明的实施方案中， 可以向患者施用含有治疗有效量为 200-3000μ§ 所述肽或其衍生物的药物，进一步可以是 300-2000μ§的所述肽或其衍生物的 药物。 本文中所述治疗有效量是针对一般成人体重、 单次施用的药剂中所 述有效成分的量。
在本发明的一个实施方式中， 以注射方式施用含有治疗有效量的所述 肽或其衍生物的药物。 更好地是对患者以单位制剂的剂量给药， 所述单位 制剂为满足一次给药所需有效成分的制剂， 常见的单位制剂如一单位（片） 片剂、 一单位（针）针剂或粉针剂等。 患者一次施用所需的药物的量可以 方便地通过计算患者的体重和该患者一次用药所需单位体重剂量的乘积得 到。 例如， 在制备药物的过程中， 一般认为成人体重为 50-90kg, 可以最初 可以通过实验动物与人的单位体重剂量之间的等效剂量换算关系来确定用 药量。 例如， 可以根据 FDA、 SFDA等药品管理机构提出的指导意见， 也可 参考（黄继汉等， "药理试验中动物间和动物与人体间的等效剂量换算" ， 《中国临床药理学与治疗学》 , 2004 Sep ;9(9) : 1069 - 1072 )来确定。 在本发 明的实施方式中， 可以使用按照人和大鼠的体表面积折算系数 0.018来换算 人和大鼠的剂量。 根据本发明实施方式， 所述单位制剂中 SP肽或其衍生物 以 20-30(^g/kg大鼠剂量施用至大鼠时治疗效果较好， 当以 40-20(^g/kg大鼠 剂量， 例如 18(^g/kg、 9(^g/kg, 以及 45 g/kg大鼠剂量施用至大鼠时治疗 效果更佳。 制药商可以根据上述换算方法得到用于人的单位制剂中的有效 成分含量， 用以应用于其制药过程中。 在本发明的技术方案中， 根据等效 剂量换算关系以及人的常规体重， 并且综合用药安全、 成本和药效， 优选 的，所述单位制剂中含有 200-3000μ§剂量的所述肽或其衍生物，更优选含有 300-2000μ§剂量的所述肽或其衍生物。
研究结果显示， SP肽或其衍生物能够有效降低肝病患者、 肺病患者或 肾病患者 IL-13水平， 尤其是降低肝纤维化患者， 肺纤维化患者， 或肾间质 纤维化患者的 IL-13水平， 临床表现为有效改善了这些患者的病变症状。
在本发明的实施例中使用本领域常规手段诱导出肝纤维化、 肺纤维化、 或肾间质纤维化症状的大鼠模型 (其中， 所述肝纤维化可以为 BSA免疫佐剂 诱导的肝纤维化， 所述肺纤维化可以为博莱霉素诱导的肺纤维化， 所述肾 间质纤维化可以为单侧输尿管梗阻诱导的肾间质纤维化)，从数据可以看出， 施用所述肽或其衍生物的组（SP肽高、 中、 低剂量组） , 相比于模型组， 均表现出显著降低的 IL-13水平， 以及显著改善的组织病变程度。
为了便于理解， 以下将通过具体的实施例对本发明进行详细地描述。 需要特别指出的是， 具体实例仅是为了说明， 并不构成对本发明范围的限 制。 显然本领域的普通技术人员可以根据本文说明， 在本发明的范围内对 本发明做出各种各样的修正和改变， 这些修正和改变也纳入本发明的范围 内。 另外， 本发明引用了公开文献， 这些文献也是为了更清楚地描述本发 明， 它们的全文内容均纳入本发明而作为本发明说明书的一部分。 附图说明
图 1 显示了 SP肽对各组大鼠的肝脏血清中 IL-13水平的影响。
图 2显示了各组大鼠的肝组织纤维化病变程度综合评分。
图 3 显示了 SP肽对各组大鼠的肾脏血清中 IL-13水平的影响。
图 4显示了各组大鼠的肾间质纤维化病变程度综合评分。
图 5 显示了 SP肽对各组大鼠的肺血清中 IL-13水平的影响。
图 6显示了 SP肽对各组大鼠的肺组织匀浆中羟脯氨酸水平的影响。 图 7显示了各组大鼠的肺纤维化病变程度综合评分。 具体实施方式
实施例 1 SP肽降低肝病动物 IL-13水平以及改善肝纤维化作用
1. 实验材料
1.1 动物：
大鼠 60只， 体重 18g ~ 22g, 雌雄各半， 购自南通大学实验动物中心。 1.2药物、 试剂及仪器：
使用通过固相肽合成方法，由 413A型自动肽合成仪（购自 Perkin Elmer 公司）合成的以下序列的肽： GQTYTSG (以下称为 SP肽）， 具体的合成步 骤请参见 PCT/CN2006/001176中实施例 1的记载，使用时用生理盐水溶解。
阳性对照药： 氢化可的松注射液， 商购获得， 标称人日用量 5mg /kg-d。
BSA免疫佐剂购自 Sigma公司。
大鼠 IL-13 ELISA Kit, 购自上海蓝基生物科技有限公司。
酶标仪： 美国热电雷勃 MK3。 1.3分组及药物剂量
将上述 60只大鼠随机分为 6组， 大鼠每组 10只， 分别为：
1 ) 空白对照组（施用生理盐水）；
2 )模型组（施用 CC14花生油和使用生理盐水配制的 BSA免疫佐剂）；
3 ) 阳性药物组（施用 CC14花生油和使用生理盐水配制的 BSA免疫佐 剂， 以及氢化可的松注射液， 剂量为 5mg /kg.d );
4 ) SP肽高剂量组、 5 ) SP肽中剂量组、 6 ) SP肽低剂量组（施用 CC14 花生油和使用生理盐水配制的 BSA免疫佐剂，以及剂量分别为 18(^g/kg-d, 9(^g/kg d, 45 g/kg d的 SP肽，用生理盐水配置成需要浓度的 SP肽溶液）。
2.试 3全方法
2.1 实验方案：
将 BSA免疫佐剂用生理盐水配置为 0.8wt %浓度， 现配现用。 同时配置 40%(v/v)CCl4花生油。
按照以下方式对各组大鼠进行给药， 给药期间， 每天向各组大鼠正常 喂食。
在第 1天， 对除空白对照组外的其余各组大鼠， 腹腔注射 40 % CC14花生 油溶液 0.3 ml/100 g大鼠体重， 同时皮下注射 0.8 % BSA免疫佐剂 0.3 ml/100 g 大鼠体重。 空白对照组腹腔注射等体积的生理盐水， 共进行 5周。 然后对各 组动物分别随机取 1只， 腹腔注射 1 %戊巴比妥钠麻醉后称体重， 取肝脏， 观察其形态及病理学改变， 其中除空白对照组外其余组的大鼠均表现出明 显的肝纤维化， 说明造模成功。
从第 36天起， 对空白对照组和模型组大鼠皮下注射生理盐水（大鼠每 100g体重给予 0.1 ml生理盐水）， 隔天给药一次； 对 SP肽高、 中、 低剂量 组皮下注射相应剂量的 SP肽， 大鼠每 100g体重给药 0.1 ml的 SP肽溶液， 隔天给药一次； 对阳性药物组大鼠腹腔注射氢化可的松， 大鼠每 100g体重 给药 0.1 ml, 隔天给药一次。 各组动物按照上述方式共给药 15次。
最后一次给药结束后第 2天， 将空白对照组， 模型组， SP肽高、 中、 低 剂量组， 以及阳性药物组的大鼠， 分别腹腔注射 10 %水合氯醛进行麻醉， 取血收集血清， 根据试剂盒的操作要求检测血清中 IL-13含量， 将 IL-13标准 品用试剂盒中的稀释液进行倍比稀释， 然后按照试剂盒说明书进行操作。 计算出标准曲线， 从标准曲线上读出所测样本的数值， 再乘以稀译倍数， 即得出样本血清中 IL- 13的水平值。
收集完血清后， 将大鼠肝使用冰生理盐水清洗组织表面， 滤纸吸干， 置 于 10 %中性福尔马林溶液中固定 24小时， 常规石蜡包埋， 5 μιη切片， HE染 色， 光学显微镜下进行病理组织学检查， 包括： 检查肝细胞结构是否完整、 肝小叶是否正常， 以及肝细胞间连接是否紧密， 肝细胞索排列是否规则等。
根据肝组织病变由轻到重的程度分别标记为 1分， 2分， 3分， 4分， 无 明显病变为 0分， 极轻度病变为 0.5分， 累加所有分数， 并计算出每组每只动 物的均分（±SD ), 分值越高提示病变程度越重。 对病变评分结果进行两个 样本比较的秩和检验， 与模型组进行比较。
2.2数据处理：对所有数据进行数据处理，对病理学评分采用秩和检验， 其他数据采用 t检验， 并统计分析结果。
3.结果
3.1 大鼠肝脏血清中 IL-13水平
图 1表示了 SP肽对各组大鼠的血清中 IL-13水平的影响， 以平均值表 示， 其中给药量如上述 1.3中所述。 从图 1可以看出， 由 BSA免疫佐剂引 起的复合性免疫损伤肝纤维化大鼠模型组的血清中 IL-13 水平相比空白对 照组显著升高， 根据下面病理组织学检查可知， IL-13水平升高诱发了大鼠 肝组织纤维化病变； 而 SP肽高、 中、 低剂量组大鼠的血清中 IL-13相比模 型组均有显著降低（* < 0.05 ** < 0.01 ); 并且 SP肽高剂量组相比阳 性药物组有更好的效果， SP肽中、 低剂量组与阳性药物组效果相当。
3.2肺组织切片结果分析
空白对照组大鼠肝细胞结构完整， 肝小叶正常， 肝细胞索排列规则， 肝细胞间连接紧密。
模型组大鼠肝细胞破坏严重， 胞浆发空， 核固缩或溶解， 肝小叶结构 破坏， 有假小叶形成， 从汇管区到肝中央静脉可见成纤维细胞数量增加， 肝细胞内可见大量的空泡结构， 同时还有细胞浊肿、 水样变等细胞变性坏 死表现。
阳性药物组大鼠肝细胞破坏减轻， 从汇管区到肝中央静脉可见成纤维 细胞数量有所减少。
SP肽高、 中、 低剂量组大鼠肝细胞破坏明显减轻， 肝小叶结构破坏明 显减少， 从汇管区到肝中央静脉可见成纤维细胞数量显著减少。
3.3 将各组大鼠的肝组织病变轻重程度按照上述评分规则，使用秩和检 验方法获得各病理学检查指标的综合评分结果， 如图 2所示， 病理组织学 检查表明， SP肽给药的各组大鼠，均表现出肝组织纤维化病变的显著减轻， 相比于模型组均有统计学显著性差异 ( * P < 0.05或 ** < 0.01 )。 实施例 2 SP肽降低肾病动物 IL-13水平以及改善肾间质纤维化作用
1. 实验材料
1.1 动物： 同实施例 1.
大鼠 60只， 体重 18g ~ 22g, 雌雄各半， 购自南通大学实验动物中心。 1.2药物、 试剂及仪器： 同实施例 1。
1.3分组及药物剂量
将上述 60只大鼠随机分为 6组， 大鼠每组 10只， 分别为：
1 ) 空白对照组（施用生理盐水）；
2 )模型组（手术使大鼠单侧输尿管梗阻）；
3 )阳性药物组（手术使大鼠单侧输尿管梗阻，施用氢化可的松注射液， 剂量为 5mg /kg d );
4 ) SP肽高剂量组、 5 ) SP肽中剂量组、 6 ) SP肽低剂量组（手术使大 鼠单侧输尿管梗阻， 施用剂量分别为 18(^g/kg.d, 9(^g/kg.d, 45 g/kg-d 的 SP肽， 用生理盐水配置成需要浓度的 SP肽溶液）。
2.试 3全方法
2.1 实验方案：
手术使大鼠单侧输尿管梗阻包括： 将大鼠用乙醚吸入麻醉后， 于右侧 中腹部切开， 剥离右侧输尿管， 使之处于游离状态， 分别在肾盂处和输尿 管上三分之一处用丝线分别结扎， 后切断结扎处的输尿管， 逐层缝合皮肤。 按照以下方式对各组大鼠进行给药， 给药期间， 每天向各组大鼠正常 喂食。
在第 1天， 对除空白对照组外的其余各组大鼠 (模型组， SP肽高、 中、 低剂量组， 阳性药物组)， 采用手术使大鼠单侧输尿管梗阻。 然后常规饲养
24天， 对各组动物分别随机取 1只， 腹腔注射 1 %戊巴比妥钠麻醉后称体重， 取肝脏， 观察其形态及病理学改变， 其中除空白对照组外其余组的大鼠均 表现出明显的右肾间质纤维化， 说明造模成功。
从第 25天起， 对空白对照组和模型组大鼠皮下注射生理盐水（大鼠每 100g体重给予 0.1 ml生理盐水）， 隔天给药一次； 对 SP肽高、 中、 低剂量 组皮下注射相应剂量的 SP肽， 大鼠每 100g体重给药 0.1 ml的 SP肽溶液， 隔天给药一次； 对阳性药物组大鼠腹腔注射氢化可的松， 大鼠每 100g体重 给药 0.1 ml的阳性药物； 隔天给药一次。 各组动物按照上述方式共给药 15 次。
最后一次给药结束后第 2天， 将空白对照组， 模型组， SP肽高、 中、 低 剂量组， 以及阳性药物组的大鼠， 分别腹腔注射 10 %水合氯醛进行麻醉， 取血收集血清， 根据试剂盒的操作要求检测血清中 IL-13含量， 将 IL-13标准 品用试剂盒中的稀释液进行倍比稀释， 然后按照试剂盒说明书进行操作。 计算出标准曲线， 从标准曲线上读出所测样本的数值， 再乘以稀译倍数， 即得出样本血清中 IL- 13的水平值。
收集完血清后， 将大鼠肾脏使用冰生理盐水清洗组织表面， 滤纸吸干， 置于 10 %中性福尔马林溶液中固定 24小时， 常规石蜡包埋， 5 μιη切片， HE 染色， 光学显微镜下进行肾脏病理组织学检查， 包括： 检查肾盂及肾小管 结构是否正常、 肾小体体积是否变化等。
根据肾脏组织病变由轻到重的程度分别标记为 1分， 2分， 3分， 4分， 无明显病变为 0分， 极轻度病变为 0.5分， 累加所有分数， 并计算出每组每只 动物的均分（±SD ), 分值越高提示病变程度越重。 对病变评分结果进行两 个样本比较的秩和检验， 与模型组进行比较。
2.2数据处理：对所有数据进行数据处理，对病理学评分采用秩和检验， 其他数据采用 t检验， 并统计分析结果。
3.结果
3.1 大鼠肾脏血清中 IL-13水平
图 3表示了 SP肽对各组大鼠的血清中 IL-13水平的影响， 以平均值表 示， 其中给药量如上述 1.3中所述。 从图 3可以看出， 由单侧输尿管梗阻引 起的右肾间质纤维化大鼠模型组的血清中 IL-13 水平相比空白对照组显著 升高， 才艮据下面病理组织学检查可知， IL-13水平升高诱发了大鼠右肾间质 纤维化病变。 而 SP肽高、 中、 低剂量组大鼠的血清中 IL-13相比模型组均 有显著降低（* p o.os或 ** P O.Ol ); 并且 SP肽高、 中、 低剂量组相比 阳性药物组均具有更好的效果。
3.2 肾组织切片结果分析
空白对照组大鼠肾盂及肾小管结构正常、 肾小体体积无明显变化。 模型组大鼠肾脏内表面肾盂的移行上皮被扩张， 表现为极薄而扁平， 单行排列， 其内侧的平滑肌和结締组织明显增多； 肾小体体积变小， 呈扁 椭圓型， 血管球数量明显减少， 并呈单层样分布在嚢壁内； 肾小管正常结 构少见， 多数被大量的间质成分所取代。
阳性药物组大鼠表现为肾盂内侧的平滑肌和结締组织相比于模型组有 所减少。
SP肽高、 中、 低剂量组大鼠的肾组织相比于模型组表现为轻度的纤维 化和肾小管增厚， 有一定的浸润细胞增多。
3.3 将各组大鼠的肾组织病变轻重程度按照上述评分规则，使用秩和检 验方法获得各病理学检查指标的综合评分结果， 如图 4所示， 病理组织学 检查表明， SP肽给药的各组大鼠， 均表现出大鼠肾间质纤维化病变的显著 减轻， 相比于模型组均有统计学显著性差异 ^ ^〈(^(^或^ /5 。^^。 实施例 3 SP肽降低肺病动物 IL-13水平以及改善肺纤维化作用
1. 实验材料
1.1 动物： 同实施例 1。
1.2药物、 试剂及仪器： 使用的 SP肽， 阳性药物同实施例 1。
大鼠 IL-13 ELISA Kit, 购自上海蓝基生物科技有限公司。
酶标仪： 美国热电雷勃 MK3。
博莱霉素， 商购获得。
1.3分组及药物剂量
将上述 60只大鼠随机分为 6组， 大鼠每组 10只， 分别为：
1 ) 空白对照组（施用生理盐水）；
2 )模型组（施用含博莱霉素的生理盐水）；
3 )阳性药物组（施用含博莱霉素的生理盐水， 以及氢化可的松注射液， 剂量为 5mg /kg，d );
4 ) SP肽高剂量组、 5 ) SP肽中剂量组、 6 ) SP肽低剂量组（施用含博 莱霉素的生理盐水， 以及剂量分别为 18(^g/kg.d, 9(^g/kg.d, 45 g/kg-d 的 SP肽， 用生理盐水配置成需要浓度的 SP肽溶液）。
2.试 3全方法
2.1 实验方案：
以 lkg动物体重给予 5mg博莱霉素的剂量造模， 博莱霉素用生理盐水现 配现用。
按照以下方式对各组大鼠进行给药， 给药期间， 每天向各组大鼠正常 喂食。
第 1天， 对除空白对照组外的其余各组大鼠 (模型组， SP肽高、 中、 低 剂量组， 阳性药物组)， 禁食不禁水 12小时， 将大鼠用 10 %水合氯醛腹腔注 射麻醉 (0.35ml/100g)后， 仰卧于固定台上， 暴露气管， 每只大鼠用 l mL注射 器向气管内一次性注入博莱霉素生理盐水溶液， 大鼠每 100g体重给予 0.25 mL。 空白对照组腹腔注射等体积的生理盐水。 实验期间各组大鼠均正常喂 食， 6天后对各组动物分别随机取 1只， 腹腔注射 1 %戊巴比妥钠麻醉后称体 重， 取左肺组织， 观察其形态及病理学改变， 其中除空白对照组外其余组 的大鼠均表现出明显的肺纤维化， 说明造模成功。
从第 8天起， 对空白对照组和模型组大鼠皮下注射生理盐水（大鼠每 100g体重给予 0.1 ml生理盐水）， 隔天给药一次； 对 SP肽高、 中、 低剂量 组皮下注射相应剂量的 SP肽， 大鼠每 100g体重给药 0.1 ml的 SP肽溶液， 隔天给药一次； 对阳性药物组大鼠腹腔注射氢化可的松， 大鼠每 100g体重 给药 0.1 ml, 隔天给药一次。 各组动物按照上述方式共给药 15次。
最后一次给药结束后第 2天， 将空白对照组， 模型组， SP肽高、 中、 低 剂量组， 以及阳性药物组的大鼠， 分别腹腔注射 10 %水合氯醛进行麻醉， 取血收集血清， 根据试剂盒的操作要求检测血清中 IL-13含量， 将 IL-13标准 品用试剂盒中的稀释液进行倍比稀释， 然后按照试剂盒说明书进行操作。 计算出标准曲线， 从标准曲线上读出所测样本的数值， 再乘以稀译倍数， 即得出样本血清中 IL- 13的水平值。
收集完血清后， 将打开胸腔取出整个肺脏，使用冰生理盐水清洗组织表 面， 滤纸吸干， 取右肺组织置于 10 %中性福尔马林溶液中固定 24小时， 常 规石蜡包埋， 5 μιη切片， HE染色， 光学显微镜下进行肺组织病理组织学检 查， 包括： 检查肺内结构是否清晰， 是否有水肿、 无炎症及纤维化表现， 肺泡间隔是否增厚等。
肺组织匀浆的制备： 取左肺组织 10 %匀浆， 以 3000rpm离心 10分钟， 取 上清液， 根据试剂盒的操作要求检测肺组织中羟脯氨酸 (HyP)水平， 根据试 剂盒的操作要求检测肺组织中羟脯氨酸 (HyP)水平。
根据肺组织组织病变由轻到重的程度分别标记为 1分， 2分， 3分， 4分， 无明显病变为 0分， 极轻度病变为 0.5分， 累加所有分数， 并计算出每组每只 动物的均分（±SD ), 分值越高提示病变程度越重。 对病变评分结果进行两 个样本比较的秩和检验， 与模型组进行比较。
2.2数据处理：对所有数据进行数据处理，对病理学评分采用秩和检验， 其他数据采用 t检验， 并统计分析结果。
3.结果
3.1 大鼠肺部血清中 IL-13水平
图 5表示了 SP肽对各组小鼠的血清中 IL-13水平的影响， 以平均值表 示， 其中给药量如上述 1.3中所述。 从图 5可以看出， 博莱霉素引起的肺纤 维化大鼠模型组的血清中 IL-13水平相比空白对照组显著升高，根据下面病 理组织学检查可知， IL-13水平升高诱发了大鼠肺纤维化病变。 而 SP肽高、 中、低剂量组小鼠的血清中 IL-13水平相比模型组均有显著降低（* P < 0.05 P < 0.0l ), 表明 SP肽可通过降低 IL-13进行大鼠肺纤维化的治疗。 并 且 SP肽高剂量组相比阳性药物组有更好的效果， SP肽中剂量组与阳性药 物组效果相当。
图 6表示了 SP肽对各组小鼠的肺组织勾浆中羟脯氨酸水平的影响， 以 平均值表示， 其中给药量如上述 1.3中所述。 从图 6可以看出， 博莱霉素引 起的肺纤维化大鼠模型组的肺组织匀浆中羟脯氨酸水平相比空白对照组显 著升高， 羟脯氨酸水平是肺纤维化病变的重要生化指标。 而 SP肽高、 中、 低剂量组小鼠肺组织匀浆中羟脯氨酸水平相比模型组均有显著降低 ( * P < 0.05 ** < 0.01 ); 并且 SP肽高剂量组相比阳性药物组有更好的效果， SP 肽中剂量组与阳性药物组效果相当。
3.2肺组织切片结果分析
空白对照组大鼠肺内结构清晰， 无水肿、 无炎症及纤维化表现， 肺泡 间隔未见增厚。
模型组大鼠肺泡腔及肺间质内有大量炎性细胞浸润， 肺泡水肿， 肺泡 间隔明显增宽， 成纤维细胞增生， 胶原纤维增生呈带状， 肺组织以胶原沉 积、 纤维化改变为主。
阳性药物组大鼠肺泡炎、 纤维化程度均较模型组轻， 肺泡间隔增厚程 度均较模型组轻， 炎症细胞浸润少， 病变范围小。
SP肽高、 中、低剂量组大鼠的肺泡炎症状及纤维化程度均较模型组轻， 肺内结构较清晰， 肺泡间隔增厚程度较轻， 炎性细胞浸润范围小。
3.3 将各组大鼠的肺组织病变轻重程度按照上述评分规则，使用秩和检 验方法获得各病理学检查指标的综合评分结果， 如图 7所示， 病理组织学 检查表明， SP肽给药的各组大鼠，均表现出大鼠肺纤维化病变的显著减轻， 相比于模型组均有统计学显著性差异（ ^^ P O.OS ^ p < o )。

Claims

权 利 要求
1、 如式 I所示的肽或其衍生物在制备降低 IL-13水平的药物中的应用： Xaal-Gln-Xaa2-Xaa3-Thr-Ser-Gly-Xaa4 (式 I ) 其中，
Xaal为缺失、 Ala、 Gly、 Val、 Leu或 lie,
Xaa2为 Thr或 Ser,
Xaa3为 Tyr、 Phe或 Trp, 而且
Xaa4为缺失、 Ala、 Gly、 Val、 Leu, lie或 Pro;
所述衍生物包括所述肽在药学上可接受的盐或酯。
2、 根据权利要求 1所述的应用， 其中所述肽或其衍生物为式 II所示的 肽或其药学上可接受的盐或酯：
Gly-Gln-Thr-Tyr-Thr-Ser-Gly (式 II ) 。
3、 根据权利要求 1所述的应用， 所述药物为单位制剂。
4、 根据权利要求 1或 3所述的应用， 所述药物为注射制剂。
5、根据权利要求 1-4任一项所述的应用， 其中所述药物为肝病、肺病或 肾病治疗用药。
6、 根据权利要求 5所述的应用， 所述肝病为肝纤维化疾病， 所述肺病 为肺纤维化疾病， 所述肾病为肾间质纤维化疾病。
7、 根据权利要求 3或 4所述的应用， 其中所述单位制剂或注射制剂中含 有治疗有效量为 200-3000μ§ 的所述肽或其衍生物。
8、 根据权利要求 7所述的应用， 其中所述单位制剂或注射制剂中含有 治疗有效量为 300-2000μ§ 的所述肽或其衍生物。
9、 一种降低患者 IL-13水平的方法， 包括， 向患者施用含有治疗有效 量的式 I所示的肽或其衍生物的药物：
Xaal-Gln-Xaa2-Xaa3-Thr-Ser-Gly-Xaa4 (式 I ) 其中，
Xaal为缺失、 Ala、 Gly、 Val、 Leu或 lie,
Xaa2为 Thr或 Ser, Xaa3为 Tyr、 Phe或 Trp, 而且
Xaa4为缺失、 Ala、 Gly、 Val、 Leu, lie或 Pro;
所述衍生物包括所述肽在药学上可接受的盐或酯。
10、 根据权利要求 9所述的方法， 所述肽或其衍生物为下式 II所示的 SP 肽或其药学上可接受的盐或酯：
Gly-Gln-Thr-Tyr-Thr-Ser-Gly (式 II ) 。
11、 根据权利要求 9所述的方法， 其中所述患者为肝病患者、 肺病患者 或肾病患者。
12、 根据权利要求 11所述的方法， 所述肝病患者为肝纤维化患者， 所 述肺病患者为肺纤维化患者， 所述肾病患者为肾间质纤维化患者。
13、 根据权利要求 12所述的方法， 所述药物含有治疗有效量为
200-3000μ§的所述肽或其衍生物。
14、 根据权利要求 13所述的方法， 其中， 所述药物含有治疗有效量为 300-2000μ§的所述肽或其衍生物。
15、根据权利要求 9-14任一项所述的方法， 其中， 对患者以单位制剂的 剂量给药。
16、根据权利要求 15所述的方法，其中，所述单位制剂中含有 200-3000μ§ 的所述肽或其衍生物。
17、根据权利要求 16所述的方法，其中，所述单位制剂中含有 300-2000μ§ 的所述肽或其衍生物。
18、 根据权利要求 9-17任一项所述的方法， 包括以注射方式给药。
PCT/CN2013/071525 2013-02-07 2013-02-07 Sp肽或其衍生物在制备降低il-13水平的药物中的应用 WO2014121492A1 (zh)

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