WO2019047904A1 - 口服降血糖多肽、其脂肪酸修饰衍生物以及用途 - Google Patents

口服降血糖多肽、其脂肪酸修饰衍生物以及用途 Download PDF

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WO2019047904A1
WO2019047904A1 PCT/CN2018/104501 CN2018104501W WO2019047904A1 WO 2019047904 A1 WO2019047904 A1 WO 2019047904A1 CN 2018104501 W CN2018104501 W CN 2018104501W WO 2019047904 A1 WO2019047904 A1 WO 2019047904A1
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高向东
赛文博
姚文兵
杨康敏
魏青青
宋潇达
田浤
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China Pharmaceutical University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/57563—Vasoactive intestinal peptide [VIP]; Related peptides
    • 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
    • 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
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • 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 present invention relates to an oral hypoglycemic polypeptide, a fatty acid modified derivative of the polypeptide, and to their use; the polypeptide and its fatty acid modified derivative have intestinal protease proteolytic resistance, and have good intestinal permeability. Sex; belongs to the field of medical biotechnology.
  • Diabetes is a lifelong metabolic disease characterized by multiple hyperglycemia caused by multiple causes. Its complications involve various tissues and organs such as cardiovascular and cerebrovascular tissues, kidney tissues, eyes and feet. It is difficult to reverse. Pathologically, diabetes can be divided into two types: type I diabetes, which is characterized by decreased or completely lost insulin secretion, and is characterized by type 2 diabetes in which the body cannot effectively use insulin. Among them, type II diabetes accounts for 90% of the total number of patients currently. It is estimated that by 2025, the total number of diabetic patients worldwide will reach 300 million, which will not only bring the physical and psychological burden to the patients themselves, but may even cause serious social problems.
  • Glucagon-like peptide-1 receptor agonist drugs have unparalleled advantages in reducing hypoglycemia crisis, protecting and promoting islet tissue regeneration, weight control, and cardiovascular benefits to patients, but such drugs
  • the only effective route of administration is subcutaneous injection, which not only brings difficulties and pain to patients, requires appropriate technology and equipment, but also seriously affects patient compliance.
  • the glucagon-like peptide-1 receptor agonist drug exenatide which is a linear polypeptide consisting of 39 amino acids, is isolated from the saliva of the South American giant lizard. Exenatide has 53% homology with mammalian glucagon-like peptide 1, which activates glucagon-like peptide receptors to activate a variety of physiological functions: (1) glucose dependence Insulin release; (2) stimulate the release of somatostatin to inhibit the secretion of glucagon; (3) inhibit gastric cell secretion of gastric acid, prolong gastric emptying; (4) increase satiety, suppress appetite, reduce energy Ingestion; (5) enhance islet beta cell function and promote its proliferation.
  • saliva the US FDA in 2005. Long-acting injections have been marketed in the US in 2012.
  • Biotech drugs are mostly peptides. Biomacromolecules such as proteins, which have large molecular weights and poor fat solubility, are difficult to absorb in the intestines; 2. There are a large number of proteases in the intestines, which rapidly degrade biomolecules entering the intestinal tract, so that the drugs in the intestine cannot be maintained. The concentration required for treatment. It is urgent to develop an exenatide analog which has both anti-enzymatic properties and good intestinal permeability.
  • the inventors have obtained some research results in this field, including the Chinese invention patent on December 17, 2013, the patent number CN201310694475.0, and the authorization announcement number CN103665148B, which can be used for oral administration of hypoglycemic polypeptides. It is obtained by subjecting the amino acids 12, 20 and 27 of the Exendin-4 amino acid sequence to non-basic amino acid substitution. At present, the inventors have obtained new results in further research.
  • the technical problem to be solved by the present invention is to provide an oral hypoglycemic polypeptide based on the latest research results of the inventors, which has good anti-enzymatic properties. Also provided are fatty acid modified derivatives of the polypeptides, as well as the use of the polypeptides and derivatives thereof.
  • the main research process of the present invention is as follows: On the basis of the polypeptides in the aforementioned invention patents, the inventors continue to reduce the amino acid sites which can be degraded by proteases in the intestinal tract by amino acid mutation, and screen out a class of trypsin and chymotrypsin. , elastase resistant polypeptide; afterwards, a series of tests proved that such polypeptides can be administered by the oral route and have significant hypoglycemic activity.
  • Xaa1 is Met, Val, Ser or Lys
  • Xaa2 is Leu, Tyr, Val or Lys
  • Xaa3 is Glu or Leu
  • Xaa4 is Met, Val or Lys.
  • amino acid sequence is one of the following sequences:
  • the oral hypoglycemic polypeptide described above is for use in the preparation of a medicament or a pharmaceutical composition for preventing or treating diabetes, or for the preparation of a hypoglycemic medicament or a pharmaceutical composition.
  • an oral hypoglycemic polypeptide fatty acid modified derivative characterized in that the polypeptide is an oral hypoglycemic polypeptide as described above; the fatty acid modification site is one of Xaa1, Xaa2, Xaa4 or any combination thereof; when Xaa1 is modified Xaa1 is Lys at the site, Xaa2 is Lys when Xaa2 is a modification site, and Lys is Xaa3 when Xaa3 is a modification site.
  • the fatty acid is 10-(4-carboxyphenoxy)decanoic acid or 17-carboxyheptadecanoic acid; the fatty acid is [2-(2- ⁇ 2-[2-(2- ⁇ 2-) [-4-carboxy-4-(amino)butyrylamino]ethoxy ⁇ ethoxy)acetylamino]ethoxy ⁇ ethoxy)acetyl] is attached to the amino nitrogen atom of the amino acid of the modification site.
  • the oral hypoglycemic polypeptide fatty acid modified derivative described above is for use in the preparation of a medicament or a pharmaceutical composition for preventing or treating diabetes, or for the preparation of a hypoglycemic medicament or a pharmaceutical composition.
  • a pharmaceutical composition comprising an oral hypoglycemic polypeptide as described above and/or an oral hypoglycemic polypeptide fatty acid modified derivative as hereinbefore described.
  • the pharmaceutical composition as described above is for use in the preparation of a medicament for preventing or treating diabetes, or for the preparation of a hypoglycemic agent.
  • the dosage form of the medicament is a gastrointestinal administration dosage form or a parenteral administration dosage form.
  • the diabetes is type I or type II diabetes.
  • the polypeptide of the present invention and the fatty acid modified derivative thereof have the enzymatic resistance against more proteases, can better avoid the enzymatic hydrolysis in the gastrointestinal tract, and are more suitable as oral hypoglycemic drugs. .
  • Fig. 1 is a graph showing the results of measurement of cAMP content activated by IPCM1 and IPCM5 in Example 5 of the present invention.
  • Example 2 is a graph showing the results of detecting blood sugar control ability of IPCM1 and IPCM5 in an acute intraperitoneal injection glucose tolerance test according to Example 6 of the present invention.
  • Figure 3 is a graph showing the results of detection of glycated hemoglobin levels in db/db mice by IPCM1 and IPCM5 in Example 7 of the present invention.
  • Fig. 4 is a graph showing the results of detecting the improvement of insulin resistance by IPCM1 and IPCM5 in db/db mice in Example 8 of the present invention.
  • Fig. 5 is a graph showing the results of examining the effects of IPCM1 and IPCM5 on serum insulin levels in db/db mice in Example 9 of the present invention.
  • Fig. 6 is a view showing the pancreas section of IPCM1 and IPCM5 in the db/db mouse after treatment in Example 10 of the present invention.
  • A normal group
  • B saline group
  • C IPCM1 administration group
  • D IPCM5 treatment group
  • Figure 7 is a diagram showing the observation of kidney sections of IPCM1 and IPCM5 in the db/db mice after treatment in Example 11 of the present invention.
  • A normal group
  • B saline group
  • C IPCM1 administration group
  • D IPCM5 treatment group
  • Figure 8 is a view showing the liver section of IPCM1 and IPCM5 after treatment with db/db mice in Example 12 of the present invention.
  • A normal group
  • B saline group
  • C IPCM1 administration group
  • D IPCM5 treatment group
  • sequence IPCM1-8 is first used as a representative illustration.
  • polypeptide of the present invention has the general formula shown in SEQ ID NO. 1; the sequence of IPCM1-8 is as follows:
  • IPCM1 SEQ ID NO. 2:
  • IPCM2 SEQ ID NO. 3:
  • IPCM3 SEQ ID NO. 4:
  • IPCM4 SEQ ID NO. 5:
  • IPCM5 SEQ ID NO. 6:
  • IPCM6 SEQ ID NO. 7:
  • IPCM7 SEQ ID NO. 8:
  • IPCM8 SEQ ID NO. 9:
  • the first step three kinds of protease solutions of 0.005mg/mL were prepared.
  • the three proteases were trypsin, chymotrypsin and elastase respectively.
  • the protease solutions were incubated at 37 ° C for 15 min.
  • Exendin-4C ie, Exenatide
  • the third step using RP-HPLC detection: the column is Zorbax Eclipse Plus C18 reversed-phase column; the mobile phase A phase is double distilled water containing 0.065% TFA, and the B phase is acetonitrile solution containing 0.05% TFA: injection volume It is 50 ⁇ L; the pump flow rate is 1 mL/min; the detection wavelength is 215 nm, and the detection procedure is as follows:
  • IPCM1-8 has different degrees of resistance to the above three key proteases, and the improvement of trypsin resistance is most significant, IPCM1, IPCM2, IPCM5, IPCM6 and The residual rate of IPCM8 is over 80%.
  • the resistance of IPCM1-8 to elastase and chymotrypsin was also improved to some extent. Among them, IPCM1, IPCM3, IPCM5 and IPCM7 were more prominent for elastase resistance; IPCM1 and CM protease were more important for chymotrypsin resistance. IPCM5.
  • the first step CHO/GLP-1R-CRE chemiluminescent cell line was resuscitated, transferred to a culture flask containing 5 mL of cell growth medium, and cultured in a 37 ° C, 5% CO 2 incubator.
  • the cells were adherently grown to about 80% of the cell-attached surface, they were passaged with 0.25% trypsin and subcultured once every 3 days at a ratio of 1:3.
  • the in vitro activity assay was performed using the luciferase reporter gene method.
  • Step 2 In the experimental determination, the cells were first digested with 0.25% trypsin and then added to the assay medium. The cell liquid was seeded in a 96-well plate at a dose of 2 ⁇ 10 5 cells/mL, and 100 ⁇ L of each well was seeded, and cultured in a 37 ° C, 5% CO 2 incubator for 4 hours.
  • the third step 96-well plates were removed, and 20 ⁇ L of IPCM1-8 solution/Exendin-4 positive control was added to each well.
  • Exendin-4 was diluted with an initial concentration of 280 nmol/L and diluted by 4 times. A total of 10 gradients were diluted.
  • IPCM1-8 was diluted at an initial concentration of 250 nmol/L and diluted 7 times to a total of 10 gradients. Three replicate wells were placed for each concentration of drug. Mix gently by shaking for 30 seconds, and then incubate for 4 h in a 37 ° C, 5% CO 2 incubator.
  • Step 4 Remove the 96-well plate, add a chemiluminescent substrate (Steady-Glo) reagent, 100 ⁇ L/well, and place the 96-well plate on the shaker at room temperature for 40 minutes.
  • a chemiluminescent substrate (Steady-Glo) reagent
  • Step 5 Determine the relevant chemiluminescence unit (RLU) per well using a chemiluminescent microplate reader.
  • the sixth step taking the log10 (concentration) of the sample as the abscissa, the average RLU of each concentration is the ordinate, and plotting the dose-effect curve.
  • the EC50 values of each curve were determined by Graphpad 5.0 nonlinear fitting, and the relative titers of the candidate molecule and the Exendin-4 positive control were calculated. The results are shown in Table 2.
  • the experimental results were measured from the half effective concentration EC 50 and the maximum response Max Respones, indicating that IPCM1-8 has the ability to activate the glucagon-like peptide-1 receptor compared to the prototype molecule Exednin-4.
  • the half effective concentrations of IPCM1 and IPCM5 are 0.75nM and 0.83nM, respectively, which are within the same order of magnitude as the prototype molecules.
  • the maximum reaction values are 107.36 ⁇ 5.94% and 103.60 ⁇ 6.91%, respectively, which are slightly higher than the prototype molecules.
  • IPCM1 and IPCM5 retain the biological activity of the prototype molecule to the greatest extent while increasing key protease resistance.
  • IPCM9-96 had different degrees of resistance to three proteases compared with Exendin-4C. At the same time, IPCM9-96 had the ability to activate glucagon-like peptide-1 receptor.
  • the amino nitrogen atom of the fatty acid and the amino acid of the modification site passes through [2-(2- ⁇ 2-[2-(2- ⁇ 2-[-4-carboxy-4-(amino))
  • the acylamino]ethoxy ⁇ ethoxy)acetylamino]ethoxy ⁇ ethoxy)acetyl group is linked, and the specific fatty acids and modification sites in each derivative are shown in the following table.
  • the above fatty acid-modified derivative was tested for its anti-enzymatic ability by the method of Example 1, except that in the second step, 50 ⁇ L of the reaction liquid was taken out only at a time point of 60 min after mixing with the protease.
  • the above fatty acid modified derivative was assayed for the ability to activate the glucagon-like peptide-1 receptor by the method of Example 2.
  • the first step using the CHO cell line constructed in the laboratory to stably express the GLP-1 receptor on the cell membrane surface and ligating the luciferase reporter gene, the cAMP activation content of the GLP-1 receptor downstream signaling molecule was determined.
  • Cell resuscitation and culture conditions are as described above.
  • Step 2 When measuring, the cells were digested with digestive juice, and then according to the cell growth condition, an appropriate amount of assay medium containing 0.25% fetal bovine serum and 0.5 mM IBMX was added to bring the cell concentration to 2 ⁇ 10 5 /mL. 100 ⁇ L of the cell solution was added to each well of a 96-well plate, and cultured in the above-mentioned conditional incubator for 4 hours.
  • the third step adding an analog solution of different concentration gradients and a positive drug Exendin-4 solution to each well.
  • the analog used an initial concentration of 10 ⁇ M, diluted 7 gradients at 10 fold, Exendin-4 at an initial concentration of 1 ⁇ M, and diluted 7 gradients at 10 fold.
  • Three replicate wells were placed for each concentration of drug. Mix gently by shaking for 30 seconds, then put in the incubator and continue to incubate for 4 hours.
  • Step 4 After the end, the 96-well plate was taken out, and the cells were repeatedly frozen and thawed in a -80 ° C refrigerator and a 37 ° C incubator to break the cells. After centrifugation, the supernatant was taken, cAMP content was measured according to the operation requirements in the ELISA kit, and the results were subjected to nonlinear regression analysis.
  • the first step 32 male C57 mice of 8-12 weeks old and male were randomly divided into 4 groups, PBS control group, Exendin-4 (0.10 mg/kg) subcutaneous injection group and IPCM1 high dose (2.11). Mg/kg) gavage administration group, IPCM5 high dose (2.82mg/kg) gavage administration group, 8 mice in each group.
  • the second step the mice were fasted for 12 hours in advance, and the drinking water was freely ingested.
  • the fasting blood glucose of each group of mice was measured at the beginning of the experiment, and the administration was started in groups after completion.
  • the third step 30 minutes later, the blood glucose level of each group was measured by blood sampling from the tail vein. After the measurement, the mice were intraperitoneally injected with glucose solution (2g/kg), and then 15 minutes, 30 minutes, 60 minutes, 90 minutes, Mice blood glucose levels were monitored at 120 minutes.
  • Step 4 Draw the blood glucose-time curve and calculate the area under the curve (AUC).
  • the first step animal grouping and dosing schedule.
  • Db/db spontaneous diabetic mice 6-7 weeks old, male, 35, fasting blood glucose was measured after 2 weeks of adaptation to the environment, and divided into 4 groups: PBS control group, Exendin-4 subcutaneous injection group, IPCM1 (2.11) Mg/kg) gavage administration group, IPCM5 (2.82 mg/kg) intragastric administration group, 7 rats in each group.
  • Another 8 mice in the db/m control group were used as normal controls.
  • Each of the administration groups was administered once a day in the morning and evening.
  • Step 2 Blood was taken from the orbital venous plexus of each group of mice in the first week, the fourth week and the eighth week, and added to the pre-treated anticoagulation tube. After sampling, the cells were rapidly centrifuged at low temperature, and the supernatant was taken as follows. The total hemoglobin and glycated hemoglobin levels were measured and the ratio was calculated.
  • the third step the detection of total hemoglobin in mice using the kit.
  • Standards with an initial concentration of 480 pg/mL were diluted to 240 pg/mL, 120 pg/mL, 60 pg/mL, and 30 pg/mL, respectively.
  • the fourth step respectively set a blank hole (no sample and enzyme standard reagent, the other steps are the same operation), standard hole, sample hole to be tested. Accurately load 50 ⁇ L of the standard on the enzyme-labeled plate, add 40 ⁇ L of the sample dilution to the well to be tested, and then add 10 ⁇ L of the sample to be tested. When adding the sample, try to add the sample to the bottom of the well of the microplate. Do not touch the wall of the well and shake it gently.
  • Step 5 After sealing with a sealing film, incubate at 37 ° C for 30 minutes. At this time, the concentrated washing solution was diluted 30 times and used.
  • Step 6 Gently remove the sealing film, discard the liquid, dry it, fill each well with the washing solution, let stand for 30 seconds, discard it, repeat 5 times and pat dry.
  • Step 7 In addition to the blank wells, 50 ⁇ L of the enzyme labeling reagent was added to each well, and the step 3 incubation and the step 4 washing were repeated.
  • Step 8 Add 50 ⁇ L of the developer A to each well, add 50 ⁇ L of the color developing solution B, shake gently, shake it at 37 ° C for 10 minutes.
  • the ninth step 50 ⁇ L of the stop solution was added to each well, and the reaction was immediately terminated.
  • the sample plate was placed in a multi-function plate reader within 15 minutes, and the blank hole was zeroed to detect the absorbance at a wavelength of 450 nM.
  • Step 10 With the standard concentration as the abscissa and the absorbance as the ordinate, draw a standard curve. Calculate the corresponding concentration from the standard curve based on the absorbance of the sample and multiply by the dilution factor.
  • Step 11 Detection of mouse glycated hemoglobin using a kit.
  • the glycosylated hemoglobin ELISA test kit was used, and the above steps 1-8 were repeated to calculate the glycated hemoglobin concentration of the different samples and divide by the total hemoglobin concentration.
  • the results of the analysis showed that only the glycated hemoglobin level of db/db mice had a significant therapeutic effect under high-dose administration conditions, and the therapeutic effect was consistent with the positive drug group, and there was no significant difference between the two groups.
  • mice were fasted for 6 hours and intraperitoneally injected with 1 U/kg of insulin.
  • the second step at 0, 15, 30, 60, 90, 120 minutes, the mice were subjected to rapid hypothermia after taking blood from the orbital venous plexus, and the supernatant was taken and the blood glucose level was determined according to the glucose oxidase coupling colorimetric method. Draw a blood glucose concentration-time curve.
  • the results of the insulin tolerance test after the end of the eight-week treatment are shown in Figure 4.
  • Step 2 First equilibrate the kit for 30 min at room temperature. Standards with an initial concentration of 172.8 mIU/L were diluted to 86.4 mIU/L, 43.2 mIU/L, 21.6 mIU/L, 10.8 mIU/L, and 5.4 mIU/L, respectively.
  • the third step respectively set blank holes (no sample and enzyme standard reagents, the other steps are the same operation), standard holes, sample holes to be tested.
  • Blank holes no sample and enzyme standard reagents, the other steps are the same operation
  • sample holes to be tested Accurately add 50 ⁇ L of the standard on the enzyme-labeled plate (the biotin antibody has been integrated in the standard, so it is not added).
  • 50 ⁇ L of streptomycin-HRP was added to each well. When adding the sample, try to add the sample to the bottom of the well of the microplate. Do not touch the wall of the well and shake it gently.
  • the fourth step the plate was sealed with a sealing film and incubated at 37 ° C for 60 minutes. At this point, the concentrated washing solution is diluted and used.
  • Step 5 Gently remove the sealing film, discard the liquid, dry it, fill each well with the washing solution, let stand for 30 seconds, discard it, repeat 5 times and pat dry.
  • Step 6 Add 50 ⁇ L of the developer A to each well, add 50 ⁇ L of the color developing solution B, shake gently, shake it at 37 ° C for 10 minutes.
  • Step 7 Add 50 ⁇ L of stop solution to each well and immediately terminate the reaction.
  • the sample plate was placed in a multi-function plate reader within 10 minutes, and the blank hole was zeroed to detect the absorbance at a wavelength of 450 nm.
  • the eighth step the standard concentration is the abscissa, the absorbance is the ordinate, the standard curve is drawn, and the insulin concentration in the corresponding sample is calculated from the standard curve according to the absorbance of the sample.
  • the results of serum insulin levels after the end of the eight-week administration are shown in Figure 5.
  • Example 10 Pancreatic section of IPCM1 and IPCM5 after treatment with db/db mice
  • Example 7 Animal grouping and dosing schedules are shown in Example 7. After the mice were sacrificed, the pancreas was excised and stored in 10% neutral formaldehyde. The tissue section was 6 ⁇ m thick, and then stained with hematoxylin-eosin stain according to standard procedures, and sections were observed with an optical microscope. The grouping situation was A: normal group; B: saline group; C: IPCM1 administration group; D: IPCM5 administration group.
  • the results are shown in Fig. 6.
  • the islets of the normal control group were normal in size and intact in morphology, and the islet ⁇ cells were normal.
  • the islet tissue was atrophied, and most of the islet ⁇ cells disappeared or only a few remained.
  • the islet tissue structure and islet ⁇ -cell lesions were all attenuated in the IPCM1 and IPCM5-administered groups, and the islet tissue area was also larger, indicating that IPCM1 and IPCM5 can protect the pancreatic ⁇ -cells of diabetic mice and improve islet function.
  • Example 7 Animal grouping and dosing schedules are shown in Example 7. After the mice were sacrificed, the kidneys were excised and stored in 10% neutral formaldehyde. The tissue section was 6 ⁇ m thick, and then stained with hematoxylin-eosin stain according to standard procedures, and sections were observed with an optical microscope. The grouping situation was A: normal group; B: saline group; C: IPCM1 administration group; D: IPCM5 administration group.
  • Example 12 Liver sections of IPCM1 and IPCM5 after treatment with db/db mice
  • Example 7 Animal grouping and dosing schedules are shown in Example 7. After the mice were sacrificed, the liver was excised and stored in 10% neutral formaldehyde. The tissue section was 6 ⁇ m thick, and then stained with hematoxylin-eosin stain according to standard procedures, and sections were observed with an optical microscope. The grouping situation was A: normal group; B: saline group; C: IPCM1 administration group; D: IPCM5 administration group.
  • Fig. 8 The results are shown in Fig. 8.
  • the liver cell morphology was normal, the cell nucleus was dense, and the cord-like structure was clear.
  • the hepatocytes in the PBS group lost the normal cord-like structure, the hepatic sinus disappeared, the hepatocytes were significantly swollen, and the cytoplasm was almost complete.

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Abstract

本发明提供了一种口服降血糖多肽、其脂肪酸修饰衍生物以及用途。所述多肽的序列如SEQ ID NO：1所示。所述脂肪酸修饰衍生物的修饰位点为Xaa1、Xaa2、Xaa4之一或其组合。所述多肽及其脂肪酸修饰衍生物可用于制备预防或治疗糖尿病、降血糖的药物或药物组合物。本发明的多肽及其脂肪酸修饰衍生物具有针对更多种蛋白酶的酶解抗性，适宜作为口服降血糖药物。

Description

口服降血糖多肽、其脂肪酸修饰衍生物以及用途 技术领域
本发明涉及一种口服降血糖多肽、该多肽的脂肪酸修饰衍生物，还涉及它们的用途；该多肽及其脂肪酸修饰衍生物具有肠道主要蛋白酶酶解抗性，同时具有良好的肠道通透性；属于医药生物技术领域。
背景技术
糖尿病是一种由多病因所导致的以持续性高血糖为特征的终身性代谢类疾病，其并发症涉及心脑血管组织、肾脏组织、眼部、足部等多种组织器官，且一旦发生，很难逆转。从病理上，糖尿病可以分为两种：表现为胰岛素分泌能力降低或彻底丧失的I型糖尿病，表现为机体无法有效利用胰岛素的II型糖尿病。其中，II型糖尿病占目前患者总数的90％。预计到2025年，全球糖尿病患者总数将达到3亿，这不仅为患者本人带来生理和心理的双重负担，甚至有可能引发严重的社会问题。目前，传统的治疗模式一般包括饮食控制、小分子降糖药物以及外源性胰岛素摄入等阶梯式治疗。而胰高血糖素样肽-1受体激动剂药物在减少低血糖危象、保护并促进胰岛组织再生、体重控制及给患者带来心血管收益方面具有无可比拟的优势，但此类药物目前唯一有效的给药途径为皮下注射，这不仅给患者带来困难和疼痛，需要适当的技术与设备等，而且会严重的影响患者的依从性。
例如，胰高血糖素样肽-1受体激动剂药物艾塞那肽，它是一种从南美巨蜥蜴的唾液中分离发现、由39个氨基酸组成的直链多肽。艾塞那肽与哺乳动物分泌的胰高血糖素样肽1具有53％的同源性，它可以通过激活胰高血糖素样肽受体进而激活多种生理学功能：(1)葡萄糖依赖性促胰岛素释放作用；(2)刺激生长抑素释放来抑制胰高血糖素的分泌；(3)抑制胃壁细胞分泌胃酸，延长胃的排空；(4)增加饱足感，抑制食欲，降低能量的摄取；(5)增强胰岛β细胞功能并促进其增殖。艾塞那肽的注射型药物百泌达已于2005年经美国FDA批准上市，长效型注射液已于2012年在美国上市。
如前所述，给药途径极大限制了艾塞那肽的临床应用及患者的依从性。因此，口服给药作为目前应用最广，使用最方便的给药方式，已成为生物技术药物的持续努力方向。但由于生物技术药物口服利用度低，一直以来并未获得实用化的成果，目前仍未有可口服给药的相关生物技术药物上市，其主要原因在于两方面：1.生物技术药物多为多肽、蛋白等生物大分子，其分子量大，脂溶性较差，在肠道内难以吸收；2.肠道内存在大量蛋白酶，会迅速降解进入肠道的生物大分子，以至于肠道内的药物无法维持在治疗需要的浓度。亟待研发出既具有抗酶解特性，又具有良好肠道通透性的艾塞那肽类似物。
发明人在该领域已经获得了一些研究成果，其中包括申请日2013年12月17日、专利号CN201310694475.0、授权公告号CN103665148B的中国发明专利，该专利中涉及的可口服给药降血糖多肽是通过将Exendin-4氨基酸序列中第12、20和27位氨基酸进行非碱性氨基酸取代获得的。目前发明人在进一步研究中已获得了新的成果。
发明内容
本发明所要解决的技术问题是：基于发明人的最新研究成果，提供一种口服降血糖多肽，具有良好的抗酶解特性。还提供该多肽的脂肪酸修饰衍生物，以及该多肽及其衍生物的用途。
本发明的主要研究过程如下：在前文所述发明专利中多肽的基础上，发明人继续通过氨基酸突变来减少可被肠道中蛋白酶降解的氨基酸位点，并筛选出一类具有胰蛋白酶、糜蛋白酶、弹性蛋白酶抗性的多肽；之后，通过一系列检测证明此类多肽可以通过口服途径给药，且具有明显的降糖活性。
本发明的技术方案如下：
一种口服降血糖多肽，其特征是，具有如下氨基酸序列：
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- Xaa1-Gln-Met-Glu-Glu-Glu-Ala-Val- Xaa2- Xaa3-Leu-Ile-Glu-Trp-Leu- Xaa4-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys，其中，
Xaa1为Met、Val、Ser或Lys；
Xaa2为Leu、Tyr、Val或Lys；
Xaa3为Glu或Leu；
Xaa4为Met、Val或Lys。
优选地，所述氨基酸序列为以下序列之一：
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- Met-Gln-Met-Glu-Glu-Glu-Ala-Val- Leu- Leu-Leu-Ile-Glu-Trp-Leu- Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys；
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- Met-Gln-Met-Glu-Glu-Glu-Ala-Val- Tyr- Glu-Leu-Ile-Glu-Trp-Leu- Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys；
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- Val-Gln-Met-Glu-Glu-Glu-Ala-Val- Val- Leu-Leu-Ile-Glu-Trp-Leu- Val-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys；
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- Ser-Gln-Met-Glu-Glu-Glu-Ala-Val- Lys- Glu-Leu-Ile-Glu-Trp-Leu- Val-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys；
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- Lys-Gln-Met-Glu-Glu-Glu-Ala-Val- Lys- Glu-Leu-Ile-Glu-Trp-Leu- Val-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys；
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- Ser-Gln-Met-Glu-Glu-Glu-Ala-Val- Lys- Glu-Leu-Ile-Glu-Trp-Leu- Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys；
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- Ser-Gln-Met-Glu-Glu-Glu-Ala-Val- Lys- Glu-Leu-Ile-Glu-Trp-Leu- Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys；
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- Lys-Gln-Met-Glu-Glu-Glu-Ala-Val- Leu- Leu-Leu-Ile-Glu-Trp-Leu- Val-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys。
前文所述的口服降血糖多肽用于制备预防或治疗糖尿病药物或药物组合物、或用于制备降血糖药物或药物组合物的用途。
一种口服降血糖多肽脂肪酸修饰衍生物，其特征是，所述多肽为前文所述的口服降血糖多肽；脂肪酸的修饰位点为Xaa1、Xaa2、Xaa4之一或其任意组合；当Xaa1为修饰位点时Xaa1为Lys，当Xaa2为修饰位点时Xaa2为Lys，当Xaa3为修饰位点时Xaa3为Lys。
优选地，所述脂肪酸为10-(4-羧基苯氧基)癸酸或17-羧基十七烷酸；所述脂肪酸经[2-(2-{2-[2-(2-{2-[-4-羧基-4-(氨基)丁酰基氨基]乙氧基}乙氧基)乙酰基氨基]乙氧基}乙氧基)乙酰基]与修饰位点氨基酸的氨基氮原子相连。
前文所述的口服降血糖多肽脂肪酸修饰衍生物用于制备预防或治疗糖尿病药物或药物组合物、或用于制备降血糖药物或药物组合物的用途。
一种药物组合物，其特征是，含有前文所述的口服降血糖多肽和/或前文所述的口服降血糖多肽脂肪酸修饰衍生物。
前文所述的药物组合物用于制备预防或治疗糖尿病药剂、或用于制备降血糖药剂的用途。
其中，所述药剂的剂型为经胃肠道给药剂型或非经胃肠道给药剂型。
在前文所述的用途中，所述糖尿病为Ⅰ型或Ⅱ型糖尿病。
与现有技术相比，本发明的多肽及其脂肪酸修饰衍生物具有针对更多种蛋白酶的酶解抗性，能更好地避免在胃肠道中被酶解失效，更加适宜作为口服降血糖药物。
附图说明
图1为本发明实施例5中IPCM1与IPCM5激活cAMP含量的测定结果图。
图2为本发明实施例6中IPCM1与IPCM5在急性腹腔注射糖耐量实验中对血糖控制能力的检测结果图。
图3为本发明实施例7中IPCM1与IPCM5在db/db小鼠中对糖化血红蛋白水平的检测结果图。
图4为本发明实施例8中IPCM1与IPCM5在db/db小鼠中对胰岛素抵抗改善水平的检测结果图。
图5为本发明实施例9中IPCM1与IPCM5在db/db小鼠中对血清胰岛素水平影响检测结果图。
图6为本发明实施例10中IPCM1与IPCM5在db/db小鼠治疗结束后的胰脏切片观察图。(A：正常组；B：生理盐水组；C：IPCM1给药治疗组；D：IPCM5给药治疗组)
图7为本发明实施例11中IPCM1与IPCM5在db/db小鼠治疗结束后的肾脏切片观察图。(A：正常组；B：生理盐水组；C：IPCM1给药治疗组；D：IPCM5给药治疗组)
图8为本发明实施例12中IPCM1与IPCM5在db/db小鼠治疗结束后的肝脏切片观察图。(A：正常组；B：生理盐水组；C：IPCM1给药治疗组；D：IPCM5给药治疗组)
具体实施方式
下面参照附图并结合实施例对本发明作进一步详细描述。但是本发明不限于所给出的例子。
在本发明具体实施的说明中，首先采用序列IPCM1-8作代表性阐述。
本发明多肽的通式如SEQ ID NO.1所示；IPCM1-8的序列如下：
IPCM1，即SEQ ID NO.2：
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- Met-Gln-Met-Glu-Glu-Glu-Ala-Val- Leu- Leu-Leu-Ile-Glu-Trp-Leu- Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys。
IPCM2，即SEQ ID NO.3：
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- Met-Gln-Met-Glu-Glu-Glu-Ala-Val- Tyr- Glu-Leu-Ile-Glu-Trp-Leu- Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala -Pro-Pro-Pro-Ser-Cys。
IPCM3，即SEQ ID NO.4：
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- Val-Gln-Met-Glu-Glu-Glu-Ala-Val- Val- Leu-Leu-Ile-Glu-Trp-Leu- Val-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys。
IPCM4，即SEQ ID NO.5：
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- Ser-Gln-Met-Glu-Glu-Glu-Ala-Val- Lys- Glu-Leu-Ile-Glu-Trp-Leu- Val-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys。
IPCM5，即SEQ ID NO.6：
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- Lys-Gln-Met-Glu-Glu-Glu-Ala-Val- Lys- Glu-Leu-Ile-Glu-Trp-Leu- Val-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys。
IPCM6，即SEQ ID NO.7：
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- Ser-Gln-Met-Glu-Glu-Glu-Ala-Val- Lys- Glu-Leu-Ile-Glu-Trp-Leu- Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys。
IPCM7，即SEQ ID NO.8：
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- Ser-Gln-Met-Glu-Glu-Glu-Ala-Val- Lys- Glu-Leu-Ile-Glu-Trp-Leu- Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys。
IPCM8，即SEQ ID NO.9：
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- Lys-Gln-Met-Glu-Glu-Glu-Ala-Val- Leu- Leu-Leu-Ile-Glu-Trp-Leu- Val-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys。
实施例1 IPCM1-8抗酶解能力测定
采用高效液相色谱法来测定IPCM1-8的抗酶解能力：
第一步：配制三种0.005mg/mL的蛋白酶溶液，三种蛋白酶分别为胰蛋白酶、糜蛋白酶、弹性蛋白酶，将各蛋白酶溶液分别于37℃孵育，孵育时间为15min。
第二步：将500μL 0.5mg/mL的IPCM1-8溶液与500μL各蛋白酶溶液分别进行混合， 即每种蛋白酶：IPCM1-8＝1:100(w/w)，于37℃进行反应，然后在各预定的时间点(0，5，15，30，60min)取出50μL反应液，加入50μL 1％TFA溶液终止反应。12000rpm离心5min后，收集上清液，利用RP-HPLC对上清检测。同时，采用Exendin-4C(即艾塞那肽)作为对照样品，以相同体积和浓度进行处理并检测。
第三步：利用RP-HPLC检测：色谱柱为Zorbax Eclipse Plus C18反相色谱柱；流动相A相为含0.065％TFA的双蒸水，B相为含0.05％TFA的乙腈溶液：进样量为50μL；泵流速为1mL/min；检测波长为215nm，检测程序如下：
Figure PCTCN2018104501-appb-000001
检测结果如表1所示。
表1 IPCM1-8抗酶解能力检测结果
Figure PCTCN2018104501-appb-000002
由该结果可知，与Exendin-4C相比，IPCM1-8针对上述三种关键蛋白酶的抗性均有不同程度的提高，其中针对胰蛋白酶抗性的提高最为显著，IPCM1、IPCM2、IPCM5、IPCM6以及IPCM8的残余率均达80％以上。同时IPCM1-8针对弹性蛋白酶和糜蛋白酶的抗性也有不同程度的提高，其中针对弹性蛋白酶抗性提高较为显著的为IPCM1、IPCM3、IPCM5以及IPCM7；针对糜蛋白酶抗性提高较为显著的为IPCM1和IPCM5。
实施例2 IPCM1-8激活胰高血糖素样肽-1受体能力的测定
第一步：CHO/GLP-1R-CRE化学发光细胞株复苏后，转入含5mL细胞生长培养基的培养瓶中，于37℃、5％CO 2培养箱中培养。待细胞贴壁生长至细胞贴壁面的80％左右时，用0.25％胰蛋白酶消化传代，每3天按1：3比例传代1次。细胞传至第三代后使用荧光素酶报告基因法进行体外活性测定。
第二步：实验测定时，先用0.25％胰蛋白酶消化细胞，再加入分析培养基。以2×10 5个细胞/mL的量将细胞液接种于96孔板，每孔接种100μL，于37℃、5％CO 2培养箱中培养4h。
第三步：取出96孔板，每孔加入20μL的IPCM1-8溶液/Exendin-4阳性对照。Exendin-4采用280nmol/L的初始浓度，4倍倍比稀释，一共稀释10个梯度；IPCM1-8采用则250nmol/L的初始浓度，7倍倍比稀释，一共稀释10个梯度。每种浓度的药物设置3个复孔。轻轻振荡混匀30秒后放入37℃、5％CO 2培养箱中继续培养4h。
第四步：取出96孔板，加入化学发光底物(Steady-Glo)试剂，100μL/孔，室温下将96孔板放于振荡器上轻轻振荡40分钟。
第五步：利用化学发光酶标仪，测定每孔的相关化学发光单位(RLU)。
第六步：以样品的log10(浓度)为横坐标，每一浓度的平均RLU为纵坐标，绘制量效曲线。通过Graphpad 5.0非线性拟合求出各曲线的EC50值，计算候选分子以及Exendin-4阳性对照的相对效价。结果如表2所示。
表2 IPCM1-8激活胰高血糖素样肽-1受体能力测定结果
Figure PCTCN2018104501-appb-000003
实验结果从半数有效浓度EC 50和引起的最大反应值Max Respones两方面衡量，表明与原型分子Exednin-4相比，IPCM1-8均具有激活胰高血糖素样肽-1受体的能力，其中，IPCM1和IPCM5的半数有效浓度分别为0.75nM和0.83nM，与原型分子处于同一数量级范围内，最大反应值则分别为107.36±5.94％和103.60±6.91％，两者略高于原型分子，说明IPCM1和 IPCM5在提高关键蛋白酶抗性的同时最大程度的保留了原型分子的生物学活性。
实施例3 除IPCM1-8外其余序列的抗酶解能力和激活胰高血糖素样肽-1受体能力
除IPCM1-8外，本发明多肽通式所包括其余序列的各位点所取氨基酸如下表所示：
Figure PCTCN2018104501-appb-000004
Figure PCTCN2018104501-appb-000005
Figure PCTCN2018104501-appb-000006
以上序列按实施例1方法测定抗酶解能力，不同之处仅在于：第二步中，与蛋白酶混合后仅于时间点60min取出50μL反应液。
以上序列按实施例2方法测定激活胰高血糖素样肽-1受体能力。
受篇幅所限，此处不再一一列出具体实验数据。结果表明：与Exendin-4C相比，IPCM9-96针对三种蛋白酶的抗性均有不同程度的提高；同时，IPCM9-96均具有激活胰高血糖素样肽-1受体的能力。
实施例4 IPCM1-96脂肪酸修饰衍生物的抗酶解能力和激活胰高血糖素样肽-1受体能力
IPCM1-96脂肪酸修饰衍生物中，脂肪酸与修饰位点氨基酸的氨基氮原子通过[2-(2-{2-[2-(2-{2-[-4-羧基-4-(氨基)丁酰基氨基]乙氧基}乙氧基)乙酰基氨基]乙氧基}乙氧基)乙酰基]相连，各衍生物中具体脂肪酸和修饰位点如下表所示。
Figure PCTCN2018104501-appb-000007
Figure PCTCN2018104501-appb-000008
Figure PCTCN2018104501-appb-000009
Figure PCTCN2018104501-appb-000010
Figure PCTCN2018104501-appb-000011
Figure PCTCN2018104501-appb-000012
Figure PCTCN2018104501-appb-000013
以上脂肪酸修饰衍生物按实施例1方法测定抗酶解能力，不同之处仅在于：第二步中，与蛋白酶混合后仅于时间点60min取出50μL反应液。
以上脂肪酸修饰衍生物按实施例2方法测定激活胰高血糖素样肽-1受体能力。
受篇幅所限，此处不再一一列出具体实验数据。结果表明：与Exendin-4C相比，EX1-208针对三种蛋白酶的抗性均有不同程度的提高；同时，EX1-208均具有激活胰高血糖素样肽-1受体的能力。
由此可见，对于如SEQ ID NO.1所示通式，当Xaa1、Xaa2、Xaa4之一或其任意组合为 Lys时，以10-(4-羧基苯氧基)癸酸或17-羧基十七烷酸对其进行修饰，所得脂肪酸修饰衍生物仍然具备蛋白酶抗性和激活胰高血糖素样肽-1受体的能力。
实施例5 IPCM1与IPCM5激活cAMP含量的测定
第一步：利用本实验室前期构建的可于细胞膜表面稳定表达GLP-1受体并连接荧光素酶报告基因的CHO细胞株进行GLP-1受体下游信号分子cAMP激活含量的测定。细胞复苏及培养条件如前所述。
第二步：测定时，先使用消化液消化细胞，再按细胞生长状况，加入适量含0.25％胎牛血清和0.5mM IBMX的分析培养基，使细胞浓度达到2×10 5个/mL。向96孔板中每孔加入100μL细胞液，置于上述条件培养箱中培养4小时。
第三步：向每孔加入不同浓度梯度的类似物溶液及阳性药Exendin-4溶液。类似物采用10μM的初始浓度，10倍倍比稀释7个梯度浓度，Exendin-4采用1μM的初始浓度，10倍倍比稀释7个梯度浓度。每个浓度的药物设置3个复孔。轻轻振荡混匀30秒后放入培养箱中继续培养4小时。
第四步：结束后取出96孔板，于-80℃冰箱和37℃培养箱中反复冻融，破碎细胞。离心后取上清，按照ELISA试剂盒中的操作要求检测cAMP含量，并对结果进行非线性回归分析。
结果如图1所示，原型分子Exendin-4激活cAMP的EC 50＝4.03nM，而IPCM1和IPCM5激活下游cAMP的EC 50分别为6.43nM和9.48nM，三者均处于同一数量级范围内。这三种分子在刺激cAMP产生时的最大反应值分别为Exendin-4:0.74±0.04mM(n＝3)；IPCM1:0.78±0.04mM(n＝3,P>0.05)；IPCM5:0.70±0.02mM(n＝3,P>0.05)。这说明IPCM1和IPCM5与对照组Exendin-4无显著性差异。
实施例6 IPCM1与IPCM5在急性腹腔注射糖耐量实验中对血糖控制能力的检测
第一步：实验选取8-12周龄、雄性的C57小鼠32只，随机分为4组，PBS对照组、Exendin-4(0.10mg/kg)皮下注射给药组和IPCM1高剂量(2.11mg/kg)灌胃给药组，IPCM5高剂量(2.82mg/kg)灌胃给药组，每组8只小鼠。
第二步：实验提前12小时对小鼠禁食，饮水自由摄取。实验开始时测定各小组小鼠空腹血糖，完成后开始按分组给药。
第三步：30分钟后利用尾静脉取血测定各组小鼠血糖水平，测量完成后对小鼠腹腔注射葡萄糖溶液(2g/kg)，然后于15分钟、30分钟、60分钟、90分钟、120分钟处监测小鼠血糖水平。
第四步：绘制血糖—时间曲线并计算曲线下面积(Area Under the Curve,AUC)。
实验结果如图2中A所示，各组小鼠的血糖水平均于15分钟时达到峰值，其中PBS组峰值显著高于另外三组小鼠，表明PBS经灌胃给药不具有血糖控制的效果，而IPCM1和IPCM5经灌胃给药的小鼠血糖峰值显著低于前者且接近与阳性药Exendin-4皮下注射组，并在120分钟内维持的显著的血糖控制效果。B为AUC统计结果，结果表明阳性药Exendin-4皮下注射组小鼠获得最好血糖控制效果，IPCM1和IPCM5则显著优于PBS对照组，略高于阳性药治疗组小鼠。
实施例7 IPCM1与IPCM5在db/db小鼠中对糖化血红蛋白水平的检测
第一步：动物分组及给药方案。db/db自发糖尿病小鼠，6-7周龄，雄性，35只，适应环境饲养2周后测定空腹血糖，并平均分至4组：PBS对照组、Exendin-4皮下注射组、IPCM1(2.11mg/kg)灌胃给药组、IPCM5(2.82mg/kg)灌胃给药组，每组7只。另设db/m对照组小鼠8只作为正常对照。各给药组小鼠每天早晚各给药一次。
第二步：分别于第一周、第四周及第八周对各组小鼠进行眼眶静脉丛取血，加入预先处理的抗凝管中，取样后迅速低温离心，取上清按以下步骤分别测定血红蛋白总数和糖化血红蛋白水平，并计算比例。
第三步：使用试剂盒对小鼠总血红蛋白的检测。将初始浓度为480pg/mL的标准品按比例分别稀释至240pg/mL、120pg/mL、60pg/mL和30pg/mL。
第四步：分别设空白孔(不加样品及酶标试剂，其余各步操作相同)、标准孔、待测样品孔。在酶标包被板上标准品精确加样50μL，待测样品孔中先加样品稀释液40μL，再加入待测样品10μL。加样时尽量将样品加于酶标板孔底部，不可触及孔壁，轻轻晃动摇匀。
第五步：用封板膜封板后置37℃孵育30分钟。此时将浓缩洗涤液稀释30倍后备用。
第六步：轻轻揭掉封板膜，弃去液体，甩干，每孔加满洗涤液，静置30秒后弃去，重复5次并拍干。
第七步：除空白孔外，每孔中加入酶标试剂50μL后重复步骤3温育和步骤4洗涤。
第八步：每孔加入显色剂A 50μL，再加入显色液B 50μL，轻轻震荡摇匀，37℃避光显色10分钟。
第九步：每孔加入终止液50μL，立即终止反应。15分钟内将样品板放入多功能读板仪，以空白孔调零，检测450nM波长处吸光值。
第十步：以标准品浓度为横坐标，吸光值为纵坐标，绘制标准曲线，根据样品的吸光值由标准曲线计算出相应浓度并乘以稀释倍数。
第十一步：使用试剂盒对小鼠糖化血红蛋白进行检测。使用糖化血红蛋白ELISA检测试 剂盒，重复以上1-8步骤，计算不同样品的糖化血红蛋白浓度并除以总血红蛋白浓度。
至八周治疗结束时，db/m组小鼠6.29±0.42％(n＝7)，仍处于正常范围内。PBS对照组小鼠达到11.10±0.56％(n＝7)。阳性药Exendin-4皮下注射给药组小鼠为8.22±0.89％(n＝7，P＝2.32×10 -2)，具有极显著的治疗效果。IPCM1和IPCM5灌胃给药组小鼠治糖化血红蛋白水平分别为8.90±0.66％(n＝7，P＝2.95×10 -2)和8.50±0.68％(n＝7，P＝1.51×10 -2)。分析结果可知，只有高剂量给药条件下db/db小鼠的糖化血红蛋白水平具有显著的治疗效果，且治疗效果与阳性药组一致，两组间无显著性差异。
实施例8 IPCM1与IPCM5在db/db小鼠中对胰岛素抵抗改善水平的检测
第一步：动物分组及给药方案如实施例7所示。实验前，小鼠禁食6小时，腹腔注射1U/kg胰岛素。
第二步：于0、15、30、60、90、120分钟处对小鼠进行眼眶静脉丛取血后迅速低温离心，取上清并按葡萄糖氧化酶偶联比色法要求测定血糖水平，绘制血糖浓度-时间曲线。
八周治疗结束后胰岛素耐量实验结果如图4所示。以各组小鼠正常状态下血糖水平为100％。胰岛素注射后30分钟，各组小鼠血糖水平迅速下降；至60分钟时各组小鼠血糖水平达到最低值，db/m组小鼠为30.62±5.71％(n＝7)，PBS对照组为91.76±7.63％(n＝7)，阳性药Exendin-4皮下注射组为：64.39±5.63％(n＝7，P＝1.43×10 -3)，获得了较好的治疗效果。IPCM1和IPCM5治疗组小鼠对胰岛素的响应与PBS组间产生显著性差异前者达到69.50±7.39％(n＝7，P＝4.01×10 -2)和65.74±7.66％(n＝7，P＝4.09×10 -2)。60分钟至120分钟时，各组小鼠血糖水平逐步恢复至胰岛素注射前水平。
实施例9 IPCM1与IPCM5在db/db小鼠中对血清胰岛素水平影响检测
第一步：动物分组及给药方案如实施例7所示。对各组小鼠进行眼眶静脉丛取血，加入预先处理的抗凝管中，取样后迅速低温离心。
第二步：先将试剂盒在室温下平衡30min。将初始浓度为172.8mIU/L的标准品按比例分别稀释至86.4mIU/L、43.2mIU/L、21.6mIU/L、10.8mIU/L和5.4mIU/L。
第三步：分别设空白孔(不加样品及酶标试剂，其余各步操作相同)、标准孔、待测样品孔。在酶标包被板上标准品精确加样50μL(标准品中已事先整合好生物素抗体，故不加)，待测样品孔中先加待测样品40μL，再加入抗-INS抗体10μL，再在每孔中加入链霉素-HRP 50μL。加样时尽量将样品加于酶标板孔底部，不可触及孔壁，轻轻晃动摇匀。
第四步：用封板膜封板后置37℃孵育60分钟。此时将浓缩洗涤液稀释后备用。
第五步：轻轻揭掉封板膜，弃去液体，甩干，每孔加满洗涤液，静置30秒后弃去，重 复5次并拍干。
第六步：每孔加入显色剂A 50μL，再加入显色液B 50μL，轻轻震荡摇匀，37℃避光显色10分钟。
第七步：每孔加入终止液50μL，立即终止反应。10分钟内将样品板放入多功能读板仪，以空白孔调零，检测450nm波长处吸光值。
第八步：以标准品浓度为横坐标，吸光值为纵坐标，绘制标准曲线，根据样品的吸光值由标准曲线计算出相应样品中的胰岛素浓度。
八周给药治疗结束后血清胰岛素水平检测结果如图5所示。db/m小鼠血清中胰岛素含量为13.59±1.14mIU/L(n＝7)，各组db/db小鼠血清胰岛素水平均稍低于或显著低于这一水平。其中PBS对照组小鼠为8.10±0.75mIU/L(n＝7,P＝6.08×10 -3)；阳性药Exendin-4皮下注射组为11.58±0.36mIU/L(n＝7,P＝0.16)，已接近db/m组小鼠且与后者无组间显著性差异；IPCM1和IPCM5治疗组小鼠血清胰岛素水平分别为11.17±0.82mIU/L(n＝7,P＝0.15)和12.18±0.43mIU/L(n＝7,P＝0.36)，两者均已接近db/m组小鼠且与后者无显著性差异，具有极显著的治疗效果。
实施例10 IPCM1与IPCM5在db/db小鼠治疗结束后的胰脏切片
动物分组及给药方案如实施例7所示。将小鼠处死后，切除胰腺，储存于10％的中性甲醛中。组织切片厚度为6μm，然后按照标准步骤用苏木精-伊红染色法进行染色，并用光学显微镜观察切片。其中分组情况为A：正常组；B：生理盐水组；C：IPCM1给药治疗组；D：IPCM5给药治疗组。
结果如图6所示，正常对照组小鼠的胰岛大小正常，形态完整，胰岛β细胞均正常；PBS对照组小鼠可见胰岛组织出现萎缩，大多数胰岛β细胞消失或仅有少数残留。IPCM1及IPCM5给药组与PBS组相比胰岛组织结构、胰岛β细胞病变均有所减轻，并且胰岛组织面积也较大，说明IPCM1与IPCM5能够保护糖尿病小鼠胰腺β细胞，改善胰岛功能。
实施例11 IPCM1与IPCM5在db/db小鼠治疗结束后的肾脏切片
动物分组及给药方案如实施例7所示。将小鼠处死后，切除肾脏，储存于10％的中性甲醛中。组织切片厚度为6μm，然后按照标准步骤用苏木精-伊红染色法进行染色，并用光学显微镜观察切片。其中分组情况为A：正常组；B：生理盐水组；C：IPCM1给药治疗组；D：IPCM5给药治疗组。
结果如图7所示，正常对照组小鼠的肾小球形态正常，肾小囊腔清晰；PBS组中肾小囊腔有所缩小，肾小管上皮细胞出现细胞水肿，并伴有明显的炎细胞浸润；IPCM1与IPCM5 给药组肾小球形态良好，肾小囊腔清晰。说明两个月的给药周期对db/db小鼠的肾功能没有明显的损害，且其通过降低血糖从而减轻肾负荷的途径改善了肾小球的形态。
实施例12 IPCM1与IPCM5在db/db小鼠治疗结束后的肝脏切片
动物分组及给药方案如实施例7所示。将小鼠处死后，切除肝脏，储存于10％的中性甲醛中。组织切片厚度为6μm，然后按照标准步骤用苏木精-伊红染色法进行染色，并用光学显微镜观察切片。其中分组情况为A：正常组；B：生理盐水组；C：IPCM1给药治疗组；D：IPCM5给药治疗组。
结果如图8所示，正常对照组小鼠肝脏细胞形态大小正常，细胞核致密，索状结构清晰；PBS组肝细胞失去正常的索状结构，肝窦消失，肝细胞显著肿胀，胞浆几乎完全透明，普遍发生明显的气球样变及脂肪变性；IPCM1与IPCM5给药组肝脏细胞呈现轻微的细胞肿大及胞浆疏松化，但索状结构仍较清晰。
除上述实施例外，本发明还可以有其他实施方式。凡采用等同替换或等效变换形成的技术方案，均落在本发明要求的保护范围。
Figure PCTCN2018104501-appb-000014
Figure PCTCN2018104501-appb-000015
Figure PCTCN2018104501-appb-000016
Figure PCTCN2018104501-appb-000017

Claims (9)

  1. 一种口服降血糖多肽，其特征是，具有如下氨基酸序列：
    His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser -Xaa1-Gln-Met-Glu-Glu-Glu-Ala-Val- Xaa2- Xaa3-Leu-Ile-Glu-Trp-Leu- Xaa4-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys，其中，
    Xaa1为Met、Val、Ser或Lys；
    Xaa2为Leu、Tyr、Val或Lys；
    Xaa3为Glu或Leu；
    Xaa4为Met、Val或Lys。
  2. 根据权利要求1述及的口服降血糖多肽，其特征是，所述氨基酸序列为以下序列之一：
    His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- Met-Gln-Met-Glu-Glu-Glu-Ala-Val- Leu- Leu-Leu-Ile-Glu-Trp-Leu- Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys；
    His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- Met-Gln-Met-Glu-Glu-Glu-Ala-Val- Tyr- Glu-Leu-Ile-Glu-Trp-Leu- Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys；
    His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- Val-Gln-Met-Glu-Glu-Glu-Ala-Val- Val- Leu-Leu-Ile-Glu-Trp-Leu- Val-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys；
    His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- Ser-Gln-Met-Glu-Glu-Glu-Ala-Val- Lys- Glu-Leu-Ile-Glu-Trp-Leu- Val-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys；
    His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- Lys-Gln-Met-Glu-Glu-Glu-Ala-Val- Lys- Glu-Leu-Ile-Glu-Trp-Leu- Val-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys；
    His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- Ser-Gln-Met-Glu-Glu-Glu-Ala-Val- Lys- Glu-Leu-Ile-Glu-Trp-Leu- Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys；
    His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- Ser-Gln-Met-Glu-Glu-Glu-Ala-Val- Lys- Glu-Leu-Ile-Glu-Trp-Leu- Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys；
    His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- Lys-Gln-Met-Glu-Glu-Glu-Ala-Val- Leu- Leu-Leu-Ile-Glu-Trp-Leu- Val-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys。
  3. 权利要求1或2述及的口服降血糖多肽用于制备预防或治疗糖尿病药物或药物组合物、或用于制备降血糖药物或药物组合物的用途，所述糖尿病为Ⅰ型或Ⅱ型糖尿病。
  4. 一种口服降血糖多肽脂肪酸修饰衍生物，其特征是，所述多肽为权利要求1或2述及的口服降血糖多肽；脂肪酸的修饰位点为Xaa1、Xaa2、Xaa4之一或其任意组合；当Xaa1为修饰位点时Xaa1为Lys，当Xaa2为修饰位点时Xaa2为Lys，当Xaa3为修饰位点时Xaa3为Lys。
  5. 根据权利要求4述及的多肽脂肪酸修饰衍生物，其特征是，所述脂肪酸为10-(4-羧基苯氧基)癸酸或17-羧基十七烷酸；所述脂肪酸经[2-(2-{2-[2-(2-{2-[-4-羧基-4-(氨基)丁酰基氨基]乙氧基}乙氧基)乙酰基氨基]乙氧基}乙氧基)乙酰基]与修饰位点氨基酸的氨基氮原子相连。
  6. 权利要求4或5述及的口服降血糖多肽脂肪酸修饰衍生物用于制备预防或治疗糖尿病药物或药物组合物、或用于制备降血糖药物或药物组合物的用途，所述糖尿病为Ⅰ型或Ⅱ型糖尿病。
  7. 一种药物组合物，其特征是，含有权利要求1或2述及的口服降血糖多肽和/或权利要求4或5述及的口服降血糖多肽脂肪酸修饰衍生物。
  8. 权利要求7述及的药物组合物用于制备预防或治疗糖尿病药剂、或用于制备降血糖药剂的用途，所述糖尿病为Ⅰ型或Ⅱ型糖尿病。
  9. 根据权利要求8述及的用途，其特征是，所述药剂的剂型为经胃肠道给药剂型或非经胃肠道给药剂型。
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