WO2018165933A1 - 调节糖代谢的多肽及其用途 - Google Patents

调节糖代谢的多肽及其用途 Download PDF

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WO2018165933A1
WO2018165933A1 PCT/CN2017/076882 CN2017076882W WO2018165933A1 WO 2018165933 A1 WO2018165933 A1 WO 2018165933A1 CN 2017076882 W CN2017076882 W CN 2017076882W WO 2018165933 A1 WO2018165933 A1 WO 2018165933A1
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seq
polypeptide
isap
pro
amino acid
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PCT/CN2017/076882
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English (en)
French (fr)
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任培根
张键
滕斌
李健
姚振宇
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深圳先进技术研究院
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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
    • A61K38/16—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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the present invention relates to the field of biomedicine, and in particular to polypeptides which modulate glucose metabolism and their use in the manufacture of a medicament for the treatment of diseases associated with abnormal glucose metabolism.
  • Diabetes is a major and growing health problem worldwide. Diabetes is a chronic disease in which the metabolism of carbohydrates, fats, and proteins is caused by a relative or absolute deficiency of insulin in the body or a decrease in insulin sensitivity of the target cells, or a structural defect in the insulin itself. Diabetes is clinically characterized by polydipsia, polyphagia, polyuria, and weight loss, with various complications such as ketoacidosis, limb gangrene, polyneuritis, blindness, and renal failure. According to the pathogenesis, diabetes can be divided into type 1 and type 2, of which 95% are type 2. Type 2 diabetes, also known as non-insulin-dependent diabetes mellitus, occurs more frequently after 35-40 years of age. The ability to produce insulin in the body is not completely lost or even modernized, but insulin resistance is produced in the body, resulting in relative deficiency.
  • Osteocalcin is a vitamin K-dependent calcium-binding protein synthesized and secreted by osteoblasts, a non-collagen acidic glycoprotein whose vitamin K-dependent glutamate residues are osteocalcin and Ca 2 + combines important functional groups [1, 2] .
  • the present inventors have unexpectedly discovered a polypeptide derived from osteocalcin which regulates glucose metabolism; it has the ability to promote insulin secretion, and can maintain blood glucose homeostasis, especially in the case of oral administration. Effect.
  • Some aspects of the invention relate to polypeptides that modulate sugar metabolism, represented by the formula M 1 -Z a -M 2 , wherein:
  • M 1 and M 2 are each independently a polypeptide segment having no more than 5, 4, 3, 2 or 1 amino acid residues or absent;
  • Z a is Tyr-Leu-X 1 -X 2 -X 3 -X 4 -Gly-Ala-X 5 -X 6 -Pro-X 7 -Pro-Asp-X 8 -Leu-Glu-Pro-X 9 , among them:
  • X 1 is Tyr, Asn, Asp or absent
  • X 2 is Gln, Asn, His, Pro, Ser or does not exist
  • X 3 is Trp, Gly or does not exist
  • X 4 is Leu or does not exist
  • X 5 is Pro or Ser
  • X 6 is Ala or Val
  • X 7 is Tyr or Ser
  • X 8 is Thr or Pro
  • the Z a may optionally have a medium amino acid substitution, insertion or deletion and the total number of amino acid substitutions, insertions and deletions does not exceed 4, preferably does not exceed 3, more preferably does not exceed 2, and most preferably does not exceed 1.
  • SEQ ID NO. 1 YLGASVPSPDPLEP
  • SEQ ID NO. 2 YLYQWLGAPVPYPDPLEP
  • SEQ ID NO. 3 YLYQWLGAPVPYPDPLEP
  • SEQ ID NO. 4 YLNNGLGAPAPYPDPLEP
  • SEQ ID NO. 5 YLYQWLGAPVPYPDTLEP
  • SEQ ID NO. 6 YLYQWLGAPVPYPDPLEP
  • SEQ ID NO. 7 YLDHWLGAPAPYPDPLEP
  • SEQ ID NO. 8 YLDPGLGAPAPYPDPLEP
  • SEQ ID NO. 9 YLDHGLGAPAPYPDPLEP
  • SEQ ID NO. 10 YLDQGLGAPAPAPDPLEP
  • SEQ ID NO. 11 YLDSGLGAPVPYPDPLEP.
  • Z a is YLYQWLGAPVPYPDPLEP, M 1 is absent and M 2 is present in the polypeptide Is Arg, ie the amino acid sequence of the polypeptide is set forth in SEQ ID NO. 3; in still other embodiments, Z a is YLGASVPSPDPLEP in the polypeptide, M 1 is absent and M 2 is Thr, ie the amino acid sequence of the polypeptide As shown in SEQ ID NO.
  • polypeptides which modulate glucose metabolism comprising at least 6 contiguous amino acids selected from any of the following sequences and having a total number of amino acid residues of no more than 18, such as no more than 17, 16, 15, 14:
  • SEQ ID NO. 1 YLGASVPSPDPLEP
  • SEQ ID NO. 2 YLYQWLGAPVPYPDPLEP
  • SEQ ID NO. 3 YLYQWLGAPVPYPDPLEP
  • SEQ ID NO. 4 YLNNGLGAPAPYPDPLEP
  • SEQ ID NO. 5 YLYQWLGAPVPYPDTLEP
  • SEQ ID NO. 6 YLYQWLGAPVPYPDPLEP
  • SEQ ID NO. 7 YLDHWLGAPAPYPDPLEP
  • SEQ ID NO. 8 YLDPGLGAPAPYPDPLEP
  • SEQ ID NO. 9 YLDHGLGAPAPYPDPLEP
  • SEQ ID NO. 10 YLDQGLGAPAPAPDPLEP
  • SEQ ID NO. 11 YLDSGLGAPVPYPDPLEP.
  • SEQ ID NO. 12 PVPYPDPLEP
  • SEQ ID NO. 14 PDPLEP
  • SEQ ID NO. 15 SVPSPDPLEP
  • SEQ ID NO. 16 PSPDPLEP.
  • SEQ ID NO. 12 PVPYPDPLEP
  • SEQ ID NO. 14 PDPLEP
  • SEQ ID NO. 15 SVPSPDPLEP
  • SEQ ID NO. 16 PSPDPLEP.
  • Some aspects of the invention relate to pharmaceutically acceptable salts of the polypeptides of the invention.
  • Some aspects of the invention relate to such a polypeptide of the invention, or a pharmaceutically acceptable salt thereof, having activity to increase insulin levels and lower blood glucose.
  • Some aspects of the invention relate to polynucleotides encoding the polypeptides of the invention.
  • Some aspects of the invention relate to vectors comprising the polynucleotides described above.
  • Some aspects of the invention relate to host cells transfected with the aforementioned vectors and capable of producing a polypeptide of the invention under conditions in which the protein can be expressed.
  • Some aspects of the invention are directed to a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide of the invention described above, or a pharmaceutically acceptable salt thereof.
  • Some aspects of the invention relate to the use of a polypeptide of the invention, or a pharmaceutically acceptable salt or pharmaceutical composition thereof, for the manufacture of a medicament for the treatment of a disorder associated with abnormal glucose metabolism, which may benefit from elevated insulin, blood glucose
  • the reduced disease is preferably type 2 diabetes, insulin resistance, and hyperglycemia.
  • Some aspects of the invention relate to methods of treating a disorder associated with abnormal glucose metabolism comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide of the invention, or a pharmaceutically acceptable salt or pharmaceutical composition thereof.
  • Some aspects of the invention relate to methods of treating a condition which may benefit from elevated insulin, reduced blood glucose, comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide of the invention, or a pharmaceutically acceptable salt or pharmaceutical composition thereof,
  • the disease is preferably type 2 diabetes.
  • a polypeptide of the invention can be used to treat a disorder associated with abnormal glucose metabolism, preferably a disease that benefits from elevated insulin, decreased blood glucose, and most preferably type 2 diabetes.
  • the pharmaceutical compositions of the invention can be administered with any of the pharmaceutical and method compositions known for treating diseases associated with abnormal glucose and fat metabolism.
  • a polypeptide of the invention can Administration by various conventional means is preferably oral. Oral administration reduces the inconvenience of conventional polypeptide administration, and the polypeptide of the present invention has few residues, greatly reduces the cost, and has a very significant potential advantage as a drug.
  • Figure 1 shows that in vitro experiments demonstrated that ISAP 1 promotes the expression of more insulin mRNA in ⁇ TC-6 cells, and real-time quantitative PCR analysis of insulin mRNA expression.
  • the ⁇ TC-6 cells were starved for 4 hours in the sugar-free medium containing 1% FBS before stimulation with the polypeptide, and stimulated with different concentrations of the polypeptide for 1 hour. All experiments were juxtaposed in groups and repeated 3 times. Results: mean ⁇ standard error *: P ⁇ 0.05, **: P ⁇ 0.01 (t test).
  • FIG. 2 shows that ISAP 1 promotes islet expression of more insulin mRNA real-time quantitative PCR analysis of insulin mRNA expression. Islet was starved for 4 hours in the sugar-free medium containing 1% FBS before polypeptide stimulation for 1 hour with different concentrations of polypeptide. All experiments were juxtaposed in groups and repeated 3 times. Results: mean ⁇ standard error *: P ⁇ 0.05, **: P ⁇ 0.01 (t test).
  • Figure 3 shows that in the glucose tolerance test, ISAP 1 causes a decrease in blood glucose levels in mice; (A) a trend plot at each time point, and (B) a comparison of the area under the curve in the results of A.
  • Figure 4 shows the effect of intraperitoneal injection of ISAP 1 on fasting blood glucose in obese-T2DM mice. *P ⁇ 0.05, **P ⁇ 0.01; compared to the HFD+ vehicle group.
  • Figure 5 shows the effect of intraperitoneal injection of ISAP 1 on insulin levels in serum of obese-T2DM mice. *P ⁇ 0.05, **P ⁇ 0.01.
  • FIG. 6 shows islet enlargement in T2DM mice treated with ISAP 1 , OCN (mouse osteocalcin). The quantitative statistics of AE are shown in F, and both ISAP 1 and OCN promote the islet area. ISAP 1 was able to significantly restore the size of islets in the pancreas of obese-T2DM mice.
  • A feeding normal feed (ND) and
  • B high fat feed (HFD) and HFD plus daily intraperitoneal injection
  • C OCN (6 pmol/g/d) or
  • D ISAP 1 (20 pmol/g/d) Or
  • E ISAP 1 (2 pmol/g/d)
  • Figure 7 shows the effect of intragastric administration of OCN, ISAP 1 , ISAP 2 , and ISAP3 on blood glucose levels in high fat diet (HFD) mice.
  • Figure 8 shows the combination of ISAP 1 and ISAP 2 with human GPRC6A and its internalization.
  • Figure 9 shows the effect of intragastric administration of ISAP 1 , ISAP 4 , ISAP 5 , and ISAP 6 on blood glucose in mice fed a high-fat diet: (A) effects on blood glucose, and (B) effects on GLP1.
  • Figure 10 shows a comparison of the sequences of ISAP from different species.
  • polysaccharide-modulating polypeptide of the present invention is also referred to as "Insulin Secretion Association Peptide (ISAP)", and refers to a polypeptide derived from osteocalcin or derived therefrom capable of regulating sugar metabolism.
  • IFP Insulin Secretion Association Peptide
  • conservative amino acid substitution refers to the replacement of the original amino acid sequence with another amino acid residue having similar properties.
  • lysine residues, arginine residues, and histidine residues are similar in that they have a basic side chain.
  • aspartic acid residues and glutamic acid residues are similar in that they have acidic side chains.
  • asparagine residues, glutamine residues, serine residues, threonine residues, tyrosine residues and cysteine residues are similar in that they have uncharged polar side chains
  • a glycine residue, an alanine residue, a proline residue, a leucine residue, an isoleucine residue, a proline residue, a tryptophan residue, a phenylalanine residue, and a Thionine residues are similar in that they have non-polar side chains.
  • the tyrosine residue, the phenylalanine residue, the tryptophan residue, and the histidine residue are similar in having an aromatic side chain.
  • diabetes characterized by a genetic or environmental or both, characterized by a glyco-thankton disorder or a complication thereof such as, but not limited to, type 2 diabetes, hyperglycemia, Insulin resistance and so on.
  • DMEM medium was purchased from Sigma, supplemented with 10% FBS (fetal calf serum), 1% non-essential amino acids, 1 g glucose, 0.75 g sodium bicarbonate, 0.1 g bovine serum albumin and 1.5 ml HEPES per 500 ml medium. - hydroxyethylpiperazineethanesulfonic acid).
  • the ISAP 1 sequence is Tyr-Leu-Gly-Ala-Ser-Val-Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro-Thr (SEQ ID NO. 22).
  • the sequence of ISAP 2 is Tyr-Leu-Tyr-Gln-Trp-Leu-Gly-Ala-Ser-Val-Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro (SEQ ID NO. 2).
  • the sequence of ISAP 3 is Tyr-Leu-Tyr-Gln-Trp-Leu-Gly-Ala-Ser-Val-Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro-Arg (SEQ ID NO. 3).
  • ISAP 4 sequence is Ser-Val-Pro-Ser- Pro-Asp-Pro-Leu-Glu-Pro (SEQ ID NO.15).
  • the ISAP 5 sequence was Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro (SEQ ID NO. 16).
  • the ISAP 6 sequence was Pro-Asp-Pro-Leu-Glu-Pro (SEQ ID NO. 14).
  • ⁇ TC-6 was purchased from the American Type Culture Collection (ATCC), a cell line derived from mouse pancreatic cancer and into which a large T antigen of SV40 ( monkey vacuolar virus 40) was introduced into the genome, which has the function of secreting insulin. .
  • ATCC American Type Culture Collection
  • SV40 monkey vacuolar virus 40
  • the ⁇ TC-6 cell suspension was plated at a density of 1.6 ⁇ 10 5 /mL in a 24-well plate at 0.5 mL per well, and cultured at 37 ° C, 5% CO 2 for 24 hours.
  • ISAP 1 was diluted to 2 nmol/L, 200 pmol/L, 20 pmol/L, 2 pmol/L in an ice bath with sugar-free DMEM.
  • mice Primary islets were isolated from mice according to the method described by Gregory L. Szot et al., Murine Pancreatic Islet Isolation, Journal of Visualized Experiments, 2007, and the primary islets were subjected to starvation pretreatment using KRBB buffer (129 g NaCl, 5 g NaHCO 3 ).
  • KRBB buffer 129 g NaCl, 5 g NaHCO 3 .
  • 4.8 g KCl, 1.2 g KH 2 PO 4 , 1.2 g MgSO 4 , 2.5 g CaCl 2 , 10 mM HEPES and 0.1% BSA were treated at pH 7.4 for 4 hours.
  • mice Healthy 6-week-old male C57BL/6SPF mice were purchased from experimental animals in Guangdongzhou. Heart, body weight 18-22g. Divided into two groups and housed in the SPF animal room of the Institute.
  • Control group Normal feed (ND) was administered to mice (normal feed: fat, 5%; carbohydrate, 53%; protein, 23%; total calories 25 J/g), free feeding and drinking, and fed for 12 weeks.
  • Experimental group mice were given high fat diet (HFD) (high fat diet D12451, Research Diets, Inc.), fed freely and drink, and fed for 12 weeks. Detect its physiological indicators. The mice fed the high-fat diet weighed more than 40 g, and the type 2 diabetes model (T2DM) was considered to be successful.
  • HFD high fat diet
  • T2DM type 2 diabetes model
  • Glucose tolerance tests after fasting T2DM mice for 16 hours, immediately after intraperitoneal injection of 20% glucose solution, immediately after injection of normal saline, ISAP 1 or OCN for 10 minutes, 30 minutes, 60 minutes, 90 minutes to detect changes in blood sugar.
  • ISAP 1 and has a short OCN role in T2DM mice.
  • the blood glucose levels were lowered at each time point in the OCN and ISAP 1 compared with the control group (injected with physiological saline). It demonstrates the role of ISAP 1 in lowering blood sugar.
  • T2DM mice were used and divided into 5 groups of 6 each. Use the corresponding feed and application method; the first group, normal feed (ND) + carrier; the second group, high fat feed (HFD) + carrier; the third group, HFD + OCN6pmol / g / d + carrier; Four groups, HFD + ISAP 1 20pmol / g / d + carrier; the fifth group, HFD + ISAP 1 2pmol / g / d + carrier; d represents daily intraperitoneal injection. Blood glucose levels were measured at weeks 0, 2, and 4, respectively, and approximately 10 microliters of blood was taken from the tip of the tail and measured using a Roche blood glucose meter according to the manufacturer's instructions. As a result, referring to Fig.
  • mice were euthanized with 90% carbon dioxide, the pancreas was removed, and the small pieces were collected and fixed in 4% paraformaldehyde solution for 24 hours, embedded in paraffin and sliced at a thickness of 5 ⁇ m (Leica RM2235). , Germany), then stained and observed with hematoxylin and eosin (C0105, Beyotime, China).
  • the experimental results are shown in Figure 6, using 40X magnification, AE to make the same scale; F is a quantitative analysis of A to E. Compared with the control, the islet area of the experimental group increased significantly, but the cell volume did not change significantly, indicating that ISAP 1 can promote islet proliferation.
  • hGPRC6A overexpression vector (pReceiver-M61) was transfected into HeLa cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions, and the normal medium was changed 4 hours after transfection.
  • ISAP 1 has a blood sugar lowering effect as compared with the control.
  • ISAP 2 features ISAP 2 combined with human GPRC6A
  • hGPRC6 overexpression vector (pReceiver-M61) was transfected into HeLa cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions, and the normal medium was changed 4 hours after transfection.
  • Cy5-tagged OCN, hOCN-22, ISAP 2 promotes internalization of GPRC6A in GPRC6A overexpressing HeLa cells.
  • the hGPRC6A-Hela cell suspension was plated at a density of 1.6 ⁇ 10 5 /mL in a 24-well plate pre-placed with gelatin-coated coverslips, 0.5 mL DMEM per well, and cultured at 37 ° C, 5% CO 2 . 24 hours.
  • the cells were starved for 4 hours in serum-free medium before treatment, and 100 nM of Cy5-OCN, Cy5-hOCN22, Cy5-ISAP 2 were added to each well, and incubated at 37 ° C for 30 minutes, and the cells were fixed with polymethanol for 30 minutes and added.
  • ISAP 2 and ISAP 3 were administered daily by a gavage method well known to those skilled in the art, and the polypeptide was dissolved in physiological saline using a 1 ml syringe and a 12-gauge needle at a gavage volume of 10 ⁇ L/g.
  • ISAP 3 has an effect of lowering blood sugar as compared with the control.
  • Binding embodiments experimental results of Examples 1 and 2, Comparative sequence ISAP 1, ISAP 2, ISAP 3 is found: with respect to 2, two alternative, and a deletion has four insertion ISAP 1, ISAP; relative ISAP 1, ISAP 3 has 4 insertions, 3 substitutions; relative to ISAP 2 , ISAP 1 has 4 deletions, 2 substitutions, and 1 insertion; in summary, it can be considered that the three polypeptides are variants of each other, presumably can be Based on the three sequences, conservative substitutions, insertions and deletions of amino acids well known to those skilled in the art are performed, provided that their ability to modulate glucose metabolism is not significantly reduced, for example, by no more than 40%, 30%, 20%, 10%.
  • SEQ ID NO. 2 YLYQWLGAPVPYPDPLEP
  • SEQ ID NO. 4 YLNNGLGAPAPYPDPLEP
  • SEQ ID NO. 5 YLYQWLGAPVPYPDTLEP
  • SEQ ID NO. YLYQWLGAPVPYPDPLEP SEQ ID NO. 7: YLDHWLGAPAPYPDPLEP
  • SEQ ID NO. 8 YLDPGLGAPAPYPDPLEP
  • SEQ ID NO. 9 YLDHGLGAPAPYPDPLEP
  • SEQ ID NO. 10 YLDQGLGAPAPAPDPLEP
  • SEQ ID NO. 1 has only one amino acid residue deleted in comparison with SEQ ID NO. 22, so it is inferred that SEQ ID NO. 1 also has a function similar to SEQ ID NO.
  • the experimental results of Examples 2 and 3 embodiment, with respect to the ISAP 2, ISAP 1 and 3 equivalent of the ISAP 2 having an insertion end of the ISAP; may be added to indicate that several amino acid residues at the terminus of a polypeptide, with the proviso that not to Its ability to regulate glucose metabolism is significantly reduced, for example, by no more than 40%, 30%, 20%, 10%, preferably no more than 5, such as 4, 3, 2, 1 or 0 amino acids. Residues.
  • mice Eighteen healthy 6-week-old male C57BL/6 SPF mice were selected in the same manner as in Example 1 and divided into 6 groups of 3 animals each. One group was the control group, and the mice were given normal feed (ND); The remaining 5 groups were given high-fat diet (HFD), freely fed and watered. The mice fed the high-fat diet weighed more than 40g and performed the following experiments:
  • mice 3-6 to the first group of mice (the HFD), to 2 pmol/g weight daily, orally administered respectively ISAP 1, ISAP 4, ISAP 5 , ISAP 6; to Group 1 mice (ND), a second group of small Rats (HFD) were intragastrically administered with an equal volume of saline.
  • HFD small Rats
  • the length of the polypeptide is closely related to the length of the synthesis.
  • the market price at the time of the present example is 4 mg
  • the synthesis price of the 6 amino acid residue polypeptide is 225 yuan (RMB); 8 amino acid residue polypeptide (such as ISAP 5 ) synthesis price of 300 yuan (RMB); 10 amino acid residue polypeptide (such as ISAP 4 ) synthesis price of 375 yuan (RMB); 15 amino acid residue polypeptide (such as ISAP 1 ) synthesis price of 562 yuan (RMB); the synthesis price of 46 amino acid residue polypeptides (such as mouse OCN) is 1675 yuan (RMB); considering the difference in molecular weight, the price of equimolar ISAP 6 is about one-sixtieth of that of mouse OCN. Large cost reduction, very suitable for large-scale manufacturing and use.
  • the polypeptide of the present invention can function orally.
  • most of the polypeptides administered by injection greatly facilitate the patient and improve the patient compliance, and thus have the medical field. Great application prospects.

Abstract

调节糖代谢的多肽及其在制备用于治疗与糖代谢有关之疾病的药物中用途。所述的多肽能够降低血糖，促进胰岛素分泌；可以用于制备用于治疗糖尿病的药物，并且可以与现有的治疗方案组合施用。所述的多肽具有治疗有效剂量低，可以经口施用等显著优点。

Description

调节糖代谢的多肽及其用途 技术领域
本发明涉及生物医药领域，具体地涉及调节糖代谢的多肽及其在制备用于治疗与糖代谢异常有关之疾病的药物中的用途。
发明背景
糖尿病是世界范围内主要且日益增长的健康问题。糖尿病是一种体内胰岛素相对或绝对不足或靶细胞对胰岛素敏感性降低，或胰岛素本身存在结构上的缺陷而引起的碳水化合物、脂肪和蛋白质代谢紊乱的一种慢性疾病。糖尿病在临床上表现为多饮、多食、多尿和体重减少，并伴随各种并发症如酮症酸中毒、肢体坏疽、多发性神经炎、失明和肾功能衰竭等。根据发病机理，将糖尿病可分为1型和2型，其中95％患者为2型。2型糖尿病又称非胰岛素依赖型糖尿病，多在35-40岁之后发病，其体内产生胰岛素的能力并非完全丧失甚至出现代偿性增高，但体内产生胰岛素抵抗，造成相对缺乏。
现有治疗2型糖尿病并没有可彻底治愈的方法出现，而且治疗药物必须伴随一生，并会伴有其他副作用，所以目前糖尿病的治疗还有很大的缺陷，需要开发性能更优的新药。
骨钙素是由成骨细胞合成和分泌的一种维生素K依赖性钙结合蛋白，一种非胶原酸性糖蛋白，其分子中的维生素K依赖性谷氨酸残基是骨钙素和Ca2+结合的重要功能基团[1,2]。
发明内容
本发明人出人意料的发现了一种来源于骨钙素的能够调节糖代谢的多肽；其具有促进胰岛素分泌的能力，能够维持血糖稳态，特别是在经口服用的情况下，同样可以取得良好的效果。
本发明的一些方面涉及调节糖代谢的多肽，所述多肽由式M1-Za-M2表示，其中：
M1、M2各自独立地为具有不超过5、4、3、2或1个氨基酸残基的多肽区段或者不存在；
Za为Tyr-Leu-X1-X2-X3-X4-Gly-Ala-X5-X6-Pro-X7-Pro-Asp-X8-Leu-Glu-Pro-X9，其中：
X1为Tyr、Asn、Asp或不存在，
X2为Gln、Asn、His、Pro、Ser或不存在，
X3为Trp、Gly或不存在，
X4为Leu或不存在，
X5为Pro或Ser，
X6为Ala或Val，
X7为Tyr或Ser，
X8为Thr或Pro，
并且所述Za可任选地具有中氨基酸替换、插入或缺失并且所述氨基酸替换、插入和缺失的总数不超过4，优选不超过3，更优选不超过2，最优选不超过1。
进一步地，本发明的一些实施方案中，所述多肽中的Za选自以下之一：
SEQ ID NO.1：YLGASVPSPDPLEP，
SEQ ID NO.2：YLYQWLGAPVPYPDPLEP
SEQ ID NO.3：YLYQWLGAPVPYPDPLEP，
SEQ ID NO.4：YLNNGLGAPAPYPDPLEP，
SEQ ID NO.5：YLYQWLGAPVPYPDTLEP，
SEQ ID NO.6：YLYQWLGAPVPYPDPLEP，
SEQ ID NO.7：YLDHWLGAPAPYPDPLEP，
SEQ ID NO.8：YLDPGLGAPAPYPDPLEP，
SEQ ID NO.9：YLDHGLGAPAPYPDPLEP，
SEQ ID NO.10：YLDQGLGAPAPAPDPLEP，
SEQ ID NO.11：YLDSGLGAPVPYPDPLEP。
进一步地，本发明的另一些实施方案中，所述多肽中的M1、M2均不存在；或者在又一些实施方案中，所述多肽中Za为YLYQWLGAPVPYPDPLEP、M1不存在且M2为Arg，即该多肽的氨基酸序列如SEQ ID NO.3所示；在又一些实施方案中，所述多肽中Za为YLGASVPSPDPLEP，M1不存在且M2为Thr，即该多肽的氨基酸序列如SEQ ID NO.22所示。
本发明的另一些方面涉及调节糖代谢的多肽，其包含选自以下任一序列之至少6个连续氨基酸且其氨基酸残基总数不超过18，例如不超过17、16、15、14：
SEQ ID NO.1：YLGASVPSPDPLEP，
SEQ ID NO.2：YLYQWLGAPVPYPDPLEP
SEQ ID NO.3：YLYQWLGAPVPYPDPLEP，
SEQ ID NO.4：YLNNGLGAPAPYPDPLEP，
SEQ ID NO.5：YLYQWLGAPVPYPDTLEP，
SEQ ID NO.6：YLYQWLGAPVPYPDPLEP，
SEQ ID NO.7：YLDHWLGAPAPYPDPLEP，
SEQ ID NO.8：YLDPGLGAPAPYPDPLEP，
SEQ ID NO.9：YLDHGLGAPAPYPDPLEP，
SEQ ID NO.10：YLDQGLGAPAPAPDPLEP，
SEQ ID NO.11：YLDSGLGAPVPYPDPLEP。
进一步地，其包含以下任一：
SEQ ID NO.12：PVPYPDPLEP，
SEQ ID NO.13：PYPDPLEP，
SEQ ID NO.14：PDPLEP，
SEQ ID NO.15：SVPSPDPLEP，
SEQ ID NO.16：PSPDPLEP。
更进一步地，其为
SEQ ID NO.12：PVPYPDPLEP，
SEQ ID NO.13：PYPDPLEP，
SEQ ID NO.14：PDPLEP，
SEQ ID NO.15：SVPSPDPLEP，
SEQ ID NO.16：PSPDPLEP。
本发明的一些方面涉及本发明的多肽的可药用盐。
本发明的一些方面涉及这样的本发明多肽或其可药用盐，其具有提高胰岛素水平、降低血糖的活性。
本发明的一些方面涉及多核苷酸，其编码本发明的多肽。
本发明的一些方面涉及载体，其包含前述的多核苷酸。
本发明的一些方面涉及宿主细胞，其转染有前述的载体并且能够在可表达蛋白质的条件下产生本发明的多肽。
本发明的一些方面涉及药物组合物，其包含治疗有效量的前述本发明多肽或其可药用盐。
本发明的一些方面涉及本发明的多肽或其可药用盐或药物组合物在制备用于治疗与糖代谢异常有关之疾病的药物中的用途，所述疾病为可受益于胰岛素升高、血糖降低的疾病，优选为2型糖尿病、胰岛素抵抗、高血糖。
本发明的一些方面涉及治疗与糖代谢异常有关之疾病的方法，其包括向由此需要的对象施用治疗有效量的本发明的多肽或其可药用盐或药物组合物。
本发明的一些方面涉及治疗可受益于胰岛素升高、血糖降低之疾病的方法，其包括向由此需要的对象施用治疗有效量的本发明的多肽或其可药用盐或药物组合物，所述疾病优选为2型糖尿病。
在一些实施方案中，本发明的多肽或其可药用盐或药物组合物可用于治疗与糖代谢异常有关之疾病，优选受益于胰岛素升高、血糖降低的疾病，最优选2型糖尿病。
在一些实施方案中，本发明的药物组合物可与任何已知用于治疗与糖和脂肪代谢异常有关之疾病的药物和方法组合物一起施用。
在一些实施方案中，本发明的多肽或其可药用盐或药物组合物可以 通过各种常规的方式施用，优选经口施用。经口施用减少了常规多肽施用的诸多不便，而且本发明多肽的残基很少，大大降低了成本，具有作为药物的非常显著的潜在优势。
附图简述
图1示出了在体外实验中证明ISAP1可促进βTC-6细胞表达更多的胰岛素mRNA，实时定量PCR分析胰岛素mRNA的表达。βTC-6细胞在多肽刺激前在含1％FBS的无糖培养基中饥饿4小时后用不同浓度的多肽刺激1小时。所有实验两个并列组、重复3次。结果：平均值±标准误*:P<0.05,**:P<0.01(t检验)。
图2示出了ISAP1可促使胰岛表达更多的胰岛素mRNA实时定量PCR分析胰岛素mRNA的表达。胰岛在多肽刺激前在含1％FBS的无糖培养基中饥饿4小时后用不同浓度的多肽刺激1小时。所有实验两个并列组、重复3次。结果：平均值±标准误*:P<0.05,**:P<0.01(t检验)。
图3示出了糖耐量检测中，ISAP1导致小鼠的血糖水平降低；(A)各时间点的趋势图，(B)A中结果的曲线下面积比较。
图4示出了腹腔注射ISAP1对肥胖-T2DM小鼠空腹血糖的影响。*P<0.05,**P<0.01；与HFD+载剂组相比。
图5示出了腹腔注射ISAP1对肥胖-T2DM小鼠血清中胰岛素水平的影响。*P<0.05,**P<0.01。
图6示出了经ISAP1、OCN(小鼠骨钙素)处理过的T2DM小鼠胰岛增大。F中示出A-E的定量统计结果，ISAP1、OCN均促使胰岛面积增大。ISAP1能够显著回复肥胖-T2DM小鼠胰腺中胰岛的大小。(A)饲喂普通饲料(ND)及(B)高脂饲料(HFD)以及HFD加之每日腹腔注射(C)OCN(6pmol/g/d)或(D)ISAP1(20pmol/g/d)或(E)ISAP1(2pmol/g/d),(F)双盲条件下计算出的连续切片中胰岛面积。放大倍数均为40×。*:P<0.05,**:P<0.01,***:P<0.001，与HFD+载剂组相比；每组6只小鼠。
图7示出了灌胃施用OCN、ISAP1、ISAP2、ISAP3对高脂饲料(HFD)的小鼠的血糖水平的影响。
图8示出了ISAP1和ISAP2分别与人GPRC6A的结合及其内化。A)人GPRC6A在Hela细胞中过表达；(B)ISAP1、OCN和OCN-22；以及 ISAP2、hOCN和hOCN-22；与huGPRC6A过表达的Hela细胞的细胞膜的结合实验；(C)带有Cy-5标记的OCN(Cy5-OCN)、带有Cy-5标记的OCN-22(Cy5-Ocn22)，带有Cy-5标记的ISAP2(Cy5-ISAP2)在GPRC6A过表达的Hela细胞中对GPRC6A内化的影响。
图9示出了灌胃施用ISAP1、ISAP4、ISAP5、ISAP6对于饲喂高脂饲料的小鼠的血糖的影响：(A)对血糖的影响，(B)对GLP1的影响。
图10示出了来自不同物种的ISAP的序列的比较。
具体实施方式
定义
本发明的“调节糖代谢的多肽”也称作“胰岛素分泌相关多肽(Insulin Secretion Association Peptide，ISAP)”，是指来源于骨钙素或由其衍生的能够调节糖代谢的多肽。
本文所使用的术语“保守氨基酸替换”是指用具有相似性质的另一个氨基酸残基对原氨基酸序列进行的替换。例如，赖氨酸残基、精氨酸残基和组氨酸残基在具有碱性侧链方面是相似的。此外，天冬氨酸残基和谷氨酸残基在具有酸性侧链方面是相似的。此外，天冬酰胺残基、谷氨酰胺残基、丝氨酸残基、苏氨酸残基、酪氨酸残基和半胱氨酸残基在具有不带电的极性侧链方面是相似的，并且甘氨酸残基、丙氨酸残基、缬氨酸残基、亮氨酸残基、异亮氨酸残基、脯氨酸残基、色氨酸残基、苯丙氨酸残基和甲硫氨酸残基在具有非极性侧链方面是相似的。此外，酪氨酸残基、苯丙氨酸残基、色氨酸残基和组氨酸残基在具有芳族侧链方面是相似的。因此，对本领域技术人员显而易见的是，在具有上述相似性质的氨基酸组中进行的氨基酸替换不会引起性质的任何变化。
本文中所使用的氨基酸简写及中文名称对照如下
中文名称 三字母简写 单字母简写
丝氨酸 Ser S
苏氨酸 Thr T
天冬酰胺 Asn N
谷氨酰胺 Gln Q
酪氨酸 Tyr Y
半胱氨酸 Cys C
天冬氨酸 Asp D
谷氨酸 Glu E
组氨酸 His H
赖氨酸 Lys K
精氨酸 Arg R
甘氨酸 Gly G
丙氨酸 Ala A
缬氨酸 Val V
亮氨酸 Leu L
异亮氨酸 Ile I
苯丙氨酸 Phe F
甲硫氨酸 Met M
脯氨酸 Pro P
色氨酸 Trp W
本文所使用的术语“糖代谢异常有关的疾病”是指有遗传或环境或二者共同导致的以糖代谢谢紊乱为特征的疾病或其并发症，例如但不限于2型糖尿病、高血糖、胰岛素抵抗等。
具体实施方式
DMEM培养基购自Sigma，其中每500ml培养基中补充10％FBS(胎牛血清)，1％非必须氨基酸，1g葡萄糖，0.75g碳酸氢钠，0.1g牛血清白蛋白以及1.5ml HEPES(4-羟乙基哌嗪乙磺酸)。
ISAP1序列为Tyr-Leu-Gly-Ala-Ser-Val-Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro-Thr(SEQ ID NO.22)。ISAP2的序列为Tyr-Leu-Tyr-Gln-Trp-Leu-Gly-Ala-Ser-Val-Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro(SEQ ID NO.2)。ISAP3的序列为Tyr-Leu-Tyr-Gln-Trp-Leu-Gly-Ala–Ser-Val-Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro-Arg(SEQ ID NO.3)。ISAP4序列为Ser-Val-Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro(SEQ ID NO.15)。ISAP5序列为Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro(SEQ ID NO.16)。ISAP6序列为Pro-Asp-Pro-Leu-Glu-Pro(SEQ ID NO.14)。
βTC-6购自美国典型培养物保藏中心(ATCC)，这一细胞系来源于小鼠胰腺癌并向其基因组内导入了SV40(猴空泡病毒40)的大T抗原，有分泌胰岛素的功能。
所有的动物实验均通过中国科学院先进技术研究院动物伦理委员会批准，按照伦理委员会的要求进行。
实施例1
ISAP1的功能
1、ISAP1对βTC-6细胞的影响。
实验步骤如下：
1.细胞铺板：将βTC-6细胞悬液以1.6×105/mL的密度铺板在24孔板中，每孔0.5mL，于37℃，5％的CO2中培养24小时。
2.用无糖的DMEM将ISAP1分别稀释至2nmol/L、200pmol/L、20pmol/L、2pmol/L冰浴保存。
3.对βTC-6细胞进行饥饿预处理，使用KRBB缓冲液(129g NaCl,5g NaHCO3,4.8g KCl,1.2g KH2PO4,1.2g MgSO4,2.5g CaCl2,10mM HEPES和0.1％BSA在pH 7.4)处理4小时。
4.弃去细胞培养液，向实验组和阳性对照组分别加入含有不同浓度的ISAP1和葡萄糖的培养液，阴性对照组仅加入培养基(空白)，于37℃，5％的CO2中作用1小时。
5.用无菌PBS清洗板底细胞两次，添加300μL的Trizol溶液，按 照RNAiso Plus(TaKaRa)说明书提取细胞RNA，经DNA酶处理后,用SuperScriptT(Invitrogen公司,加拿大)试剂盒经DNAEngine反转录成cDNA。以cDNA为模板,用SYBRGreen法对各时间点的荧光PCR产物进行实时监测分析(Light Cycler Roche公司,德国)，以检测胰岛素基因的表达量。
实时定量PCR使用的引物，鼠Ins-1：
正向5’-CCAGCTATAATCAGAGACCA-3’，
反向5’-CCAGGTGGGGACCACAAAGA-3’。
GAPDH作为内参
正向5’-AGCAGTCCCGTACACTGGCAAAC-3’
反向5’-TCTGTGGTGATGTAAATGTCCTCT--3’。
实验结果见图1，不同浓度的ISAP1均可促进βTC-6细胞表达更多的胰岛素mRNA。
2、ISAP1对胰岛的影响。
按照Gregory L.Szot等，Murine Pancreatic Islet Isolation，Journal of Visualized Experiments，2007所述的方法，从小鼠分离原代胰岛，对原代胰岛进行饥饿预处理，使用KRBB缓冲液(129g NaCl,5g NaHCO3,4.8g KCl,1.2g KH2PO4,1.2g MgSO4,2.5g CaCl2,10mM HEPES和0.1％BSA在pH 7.4)处理4小时。弃去KRBB液，向阳性对照组加入含16.8mM葡萄糖的DMEM培养基，实验组则加入含有不同浓度的ISAP1无糖DMEM培养液，阴性对照组仅加入无糖DMEM培养基(空白)，于37℃，5％的CO2中作用1小时。实验结果参照图2、不同浓度的ISAP1均可促进胰岛表达更多的胰岛素mRNA。
3、体内实验中ISAP1对糖代谢的影响。
3.1高脂喂养建立2型糖尿病模型
健康6周龄雄性C57BL/6SPF级小鼠购买自广东省实验动物中 心，体质量18-22g。分两组，饲养于本研究院SPF级动物房。对照组：给予小鼠正常饲料(ND)(正常饲料：脂肪，5％；碳水化合物，53％；蛋白，23％；总热量25J/g)，自由摄食和饮水，喂养12周。实验组：给予小鼠高脂饲料(HFD)(高脂饲料D12451，Research Diets,Inc.)，自由摄食和饮水，喂养12周。检测其生理指标。高脂饲料喂养的小鼠体重超过40g，认为2型糖尿病模型(T2DM)构建成功。
3.2代谢实验检测
糖耐量检测(Glucose tolerance tests，GTT)，在将T2DM小鼠禁食16个小时后，经腹腔注射20％葡萄糖溶液后1分钟立刻注射生理盐水、ISAP1或OCN，于10分钟，30分钟，60分钟，90分钟检测其血糖变化。ISAP1和OCN在T2DM小鼠中具有短时间的作用。实验结果如图3中所示，OCN和ISAP1与对照组(注射生理盐水)相比，在各时间点，血糖水平均降低。证明了ISAP1降低血糖的作用。
3.3长期施用ISAP1对糖代谢的影响
使用T2DM小鼠，分5组，每组6只。采用相应的饲料和施用方式；第一组，正常饲料(ND)+载剂；第二组，高脂饲料(HFD)+载剂；第三组，HFD+OCN6pmol/g/d+载剂；第四组，HFD+ISAP120pmol/g/d+载剂；第五组，HFD+ISAP1 2pmol/g/d+载剂；d代表每天腹腔注射。分别在第0、2、4周时测量血糖水平，从尾尖采取约10微升血液，使用罗氏血糖仪，按照制造商的说明书进行测量。结果参见图4，与对照组(第二组)相比，血糖水平降低。在第0和6周使用Ultra Sensitive Mouse Insulin ELISA Kit(cystalchem公司，美国)和MultiskanTM FC酶标仪(Thermo公司，美国)测量血清中的胰岛素水平。结果参见图5，ISAP1改善了血清中胰岛素水平。
3.4组织形态学分析
完成步骤3.3的试验后，使用90％浓度的二氧化碳将小鼠安乐死，取出胰脏，采集小块组织固定于4％多聚甲醛溶液中24小时，石蜡包埋并以5μm厚度进行切片(Leica RM2235,Germany)，之后用苏木精和伊红(C0105,Beyotime,中国)对组织染色并观察。实验结果参见附图6，使用40X放大倍数，A-E使相同的比例尺；F是对A至E的定量分析。与对 照相比，实验组的胰岛面积显著增加，而细胞体积并无明显改变，说明ISAP1可以促使胰岛增殖。
4、ISAP1与人GPRC6A的结合
4.1在Hela细胞中过表达hGPRC6A 1)细胞铺板：将Hela细胞悬液以1.6×105/mL的密度铺板在6孔板中，每孔2mL DMEM培养基，于37℃，5％的CO2中培养24小时。
2)使用Lipofectamine2000(Invitrogen)，按照厂商的说明书将hGPRC6A过表达载体(pReceiver-M61)转染到Hela细胞中，转染后4小时更换正常培养基。
3)转染后48小时，弃掉培养基，用无菌PBS清洗板底细胞两次，添加300μL的Trizol溶液，按照RNAiso Plus(TaKaRa)说明书提取细胞RNA，经DNA酶处理后,用SuperScriptT(Invitrogen公司,加拿大)试剂盒经DNAEngine反转录成cDNA。以cDNA为模板,用SYBRGreen法对各时间点的荧光PCR产物进行实时监测分析(Light Cycler Roche公司,德国)，以检测hGPRC6A的表达量。如有需要，使用嘌呤霉素筛选得到稳定表达细胞株，之后使用qPCR检测其基因表达，实验结果如图6A所示，人GPRC6A在Hela中稳定大量表达实验结果如图8A所示，人GPRC6A在Hela中稳定大量表达。
4.2 ISAP1与过表达hGPRC6A的Hela细胞的细胞膜的结合实验，
实验结果见图8B，与OCN相比，ISAP1具有几乎一致的膜结合能力，而OCN-22不具备这种能力，证明ISAP1是OCN的核心结构域，与受体hGPRC6A相互作用。
5、灌胃施用ISAP1对高脂饲料(HFD)的小鼠的血糖水平的影响
健康6周龄雄性C57BL/6小鼠，体质量18-22g，分6组，每组5只，第一组，正常饲料(ND)；第二组，高脂饲料(HFD)；第三组，HFD+OCN2pmol/g；第四组，HFD+ISAP1 2pmol/g；第五组，HFD+ISAP2 2pmol/g；第六组，HFD+ISAP3 2pmol/g；其中OCN和ISAP1、ISAP2、ISAP3通过本领域技术人员公知的灌胃方法每天施用，使用1ml注射器和12号针头，将多肽溶解在生理盐水中，灌胃体积为10μL/g。
四周后，从尾尖采取约10微升血液，使用罗氏血糖仪，按照制造商的说明书进行测量。结果参见图7，与对照相比，ISAP1具有降低血糖的作用。
实施例2
ISAP2的功能ISAP2与人GPRC6A的结合
2.1在Hela细胞中过表达hGPRC6A 1)细胞铺板：将Hela细胞悬液以1.6×105/mL的密度铺板在6孔板中，每孔2mL DMEM培养基，于37℃，5％的CO2中培养24小时。
2)使用Lipofectamine2000(Invitrogen)，按照厂商的说明书将hGPRC6过表达载体(pReceiver-M61)转染到Hela细胞中，转染后4小时更换正常培养基。
3)转染后48小时，弃掉培养基，用无菌PBS清洗板底细胞两次，添加300μL的Trizol溶液，按照RNAiso Plus(TaKaRa)说明书提取细胞RNA，经DNA酶处理后,用SuperScriptT(Invitrogen公司,加拿大)试剂盒经DNAEngine反转录成cDNA。以cDNA为模板,用SYBRGreen法对各时间点的荧光PCR产物进行实时监测分析(Light Cycler Roche公司,德国)，以检测hGPRC6A的表达量。如有需要，使用嘌呤霉素筛选得到稳定表达细胞株，之后使用qPCR检测其基因表达，实验结果如图6A所示，人GPRC6A在Hela中稳定大量表达实验结果如图8A所示，人GPRC6A在Hela中稳定大量表达。
2.2 ISAP2与过表达hGPRC6A的Hela细胞的细胞膜的结合实验
实验结果见图8B，与hOCN相比，ISAP2具有几乎一致的膜结合能力，而hOCN-22不具备这种能力，结合实施例1中项4.2的结果，证明ISAP1和ISAP2分别是OCN和hOCN的核心结构域，均与受体hGPRC6A相互作用，从而推定ISAP1和ISAP2具有相同的功能，通过hGPRC6A引起后续的信号传导和生物学事件。
2.3带有Cy5标记的OCN、hOCN-22，ISAP2在GPRC6A过表达的Hela细胞中促进GPRC6A的内化。
将表达hGPRC6A-Hela细胞悬液以1.6×105/mL的密度铺板在预先放置明胶涂层盖玻片的24孔板中，每孔0.5mL DMEM，于37℃，5％的CO2中培养24小时。细胞在处理前，用无血清培养基饥饿4小时后，每孔添加100nM的Cy5-OCN、Cy5-hOCN22，Cy5-ISAP2，37℃孵育30分钟，使用多聚甲醇固定细胞30分钟，并加入Triton X-100(sigma)孵育10分钟，DAPI(Sigma)10秒按照厂商的说明书，对细胞和染色，使用荧光共聚焦显微镜观察并拍照，实验结果参见图6C。可见，Cy5-OCN和Cy5-ISAP2细胞内部有分布，而Cy5-hOCN22则分布在胞外，再次说明了Cy5-OCN和Cy5-ISAP2均可与受体结合并通过内在化作用进入细胞内，从而发挥调节能量代谢的作用。
2.4灌胃施用ISAP2对高脂饲料(HFD)的小鼠的血糖水平的影响
[01]健康6周龄雄性C57BL/6小鼠，体质量18-22g，分6组，每组5只，第一组，正常饲料(ND)；第二组，高脂饲料(HFD)；第三组，HFD+OCN2pmol/g；第四组，HFD+ISAP1 2pmol/g；第五组，HFD+ISAP2 2pmol/g；第六组，HFD+ISAP3 2pmol/g；其中OCN和ISAP1、ISAP2、ISAP3通过本领域技术人员公知的灌胃方法每天施用，使用1ml注射器和12号针头，将多肽溶解在生理盐水中，灌胃体积为10μL/g。
[02]四周后，从尾尖采取约10微升血液，使用罗氏血糖仪，按照制造商的说明书进行测量。结果参见图7，与对照相比，ISAP2具有降低血糖的作用。
实施例3
ISAP3的功能
灌胃施用ISAP3对高脂饲料(HFD)的小鼠的血糖水平的影响
健康6周龄雄性C57BL/6小鼠，体质量18-22g，分6组，每组5只，第一组，正常饲料(ND)；第二组，高脂饲料(HFD)；第三组，HFD+OCN2pmol/g；第四组，HFD+ISAP1 2pmol/g；第五组，HFD+ISAP2 2pmol/g；第六组，HFD+ISAP3 2pmol/g；其中OCN和ISAP1、ISAP2、ISAP3通过本领域技术人员公知的灌胃方法每天施用，使用1ml注射器和12号针头，将多肽溶解在生理盐水中，灌胃体积为10μL/g。
四周后，从尾尖采取约10微升血液，使用罗氏血糖仪，按照制造商的说明书进行测量。结果参见图7，与对照相比，ISAP3具有降低血糖的作用。
结合实施例1和2的实验结果，对比ISAP1、ISAP2、ISAP3的序列可知：相对于ISAP1,ISAP2具有4个插入，2个替换，和1个缺失；相对于ISAP1，ISAP3具有4个插入，3个替换；相对于ISAP2，ISAP1具有4个缺失，2个替换，和1个插入；综上所述，可以认为三个多肽互为变体，推定可以以这三个序列为基础，进行本领域技术人员公知的氨基酸保守替换、插入和缺失，条件是不使其调节糖代谢的能力显著降低，例如降低不超过40％、30％、20％、10％。参见附图10中对多种不同生物来源的同源序列进行了对比，其中SEQ ID NO.2：YLYQWLGAPVPYPDPLEP，SEQ ID NO.4：YLNNGLGAPAPYPDPLEP，SEQ ID NO.5：YLYQWLGAPVPYPDTLEP，SEQ ID NO.6：YLYQWLGAPVPYPDPLEP，SEQ ID NO.7：YLDHWLGAPAPYPDPLEP，SEQ ID NO.8：YLDPGLGAPAPYPDPLEP，SEQ ID NO.9：YLDHGLGAPAPYPDPLEP，SEQ ID NO.10：YLDQGLGAPAPAPDPLEP，SEQ ID NO.11：YLDSGLGAPVPYPDPLEP之间差异很小，两两比较时，多数情况下差异不超过四个氨基酸的替换，因此可以推定这些序列具有类似的生物学功能，也应当包含在本发明的范围内，优选地，其中缺失、替换和插入的总数不超过4，例如不超过3、2、1。另外SEQ ID NO.1与SEQ ID NO.22相比仅缺失了一个氨基酸残基，因此推断SEQ ID NO.1也具有与SEQ ID NO.22类似的功能。
同时，结合实施例2和3的实验结果,相对于ISAP2，ISAP1和ISAP3相当于在ISAP2末端具有1个插入；表明可以在多肽的末端添加若干个氨基酸残基，条件是不使其调节糖代谢的能力显著降低，例如降低不超过40％、30％、20％、10％，优选地末端添加不超过5个，例如4个、3个、2个、1个或0个氨基酸残基。
实施例4
类似实施例1中所述方法，选取健康6周龄雄性C57BL/6SPF级小鼠18只，分为6组，每组3只。一组为对照组，给予小鼠正常饲料(ND)； 其余5组给予小鼠高脂饲料(HFD)，自由摄食和饮水，高脂饲料喂养的小鼠体重超过40g后进行以下实验：
给第3-6组小鼠(HFD)，每日以2pmol/g体重、分别灌胃施用ISAP1、ISAP4、ISAP5、ISAP6；给第1组小鼠(ND)、第2组小鼠(HFD)分别灌胃施用等体积盐水。持续进行7周，每周从尾尖采取约10微升血液，使用罗氏血糖仪监测一次血糖水平，实验结果参照图9A，灌胃施用ISAP1、ISAP4、ISAP5、ISAP6均导致血糖水平的显著降低。
同时，在实验结束后，对血液中GLP1的水平，使用针对GLP1的抗体进行检测，实验结果参见图9B。与两组对照组相比，ISAP1、ISAP4、ISAP5、ISAP6均导致GLP1水平大幅度提高。证明了他们和ISAP1、ISAP2、ISAP3在生物学功能上相当，同时，参照图10的序列对比，也可以看出SEQ ID NO.12：PVPYPDPLEP与SEQ ID NO.15：SVPSPDPLEP，SEQ ID NO.13：PYPDPLEP与SEQ ID NO.16：PSPDPLEP在序列上相似度非常高，同时在各个物种之间也是非常保守的序列，因而应当具有类似的生物活性，应涵盖在本发明的范围内。
本领域人员公知的，多肽的长度与合成长度有密切联系，以本实施例进行时的市价为例，重量均为4mg，6氨基酸残基多肽(例如ISAP6)合成价格225元(人民币)；8氨基酸残基多肽(例如ISAP5)合成价格300元(人民币)；10氨基酸残基多肽(例如ISAP4)合成价格375元(人民币)；15氨基酸残基多肽(例如ISAP1)合成价格562元(人民币)；46氨基酸残基多肽(例如小鼠OCN)合成价格1675元(人民币)；考虑到分子量的区别，等摩尔的ISAP6的价格约为小鼠OCN的约六十分之一，极大的降低了成本，非常适合大规模制造和使用。
同时，本发明的多肽口服即可起到作用，相比现有技术中，绝大部分以注射施用的多肽而言，极大的方便了患者，改善了患者依从性，因此是具有医药领域的巨大应用前景。
通过引用并入
本文中提及的所有出版物和专利通过引用全文并入本文，如同每个单独的出版物或专利被具体地且单独地表明通过引用并入。在冲突的情 况下，以本申请(包括本文中的任何定义)为准。
等同方案
虽然本文中已经明确地公开了本发明的一些具体实施方案，以上说明书是举例说明性的而非限制性的。对于本领域的技术人员来说，通过浏览本说明书和所附权利要求，本发明的许多变化形式将变得明显。本发明的全部范围应通过参照权利要求、其等同方案的全部范围、以及本说明书和这样的变化来确定。

Claims (16)

  1. 调节糖代谢的多肽，所述多肽由式M1-Za-M2表示，其中：
    M1、M2各自独立地为具有不超过5、4、3、2或1个氨基酸残基的多肽区段或者不存在；
    Za为Tyr-Leu-X1-X2-X3-X4-Gly-Ala-X5-X6-Pro-X7-Pro-Asp-X8-Leu-Glu-Pro，其中：
    X1为Tyr、Asn、Asp或不存在，
    X2为Gln、Asn、His、Pro、Ser或不存在，
    X3为Trp、Gly或不存在，
    X4为Leu或不存在，
    X5为Pro或Ser，
    X6为Ala或Val，
    X7为Tyr或Ser，
    X8为Thr或Pro，
    并且所述Za可任选地具有中氨基酸替换、插入或缺失并且所述氨基酸替换、插入和缺失的总数不超过4。
  2. 根据权利要求1所述的多肽，其中所述Za选自以下之一：
    SEQ ID NO.1：YLGASVPSPDPLEP，
    SEQ ID NO.2：YLYQWLGAPVPYPDPLEP
    SEQ ID NO.4：YLNNGLGAPAPYPDPLEP，
    SEQ ID NO.5：YLYQWLGAPVPYPDTLEP，
    SEQ ID NO.6：YLYQWLGAPVPYPDPLEP，
    SEQ ID NO.7：YLDHWLGAPAPYPDPLEP，
    SEQ ID NO.8：YLDPGLGAPAPYPDPLEP，
    SEQ ID NO.9：YLDHGLGAPAPYPDPLEP，
    SEQ ID NO.10：YLDQGLGAPAPAPDPLEP，
    SEQ ID NO.11：YLDSGLGAPVPYPDPLEP。
  3. 根据权利要求2所述的多肽，其中所述M1、M2均不存在。
  4. 根据权利要求2所述的多肽，其中Za为YLYQWLGAPVPYPDPLEP，M1不存在且M2为Arg；或者其中Za为YLGASVPSPDPLEP，M1不存在且M2为Thr。
  5. 调节糖代谢的多肽，其包含权利要求3所述的多肽的至少6个连续氨基酸且其氨基酸残基总数不超过18。
  6. 根据权利要求5所述的多肽，其包含以下之一：
    SEQ ID NO.12：PVPYPDPLEP，
    SEQ ID NO.13：PYPDPLEP，
    SEQ ID NO.14：PDPLEP，
    SEQ ID NO.15：SVPSPDPLEP，
    SEQ ID NO.16：PSPDPLEP。
  7. 根据权利要求6所述的多肽，其为
    SEQ ID NO.12：PVPYPDPLEP，
    SEQ ID NO.13：PYPDPLEP，
    SEQ ID NO.14：PDPLEP，
    SEQ ID NO.15：SVPSPDPLEP，
    SEQ ID NO.16：PSPDPLEP。
  8. 权利要求1至7中任一项所述的多肽的可药用盐。
  9. 多核苷酸，其编码权利要求1至7中任一项所述的多肽。
  10. 载体，其包含权利要求9所述的多核苷酸。
  11. 宿主细胞，其转染有权利要求10所述的载体并且能够在可表达蛋白质的条件下产生权利要求1至7中任一项所述的多肽。
  12. 药物组合物，其包含治疗有效量的权利要求1至7中任一项所述的多肽或权利要求8所述的可药用盐。
  13. 权利要求1至7中任一项所述的多肽或权利要求8所述的可药 用盐或权利要求12所述的药物组合物在制备用于治疗与糖代谢异常有关之疾病的药物中的用途。
  14. 权利要求13所述的用途，其中所述疾病为可受益于胰岛素升高、血糖降低的疾病。
  15. 权利要求14所述的用途，其中所述疾病可以为2型糖尿病、胰岛素抵抗、高血糖。
  16. 权利要求1至7中任一项所述的多肽或权利要求8所述的可药用盐或权利要求12所述的药物组合物，其特征在于可经口施用。
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