WO2019200594A1 - 酰化的glp-1衍生物 - Google Patents

酰化的glp-1衍生物 Download PDF

Info

Publication number
WO2019200594A1
WO2019200594A1 PCT/CN2018/083789 CN2018083789W WO2019200594A1 WO 2019200594 A1 WO2019200594 A1 WO 2019200594A1 CN 2018083789 W CN2018083789 W CN 2018083789W WO 2019200594 A1 WO2019200594 A1 WO 2019200594A1
Authority
WO
WIPO (PCT)
Prior art keywords
glp
analog
pharmaceutically acceptable
acceptable salt
derivative
Prior art date
Application number
PCT/CN2018/083789
Other languages
English (en)
French (fr)
Inventor
许峥
李峰
宋瑞
郭万军
潘海
Original Assignee
杭州先为达生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 杭州先为达生物科技有限公司 filed Critical 杭州先为达生物科技有限公司
Priority to PCT/CN2018/083789 priority Critical patent/WO2019200594A1/zh
Priority to JP2021506033A priority patent/JP7164247B2/ja
Priority to PCT/CN2019/083383 priority patent/WO2019201328A1/zh
Priority to KR1020227008742A priority patent/KR102653113B1/ko
Priority to AU2019256245A priority patent/AU2019256245B2/en
Priority to MX2020011007A priority patent/MX2020011007A/es
Priority to KR1020207033205A priority patent/KR102464605B1/ko
Priority to CA3135910A priority patent/CA3135910C/en
Priority to CA3202015A priority patent/CA3202015A1/en
Priority to BR112020021245A priority patent/BR112020021245A8/pt
Priority to US17/048,550 priority patent/US11612640B2/en
Priority to EP19789171.6A priority patent/EP3783014A4/en
Publication of WO2019200594A1 publication Critical patent/WO2019200594A1/zh
Priority to AU2021204749A priority patent/AU2021204749B2/en
Priority to JP2022164849A priority patent/JP2022191398A/ja
Priority to US18/186,069 priority patent/US20230330189A1/en

Links

Images

Classifications

    • 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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • 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
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides

Definitions

  • the invention belongs to the field of polypeptide technology.
  • the invention relates to fatty acid modified derivatives of GLP-1 (7-37) polypeptide analogs.
  • the present invention relates to a method for producing the peptide derivative, a medicament containing the peptide derivative, a use in the preparation of a medicament, and the like.
  • Diabetes is a disorder of glucose metabolism caused by various factors such as genetics and environment. It has become the third major disease that threatens human health and life after cancer and cardiovascular and cerebrovascular diseases. Diabetes itself does not necessarily cause harm, but long-term blood sugar is increased, large blood vessels, micro-vessels are damaged and endanger the heart, brain, kidney, peripheral nerves, eyes, feet, etc. According to the World Health Organization, diabetes complications are as high as 100 kinds. One of the diseases with the most complications. More than half of the deaths due to diabetes are caused by cardiovascular and cerebrovascular diseases, and 10% are caused by nephropathy. Amputation due to diabetes is 10 to 20 times that of non-diabetes. To treat diabetes and prevent its complications is a social issue that is crucial.
  • Diabetes can be divided into several types due to different mechanisms of disease. Most of them belong to type 2 diabetes (about 90%), mainly due to overweight and lack of physical activity. Patients with type 2 diabetes often have abnormalities in insulin resistance and insufficient insulin secretion. Apoptosis of islet ⁇ cells often occurs in the middle and late stages of the disease. At present, the mechanism of action of oral hypoglycemic agents used in clinical practice is to enhance insulin sensitivity, or to promote insulin secretion to stabilize blood sugar, and the problem of ⁇ -cell apoptosis cannot be solved.
  • Glucagon-like peptide-1 (GLP-1) and its analogues have been used to treat type 2 diabetes by slowing the apoptosis of ⁇ -cells, enhancing their regeneration, and promoting the differentiation and proliferation of islet ⁇ cells. Focus.
  • glucagon-like peptide-1 BELL G I, SANCHEZ-
  • PG proglucagon
  • PESCADOR R LAYBOURN P J, et al. Exon duplication and divergence in the human preproglucagon gene [J]. Nature, 1983, 304 (5924): 368-371).
  • the PG gene sequence consists of 6 exons and 5 introns and contains 3 major domains: glucagon (33-61), GLP-1 (72-108) and GLP-2 (126 ⁇ ). 158).
  • the mRNA of PG is expressed in pancreatic A cells, intestinal L cells and brain, and specific translational modifications are made in these tissue cells to finally form different end products.
  • GLP-1 analogue is a polypeptide hormone secreted by Langerhans's cell in the terminal jejunum, ileum and colon. It has glucose-dependent insulin secretion and biosynthesis, inhibits glucagon secretion and It inhibits various functions such as gastric emptying.
  • GLP-2 analogues are synthesized in the intestinal tissue and in the brainstem and hypothalamic neurons of the central nervous system, mainly promoting the growth of normal small intestine and repairing intestinal mucosal damage (Fu Gang, Gong Yu, Xu Weiren. Pancreas Research progress of glucagon-like peptide 1 and its receptor agonist [J]. Tianjin Medicine, 2012, 40 (2): 181-184.).
  • GLP-1 is an endogenous hormone that promotes insulin secretion, mainly secreted by intestinal L-cells, and plays a role in balancing insulin and glucose levels.
  • GLP-1 histidine (His)-asparagine (Asn)-glutamic acid (Glu)-phenylalanine (Phe)-glutamic acid (Glu)-arginine (Arg - histidine (His)-alanine (Ala)-glutamic acid (Glu)-glycine (Gly)-threonine (Thr)-phenylalanine (Phe)-threonine (Thr)- Serine (Ser)-aspartic acid (Asp)-valine (Val)-serine (Ser)-serine (Ser)-tyrosine (Tyr)-leucine (Leu)-glutamic acid (Glu) - glycine (Gly)-glutamine (Gln)-alanine (Ala)-alanine (Ala)-lysine (Lys)-glutamic acid (Glu)-phenylalanine (Phe)-iso Leucine (Ile)
  • DDP-IV can rapidly degrade histidine (H)-alanine (A) at the 7th to 8th positions of the N-terminus.
  • DDP-IV mainly acts as a peptide chain end hydrolase, and the 8th position is alanine or guanidine.
  • Acid which degrades and rapidly inactivates GLP-1 (AERTGEERTS K, YE S, TENNANT MG, et al.Crystal structure of human dipeptidyl peptidase IV in complex with a dipeptide peptidase reveals details on substrate specificity and Tetrahedral intermediate [J]. Protein Sci, 2004, 13(2): 412-421).
  • SARRAUSTE DE MENTHIERE and other GLP-1 models were constructed to observe the changes in affinity and intrinsic activity of GLP-1 analogues and receptors after amino acid substitution.
  • the histidine at position 7 is the determinant of affinity and intrinsic activity.
  • the ring is smaller than tryptophan and does not have any polar substituents; the polar group in the alanine side chain at the 8th position affects the activity of GLP-1, and the volume of the side chain cannot be too large, beyond a certain limit.
  • the activity will decrease; when glutamic acid at position 9 is replaced by certain amino acids, such as acidic, polar and hydrophobic amino acids, the activity does not change, and the activity is decreased or even inactivated when replaced with basic amino acids; GLP-1 Binding to the receptor, if the amino acid residue has an ionic bond, it occurs between amino acids 7-15, forming a ring structure, and Ala8-Glu9-Gly10-Thr11 forms a ⁇ -turn, making the 7th position Three aromatic nucleuses, such as histidine, phenylalanine at position 12, and tyrosine at position 19, interact with the hydrophobic pockets of aromatic clusters present on the receptor, presumably they Activate the role of the receptor; on the 22nd Amino acid glycine is a flexible, flexible connection play role in maintaining the coiled-coil shape.
  • amino acids such as acidic, polar and hydrophobic amino acids
  • Destruction of glycine causes all aromatic amino acids to be clustered, but the affinity for the receptor is reduced by 1/40 (SARAUSTE DE MENTHIEREC, CHAVANIEUA, GRASSYG, et al.Structural requirements of the N-terminal region of GLP-1-[7- 37]-NH 2 for receptor interaction and cAMP production [J]. Eur J Med Chem, 2004, 39(6): 473-480).
  • GLP-1 includes molecular forms such as GLP-1 (1-37), GLP-1 (1-36), GLP-1 (7-37) glycine derivatives, and GLP-1 (7-36) NH 2 . It is generally believed that the latter two have the same biological activity. GLP-1 (1-37) secreted by intestinal mucosal L cells is inactive and requires further hydrolysis to excise the N-terminal 6 amino acids to become active GLP-1 (7-37). GLP-1 (7-37) is present in the body for a short period of time and is rapidly degraded. Accordingly, various studies have been conducted on GLP-1 analogs having anti-DPP IV. For example, U.S. Patent No.
  • 5,554,618 describes the modification of the N-terminus with an alkyl group or an acyl group
  • Gallwitz et al. describe the seventh-position His for N. Methylation or a methylation, or substitution of imidazole with the entire His to increase resistance to DPP-IV and maintain physiological activity.
  • the GLP-1 analogue exendin-4 (US Pat. No. 5,424,686) purified from the salivary glands of the Hieratolus has resistance to DPP IV and is higher than GLP-1. Physiological activity. Therefore, it has an in vivo half-life of 2 to 4 hours longer than the half-life of GLP-1.
  • DPP IV resistance is applied, physiological activity cannot be sufficiently maintained, and in the case of using commercially available exenatide, it requires injection twice a day to the patient, which It is still very painful for the patient.
  • insulinotropic peptides have a small molecular weight and are therefore rapidly excreted from the kidneys.
  • Some scientists have used chemical methods to add high solubility polymers such as polyethylene glycol to the surface of peptides to inhibit their loss in the kidney.
  • U.S. Patent No. 692,464 describes the binding of PEG to the lysine residue of Exendin-4 to increase its residence time in vivo.
  • this method while increasing the residence time of the peptide drug in vivo, With the increase in molecular weight, the concentration of the peptide drug is significantly reduced, and the reactivity to the peptide is also lowered.
  • WO 96/29342 discloses peptide hormone derivatives modified by introducing a lipophilic substituent at the C-terminal amino acid residue of the parent peptide hormone or at the N-terminal amino acid residue.
  • WO 98/08871 discloses a GLP-1 derivative (liraglutide) in which at least one amino acid residue of a parent peptide is linked to a lipophilic substituent.
  • WO 99/43708 discloses lipophilic substituents GLP-1 (7-35) and GLP-1 (7-36) derivatives having an amino acid residue linked to the C-terminus.
  • WO 00/34331 discloses bisacylated GLP-1 analogs.
  • WO 00/69911 discloses activated insulinotropic peptides for injection, which are believed to react with blood components to form conjugates in patients, prolonging the duration of action in vivo.
  • WO2006/097537 discloses another acylated GLP-1 analogue (semaglutide) having a mutated amino acid at position 8 to an unnatural amino acid, compared to the acylated GLP-1 (liraglutide) of WO 98/08871 Longer half-life.
  • WO 02/046227 discloses the use of genetic recombination techniques to prepare fusion proteins by binding GLP-1, Exendin-4 or an analog thereof to human serum albumin or immunoglobulin region (Fc), which can be solved, for example, by polyethylation
  • the diolization yields are low and non-specific, but their effect on increasing half-life in blood is still not as significant as expected.
  • various types of peptide connectors have been tried, but the problem faced by this method is that it may cause an immune response.
  • CN107033234A discloses a fatty acid modified conjugate of a GLP-1 analog, the fatty acid modification site being on Lys 26 , which can suitably prolong the in vivo action time of the GLP-1 analogue, but the prolonged time is still not ideal.
  • Exenatide-4 isolated from lizard saliva, and human GLP-1 similar to fatty acid, antibody Fc fragment or serum albumin modification. Things.
  • the half-life of Exenatide-4 is too short, only 2-4 hours, requiring at least two injections a day.
  • Novo Nordisk's fatty acid-modified liraglutide is most effective in reducing hemoglobin glycosylation with fewer side effects, but the disadvantage is that the in vivo half-life is only 13 hours and requires daily dosing.
  • amino acid sequence mutants and modified long-acting GLP-1 analogs such as FC, fatty acid or albumin have been developed.
  • FC fatty acid
  • albumin such as Lula Rubide from Lilly and the Semaglutide from Novo Nordisk.
  • the half-life of these long-acting GLP-1 analogs in humans can be extended to varying degrees, up to the frequency of dosing once a week. Since GLP-1 analogs require long-term injection administration, attempts have been made to find longer-acting drugs to further improve patient compliance.
  • the inventors of the present application have developed a new GLP-1 analogue and its derivative after long-term research, under the same experimental conditions, compared with the currently recognized best drug somaglutide, in ordinary small In the mouse and diabetic mouse models, the duration of hypoglycemic activity in the body can be increased by about 1 time, meaning that administration can be achieved in the human body at least weekly intervals, even every two weeks or longer intervals. Frequency has a significant application prospect.
  • the present invention provides a derivative of a GLP-1 (7-37) analog, or a pharmaceutically acceptable salt thereof, wherein the GLP-1 analogue comprises an amino acid sequence of the following formula Peptide:
  • HVEGTFTSDVSSX 19 LEEX 23 AAX 26 X 27 FIX 30 WLVX 34 GX 36 X 37
  • X 19 is Y or K
  • X 23 is Q or K
  • X 26 is R or K
  • X 27 is E or K
  • X 30 is A or K
  • X 34 is R or K
  • X 36 is R or K
  • X 37 is G or K
  • the derivative comprises an extension joined to the K residue, wherein the extension is
  • x is an integer from 4 to 38.
  • the extension is preferably: HOOC(CH 2 ) 14 CO-, HOOC(CH 2 ) 15 CO-, HOOC(CH 2 ) 16 CO-, HOOC(CH 2 ) 17 CO-, HOOC(CH 2 ) 18 CO -, HOOC(CH 2 ) 19 CO-, HOOC(CH 2 ) 20 CO-, HOOC(CH 2 ) 21 CO- and HOOC(CH 2 ) 22 CO-, more preferably HOOC(CH 2 ) 16 CO-.
  • the extension of a derivative of a GLP-1 analogue of the invention, or a pharmaceutically acceptable salt thereof, is linked to the K residue of GLP-1 via a linker.
  • the joint may be of the following structure:
  • n 0, 1, 2 or 3
  • n 1, 2 or 3
  • s is any integer from 0 to 6
  • p is an arbitrary integer from 1 to 8.
  • the joint is:
  • the joint is:
  • n 1 or 2.
  • the invention also relates to GLP-1 (7-37) analogs, the analogs comprising
  • HVEGTFTSDVSSX 19 LEEX 23 AAX 26 X 27 FIX 30 WLVX 34 GX 36 X 37 sequence, which contains a mutation selected from one or more of the following positions:
  • the amino acid residue at position 19 is Y or K
  • the amino acid residue at position 23 is Q or K
  • the amino acid residue at position 27 is E or K
  • the amino acid residue at position 30 is A or K
  • the 34th amino acid residue is R or K
  • the 36th amino acid residue is R or K
  • the 37th amino acid residue is G or K, provided that the 19th, 23rd, 27th, and 30th positions Only one of the 34, 36 or 37 bits can be a K residue.
  • the acylated derivative of the above GLP-1 analog can obtain a longer duration of hypoglycemic activity in mice and inhibit OGTT in diabetic mice compared to the same acylated GLP-1 product somaglutide.
  • the rate effect is significantly better than somatoglutide.
  • the GLP-1 (7-37) analog of the present invention can be produced by a method comprising expressing a DNA sequence encoding the polypeptide in a host cell under conditions allowing expression of the peptide, and then recovering the produced peptide.
  • the medium used to culture the cells may be any conventional medium for culturing the host cells, such as a minimal medium or a complex medium containing suitable additives.
  • a suitable medium can be obtained by commercially available or a suitable medium can be prepared according to the published method.
  • the polypeptide produced by the host cell can then be recovered from the culture medium by a conventional method, for example, the supernatant of the supernatant or the protein component in the filtrate is precipitated with a salt such as ammonium sulfate, and various chromatographic methods are selected depending on the kind of the peptide of interest. Further purification is carried out by exchange chromatography, gel filtration chromatography, affinity chromatography or the like.
  • the above-described coding DNA sequence can be inserted into any suitable vector.
  • the choice of vector will often depend on the host cell into which the vector is to be introduced.
  • the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, such as a plasmid.
  • the vector may be of a type which, when introduced into a host cell, will integrate into the host cell genome and replicate along with the chromosome into which it is integrated.
  • the vector is preferably an expression vector in which the DNA sequence encoding the peptide is operably linked to other segments required for transcription of the DNA, such as a promoter.
  • promoters suitable for directing transcription of DNA encoding the peptides of the invention in a variety of host cells are well known in the art, see, for example, Sambrook, J, Fritsch, EF and Maniatis, T, Molecular Cloning: A Guide to Experimental Procedures, Cold Spring Harbor Laboratory Press, New York, 1989.
  • the vector may also contain a selection marker, such as a gene whose gene product will compensate for a defect in the host cell or confer a drug such as ampicillin, doxorubicin, tetracycline, chloramphenicol, neomycin Resistance to streptomycin or methotrexate.
  • a selection marker such as a gene whose gene product will compensate for a defect in the host cell or confer a drug such as ampicillin, doxorubicin, tetracycline, chloramphenicol, neomycin Resistance to streptomycin or methotrexate.
  • a secretion signal sequence (also referred to as a leader sequence) can be provided in the recombinant vector.
  • the secretion signal sequence is ligated in the correct reading frame to the DNA sequence encoding the peptide.
  • the secretion signal sequence is usually located on the 5' side of the DNA sequence encoding the peptide.
  • the secretion signal sequence may be a secretion signal sequence that is normally linked to the peptide, or may be derived from a gene encoding another secreted protein.
  • the host cell into which the DNA sequence or recombinant vector is introduced may be any cell capable of producing the peptide of the present invention, including bacteria, yeast, fungi, and higher eukaryotic cells.
  • suitable host cells include, but are not limited to, E. coli, Saccharomyces cerevisiae, or mammalian BHK or CHO cell lines.
  • the present invention relates to a pharmaceutical or pharmaceutical composition
  • a pharmaceutical or pharmaceutical composition comprising the above-mentioned GLP-1 (7-37) analog, and to the use of the analog in the preparation of a medicament, for example, in the preparation of a prophylactic or therapeutic diabetes (preferably type 2 diabetes), diabetes complicated Use in diseases such as diabetic nephropathy, diabetic heart disease, drugs that lower blood sugar or increase glucose tolerance.
  • a prophylactic or therapeutic diabetes preferably type 2 diabetes
  • diabetes preferably type 2 diabetes
  • diabetes complicated Use in diseases such as diabetic nephropathy, diabetic heart disease, drugs that lower blood sugar or increase glucose tolerance.
  • the invention also relates to the prevention or treatment of diabetes (preferably type 2 diabetes) by administering to a subject a GLP-1 (7-37) analog or a derivative of the above GLP-1 (7-37) analog , diabetes complications (such as diabetic nephropathy, diabetes heart disease), ways to lower blood sugar or increase glucose tolerance.
  • diabetes preferably type 2 diabetes
  • diabetes complications such as diabetic nephropathy, diabetes heart disease
  • the present invention also relates to the above-mentioned GLP-1 (7-37) analog or a derivative of the above GLP-1 (7-37) analog in the preparation of a prophylactic or therapeutic diabetes (preferably type 2 diabetes), diabetic complications (For example, diabetic nephropathy, diabetic heart disease), use in drugs that lower blood sugar or increase glucose tolerance.
  • a prophylactic or therapeutic diabetes preferably type 2 diabetes
  • diabetic complications For example, diabetic nephropathy, diabetic heart disease
  • the invention features a pharmaceutical composition, article or kit comprising the above-described GLP-1 (7-37) analog.
  • the invention further relates to a pharmaceutical composition, article or kit comprising a derivative of the above GLP-1 (7-37) analog.
  • the pharmaceutical composition of the present invention comprises, in addition to the active ingredient GLP-1 (7-37) analog or a derivative of the GLP-1 (7-37) analog or a salt thereof, a pharmaceutically acceptable adjuvant.
  • Pharmaceutically acceptable excipients such as non-toxic fillers, stabilizers, diluents, carriers, solvents or other formulation excipients are well known to those skilled in the art.
  • diluents such as microcrystalline cellulose, mannitol, etc.
  • fillers such as starch, sucrose, etc.
  • binders such as starch, cellulose derivatives, alginates, gelatin and/or polyethylene Pyrrolidone
  • a disintegrating agent such as calcium carbonate and/or sodium hydrogencarbonate
  • an absorption enhancer such as a quaternary ammonium compound
  • a surfactant such as cetyl alcohol
  • a carrier a solvent such as water, physiological saline, kaolin, soap clay, etc.
  • Lubricants such as talc, calcium/magnesium stearate, polyethylene glycol, and the like.
  • the pharmaceutical composition of the present invention is preferably an injection.
  • the present invention also relates to a method of reducing islet ⁇ -cell apoptosis, enhancing islet ⁇ -cell function, increasing islet ⁇ -cell number, and/or restoring glucose sensitivity of islet ⁇ -cells, including administering a subject in need thereof An effective amount of the above analog, derivative or drug, pharmaceutical composition.
  • the present invention also relates to the preparation of the above analogs, derivatives or drugs, pharmaceutical compositions for reducing islet ⁇ -cell apoptosis, enhancing islet ⁇ -cell function, increasing islet ⁇ -cell number and/or restoring islet ⁇ -cell glucose sensitivity Use in sexual drugs.
  • a GLP-1 (7-37) polypeptide, a GLP-1 (7-37) polypeptide analog, and a GLP-1 (7-37) analog are used interchangeably to indicate an amino acid sequence: HVEGTFTSDVSSX 19 LEEX 23 AAX 26 X 27 FIX 30 WLVX 34 GX 36 X 37 polypeptide, wherein X 19 is Y or K, X 23 is Q or K, X 26 is R or K, X 27 is E or K, X 30 is A or K, X 34 is R or K, X 36 is R or K, and X 37 is G or K.
  • the GLP-1 (7-37) polypeptide analog forms a derivative of the GLP-1 (7-37) polypeptide analog by attachment to an extension.
  • the invention relates to acylated derivatives of GLP-1 (7-37) analogs.
  • the acylated derivative not only has a remarkable therapeutic effect, but also has an in vivo activity duration of about 1 time higher than that of the currently recognized best drug somaglutide, meaning that at least weekly can be achieved in the human body. The frequency of dosing administered at intervals, even at intervals of two or more weeks.
  • a derivative of the GLP-1 (7-37) analog of the present invention, an acylated derivative of the GLP-1 (7-37) analog, a GLP-1 (7-37) derivative, and a GLP-1 derivative may be mutually Change to use.
  • the present invention is also a process for the preparation of the above derivative or a pharmaceutically acceptable salt thereof, comprising:
  • the above method comprises adding triethylamine to a solution of the GLP-1 analog.
  • the extension e.g., fatty acid
  • the extension is a solution in which acetonitrile is dissolved.
  • An exemplary preparation method of the present invention comprises (1) providing a GLP-1 (7-37) analog solution, adjusting the pH to 9-12;
  • the present invention relates to a preparation of a pharmaceutical composition comprising a derivative of GLP-1 (7-37) analog or a pharmaceutically acceptable salt thereof.
  • a derivative of the invention comprising a GLP-1 (7-37) analog, or a pharmaceutically acceptable salt thereof, is present at a concentration of from 0.1 mg/ml to 25 mg/ml, preferably at 0.1 mg/ml It is present at a concentration of 10.0 mg/ml.
  • the pharmaceutical composition has a pH of from 3.0 to 9.0.
  • the pharmaceutical composition may further comprise a buffer system, a preservative, a surface tensioning agent, a chelating agent, a stabilizer, and a surfactant.
  • the medicaments or formulations of the invention are aqueous medicaments or formulations, for example, they may generally be solutions or suspensions.
  • the medicament or formulation is a stable aqueous solution.
  • the medicament or formulation is a lyophilized formulation to which a solvent and/or diluent is added prior to use.
  • the invention further relates to a kit or kit comprising the above pharmaceutical composition, formulation, medicament.
  • a kit or kit comprising the above pharmaceutical composition, formulation, medicament.
  • other drugs, pharmaceutical compounds or compositions which can be used in combination with the pharmaceutical composition, preparation, or drug for example, the other drugs and drug compounds are included.
  • the composition may be selected from the group consisting of anti-diabetic drugs, drugs for treating and/or preventing complications caused by or associated with diabetes.
  • drugs include: insulin, sulfonylureas, biguanides, meglitinides, glucosidase inhibitors, glucagon antagonists, inhibitors of liver enzymes involved in stimulating gluconeogenesis and/or glycogenolysis , glucose uptake regulator, NPY antagonist, PYY agonist, PYY2 agonist, PYY4 agonist, TNF agonist, cortisol releasing factor agonist, 5HT, bombesin agonist, gangliopeptide antagonist, growth hormone , thyroid stimulating hormone releasing hormone agonist, TR ⁇ agonist; histamine H3 antagonist, lipase/amylase inhibitor, gastric inhibitory polypeptide agonist or antagonist, gastrin and gastrin analog, and the like.
  • the pharmaceutical compositions, formulations, medicaments, and other drugs, pharmaceutical compounds, or compositions of the invention are each placed in separate containers.
  • the present invention also relates to a method for preventing or treating diabetes (preferably type 2 diabetes), diabetic complications (such as diabetic nephropathy, diabetic heart disease), comprising administering to a subject in need thereof the above-mentioned analog, derivative or drug, a pharmaceutical composition, wherein the analog, derivative or drug, pharmaceutical composition is used in combination with other drugs, pharmaceutical compounds or compositions, for example, the other drug, pharmaceutical compound or composition may be selected from an anti-diabetic drug, A medicament for treating and/or preventing complications caused by or associated with diabetes.
  • diabetes preferably type 2 diabetes
  • diabetic complications such as diabetic nephropathy, diabetic heart disease
  • drugs include: insulin, sulfonylureas, biguanides, meglitinides, glucosidase inhibitors, glucagon antagonists, inhibitors of liver enzymes involved in stimulating gluconeogenesis and/or glycogenolysis , glucose uptake regulator; CART agonist, NPY antagonist, PYY agonist, PYY2 agonist, PYY4 agonist, TNF agonist, cortisol releasing factor agonist, 5HT, bombesin agonist, ganglion peptide antagonism Agent, growth hormone, thyroid stimulating hormone releasing hormone agonist, TR ⁇ agonist; histamine H3 antagonist, lipase/amylase inhibitor, gastric inhibitory polypeptide agonist or antagonist, gastrin and gastrin analog Wait.
  • the diabetes is type 2 diabetes or diabetic nephropathy.
  • the "diabetic complication" as used in the present invention refers to damage or dysfunction of other organs or tissues of the body caused by poor glycemic control during diabetes, including damage or function of the liver, kidney, heart, retina, nervous system. Obstacles and so on.
  • the complications of diabetes can be divided into five aspects: 1. Cardiovascular disease: including microvascular disease on the heart and large blood vessels, myocardial lesions, cardiac autonomic neuropathy, the leading cause of death in diabetic patients. 2. Cerebrovascular disease: refers to intracranial large blood vessels and microvascular lesions caused by diabetes, mainly manifested as cerebral arteriosclerosis, ischemic cerebrovascular disease, cerebral hemorrhage, brain atrophy. 3.
  • Renal vascular disease The main form is diabetic nephropathy, which is one of the most important complications of diabetes patients. 4. Lower extremity arterial disease: mainly manifested as diabetic foot. 5. Fundus microvascular disease: mainly manifested as diabetic retinopathy.
  • FIG 1 shows the hypoglycemic effect of different GLP-1 derivatives on ICR mice.
  • the 6-His tag, the SUMO tag and the Val 8 Glu 22 Lys 23 Arg 26,34 -GLP-1 (7-37) coding gene sequence (SEQ ID NO: 7) were sequentially fused in tandem, and the gene was obtained by chemical synthesis. Fragment (SEQ ID NO: 18). The above fragment was inserted into the prokaryotic expression plasmid pET-24(+) by BamHI and XhoI sites and verified by sequencing. The resulting expression plasmid for transformation assay was designated pET-24(+)-His-SUMO-Val 8 Glu 22 Lys 23 Arg 26,34 -GLP-1 (7-37).
  • Val 8 Glu 22 Lys 26 Arg 34 -GLP-1 (7-37) ( gene encoding SEQ ID NO: 3), Val 8 Glu 22 Lys 30 Arg 26,34 -GLP-1 (7 -37) (coding gene is SEQ ID NO: 11), Val 8 Glu 22 Lys 19 Arg 26, 34 - GLP-1 (7-37) (coding gene is SEQ ID NO: 5), Val 8 Glu 22 Lys 27 Arg 26,34 -GLP-1 (7-37) (coding gene is SEQ ID NO: 9), Val 8 Glu 22 Lys 34 Arg 26 -GLP-1 (7-37) (coding gene is SEQ ID NO: 13 ), Val 8 Glu 22 Arg 26,34 Lys 36 -GLP-1 (7-37) (coding gene is SEQ ID NO: 15), Val 8 Glu 22 Arg 26, 34 Lys 37 -GLP-1 (7-37) (The corresponding gene encoding the gene is SEQ ID NO: 17).
  • Expression of the fusion protein was carried out using the DNA construction described in Example 1, and the target protein was obtained by expressing the cell BL21 (Trabs Gen Biotech., catalog #CD601). 50 ⁇ l of BL21 competent cells were thawed on an ice bath, DNA of interest was added, gently shaken, and left in an ice bath for 30 minutes. Then heat in a 42 ° C water bath for 30 seconds, then quickly transfer the tube to the ice bath for 2 minutes, do not shake the tube. 500 ⁇ l of sterile LB medium (without antibiotics) was added to the centrifuge tube, mixed and placed at 37 ° C, and cultured at 180 rpm for 1 hour to resuscitate the bacteria.
  • bacterial solution (expressing GLP-1 bacterial solution) was added to 50 ml of LB medium, and 50 ⁇ l of kanamycin was added thereto, mixed, and placed in a 30 ° C constant temperature shaker, and inoculated overnight. 10 ml of the overnight inoculum was added to 1000 ml of LB medium while 1000 ⁇ l of kanamycin was added. After shaking, the cells were placed in a 37 ° C shaker at 200 rpm. After inoculation for 4 hours, IPTG was added to the medium at a final concentration of 0.1 mol/L, shaken, placed in a shaker at 30 ° C, and induced to express overnight at 180 rpm. The overnight expressed bacterial solution was centrifuged at 13,000 g for 60 min. The bacterial cell yield is about 4g bacteria/L fermentation broth, and the expression of the target protein by SDS-PAGE is about 40%.
  • 100 g of the cell slurry was weighed and resuspended in 500 ml of 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, and sonicated for 30 min in an ultrasonic cell pulverizer to disrupt the cells.
  • the homogenate was centrifuged at 13,000 g for 60 min at 4 ° C. After centrifugation, the supernatant was collected and the sample was a Ni column chromatography.
  • the resulting supernatant was concentrated by Chelating Sepharose FF previously equilibrated with 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole (Equilibration 1). After the equilibration solution 1 was rinsed, it was further eluted with 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 0.3 M imidazole (eluent). The purity of the GLP-1 intermediate produced by the above purification process was higher than 70% by SDS-PAGE analysis.
  • the Sumo tag sequence was excised using the ULP enzyme: the intermediate product was diluted three times by adding 20 mM PB, pH 7.4 buffer, and mixed with ULP according to ULP enzyme: intermediate product 1:150 at 4 ° C, and then cleaved overnight.
  • the enzyme digestion rate was nearly 100% by SDS-PAGE.
  • GLP-1 analogue The product obtained after digestion was concentrated by Tosoh Butyl 550C medium previously equilibrated with 20 mM Na 2 HPO 4 , 0.7 M NaCl (equilibrium solution 2). After the equilibration solution 2 was rinsed, it was eluted with 20% ethanol, and the purity by SDS-PAGE was about 90%.
  • Fatty acid modification Water was added to the precipitate of Val 8 Glu 22 Lys 23 Arg 26, 34 -GLP-1 (7-37) prepared and collected in the above examples to prepare a 4-6 mg/ml solution, and 1 M hydroxide was added. The sodium was adjusted to pH 11.0-11.5, shaken to completely dissolve the protein, and the polypeptide concentration was quantified by HPLC. The fatty acid powder was weighed into acetonitrile at a molar ratio of 1:4 to the fatty acid (structure as follows). To the polypeptide solution, triethylamine in a volume of two thousandths was added and mixed with the fatty acid solution, and the mixture was allowed to stand at 4 ° C for one hour.
  • the sample was diluted with water 5 times, the pH was adjusted to 4.8 with 1 M citric acid (or 10% acetic acid) to terminate the reaction, and the acid solution was allowed to stand at 4 ° for 10 min, after centrifugation, centrifuged 13000 g, centrifuged at 4 ° C for 30 min, and the precipitate was placed at -80. °C save.
  • TFA was added to the acid precipitation sample to a final concentration of about 10 mg/ml of the polypeptide.
  • the precipitate was dissolved by shaking, left to stand at room temperature for 30 min, and 4 M NaOH was added dropwise to the reaction solution to adjust the pH to 7.5. -8.5 Terminate the reaction.
  • the reaction solution was stopped at a flow rate of 4 ml/min, and pumped into UniSil10-120C18 (purchased from Suzhou Na.) previously equilibrated with 10 mM ammonium acetate and 20% ethanol (equilibration solution 3). Micro Technology Co., Ltd.) is concentrated. After the equilibration solution 3 was rinsed, it was further eluted with a gradient of 0-100% eluent (10 mM ammonium acetate, 80% ethanol), and the eluted peak was collected by RP-HPLC to a purity of about 90%.
  • the elution peak was diluted 3 times with water, the acid was adjusted to pH 4.80, and the acid was precipitated at 4 ° C for 30 min. After centrifugation, the precipitate was reconstituted by adding PBST buffer (pH 7.0), and then frozen at -80 °C.
  • RIN-m5F cells in good culture were selected.
  • the cells were collected, counted, and RPMI1640 base medium was used to prepare a cell suspension of 1 ⁇ 10 5 cells/ml.
  • the cell suspension was seeded in a 96-well cell culture plate at 100 ⁇ l per well, and cultured overnight at 37 ° C under 5% CO 2 .
  • the activity was detected by the promega detection kit: the diluted sample of the culture medium (Aib, M0, M1, M2, M3, M4, M5, M6, M7) was prepared to 300 ng/ml, and then subjected to 3-fold gradient dilution in a 96-well plate. A total of 8 concentrations, 2 duplicate wells per dilution. M0, M1, M2, M3, M4, M5, M6, M7 were prepared as described above.
  • Aib is
  • the prepared cell plate was taken out, the medium was discarded, and the filter paper was blotted dry.
  • the sample solution was correspondingly transferred into the cell plate at 40 ⁇ l/well.
  • the lid was opened for 15 min at 37 ° C under 5% CO 2 .
  • the cell culture plate was taken out from the incubator, 10 ⁇ l of CD solution (Promega kit) was added to each well, and the cell plate was placed at 22 ° C - 25 ° C and shaken at 500 rpm for 20 min.
  • 50 ⁇ l of KG solution (Promega kit) was added to each well, and the mixture was shaken at a temperature of 22 ° C to 25 ° C and 500 rpm for 10 min.
  • the chemiluminescence values were read using a Molecular Devices SpectraMax L chemiluminometer and the assay was completed in 30 min.
  • Sample EC50 was calculated using a four parameter regression in softmax Pro software software.
  • mice Twenty-eight healthy CD-1 female mice, aged 4-6 weeks, were divided into 4 groups, subcutaneously injected with M2, M4, M0 and somaglutide (Aib) at a dose of 0.15 mg/kg body weight.
  • 20% glucose was administered at intervals of 6h, 1 day, 2 days, 3 days and 4 days after the administration, the dose was 2g/kg body weight, fasted for 6h before sugar feeding, and 0 after sugar feeding.
  • blood was taken from the tip of the tail, and the blood glucose level was measured in real time using Roche blood glucose test paper, and the blood glucose AUC (area under the blood glucose to time curve) was calculated within 0 to 120 minutes, and the blood glucose suppression rate was calculated (Table 3).
  • hypoglycemic activity of somaglutide in normal mice lasted about 2 days
  • hypoglycemic activity of M0 in normal mice lasted about 3 days
  • M2 and M4 were in normal mice.
  • the hypoglycemic activity still showed significant activity on the fourth day, and the time to maintain the sustained hypoglycemic activity in the body was significantly longer than that of the somaglutide or M0, and the hypoglycemic effect of M2 and M4 at each time point after the third day of administration. Also significantly stronger than somaglutide or M0.
  • mice Twenty-eight healthy CD-1 female mice, aged 4-6 weeks, were divided into 4 groups, subcutaneously injected with M4, M5, M7 and M0, respectively at a dose of 0.15 mg/kg body weight.
  • 20% glucose was administered at intervals of 6h, 1 day, 2 days, 3 days and 4 days after the administration, the dose was 2g/kg body weight, fasted for 6h before sugar feeding, and 0 after sugar feeding.
  • blood glucose was taken from the tip of the tail and blood glucose was measured in real time using Roche blood glucose test paper, and blood glucose AUC (area under blood glucose to time curve) was calculated within 0 to 120 minutes, and the blood glucose suppression rate was calculated (Table 4).
  • ICR mouse OGTT test 30 ICR mice aged 4-6 weeks were divided into 6 groups, 5 rats/group, subcutaneously injected with M0, somaglutide, M2, M4, M5 and M7, respectively.
  • 1d, 2d, 3d, 4d, 5d, 20% glucose was administered daily, the dose was 2g/kg body weight, fasted for 6h before sugar, and 0, 0.5, 1, 2 hours after sugar supply. Blood was taken from the tip of the tail and blood glucose was measured in real time using Roche blood glucose test strips.
  • the tail tip was taken and the blood glucose level was measured in real time using a Roche blood glucose test strip, and the blood glucose AUC (area under the blood glucose to time curve) in 0 to 120 minutes was calculated, and the blood sugar suppression rate was calculated (Table 5, Fig. 1).
  • hypoglycemic maintenance effect M4, M5, M2, M7 can maintain the hypoglycemic effect for at least 4 days, while M0 only lasts for 3 days, and somaglutide only lasts for 2 days. Both were statistically significant.
  • mice Twenty-eight healthy ob/ob male mice aged 4-6 weeks were divided into 4 groups (7/group). The experimental group was injected subcutaneously with M2, M4 and somaglutide (Aib) at a dose of 0.05. Mg/kg body weight, once daily. Before administration, 20% glucose was administered at a time interval of 15 days after the administration, and the dose was 2 g/kg body weight. The rats were fasted overnight before the sugar, and the blood was collected at the tail tips at 0, 30 min, 60 min, and 120 min after the sugar administration. The blood glucose level (OGTT) was measured in real time using Roche blood glucose test strips, and the blood glucose AUC (area under the blood glucose to time curve) was calculated from 0 to 120 minutes using the trapezoidal area method.
  • the blood glucose inhibition rate formula is as follows:

Abstract

本发明提供了GLP-1(7-37)多肽类似物、该类似物的脂肪酸修饰的衍生物和包含该衍生物的药物。另外，本发明还提供了该衍生物的制备方法以及该衍生物在制备药物中的用途。

Description

酰化的GLP-1衍生物 技术领域
本发明属于多肽技术领域。具体而言，本发明涉及GLP-1(7-37)多肽类似物的脂肪酸修饰的衍生物。另外，本发明还涉及该肽衍生物的制备方法、含该肽衍生物的药物以及在制备药物中的用途等。
背景技术
糖尿病是一种由遗传和环境等多种因素引起的糖代谢紊乱疾病，现已成为继肿瘤、心脑血管疾病之后威胁人类健康和生命安全的第三位重大疾病。糖尿病本身不一定造成危害，但长期血糖增高，大血管、微血管受损并危及心、脑、肾、周围神经、眼睛、足等，据世界卫生组织统计，糖尿病并发症高达100多种，是目前已知并发症最多的一种疾病。因糖尿病死亡者有一半以上是心脑血管所致，10％是肾病变所致。因糖尿病截肢是非糖尿病的10～20倍。为此治疗糖尿病进而预防其并发症是至关重要的社会问题。
糖尿病由于患病机理不同可分为几种类型。其中绝大部分属于二型糖尿病(约90％)，主要是因体重过重和缺乏身体活动所致。II型糖尿病患者多存在胰岛素抵抗和胰岛素分泌不足两方面异常，在发病的中晚期往往出现胰岛β细胞凋亡。目前，临床使用的口服降糖药的作用机理多为增强胰岛素敏感性，或促进胰岛素分泌以稳定血糖，均无法解决β细胞凋亡这一难题。而胰高血糖素样肽-1(GLP-1)及其类似物药物由于具有减缓β细胞凋亡，增进其再生，促使胰岛β细胞分化和增殖的作用，使其成为治疗II型糖尿病的研究重点。
1983年，BELL等在分析胰高血糖素原(proglucagon，PG)的基因序列时发现了胰高血糖素样肽-1(glucagon-like peptide-1)即GLP-1(BELL G I，SANCHEZ-PESCADOR R，LAYBOURN P J，et al.Exon duplication and divergence in the human preproglucagon gene[J].Nature，1983，304(5924)：368-371)。PG基因序列由6个外显子和5个内含子组成，包含3个主要的结构域：胰高血糖素(33～61)、GLP-1(72～108)和GLP-2(126～158)。PG的mRNA在胰腺A细胞、肠道L细胞和脑中均有表达，在这些组织细胞中进行特异性的翻译修饰，最终形成不同的终产物。
GLP有两种亚型，GLP-1类似物和GLP-2类似物，它们和胰高血糖素的氨基酸序列有近一半相同，二者之间也有约35％的同源性。GLP-1类似物是由末端空肠、回肠和结肠的朗格汉斯细胞(Langerhans’scell)分泌的一种多肽激素，具有葡萄糖依赖性的促胰岛素分泌和生物合成、抑制胰高血糖素分泌及抑制胃排空等多种功能。GLP-2类似物在肠道组织和中枢神经***的脑干和视丘下部神经原细胞均有合成，主要促进正常小肠的生长和肠黏膜损伤的修复(付刚，龚珉，徐为人.胰高血糖素样肽1及其受体激动剂研究进展[J].天津医药，2012，40(2)：181-184.)。
GLP-1是一种内源性促进胰岛素分泌的激素，主要是由肠内L-细胞分泌，在平衡胰岛素和葡萄糖水平中发挥作用。
GLP-1的一级结构为：组氨酸(His)-天冬酰胺(Asn)-谷氨酸(Glu)-苯丙氨酸(Phe)-谷氨酸(Glu)-精氨酸(Arg)-组氨酸(His)-丙氨酸(Ala)-谷氨酸(Glu)-甘氨酸(Gly)-苏氨酸(Thr)-苯丙氨酸(Phe)-苏氨酸(Thr)-丝氨酸(Ser)-天冬氨酸(Asp)-缬氨酸(Val)-丝氨酸(Ser)-丝氨酸(Ser)-酪氨酸(Tyr)-亮氨酸(Leu)-谷氨酸(Glu)-甘氨酸(Gly)-谷氨酰胺(Gln)-丙氨酸(Ala)-丙氨酸(Ala)-赖氨酸(Lys)-谷氨酸(Glu)-苯丙氨酸(Phe)-异亮氨酸(Ile)-丙氨酸(Ala)-色氨酸(Trp)-亮氨酸(Leu)-缬氨酸(Val)-赖氨酸(Lys)-甘氨酸(Gly)-精氨酸(Arg)-甘氨酸(Gly)。DDP-IV能够快速降解N端7～8位上的组氨酸(H)-丙氨酸(A)，DDP-IV主要起肽链端水解酶作用，凡是第8位是丙氨酸或脯氨酸，该酶就会发挥降解作用，使GLP-1快速失去活性(AERTGEERTS K，YE S，TENNANT M G，et al.Crystal structure of human dipeptidyl peptidase IV in complex with a dipeptide peptidase reveals details on substrate specificity and tetrahedral intermediate[J].Protein Sci，2004，13(2)：412-421)。SARRAUSTE DE MENTHIERE等建GLP-1模型，通过氨基酸替换后观察GLP-1类似物与受体亲和力和内在活性的变化，第7位上的组氨酸是亲和力和内在活性的决定因素，其上面芳环比色氨酸小，而且没有任何极性取代基；第8位上的丙氨酸侧链有极性基团会影响GLP-1的活性，侧链的体积不能过大，超过一定限度时，活性就会下降；第9位上的谷氨酸被某些氨基酸，如酸性、极性和疏水性氨基酸替换时，活性不发生变化，被碱性氨基酸替换时活性下降甚至失活；GLP-1与受体呈结合状态，如果其中的氨基酸残基有离子键作用，那么就发生在7～15位氨基酸之间，形成环结构，Ala8-Glu9-Gly10-Thr11会形成β 转角，使得第7位上的组氨酸、第12位上的苯丙氨酸和第19位上的酪氨酸等3个芳香核发生相互作用，与受体上存在的芳香簇疏水口袋相对应，推测它们起到激活受体的作用；第22位上的甘氨酸为柔性氨基酸，起到柔性连接作用，维持螺旋卷曲状。破坏甘氨酸会使所有的芳香氨基酸簇集，但是与受体的亲和力却减少1/40(SARAUSTE DE MENTHIEREC，CHAVANIEUA，GRASSYG，et al.Structural requirements of the N-terminal region of GLP-1-[7-37]-NH 2 for receptor interaction and cAMP production[J].Eur J Med Chem，2004，39(6)：473-480)。
GLP-1包括GLP-1(1-37)、GLP-1(1-36)、GLP-1(7-37)甘氨酸衍生物和GLP-1(7-36)NH 2等分子形式。一般认为，后两者具有相同的生物活性。肠粘膜L细胞分泌的GLP-1(1-37)无活性，需要进一步水解切除N端6个氨基酸，成为有活性的GLP-1(7-37)。GLP-1(7-37)在体内存在的时间较短，很快就被降解。因此，人们进行了各种关于具有抗DPP IV的GLP-1类似物的研究，例如，美国专利第5545618号描述了用烷基或酰基修饰N末端，并且Gallwitz等描述了第7位His进行N甲基化或a甲基化，或者用整个His用咪唑取代来增加对DPP-IV的抗性并保持生理活性。
除了这些修饰，从希拉毒蜥蜴的唾液腺纯化的GLP-1类似物艾塞那肽-4(exendin-4)(美国专利第5424686号)具有对DPP IV的抗性并比GLP-1具有更高的生理活性。因此，它具有比GLP-1的半衰期更长的2至4小时的体内半衰期。然而，仅适用增加DPP IV抗性的方法，生理活性不能被充分地保持，并且在使用商业可获得的艾塞那肽-4(exenatide)的情况下，它需要一天两次注射给病人，这对病人仍然是很痛苦的。
这些促胰岛素肽分子量很小，因此被很快从肾脏排出体外。有科学家使用化学方法在肽的表面添加高溶解度的聚合物例如聚乙二醇来抑制其在肾中的损失。例如美国专利第692464号描述了PEG结合到艾塞那肽-4的赖氨酸残基来增加其体内停留时间，然而，这种方法尽管增加了肽类药物在体内的停留时间，但同时随着分子量的增加，该肽类药物的浓度显著减少，对肽的反应性也降低了。
此外，还有一系列其它方法用于修饰胰高血糖素样肽-1化合物的结构试图延长其作用持续时间。例如WO96/29342公开了在亲本肽激素C-端氨基酸残基处或在N-端氨基酸残基处引入亲脂性取代物而修饰的肽激素衍生物。 WO98/08871公开了其中亲本肽的至少一个氨基酸残基连接有亲脂性取代物的GLP-1衍生物(liraglutide)。WO99/43708公开了具有连接至C-端氨基酸残基的亲脂性取代物GLP-1(7-35)和GLP-1(7-36)衍生物。WO00/34331公开了双酰化的GLP-1类似物。WO 00/69911公开了用于注射的活化的促胰岛素肽，据认为在患者体内，它们与血液成分反应形成缀合物，延长了体内作用持续时间。
WO2006/097537公开了另一种酰化的GLP-1类似物(semaglutide)，通过将第8位氨基酸突变为非天然氨基酸，与WO98/08871的酰化的GLP-1(liraglutide)相比，具有更长的半衰期。
WO02/046227公开了使用基因重组技术通过将GLP-1，艾塞那肽-4或其类似物与人血清白蛋白或免疫球蛋白区(Fc)结合来制备融合蛋白，这可以解决如聚乙二醇化产量低和非特异性的问题，但是它们增加血液中半衰期的效果仍然不像期待中的那么显著。为了使增加血液中半衰期的效果最大化，人们试图使用各种类型的肽连接器，但是这种方法面临的问题是可能引起免疫反应。
CN107033234A公开了GLP-1类似物的脂肪酸修饰的缀合物，脂肪酸修饰位点是在Lys 26上，该方式可以适当地延长GLP-1类似物的体内作用时间，但延长的时间仍然不理想。
目前市场上获批的GLP-1药物主要有从蜥蜴唾液中分离出的艾塞那肽-4(Exenatide-4)，以及采用脂肪酸，抗体Fc段或血清白蛋白修饰的人源GLP-1类似物。艾塞那肽-4半衰期太短，仅2-4小时，一天需要至少两次注射。诺和诺德公司的脂肪酸修饰的利拉鲁肽在降血红蛋白糖基化方面最有效且副作用较少，但其不足之处在于体内半衰期只有13小时，需要每天给药。为了进一步延长体内半衰期，减少给药频率，近年来陆续开发了氨基酸序列突变体和FC、脂肪酸或白蛋白等修饰的长效GLP-1类似物。如礼来公司的杜拉鲁肽和诺和诺德公司的索玛鲁肽(Semaglutide)。这些长效化的GLP-1类似物在人体内的半衰期可被不同程度地延长，最长可实现每周给药一次的给药频率。由于GLP-1类似物需要长期注射给药，所以人们试图寻找更长效的药物，从而进一步提高患者的依从性。
本申请的发明人经过长期研究，开发了一种新的GLP-1类似物及其衍生物，在相同实验条件下，与现有公认的最好的药物索玛鲁肽相比，在普通小 鼠及糖尿病小鼠模型上，其体内降糖活性持续时间可提高1倍左右，意味着在人体内可实现至少每周间隔给药、甚至每两周间隔或更长时间间隔给药的给药频率，具有显著的应用前景。
发明内容
本发明的目的在于提供一种新的GLP-1(7-37)类似物、该类似物的酰化衍生物。另外，本发明还提供了该类似物或衍生物的制备方法、包含该类似物或衍生物的药物组合物、制品以及它们在预防和治疗疾病中的用途。
具体而言，一方面，本发明提供了一种GLP-1(7-37)类似物的衍生物或其药学上可接受的盐，其中所述GLP-1类似物包含以下式氨基酸序列组成的多肽：
HVEGTFTSDVSSX 19LEEX 23AAX 26X 27FIX 30WLVX 34GX 36X 37
其中X 19为Y或K，X 23为Q或K，X 26为R或K，X 27为E或K，X 30为A或K，X 34为R或K，X 36为R或K，X 37为G或K，
条件是，在X 19、X 23、X 26、X 27、X 30、X 34、X 36或X 37中只有一个是K残基，
所述衍生物包含与所述K残基连接的延长部分，其中所述延长部分为
Figure PCTCN2018083789-appb-000001
其中x是4-38的整数。
其中，延长部分优选为：HOOC(CH 2) 14CO-、HOOC(CH 2) 15CO-、HOOC(CH 2) 16CO-、HOOC(CH 2) 17CO-、HOOC(CH 2) 18CO-、HOOC(CH 2) 19CO-、HOOC(CH 2) 20CO-、HOOC(CH 2) 21CO-和HOOC(CH 2) 22CO-，更优选为HOOC(CH 2) 16CO-。
在优选实施方案中，本发明所述GLP-1类似物的衍生物或其药学上可接受的盐的延长部分经一接头与GLP-1的K残基连接。所述接头可以是如下结构：
Figure PCTCN2018083789-appb-000002
Figure PCTCN2018083789-appb-000003
其中m是0、1、2或3；n是1、2或3；s是0-6的任意整数；p是1-8的任意整数。
优选地，接头为：
Figure PCTCN2018083789-appb-000004
其中m是1或2；n是1或2；p是1-5的任意整数。
更优选：接头为：
Figure PCTCN2018083789-appb-000005
其中m是1，n是1或2。
本发明也涉及GLP-1(7-37)类似物，该类似物包含
HVEGTFTSDVSSX 19LEEX 23AAX 26X 27FIX 30WLVX 34GX 36X 37序列，该序列包含选自如下一个或多个位点的突变：
第19位、23位、27位、30位、34位、36位和37位。在一个优选的实施方案中，第19位氨基酸残基为Y或K，第23位氨基酸残基为Q或K，第27位氨基酸残基为E或K，第30位氨基酸残基为A或K，第34位氨基酸残基为R或K，第36位氨基酸残基为R或K，第37位氨基酸残基为G 或K，条件是，第19位、23位、27位、30位、34位、36位或37位只能有一位为K残基。
上述GLP-1类似物经过酰化后的衍生物，与同样为酰化的GLP-1产品索玛鲁肽相比，小鼠体内可以获得更长的降糖活性持续时间，对糖尿病鼠OGTT抑制率效果显著优于索玛鲁肽。
可以通过这样一种方法制备本发明GLP-1(7-37)类似物，该方法包括在允许肽表达的条件下，在宿主细胞中表达编码该多肽的DNA序列，然后回收产生的肽。
用来培养细胞的培养基可以是用于培养该宿主细胞的任何常规培养基，如基本培养基或含有适宜添加物的复合培养基。可以通过市售得到适宜的培养基，或根据已公开的制法制备适宜的培养基。然后可以通过常规方法从培养基中回收由所述宿主细胞产生的多肽，例如用盐如硫酸铵沉淀上清液或滤液中的蛋白质成分，根据目的肽的种类而选用各种层析方法如例子交换层析、凝胶过滤层析、亲和层析等进行进一步纯化。
可以将上述编码DNA序列***任何适当的载体中。通常，载体的选择常常取决于该载体将要被引入的宿主细胞，因此，载体可以是一种自主复制型载体，即作为染色体外实体存在的载体，其复制不依赖于染色体复制，如质粒。或者，载体可以是这样一种类型，当将其引入宿主细胞时，它将整合到宿主细胞基因组中，并与它所整合入的染色体一起复制。
载体优选是一种表达载体，其内编码所述肽的DNA序列与该DNA转录所需的其它区段(如启动子)有效相连。本领域熟知适合于在多种宿主细胞中指导编码本发明肽的DNA进行转录的启动子例子，参见例如Sambrook，J，Fritsch，EF和Maniatis，T，分子克隆：实验操作指南，Cold Spring Harbor Laboratory Press，纽约，1989中所述。
载体还可以含有选择标记，如可以是这样的一种基因，其基因产物将弥补宿主细胞内的一个缺陷，或者能赋予对药物如氨苄青霉素、阿霉素、四环素、氯霉素、新霉素、链霉素或氨甲喋呤等的抗性。
为将本发明表达的肽引入宿主细胞的分泌途径，可以在重组载体中提供分泌信号序列(也称之为前导序列)。分泌信号序列以正确读框与编码该肽的DNA序列连接。分泌信号序列通常位于编码该肽的DNA序列的5’侧。 分泌信号序列可以是正常地与该肽连接的分泌信号序列，或可以源于编码另一种分泌蛋白质的基因。
用于分别连接编码本发明肽的DNA序列，启动子和可选择的终止子和/或分泌信号肽序列，并将其***到适宜的含有复制所必需的信息的载体中的方法，对本领域的技术人员是已知的。
将导入DNA序列或重组载体的宿主细胞可以是能够产生本发明肽的任何细胞，包括细菌、酵母、真菌和高等真核生物细胞。本领域技术人员熟知并使用的适宜的宿主细胞的例子包括但不限于：大肠杆菌、酿酒酵母、或哺乳动物BHK或CHO细胞系。
本发明涉及包含上述GLP-1(7-37)类似物的药物或药物组合物，还涉及该类似物在制备药物中的用途，例如在制备预防或治疗糖尿病(优选2型糖尿病)、糖尿病并发症(例如糖尿病肾病，糖尿病心脏病)、降低血糖或提高糖耐量的药物中的用途。
另一方面，本发明也涉及通过给药受试者上述GLP-1(7-37)类似物或上述GLP-1(7-37)类似物的衍生物预防或治疗糖尿病(优选2型糖尿病)、糖尿病并发症(例如糖尿病肾病，糖尿病心脏病)、降低血糖或提高糖耐量的方法。
另一方面，本发明也涉及上述GLP-1(7-37)类似物或上述GLP-1(7-37)类似物的衍生物在制备预防或治疗糖尿病(优选2型糖尿病)、糖尿病并发症(例如糖尿病肾病，糖尿病心脏病)、降低血糖或提高糖耐量的药物中的用途。
另一方面，本发明涉及包含上述GLP-1(7-37)类似物的药物组合物，制品或试剂盒。
本发明还涉及包含上述GLP-1(7-37)类似物的衍生物的药物组合物，制品或试剂盒。
本发明所述的药物组合物除包含活性成分GLP-1(7-37)类似物或GLP-1(7-37)类似物的衍生物或其盐外，还包含药学上可接受的辅料。本领域技术人员熟知药学上可接受的辅料，例如无毒的填充剂、稳定剂、稀释剂、载体、溶剂或其他制剂辅料。例如，稀释剂、赋形剂，如微晶纤维素、甘露醇等；填充剂，如淀粉、蔗糖等；粘合剂，如淀粉、纤维素衍生物、藻酸盐、明胶和/或聚乙烯吡咯烷酮；崩解剂，如碳酸钙和/或碳酸氢钠；吸收促进剂， 如季铵化合物；表面活性剂，如十六烷醇；载体、溶剂，如水、生理盐水、高岭土、皂粘土等；润滑剂，如滑石粉、硬脂酸钙/镁、聚乙二醇等。另外，本发明的药物组合物优选为注射剂。
本发明还涉及一种减少胰岛β-细胞凋亡、增强胰岛β-细胞功能、增加胰岛β-细胞数量和/或恢复胰岛β-细胞的葡萄糖敏感性的方法，包括给药有需要的受试者有效量的上述类似物、衍生物或药物、药物组合物。
本发明还涉及上述类似物、衍生物或药物、药物组合物在制备减少胰岛β-细胞凋亡、增强胰岛β-细胞功能、增加胰岛β-细胞数量和/或恢复胰岛β-细胞的葡萄糖敏感性的药物中的用途。
在本发明中，GLP-1(7-37)多肽、GLP-1(7-37)多肽类似物、GLP-1(7-37)类似物可以互换使用，表示含有氨基酸序列：HVEGTFTSDVSSX 19LEEX 23AAX 26X 27FIX 30WLVX 34GX 36X 37的多肽，其中X 19为Y或K，X 23为Q或K，X 26为R或K，X 27为E或K，X 30为A或K，X 34为R或K，X 36为R或K，X 37为G或K。该GLP-1(7-37)多肽类似物通过与延长部分连接，形成该GLP-1(7-37)多肽类似物的衍生物。具体地，本发明涉及GLP-1(7-37)类似物的酰化衍生物。该酰化衍生物不但具有显著的治疗效果，而且与现有公认的最好的药物索玛鲁肽相比，其体内活性持续时间可提高1倍左右，意味着在人体内可实现至少每周间隔给药、甚至每两周间隔或更长时间间隔给药的给药频率。
本发明GLP-1(7-37)类似物的衍生物、GLP-1(7-37)类似物的酰化衍生物，GLP-1(7-37)衍生物、GLP-1衍生物可以互换使用。
另一方面，本发明还涉及制备上述衍生物或其药学上可接受的盐的方法，包括：
(1)将溶解有上述的GLP-1类似物的溶液与溶解有延长部分(例如脂肪酸)的溶液混合；
(2)调节pH至4-5终止反应，静置，至沉淀产生，取沉淀；和
(3)向沉淀中加入TFA，调节pH至7.5-8.5终止反应。
在一个优选实施方案中，上述方法包括向GLP-1类似物的溶液中加入三乙胺。
在一个优选实施方案中，上述延长部分(例如脂肪酸)是乙腈溶解的溶液。
本发明例示性的制备方法包括(1)提供GLP-1(7-37)类似物溶液，调整pH至9-12；
(2)然后向步骤(1)获得的溶液中加入三乙胺；
(3)称取不低于该GLP-1类似物2倍量(摩尔比)的如下结构的脂肪酸，优选为不低于3倍量的GLP-1类似物，溶于乙腈中；
Figure PCTCN2018083789-appb-000006
(4)将步骤(2)获得的GLP-1类似物溶液与步骤(3)获得的脂肪酸溶液混合，低温静置，例如一小时；
(5)调节pH至4-5终止反应，低温静置酸沉，收取沉淀；
(6)向步骤(5)获得的酸沉样品中加入TFA至多肽终浓度5-15mg/ml，静置0.5-2小时，向反应液中滴入碱性溶液，例如NaOH，调节pH至7.5-8.5终止反应；
(7)分离纯化所得产物。
本发明涉及包含GLP-1(7-37)类似物的衍生物或其药学上可接受的盐的药物组合物的制剂。在一些实施方案中，本发明的包含GLP-1(7-37)类似物的衍生物或其药学上可接受的盐以0.1mg/ml至25mg/ml的浓度存在，优选以0.1mg/ml至10.0mg/ml的浓度存在。在优选实施方案中，所述药物组合物具有3.0至9.0的pH。在优选实施方案中，所述药物组合物可进一步包含缓冲***、防腐剂、表面张力剂、螯合剂、稳定剂和表面活性剂。在一些实施方案中，本发明所述的药物或制剂是含水药物或制剂，例如，他们通常可以是溶液或悬浮液。在本发明的具体实施方案中，所述的药物或制剂是稳定的含水溶液。在本发明的另一些具体实施方案中，所述药物或制剂是一种冻干制剂，在使用前将溶剂和/或稀释液加入其中。
本发明还涉及包含上述药物组合物、制剂、药物的药盒或试剂盒。在该药盒或试剂盒中，除包含上述药物或制剂外，还包括可以与该药物组合物、制剂、药物联合使用的其它药物、药物化合物或组合物，例如，所述其它药 物、药物化合物或组合物可以选自抗糖尿病药物、用于治疗和/或预防由糖尿病引发或与其相关的并发症的药物。这些药物的实例包括：胰岛素、磺脲类、双胍、氯茴苯酸类、葡萄糖苷酶抑制剂、胰高血糖素拮抗剂、涉及刺激糖异生和/或糖原分解的肝脏酶的抑制剂、葡萄糖摄取调节剂、NPY拮抗剂、PYY激动剂、PYY2激动剂、PYY4激动剂、TNF激动剂、促皮质素释放因子激动剂、5HT、蛙皮素激动剂、神经节肽拮抗剂、生长激素、促甲状腺激素释放激素激动剂、TRβ激动剂；组胺H3拮抗剂、脂肪酶/淀粉酶抑制剂、胃抑制性多肽激动剂或拮抗剂、胃泌素和胃泌素类似物等。在一些实施方案中，本发明所述的药物组合物、制剂、药物与其它的药物、药物化合物或组合物分别放置在不同的容器中。
本发明也涉及一种预防或治疗糖尿病(优选2型糖尿病)、糖尿病并发症(例如糖尿病肾病，糖尿病心脏病)的方法，包括给药有需要的受试者上述类似物、衍生物或药物、药物组合物，其中所述类似物、衍生物或药物、药物组合物与其它药物、药物化合物或组合物联合使用，例如，所述其它药物、药物化合物或组合物可以选自抗糖尿病药物、用于治疗和/或预防由糖尿病引发或与其相关的并发症的药物。这些药物的实例包括：胰岛素、磺脲类、双胍、氯茴苯酸类、葡萄糖苷酶抑制剂、胰高血糖素拮抗剂、涉及刺激糖异生和/或糖原分解的肝脏酶的抑制剂、葡萄糖摄取调节剂；CART激动剂、NPY拮抗剂、PYY激动剂、PYY2激动剂、PYY4激动剂、TNF激动剂、促皮质素释放因子激动剂、5HT、蛙皮素激动剂、神经节肽拮抗剂、生长激素、促甲状腺激素释放激素激动剂、TRβ激动剂；组胺H3拮抗剂、脂肪酶/淀粉酶抑制剂、胃抑制性多肽激动剂或拮抗剂、胃泌素和胃泌素类似物等。在优选实施方案中，所述糖尿病为2型糖尿病或糖尿病肾病。
本发明所述的“糖尿病并发症”是指由糖尿病过程中血糖控制不良导致的身体其他器官或组织的损害或功能障碍性疾病，其中包括肝脏、肾脏、心脏、视网膜、神经***的损害或功能障碍等。糖尿病的并发症可分为五个方面：1.心血管病变：包括心脏和大血管上的微血管病变、心肌病变、心脏自主神经病变，引起糖尿病患者死亡的首要病因。2.脑血管病变：是指由糖尿病所引起的颅内大血管和微血管病变，主要表现为脑动脉硬化、缺血性脑血管病、脑出血、脑萎缩等。3.肾血管病变：主要表为糖尿病肾病，是糖尿病 患者最重要的合并症之一。4.下肢动脉病变：主要表现为糖尿病足。5.眼底微血管病变：主要表现为糖尿病性视网膜病变。
本发明通过下面实施例来进一步阐述，然而，所述的实施例不应理解为限制本专利的保护范围，在前面描述和下列实施例中公开的特征(个别地和它们的任何组合)，可以是用于以基本不同形式实现本发明的材料，他们可以任意组合。另外，本发明引用了公开文献，这些文献是为了更清楚地描述本发明，它们的全文内容均纳入本文进行参考，就好像它们的全文已经在本文中重复叙述过一样。
附图简述
图1为不同GLP-1衍生物对ICR小鼠体内降糖效果。
实施例
以下本文将通过具体的实施例来描述发明。如未特别指明之处，可根据本领域技术人员所熟悉的《分子克隆实验指南》、《细胞实验指南》等实验手册以及CFDA的试验指引等所列方法来实施。其中，所用的试剂原料均为市售品，可以通过公开渠道购买获得。
实施例1 GLP-1类似物表达质粒的构建
构建Val 8Glu 22Lys 23Arg 26，34-GLP-1(7-37)的DNA
将6-His标签、SUMO标签和Val 8Glu 22Lys 23Arg 26，34-GLP-1(7-37)编码基因序列(SEQ ID NO：7)依次串联融合，并使用化学合成的方式获得基因片段(SEQ ID NO：18)。通过BamHI和XhoI位点，将上述片段***原核表达质粒pET-24(+)中并测序验证。得到的用于转化测定的表达质粒，称作pET-24(+)-His-SUMO-Val 8Glu 22Lys 23Arg 26，34-GLP-1(7-37)。
依据上述方法，依次构建Val 8Glu 22Lys 26Arg 34-GLP-1(7-37)(编码基因为SEQ ID NO：3)、Val 8Glu 22Lys 30Arg 26，34-GLP-1(7-37)(编码基因为SEQ ID NO：11)、Val 8Glu 22Lys 19Arg 26，34-GLP-1(7-37)(编码基因为SEQ ID NO：5)、Val 8Glu 22Lys 27Arg 26，34-GLP-1(7-37)(编码基因为SEQ ID NO：9)、Val 8Glu 22Lys 34Arg 26-GLP-1(7-37)(编码基因为SEQ ID NO：13)、Val 8Glu 22Arg 26，34Lys 36-GLP-1(7-37)(编码基因为SEQ ID NO：15)、 Val 8Glu 22Arg 26，34Lys 37-GLP-1(7-37)(编码基因为SEQ ID NO：17)的相应表达质粒。
实施例2 融合蛋白表达
使用实施例1中所述DNA构建进行融合蛋白的表达，通过表达细胞BL21(TrabsGenBiotech.，catalog#CD601)获得目的蛋白。将BL21感受态细胞50μl置于冰浴上融化，加入目的DNA，轻轻摇匀，并在冰浴中放置30分钟。继而42℃水浴热激30秒，然后快速将离心管转移到冰浴中放置2分钟，该过程不要摇动离心管。向离心管中加入500μl无菌的LB培养基(不含抗生素)，混匀后置于37℃，180rpm培养1小时，使细菌复苏。吸取200μl已转化的感受态细胞加到含有卡那霉素抗性的LB琼脂培养基平板上，将细胞均匀涂开。将平板置于37℃至液体被吸收，倒置平板，37℃过夜培养。次日，使用接种环挑取转化平皿中的单克隆菌落，并接种于15ml的无菌LB培养基(含抗生素)，30℃过夜培养。
实施例3 重组GLP-1类似物的发酵
向50ml的LB培养基中加入50μl菌液(表达GLP-1菌液)，同时加入50μl卡那霉素，混匀后放30℃恒温振荡器中，接种过夜。取过夜接种的菌液10ml加入1000ml的LB培养基中，同时加入1000μl卡那霉素。摇匀后放于37℃摇床内，200rpm，接种4h后向培养基中接入终浓度为0.1mol/L的IPTG，摇匀后放于30℃摇床内，180rpm，过夜诱导表达。将过夜表达的菌液以13000g离心60min。菌体收率约为4g菌/L发酵液，SDS-PAGE测定目的蛋白表达量约可达40％。
实施例4 重组GLP-1类似物的纯化
称取100g细胞浆重悬于500ml的50mM Tris-HCl、pH8.0，50mM NaCl中，用超声波细胞粉碎机中超声30min，以使细胞破碎。所述匀浆在4℃下以13000g离心60min，离心完成后收集上清即为Ni柱层析样品。
将所得上清液经事先用50mM Tris-HCl、pH8.0，500mM NaCl，10mM咪唑(平衡液1)平衡过的Chelating Sepharose FF浓缩。平衡液1淋洗后，再用50mM Tris-HCl、pH8.0，50mM NaCl，0.3M咪唑(洗脱液)洗脱。经SDS-PAGE分析，由上述纯化过程生成GLP-1中间产物纯度高于70％。
使用ULP酶将Sumo标签序列切除：中间产物中加入20mM PB、pH7.4缓冲液使其稀释三倍，4℃条件下按照ULP酶：中间产物为1∶150加入ULP混匀后酶切过夜。经SDS-PAGE分析酶切率近100％。
GLP-1类似物的精纯：将酶切后所得产物经事先用20mM Na 2HPO 4，0.7M NaCl(平衡液2)平衡过的东曹Butyl 550C介质浓缩。平衡液2淋洗后，再用20％乙醇洗脱，经SDS-PAGE纯度约为90％。
洗脱样品中加入0.2M Na 2HPO 4使其终浓度为20mM Na 2HPO 4，用1M柠檬酸调节pH至4.8-5.0，4℃酸沉过夜。SDS-PAGE检测收率90％以上。4℃下以13000g离心30min，收取沉淀放于-20℃保存。
实施例5 GLP-1类似物的衍生物的制备
如下所示的GLP-1类似物的衍生物，N-ε 23-[2-(2-[2-(2-[2-(2-[4-(17-羧基十七烷酰氨基)-4(s)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰氨基)乙氧基]乙氧基)乙酰基](Val 8Glu 22Lys 23Arg 26，34-GLP-1(7-37))肽(简称M2)的制备
Figure PCTCN2018083789-appb-000007
1.脂肪酸修饰：向上述实施例制备、收集的Val 8Glu 22Lys 23Arg 26， 34-GLP-1(7-37)沉淀中加水，配制为4～6mg/ml溶解液，加入1M氢氧化钠调整pH至11.0-11.5，摇匀使蛋白完全溶解，HPLC定量多肽浓度。按多肽与脂肪酸(结构如下)摩尔比1∶4称取脂肪酸粉末溶于乙腈中。向该多肽溶液中加入体积为千分之二的三乙胺，并与脂肪酸溶液混合，将混合液于4℃静置一小时。
Figure PCTCN2018083789-appb-000008
样品加水稀释5倍，用1M柠檬酸(或10％乙酸)调pH至4.8终止反应，放于4度静置酸沉10min，酸沉后离心13000g，4℃离心30min，将沉淀放于-80℃保存。
2.脂肪酸脱保护与纯化：向酸沉样品中加入TFA至多肽终浓度约10mg/ml，震荡使沉淀溶解，置于室温静置脱保护30min，向反应液中滴入4M NaOH调节pH至7.5-8.5终止反应。
用制备液相仪(岛津LC-8A)将终止后反应液按4ml/min流速，泵入事先用10mM乙酸铵，20％乙醇(平衡液3)平衡过的UniSil10-120C18(购自苏州纳微科技有限公司)进行浓缩。平衡液3淋洗后，再按0-100％洗脱液(10mM乙酸铵，80％乙醇)梯度洗脱，收集洗脱峰经RP-HPLC检测纯度约为90％。
洗脱峰用水稀释3倍，酸沉调整pH至4.80，4℃酸沉30min。离心后沉淀中加入PBST缓冲液(pH7.0)复溶后-80℃冻存。
依据上述方法，依次制备N-ε 26-[2-(2-[2-(2-[2-(2[4-(17-羧基十七烷酰氨基)-4(s)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰氨基)乙氧基]乙氧基)乙酰基]Val 8Glu 22Lys 26Arg 34-GLP-1(7-37)(M0)，N-ε 30-[2-(2-[2-(2-[2-(2-[4-(17-羧基十七烷酰氨基)-4(s)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰氨基)乙氧基]乙氧基)乙酰基](Val 8Glu 22Lys 30Arg 26，34-GLP-1(7-37))肽(M4)、N-ε 19-[2-(2-[2-(2-[2-(2-[4-(17-羧基十七烷酰氨基)-4(s)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰氨基)乙氧基]乙氧基)乙酰基](Val 8Lys 19Glu 22Arg 26，34-GLP-1(7-37))肽(M1)、N-ε 27-[2-(2-[2-(2-[2-(2-[4-(17-羧基十七烷酰氨基)-4(s)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰氨基)乙氧基]乙氧基)乙酰基](Val 8Glu 22Lys 27Arg 26，34-GLP-1(7-37))肽(M3)、N-ε 34-[2-(2-[2-(2-[2-(2-[4-(17-羧基十七烷酰氨基)-4(s)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰氨基)乙氧基]乙氧基)乙酰基](Val 8Glu 22Arg 26Lys 34-GLP-1(7-37))肽(M5)、N-ε 36-[2-(2-[2-(2-[2-(2-[4-(17-羧基十七烷酰氨基)-4(s)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰氨基)乙氧基]乙氧基)乙酰基](Val 8Glu 22Arg 26，34Lys 36-GLP-1(7-37))肽(M6)、N-ε 37-[2-(2-[2-(2-[2-(2-[4-(17-羧基十七烷酰氨基)-4(s)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰氨基)乙氧基]乙氧基)乙酰基](Val 8Glu 22Arg 26，34Lys 37-GLP-1(7-37))肽(M7)。
表1 GLP-1(7-37)类似物及其相应衍生物对照表
Figure PCTCN2018083789-appb-000009
实施例6 GLP-1类似物的衍生物体外活性测试
选取培养状态良好的RIN-m5F细胞。收集细胞，计数，用RPMI1640基础培养液配成1×10 5个细胞/ml的细胞悬液。接种细胞悬液于96孔细胞培养板中，每孔100μl，37℃、5％CO 2条件下培养过夜。利用promega检测试剂盒检测活性：配制测定培养液稀释样品(Aib、M0、M1、M2、M3、M4、M5、M6、M7)至300ng/ml，之后在96孔板中进行3倍梯度稀释，共8个浓度，每个稀释度做2个复孔。M0、M1、M2、M3、M4、M5、M6、M7如上所述制备。Aib为
N-ε 26-[2-(2-[2-(2-[2-(2[4-(17-羧基十七烷酰氨基)-4(s)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰氨基)乙氧基]乙氧基)乙酰基][Aib 8，Arg 34]GLP-1-(7-37)肽(参见CN101133082B实施例4)，商品名为索玛鲁肽，依据专利CN101133082B所公开的方法制备。
取出已制备好的细胞板，弃掉培养基，滤纸上吸干。将样品溶液对应移入细胞板中，40μl/孔。于37℃、5％CO 2条件下开盖处理15min。从培养箱内取出细胞培养板，在各孔加入10μl CD溶液(Promega试剂盒)，将细胞板放在22℃-25℃、500rpm水平振荡放置20min。各孔加入50μl KG溶液(Promega试剂盒)，22℃-25℃、500rpm水平振荡避光放置10min。用Molecular Devices SpectraMax L化学发光仪读取化学发光数值，30min完成检测。利用softmax Pro software软件中的四参数回归计算样品EC50。
表2 体外活性实验结果
样品 Aib M0 M1 M2 M3 M4 M5 M6 M7
EC50 2.437 10.68 5.386 1.996 5.387 2.322 3.043 7.650 3.208
体外药效学表明，M2的EC50值低于Aib、M4的EC50值与Aib相当，M5和M7略高于Aib，M1、M3、M6相对更高。
实施例7 GLP-1类似物的脂肪酸修饰的衍生物在正常小鼠体内的降糖研究
选用28只周龄4～6周的健康CD-1雌性小鼠，分为4组，分别皮下注射M2、M4、M0和索玛鲁肽(Aib)，剂量分别为0.15mg/kg体重。按照给药前，给药后6h、1天、2天、3天、4天的时间间隔灌胃20％葡萄糖，剂量为2g/kg体重，给糖前禁食6h，并在给糖后0、0.5、1、2h分别尾尖取血并使用罗氏血糖试纸实时检测血糖值，并计算出0～120分钟内的血糖AUC(血糖～时间曲线下面积)，算出血糖抑制率(表3)。
Figure PCTCN2018083789-appb-000010
表3 正常小鼠体内降糖效果比较
Figure PCTCN2018083789-appb-000011
Figure PCTCN2018083789-appb-000012
P值：与给药前血糖比较
从表3中可以看出，索玛鲁肽在正常小鼠体内的降糖活性持续大约2天，M0在正常小鼠体内的降糖活性持续大约3天，而M2及M4在正常小鼠体内的降糖活性在第4天仍然表现出明显活性，体内维持持续降糖活性的时间明显长于索玛鲁肽或M0，并且在给药第3天后的各个时间点，M2及M4的降糖效果也都明显强于索玛鲁肽或M0。
选用28只周龄4～6周的健康CD-1雌性小鼠，分为4组，分别皮下注射M4、M5、M7和M0，剂量分别为0.15mg/kg体重。按照给药前，给药后6h、1天、2天、3天、4天的时间间隔灌胃20％葡萄糖，剂量为2g/kg体重，给糖前禁食6h，并在给糖后0、0.5、1、2h分别尾尖取血并使用罗氏血糖试纸实时检测血糖值，并计算出0～120分钟内的血糖AUC(血糖～时间曲线下面积)，算出血糖抑制率(表4)。
Figure PCTCN2018083789-appb-000013
表4 正常小鼠体内降糖效果比较
Figure PCTCN2018083789-appb-000014
Figure PCTCN2018083789-appb-000015
从表3和表4结果看，M2、M4效果优于M0及Aib，M2、M4、M5、M7之间效果相当，无显著差异。
实施例8 使用ICR小鼠的降糖效果研究
ICR小鼠OGTT试验：选用30只周龄4～6周的ICR小鼠，分为6组，5只/组，分别皮下注射M0、索玛鲁肽、M2、M4、M5和M7，剂量分别为0.15mg/kg体重，单次给药。按照4h、1d、2d、3d、4d、5d的时间，每天灌胃20％葡萄糖，剂量为2g/kg体重，给糖前禁食6h，并在给糖后0、0.5、1、2小时分别尾尖取血并使用罗氏血糖试纸实时检测血糖值。尾尖取血并使用罗氏血糖试纸实时检测血糖值，并计算出0～120分钟内的血糖AUC(血糖～时间曲线下面积)，算出血糖抑制率(表5、图1)。
Figure PCTCN2018083789-appb-000016
表5 ICR小鼠体内降糖效果比较
Figure PCTCN2018083789-appb-000017
从表5和图1结果可以看出，降糖维持作用：M4、M5、M2、M7的降糖作用均可维持至少4天，而M0仅维持3天，索玛鲁肽仅维持2天，均有统计学意义。
实施例9 使用ob/ob II型糖尿病小鼠的降糖效果研究
选用28只周龄4～6周的健康ob/ob雄性小鼠，分为4组(7只/组)，实验组分别皮下注射M2、M4和索玛鲁肽(Aib)，剂量均为0.05mg/kg体重，每天给药一次。按照给药前，给药后15天的时间间隔灌胃20％葡萄糖，剂量为2g/kg体重，给糖前过夜禁食，并在给糖后0、30min、60min、120min分别尾尖取血并使用罗氏血糖试纸实时检测血糖值(OGTT)，并用梯形的面积法，计算出0～120分钟内的血糖AUC(血糖～时间曲线下面积)，血糖抑制率公式如下：
Figure PCTCN2018083789-appb-000018
表6 ob/ob小鼠OGTT实验结果
Figure PCTCN2018083789-appb-000019
从ob/ob小鼠的OGTT数据看，给药15天后，M2和M4对OGTT的抑制率明显优于Aib。

Claims (21)

  1. 一种GLP-1(7-37)类似物的衍生物或其药学上可接受的盐，其中GLP-1(7-37)类似物包含下式的氨基酸序列：
    HVEGTFTSDVSSX 19LEEX 23AAX 26X 27FIX 30WLVX 34GX 36X 37
    其中X 19为Y或K，X 23为Q或K，X 26为R或K，X 27为E或K，X 30为A或K，X 34为R或K，X 36为R或K，X 37为G或K，
    条件是，在X 19、X 23、X 26、X 27、X 30、X 34、X 36或X 37中只有一个是K残基，
    所述衍生物包含与所述GLP-1(7-37)类似物的K残基连接的延长部分，其中所述延长部分为
    Figure PCTCN2018083789-appb-100001
    Figure PCTCN2018083789-appb-100002
    其中x是4-38的整数。
  2. 根据权利要求1所述的衍生物或其药学上可接受的盐，其中所述延长部分选自：
    HOOC(CH 2) 14CO-、HOOC(CH 2) 15CO-、HOOC(CH 2) 16CO-、HOOC(CH 2) 17CO-、HOOC(CH 2) 18CO-、HOOC(CH 2) 19CO-、HOOC(CH 2) 20CO-、HOOC(CH 2) 21CO-和HOOC(CH 2) 22CO-。
  3. 根据权利要求1或2所述的衍生物或其药学上可接受的盐，其中所述延长部分通过接头与GLP-1(7-37)类似物的K残基连接。
  4. 根据权利要求3所述的衍生物或其药学上可接受的盐，其中接头为：
    Figure PCTCN2018083789-appb-100003
    Figure PCTCN2018083789-appb-100004
    Figure PCTCN2018083789-appb-100005
    其中m是0、1、2或3；n是1、2或3；s是0-6的任意整数；p是1-8的任意整数。
    优选地，接头为：
    Figure PCTCN2018083789-appb-100006
    Figure PCTCN2018083789-appb-100007
    其中m是1或2；n是1或2；p是1-5的任意整数。
  5. 根据权利要求4所述的衍生物或其药学上可接受的盐，其中接头为：
    Figure PCTCN2018083789-appb-100008
    其中m是1，n是1或2。
  6. 制备权利要求1-5中任意一项所述的衍生物或其药学上可接受的盐的方法，包括：
    (1)将溶解有上述权利要求任一项中所述的GLP-1类似物的溶液与溶解有上述权利要求任一项所述的延长部分的溶液混合；
    (2)调节pH至4-5终止反应，静置，至沉淀产生，取沉淀；和
    (3)向沉淀中加入TFA，调节pH至7.5-8.5终止反应。
  7. 权利要求6的方法，还包括在与溶解有上述权利要求任一项所述延长部分的溶液混合前，向溶解有GLP-1类似物的溶液中加入三乙胺。
  8. 权利要求6或7的方法，其中上述权利要求任一项所述的延长部分的溶液是乙腈溶解的。
  9. 一种药物组合物，其包括权利要求1-5中任意一项权利要求所述的衍生物或其药学上可接受的盐，以及药学上可接受的辅料。
  10. 权利要求1-5中任意一项权利要求所述的衍生物或其药学上可接受的盐在制备预防和/或治疗糖尿病或糖尿病并发症的药物中的用途。
  11. 权利要求10的用途，其中所述糖尿病并发症为糖尿病性肾病。
  12. 权利要求1-5中任意一项权利要求所述的衍生物或其药学上可接受的盐在制备减少胰岛β-细胞凋亡、增强胰岛β-细胞功能、增加胰岛β-细胞数量和/或恢复胰岛β-细胞的葡萄糖敏感性的药物中的用途。
  13. 一种预防和/或治疗糖尿病或糖尿病并发症的方法，包括给药受试者预防或治疗有效量的权利要求1-5中任意一项所述的衍生物或其药学上可接受的盐。
  14. 权利要求13的方法，其中所述糖尿病并发症为糖尿病性肾病。
  15. 一种减少胰岛β-细胞凋亡、增强胰岛β-细胞功能、增加胰岛β-细胞数量和/或恢复胰岛β-细胞的葡萄糖敏感性的方法，包括给药受试者治疗有效量的权利要求1-5中任意一项权利要求所述的衍生物或其药学上可接受的盐。
  16. 一种GLP-1(7-37)类似物，包含由如下氨基酸序列组成的多肽：
    HVEGTFTSDVSSX 19LEEX 23AAX 26X 27FIX 30WLVX 34GX 36X 37
    其中X 19为Y或K，X 23为Q或K，X 26为R或K，X 27为E或K，X 30为A或K，X 34为R或K，X 36为R或K，X 37为G或K，并且，在X 19、X 23、X 26、X 27、X 30、X 34、X 36或X 37中只有一个是K。
  17. 包含权利要求16的类似物的药物组合物。
  18. 权利要求16的类似物在制备预防或治疗糖尿病、糖尿病并发症的药物中的用途。
  19. 一种制品，包括其中装有权利要求9或权利要求17的药物组合物的容器和包装插页，其中该包装插页载有所述药物组合物的使用说明。
  20. 权利要求19的制品，还包含装有其它药物的容器。
  21. 权利要求20的制品，其中所述其它药物为治疗糖尿病或糖尿病并发症的其它药物。
PCT/CN2018/083789 2018-04-19 2018-04-19 酰化的glp-1衍生物 WO2019200594A1 (zh)

Priority Applications (15)

Application Number Priority Date Filing Date Title
PCT/CN2018/083789 WO2019200594A1 (zh) 2018-04-19 2018-04-19 酰化的glp-1衍生物
CA3135910A CA3135910C (en) 2018-04-19 2019-04-19 Acylated glp-1 derivative
CA3202015A CA3202015A1 (en) 2018-04-19 2019-04-19 Acylated glp-1 derivative
KR1020227008742A KR102653113B1 (ko) 2018-04-19 2019-04-19 아실화된 glp-1 유도체
AU2019256245A AU2019256245B2 (en) 2018-04-19 2019-04-19 Acylated GLP-1 derivative
MX2020011007A MX2020011007A (es) 2018-04-19 2019-04-19 Derivado de péptido-1 (glp-1) similar al glucagón acilado.
KR1020207033205A KR102464605B1 (ko) 2018-04-19 2019-04-19 아실화된 glp-1 유도체
JP2021506033A JP7164247B2 (ja) 2018-04-19 2019-04-19 アシル化ｇｌｐ－１誘導体
PCT/CN2019/083383 WO2019201328A1 (zh) 2018-04-19 2019-04-19 酰化的glp-1衍生物
BR112020021245A BR112020021245A8 (pt) 2018-04-19 2019-04-19 Derivado de um análogo de peptídeo semelhante a glucagon 1, métodos para preparar o derivado, para prevenir e/ou tratar diabetes ou complicações diabéticas e para reduzir glicose no sangue, aumentar tolerância à glicose, reduzir apoptose de célula beta de ilhota, intensificar função de célula beta de ilhota, aumentar número de célula beta de ilhota e/ou restaurar sensibilidade à glicose de célula beta de ilhota, composição farmacêutica, usos do derivado e do análogo, análogo de peptídeo semelhante a glucagon 1, e, produto.
US17/048,550 US11612640B2 (en) 2018-04-19 2019-04-19 Acylated GLP-1 derivative
EP19789171.6A EP3783014A4 (en) 2018-04-19 2019-04-19 ACYLATED GLP-1 DERIVATIVES
AU2021204749A AU2021204749B2 (en) 2018-04-19 2021-07-07 Acylated glp-1 derivative
JP2022164849A JP2022191398A (ja) 2018-04-19 2022-10-13 アシル化ｇｌｐ－１誘導体
US18/186,069 US20230330189A1 (en) 2018-04-19 2023-03-17 Acylated glp-1 derivative

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/083789 WO2019200594A1 (zh) 2018-04-19 2018-04-19 酰化的glp-1衍生物

Publications (1)

Publication Number Publication Date
WO2019200594A1 true WO2019200594A1 (zh) 2019-10-24

Family

ID=68239398

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/CN2018/083789 WO2019200594A1 (zh) 2018-04-19 2018-04-19 酰化的glp-1衍生物
PCT/CN2019/083383 WO2019201328A1 (zh) 2018-04-19 2019-04-19 酰化的glp-1衍生物

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/083383 WO2019201328A1 (zh) 2018-04-19 2019-04-19 酰化的glp-1衍生物

Country Status (9)

Country Link
US (2) US11612640B2 (zh)
EP (1) EP3783014A4 (zh)
JP (2) JP7164247B2 (zh)
KR (2) KR102464605B1 (zh)
AU (2) AU2019256245B2 (zh)
BR (1) BR112020021245A8 (zh)
CA (2) CA3202015A1 (zh)
MX (1) MX2020011007A (zh)
WO (2) WO2019200594A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021136303A1 (zh) * 2019-12-30 2021-07-08 甘李药业股份有限公司 长效glp-1化合物
WO2023151594A1 (en) * 2022-02-11 2023-08-17 Hangzhou Sciwind Biosciences Co., Ltd. A composition comprising gip receptor agonists and glp-1 receptor agonists and use thereof

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20230015414A (ko) 2020-05-29 2023-01-31 항저우 사이윈드 바이오사이언시즈 컴퍼니 리미티드 칼륨 n-[8-(2-히드록시벤조일)아미노]옥타노에이트결정 다형체 및 이의 제조 방법과 용도
CN115536739B (zh) * 2022-07-04 2023-04-14 北京惠之衡生物科技有限公司 一种glp-1受体和gcg受体共激动多肽衍生物的制备方法
CN115786426B (zh) * 2022-10-31 2023-09-08 杭州先为达生物科技有限公司 高特异性肠激酶酶切方法及乙醇在提高肠激酶酶切特异性中的用途
CN117247422A (zh) * 2022-12-06 2023-12-19 杭州先为达生物科技股份有限公司 一种多肽衍生物及其制备方法

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005023291A2 (en) * 2003-09-11 2005-03-17 Novo Nordisk A/S Use of glp1-agonists in the treatment of patients with type i diabetes
WO2006002532A1 (en) * 2004-07-01 2006-01-12 Waratah Pharmaceuticals, Inc. Methods and compositions using cd3 agonists
CN1882356A (zh) * 2003-11-20 2006-12-20 诺沃挪第克公司 对于生产和用于注射装置中是最佳的含有丙二醇的肽制剂
CN1938334A (zh) * 2004-01-30 2007-03-28 瓦拉塔药品公司 Glp－1激动剂和胃泌素化合物的联合使用
CN101133082A (zh) * 2005-03-18 2008-02-27 诺和诺德公司 酰化的glp-1化合物
CN104411322A (zh) * 2012-05-08 2015-03-11 诺和诺德A/S（股份有限公司） 双酰化glp-1衍生物
CN104519902A (zh) * 2012-05-08 2015-04-15 诺和诺德A/S（股份有限公司） 双酰化glp-1衍生物
CN105451776A (zh) * 2013-08-15 2016-03-30 诺和诺德股份有限公司 Glp-1衍生物及其用途
CN107033234A (zh) * 2017-01-03 2017-08-11 北京凯因科技股份有限公司 酰化的glp‑1衍生物

Family Cites Families (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US692464A (en) 1901-05-04 1902-02-04 Daniel Mackenzie Combined baby carriage and sleigh.
US5545618A (en) 1990-01-24 1996-08-13 Buckley; Douglas I. GLP-1 analogs useful for diabetes treatment
US5424686A (en) 1994-04-20 1995-06-13 Philips Electronics North America Corporation Negative-resistance-compensated microwave buffer
US5869602A (en) 1995-03-17 1999-02-09 Novo Nordisk A/S Peptide derivatives
BRPI9711437B8 (pt) 1996-08-30 2021-05-25 Novo Nordisk As derivados de glp-1
EP1056775B1 (en) 1998-02-27 2010-04-28 Novo Nordisk A/S Glp-1 derivatives of glp-1 and exendin with protracted profile of action
NZ527241A (en) 1998-12-07 2004-12-24 Sod Conseils Rech Applic Analogues of GLP-1
US6329336B1 (en) 1999-05-17 2001-12-11 Conjuchem, Inc. Long lasting insulinotropic peptides
WO2002046227A2 (en) 2000-12-07 2002-06-13 Eli Lilly And Company Glp-1 fusion proteins
BRPI0507189A (pt) 2004-01-30 2007-06-26 Waratah Pharmaceuticals Inc uso combinado de um agonista de glp-1 e compostos de gastrina
WO2006097538A1 (en) 2005-03-18 2006-09-21 Novo Nordisk A/S Extended glp-1 compounds
EP2190460B1 (en) 2007-09-05 2014-12-17 Novo Nordisk A/S Peptides derivatized with a-b-c-d- and their therapeutical use
EP2679597A1 (en) 2007-09-05 2014-01-01 Novo Nordisk A/S Glucagon-like peptide-1 derivatives and their pharmaceutical use
CN101367873B (zh) 2008-10-08 2011-05-04 南开大学 一种改构的胰高血糖素样肽-1的类似物和修饰物及其应用
JP6139712B2 (ja) 2013-07-04 2017-05-31 ノヴォ ノルディスク アー／エス Ｇｌｐ−１様ペプチドの誘導体及びその使用
WO2015155151A1 (en) * 2014-04-07 2015-10-15 Novo Nordisk A/S Double-acylated glp-1 compounds
ES2739289T3 (es) 2014-11-27 2020-01-30 Novo Nordisk As Derivados de GLP-1 y sus usos
US10392428B2 (en) 2014-12-17 2019-08-27 Novo Nordisk A/S GLP-1 derivatives and uses thereof

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005023291A2 (en) * 2003-09-11 2005-03-17 Novo Nordisk A/S Use of glp1-agonists in the treatment of patients with type i diabetes
CN1882356A (zh) * 2003-11-20 2006-12-20 诺沃挪第克公司 对于生产和用于注射装置中是最佳的含有丙二醇的肽制剂
CN1938334A (zh) * 2004-01-30 2007-03-28 瓦拉塔药品公司 Glp－1激动剂和胃泌素化合物的联合使用
WO2006002532A1 (en) * 2004-07-01 2006-01-12 Waratah Pharmaceuticals, Inc. Methods and compositions using cd3 agonists
CN101133082A (zh) * 2005-03-18 2008-02-27 诺和诺德公司 酰化的glp-1化合物
CN104411322A (zh) * 2012-05-08 2015-03-11 诺和诺德A/S（股份有限公司） 双酰化glp-1衍生物
CN104519902A (zh) * 2012-05-08 2015-04-15 诺和诺德A/S（股份有限公司） 双酰化glp-1衍生物
CN105451776A (zh) * 2013-08-15 2016-03-30 诺和诺德股份有限公司 Glp-1衍生物及其用途
CN107033234A (zh) * 2017-01-03 2017-08-11 北京凯因科技股份有限公司 酰化的glp‑1衍生物

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021136303A1 (zh) * 2019-12-30 2021-07-08 甘李药业股份有限公司 长效glp-1化合物
CN114901680A (zh) * 2019-12-30 2022-08-12 甘李药业股份有限公司 长效glp-1化合物
WO2023151594A1 (en) * 2022-02-11 2023-08-17 Hangzhou Sciwind Biosciences Co., Ltd. A composition comprising gip receptor agonists and glp-1 receptor agonists and use thereof

Also Published As

Publication number Publication date
EP3783014A1 (en) 2021-02-24
CA3202015A1 (en) 2019-10-24
AU2021204749A1 (en) 2021-07-29
AU2021204749B2 (en) 2022-03-31
EP3783014A4 (en) 2021-07-07
US20210393744A1 (en) 2021-12-23
EP3783014A9 (en) 2021-05-19
US20230330189A1 (en) 2023-10-19
KR20220035996A (ko) 2022-03-22
AU2019256245B2 (en) 2021-08-05
AU2019256245A1 (en) 2020-11-19
KR102464605B1 (ko) 2022-11-10
BR112020021245A2 (pt) 2021-02-02
KR102653113B1 (ko) 2024-04-02
JP7164247B2 (ja) 2022-11-01
CA3135910C (en) 2023-08-22
US11612640B2 (en) 2023-03-28
MX2020011007A (es) 2021-01-20
JP2022191398A (ja) 2022-12-27
KR20210005093A (ko) 2021-01-13
CA3135910A1 (en) 2019-10-24
JP2021522318A (ja) 2021-08-30
WO2019201328A1 (zh) 2019-10-24
BR112020021245A8 (pt) 2022-05-10

Similar Documents

Publication Publication Date Title
WO2019200594A1 (zh) 酰化的glp-1衍生物
US8809499B2 (en) Fusion protein of human fibroblast growth factor-21 and exendin-4
CA2909045C (en) Therapeutic peptides
JP2002506792A (ja) Ｎ末端修飾ｇｌｐ−１誘導体
US11713344B2 (en) Acylated oxyntomodulin peptide analog
JP2017509698A (ja) プレプログルカゴンから誘導可能なペプチドホルモン類似体
CN110386975B (zh) 酰化的glp-1衍生物
US7608587B2 (en) Exendin 4 polypeptide fragment
CN115814063A (zh) Glp-1衍生物及其治疗用途
CN107033234A (zh) 酰化的glp‑1衍生物
KR20230008846A (ko) 이중 수용체 아고니즘 작용을 갖는 폴리펩티드 유도체 및 그 용도
WO2019201333A1 (zh) Glp-1衍生物及其治疗用途
RU2773242C2 (ru) Ацилированное производное GLP-1
WO2023151594A1 (en) A composition comprising gip receptor agonists and glp-1 receptor agonists and use thereof
WO2020108228A1 (zh) 艾塞纳肽类似物

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18915272

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18915272

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 25.03.2021)

122 Ep: pct application non-entry in european phase

Ref document number: 18915272

Country of ref document: EP

Kind code of ref document: A1