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WO2013086786A1 - Composé réduisant le taux de glycémie, composition et applications associées - Google Patents

Composé réduisant le taux de glycémie, composition et applications associées Download PDF

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WO2013086786A1
WO2013086786A1 PCT/CN2012/001700 CN2012001700W WO2013086786A1 WO 2013086786 A1 WO2013086786 A1 WO 2013086786A1 CN 2012001700 W CN2012001700 W CN 2012001700W WO 2013086786 A1 WO2013086786 A1 WO 2013086786A1
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molecular weight
formula
compound
deletion
lysine
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Chinese (zh)
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秦树林
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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
    • A61K38/22—Hormones
    • A61K38/30—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • 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
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention belongs to the field of biopharmaceuticals, and particularly relates to compounds, compositions and uses thereof having hypoglycemic effects. Background technique
  • insulin has undergone three generations of historical evolution:
  • the first generation of products was extracted from the pancreas of animals such as pigs or cattle. Due to heterogeneous allergic reactions, these products are less effective.
  • the second generation of products is recombinant human insulin, which is obtained by extracting insulin genes from human cells, and then inserting them into yeast or E. coli for cultivation through complex modern biological gene technology.
  • the third generation is an insulin analogue obtained by structural modification of human insulin, including fast-acting insulin and long-acting insulin.
  • ADA American Diabetes Association
  • EASD European Diabetes Association
  • Basal insulin is used to maintain normal blood glucose secretion on an empty stomach. Many metabolic studies have found that maintaining a basic insulin level between meals and at night can reduce the breakdown of triglyceride, inhibit the liver's output of glucose, and stabilize the fasting blood glucose, thereby reducing overall blood sugar levels. Ideal basal insulins, such as long-acting insulin analogues, should be able to mimic physiological insulin secretion patterns, avoid hypoglycemia, especially nocturnal hypoglycemia, and do not increase body weight.
  • the most long-acting insulins currently used can be divided into three major categories.
  • the first type is a suspension of crystals formed by human insulin with zinc ions or basal protamine, such as NPH insulin, lente insulin, and the like. These insulin preparations are unstable and are being replaced by long-acting insulin analogues.
  • the second is detemir. It is an insulin analog of myristic acid linked to B29 lysine. Detemir is slowly absorbed after injection. The disappearance time T 50% at the injection site is approximately 10 hours. It binds to albumin in the blood through the fatty acid at position 29 and then slowly dissociates from the complex. Dihexamerization, hexamer and dimer binding to albumin prolongs the retention time of insulin detemir at the injection site. After the insulin enters the blood circulation, it binds to albumin, further prolonging the residence time in the body.
  • the third is that the insulin glargine drug dissolves in the formulation at pH 3.0, and crystallizes when the pH rises to about 7.4 after injection.
  • the slow decomposition of the injection site brings about a delayed effect.
  • absorption properties and pharmacokinetics vary widely in the population and within the individual.
  • Insulin glargine and insulin detemir are the only two long-acting insulin analogues on the market, with a maximum duration of action of no more than 24 hours. Insulin glargine is much more active on the insulin-like growth factor-1 receptor (IGF-1R) than native human insulin. Because insulin-like growth factor-1 receptor is closely related to the development of many types of cancer, it has been controversial whether long-term use of insulin glargine increases the risk of cancer in patients. The biological activity of insulin detemir in the human body is approximately the confirmation of natural human insulin. 20%, so it is used at a dose five times the conventional insulin dose, which significantly increases production and use costs.
  • IGF-1R insulin-like growth factor-1 receptor
  • Recombinant human insulin is difficult to meet the insulin requirements of the meal.
  • the human insulin molecule usually forms a hexamer structure, which is gradually depolymerized into a dimer after subcutaneous injection, and further dissociates into a monomer to enter the circulation through the capillaries, thereby exerting a hypoglycemic effect. Due to the depolymerization and absorption process, recombinant human insulin takes about 30 minutes after subcutaneous injection, and the peak time is long, and the effect lasts for about 6-7 hours. Moreover, due to individual differences, there is a significant difference in the amount of final influx after injection of the same dose of human insulin.
  • Rapid-acting insulin analogues such as insulin aspart, insulin lispro, etc.
  • Fast-acting insulin analogues absorb quickly, have a short peak time, have a higher peak value, and have a peak concentration of 1 to 3 hours.
  • the duration of action is 3 to 5 hours, which is significantly better than human insulin.
  • the onset time of insulin aspart and insulin lispro is about 20 minutes, which is still not convenient for diabetics, and there is still much room for improvement.
  • a first aspect of the invention provides a compound having hypoglycemic effect, the amino acid sequence of said compound being:
  • Xl07 is a phenine-valine-asparagine-glutamine tetrapeptide, a proline-asparagine-glutamine tripeptide, an asparagine-glutamine dipeptide, or glutamine, or a sequence in which the amino acid residue of any of the above-mentioned di-, tri-, or tetrapeptide sequences is substituted with lysine or arginine, or a deletion;
  • X 1 () 8 is histidine, phenylalanine, arginine or Glutamine;
  • X 1Q9 is arginine, alanine, glutamic acid or aspartic acid;
  • Xuo is phenylalanine, tyrosine or histidine;
  • X 14 is threonine, asparagine, glutamic acid, aspartic acid or deletion;
  • X 113 is valine, lysine
  • the present invention further provides a compound having a hypoglycemic effect and modified on a polypeptide basis to further improve the solubility, stability, in vivo circulation time, and the like of the compound.
  • the modification is an ⁇ -amino group which links the modified side chain to the N-terminal amino acid residue of the compound of the present invention, or an ⁇ -amino group which is linked to the lysine present in the compound of the present invention.
  • the structure of the compound is:
  • X 30 o is phenylalanine or U L -phenylalanine;
  • X 3 () 1 is phenylalanine, histidine or tyrosine;
  • X 3 o2 is tyrosine, benzene 2 001700
  • X 3 03 is threonine, asparagine, glutamic acid, aspartic acid or deletion;
  • X 3 04 is valine, lysine, glutamic acid, aspartic acid or Missing; X 3 .
  • X 3 o 6 is threonine, structure or deletion of formula (I)
  • X 3()7 is lysine, serine, alanine, glycine, structure or deletion of formula (I);
  • X 3Q8 is glycine, structure or deletion of formula (I);
  • X 3G9 is lysine, glycine , serine, structure or deletion of formula (I);
  • X 31Q is lysine, glycine, serine, structure or deletion of formula (I);
  • x 311 is lysine, glycine, serine, alanine, formula ( I) structure or deletion;
  • x 312 is lysine, arginine, alanine, valine, glycine, structure or deletion of formula (I);
  • x 313 is glycine, alanine, arginine, lysine Acid, glut
  • a third aspect of the present invention provides a pharmaceutical composition which is prepared by mixing a hypoglycemic compound of the present invention and a pharmaceutically acceptable carrier, and the mixing ratio may be about 90/10%, about 80/20%. , about 70/30%, about 60/40%, about 50/50%, about 40/60%, about 30/70%, about 20/80%, or about 10/90%; preferably, the composition Further comprising a fast-acting insulin analog; the fast-acting insulin analog can be Asp B28 human insulin, Lys B28 Pro B29 human insulin or Lys B3 Glu B29 human insulin.
  • a fourth aspect of the invention provides the use of a compound of the invention in the manufacture of a medicament for the treatment of diabetes or hyperglycemia.
  • a fifth aspect of the invention provides a method of treating diabetes or hyperglycemia, etc., comprising administering a compound or composition of the invention to a patient in need thereof.
  • the compound of the present invention Compared with the existing insulin and its analogs, the compound of the present invention has good water solubility, high activity in binding to the insulin receptor, low toxic side effects, and easy preparation. The cycle time of the modified compound in the body is significantly prolonged. DRAWINGS
  • Figure 1 is a graph showing changes in blood glucose with time after subcutaneous injection of physiological saline, human insulin and a compound of the invention II-2
  • Fig. 2 is a graph showing changes in blood glucose with time after subcutaneous injection of physiological saline and a compound of the invention II-17 in mice. ;
  • Figure 3 is a graph showing changes in blood glucose over time following subcutaneous injection of physiological saline, human insulin, and the compound II-11 of the present invention in mice.
  • Amino acid refers to any molecule that contains both amino and carboxyl functional groups, the amino and carboxyl groups of the alpha-amino acid being attached to the same carbon atom (alpha carbon).
  • the alpha carbon may have 1-2 organic substituents.
  • Amino acids contain L and D isomers and racemic mixtures 0
  • amino acid residues in the polypeptide sequence of the present invention are L isomers, that is, L-amino acids, and D-amino acids are represented by a lowercase letter "d" before the amino acid name or abbreviation, such as dK.
  • encodeable amino acid or “encodeable amino acid residue” is used to mean an amino acid or amino acid residue which may be encoded by a nucleotide triplet.
  • hGlu is homoglutamic acid
  • -hGlu is - the L isomer of HNCH(CO-)CH 2 CH 2 CH 2 COOH;
  • ⁇ -hGlu is the L isomer of -H CH(COOH)CH 2 CH 2 CH 2 CO-;
  • a-Asp is the L isomer of H CH(CO-)CH 2 COOH;
  • ⁇ -Asp is the L isomer of -HNCH(COOH)C3 ⁇ 4CO-;
  • a-Glu is the L isomer of -HNCH(CO-)CH 2 CH 2 COOH;
  • ⁇ -Glu is the L isomer of -HNCH(COOH)CH 2 CH 2 CO-;
  • ⁇ -Ala is — HN-CH 2 —CH 2 —COOH
  • Sar is sarcosine.
  • Amino acid residues can be represented by three-letter amino acid codes or single-letter amino acid codes; the amino acid tables are as follows: Table 1: Amino acid names and cartridges
  • Natural insulin refers to mammalian insulin (such as human insulin, bovine insulin, porcine insulin, etc.) derived from natural, chemical synthesis, genetic engineering. Human insulin comprises an A chain consisting of 21 amino acids and a B chain consisting of 30 amino acids. The two strands are linked by three disulfide bonds: A7 and B7, A20 and B19, A6 and AI L B7, A7 refer to the amino acid residue of position 7 (from the N-terminus) of the native insulin B chain and the position of the insulin A chain. 7 (from the N-terminus) of amino acid residues.
  • Insulin analogs are generic terms for modified insulin polypeptides, including double-stranded molecules consisting of A and B chains with homologous sequences to native insulin, and single chain insulin analogs. "Insulin analogs, which have partial, total or enhanced activity of natural insulin, or which can be converted in vivo or in vitro to a polypeptide that has partial, total or enhanced activity of natural insulin, for example one that increases, decreases or replaces one or more than native insulin. Polypeptides of amino acid residues.
  • Insulin analogs Human, animal, and even non-mammalian proinsulin, pro-proinsulin, insulin precursors, single-chain insulin precursors, and the like are all called “insulin analogs.” Many insulin analogs are found in The "insulin analog” broadly includes natural insulin and insulin analogs, unless otherwise stated.
  • the insulin referred to in this application refers to human insulin.
  • the human insulin A chain sequence is the sequence set forth in SEQ ID NO: 124
  • the human insulin B chain sequence is the sequence set forth in SEQ ID NO: 125. 1700
  • a single-chain compound refers to a polypeptide sequence having a general structural B chain-C L -A chain or a modified polypeptide sequence, wherein the B chain is the B chain or analog of insulin, and the A chain is the A chain or analog of insulin, C L is a peptide chain which links the C-terminal amino acid residue of the B chain to the N-terminus of the A chain.
  • the amino acid indicated by the position of the A chain or the B chain in the present application such as A14, B28, etc., represents an amino acid corresponding to the A chain or B chain of insulin or a change thereof, wherein the A chain or B chain of insulin The number starts from 1.
  • the insulin A and B chain sequences are found in SEQ ID NOS: 124 and 125, respectively.
  • cysteines in each compound of the present invention are numbered, respectively, C n] ⁇ C [6] , which in turn correspond to six cysteines of the single-chain compound from the N-terminus to the C-terminus.
  • the compound of the present invention is an insulin-based structure, and therefore the disulfide bond is contained in the tertiary structure of any of the compounds of the present invention, and a disulfide bond is formed in the same manner as insulin, that is, C n] and C[ 4] are formed.
  • Disulfide bond, . [2 ] and. [6 ] forms a disulfide bond, and C [3] and C [5] form a disulfide bond.
  • the insulin analog may comprise one or more modifying groups.
  • the modifying group is capable of providing the characteristics required for the insulin analog.
  • a modifying group can reduce the rate of degradation of an insulin analog in various environments (e.g., digestive tract, blood).
  • Preferred modifying groups are those which allow the insulin analog to retain comparable insulin receptor binding activity.
  • Preferred modifying groups include amphoteric groups, water soluble groups, or groups which render the insulin analog less lipophilic, more lipophilic, and more water soluble than the unmodified analog.
  • the modifying group can comprise a degradable linker.
  • PAG may include a readily hydrolyzable linker such as lactide, glycolide, carbonic acid, ester, amino phthalate. This method can degrade the polymer into small molecular weight fragments.
  • the modifying group may include one or more hydrophilic groups, lipophilic groups, amphoteric groups, salt-forming groups, spacer groups, linking groups, capping groups, or a combination of these groups.
  • the various groups may be linked together by covalent bonds or by hydrolyzable or non-hydrolyzable bonds. Representative hydrophilic groups and lipophilic groups are described below.
  • hydrophilic group examples include a PAG group, a polysaccharide, a polysorbate, and a combination of these groups.
  • Polyalkylene Glycol consists of a plurality of alkylene glycol monomers. In one embodiment, all monomers are the same (e.g., polyethylene glycol (PEG) or polypropylene glycol (PPG)). In another embodiment, the alkylene glycols are different.
  • the polymer may be a random copolymer such as a copolymer of ethylene oxide and propylene oxide, or a branched or graft copolymer.
  • PEG polyethylene glycol refers to any water soluble polyethylene glycol or polyethylene oxide.
  • the chemical formula of polyethylene glycol is -(CH 2 CH 2 0) n -, wherein n may be an integer from 2 to 2,000.
  • One end of the PEG is usually a relatively inactive functional group such as an alkyl group or an alkoxy group.
  • Alkyl groups include saturated straight or branched chain hydrocarbon groups. Representative examples of alkoxy are decyloxy, ethoxy, propoxy (e.g., 1-propoxy and 2-propoxy), butoxy (e.g., 1-butoxy, 2-butoxy). And 2-methyl-2-propoxy), pentyloxy, hexyloxy and the like.
  • the methoxy-terminated PEG is designated mPEG, the structural formula CH 3 0(CH 2 CH 2 0) n -, but is still generally referred to as PEG.
  • PEG20K refers to a molecular weight of 20,000 polyethylene glycol molecules.
  • the other end of the PEG is usually an activating functional group or a functional group which is liable to form a covalent bond, such as an amino group, a carboxyl group, a hydroxyl group, a thiol group, and the like.
  • PEG-maleimide, PEG-vinyl sulfone and PEG-iodoacetyl (CO-CH 2 -I ) can be combined with cyste 0
  • the thiol-SH reaction of the side chain of the acid forms a stable covalent bond;
  • PEG-NHS succinimide
  • acylation nucleophilic substitution reaction
  • the PEG-aldehyde and the amino group of the polypeptide can be joined by a reductive alkylation reaction under the action of a reducing agent such as sodium cyanoborohydride.
  • the PEG molecule in the present invention may be a linear, branched, bifurcated or dumbbell-shaped PEG.
  • the branched PEG can be represented by the formula m R (-PEG- n OH), where R (typically polyhydric) of the core group, such as pentaerythritol, a sugar, lysine or glycerol.
  • R typically polyhydric
  • m represents the number of branches, which may be the maximum number of attachment sites from 2 to the core group
  • n represents the number of PEG fragments, and the number of PEG fragments per branch may vary. In general, n is an integer from 2 to 1800.
  • the branched PEG can be represented by the formula (CH 3 0-PEG- n ) p RZ, p is equal to 2 or 3, R is lysine or glycerol, and Z represents an activating functional group capable of undergoing a reaction.
  • the bifurcated PEG is represented by the general formula PEG(-LX) n , L is a linking group, and X is a terminal activating functional group.
  • PEG is generally polydisperse, with multiple ⁇ I moieties less than 1.05.
  • the PEG group can also be mono-:.
  • Monodisperse means that PEG has a single length (molecular weight) rather than a mixture of various lengths (molecular weight).
  • Representative sugar groups include, but are not limited to, glycerin, monosaccharides, disaccharides, trisaccharides, oligosaccharides, and polysaccharides such as starch, glycogen, cellulose, and/or polysaccharide gums.
  • Particular monosaccharides include C6 and above (especially C6 and C8) sugars such as glucose, fructose, mannose, galactose, nucleic acid sugar or sedose heptose; disaccharides and trisaccharides include two or three monosaccharide units ( In particular, groups of C5 to C8), such as sucrose, cellobiose, maltose, lactose and/or raffinose.
  • Biocompatible polycationic groups include polyamine groups having a plurality of amino groups on the backbone or side chain, such as polylysine and other natural or synthetic amino acids having a plurality of positively charged amino acid polymers, including poly birds.
  • Biocompatible polyanionic groups include groups having a plurality of carboxyl groups on the backbone or side chain, such as polyaspartic acid, polyglutamic acid, and the like.
  • Other hydrophilic groups include natural or synthetic polysaccharides such as chitosan, dextran, and the like.
  • Certain hydrophilic groups have potential bioadhesive properties. An example of this can be found in U.S. Patent 6,197,346. These polymers having a plurality of carboxyl groups exhibit bioadhesive properties. Rapid biodegradable polymers that exhibit multiple carboxyl groups upon degradation, such as lactic acid glycolic acid copolymers, polyanhydrides, and polyorthoesters, are also bioadhesives. These polymers can be used to administer insulin analogs to the gastrointestinal tract. The carboxyl groups exposed during polymer degradation can be firmly attached to the gastrointestinal tract and assist in the administration of insulin analogs.
  • the modifying group includes one or more lipophilic groups.
  • Lipophilic groups can be well known to those skilled in the art and include, but are not limited to: alkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, fatty acid, cholesterol, and lipophilic polymers And oligomers.
  • the hydrocarbyl group can be a saturated, unsaturated, linear, branched or cyclic hydrocarbon having one or more carbon atoms.
  • the hydrocarbon group has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more carbon atoms.
  • the hydrocarbyl group may be unsubstituted or have one or more substituents which preferably do not lose the biological activity of the conjugate.
  • the lipophilic group can also be a fatty acid such as a natural, synthetic, saturated, unsaturated, linear or branched fatty acid.
  • the fatty acids are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more carbon atoms. Conjugation strategy
  • an insulin analog has one, two, three, four or more modifying groups after modification.
  • the binding site may include an amino acid residue, such as a lysine residue.
  • the insulin conjugate is a single conjugate.
  • the insulin conjugate is a multi-conjugate.
  • the insulin conjugate is a mixture of monoconjugates, biconjugates, triconjugates, tetraconjugates, and the like.
  • the modifier groups can be the same or different.
  • one or more modifying groups are preferably attached to the insulin conjugate via a hydrolyzable linkage and the other one or more modifying groups are preferably linked to the insulin conjugate via a non-hydrolyzable linkage.
  • all of the modifying groups are linked to the insulin conjugate via a hydrolyzable bond, but the rate of hydrolysis of each modifying group in vivo is fast and slow.
  • binding sites include an N-terminal alpha amino group and a lysine side chain amino group. Alternatively, it can be created by embedding a natural or non-natural number of acid residues having an amino group or a thiol group in a linking fragment of a single-chain compound or an oxime chain or an oxime chain. Other binding sites.
  • the modifying group and the insulin analog can be combined by a hydrolyzable bond such as an ester, carbonic acid, hydrolyzable aminodecanoate.
  • the hydrolyzable bond allows the insulin conjugate to have the effect of a prodrug.
  • a prodrug strategy is the preferred method if it is desired that the modifying group is inactive with the insulin conjugate, such as the binding site of the modifying group in the insulin analog to insulin receptor binding region.
  • the insulin analog is linked to the modifying group by a non-hydrolyzable linkage (eg, an amide linkage, an ether linkage).
  • a non-hydrolyzable linkage eg, an amide linkage, an ether linkage.
  • non-hydrolyzed bonds help to prolong the circulation time of the insulin conjugate in plasma.
  • Insulin homologs can be attached to the modifying group by various nucleophilic functional groups including, but not limited to, nucleophilic hydroxyl or amino groups. For example, serine, threonine, tyrosine have a nucleophilic hydroxyl group, histidine, lysine or insulin analog A chain, B chain N-terminus has a nucleophilic amino group. Insulin homologs can also be attached to the modifying group via free thiol-SH, for example to form succinate, thioether, sulfonamide linkages.
  • a common method is to form a hydrolyzable or non-hydrolyzable bond with a natural or synthetic macromolecule.
  • Biomacromolecules include albumin, polysaccharides, antibodies (such as IgG), and the like. 70% of the albumin in the blood vessels is mercaptalbumin, and the side chain thiol of cysteine-34 is the most active sulfhydryl group in plasma.
  • the insulin analog can be reacted with a linker having an activating functional group such as maleimine at one end to form an insulin-albumin conjugate.
  • the linker can be a long chain fatty acid or a PEG molecule. Specific examples can be found in Bioconjugate Chem. 2005, 16, 1000-1008. Synthetic macromolecules include polyethylene glycol and dextran. Another way is fatty acid acylation, which will be discussed in the section on acylated insulin analogs.
  • Sortase is a transpeptidase that mediates the covalent binding of Gram-positive bacterial cell walls to surface proteins, mainly in Gram-positive bacteria.
  • SrtA or SrtA Stap h Staphylococcus aureus sortase A
  • isoform isoform
  • SrtA recognizes a substrate comprising the LPXTG (Leu-Pro-X-Thr-Gly) motif, and the cysteine at position 184 acts as a nucleophilic group to attack the peptide bond Thr-Gly in the LPXTG motif, thereby producing a Acyl-enzyme intermediate.
  • the thioester intermediate of the threonine carboxyl group undergoes a nucleophilic reaction with the amino group of the oligoglycine of the substrate (penta-glycine (Gly 5 ) which is a bridge of the precursor of the branched lipid II in S. aureus) , creating new connection products.
  • SrtA strep Another related ⁇ Str ⁇ ptococciw / ⁇ ogew y sortase can accept a nucleophilic group consisting of two alanines, but the aureus enzyme cannot.
  • This sortase (SrtA strep ) cleaves the peptide bond Thr-Ala in the LPXTA motif, allowing an alanine-based nucleophilic group.
  • SrtA strep also recognizes LPXTG motifs but is less active. The LPXTA motif is not cut by SrtA Staph .
  • SrtA Staph For the sake of simplicity, the following methods are discussed with SrtA Staph as an example, but the SrtA strep equivalent can also be used in the same or similar manner.
  • SrtA is highly specific for the LPXTG motif and the glycine repeat (a-Gly n ) with a free amino group at the N-terminus.
  • the X position can be all natural amino acids other than cysteine and tryptophan (not yet tested).
  • the non-natural functional group can be a small molecule, a synthetic polypeptide or protein, a polymer, and the like. These functional groups can be combined with LPXTG or a-Gly n to form a substrate for SrtA.
  • Specific methods and reaction conditions can be referred to, literature (: ⁇ mouth Tsukiji et al, "Sortase-Mediated Ligation: A Gift from Gram-Positive Bacteria to Protein Engineering", ChemBioChem, 2009, 10, 787-798; Popp et al, "Sortase- Catalyzed transformations that improve the properties of cytokines", PNAS, 2011, 108, 3169-3174).
  • SrtA is capable of introducing non-natural functional groups on insulin analogs, depending on the structure of the insulin analog.
  • the binding site that affects biological activity less is the N-terminus of the B-chain or the C-terminus of the B-chain.
  • the N-terminus of the insulin B chain preferably introduces a plurality of glycines, such as the GGGGG-insulin B chain, and the modified group to be introduced, such as PEG, long-chain fatty acids or albumin.
  • the C-terminus has a LPXTG motif such as PEG-LPATGGGG, albumin-LPETGGG or fatty acid LPGTGGGGG.
  • the binding site is the C-terminus of the A or B chain
  • the C-terminal amino acid sequence of the A or B chain comprises an LPXTG motif, such as insulin A-LPATGGGGG or insulin B-LPGTGGGG, etc.
  • the modified group to be introduced Groups such as PEG, long-chain fatty acids or albumin have one or more glycines at the N-terminus, such as GGG-PEG, GGGG-long-chain fatty acids, GGGGG-albumin, and the like.
  • the easiest binding site is the N-terminus of the B-chain or the C-terminus of the A-chain, and the method and the double-stranded analog are substantially identical.
  • Single-chain compound single-chain compound based on insulin
  • Proinsulin is a single-stranded precursor of 86 amino acids constructed as: ⁇ chain -ArgArg-C peptide -LysArg-A chain.
  • the C peptide is a "linker peptide" composed of 31 amino acids.
  • Arg-Arg and Lys-Arg are the proteolytic enzymes that cause the C-peptide to split from the A and B chains.
  • Proteolytic enzymes are known to be prohormone convertases (PC1 and PC2), and exoproteinase carboxypeptidase 5 . These changes in proinsulin remove the C-peptide, and the remaining B-chain and A-chain are bound together by disulfide bonds.
  • the double-stranded structure of insulin allows insulin to have multiple conformations.
  • Insulin has the potential for considerable conformational changes, and limitations on these changes significantly reduce the affinity of the insulin receptor for ligands. Blocking the amino terminus of GlyAl also attenuates receptor binding ability.
  • Proinsulin has an affinity for insulin receptors of only 1-2% of insulin.
  • C-peptide in proinsulin folding is still unclear.
  • the length of the C peptide varies between 26 and 38 amino acids in different animal species.
  • the dibasic amino acid residues at the junction of the B-chain-C peptide (B-C) and the C-peptide-A chain (C-A) are conserved and the need for insulin conservation is considered to be minimal.
  • the three-dimensional structure of insulin shows that the A chain and the B chain can be bound by a linker peptide which is much smaller than the 31 amino acid C peptide.
  • the intrinsic physical and chemical stability of insulin molecules is a prerequisite for insulin therapy for diabetes, and is also the basis for insulin conformation, applicable insulin delivery methods, and shelf life and storage conditions for pharmaceutical formulations.
  • insulin administration 1700 The use of solutions to expose insulin molecules to a variety of factors, such as elevated temperatures, gas-liquid-solid phase changes, and shear forces, may result in irreversible conformational changes in insulin molecules, such as fibrillation.
  • This is closely related to the insulin solution in the syringe pump, because insulin molecules are exposed to these factors, both externally and implanted, as well as from the shear forces generated during long-term movement of the pump. Therefore, fibrillation is a big problem when using a syringe pump as an insulin delivery system.
  • solubility of insulin is affected by a variety of factors and is significantly reduced in the range of pH 4.2-6.6.
  • the pH settling zone typically imposes limitations on the formulation.
  • the present invention addresses these problems by providing a stable single-stranded compound by introducing a C-peptide between the B and A chains, reducing molecular flexibility while reducing fibrillation tendency, limiting or modifying the pH settling zone, thereby providing more formulation and formulation. Broad choice.
  • the current genetic engineering and then the pancreatin precursor is digested to generate double-stranded islets ⁇ :. If the single-chain insulin analog is directly produced, the production process is greatly simplified and the cost is reduced.
  • insulin-like growth factor-1 is a single-chain peptide having 70 amino acid residues, including the A, B, C, and D domains.
  • the basic structure of the A and B domains of IGF-1 is highly similar to the A and B chains of insulin, with 52% and 45% homology, respectively. Their three-dimensional structure is also very similar.
  • the C domain of IGF-1 plays a minor role in insulin receptor binding. Removal of all IGF-1 C domains, replaced by a bridge of four glycines, resulted in a twofold increase in insulin receptor binding compared to wild type, while addition of the IGF-1 C domain to the C-terminus of the insulin B chain causes insulin Receptor affinity was reduced by a factor of 3.5 compared to wild type. The insulin binding capacity of the single-chain insulin/IGF-1 mixture consisting of insulin and the IGF-1 C domain was not significantly different from that of native human insulin. Interestingly, the IGF-1 CII hybrid has increased affinity for both IR-A and IR-B, while IGF-2 CI has a weaker affinity, indicating that the C domain determines IR binding specificity.
  • Tyr31 in IGF-1 is essential for maintaining high affinity for IGF-1 receptors, but it appears to block binding to the insulin receptor, as tyrosine is replaced by alanine, resulting in a small but distinct human placenta A double increase in insulin receptor binding.
  • the inventors have found in the research that a single-stranded compound formed by ligation of a double strand of an insulin molecule by a linker has the same insulin activity and has the advantages of being easy to prepare, providing more polypeptide modification sites, and the like.
  • the ligation fragment CL is a peptide sequence consisting of 6-60 amino acids, wherein each amino acid is independently selected from the group consisting of glycine, alanine, serine, threonine, and valine.
  • a suitable connecting segment C L has a three-point feature: First, the connecting segment requires an appropriate length. When the B chain is 30 amino acids in length, the length of the ligated fragment is preferably not less than 6 amino acids; when the B chain is 25 amino acids, the length of the ligated fragment is preferably not less than 10 amino acids.
  • the insulin receptor binding ability of the single-chain analog has a decreasing tendency;
  • the ligated fragment preferably has no secondary structure, and the spatial conformation can be flexibly changed;
  • the ligation fragment itself is not biologically active, but can provide polypeptide modification sites such as acylation, glycosylation and the like.
  • C L may be substituted by amino acid residues and comprising at least one insert or an aspartic acid, glutamic acid, arginine, lysine, cysteine or asparagine.
  • C L may include 1, 2, 3, 4 aspartic acid, glutamic acid, arginine or lysine to regulate the charge balance of the polypeptide sequence and improve solubility.
  • the sequence may comprise 1, 2, 3, 4, 5 asparagine and the same amount of serine or threonine to form the NXS/T consensus sequence required for N-glycosylation (X is a codeable natural amino acid) ).
  • the peptide may further comprise 1, 2, 3 or 4 lysine or cysteine, and the side chain amino group or sulfhydryl group may be a natural or synthetic modifying group such as a fatty acid, polyethylene glycol or albumin.
  • the linked insulin molecules have different physical, chemical and biological properties by being linked by a hydrolysis bond or a non-hydrolysis bond.
  • the C-terminal amino acid of C L may be selected from the group consisting of glycine-lysine, glycine-arginine, arginine-arginine, lysine-lysine, arginine-lysine a group consisting of lysine-arginine, valine-glutamine-threonine, valine-glutamine-lysine, or valine-glutamine-arginine.
  • the terminal amino acid is selected from lysine or arginine.
  • C L may be all or part of the sequence of the following polypeptide fragments, or may differ from the following polypeptide fragments by 1, 2 or 3 amino acid residues, or 70%, 80% of the following polypeptide fragments, 90% similar, or 1, 2, 3, 4 or 5 repeats of all or part of the sequence of the following polypeptide fragments:
  • GASPGGSSGS (GASPGGSSGS)FortunatelyGR, where n is 1, 2, 3, 4 or 5; GSSGSSGPGSSR; GSSGSGSSAPQT;
  • GSGGAPSRSGSSR GGSGGSGGR; GSSPATSGSPQR; GASSSATPSPQR; GSGSSSRAPPSAPSPQR; GSSSESPSGAPQT; GAGTPASGSAPGR; GSSPSGGSSAPQT;
  • GSGSSSAAAPQT GSGSSSAAPQT ; GASPGTSSTSGR ; GSGSSSAPQT ; GSGSSSRRA ;
  • GSPAGSPTSTSR GSGPSSATPASR
  • GSGSSSRGR GSGGPSTRSAPQR
  • GPETPSGPSSAPQT GSPAGSPTSTSR
  • GSGPSSATPASR GSGSSSRGR
  • GGPSTRSAPQR GSGSSSRGR
  • GSGPSTRSAPQR GSGSSSRGR
  • GPETPSGPSSAPQT GSPAGSPTSTSR; GSGPSSATPASR; GSGSSSRGR; GSGPSTRSAPQR; GPETPSGPSSAPQT;
  • GAGSSSRAPPPSAPSPSRAPGPSAPQR GSGSSAGR; GASSPSTSRPGR; GSSSGSSGSPSGR; GSSPSASTGTGR; GAGSSSAPSAPSPSRAPGPSAPQR; GSGSGSGR; GSPSSPTRGSAPQT;
  • GASTSSRGAPSR GASTSSRGAPSR; GSGSSSAGR; GPSGTSTSAPGR; GAGSSSAPQT; SSSSAPSAPSPSRPQR;
  • GSGASSPTSPQR GAGGSGSGR; GSSPATSATPQT; GAGSSSAPPPSAPSPSRAPGPSAPQR;
  • GASTGSSRPSGR GSTAGSRTSTGR; GSTAGSRTSPQR; GSGTATSGSPQT; GASSSATSASGR;
  • GAGSATRGSASR GAGSATRGSASR; GSSSRSPSGSGR; SSSSAPPPSAPSPSRAPGPSAPQR; GSSPSG SSSPGR; GSPAGSPSSSAGSSASASPASPGR; GSPAGSPSSSAGSSASASPASGPGSSSAPSAGSPGR;
  • SSSAPPPSAPSPSRAPGPSPQR SAASSSASSSSASSASAGR; GAGGPSSGAPPPSPQT;
  • GSGSSGGR GAGSPAAPASPAPAPS AGR; SSSAPSPSRSPGPSPQR; SSSAPSAPSPSPQR;
  • GSGSSSRRAPQT SSSSAASAASASSSASGR; SSSRAPPSAPSPQR; GGPSSGAPPPSR; SSSSGAPPPGR; GPSSGAPSR; GPSSGAPQT; GGPSSGAPPPSPQT; SSSAPPPSAPSPSRAPQT;
  • GAGPSSGAPPPSPQT GGGGAPQT; GAGGPSSGAPPPQT; GGPSSGAPPPSPSPSRPGPSPQR;
  • SSASSASSSSAGR GAGSSR
  • SSASSSAASSSASSSASGR SSSGAPPPSPSRAPGPSPQR
  • GSGSASRGR SSSSAASSASGR ; SASASASASSASSGR ; SASSPSPSAPSSPSPAS ;
  • GPSSPSPSAPSSPSPASPSSGR SSSAPPPASPSPSRAPGPQR ; SASASASASASSAGR ; GSGASSRGR ; GSGAAPASPAAPAPS AGR; GGPSSGAPPPSGR ; SSPSASPSSPASPSSGR ;
  • GAPASPAPSAPAPAAPSGR GPSSPSPSAPSSPSPASPSSAPQT
  • SSASSASSSSSASAGR SSASSASSSSSASAGR
  • SAPSSPSPSAPSSPSASPSGR SSSAPPPSAPSPSAPQR ; GASSPSPSAPSSPSPASGR ;
  • GSGSSR GSGSSSAR
  • GSGSSSGR GSGAPQR
  • SSSSAPSAPSPSRAPGPSPAPQR GSGSSSR; GSGSSAPQT; GGGGAPQR; GSGSSSAAR; GSGSSAAPQR; SSSSRRAPQR;
  • GSGSSSAAAPQR GSGSSSAAAPQR
  • GAGSSSAAAPQR GAGSSSAAAPQT
  • GSSGGSGR GAGGGSSGR;
  • SSSSRAPPPSAPSPSRAPGPSAPQR GGGSSR; GSGSSSAAPQR; GASPGGSSGSSR; GSGSSSRSGR; GTGPSSATPASR; GAGPSGTASPSS; SSSSAPSAPSPSRAPQR; GSPSSPTRGSAT ; GPETPSGPSSAT ; GSSPATSGTPQT ;
  • GSGSSSRAPPPSAPSPSRAPGPSPAPQR GSGSSSRAPPPSAPSPSRAPGPSPAPQR; GSSTPSGAGPQT; GSGSSSRAPPPSAPSPSRAPQR; GSPAGSPSSSAGSSASASPASGPGSSSAPSAGSPAR; GAGSSSRAPPPSAPSPSRAPGPSPQR; GSGSSSRAPPSAPSAPQR; GSTAGSRTSTAR; GSSPSGRSSSPAR; SSASSASSSSSAASAGR; GSSSGSSGSPSAR; SSSAPSPSRAPGPSPQR; GAGSSSRAPPPSAPSPSRAPQR; GSPAAPAPASPAAPAPSAGR; SSSAPSAPSPSAPQR; GGPSSGAPPPSPSPSRPGPSDTPPQR; SASASASASASASSASSGR; SASSPSPSAPSSPSPASGR; SASASASASASASSAGR; SSPSASPSSPASPSPSSGR; GAPASPAPAAPSAPAPAAPSGR; GAGSPAAPAPASPAPAPSAGR; SSSRAPPPSAPSPSAPQT
  • the present invention provides a single-chain compound having a hypoglycemic effect, the compound being modified according to the structure of the insulin, the structure of which is:
  • X 1Q7 is phenylalanine-valine-asparagine-glutamine tetrapeptide, proline -asparagine-glutamine tripeptide, asparagine-glutamine dipeptide, or glutamine , or a sequence in which the amino acid residue of any of the above two, three, or tetrapeptide sequences is substituted with lysine or arginine, or a deletion;
  • X 1 () 8 is histidine, phenylalanine, spermine Acid or glutamine;
  • ⁇ ) 9 is arginine, alanine, glutamic acid or aspartic acid;
  • X 110 is phenylalanine, tyrosine or histidine;
  • X m is tyrosine, Phenylalanine or deletion;
  • X 112 is threonine, asparagine, glutamic acid, aspartic acid or deletion;
  • X 113 is valine,
  • cysteine in the single chain forms a double bond, specifically: C n] and C [4 ] form a double bond, and C m and C[ 6] form two Sulfur bond, C [3 ] and. [ 5] Formation of a disulfide bond.
  • X 108 , X 10 9 , X , X , X are related to whether the compound produces self association like human insulin.
  • Human insulin is typically stored in islet beta cells by self-association to form hexamers. After subcutaneous injection of recombinant human insulin molecules, the hexamers are gradually depolymerized into dimers, and further dissociated into monomers to enter the circulation through the capillaries, and play a hypoglycemic effect.
  • Recombinant human insulin has a long-lasting effect after subcutaneous injection due to the presence of depolymerization and absorption processes (Brange et al., "Monomelic insulins and their experimental and clinical implications" Diabetes Care, Vol 13 No.
  • X 108 is histidine, it is advantageous for the compound to form a hexamer structure with the aid of zinc ions.
  • X 1 () 8 is an amino acid residue such as aspartic acid, glutamic acid, phenylalanine, glutamine or arginine, a stable hexamer structure cannot be formed.
  • the amino acid residues of ⁇ ) 9 , X 112 , X 113 , X U4 and the like are aspartic acid or glutamic acid, stable self-association is not easily formed. Therefore, if X 108 is an unhistidine amino acid residue, or Xl09,
  • One or more of the amino acid residues of X, Xii3, X and the like are aspartic acid or glutamic acid, and the corresponding compound is more likely to exist in the form of dimer or monomer, and rapidly enters the blood after subcutaneous injection. , can achieve the effect of lowering blood sugar in a short time.
  • the structure of the single chain compound is:
  • x is tyrosine or deletion; x 112 is threonine or deletion; x 113 is valine or deletion; x 114 is lysine or deletion; X U5 is threonine or a deletion; C L is a linker fragment as defined herein.
  • the structure of the single chain compound is:
  • the single-chain compound of the present invention having hypoglycemic action is selected from the group consisting of the following compounds: I -1:
  • NYCN SEQ ID NO: 44 ;
  • NYCN SEQ ID NO: 45 ;
  • FVNC CN (SEQIDNO: 51 );
  • NYCN SEQ ID NO: 84 ;
  • SICSLYQLENYCN SEQ ID NO: 110
  • FVNQP NYCN (SEQ ID NO: 115);
  • I-119 ENYCN (SEQ ID NO: 119); I -120:
  • Polypeptide chemists have used several methods to solve the problem of rapid clearance of drug molecules with a molecular weight of less than 67 kDa in the kidney by plasma.
  • the injection site is built with "depot”; 2. It is combined with non-covalent bond of plasma carrier protein to prevent glomerular filtration; 3. Covalently linked with carrier protein; 4. Large molecular weight Modification group binding, such as large molecular weight PEG, polysaccharides, etc. (as disclosed in the previous section of this application).
  • "Hydrophobic depoting" greatly increases the hydrophobicity of the peptide to reduce solubility and make it in the injection section.
  • the polypeptide binds to the cell membrane and/or the systemic carrier protein (such as albumin, etc.)
  • the systemic carrier protein such as albumin, etc.
  • the molecular weight of the carrier protein is greater than the maximum molecular weight of the glomerular filtration, so it is not easily removed by the kidney.
  • the plasma is circulated for many days. Therefore, the polypeptide bound to the carrier protein is not easily filtered by the glomerulus or degraded by the protease on the inner membrane.
  • Fatty acids generally extend the time of action of the polypeptide in vivo in three ways.
  • the fatty acid can bind non-covalently to albumin at the site of drug injection, and the formed polypeptide-fatty acid-albumin macromolecule conjugate is slowly dried.
  • the large molecular weight of the polypeptide-fatty acid-albumin conjugate is The renal clearance rate is reduced;
  • albumin provides protection for the polypeptide and is not easily degraded by proteases.
  • fatty acids reduce the immunogenicity of the polypeptide. The latter three features are similar to the long-chain PEG modification. Further mechanisms and experimental support can be found in Biochem. J.
  • modification of insulin with macromolecules such as polyethylene glycol (PEG, molecular weight not less than 20K) and human albumin can also achieve an effect of prolonging the in vivo action time similar to the above fatty acid modification. Therefore, all of the sites which can be acylated with fatty acids can be modified with macromolecules such as polyethylene glycol or human albumin.
  • the present invention is based on the recognition that the overall hydrophobicity of the hypoglycemic compound of the present invention plays an important role in the in vivo efficacy of the compound.
  • the present invention further provides a compound having a hypoglycemic effect and modified on a polypeptide basis to further increase the in vivo circulation time of the compound.
  • the modification is an ⁇ -amino group which links the modified side chain to the N-terminal amino acid residue of the single-chain compound of the present invention, or an ⁇ -amino group of lysine which is linked to the single-chain compound.
  • the modified compounds of the invention may also be formed based on modification of insulin or an analog thereof.
  • Ben The invention provides a modified single chain compound based on insulin, the structure of which is:
  • ⁇ 30 ⁇ is phenylalanine or phenylalanine;
  • X 3 oi is phenylalanine, histidine or tyrosine;
  • X 302 is tyrosine, phenylalanine or deletion;
  • X 3 03 is sul Acid, asparagine, glutamic acid, aspartic acid or deletion;
  • X30 4 is valine, lysine, glutamic acid, aspartic acid or deletion;
  • X 3Q5 is aspartic acid, glutamine Acid, valine, arginine, lysine or deletion, or structure of formula (I);
  • X 3 o 6 is threonine, structure or deletion of formula (I);
  • X 3 () 7 is lysine Acid, serine, alanine, glycine, structure or deletion of formula (I);
  • s is glycine, structure or deletion of formula (I);
  • X 3 o 9 is lysine, glycine, serine, structure or deletion of formula (I);
  • X 31Q is lysine, glycine, serine, formula (I) Structure or deletion;
  • X 311 is lysine, glycine, serine, alanine, structure or deletion of formula (I);
  • X 312 is lysine, arginine, alanine, valine, glycine, Structure or deletion of formula (I);
  • X 3 13 is glycine, alanine, arginine, lysine, glutamine, valine, structure or deletion of formula (I);
  • X 314 is arginine , alanine, valine, threonine, glutamine, glycine, structure or deletion of formula (I);
  • X 315 is valine, glutamine, arginine, glycine or deletion or
  • X 325 is asparagine or a structure of formula (I);
  • x 326 is alanine, glycine or asparagine;
  • x 327 is lysine, arginine-lysine dipeptide or deletion Or a structure of the formula (I); when x 327 is a dipeptide, one of the amino acids is a structure of the formula (I);
  • the pass structure is:
  • U L is -WXYZ structure, fatty acid, polyethylene glycol, albumin, L Principal-M L structure, hydrogen atom or N a -(N a -(HOOC(CH 2 ) n CO)-Y-Glu)- , N a -(N a -(CH 3 (CH 2 ) n CO)-Y-Glu)- , wherein ⁇ is an integer from 8 to 20, such as 8, 10, 12, 14, 16, 18 or 20, ⁇ ⁇
  • the structure of the formula ( ⁇ ) is:
  • J is a -WXYZ structure, an L n -M t structure or a hydrogen atom.
  • M L is a modifying group including, but not limited to, -WXYZ, a fatty acid, polyethylene glycol, albumin, IgGFc, a sugar group, and the like. In the present invention, it is an optional linker, covalent bond or absent.
  • Optional linkers include, but are not limited to: A long chain formed by covalent bonding of polyethylene glycol, long chain fatty acids or one or more polyethylene glycol molecules and long chain fatty acid molecules.
  • List can be -NH-(CH 2 ) n -£0-, -NH-(CH 2 CH 2 0) n -CH 2 -CO- , -NH-(CH 2 CH 2 0) faced-(CH 2 r -CO-, n is an integer from 1 to 20, and r is an integer from 1 to 10; in one embodiment, is -NH-(C3 ⁇ 4CH 2 0) 2 -CH 2 -CONH -(CH 2 CH 2 0) 2 -CH 2 -CO-; In one embodiment, -NH-iCHz ⁇ -C CHzCHzO CH nj- O-, nl, n2, ⁇ 3 are integers from 1 to 16 respectively; Wherein, L n is -NH-(CH 2 ) nl -(OCH 2 CH 2 ) deliberately2-£0-, nl, n2 are integers of 1 to 16 respectively.
  • Lache is underlined by The bond of the carbonyl carbon forms an amide bond with the amino group of the polypeptide compound, and the other end forms a covalent bond with M L .
  • L amide forms an amide bond with the amino group of the polypeptide compound via a bond from the underlined carbonyl carbon and an amide bond with -WXYZ at the other end.
  • W is an ⁇ -amino acid residue having a carboxyl group in a side chain which has a carboxyl group and an ⁇ -amino group of the ⁇ -terminal amino acid residue of the polypeptide compound of the present invention or an ⁇ -amino group of a lysine residue of the polypeptide compound.
  • W is a chain linked by 2, 3 or 4 ⁇ -amino acid residues via an amide bond, the chain being linked to the ⁇ -amino group of the ⁇ -terminal amino acid residue of the polypeptide compound or the lysine of the polypeptide compound by an amide bond
  • the ⁇ -amino group of the residue, the amino acid residue of W is selected from the group consisting of an amino acid residue having a neutral side chain and an amino acid residue having a carboxyl group in a side chain, such that W contains at least one amino acid residue having a carboxyl group in a side chain.
  • W is a covalent bond from X to the ?-amino group of the ?-terminal amino acid residue of the polypeptide compound or to the ?-amino group of the lysine residue of the polypeptide compound;
  • X is -£0-, -CH(COOH)CO-, -N(CH 2 COOH)CH 2 CO- , -N(CH 2 COOH)CH 2 CON (CH 2 COOH)CH 2 CO- , -N( CH 2 CH 2 COOH)CH 2 CH 2 CO- , -N(CH 2 CH 2 COOH)CH 2 CH 2 CON(CH 2 CH 2 COOH)CH 2 CH 2 CO-, -NHCH(COOH)(CH 2 ) 4 NHCO-, -N(CH 2 CH 2 COOH)CH 2 £0- or -N (CH 2 COOH)CH 2 CH 2 CO- , wherein
  • ⁇ is -(CH 2 ) m , where m is an integer from 6 to 32;
  • Z is -COOH, -CO- Asp, -CO-Glu , -CO-Gly, -CO-Sar, -CH (COOH) 2, -N (CH 2 COOH) 2, -S0 3 H, -P0 3 H Or absent; condition is that when W is a covalent bond and X is -CO-, Z is not -COOH.
  • the middle W of the side chain -W-X-Y-Z can be a covalent bond.
  • W may be an ⁇ -amino acid residue having a carboxyl group in the side chain, including a total of 4 to 10 carbon atoms.
  • W may be an alpha-amino acid residue encoded by a genetic code.
  • W may be selected from the group consisting of a-Asp, ⁇ -Asp, ⁇ -Glu, and ⁇ -Glu.
  • Other choices for W are, for example, ⁇ -hGlu or 5-hGlu.
  • W is a chain consisting of two alpha-amino acid residues, wherein one alpha-amino acid residue has 4-10 carbon atoms and the side chain has a carboxyl group and the other has 2-11 carbons Atom but no free carboxyl group.
  • the alpha-amino acid residue without a free carboxyl group may be a neutral, codeable alpha-amino acid residue.
  • Examples of W according to this embodiment are: a-Asp-Gly, Gly-a-Asp, ⁇ -Asp-Gly, Gly-P.Asp, a-Glu-Gly, Gly-a-Glu, y-Glu -Gly Gly-y-Glu> a-hGlu-Gly.
  • W is a chain consisting of two alpha-amino acid residues, each having from 4 to 10 carbon atoms and having a carboxyl group in the side chain.
  • ⁇ -amino acid residues may be an encoded ⁇ -amino acid residue.
  • W examples are: a-Asp-a-Asp, a-Asp-a-Glu, a-Asp-a-hGlu, a-Asp-P-Asp, a-Asp-y-Glu , a-Asp-6-hGlu, ⁇ -Asp-a-Asp, ⁇ -Asp-a-Glu, ⁇ -Asp-a-hGlu, ⁇ - ⁇ - ⁇ - ⁇ , ⁇ -Asp-y-Glu, - Asp-6-hGlu, a-Glu-a-Asp, a-Glu-a-Glu, a-Glu-a-hGlu, a-Glu-P-Asp a-Glu-y-Glu, a-Glu-5 -hGlu, ⁇ -Glu-a-Asp, ⁇ -Glu-a-Glu, ⁇ -Glu-a-hGlu, y-Glu-P_Asp, ⁇ -Glu-y-Glu, ⁇ -Glu
  • W is a chain consisting of three a-amino acid residues each having 4 to 10 carbon atoms, the amino acid residue of the chain being selected from the group consisting of residues having a neutral side chain and side chains having The residue of the carboxyl group is such that the chain contains at least one residue having a carboxyl group in its side chain.
  • the amino acid residue is a codeable residue.
  • W is a chain consisting of four a-amino acid residues each having 4 to 10 carbon atoms, the amino acid residue of the chain being selected from the group consisting of a residue having a neutral side chain and a side chain having The residue of the carboxyl group is such that the chain contains at least one residue having a carboxyl group in its side chain.
  • the amino acid residue is a codeable residue.
  • W in -W-X-Y-Z can be attached to the ⁇ -amino group of the lysine residue by a urea derivative.
  • X in the side chain -WXYZ may be a group of the formula -£0-, forming an amide bond with an amino group in W by a bond from an underlined carbonyl carbon; or when W is a covalent bond, X is derived from The underlined carbonyl carbon bond forms an amide bond with the ⁇ -terminal a-amino group of the polypeptide compound or with the ⁇ -amino group of the lysine residue in the polypeptide compound.
  • X in the side chain -WXYZ may be a group of the formula -CH(COOH) £0-, forming an amide bond with an amino group in W by a bond from an underlined carbonyl carbon; Or when W is a covalent bond, X forms an amide bond with the a-amino group at the N-terminus of the polypeptide compound or the ⁇ -amino group of the lysine residue in the polypeptide compound by a bond from the underlined carbonyl carbon.
  • X in the side chain -WXYZ may be a group of the formula -N(CH 2 COOH)CH 2 £0-, which forms an amide with an amino group from W by a bond from an underlined carbonyl carbon. Key; or when W is a covalent bond, X forms an amide bond with the a-amino group at the N-terminus of the polypeptide compound or the ⁇ -amino group of the lysine residue in the polypeptide compound by a bond from the underlined carbonyl carbon. .
  • the X in the side chain -WXYZ may be a group of the formula -N(CH 2 CH 2 COOH)CH 2 £0-, by a bond from the underlined carbonyl carbon and an amino group in W Forming an amide bond; or when W is a covalent bond, X is formed by a bond from an underlined carbonyl carbon to the N-terminal a-amino group of the polypeptide compound or to the ⁇ -amino group of the lysine residue in the polypeptide compound Amide bond.
  • X in -WXYZ may be a group of the formula -N(CH 2 COOH) CH 2 CH 2 £0-, formed by an bond from the underlined carbonyl carbon and an amide bond in W Or when W is a covalent bond, X forms an amide bond with the a-amino group at the N-terminus of the polypeptide compound or the ⁇ -amino group of the lysine residue in the polypeptide compound by a bond from the underlined carbonyl carbon.
  • X in -WXYZ may be a group of the formula -N(CH 2 COOH) CH 2 CON(CH 2 COOH)CH 2 £0-, by a bond from an underlined carbonyl carbon
  • the amino group in W forms an amide bond; or when W is a covalent bond, X passes through the bond from the underlined carbonyl carbon to the N-terminal a-amino group of the polypeptide compound or to the lysine residue in the polypeptide compound
  • the ⁇ -amino group forms an amide bond.
  • X in -WXYZ can be of the formula -N(CH 2 C3 ⁇ 4COOH) C3 ⁇ 4CH 0- a group, which forms an amide bond with an amino group in W by a bond derived from an underlined carbonyl carbon; or when W is a covalent bond, X passes through a bond derived from an underlined carbonyl carbon and an N-terminal ⁇ -amino group of the polypeptide compound Or forming an amide bond with the ⁇ -amino group of the lysine residue in the polypeptide compound.
  • X in -WXYZ can be a group of the formula -N(CH 2 CH 2 COOH) CH 2 CH 2 CON(CH 2 CH 2 COOH) CH 2 CH 2 £0-,
  • the underlined carbonyl carbon bond forms an amide bond with the amino group in W; or when W is a covalent bond, X passes through the bond from the underlined carbonyl carbon to the ⁇ -terminal ⁇ -amino group of the polypeptide compound or to the polypeptide compound
  • the ⁇ -amino group of the lysine residue in the amide bond forms an amide bond.
  • the oxime in the side chain -WXYZ may be a group of the formula -(CH 2 ) m wherein m is an integer of 6-32, 8-20, 12-20 or 12-16.
  • the number of groups satisfying the total number of carbon atoms in the hydrocarbon chain is in the range of 6-32, 10-32, 12-20 or 12-16.
  • Y in -WXYZ is a divalent hydrocarbon chain of the formula -(CH 2 ) V C 6 H 4 (CH 2 ) w -, wherein v and w are integers, or one of them is Zero, such that the sum of V and w ranges from 6-30, 10-20 or 12-16.
  • Z in the side chain -W-X-Y-Z is -COOH, provided that when W is a covalent bond and X is -CO-, Z is not -COOH.
  • -WXYZ in Z is -CO-Asp, -CO-Glu, -CO-Gly, -CO-Sar, -CH (COOH) 2, -N (CH 2 COOH) 2, -S0 3 H or - P0 3 H.
  • W in -WXYZ is a-Asp, p-Asp> a-Glu or ⁇ -Glu;
  • X is -CO- or -CH(COOH)CO-;
  • Y is -(CH 2 ) m , wherein m is an integer from 12 to 18;
  • Z is -COOH -, -CH(COOH) 2 or is absent.
  • W in -WXYZ is a-Asp, ⁇ -Asp, a-Glu or ⁇ -Glu; -XYZ is -CO(CH 2 ), through a bond from the underlined carbonyl carbon An amide bond is formed with an amino group in W, wherein n is an integer in 10-20.
  • W in -W-X-Y-Z is a-Asp, ⁇ -Asp, a-Glu or ⁇ -Glu; -X-Y-Z is
  • W in -WXYZ is a-Asp, ⁇ -Asp, a-Glu or ⁇ -Glu; -XYZ is -CO(CH 2 ) 16 .
  • W in -WXYZ is a-Asp, ⁇ -Asp, a-Glu or ⁇ -Glu; -XYZ is -CO(CH 2 ) 18 .
  • W in -WXYZ is a-Asp, ⁇ -Asp, a-Glu or ⁇ -Glu;
  • -XYZ is cholesterol, bile acid (such as cholic acid, chenodeoxycholic acid, hepatobiliary acid, Taurocholic acid, deoxycholic acid, lithocholic acid).
  • [1] - [6] represent the number of cysteine.
  • a disulfide bond in the chain is formed by the structure of insulin, specifically: A disulfide bond is formed with C [4 ], a disulfide bond is formed by C [2 ] and C [6], and a ruthenium bond is formed by ⁇ [ 31 and [5] .
  • the structure of the compound is:
  • modified single chain compound of the invention is selected from the group consisting of
  • ⁇ ⁇ represents an ⁇ -amino group of an amino acid or an amino acid residue
  • ⁇ ⁇ represents an ⁇ -amino group of an amino acid or an amino acid residue, such as an ⁇ -amino group of a lysine side chain.
  • the hypoglycemic compound of the present invention can be provided in the form of a substantially zinc-free compound or a complex.
  • a zinc complex of the compound of the present invention wherein the compound of the present invention can form a hexamer, each hexamer can bind 2 ⁇ 2+ , 3 ⁇ 2+ or 4 ⁇ ⁇ 2+ .
  • a pharmaceutical composition comprising a therapeutically effective amount of a compound according to the invention and a pharmaceutically acceptable carrier for the treatment of type 1 diabetes, 2 Type 2 diabetes and other conditions that cause hyperglycemia.
  • the insulin receptor binding analog according to the present invention can be used for the preparation of a pharmaceutical composition for the treatment of type 1 diabetes, type 2 diabetes, and other conditions which cause hyperglycemia.
  • a pharmaceutical composition for treating type 1 diabetes, type 2 diabetes and other conditions which cause hyperglycemia comprising a therapeutically effective amount of a compound according to the invention, An insulin or insulin analog having a rapid action effect, and a pharmaceutically acceptable carrier and additive are mixed.
  • compositions of the insulin analogs of the invention can be prepared using conventional techniques of the pharmaceutical industry, including dissolving and mixing the appropriate ingredients to provide the desired final product.
  • the insulin analog of the present invention is dissolved in a quantity of water having a volume slightly lower than the final volume of the composition to be prepared.
  • the volume of the solution is finally adjusted to the desired concentration with water.
  • the buffering agent is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, phosphoric acid Disodium hydrogen, sodium phosphate and tris(hydroxyindenyl)-aminodecane, hydrazine-bis(hydroxyethyl)glycine, hydrazine-(hydroxyindole) decylglycine, malic acid, succinate, maleic acid, Fumaric acid, tartaric acid, aspartic acid or a mixture thereof.
  • Each of these specific buffers constitutes an alternative embodiment of the invention.
  • the formulation comprises a pharmaceutically acceptable preservative selected from the group consisting of phenol, o-nonanol, m-nonylphenol, p-nonylphenol, p-hydroxybenzoate, Ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate, 2-phenoxyethanol, benzyl alcohol, chlorobutanol, thimerosal, bromide, benzoic acid, imidate, Dichlorobenzonitrile, sodium dehydroacetate, chlorpheniramine, benzethonamine, chlorophenylglycine or a mixture thereof.
  • a pharmaceutically acceptable preservative selected from the group consisting of phenol, o-nonanol, m-nonylphenol, p-nonylphenol, p-hydroxybenzoate, Ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, butyl p-hydroxybenz
  • the concentration of the preservative is from 0.1 mg/mL to 20 mg/mL. In another embodiment of the invention, the concentration of the preservative is from 0.1 mg/mL to 5 mg/mL. In another embodiment of the invention, the concentration of the preservative is from 5 mg/mL to 10 mg/mL.
  • concentration of the preservative is from 0.1 mg/mL to 20 mg/mL. In another embodiment of the invention, the concentration of the preservative is from 0.1 mg/mL to 5 mg/mL. In another embodiment of the invention, the concentration of the preservative is from 5 mg/mL to 10 mg/mL.
  • the formulation further comprises an isotonicity agent selected from the group consisting of a salt (eg, sodium chloride), a sugar or sugar alcohol, an amino acid, a sugar alcohol (eg, glycerol, propylene glycol, 1, 3-propanediol) , 1, 3-butanediol), polyethylene glycol (eg PEG400) or a mixture thereof.
  • a salt eg, sodium chloride
  • a sugar or sugar alcohol eg, glycerol, propylene glycol, 1, 3-propanediol
  • 1, 3-butanediol 1, 3-butanediol
  • polyethylene glycol eg PEG400
  • Any sugar such as a monosaccharide, disaccharide, polysaccharide or water-soluble glucan, including, for example, sugar, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, Dextrin, cyclodextrin, soluble starch, hydroxyethyl starch and carboxymethyl cellulose-Na.
  • the sugar additive is sucrose.
  • Sugar alcohols are defined as C4-C8 hydrocarbons having at least one -OH group including, for example, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol.
  • the sugar alcohol additive is mannitol.
  • the saccharides or sugar alcohols may be used singly or in combination. There is no fixed limit to the amount used, as long as the sugar or sugar alcohol is dissolved in the liquid preparation and does not adversely affect the stabilization obtained by the method of the present invention.
  • the concentration of the sugar or sugar alcohol is from 1 mg/mL to 150 mg/mL.
  • the concentration of the isotonic agent is from 1 mg/mL to 50 mg/mL. In another embodiment, the concentration of the isotonic agent is from 1 mg/mL to 7 mg/mL. In another embodiment, the concentration of the isotonic agent is from 8 mg/mL to 24 mg/mL. In another embodiment, the concentration of the isotonic agent is from 25 mg/mL to 50 mg/mL.
  • concentration of the isotonic agent is from 1 mg/mL to 50 mg/mL. In another embodiment, the concentration of the isotonic agent is from 1 mg/mL to 7 mg/mL. In another embodiment, the concentration of the isotonic agent is from 8 mg/mL to 24 mg/mL. In another embodiment, the concentration of the isotonic agent is from 25 mg/mL to 50 mg/mL.
  • Typical isotonic agents are sodium chloride, mannitol, disulfoxide and glycerin.
  • Typical preservatives are phenol, m-nonylphenol, decyl hydroxybenzoate and benzyl alcohol.
  • surfactant examples include sodium acetate, glycylglycine, hydroxyethylpiperine. Qin ethanesulfonic acid (HEPES) and sodium phosphate.
  • HEPES Qin ethanesulfonic acid
  • ACN acetonitrile: acetonitrile
  • BOP benzotriazol-1-yloxytris(dimethylamino) phosphonium hexafluorophosphate: benzotriazole-tris(trimethylamino)-hexafluorophosphate (Carter condensate);
  • DCC ⁇ , ⁇ '-Dicyclohexylcarbodiimide: Cyclohexylcarbodiimide; DCM: dichlorodecane;
  • DEPBT 3-(Diethoxyphosphoryloxy)-l,2,3-benzotriazin-4(3H)-one:3-(diethoxyortanoyloxy)- 1, 2,3-benzotriazin-4-one;
  • DIC N, N'-Diisopropylcarbodiimide: N, N'-diisopropylcarbodiimide
  • DIPEA or DIEA
  • succinimide succinimide
  • TEA triethylamine: triethylamine
  • TFA trifluoroacetic acid trifluoroacetic acid
  • TFE 2,2,2-Trifluoroethanol trifluoroethanol
  • THF tetrahydrofuran tetrahydrofuran
  • TIS triisopropylsilane triisopropyl pit.
  • Linear polypeptides use Boc or Fmoc solid phase peptide synthesis. If Fmoc is used to synthesize a polypeptide having a C-terminal carboxyl group, a Wang resin is generally used; and a C-terminal amide is usually a Rink amide resin. If using Boc A chemically synthesized polypeptide having a C-terminal carboxyl group is generally selected from Pam resin; and a polypeptide having a C-terminal amide is usually selected from an MBHA resin.
  • the condensing agent and activator are DIC and HOBT, and other optional peptide bond condensing agents include BOP, HBTU, DEPBT and the like. A 5-fold excess of amino acids. The condensation time was 1 hour.
  • the Fmoc protecting group was removed with 50°/A3 ⁇ 4/DMF.
  • the Boc protecting group was removed using TFA.
  • the peptide bond condensation reaction was monitored with Ninhydrin (2,2-Dihydroxyindane-l, 3-dione) reagent.
  • the commonly used cleavage reagent is TFA.
  • the dry resin was placed in a shake flask, and an appropriate amount of TFA/TIS/H 2 0 (95:2.5:2.5, 10-25 mL/g resin) was added, and the lid was capped, and intermittent rotation was performed at room temperature. After 2 hours, the resin was filtered, and the resin was washed 2-3 times with a new TFA, and the filtrate was combined, and 8-10 volumes of ice diethyl ether was added dropwise. Finally, the precipitated polypeptide was collected by centrifugation.
  • the insulin-based single-chain compound is synthesized in two fragments.
  • a section of the insulin B chain fragment 1-18 was synthesized by Boc chemistry: EEEEEEM-[l-18]-COS-(CH 2 ) 2 CO-(Arg) 4 A.
  • S-tritylpropionic acid is used in the synthesis of the thioester residue.
  • the second segment includes all amino acids, C chain and A chain amino acids from B19Cys (corresponding to C[ 2] in the formula) to the C-terminus of the B chain.
  • the two peptides are solid phase synthesized, cleaved and purified by a general method.
  • the natural chemical ligation reaction is carried out in a buffer.
  • the buffer contains 6 M guanidine hydrochloride, 200 mM phosphate, 200 mM 4-carboxymethylthiophenol (MPAA, (4-carboxymethyl)thiophenol), 20 mM tris(2-decanoylethyl)phosphine ( TCEP, tris-(2-carboxyethyl)phosphine, pH 6.9, the polypeptide was dissolved at a molar ratio of 1 : 1 at a concentration of 2 mM. The reaction was detected by HPLC and purified.
  • Purified IGF (SH) 6 was dissolved in 0.5 M guanidine hydrochloride, 20 mM Tris, 8 mM cysteine, 1 mM cystine hydrochloride buffer, pH 7.8, peptide concentration 0.5 mg/mL. After HPLC showed that the folding was completed, the buffer was acidified to pH 3 with 0.1 N hydrochloric acid. The polypeptide was purified by preparative HPLC.
  • the first fragment EEEEEEMFVNQHLCGSHLVEALYLV- (COS- C3 ⁇ 4 C3 ⁇ 4-C0)-RRA was synthesized by Boc chemistry and the crude peptide was purified by RP-HPLC. Molecular weight calculated value 3419.9, mass spectrometry test molecular weight 3421.3.
  • the second fragment, CGERGFFYTPKTGSGSSSAAAPQTGIVEQCCTSICSLYQLENYCN was synthesized according to the general method. Molecular weight calculated value 4773.4., mass spectrometry test molecular weight 4775.0.
  • the first fragment 34 mg (10 ⁇ ) and the second fragment 48 mg (10 ⁇ ) were dissolved in buffer (5 mL).
  • the buffer contained 6 M guanidine hydrochloride, 200 mM phosphate, 200 mM 4-carboxypyridylbenzene, 20 mM tris(2-nonanoylethyl)phosphine, pH 6.9. The reaction was completed after 10 hours. The calculated molecular weight was 7703.6, and the molecular weight of the mass spectrum was 7704.2. The mixture was transferred to a size exclusion chromatography column with 0.5 M guanidine hydrochloride, 20 mM Tris, pH 7.8. The fraction containing the correct molecular weight of the polypeptide was collected, and 8 mM cysteine, 1 mM cystine hydrochloride buffer was added, and the polypeptide was folded after 2 hours. The buffer was acidified to pH 3 with 0.1 N hydrochloric acid and then purified by RP-HPLC. Molecular weight calculated value 7697.6, mass spectrometry test molecular weight 7699.5.
  • the polypeptide was dissolved in 70% citric acid (or 0.1 M hydrochloric acid), cyanogen bromide (30 times the amount) was added, and cultured at room temperature. After the completion of the HPLC reaction, most of the citric acid and cyanogen bromide were evaporated with nitrogen, diluted with 10% acetic acid, and then purified by RP-HPLC. Finally, the molecular weight of Compound I-2 was calculated to be 6791.7, and the molecular weight of the mass spectrum was 6792.3. Upon sequencing, the amino acid sequence of the compound is SEQ ID NO: 2.
  • insulin-based single chain compounds were synthesized in the same manner.
  • the insulin-based single-chain compound was synthesized by the above method, and the molecular weight of each compound was examined by mass spectrometry.
  • the structure of each compound was detected by sequencing, and the results were as follows:
  • I -12 molecular weight calculated value 6476.4, mass spectrometry molecular weight 6477.2; sequenced The amino acid sequence of the compound is SEQ ID NO: 12;
  • I -16 molecular weight calculated value 6405.3, mass spectrometry molecular weight 6406.2; sequenced The amino acid sequence of the compound is SEQ ID NO: 16;
  • I-21 Molecular weight calculated value 6362.3, mass spectrometry molecular weight 6362.9; sequenced The amino acid sequence of the compound is SEQ ID NO: 21;
  • I-22 molecular weight calculated value 6378.3, mass spectrometry molecular weight 6379.1; sequenced The amino acid sequence of the compound is SEQ ID NO: 22;
  • I-29 molecular weight calculated value 6378.2, molecular weight test molecular weight 6379.5; sequenced, the amino acid sequence of the compound is SEQ ID NO: 29;
  • I-32 molecular weight calculated value 6295.1, mass spectrometry molecular weight 6296.0; sequenced The amino acid sequence of the compound is SEQ ID NO: 32;
  • I-33 molecular weight calculated value 6548.4, mass spectrometry molecular weight 6549.8; sequenced The amino acid sequence of the compound is SEQ ID NO: 33;
  • I-35 molecular weight calculated value 6777.7, mass spectrometry test molecular weight 6778.2; sequenced The amino acid sequence of the compound is SEQ ID NO: 35;
  • I-37 molecular weight calculated value 6793.7, mass spectrometry molecular weight 6793.9; sequenced The amino acid sequence of the compound is SEQ ID NO: 37;
  • I-38 molecular weight calculated value 6850.8, mass spectrometry test molecular weight 6851.2; sequenced The amino acid sequence of the compound is SEQ ID NO: 38;
  • I-39 molecular weight calculated value 6845.8, mass spectrometry molecular weight 6846.6; sequenced The amino acid sequence of the compound is SEQ ID NO: 39;
  • I-42 molecular weight calculated value 6904.9, mass spectrometry molecular weight 6905.8; sequenced The amino acid sequence of the compound is SEQ ID NO: 42;
  • I-43 Molecular weight calculated value 6962.8, mass spectrometry molecular weight 6964.3; sequenced The amino acid sequence of the compound is SEQ ID NO: 43;
  • I-44 molecular weight calculated value 6856.8, mass spectrometry molecular weight 6858.5; sequenced The amino acid sequence of the compound is SEQ ID NO: 44;
  • I-46 molecular weight calculated value 6934.9, mass spectrometry molecular weight 6936.7; sequenced The amino acid sequence of the compound is SEQ ID NO: 46;
  • I-47 molecular weight calculated value 6746.7, mass test molecular weight 6747.0; sequenced The amino acid sequence of the compound is SEQ ID NO: 47;
  • I-49 molecular weight calculated value 6835.8, mass spectrometry molecular weight 6837.1; sequenced, the amino acid sequence of the compound is SEQ ID NO: 49;
  • I-57 molecular weight calculated value 6902.8 ; mass spectrometry test molecular weight 6903.5 by sequencing the amino acid sequence of the compound is SEQ ID NO: 57;
  • I-58 molecular weight calculated value 6861.8, mass spectrometry molecular weight 6863.1; sequenced, the amino acid sequence of the compound is SEQ ID NO: 58;
  • I-59 molecular weight calculated value 6930.9, mass spectrometry molecular weight 6932.6; sequenced The amino acid sequence of the compound is SEQ ID NO: 59;
  • I-60 molecular weight calculated value 8412.5, mass spectrometry molecular weight 8413.9; sequenced The amino acid sequence of the compound is SEQ ID NO: 60;
  • I-62 molecular weight calculated value 6817.7, mass spectrometry molecular weight 6818.8; sequenced The amino acid sequence of the compound is SEQ ID NO: 62;
  • I-63 Molecular weight calculated value 7315.3, quality test molecular weight 7316.5; sequenced The amino acid sequence of the compound is SEQ ID NO: 63;
  • I-64 molecular weight calculated value 8745.7, molecular weight test molecular weight 8746.9; sequenced The amino acid sequence of the compound is SEQ ID NO: 64;
  • I-65 molecular weight calculated value 6933.9, mass spectrometry molecular weight 6935.1; sequenced The amino acid sequence of the compound is SEQ ID NO: 65;
  • I-66 Molecular weight calculated value 6877.8, mass spectrometry test molecular weight 6879.3; sequenced The amino acid sequence of the compound is SEQ ID NO: 66;
  • I-67 molecular weight calculated value 6930.9, mass spectrometry molecular weight 6931.7; sequenced The amino acid sequence of the compound is SEQ ID NO: 67;
  • I-68 molecular weight calculated 6922.9, mass spectrometry molecular weight 6924.4; sequenced amino acid sequence of the compound That is SEQ ID NO: 68;
  • I-69 molecular weight calculated value 6975.9, mass spectrometry test molecular weight 6776.5; sequenced The amino acid sequence of the compound is SEQ ID NO: 69;
  • I-70 molecular weight calculated value 6821.7, mass spectrometry molecular weight 6823.2; sequenced The amino acid sequence of the compound is SEQ ID NO: 70;
  • I-71 molecular weight calculated value 6809.7, mass spectrometry molecular weight 6810.8; sequenced The amino acid sequence of the compound is SEQ ID NO:71;
  • I-72 Molecular weight calculated value 6848.8, mass spectrometry molecular weight 6850.4; sequenced The amino group of the compound ⁇ !> column is SEQ ID NO: 72;
  • I-73 Molecular weight calculated value 8228.3, mass spectrometry test molecular weight 8230.1; sequenced The amino group of the compound is SEQ ID NO: 73;
  • I-74 molecular weight calculated value 6894.8, molecular weight test molecular weight 6896.9; sequenced The amino acid sequence of the compound is SEQ ID NO: 74;
  • I-76 molecular weight calculated value 7788.7, molecular weight test molecular weight 7790.2; sequenced, the amino acid sequence of the compound is SEQ ID NO: 76;
  • I-77 molecular weight calculated value 7358.3, molecular weight test molecular weight 7360.8; sequenced The amino acid sequence of the compound is SEQ ID NO: 77;
  • I-78 molecular weight calculated value 7586.6, mass test molecular weight 7587.7; sequenced The amino acid sequence of the compound is SEQ ID NO: 78;
  • I-79 molecular weight calculated value 7142.2, mass spectrometry test molecular weight 7143.0; sequenced The amino acid sequence of the compound is SEQ ID NO: 79;
  • I-80 molecular weight calculated value 6208.1, mass spectrometry molecular weight 6209.2; sequenced The amino acid sequence of the compound is SEQ ID NO: 80;
  • I-81 molecular weight calculated value 6756.7, mass spectrometry molecular weight 6757.8; sequenced The amino acid sequence of the compound is SEQ ID NO: 81;
  • I-82 molecular weight calculated value 7296.3, mass spectrometry molecular weight 7297.5; sequenced The amino acid sequence of the compound is SEQ ID NO: 82;
  • I-83 molecular weight calculated value 7455.4, mass transfer test molecular weight 7457.6; sequenced The amino acid sequence of the compound is SEQ ID NO: 83;
  • I-84 molecular weight calculated value 6812.6, mass spectrometry molecular weight 6813.8; sequenced The amino acid sequence of the compound is SEQ ID NO: 84;
  • I-85 molecular weight calculated value 6638.4, mass spectrometry molecular weight 6639.0; sequenced The amino acid sequence of the compound is SEQ ID NO: 85;
  • I-86 molecular weight calculated value 6846.8, mass spectrometry molecular weight 6848.2; sequenced The amino acid sequence of the compound is SEQ ID NO: 86;
  • I-87 molecular weight calculated value 7136.1, mass transfer test molecular weight 7137.9; sequenced The amino acid sequence of the compound is SEQ ID NO: 87;
  • I-89 molecular weight calculated value 6605.5, mass spectrometry test molecular weight 6606.8; sequenced, the amino acid sequence of the compound is SEQ ID NO: 89;
  • I-90 molecular weight calculated value 6837.8, mass spectrometry molecular weight 6839.1; sequenced, the amino acid sequence of the compound is SEQ ID NO: 90;
  • I-91 molecular weight calculated value 6770.7, mass spectrometry test molecular weight 6772.4; sequenced, the amino acid sequence of the compound is SEQ ID NO: 91;
  • I-92 molecular weight calculated value 6586.5, mass spectrometry test molecular weight 6587.7; sequenced The amino acid sequence of the compound is SEQ ID NO: 92;
  • I-93 molecular weight calculated value 6572.5, mass spectrometry molecular weight 6573.0; sequenced The amino acid sequence of the compound is SEQ ID NO: 93;
  • I-94 molecular weight calculated value 7008.0, mass spectrometry test molecular weight 7009.2; sequenced The amino acid sequence of the compound is SEQ ID NO: 94;
  • I-95 molecular weight calculated value 6607.5, mass test molecular weight 6608.7; sequenced amino acid sequence of the compound is SEQ ID NO: 95;
  • I-96 molecular weight calculated value 7079.1, mass spectrometry molecular weight 7080.6; sequenced, the amino acid sequence of the compound is SEQ ID NO: 96;
  • I-97 molecular weight calculated value 6951.9, mass spectrometry molecular weight 6953.4; sequenced: the amino acid sequence of the compound is SEQ ID NO: 97;
  • I-98 molecular weight calculated value 8239.3, mass spectrometry molecular weight 8241.1; sequenced The amino acid sequence of the compound is SEQ ID NO: 98;
  • I-99 molecular weight calculated value 6322.1, shield test molecular weight 6322.7; sequenced The amino acid sequence of the compound is SEQ ID NO: 99;
  • I-101 molecular weight calculated value 6709.5, mass spectrometry molecular weight 6710.6; sequenced The amino acid sequence of the compound is SEQ ID NO: 101;
  • I-102 molecular weight calculated value 6235.0, mass spectrometry molecular weight 6235.9; sequenced The amino acid sequence of the compound is SEQ ID NO: 102;
  • I-103 molecular weight calculated value 7371.3, mass transfer test molecular weight 7372.5; sequenced The amino acid sequence of the compound is SEQ ID NO: 103;
  • I-104 molecular weight calculated value 7485.5, mass spectrometry molecular weight 7487.0; sequenced The amino acid sequence of the compound is SEQ ID NO: 104;
  • I-105 molecular weight calculated value 7752.7, mass spectrometry test molecular weight 7754.2; sequenced The amino acid sequence of the compound is SEQ ID NO: 105;
  • I-106 molecular weight calculated value 7387.3, mass spectrometry molecular weight 7388.7; sequenced The amino acid sequence of the compound is SEQ ID NO: 106;
  • I-109 molecular weight calculated value 7124.0, mass spectrometry test molecular weight 7124.8; sequenced, the amino acid sequence of the compound is SEQ ID NO: 109;
  • I-110 molecular weight calculated value 7640.7, molecular weight test molecular weight 7641.4; sequenced, the amino acid sequence of the compound is SEQ ID NO: 110;
  • I-111 molecular weight calculated value 7936.9, mass spectrometry molecular weight 7938.5; sequenced, the amino acid sequence of the compound is SEQ ID NO: 111;
  • I-112 molecular weight calculated value 6902.8, mass spectrometry molecular weight 6903.1; sequenced, the amino acid sequence of the compound is SEQ ID NO: 112;
  • I-114 molecular weight calculated value 7529.6, mass spectrometry test molecular weight 7531.8; sequenced, the amino acid sequence of the compound is SEQ ID NO: 114;
  • I-115 molecular weight calculated value 7041.0, mass spectrometry test molecular weight 7041.6; sequenced, the amino acid sequence of the compound is SEQ ID NO: 115;
  • I-116 molecular weight calculated value 7007.9, shield test molecular weight 7009.2; sequenced, the amino acid sequence of the compound is SEQ ID NO: 116;
  • I-117 molecular weight calculated value 7630.7, mass spectrometry test molecular weight 7631.9; sequenced, the amino acid sequence of the compound is SEQ ID NO: 117;
  • I-118 molecular weight calculated value 7421.3, mass spectrometry molecular weight 7423.3; sequenced, the amino acid sequence of the compound is SEQ ID NO: 118;
  • I-119 molecular weight calculated value 6965.9, mass spectrometry molecular weight 6967.0; sequenced, the amino acid sequence of the compound is SEQ ID NO: 119;
  • I-120 molecular weight calculated value 6375.3, molecular weight test molecular weight 6376.7; sequenced, the amino acid sequence of the compound is SEQ ID NO: 120;
  • I-121 molecular weight calculated value 6304.2, mass spectrometry molecular weight 6305.6; sequenced, the amino acid sequence of the compound is SEQ ID NO: 121;
  • I-122 molecular weight calculated value 6595.4, mass spectrometry test molecular weight 6597.8; sequenced, the amino acid sequence of the compound is SEQ ID NO: 122;
  • Hexadecandioic acid (5.72 g, 20 mmol) was dissolved in dry DMF (240 mL) and cooled with water. Add 2-mercapto-2-propanol ( 1.48 g, 20 mmol), DIC (2.7 g, 2.25 mL, 21.4 mmol), HOBT ( 2.88 g, 21.4 mmol). NMM ( 2.16 g, 2.34 mL, 21.4 mmol) > DMAP (244 mg, 2 mmol). The mixture was stirred at room temperature overnight. After adding 80 mL of water, acidified to pH 3, and extracted with ethyl acetate.
  • Fmoc-Glu-OtBu ( 4.25g, 10 mmol) was dissolved in DCM (30 mL), 3 g of 2-chlorotrityl chloride resin (sub. lmmol/g) was added, and DIPEA ( 1.29 g, 10 mmol, 1.74 mL). After the mixture was shaken for 5 minutes in the shaker, DIPEA (1.93 g, 15 mmol, 2.6 mL) was added. The mixture was shaken vigorously for 1 hour.
  • the NMR data is 'H-NMR (CDC1 3 ) ⁇ : 6.25 (d, lH), 4.53 (m, 1H), 2.42 (m, 2H), 2.21 (m, 4H), 1.92 (m, 1H), 1.58 (m, 4H) ; 1.47(s, 9H), 1.22-1.43 (m, 18H).
  • the NMR data are: 1H-NMR (CDC1 3 ) ⁇ : 6.17 (d, lH), 4.60 (m, 1H), 2.84 (s, 4H), 2.72 (m, 1H), 2.64 (m, 1H), 2.32 (m, 1H), 2.20 (m, 4H), 2.08 (m, 1H), 1.6 (m, 4H), 1.47 (s, 9H), 1.43 (s, 9H), 1.20-1.33 (m, 20H).
  • the first fragment EEEEEEMFVNQHLCGSHLVEALYLV- (COS-CH 2 CH 2 -CO)-RRA was synthesized by Boc chemistry. The molecular weight calculated was 3419.9, and the mass spectrum was tested to have a molecular weight of 3420.6. The crude peptide was purified by RP-HPLC. The molecular weight was calculated to be 4686.3, and the mass spectrum was tested to be 4687.5. The crude peptide was purified by RP-HPLC. The first fragment 34 mg ( ⁇ ) and the second fragment 47 mg ( ⁇ ) were dissolved in buffer (5 mL).
  • the buffer contained 6 M guanidine hydrochloride, 200 mM phosphate, 200 mM 4-carboxydecyl thiophenol, 20 mM tris(2-formylethyl) phosphine, pH 6.9.
  • the molecular weight calculated was 7913.9, and the mass spectrometry was 7915.7.
  • the mixture was transferred to a size exclusion chromatography column with 0.5 M guanidine hydrochloride, 20 mM Tris, pH 7.8.
  • the fraction containing the correct molecular weight of the polypeptide was collected and combined with 8 mM cysteine, 1 mM cystine hydrochloride buffer.
  • the buffer was acidified to pH 2 with 0.1 N hydrochloric acid and then purified by RP-HPLC.
  • the molecular weight calculated was 7610.5, and the mass spectrum was tested to have a molecular weight of 7611.2.
  • the polypeptide (78 mg) was dissolved in 100 mM Na 2 CO 3 (2 mL, pH 10) solution at room temperature.
  • tert-Butylhexadecandioyl-LG] u ( OSu ) -OtBu (6.5 mg) was dissolved in acetonitrile (2 mL) and added to a peptide solution. After stirring for 30 minutes, it was acidified with 50% acetic acid and purified on a RP-HPLC C5 column. Buffer A: 0.1% TFA in water, 10% acetonitrile; Buffer B: 0.1% TFA in water, 80% acetonitrile.
  • the polypeptide was dissolved in 70% citric acid (or 0.1 M hydrochloric acid), cyanogen bromide (30 times the amount) was added, and cultured at room temperature. After the completion of the HPLC reaction, most of the formic acid and cyanogen bromide were volatilized with nitrogen, diluted with 10% acetic acid, and then purified by RP-HPLC. Finally, the calculated molecular weight of II-2 was 7102.1, and the molecular weight of the mass spectrum was 7103.5.
  • the FV QHLCGSHLVEALYLVCGERGFFYTPTGKGSSSAAAPQTGIVEQC CTSICSLYQLENYCN was synthesized by the above method.
  • the molecular weight was calculated to be 6704.6, and the mass spectrum was tested to be 6706.3.
  • the polypeptide (67 mg, 10 ⁇ ) was dissolved in 100 mM Na 2 CO 3 (1 ml, pH 10) solution at room temperature.
  • Buffer A 0.1% TFA in water, 10% acetonitrile
  • Buffer B 0.1% TFA in water, 80% acetonitrile.
  • the molecular weight calculated was 7421.5, and the mass spectrum was tested to have a molecular weight of 7422.9. After analysis, the obtained compound was 11-24.
  • Fmoc-Glu-OBzl (4.59g, 10 mmol) was dissolved in DCM (30 mL), 3 g of 2-chlorotrityl chloride resin (sub. lmmol/g) was added, and DIPEA ( 1.29 g, 10 mmol, 1.74 mL). after 5 minutes the mixture was vibration shaker, was added DIPEA (1.93g, 15 mmol, 2.6 mL) 0 mixture was shaken vigorously for 1 hour.
  • HPLC grade sterol (2.4 mL) was added to the resin and mixed for 15 minutes.
  • the resin was filtered and washed with DCM (3 X 30 mL), DMF (2 X 30 mL), DCM (3 X 30 mL), decyl alcohol (3 X 30 mL) and dried in vacuo.
  • FVNQHLCGSHLVEALYLVCGERGFFYTPTGKGSSSAAAPQTGIVEQCC TSICSLYQLENYCN was synthesized by the above method. The molecular weight was calculated to be 6704.6, and the mass spectrum was tested to be 6705.9.
  • the polypeptide (67 mg, 10 ⁇ ) was dissolved in 100 mM Na 2 CO 3 (1 ml, pH 10) at room temperature.
  • Octadecandioyl-Glu(Glu(OSu)-OH)-OH was dissolved in acetonitrile (0.5 ml) and the peptide solution was added. After stirring for 30 minutes, it was acidified with acetic acid and purified on a RP-HPLC C5 column.
  • the polypeptide, mPEG20K-CHO, sodium cyanoborohydride (NaBH 3 CN) was dissolved in a pH 4.3 acetic acid solution (0.1 M NaCl, 0.2 M CH 3 COOH, 0.1 M Na 2 CO 3 ) in a 1:2:45 ratio.
  • the polypeptide concentration is 0.5-1 mg/mL.
  • the reaction was detected and purified by HPLC. The yield is about 55%.
  • the reductive alkylation reaction can selectively bind polyethylene glycol to the B1 position.
  • GIVEQCCTSICSLYQLENYCN and mPEG20K-CHO reductive alkylation reaction to give the product.
  • the mass spectrum of the compound was calculated by mass spectrometry to be 26201.0, and a broad peak was obtained by mass spectrometry with an intermediate molecular weight of 26206.3. After a small amount of compound was reduced by DTT and degraded by trypsin, the mass of GFFGSGSSSAAAPQT GIVEQCCTSICSLYQLENYCN was observed by liquid chromatography-mass spectrometry (calculated molecular weight 3737.2, mass spectrometry molecular weight 3738.5).
  • the polypeptide and mPEG20K-NHS molar ratio 2:1 were dissolved in 0.1N N,N-bis(2-hydroxyethyl)glycine solution (pH 10 ), and the peptide concentration was 0.5 mg/mL.
  • the reaction was carried out at room temperature for 1 hour and purified by HPLC. The yield was 56%.
  • the CN synthesis method is referred to I-2.
  • the polypeptide is reacted with mPEG20K-NHS according to the above acylation method to give the product.
  • mass spectrometry the molecular weight was calculated to be 26704.6, and the mass spectrometry gave a broad peak with an intermediate molecular weight of 26710.7.
  • FVNQHLCGSHLVEA LYLVCGER was observed by liquid phase color-protonation.
  • YCN is synthesized using the above natural chemical ligation method. The molecular weight calculated was 6778.6 and the mass spectrum was tested to be 6779.8.
  • the polypeptide (68 mg) was dissolved in DMF (3 mL) and Mal-dPEG 12 -NHS (8.7 mg) (quanta Biodesign) and triethylamine (30 L) were added. The reaction was stirred at room temperature for 2 hours. After evaporating the solvent under reduced pressure, the crude material was dissolved in 3 ⁇ 40/ACN (3:1) and purified by RP-HPLC. The maleimide polypeptide is dissolved in purified water at a peptide concentration of 10 mM.
  • Human albumin (665 mg) was added and incubated at 37 ° C for 30 minutes. It was then diluted to 5% human albumin with a 20 mM sodium phosphate solution containing 5 mM sodium octoate and 750 mM ammonium sulfate. Unreacted reagent, 0.05 M aqueous ammonium hydrogencarbonate solution was removed by gel filtration chromatography as an eluent. Purely obtained after vacuum freeze drying. After analysis, the molecular weight calculated value was 74001.7, the mass spectrometry test molecular weight was 74003.8, and the obtained compound was II-4.
  • II -1 molecular weight calculated value 6932.0, mass spectrometry test molecular weight 6933.8, after analysis, the synthesized substance is II -1 ;
  • II -7 molecular weight calculated value 7274.3, mass spectrometry test molecular weight 7275.5, after analysis, the synthesized substance is II -7;
  • EK Frandsen and RA Bacchus "New, simple insulin-receptor assay with universal application to solubilized insulin receptors and receptors in broken and intact cells.” Diabetes, 1987, 36, 3: 335-340) or the following method One. Unless otherwise stated, the method of preparation of the receptor is also used as a literature method using a human placental membrane. In general, 0.025 mg of placental membrane was used for the insulin receptor binding assay; 0.2 mg of placental membrane was used for the IGF-1 receptor binding assay.
  • the insulin standard and the starting concentration of the compound of the present invention are both ⁇ , and then the insulin and the compound of the present invention are diluted 3 times to obtain 7 different concentrations of the control and compound solutions (100 nM, 33.33). nM, l l.llnM, 3.70 nM, 1.23 nM, 0.41 nM, 0.13 nM, 0.04 nM).
  • the initial concentration of the compound is 500 nM.
  • the initial concentration of the IGF-1 standard is ⁇
  • the starting concentration of the compound of the present invention is ⁇
  • IGF-1 is diluted 3 times with the compound of the present invention to obtain 7 different Concentration control and compound solutions (1000 nM, 333.33 nM, ll ll lnM, 37.04 nM, 12.35 nM, 4.12 nM, 1.37 nM, 0.46 nM).
  • the initial concentration of the compound is 5000 nM.
  • IGF-1 or insulin receptor 125 I-IGF-1 (3-10 pM) or 125 1-insulin (3 pM) and a series of 3-fold diluted polypeptides added to buffer [100 mM Hepes, pH 8.0, 100 mM NaCl, 10 mM MgCl 2 , 0.5 % (w/v) BSA, 0.025 % (w/v) Triton X-100], total volume 200 ⁇ , at 4. C was cultured for 48 hours. The receptor and the receptor-bound polypeptide and ligand were precipitated with 0.2% gamma-globulin and 500 ⁇ 25 % (w/v) PEG 8000, and the radioactivity in the precipitate was measured. The concentration of the receptor is adjusted to 15-20% of the receptor binding to the ligand when no polypeptide is added.
  • the membrane-bound receptors used in the receptor binding assay were derived from BHK cells that highly expressed full length insulin or IGF-1 receptor. Equal amounts of transfected BHK cells (2000-5000) were evenly distributed in each well of a 96-well plate and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) fetal bovine serum for 24 hours. Receptor binding assays were performed. The cells were washed once with binding buffer (DMEM, containing 0.50% BSA, 20 mM Hepes, pH 7.8), and 400 L 125 I-IGF-l (6.5 pM) or 125 1-insulin (6.5 pM) was added and dissolved.
  • binding buffer DMEM, containing 0.50% BSA, 20 mM Hepes, pH 7.8
  • a series of 3-fold diluted peptides of the binding buffer At 16. C was cultured for 3 hours, and unbound polypeptide was aspirated with an aspirator and washed once with 1.2 ml of binding buffer. The cells were lysed in 500 ⁇ L of 1% (w/v) SDS, 100 mM NaCl, 25 mM Hepes (pH 7.8) and then measured. The number of cells should be adjusted so that 16-28% of the receptors bind to the ligand when no polypeptide is added.
  • IGF-1 receptor [Thr 59 ] IGF-l is used for tyrosine achiline (iodination). 125 I-IGF-1 (50-80 Ci/g, 50 fmol), human placental membrane (0.2 mg) and serial 3-fold diluted peptides were added to 0.2 ml of 0.1 M Hepes buffer, pH 8, containing 120 mM sodium chloride, 5 mM potassium chloride, 0.12 mM magnesium sulfate, and 0.1% bovine albumin, at 20. C was incubated for 1 hour. Samples were filtered using Whatman GF/F filters to isolate bound and unbound polypeptide compounds. The filter was previously treated with 0.1% polyethyleneimine.
  • the culture tubes and filters were washed 4 times with 2.5 ml of cold buffer without bovine albumin. In the absence of a placental membrane, less than 5% of the polypeptide compound is attached to the filter. In the absence of polypeptide competition, the placental membrane binds approximately 38% of the ligand. Non-specific binding to the placental membrane can be measured by adding an excess of non-iodinated [Thr 59 ] IGF-1 (0.3 ⁇ ) to the culture mixture. Non-specific binding typically accounts for 5% of the total binding of the ligand to the placental membrane.
  • Insulin receptor 125 1-insulin (30 nCi), serial 3-fold diluted polypeptide and placental membrane (0.025 mg) were incubated in 0.05 ml of the above buffer for 1 hour at 20 °C.
  • the sample was filtered through an EHWP filter, and the culture tube and filter were washed 4 times with 2.5 ml of cold buffer containing no bovine albumin.
  • less than 5% of the polypeptide compound is attached to the filter.
  • Non-specific binding to the placental membrane can be measured by adding an excess of non-iodinated insulin (1 ⁇ M) to the culture mixture.
  • Non-specific binding typically accounts for less than 1% of the total binding of the ligand to the placental membrane.
  • Specific binding percentage (binding amount - non-specific binding amount I total binding amount - non-specific binding amount) ⁇ 100.
  • the total bound amount of radiation is the total amount of radiation measured when no polypeptide is added.
  • the combined amount of radiation is the amount of radiation measured after the addition of the polypeptide.
  • IC 50 polypeptide compound using Origin software (OriginLab, Northampton, MA) is calculated.
  • Activity of the polypeptide relative to human insulin or IGF-1 standard IC 50 insulin or IGF-1 standard / IC 50 polypeptide.
  • mice 7-9 weeks old C57BL/6 male mice, with an average body weight of 20-25 g, were divided into 6 groups and banned 4 hours before the start of the experiment. Blood glucose was measured before the start of the experiment, and blood glucose was measured at various designated time points in the future.
  • the control group was saline, and the polypeptide was dissolved in physiological saline and injected subcutaneously. Observe the response of the mice throughout the experiment and record any abnormal behavior.
  • An insulin-based single-chain compound consisting of insulin A and B chains and ligated fragments, which binds to the insulin receptor close to the level of insulin, but also because the IGF-1C peptide is a key sequence that binds to the IGF-1 receptor.
  • Single-chain polypeptides bind much more strongly at the IGF-1 receptor than native insulin.
  • C2Tyr is a prominent amino acid residue in the crystal structure of IGF-1, indicating that it may be the key to binding to the IGF-1 receptor.
  • the insulin receptor activity is substantially retained, but the IGF-1 receptor activity is greatly reduced to achieve the desired effect.
  • the results show that the length of the C peptide does not necessarily require 12 amino acid residues.
  • the 3, 4, 5, and 6 amino acid residues at the C-terminus of the original IGF-1 can be deleted or replaced.
  • the removal of three amino acid residues PQT and four residues of APQT at the C-terminus of IGF-1 did not adversely affect the activity of the insulin receptor, but also reduced its IGF-1 receptor activity to insulin levels.
  • the 5 amino acid residues X111X112X113X114X115 at the C-terminus of the insulin B chain may have 1, 2, 3, 4 or all deletions. It does not reduce insulin receptor binding.
  • Further studies have shown that the IGF-1 C peptide is only one of a wide variety of ligation fragments. These data indicate that proper C-chain length, flexible C-strand conformation, and amino acid substitution at specific sites are important factors in increasing insulin receptor activity and reducing IGF-1 receptor binding.
  • PEGylation and fatty acid acylation are common methods for extending the duration of action of a polypeptide in vivo, but pegylation and fatty acid acylation generally greatly reduce biological activity. Therefore, it is sought to introduce a acylation site having an amino group such as lysine in addition to human insulin B29, and the acylation product has sufficient biological activity for the development of a long-acting polypeptide.
  • IR is an insulin receptor.
  • the delayed effect of the insulin analog of the present invention is also given in the experimental results.
  • Experiments were carried out with human insulin, compound ⁇ -2 and a negative control, and the amounts of insulin and II-2 were 70 nmol/kg, respectively.
  • the blood glucose concentration of the mice was monitored and the results are shown in Figure 1.
  • ⁇ -2 still has a significant blood sugar-suppressing effect within 5-10 hours after administration, but insulin has gradually lost its ability to inhibit blood sugar during this time.
  • Human insulin showed a typical V-shaped blood glucose lowering curve in the experiment.
  • the disadvantage of this blood glucose lowering curve is that the initial blood sugar drops too fast, which is easy to cause hypoglycemia, and later it is impossible to control blood sugar.
  • ⁇ -2 shows an L-type blood sugar lowering curve, blood sugar control is smooth and long-lasting, and the effect is significantly better than human insulin.
  • mice were monitored for changes in blood glucose levels after subcutaneous injection of three doses of II-17. The results are shown in Figure 2.
  • a dose of 11-17 as low as 25nmol/kg is sufficient to control blood glucose for at least 24 hours.
  • the dose was increased to 90 nmol/kg, the mice did not have a hypoglycemia. Therefore, II-17 is superior to human insulin in controlling blood sugar and safety.
  • mice were tested for blood glucose over time after subcutaneous injection of normal saline, human insulin, and II-11.
  • the results are shown in Figure 3. Similar to Figure 1, human insulin exhibits a typical V-shaped blood glucose lowering curve, while II -11 shows an L-shaped blood glucose lowering curve, which controls blood glucose for at least 24 hours, and is significantly better than human insulin.

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Abstract

L'invention concerne les applications d'un composé réduisant le taux de glycémie, d'une composition et du composé ou d'une composition pour le traitement du diabète sucré et de l'hyperglycémie. L'invention concerne aussi un procédé de traitement du diabète sucré et de l'hyperglycémie, entre autres, comprenant l'application du composé ou de la composition de cette invention pour une maladie nécessitant un tel traitement. L'hydrosolubilité du composé de cette invention est bonne en comparaison avec l'insuline et ses analogues, son activité alliée à un récepteur d'insuline s'en trouve donc améliorée dans un cycle prolongé in vivo, d'où une réduction sensible des effets toxiques sur les patients. De plus, la préparation est aisée.
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CN111518009A (zh) * 2019-02-01 2020-08-11 鲁南制药集团股份有限公司 一种脂肪酸衍生物及其合成方法

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JP6200806B2 (ja) 2010-05-21 2017-09-20 メリマック ファーマシューティカルズ インコーポレーティッド 二重特異的融合タンパク質
GB201315335D0 (en) 2013-08-29 2013-10-09 Of Singapore Amino diacids containing peptide modifiers
US10040840B2 (en) 2015-10-02 2018-08-07 Silver Creek Pharmaceuticals, Inc. Bi-specific annexin A5/IGF-1 proteins and methods of use thereof to promote regeneration and survival of tissue
CA3122636A1 (fr) 2018-12-11 2020-06-18 Sanofi Analogues d'insuline ayant une affinite de liaison de recepteur d'insuline reduite
CN111349155B (zh) * 2018-12-24 2022-04-05 浙江和泽医药科技股份有限公司 一种胰高血糖素类似物及其制备方法和用途
WO2020236762A2 (fr) * 2019-05-17 2020-11-26 Case Western Reserve University Analogues de l'insuline monocaténaire variante
CN114867743A (zh) * 2019-12-10 2022-08-05 赛诺菲 形成磺酰胺与多肽的缀合物的方法

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WO2015081891A1 (fr) 2013-12-06 2015-06-11 Baikang (Suzhou) Co., Ltd Pro-fragments bioréversibles pour médicaments contenant de l'azote et de l'hydroxyle
CN111518009A (zh) * 2019-02-01 2020-08-11 鲁南制药集团股份有限公司 一种脂肪酸衍生物及其合成方法
CN111518009B (zh) * 2019-02-01 2023-06-23 鲁南制药集团股份有限公司 一种脂肪酸衍生物及其合成方法

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