WO2023001313A1 - 胰高血糖素样肽-2突变体的定向化学偶联物及其应用 - Google Patents

胰高血糖素样肽-2突变体的定向化学偶联物及其应用 Download PDF

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WO2023001313A1
WO2023001313A1 PCT/CN2022/112935 CN2022112935W WO2023001313A1 WO 2023001313 A1 WO2023001313 A1 WO 2023001313A1 CN 2022112935 W CN2022112935 W CN 2022112935W WO 2023001313 A1 WO2023001313 A1 WO 2023001313A1
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peptide
glucagon
mutant
intestinal
conjugate
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French (fr)
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范开
彭永亮
陈清
张琦
何勇
覃晓兰
苏洁
梅鑫
赵文杰
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重庆派金生物科技有限公司
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Priority to US18/417,994 priority Critical patent/US20240150424A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the disclosure relates to the fields of biotechnology and pharmacy, and in particular, the disclosure relates to directed chemical conjugates of glucagon-like peptide-2 mutants and applications thereof.
  • the glucagon-like peptide-2 (GLP-2) gene is included in the proglucagon (PG) sequence.
  • the gene of proglucagon is expressed as a single-chain precursor protein containing 160 amino acids, and the precursor protein generates a series of glucagon with different biological activities under the action of prohormone convertases (PCs)
  • Proglucagon-derived peptides (PGDP) including: glucagon (glucagon), glucagon-related peptides (glicentin relative peptides, GRPP), intermediate peptide-1 (Intervening peptide-1, IP-1) , intermediate peptide-2 (Intervening peptide-1, IP-2), glucagon-like peptide-1 (glucagon-like peptide-1, GLP-1) and glucagon-like peptide-2 (glucagon-like peptide -2, GLP-2), GLP-2 is located at positions 126 to 158 of the single-chain precursor protein and consists of 33 amino acid residues
  • Glucagon-like peptide-2 (GLP-2) is an intestinal hormone secreted by human intestinal L-type cells. It has multiple biological functions in the body, mainly including: (1) stimulating the proliferation of intestinal mucosal crypt cells and inhibit its apoptosis, thereby promoting the growth of intestinal mucosa and regeneration after injury ((DG Burrin et al., Am J Physiol Gastrointest Liver Physiol 279(6):G1249-1256, 2000)); (2) stimulate the growth of small intestinal villi , thereby promoting intestinal absorption of nutrients ((PALLE BJ et al., Gastrpenterology 120:806-815, 2001); (3) increasing intestinal mucosal thickness and improving intestinal barrier function (Cameron HL et al., Am J PhysiolGastrointest Liver Physiol 284(6):G905-12, 2003); (4) inhibit the secretion of gastric acid and gastric motility ((Meier JJ et al., Gastrpenterology
  • Glucagon-like peptide-2 exists in the human body as the active form of 1-33 sequence, and there is also GLP-2 (3-33) after hydrolysis, but it has no biological activity.
  • the half-life of natural hGLP-2 in the human body is 7min (P B Jeppesen et al. Teduglutide (ALX-0600), a dipeptidyl peptidase IV resistant glucagon-like peptide 2 analogue, improves intestinal function in short bowel syndrome patients), easily digested by diacylpeptidase IV (DPP-IV) is hydrolyzed into inactive GLP-2 (3-33), and the half-life of inactive GLP-2 (3-33) in the body is only 27 minutes.
  • NPS Company mutated the second alanine to glycine on the basis of hGLP-2 1-33 .
  • the mutated GLP-2 analogue can resist DPP-IV degradation and prolong the half-life in vivo to 2h.
  • NPS named this GLP-2 analog Teduglutide, and patients can receive a subcutaneous injection once a day at a dose of 0.05mg/Kg.
  • the drug was approved by the FDA in 2012 for the treatment of short bowel syndrome in adults and was approved in 2019. It can be used to treat short bowel syndrome in children. Its administration frequency is once a day, which reduces patient compliance, and the development of long-acting GLP-2 analogues is imminent.
  • an object of the present disclosure is to provide a mutant of glucagon-like peptide-2 and a conjugate obtained by targeted chemical modification.
  • the glucagon-like peptide-2 mutant is based on the human glucagon-like peptide-2 through multiple site mutations and carboxy-terminal extensions, and the introduction of targeted chemical modification sites, so that the glucagon-like peptide Glucagon-like peptide-2 mutants have the same biological activity as natural human glucagon-like peptide-2, and at the same time significantly prolong the half-life in vivo, shorten the frequency of administration, and achieve the treatment or prevention of short bowel syndrome, chemotherapy drugs or radiotherapy Intestinal mucosal injury caused by factors, as well as the purpose of ulcerative colitis, chronic enteritis and non-inflammatory intestinal injury diseases.
  • one aspect of the present disclosure provides a mutant of glucagon-like peptide-2.
  • the 30th amino acid in the amino acid sequence of the glucagon-like peptide-2 mutant is non-lysine basic amino acids
  • amino acid sequence of the wild-type glucagon-like peptide-2 is shown in SEQ ID NO:9.
  • amino acid sequence of SEQ ID NO:9 is as follows: HADGS-FSDEM-NTILD-NLAAR-DFINW-LIQTK-ITD.
  • the glucagon-like peptide-2 mutant also has at least one of the following additional technical features:
  • the glucagon-like peptide-2 mutant has a p.Lys30Arg or p.Lys30His mutation.
  • the glucagon-like peptide-2 mutant further has a p.Ala2Gly or p.Ala2Aib mutation.
  • the inventor's creative discovery based on the wild-type hGLP-2 as shown in SEQ ID NO:9
  • a specific mutation of the 30th amino acid will not affect the biological activity of hGLP-2, and will help prolong the half-life of the hGLP-2 mutant in vivo.
  • the specific mutation of the second amino acid can further prolong the half-life of the glucagon-like peptide-2 mutant in vivo and improve the efficacy of the drug.
  • the amino acid sequence of the glucagon-like peptide-2 mutant is: HX 1 DGSFSDEMNTILDNLAARDFINWLIQTX 2 ITD,
  • X1 is Gly or Aib
  • X2 is Arg or His
  • the derivative of the glucagon-like peptide-2 mutant includes the mutant of the glucagon-like peptide-2, an extended amino acid sequence and Lysine Lys, the glucagon
  • the carboxy terminus of the peptide-like peptide-2 mutant is connected to the amino terminus of the extended amino acid sequence, and the carboxyl terminus of the extended amino acid sequence is connected to the lysine.
  • the glucagon-like peptide-2 mutant derivative also has at least one of the following additional technical features:
  • the extended amino acid sequence is selected from (G m1 S n1 ) x1 , (S n2 G m2 ) x2 , (G m3 S n3 G m4 ) x3 and (S n4 G m5 S n5 ) x4 at least one of the
  • m1 represents the number of glycine
  • n1 represents the number of serine
  • x1 represents the number of repetitions of the (G m1 S n1 ) peptide
  • m1 and n1 are any integers between 1 and 4
  • x1 is any number between 1 and 3.
  • m2 represents the number of glycine
  • n2 represents the number of serine
  • x2 represents the number of repetitions of the (S n2 G m2 ) peptide segment
  • m2 and n2 are any integers between 1 and 4
  • m3 and m4 represent the number of glycine
  • n3 represents the number of serine
  • x3 represents the number of repetitions of the (G m3 S n3 G m4 ) peptide segment
  • m3, m4, and n3 are any between 1 and 3 Integer
  • x3 is any integer between 1 and 3
  • m3+m4+n3 5.
  • m5 represents the number of glycine
  • n4 and n5 represent the number of serine
  • x4 represents the number of repetitions of the (S n4 G m5 S n5 ) peptide segment
  • m5, n4 and n5 are any between 1 and 3 Integer
  • x4 is any integer between 1 and 3
  • m5+n4+n5 5.
  • the amino acid sequence of the glucagon-like peptide-2 mutant derivative is: HX 1 DGSFSDEMNTILDNLAARDFINWLIQTX 2 ITDX 3 K,
  • X 1 is Gly or Aib
  • X 2 is Arg or His
  • X 3 extended amino acid sequence
  • m1 represents the quantity of glycine
  • n1 represents the quantity of serine
  • x1 represents (G m1 S n1 )
  • the number of repetitions of the peptide segment, m1 and n1 are any integer between 1 and 4
  • x1 is any integer between 1 and 3
  • amino acid sequence of the derivative of the glucagon-like peptide-2 mutant is shown in SEQ ID NO: 1-4.
  • nucleic acid molecule encodes the glucagon-like peptide-2 mutant or the glucagon-like peptide-2 mutant derivative.
  • the expression vector comprises the nucleic acid molecule.
  • the host cell comprises the nucleic acid molecule or the expression vector or expresses the glucagon-like peptide-2 mutant or the glucagon-like peptide-2 mutant derivatives.
  • Another aspect of the present disclosure provides a method for preparing the glucagon-like peptide-2 mutant or the derivative of the glucagon-like peptide-2 mutant. According to an embodiment of the present disclosure, the method comprises:
  • Another aspect of the present disclosure provides a GLP-2 mutant conjugate or a pharmaceutically acceptable salt thereof. According to an embodiment of the present disclosure, it includes the above-mentioned glucagon-like peptide-2 mutant derivatives and conjugates,
  • the Lys at the carboxyl end of the glucagon-like peptide-2 mutant derivative is connected to the conjugate through an amide bond.
  • the present disclosure provides a mutant conjugate of glucagon-like peptide-2, by mutating Ala2 and Lys30 of hGLP-2, extending the carboxy-terminal by 5-15 amino acids, and designing lysine at the carboxy-terminal of the extended amino acid acid for directed chemical conjugation to form a long-acting glucagon-like peptide-2 mutant conjugate with a longer in vivo half-life.
  • the glucagon-like peptide-2 mutant conjugate or a pharmaceutically acceptable salt thereof also has at least one of the following additional technical features:
  • the conjugate is selected from at least one of fatty acid conjugates and polyethylene glycol.
  • the fatty acid conjugate includes an aliphatic chain and a linker, and the aliphatic chain and the linker are connected, wherein the general structural formula of the aliphatic chain is HOOC-(CH 2 ) a -COOH, a is an integer of 12-24, preferably, a is 16 or 18.
  • the general structural formula of the linker is (17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid) b -( ⁇ Glu ) c , b is 1 or 2, c is 1 or 2, preferably, b is 1, and c is 1.
  • the fatty chain and the linker are linked by an amide bond.
  • the polyethylene glycol has a molecular weight of 5KDa-40KDa, preferably 20KDa.
  • the polyethylene glycol has a linear or branched structure, preferably a linear structure.
  • the polyethylene glycol is polyethylene glycol having an activation group selected from N-hydroxysuccinimide or acid chloride.
  • Another aspect of the present disclosure provides the glucagon-like peptide-2 mutant, the glucagon-like peptide-2 mutant derivative, the glucagon-like peptide-2 mutant Use of the conjugate or a pharmaceutically acceptable salt thereof in the preparation of medicaments.
  • the medicament is used for treating and/or preventing intestinal-related diseases.
  • the glucagon-like peptide-2 mutant provided by this disclosure is a uniquely designed molecular structure, which has the same biological activity as natural hGLP-2, and the carboxy-terminus extended backwards is designed with lysine as a conjugate-directed chemical modification position point.
  • the glucagon-like peptide-2 mutant fatty acid conjugate provided by the present disclosure has the effect of significantly prolonging the half-life in vivo by reversibly binding to albumin, shortening the administration frequency to once a week, and achieving the treatment or prevention of short bowel syndrome Intestinal mucosal injury caused by symptoms, chemotherapy drugs or radiation therapy factors, as well as ulcerative colitis, chronic enteritis and non-inflammatory bowel injury diseases.
  • the intestinal-related diseases include at least one selected from short bowel syndrome, intestinal mucosal injury caused by chemotherapy or radiotherapy, ulcerative enteritis, chronic enteritis and non-inflammatory intestinal injury.
  • the short bowel syndrome includes, but is not limited to, short bowel syndrome resulting from surgical resection.
  • the pharmaceutical composition comprises the glucagon-like peptide-2 mutant and/or the glucagon-like peptide-2 mutant derivative and/or the Glucagon-like peptide-2 mutant conjugate or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition provided by the present disclosure has the functions of stimulating the growth of intestinal mucosa, preventing intestinal injury, promoting the repair of intestinal mucosa after injury and treating intestinal related diseases.
  • the pharmaceutical composition further includes at least one of buffer salts, excipients, and protective agents.
  • the buffer salt is at least one selected from acetate, phosphate, borate, and carbonate, preferably phosphate.
  • the excipient is selected from at least one of mannitol, sucrose, maltose, and trehalose, preferably mannitol and/or trehalose.
  • the protective agent includes but not limited to His, Gly, Ala, Arg, preferably His.
  • the pharmaceutical composition further includes other drugs for treating or preventing intestinal-related diseases or cancer chemotherapy and/or radiotherapy drugs.
  • the dosage form of the pharmaceutical composition is selected from injection, pill, freeze-dried powder, tablet, capsule or granule.
  • patients suffering from intestinal-related diseases use the pharmaceutical composition provided by the present disclosure at a dose of 0.01-0.5 mg/Kg, and administer it once a week by subcutaneous injection.
  • the pharmaceutical single dosage form contains 0.1-10 mg of the glucagon-like peptide-2 mutant, the glucagon-like peptide-2 mutant derivative, the Glucagon-like peptide-2 mutant conjugate or a pharmaceutically acceptable salt thereof.
  • Another aspect of the present disclosure provides a method for treating and/or preventing intestinal-related diseases.
  • the method comprises administering to a subject having or suspected of having a gut-related disease at least one of the following:
  • the intestinal-related diseases include at least one selected from short bowel syndrome, intestinal mucosal injury caused by chemotherapy or radiotherapy, ulcerative enteritis, chronic enteritis and non-inflammatory intestinal injury.
  • Another aspect of the present disclosure provides the aforementioned glucagon-like peptide-2 mutant, the aforementioned glucagon-like peptide-2 mutant derivative, the aforementioned nucleic acid molecule, the aforementioned expression
  • the carrier, the aforementioned host cell, the aforementioned glucagon-like peptide-2 mutant conjugate or a pharmaceutically acceptable salt thereof, the aforementioned pharmaceutical composition, the aforementioned drug single dosage form Use in the treatment and/or prevention of intestinal-related diseases.
  • the intestinal-related diseases include at least one selected from short bowel syndrome, intestinal mucosal injury caused by chemotherapy or radiotherapy, ulcerative enteritis, chronic enteritis and non-inflammatory intestinal injury.
  • Figure 1 shows the LC-MS detection results of PT01
  • Figure 2 shows the LC-MS detection results of PF01
  • Figure 3 shows the LC-MS detection results of PT02
  • Figure 4 shows the detection results of the PF01 peptide map
  • Figure 5 shows the results of PF04 peptide map detection
  • Figure 6 shows the in vitro cell activity of Teduglutide, PT01, PT02, PT03 and PT04 glucagon-like peptide-2 mutants
  • Figure 7 shows the changes in CIT (citrulline) of Teduglutide (Tedu), PF01 and PF04 in intestinal chemotherapy injury animal models, and Model was used as the control group;
  • Figure 8 shows the slices of cases related to the pharmacodynamic study of Teduglutide (Ted), PF01 and PF04 on 5-FU-induced intestinal diseases;
  • Figure 9 shows the pharmacodynamic study of PF01 on intestinal diseases induced by indomethacin-body weight
  • Figure 10 shows the pharmacodynamic study of PF01 on intestinal diseases induced by indomethacin-serum citrulline content
  • Figure 11 shows the pharmacodynamic study of PF01 on intestinal diseases induced by indomethacin - statistical results of serum ⁇ -acidic protein content
  • Figure 12 shows the pharmacodynamic study of PF01 on intestinal diseases induced by indomethacin - statistical results of ⁇ -acidic protein content in small intestinal tissue supernatant, where No Indo refers to the control group without indomethacin injection ;
  • Figure 13 shows the pharmacokinetic drug-time curve of PF01 in cynomolgus monkeys
  • Figure 14 shows the chemical structure of the octadecyl fatty acid conjugate used in Example 2;
  • Figure 15 shows the structure of the polyethylene glycol conjugate used in Example 2.
  • first and second are used for descriptive purposes only, and cannot be interpreted as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features.
  • the features defined as “first” and “second” may explicitly or implicitly include at least one of these features.
  • “plurality” means at least two, such as two, three, etc., unless otherwise specifically defined.
  • the present disclosure provides a mutant conjugate of glucagon-like peptide-2.
  • the inventors have modified the amino acid sequence of the natural GLP-2 and combined it with different molecular weight and structure
  • the modifiers are chemically coupled to form candidate coupling compounds of various structures. Specifically, by mutating Ala2 and Lys30 of hGLP-2, extending the carboxy-terminal by 5-15 amino acids, and designing lysine at the carboxy-terminal of the extended amino acid for directional chemical coupling, a long-acting pancreatic hyperplasia Glucagon-like peptide-2 mutant conjugates with longer in vivo half-life.
  • the present disclosure also provides fatty acid-directed chemically conjugated conjugates and pharmaceutical compositions thereof, and their use in the treatment of short bowel syndrome, intestinal mucosal injury caused by chemotherapy or radiotherapy, ulcerative enteritis, chronic enteritis and non-inflammatory intestinal injury use in .
  • the chemical conjugate of the glucagon-like peptide-2 mutant provided by the present disclosure includes the glucagon-like peptide-2 mutant and a conjugate chemically coupled thereto.
  • the glucagon-like peptide-2 mutant has the same biological activity as the natural hGLP-2 receptor.
  • the glucagon-like peptide-2 mutant is mutated on the basis of hGLP-2, and the mutation sites include the extension of Ala2, Lys30 and the carboxy-terminal amino acid and the carboxyl group of the extended peptide. End design Lys.
  • the mutation at the Ala2 site can be a natural or unnatural amino acid, preferably Gly or Aib
  • the Lys30 site is mutated to other basic amino acids other than lysine, preferably Arg
  • the Lys added at the carboxyl terminal of the extended peptide is used for Directed chemical coupling.
  • the number of extended amino acids at the carboxy-terminal of Asp33 is 5-15, and the extended amino acids conform to (G m1 S n1 ) x1 , (S n2 G m2 ) x2 , (G m3 S n3 G m4 ) x3 Or (S n4 G m5 S n5 ) x4 .
  • m1 represents the number of glycine
  • n1 represents the number of serine
  • x1 represents the number of repetitions of the (G m1 S n1 ) peptide
  • m1 is any integer between 1 and 4
  • x1 is any integer between 1 and 3
  • m2 represents the number of glycine
  • n2 represents the number of serine
  • x2 represents the number of repetitions of the (S n2 G m2 ) peptide
  • m2 is any integer between 1 and 4
  • x2 is any integer between 1 and 3
  • m3 and m4 represent the number of glycines
  • n3 represents the number of serines
  • x3 represents the number of (G m3 S n3 G m4 ) peptides
  • m3, m4, and n3 are arbitrary integers between 1 and 3
  • x3 is an arbitrary integer between 1 and 3
  • m3+m4+n3 5.
  • m5 represents the number of glycine
  • n4 and n5 represent the number of serine
  • x4 represents the number of (S n4 G m5 S n5 ) peptides
  • m5, n4, and n5 are arbitrary integers between 1 and 3
  • x4 is an arbitrary integer between 1 and 3
  • m5+n4+n5 5.
  • the conjugate of the glucagon-like peptide-2 mutant may be a fatty acid conjugate or a polyethylene glycol conjugate.
  • the fatty acid conjugate is formed by coupling an aliphatic chain and a linker, the aliphatic chain conforms to the structure of HOOC-(CH 2 ) a -COOH, a is an integer of 12-24, Indicates the number of repetitions of the (CH 2 ) group, preferably 16 or 18; the linker corresponds to (17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid ) b -( ⁇ Glu) c structure, wherein b is an integer 1 or 2, indicating the number of 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid in series , preferably 1; c is an integer 1 or 2, representing the number of ⁇ Glu in series, preferably 1.
  • the Glu- ⁇ amino group of the linker is coupled to the fatty chain carboxyl group, and the Glu- ⁇ carboxyl group is coupled to 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid Coupling of the amino group, carboxyl group of 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid with the carboxy-terminal lysine of the glucagon-like peptide-2 mutant Amino acid epsilon amino coupling.
  • the coupling relationship between the linker and the fatty chain can be one-to-one, or two-to-one, and the coupling relationship between the glucagon-like peptide-2 mutant and the fatty acid conjugate can be one-to-one or two-to-one One to one, preferably one to one.
  • the fatty acid conjugate and the carboxy-terminal Lys of the glucagon-like peptide-2 mutant provided by the present disclosure complete directional chemical coupling by forming an amide bond to form a glucagon-like peptide-2 mutant conjugate.
  • the activating group when the ⁇ Glu is coupled with 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid is preferably N-hydroxysuccinimide or Acyl chloride
  • the activation group when the ⁇ Glu is coupled with a fatty acid is preferably N-hydroxysuccinimide or acid chloride
  • the activation group when the fatty acid conjugate is coupled with a glucagon-like peptide-2 mutant N-hydroxysuccinimide or acid chloride is preferred.
  • the polyethylene glycol has a straight-chain or branched chain structure, preferably a straight-chain structure, and the molecular weight of the polyethylene glycol is 5KDa-40KDa, preferably 20KDa, and the polyethylene glycol
  • the activating group when the diol is coupled to the glucagon-like peptide-2 mutant is preferably N-hydroxysuccinimide or acid chloride.
  • the glucagon-like peptide-2 mutant has the same biological activity as natural human glucagon-like peptide-2.
  • the pharmaceutical composition containing the glucagon-like peptide-2 mutant conjugate includes but not limited to the glucagon-like peptide-2 mutant conjugate, buffer salt, excipient Excipients, protective agents, the buffer salts include but not limited to acetate, phosphate, borate, carbonate, preferably phosphate, the excipients include but not limited to mannitol, sucrose, maltose, Trehalose, preferably mannitol and trehalose, the protective agent includes but not limited to His, Gly, Ala, Arg. Preferably it is His.
  • the pharmaceutical composition provided by the present disclosure further includes a pharmaceutically acceptable carrier, including any solvent, solid excipient, diluent, binder, disintegrant, or other liquid excipient Excipients, dispersants, flavoring agents or suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, solid binders, glidants or lubricants, etc., suitable for specific Target dosage form.
  • a pharmaceutically acceptable carrier including any solvent, solid excipient, diluent, binder, disintegrant, or other liquid excipient Excipients, dispersants, flavoring agents or suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, solid binders, glidants or lubricants, etc., suitable for specific Target dosage form.
  • the glucagon-like peptide-2 mutants, glucagon-like peptide-2 mutant derivatives, and glucagon-like peptide-2 mutant conjugates of the present disclosure can be Incorporated into pharmaceutical compositions suitable for parenteral administration (eg, intravenous, subcutaneous, intraperitoneal, intramuscular).
  • parenteral administration eg, intravenous, subcutaneous, intraperitoneal, intramuscular.
  • These pharmaceutical compositions can be prepared in various forms.
  • liquid, semi-solid and solid dosage forms and the like including but not limited to liquid solutions (eg, injection solutions and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.
  • the pharmaceutical composition can be made into injection solution or lyophilized powder, preferably lyophilized powder, which can be injected intravenously, intramuscularly or subcutaneously after reconstitution, preferably Subcutaneous injection.
  • the glucagon-like peptide-2 mutant conjugate has the functions of stimulating the growth of intestinal mucosa, preventing metabolic intestinal injury, and promoting and protecting the repair of intestinal mucosa after injury.
  • the mucosal injury diseases include but not limited to intestinal mucosal injury caused by surgical resection, intestinal mucosal injury caused by chemotherapy or radiotherapy, and ulcerative colitis.
  • the pharmaceutical composition of the glucagon-like peptide-2 mutant conjugate has a longer half-life in vivo.
  • the modified GLP-2 mutant polypeptides PT01, PT02, PT03, and PT04, have the same in vitro activity as natural human glucagon-like peptide-2, and the candidate compounds formed by chemical coupling with the conjugate, Enhanced in vivo half-life, significantly prolonging the action time of candidate compounds in vivo.
  • the half-life of hGLP-2 in humans is 7 minutes
  • the half-life of Teduglutide in rats is 0.5 h
  • the half-life in humans is 2 h
  • the half-life of PF04 in rats is 19 h
  • the half-life of PF01 in rats It is 21h
  • the half-life in cynomolgus monkeys is 30h.
  • patients with intestinal-related diseases use the pharmaceutical composition provided by the present disclosure to realize administration once a week through subcutaneous injection, and each administration dose is between 0.01mg-0.5mg/Kg between.
  • the fatty acid chemical coupling product of the glucagon-like peptide-2 mutant of the present disclosure has the effect of stimulating the growth of intestinal mucosa and promoting the repair of intestinal mucosa after administration, and the drug effect is obvious, and the dosage is 0.01mg-0.5mg/Kg There is a clear dose-effect relationship within the range.
  • the "glucagon-like peptide-2 mutant” described in the present disclosure is obtained by mutating the natural glucagon-like peptide-2 through genetic engineering technology.
  • Peptide-2 has at least 50% sequence identity
  • "natural glucagon-like peptide-2” in the term refers to human glucagon-like peptide-2, whose amino acid sequence is HADGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ ID NO: 9)
  • “mutation” in the term refers to the modification, deletion, insertion, or addition of a certain gene sequence through genetic engineering technology
  • the “genetic engineering technology” in the term is a technology well known to those skilled in the art.
  • non-natural amino acid refers to an amino acid that cannot be expressed through recombination
  • Al in this disclosure refers to 2-aminoisobutyric acid, which is an unnatural amino acid
  • non-basic amino acid refers to an amino acid that has no other amino groups other than the ⁇ amino group, or that cannot ionize NH4 + ;
  • directed chemical modification can be used interchangeably, referring to the carboxyl group of the modifier and the lysine The ⁇ amino group is covalently bonded
  • modifier refers to a polymer used for protein and polypeptide drug modification, such as fatty acid, polyethylene glycol, polypeptide derivatives, etc.
  • Activated fatty acids refer to fatty acid derivatives with functional groups (or activation groups), and are currently mainly used for protein and polypeptide drug modification.
  • the fatty acid used for targeted chemical modification of the glucagon-like peptide-2 mutant in the present disclosure is a fatty acid with an activation group.
  • the active group of fatty acid includes but not limited to N-hydroxysuccinimide, N-acyl urea, acid chloride, active ester or other highly chemically reactive groups.
  • the active group of fatty acid is N-hydroxyl succinimide.
  • the "coupling relationship between the glucagon-like peptide-2 mutant and the fatty acid conjugate can be one-to-one or two-to-one, preferably one-to-one" in the present disclosure, wherein “one-to-one” refers to one pancreatic Glucagon-like peptide-2 mutants are covalently bound to one fatty acid conjugate, wherein “two-to-one” means that two glucagon-like peptide-2 mutants are covalently bound to one fatty acid conjugate.
  • conjugate compound can be used interchangeably, and refer to the product of the glucagon-like peptide-2 mutant coupled with a modifier.
  • naked peptide in the present disclosure refers to an unmodified polypeptide or protein.
  • 18-carbon fatty acid modifier "18-carbon modifier” and “18C” mentioned in this disclosure can be used interchangeably, which means that the carbon chain skeleton is 18 carbons.
  • 16-carbon fatty acid modifier "16-carbon modifier” and “16C” mentioned in this disclosure can be used interchangeably, which means that the carbon chain skeleton is 16-carbon.
  • the "dipeptide” in the present disclosure refers to His-Aib peptide, His represents histidine, and Aib represents 2-aminoisobutyric acid.
  • treatment and prevention and words derived therefrom as used herein do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prophylaxis that would be considered by one of ordinary skill in the art to be of potential benefit or therapeutic effect. Furthermore, treatment or prevention provided by the present disclosure may include the disease being treated or prevented. Additionally, for purposes herein, “prevention” may encompass delaying the onset of a disease or a symptom or condition thereof.
  • the targeted chemical coupling products of the glucagon-like peptide-2 mutants in the present disclosure can be used alone or in combination for the treatment of intestinal diseases with decreased serum citrulline content.
  • the intestinal diseases include, but are not limited to, short bowel syndrome, intestinal injury caused by chemotherapy or radiotherapy, enteritis caused by intestinal insufficiency, and the like.
  • the glucagon-like peptide-2 mutant is used as a drug to treat enteritis caused by chemotherapy.
  • the patient can be a model animal, a child or an adult representative.
  • the conjugate can be used alone or in combination , has a significant role in promoting the enhancement of small intestine function.
  • the directional chemical coupling product of the glucagon-like peptide-2 mutant described in the present disclosure can be used as a combined drug for parenteral nutrition support, and has the effect of improving intestinal function and enhancing intestinal absorption.
  • the glucagon-like peptide-2 mutants described in the present disclosure can be prepared by recombinant expression, chemical synthesis or a combination of the two.
  • the recombinant expression strains include but not limited to Escherichia coli and yeast.
  • PT01 According to the amino acid sequence shown in SEQ ID NO: 1, combined with the preferred codons of Escherichia coli, the nucleic acid sequence of SEQ ID NO: 6 was designed, and the expression vector pet-9a plasmid SEQ ID NO: 6 was constructed with the sequence of SEQ ID NO: 6 Encoding SEQ ID NO: 1 amino acid sequence), the constructed correct expression plasmid was transformed into Escherichia coli host bacteria, the recombinant expression strain was obtained by screening, the recombinant strain was inoculated in YT culture medium, induced by IPTG, fermented and cultivated, and the fermentation broth was centrifuged to collect bacteria body, stored at -20°C for later use.
  • a series of purification chromatography columns and methods are used to separate and concentrate the PT01 protein.
  • PT01 is adsorbed on an ion exchange resin column, eluted with different salt concentrations, and the eluted samples are collected, purified by a reversed-phase column, and the organic solvent content in the mobile phase is gradually increased for linear gradient elution, and the collected samples containing The effluent of the PT01 sample was freeze-dried to obtain the raw material of PT01.
  • the purity of the PT01 raw material was estimated to be greater than 98% by HPLC, and the molecular weight and amino acid sequence of the PT01 raw material were verified to be the same as the theoretical values by LC-MS (as shown in Figure 1).
  • the preparation methods of PT03 and PT04 refer to the preparation of PT01.
  • PT02 preparation use the nucleic acid sequence SEQ ID NO: 7 to construct an expression vector plasmid (SEQ ID NO: 7 encodes the amino acid sequence shown in SEQ ID NO: 8), transform the constructed correct expression plasmid into E. coli expression host bacteria, and screen to obtain recombinant Expression strains and recombinant strains were inoculated in YT medium, induced by IPTG and then fermented. The fermentation broth was centrifuged to collect bacteria and stored at -20°C for later use.
  • the PT02 proprotein was adsorbed on an ion-exchange resin column, eluted with different salt concentrations, and the eluted samples were collected and purified by a reverse-phase column, and the organic solvent content in the mobile phase was gradually increased for linear gradient elution, The effluent containing the PT02 proprotein was collected and freeze-dried to obtain a dry powder of the PT02 proprotein.
  • the purity of the PT02 preprotein was determined to be greater than 98% by HPLC, and the molecular weight and amino acid sequence of the PT02 preprotein were verified to be the same as the theoretical values by LC-MS.
  • the buffer ionic strength is 20-200mmol/L
  • the modified pH is 9.0-11.0
  • the protein concentration is 5.0-25.0mg/ml
  • the purity of the PT02 raw material assessed by HPLC is greater than 98%
  • the molecular weight of the PT02 raw material verified by LC-MS is the same as the theoretical value (as shown in FIG. 3
  • the following table 1 shows the amino acid sequences of some glucagon-like peptide-2 mutants and Teduglutide of the present disclosure
  • SEQ ID NO:6 nucleic acid sequence (coding SEQ ID NO:1 amino acid sequence) is as follows:
  • SEQ ID NO: 7 nucleic acid sequence (coding SEQ ID NO: 8 amino acid sequence) is as follows:
  • amino acid sequence of SEQ ID NO:8 is as follows:
  • the sample was refined and purified with a reverse-phase chromatographic column. The purity of the sample was detected by RP-HPLC >98%. The molecular weight of the target product identified by LC-MS was consistent with the theoretical value ( Figure 2). Trypsin protease and V8 enzyme double-digested PF01, and LC-MS It was identified that the coupling site of the octadecyl fatty acid conjugate is the carboxy-terminal lysine (the detection result of the PFO1 peptide map in Figure 4). The PF01 sample solution was freeze-dried at low temperature, and the collected samples were stored below -15°C for future use.
  • the preparation methods of PF02 and PF03 refer to the preparation of PF01.
  • Terminate the reaction adjust the pH to 7.5 with 1mol/L Tris buffer, mount an ion-exchange chromatographic column, elute with different concentrations of salt, collect the eluted samples, purify the samples with a molecular sieve chromatographic column, and test the purity of the samples by RP-HPLC >98%, Trypsin protease digested PF04, and LC-MS identified that the coupling site of the polyethylene glycol conjugate was carboxy-terminal lysine (the detection result of the PF04 peptide map in Figure 5).
  • the PF04 sample solution was freeze-dried at low temperature, and the dry powder was collected and stored below -15°C for future use.
  • HEK-293-GLP-2R cells Take HEK-293-GLP-2R cells (HEK-293 cells were purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences, and the GLP-2R receptor gene sequence was recombined and constructed by Shanghai Shengbo Biomedical Technology Co., Ltd., Chongqing Paijin Biotechnology Co., Ltd. Co., Ltd. HEK-293-GLP-2R cells were obtained by transfection and screening) to measure the in vitro activity of glucagon-like peptide-2 mutants.
  • the glucagon-like peptide 2 mutants PT01, PT02, PT03, PT04 and Teduglutide (NPS Company) prepared in Example 1 were respectively diluted to 500 ng/ml with a diluent containing 1 mM IBMX, and the sample was diluted by 3 times. Eight gradients were serially diluted, and then transferred to cell culture wells containing recipient cells in good growth state, and incubated at 37°C, 5% CO 2 for 15 minutes.
  • HGDGSFSDEMNTILDNLAARDFINWLIQTKITD 100% PT01 HGDGSFSDEMNTILDNLAARDFINWLIQTRITDGGGGSK 125% PT02 HAibDGSFSDEMNTILDNLAARDFINWLIQTRITDGGGGSK 91% PT03 HGDGSFSDEMNTILDNLAARDFINWLIQTRITDGGGGSGGGGSK 118% PT04 HGDGSFSDEMNTILDNLAARDFINWLIQTHITDGGGGSK 109%
  • Figure 6 shows the EC50 curves of Teduglutide, PT01, PT02, PT03 and PT04, and the activity trends shown in the curves are basically consistent with those shown in the activity data in Table 3.
  • PF01 preparation Prepare phosphate buffer solution, add His protective agent and mannitol excipient and stir to dissolve.
  • the concentration of phosphate is 35mmol/L
  • the concentration of His is 50mmol/L
  • the content of mannitol is 3%.
  • the protein content was measured, after aseptic filtration, it was dispensed into vials, and freeze-dried at low temperature to obtain the PF01 preparation, which was crimped and labeled and stored below 8°C for future use.
  • the preparation method of the PF04 preparation refers to the preparation method of the PFO1 preparation (PF04 is prepared by the preparation method in Example 2).
  • Example 5 PF01, PF04 promote small intestine hyperplasia experiment
  • Teduglutide (purchased from NPS Company), PF01 preparation, PF04 preparation and placebo were used in mouse experiments to evaluate the effect of PFO1 on promoting small intestinal hyperplasia. Take 40 juvenile Kunming mice, weighing 10-20 g, and divide them into 3 groups on average, 10 in each group. The control group was set to subcutaneously administer placebo every day for 9 days, the Ted group was administered subcutaneously at 1 mg/Kg once a day for 9 days, and the PF01 and PF04 groups were administered 3 mg/Kg once every three days. 3 times. All experimental animals were weighed before administration, weighed, blood was collected, and the length and weight of the small intestine were measured on the 10th day after the administration.
  • PF01 group and PF04 were better than Teduglutide group in terms of mouse body weight, serum citrulline, small intestine length, and small intestine weight, among which PF01 was the best, and the three administration groups were all better than the control group.
  • Example 6 Pharmacodynamic study of PF01 and PF04 on intestinal diseases induced by 5-FU
  • Teduglutide (purchased from NPS Company), PF01, PF04 and placebo were used in rat experiments to evaluate the curative effect of PF01 and PF04 on the rat diarrhea and intestinal mucosal injury model induced by 5-FU.
  • the Model group was set to administer placebo subcutaneously every day for 9 consecutive days, the Ted group was administered subcutaneously once a day at 1 mg/Kg for 9 consecutive days, and the PF01 and PF04 groups were administered 3 mg/Kg once every three days for a total of 3 times. All rats were injected intraperitoneally with 50 mg/Kg of 5-FU once a day on the 4th-6d.
  • Diarrhea is marked according to the score, 0 points: no diarrhea, 1 point: mild diarrhea, feces adhered to the anus, 2 points: moderate diarrhea, feces adhered to the hind legs and tail, 3 points: severe diarrhea, front Legs and abdomen with fecal adhesions, 4 points: death. According to the experimental records, the statistical results are shown in Table 5.
  • the numerator represents the diarrhea score
  • the denominator represents the number of animals with diarrhea
  • Figure 7 shows the changes in CIT of Teduglutide, PF01 and PF04 in intestinal chemotherapy injury animal models.
  • the change trends were consistent: citrulline content in PF01 group>PF04 group>Ted group>Model group, and at 96 hours after chemotherapy, the citrulline content in PF01 group was basically the same as that in PF04 group and Ted group. Consistent, all higher than the Model group.
  • PF01 Judging from the weight of rats, diarrhea, serum citrulline content, and case slices, PF01 is more effective than Ted in preventing or treating intestinal diseases caused by 5-FU drugs, and the frequency of administration of PF01 is lower. It shows that PF01 has a significant therapeutic effect in the prevention or treatment of intestinal diseases such as diarrhea and intestinal mucosal injury caused by chemotherapy drugs.
  • Example 7 Pharmacodynamic study of PF01 on intestinal diseases induced by indomethacin
  • Teduglutide (purchased from NPS Company), PF01 and placebo were used in rat experiments to evaluate the curative effect of PF01 on indomethacin-induced rat diarrhea and intestinal mucosal injury models. Take 40 adult SD rats, weighing 200-300g, and divide them into 4 groups on average, 10 in each group. Set model group, Ted group, PF1 low-dose group, PF1 high-dose group. The model group was subcutaneously given equal volume of PBS every day for 12 days, the Ted group was given subcutaneously 0.6 mg/Kg once a day for 12 days, and the PF01 low-dose group was given 0.6 mg/Kg once every three days.
  • the PF01 high-dose group was administered 2.0 mg/Kg every three days, and administered 4 times in total. Rats in all groups were injected intraperitoneally with indomethacin at 7 mg/Kg once a day on the 4th-5th day, and the body weight changes of the rats were recorded every day after the first administration of indomethacin. The jejunum tissue was collected to determine the content of citrulline in serum, the content of ⁇ -acidic protein in serum, and the content of ⁇ -acidic protein in the supernatant of small intestine tissue.
  • PF01 and PF04 were administered subcutaneously once at 3 mg/Kg, respectively. Serum samples were collected from all rats before administration, 8h, 24h, 48h, 72h, and 96h after administration, and the plasma drug concentration was determined by ELISA.
  • the Tmax of PF01 and PF04 in rats is basically the same, the T 1/2 of PF01 in rats is 20.28h, the T 1/2 of PF04 in rats is 17.63h, and the Cmax of PF01 in rats is 67.48 ⁇ g /ml, the Cmax of PF04 in rats is 57.28 ⁇ g/ml.
  • PF01 was administered as a single dose of 0.05 mg/Kg by subcutaneous injection. Animals collected blood samples at time points of 4h, 8h, 12h, 24h, 48h, 72h, 96h, 120h, 192h, and 288h before administration and after administration, and LC-MS/MS method was used to detect the concentration of PF01 in the samples. The plasma concentration data was analyzed by the kinetic data analysis software WinNonlin, and Figure 13 shows the pharmacokinetic drug-time curve of PF01 in cynomolgus monkeys. Calculate t 1/2 , Cmax, Tmax, AUC, Cl, Vd, etc. by using non-compartmental method (NCA).
  • NCA non-compartmental method
  • the experimental results showed that the average values of T 1/2 , Cmax and Tmax of PF01 in cynomolgus monkeys were 30.92h, 458.91ng/ml and 12h, respectively.
  • the average value of Cl was 2.27ml/h/kg
  • the average value of Vd was 101.13ml/kg
  • the AUC was 21.71h ⁇ g/ml.
  • Judging from T 1/2 the in vivo half-life of the PF01 drug was approximately 100-fold longer than that of native hGLP-2.
  • the glucagon-like peptide-2 mutant conjugate provided in the present disclosure greatly improves the half-life of glucagon-like peptide-2 in animals.

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Abstract

提供了胰高血糖素样肽-2突变体的定向化学偶联物及其应用。所述胰高血糖素样肽-2突变体，在人胰高血糖素样肽-2基础上进行多个位点突变和羧基端延伸，定向化学修饰位点的引入，使得所述胰高血糖素样肽-2突变体与天然人胰高血糖素样肽-2具有一致的生物学活性，同时显著延长体内半衰期，缩短给药频率，达到治疗或预防短肠综合征、化疗药物或放射性治疗因素导致的肠粘膜损伤，以及溃疡性肠炎、慢性肠炎和非炎症性肠损伤疾病的目的。

Description

胰高血糖素样肽-2突变体的定向化学偶联物及其应用
优先权信息
本申请请求2021年07月20日向中国国家知识产权局提交的、专利申请号为202110819379.9的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本公开涉及生物技术及制药领域，具体地，本公开涉及胰高血糖素样肽-2突变体的定向化学偶联物及其应用。
背景技术
胰高血糖素样肽-2(glucagon-like peptide-2，GLP-2)基因包含于胰高血糖素原的基因(proglucagon，PG)序列。胰高血糖素原的基因翻译后表达为含有160个氨基酸的单链前体蛋白，前体蛋白在激素原转化酶(prohormoneconvertases，PCs)作用下，生成一系列具有不同生物活性的胰高血糖素原衍生肽(Proglucagon-derived peptide，PGDP)，包括：胰高血糖素(glucagon)、肠高血糖素相关肽(glicentin relative peptides，GRPP)、中间肽-1(Intervening peptide-1，IP-1)、中间肽-2(Intervening peptide-1,IP-2)、胰高血糖素样肽-1(glucagon-like peptide-1,GLP-1)和胰高血糖素样肽-2(glucagon-like peptide-2,GLP-2)，GLP-2位于单链前体蛋白的第126至158位，由33个氨基酸残基组成。
胰高血糖素样肽-2(GLP-2)是人体肠道L型细胞分泌的一种肠道激素，在体内具有多重生物学作用，主要包括：(1)刺激肠粘膜隐窝细胞的增殖和抑制其凋亡，从而促进肠粘膜的生长及损伤后的再生修复((DG Burrin等，Am J PhysiolGastrointest Liver Physiol 279(6):G1249-1256，2000))；(2)刺激小肠绒毛的增长，从而促进肠道对营养物质的吸收((PALLE BJ等，Gastrpenterology 120：806-815，2001)；(3)增加肠粘膜厚度，提高肠道的屏障功能(Cameron HL等，Am J PhysiolGastrointest Liver Physiol 284(6)：G905-12，2003)；(4)抑制胃酸的分泌和胃的运动((Meier JJ等，Gastrpenterology 130(1)：44-54，2006)；(5)增加肠道的血液供应(Bremholm L等，Scand J Gastroenterol.44(3)：314-9，2009)。
胰高血糖素样肽-2(GLP-2)在人体内以1-33序列的活性形式存在，也有水解后的GLP-2(3-33)存在，但无生物学活性。天然的hGLP-2在人体内的半衰期是7min(P B Jeppesen等Teduglutide(ALX-0600),a dipeptidyl peptidase IV resistantglucagon-like peptide 2 analogue,improves intestinalfunction in short bowel syndrome patients)，易被二酰肽肽酶IV(DPP-IV)水解成无活性的GLP-2(3-33)，无活性的GLP-2(3-33)在体内的半衰期只有27min，经过一系列降解反应最终被肾脏清除(WENDY TAVARES等Enzymatic-and renal-dependent catabolism of the intestinotropic hormone glucagon-like peptide-2 in rats)。为解决体内半衰期短的问题，NPS公司在hGLP-2 1-33基础上，将第2位的丙氨酸突变为甘氨酸，突变后的GLP-2类似物可以抵抗DPP-IV降解，延长体内半衰期至2h。NPS公司将这个GLP-2类似物命名为Teduglutide，病人可以按照0.05mg/Kg剂量每天进行一次皮下注射，该药物于2012年被FDA批准用于治疗成人短肠综合征，在2019年又被批准可用于治疗儿童短肠综合征。其给药频率为一天一次，降低了患者的依从性，长效GLP-2类似物的研发迫在眉睫。
发明内容
本公开旨在至少在一定程度上解决相关技术中的技术问题之一。为此，本公开的一个目的在于提供胰高血糖素样肽-2突变体及其定向化学修饰而成的偶联物。所述胰高血糖素样肽-2突变体，在人胰高血糖素样肽-2基础上进行多个位点突变和羧基端延伸，定向化学修饰位点的引入，使得所述胰高血糖素样 肽-2突变体与天然人胰高血糖素样肽-2具有一致的生物学活性，同时显著延长体内半衰期，缩短给药频率，达到治疗或预防短肠综合征、化疗药物或放射性治疗因素导致的肠粘膜损伤，以及溃疡性肠炎、慢性肠炎和非炎症性肠损伤疾病的目的。
为此，本公开一方面提供了一种胰高血糖素样肽-2突变体。根据本公开的实施方案，与野生型胰高血糖素样肽-2的氨基酸序列相比，所述胰高血糖素样肽-2突变体的氨基酸序列中第30位氨基酸为非赖氨酸的碱性氨基酸，
其中，所述野生型胰高血糖素样肽-2的氨基酸序列如SEQ ID NO:9所示。
SEQ ID NO:9氨基酸序列如下：HADGS-FSDEM-NTILD-NLAAR-DFINW-LIQTK-ITD。
根据本公开的实施方案，所述胰高血糖素样肽-2突变体还具有以下附加技术特征的至少之一：
根据本公开的实施方案，所述胰高血糖素样肽-2突变体具有p.Lys30Arg或p.Lys30His突变。
根据本公开的实施方案，所述胰高血糖素样肽-2突变体进一步具有p.Ala2Gly或p.Ala2Aib突变。
鉴于现有的hGLP-2类似物的半衰期较短，影响药效以及给药频率较高，降低了患者的依从性的问题，发明人创造性的发现，基于SEQ ID NO:9所示的野生型高血糖素样肽-2的氨基酸序列，对第30位氨基酸进行特定的突变，不会影响hGLP-2的生物活性，且有利于hGLP-2突变体在体内半衰期的延长，若在此基础上对第2位氨基酸进行特定的突变，能够进一步的延长该胰高血糖素样肽-2突变体在体内的半衰期，提高药效。
根据本公开的实施方案，所述胰高血糖素样肽-2突变体的氨基酸序列为：HX 1DGSFSDEMNTILDNLAARDFINWLIQTX 2ITD，
其中，X 1为Gly或Aib，X 2为Arg或His。
本公开另一方面提供了一种胰高血糖素样肽-2突变体衍生物。根据本公开的实施方案，所述胰高血糖素样肽-2突变体衍生物包括所述胰高血糖素样肽-2突变体、延伸氨基酸序列以及赖氨酸Lys，所述胰高血糖素样肽-2突变体的羧基端与所述延伸氨基酸序列的氨基端相连，所述延伸氨基酸序列的羧基端与所述赖氨酸相连。
根据本公开的实施方案，所述胰高血糖素样肽-2突变体衍生物还具有以下附加技术特征的至少之一：
根据本公开的实施方案，所述延伸氨基酸序列选自(G m1S n1) x1、(S n2G m2) x2、(G m3S n3G m4) x3和(S n4G m5S n5) x4中的至少之一，
其中，m1表示甘氨酸的数量，n1表示丝氨酸的数量，x1表示(G m1S n1)肽段的重复次数，m1、n1为1～4之间的任意整数，x1为1～3之间的任意整数，m1+n1＝5，优选地，m1＝4，n1＝1，x1＝1或2。
根据本公开的实施方案，m2表示甘氨酸的数量，n2表示丝氨酸的数量，x2表示(S n2G m2)肽段的重复次数，m2、n2为1～4之间的任意整数，x2为1～3之间的任意整数，m2+n2＝5，优选地，x2＝1。
根据本公开的实施方案，m3、m4表示甘氨酸的数量，n3表示丝氨酸的数量，x3表示(G m3S n3G m4)肽段的重复次数，m3、m4、n3为1～3之间的任意整数，x3为1～3之间的任意整数，m3+m4+n3＝5。
根据本公开的实施方案，m5表示甘氨酸的数量，n4、n5表示丝氨酸的数量，x4表示(S n4G m5S n5)肽段的重复次数，m5、n4、n5为1～3之间的任意整数，x4为1～3之间的任意整数，m5+n4+n5＝5。
根据本公开的实施方案，所述胰高血糖素样肽-2突变体衍生物的氨基酸序列为：HX 1DGSFSDEMNTILDNLAARDFINWLIQTX 2ITDX 3K，
其中，X 1为Gly或Aib，X 2为Arg或His，X 3(延伸氨基酸序列)为(G m1S n1) x1，且m1表示甘氨酸的数量，n1表示丝氨酸的数量，x1表示(G m1S n1)肽段的重复次数，m1、n1为1～4之间的任意整数，x1为1～3之间的任意整数，m1+n1＝5，优选地，m1＝4，n1＝1，x1＝1或2。
根据本公开的实施方案，所述胰高血糖素样肽-2突变体衍生物的氨基酸序列如SEQ ID NO:1-4所示。
本公开另一方面提供了一种核酸分子。根据本公开的实施方案，所述核酸分子编码所述的胰高血糖素样肽-2突变体或所述的胰高血糖素样肽-2突变体衍生物。
本公开另一方面提供了一种表达载体。根据本公开的实施方案，所述表达载体包含所述的核酸分子。
本公开另一方面提供了一种宿主细胞。根据本公开的实施方案，所述宿主细胞包含所述的核酸分子或所述的表达载体或表达所述的胰高血糖素样肽-2突变体或所述的胰高血糖素样肽-2突变体衍生物。
本公开另一方面提供了制备所述的胰高血糖素样肽-2突变体或所述的胰高血糖素样肽-2突变体衍生物的方法。根据本公开的实施方案，所述方法包括：
在适于表达所述胰高血糖素样肽-2突变体或所述胰高血糖素样肽-2突变体衍生物的条件下培养所述的宿主细胞，以便生产所述胰高血糖素样肽-2突变体或所述胰高血糖素样肽-2突变体衍生物。
本公开另一方面提供了一种胰高血糖素样肽-2突变体偶联物或其药学上可接受的盐。根据本公开的实施方案，其包括所述的胰高血糖素样肽-2突变体衍生物以及偶联物，
其中，所述胰高血糖素样肽-2突变体衍生物的羧基端的Lys与所述偶联物通过酰胺键相连。
本公开提供一种胰高血糖素样肽-2突变体偶联物，通过将hGLP-2的Ala2和Lys30进行突变，将羧基端延伸5-15个氨基酸，在延伸氨基酸的羧基末端设计赖氨酸，用于定向化学偶联，形成一种长效胰高血糖素样肽-2突变体偶联物，具有更长的体内半衰期。
根据本公开的实施方案，所述胰高血糖素样肽-2突变体偶联物或其药学上可接受的盐还具有以下附加技术特征的至少之一：
根据本公开的实施方案，所述偶联物选自脂肪酸偶联物和聚乙二醇的至少之一。
根据本公开的实施方案，所述脂肪酸偶联物包括脂肪链和连接物，所述脂肪链和连接物相连接，其中，所述脂肪链结构通式为HOOC-(CH 2) a-COOH，a为12～24的整数，优选地，a为16或18。
根据本公开的实施方案，所述连接物结构通式为(17-氨基-10-氧代-3,6,12,15-四氧杂-9-氮杂十七烷酸) b-(γGlu) c，b为1或2，c为1或2，优选地，b为1，c为1。
根据本公开的实施方案，所述脂肪链和所述连接物通过酰胺键相连。
根据本公开的实施方案，所述聚乙二醇分子量为5KDa～40KDa，优选20KDa。
根据本公开的实施方案，所述聚乙二醇为直链或支链结构，优选为直链结构。
根据本公开的实施方案，所述聚乙二醇为具有活化基团的聚乙二醇，所述活化基团选自N-羟基琥珀酰亚胺或酰氯。
本公开另一方面提供了所述的胰高血糖素样肽-2突变体、所述的胰高血糖素样肽-2突变体衍生物、所述的胰高血糖素样肽-2突变体偶联物或其药学上可接受的盐在制备药物中的用途。根据本公开的实施方案，所述药物用于治疗和/或预防肠道相关疾病。
本公开提供的胰高血糖素样肽-2突变体是独特设计的分子结构，是与天然hGLP-2相同的生物活性，向后延伸的羧基末端设计赖氨酸作为偶联物定向化学修饰位点。本公开提供的胰高血糖素样肽-2突变体脂肪酸偶联物通过与白蛋白可逆的结合具有显著延长体内半衰期的作用，缩短给药频率为一周给药一次，达到治疗或预防短肠综合征、化疗药物或放射性治疗因素导致的肠粘膜损伤，以及溃疡性肠炎、慢性肠炎和非炎症性肠损伤疾病的目的。
根据本公开的实施方案，所述肠道相关疾病包括选自短肠综合征、化疗或放疗引起的肠粘膜损伤、溃疡性肠炎、慢性肠炎和非炎症性肠损伤的至少之一。
所述短肠综合征包括但不限于由手术切除导致的短肠综合征。
本公开另一方面提供了一种药物组合物。根据本公开的实施方案，所述药物组合物包括所述的胰高 血糖素样肽-2突变体和/或所述的胰高血糖素样肽-2突变体衍生物和/或所述的胰高血糖素样肽-2突变体偶联物或其药学上可接受的盐。
本公开提供的药物组合物具有刺激肠粘膜生长、预防肠损伤、促进肠粘膜损伤后修复的作用和治疗肠道相关疾病的作用。
根据本公开的实施方案，所述药物组合物进一步包括缓冲盐、赋形剂、保护剂中的至少之一。
根据本公开的实施方案，所述缓冲盐选自醋酸盐、磷酸盐、硼酸盐、碳酸盐中的至少之一，优选为磷酸盐。
根据本公开的实施方案，所述赋形剂选自甘露醇、蔗糖、麦芽糖、海藻糖中的至少之一，优选为甘露醇和/或海藻糖。
根据本公开的实施方案，所述保护剂包括但不限于His、Gly、Ala、Arg，优选为His。
根据本公开的实施方案，所述药物组合物进一步包括其他治疗或预防肠道相关疾病的药物或癌症化疗和/或放疗药物。
根据本公开的实施方案，所述药物组合物的剂型选自注射液、丸剂、冻干粉、片剂、胶囊或颗粒。
根据本公开的实施方案，患有肠道相关疾病的病人使用本公开提供的药物组合物，按0.01-0.5mg/Kg剂量，通过皮下注射的方式实现每周给药一次。
本公开另一方面提供了一种药物单剂型。根据本公开的实施方案，所述药物单剂型含有0.1-10mg的所述的胰高血糖素样肽-2突变体、所述的胰高血糖素样肽-2突变体衍生物、所述的胰高血糖素样肽-2突变体偶联物或其药学上可接受的盐。
本公开另一方面提供一种治疗和/或预防肠道相关疾病的方法。根据本公开的实施方案，所述方法包括向患有或疑似患有肠道相关疾病的受试者施用以下中的至少之一：
前面所述的胰高血糖素样肽-2突变体；
前面所述的胰高血糖素样肽-2突变体衍生物；
前面所述的核酸分子；
前面所述的表达载体；
前面所述的宿主细胞；
前面所述的胰高血糖素样肽-2突变体偶联物或其药学上可接受的盐；
前面所述的药物组合物；
前面所述的药物单剂型。
根据本公开的实施方案，所述肠道相关疾病包括选自短肠综合征、化疗或放疗引起的肠粘膜损伤、溃疡性肠炎、慢性肠炎和非炎症性肠损伤的至少之一。
本公开另一方面提供前面所述的胰高血糖素样肽-2突变体、前面所述的胰高血糖素样肽-2突变体衍生物、前面所述的核酸分子、前面所述的表达载体、前面所述的宿主细胞、前面所述的胰高血糖素样肽-2突变体偶联物或其药学上可接受的盐、前面所述的药物组合物、前面所述的药物单剂型在治疗和/或预防肠道相关疾病中的用途。
根据本公开的实施方案，所述肠道相关疾病包括选自短肠综合征、化疗或放疗引起的肠粘膜损伤、溃疡性肠炎、慢性肠炎和非炎症性肠损伤的至少之一。
本公开的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本公开的实践了解到。
附图说明
本公开的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1显示了PT01的LC-MS检测结果；
图2显示了PF01的LC-MS检测结果；
图3显示了PT02的LC-MS检测结果
图4显示了PF01肽图检测结果；
图5显示了PF04肽图检测结果；
图6显示了Teduglutide、PT01、PT02、PT03和PT04胰高血糖素样肽-2突变体的体外细胞活性；
图7显示了Teduglutide(Tedu)、PF01与PF04在肠道化疗损伤动物模型中CIT(瓜氨酸)的变化，Model作为对照组；
图8显示了Teduglutide(Ted)、PF01与PF04在5-FU致肠道疾病上的药效学研究相关病例切片；
图9显示了PF01在吲哚美辛致肠道疾病上的药效学研究-体重；
图10显示了PF01在吲哚美辛致肠道疾病上的药效学研究-血清瓜氨酸含量；
图11显示了PF01在吲哚美辛致肠道疾病上的药效学研究-血清α-酸性蛋白含量统计结果；
图12显示了PF01在吲哚美辛致肠道疾病上的药效学研究-小肠组织上清液中α-酸性蛋白含量统计结果，其中，No Indo是指未注射吲哚美辛的对照组；
图13显示了PF01在食蟹猴体内药代动力学药时曲线；
图14显示了实施例2中所用的十八碳脂肪酸偶联物的化学结构；
图15显示了实施例2中所用的聚乙二醇偶联物结构。
公开详细描述
下面详细描述本公开的实施例。下面描述的实施例是示例性的，仅用于解释本公开，而不能理解为对本公开的限制。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本公开的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
根据本公开的一些具体实施方案，本公开提供一种胰高血糖素样肽-2突变体偶联物，发明人对天然GLP-2氨基酸序列进行了改造，并将其与不同分子量和结构的修饰剂进行化学偶联，形成多种结构的候选偶联化合物。具体地，通过将hGLP-2的Ala2和Lys30进行突变，将羧基端延伸5-15个氨基酸，在延伸氨基酸的羧基末端设计赖氨酸，用于定向化学偶联，形成一种长效胰高血糖素样肽-2突变体偶联物，具有更长的体内半衰期。本公开还提供了脂肪酸定向化学偶联的偶联物及其药物组合物，以及它们在治疗短肠综合征、化疗或放疗引起的肠粘膜损伤、溃疡性肠炎、慢性肠炎和非炎症性肠损伤中的用途。
根据本公开的一个具体的实施方案，本公开提供的胰高血糖素样肽-2突变体化学偶联物包括胰高血糖素样肽-2突变体和与其定向化学偶联的偶联物。
其中，所述胰高血糖素样肽-2突变体与天然hGLP-2受体具有相同的生物活性。
根据本公开的一个具体的实施方案，所述胰高血糖素样肽-2突变体以hGLP-2为基础进行突变，突变位点包括Ala2、Lys30及羧基末端氨基酸的延伸以及在延伸肽段羧基末端设计Lys。所述Ala2位点的突变可以是天然或非天然氨基酸，优选为Gly或Aib，Lys30位点突变为非赖氨酸的其他碱性氨基酸，优选为Arg，延伸肽段羧基末端增加的Lys用于定向化学偶联。
根据本公开的一个具体的实施方案，Asp33羧基末端延伸氨基酸数量为5-15个，延伸氨基酸符合(G m1S n1) x1、(S n2G m2) x2、(G m3S n3G m4) x3或(S n4G m5S n5) x4。
根据本公开的一个具体的实施方案，在(G m1S n1) x1结构中，m1表示甘氨酸的数量，n1表示丝氨酸的数量，x1表示(G m1S n1)肽段的重复次数，m1、n1为1～4之间的任意整数，x1为1～3之间的任意整数， m1+n1＝5，优选地，m1＝4，n1＝1，x1＝1或2。
根据本公开的一个具体的实施方案，在(S n2G m2) x2结构中，m2表示甘氨酸的数量，n2表示丝氨酸的数量，x2表示(S n2G m2)肽段的重复次数，m2、n2为1～4之间的任意整数，x2为1～3之间的任意整数，m2+n2＝5，优选地，x2＝1。
根据本公开的一个具体的实施方案，在(G m3S n3G m4) x3结构中，m3、m4表示甘氨酸的数量，n3表示丝氨酸的数量，x3表示(G m3S n3G m4)肽段的重复次数，m3、m4、n3为1～3之间的任意整数，x3为1～3之间的任意整数，m3+m4+n3＝5。
根据本公开的一个具体的实施方案，在(S n4G m5S n5) x4结构中，m5表示甘氨酸的数量，n4、n5表示丝氨酸的数量，x4表示(S n4G m5S n5)肽段的重复次数，m5、n4、n5为1～3之间的任意整数，x4为1～3之间的任意整数，m5+n4+n5＝5。
根据本公开的一个具体的实施方案，所述胰高血糖素样肽-2突变体的偶联物可以是脂肪酸偶联物或聚乙二醇偶联物。
根据本公开的一个具体的实施方案，所述脂肪酸偶联物由脂肪链和连接物偶联而成，脂肪链符合由HOOC-(CH 2) a-COOH结构，a为12～24的整数，表示(CH 2)基团的重复次数，优选为16或18；连接物符合(17-氨基-10-氧代-3,6,12,15-四氧杂-9-氮杂十七烷酸) b-(γGlu) c结构，其中b是整数1或2，表示17-氨基-10-氧代-3,6,12,15-四氧杂-9-氮杂十七烷酸串联个数，优选为1；c是整数1或2，表示γGlu串联个数，优选为1。
所述连接物的Glu-α氨基与脂肪链羧基偶联，Glu-γ羧基与17-氨基-10-氧代-3,6,12,15-四氧杂-9-氮杂十七烷酸的氨基偶联，17-氨基-10-氧代-3,6,12,15-四氧杂-9-氮杂十七烷酸的羧基与胰高血糖素样肽-2突变体羧基末端赖氨酸ε氨基偶联。所述连接物与脂肪链的偶联关系可以是一对一，或二对一，所述胰高血糖素样肽-2突变体与脂肪酸偶联物的偶联关系可以是一对一或二对一，优选为一对一。
脂肪酸偶联物与本公开提供的胰高血糖素样肽-2突变体羧基末端Lys通过形成酰胺键完成定向化学偶联成为胰高血糖素样肽-2突变体偶联物。所述γGlu与17-氨基-10-氧代-3,6,12,15-四氧杂-9-氮杂十七烷酸偶联时的活化基团优选为N-羟基琥珀酰亚胺或酰氯，所述γGlu与脂肪酸偶联时的活化基团优选为N-羟基琥珀酰亚胺或酰氯，所述脂肪酸偶联物与胰高血糖素样肽-2突变体偶联时的活化基团优选为N-羟基琥珀酰亚胺或酰氯。
根据本公开的一个具体的实施方案，所述聚乙二醇为直连或支链结构，优选为直链结构，所述聚乙二醇分子量为5KDa-40KDa，优选为20KDa，所述聚乙二醇与胰高血糖素样肽-2突变体偶联时的活化基团优选为N-羟基琥珀酰亚胺或酰氯。
所述胰高血糖素样肽-2突变体与天然人胰高血糖素样肽-2具有一致的生物学活性。
根据本公开一个具体的实施方案，含有胰高血糖素样肽-2突变体偶联物的药物组合物包含包括但不限于胰高血糖素样肽-2突变体偶联物、缓冲盐、赋形剂、保护剂，所述缓冲盐包括但不限于醋酸盐、磷酸盐、硼酸盐、碳酸盐，优选为磷酸盐，所述赋形剂包括但不限于甘露醇、蔗糖、麦芽糖、海藻糖，优选为甘露醇和海藻糖，所述保护剂包括但不限于His、Gly、Ala、Arg。优选为His。
根据本公开的另一些具体实施方案，本公开提供的药物组合物进一步包括药学上可接受的载体，包括任何溶剂、固体赋形剂、稀释剂、粘合剂、崩解剂、或其他液体赋形剂、分散剂、矫味剂或悬浮剂、表面活性剂、等渗剂、增稠剂、乳化剂、防腐剂、固体粘合剂、助流剂或润滑剂，等等，适合于特有的目标剂型。
根据本公开的一些具体实施方案，本公开的胰高血糖素样肽-2突变体、胰高血糖素样肽-2突变体衍生物、胰高血糖素样肽-2突变体偶联物可掺入适用于胃肠外施用(例如静脉内、皮下、腹膜内、肌肉内) 的药物组合物中。这些药物组合物可以被制备成各种形式。例如液体、半固体和固体剂型等，包括但不限于液体溶液(例如，注射溶液和输注溶液)、分散剂或悬浮剂、片剂、丸剂、粉末、脂质体和栓剂。
根据本公开一个具体的实施方案，所述药物组合物可以制成注射液或冻干粉，优选为冻干粉，所述冻干粉复溶后能静脉注射、肌肉注射或皮下注射，优选为皮下注射。
根据本公开一个具体的实施方案，所述胰高血糖素样肽-2突变体偶联物具有刺激肠粘膜生长、预防代谢性肠损伤、促进并保护肠粘膜损伤后修复的作用。所述粘膜损伤疾病包括但不限于手术切除导致的肠粘膜损伤，化疗或放射性治疗导致的肠粘膜损伤，以及溃疡性肠炎。所述胰高血糖素样肽-2突变体偶联物的药物组合物具有较长的体内半衰期。
发明人发现，改造后的GLP-2突变体多肽，PT01、PT02、PT03、PT04，其体外活性与天然人胰高血糖素样肽-2一致，与偶联物化学偶联形成的候选化合物，增强了体内半衰期，显著延长了候选化合物在体内的作用时间。具体地，hGLP-2在人体内的半衰期是7min，Teduglutide在大鼠体内的半衰期是0.5h，在人体内的半衰期是2h，PF04在大鼠体内的半衰期是19h，PF01在大鼠体内的半衰期是21h，在食蟹猴体内的半衰期是30h。
PT01的氨基酸序列(SEQ ID NO:1)：
Figure PCTCN2022112935-appb-000001
PT02的氨基酸序列(SEQ ID NO:2)：
Figure PCTCN2022112935-appb-000002
PT03的氨基酸序列(SEQ ID NO:3)：
Figure PCTCN2022112935-appb-000003
PT04的氨基酸序列(SEQ ID NO:4)：
Figure PCTCN2022112935-appb-000004
根据本公开一个具体的实施方案，肠道相关疾病的患者使用本公开提供的药物组合物，通过皮下注射，实现每周给药1次，每次给药剂量为0.01mg-0.5mg/Kg之间。
本公开的胰高血糖素样肽-2突变体的脂肪酸化学偶联产物给药后具有刺激肠粘膜生长、促进肠粘膜损伤后修复的作用，药效明显，在0.01mg-0.5mg/Kg剂量范围内有明显的剂量效应关系。
定义：
本公开所述的“胰高血糖素样肽-2突变体”是通过基因工程技术将天然胰高血糖素样肽-2突变而成，术语中“类似物”是指与天然胰高血糖素样肽-2至少有50％的序列一致性，术语中“天然胰高血糖素样肽-2”是指人胰高血糖素样肽-2，其氨基酸序列为HADGSFSDEMNTILDNLAARDFINWLIQTKITD(SEQ ID NO:9)，术语中“突变”指通过基因工程技术更改、缺失、插入、增加某段基因序列，术语中“基因工程技术”是本领域普通技术人员所熟知的技术。
本公开所述“突变”、“改造”可以互换使用；
本公开所述“非天然氨基酸”是指不能通过重组表达的氨基酸；
本公开所述“Aib”，指2-氨基异丁酸，为非天然氨基酸；
本公开所述“非碱性氨基酸”是指除α氨基以外无其它氨基，或不能电离出NH4 +的氨基酸；
本公开中术语“定向化学修饰”、“化学修饰”、“修饰”、“定向化学偶联”、“化学偶联”、“偶联”可以互换使用，指修饰剂的羧基与赖氨酸ε氨基共价结合，术语中“修饰剂”是指用于蛋白质和多肽药物修饰的多聚物，如脂肪酸、聚乙二醇、多肽衍生物等。
众所周知，脂肪酸两端各有一个羧基，通常会将其中一段羧基用叔丁醇封闭，另一端羧基用于活化。经过活化的脂肪酸是指带有官能团(或活化基团)的脂肪酸衍生物，目前主要用于蛋白质以及多肽药物 修饰。
本公开中用于定向化学修饰胰高血糖素样肽-2突变体的脂肪酸为带有活化基团的脂肪酸。脂肪酸的活性基团包括但不限于N-羟基琥珀酰亚胺、N-酰基脲、酰氯、活性酯或其他高化学反应基团，在特定的实施方式中，脂肪酸的活性基团为N-羟基琥珀酰亚胺。
本公开所述“胰高血糖素样肽-2突变体与脂肪酸偶联物的偶联关系可以是一对一或二对一，优选为一对一”，其中“一对一”指一个胰高血糖素样肽-2突变体与一个脂肪酸偶联物共价结合，其中“二对一”指两个胰高血糖素样肽-2突变体与一个脂肪酸偶联物共价结合。
本公开所述“候选化合物”、“偶联产物”、“偶联物”可以互换使用，指胰高血糖素样肽-2突变体偶联修饰剂后的产物。
本公开所述“裸肽”指未经过修饰的多肽或蛋白质。
本公开所述“十八碳脂肪酸修饰剂”、“十八碳修饰剂”、“18C”可以互换使用，指碳链骨架为十八碳。
本公开所述“十六碳脂肪酸修饰剂”、“十六碳修饰剂”、“16C”可以互换使用，指碳链骨架为十六碳。
本公开所述“十四碳脂肪酸修饰剂”、“十四碳修饰剂”、“14C”可以互换使用，指碳链骨架为十四碳。
本公开所述“二肽”指His-Aib肽，His表示组氨酸，Aib表示2-氨基异丁酸。
本文使用的术语“治疗”和“预防”以及源自于此的词不必暗示100％或完全治疗或预防。相反，存在不同程度的治疗或预防，本领域普通技术人员认为所述治疗或预防具有潜在的益处或治疗效果。而且，本公开提供的治疗或预防可包括正在治疗或预防的疾病。另外，为了本文的目的，“预防”可涵盖延缓疾病或其症状或病患的发作。
除非另有定义，本公开使用的所有科技术语具有本领域普通技术人员所理解的相同含义，关于本领域的定义及术语，专业人员可以参考Current Protocols in Molecular Biology(Ausubel)，氨基酸残基的缩写是本领域普通技术人员熟知的20个常用L-型氨基酸中的标准三字母或一字母代码。
本公开中胰高血糖素样肽-2突变体的定向化学偶联产物可单独或联合用于治疗血清瓜氨酸含量减少的肠道疾病。所述的肠道疾病，包括但不限于短肠综合征、化疗或放疗引起的肠损伤、肠功能不全导致的肠炎等。
本公开中胰高血糖素样肽-2突变体用作药物治疗因化疗导致的肠炎，患者可以是模式动物、儿童或成人代表，在具体的实施方案中，该偶联物可单独或联合使用，具有显著的促进小肠功能增强的作用。
本公开中所述胰高血糖素样肽-2突变体的定向化学偶联产物可以作为肠外营养支持的配合用药，具有改善肠道功能，增强肠道吸收的作用。
本公开通过下述实施例进一步阐明，但任何实施例或其组合不应当理解为对本公开的范围或实施方式的限制。本公开的范围由所附权利要求书限定，结合本说明书和本领域一般常识，本领域普通技术人员可以清楚地明白权利要求书所限定的范围。另外，本公开引用了公开文献或专利，这些文献及专利是为了更清楚地描述本公开，它们的全文内容均纳入本文进行参考，就好像它们的全文已经在本文中重复叙述过一样。
本公开所述胰高血糖素样肽-2突变体可以采用重组表达、化学合成或两者结合的方式制备，所述重组表达用菌株包括但不限于大肠杆菌、酵母。
下面详细描述本公开的实施例。下面描述的实施例是示例性的，仅用于解释本公开，而不能理解为对本公开的限制。
实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1 胰高血糖素样肽-2突变体的制备
PT01制备：根据SEQ ID NO:1所示的氨基酸序列,结合大肠杆菌偏爱密码子，设计SEQ ID NO:6核酸序列，用SEQ ID NO:6序列构建表达载体pet-9a质粒SEQ ID NO:6编码SEQ ID NO:1氨基酸序列)，将构建好的正确表达质粒转化大肠杆菌宿主菌，筛选获得重组表达菌株，重组菌株接种于YT培养液，经IPTG诱导后发酵培养，发酵液离心后收集菌体，-20℃保存备用。
发酵菌体经裂解后，利用一系列纯化层析柱和方法分离和浓缩PT01蛋白。简而言之，PT01吸附于离子交换树脂层析柱，用不同盐浓度洗脱，收集洗脱样品，通过反相色谱柱纯化，逐步提高流动相中有机溶剂含量进行线性梯度洗脱，收集含有PT01样品的流出液，冷冻干燥获得PT01原料。HPLC评估PT01原料的纯度大于98％，LC-MS验证PT01原料的分子量和氨基酸序列与理论值相同(如附图1所示)。
PT03制备方式和PT04制备方式均参考PT01的制备。
PT02制备：用核酸序列SEQ ID NO:7构建表达载体质粒(SEQ ID NO:7编码SEQ ID NO:8所示氨基酸序列)，将构建好的正确表达质粒转化大肠杆菌表达宿主菌，筛选获得重组表达菌株，重组菌株接种于YT培养基，经IPTG诱导后发酵培养，发酵液离心后收集菌体，-20℃保存备用。
发酵菌体经裂解后，利用一系列纯化层析柱和方法分离和浓缩PT02前蛋白。简而言之，PT02前蛋白吸附于离子交换树脂层析柱，用不同盐浓度洗脱，收集洗脱样品，通过反相色谱柱纯化，逐步提高流动相中有机溶剂含量进行线性梯度洗脱，收集含有PT02前蛋白的流出液，冷冻干燥获得PT02前蛋白干粉。HPLC测定PT02前蛋白的纯度大于98％，LC-MS验证PT02前蛋白的分子量和氨基酸序列与理论值相同。在碳酸盐缓冲中，缓冲离子强度为20-200mmol/L，修饰pH为9.0-11.0，蛋白浓度为5.0-25.0mg/ml，按摩尔比PT02前蛋白：二肽(His-Aib肽)＝1:1.2投入二肽(购买于成都普康生物科技有限公司)，室温下搅拌反应1-3h，通过反相硅胶柱层析除去游离的PT02前蛋白和游离的二肽，冷冻干燥获得PT02原料，HPLC评估PT02原料的纯度大于98％，LC-MS验证PT02原料的分子量与理论值相同(如图3所示)。
以下表1显示了本公开的部分胰高血糖素样肽-2突变体以及Teduglutide的氨基酸序列
名称 氨基酸序列 序列编号
PT01 HGDGS-FSDEM-NTILD-NLAAR-DFINW-LIQTR-ITD-GGGGSK SEQ ID NO:1
PT02 HAibDGS-FSDEM-NTILD-NLAAR-DFINW-LIQTR-ITD-GGGGSK SEQ ID NO:2
PT03 HGDGS-FSDEM-NTILD-NLAAR-DFINW-LIQTR-ITD-GGGGS-GGGGSK SEQ ID NO:3
PT04 HGDGS-FSDEM-NTILD-NLAAR-DFINW-LIQTH-ITD-GGGGSK SEQ ID NO:4
Teduglutide HGDGS-FSDEM-NTILD-NLAAR-DFINW-LIQTK-ITD SEQ ID NO:5
SEQ ID NO:6核酸序列(编码SEQ ID NO:1氨基酸序列)如下：
5'-CATGGTGATGGTTCTTTTTCTGATGAAATGAATACTATTCTTGATAATCTTGCTGCTCGTGATTTTATTAATTGGCTTATTCAAACTCGTATTACTGATGGTGGTGGTGGTTCTAAA-3'
SEQ ID NO:7核酸序列(编码SEQ ID NO:8氨基酸序列)如下：
5'-GATGGTTCTTTTTCTGATGAAATGAATACTATTCTTGATAATCTTGCTGCTCGTGATTTTATTAATTGGCTTATTCAAACTCGTATTACTGATGGTGGTGGTGGTTCTAAA-3'
SEQ ID NO:8氨基酸序列如下：
Figure PCTCN2022112935-appb-000005
实施例2：胰高血糖素样肽-2突变体偶联物的制备
PF01制备：称量100mg PT01原料(重庆派金生物科技有限公司生产)于100ml烧杯，用10ml pH10.5的0.1mol/L碳酸盐溶液溶解，按摩尔比PT01：十八碳脂肪酸偶联物＝1:1.2加入540mg十八碳脂肪酸偶联物(购买于成都普康生物科技有限公司，十八碳脂肪酸偶联物结构如附图14所示)，25℃条件下搅拌反应1h，1mol/L HCl溶液调pH4.5终止反应，离心收集沉淀，加入适量的三氟乙酸搅拌反应1-2h，Tris缓冲溶解后挂载离子交换色谱柱，用不同浓度的盐洗脱，收集洗脱样品，用反相色谱柱对样品进行精纯化，RP-HPLC检测样品纯度>98％，LC-MS鉴定目标产物分子量与理论值一致(图2)，Trypsin蛋白酶与V8酶双酶切PF01，LC-MS鉴定十八碳脂肪酸偶联物偶联位点是羧基末端赖氨酸(图4中PF01肽图检测结果)。将PF01样品液低温冷冻干燥，收集样品保存在-15℃以下备用。
PF02制备方式和PF03制备方式均参考PF01的制备。
PF04制备：称量100mg PT01原料(重庆派金生物科技有限公司生产)于100ml烧杯，用10ml pH9.5的0.1mol/L碳酸盐溶液溶解，按摩尔比PT01：聚乙二醇偶联物＝1:1.2加入568mg聚乙二醇偶联物(购买于厦门赛诺邦格科技有限公司，结构如附图15所示)，25℃条件下搅拌反应1h，1mol/L HCl溶液调pH4.5终止反应，1mol/L Tris缓冲调pH7.5，挂载离子交换色谱柱，用不同浓度的盐洗脱，收集洗脱样品，用分子筛色谱柱对样品进行精纯化，RP-HPLC检测样品纯度>98％，Trypsin蛋白酶酶切PF04，LC-MS鉴定聚乙二醇偶联物偶联位点是羧基末端赖氨酸(图5中PF04肽图检测结果)。将PF04样品液低温冷冻干燥，收集干粉保存在-15℃以下备用。
表2：胰高血糖素样肽-2突变体PT01以及突变体偶联物的分子量测定
名称 结构 理论分子量 实测分子量 偶联位点
PT01 SEQ ID NO:1 4223.60 4223.51 —
PF01 PT01+十八碳脂肪酸偶联物 4939.60 4938.00 Lys
PF02 PT01+十六碳脂肪酸偶联物 4590.60 4549.26 Lys
PF03 PT01+十四碳脂肪酸偶联物 4434.04 4434.97 Lys
PF04 PT01+聚乙二醇偶联物 28411.20 — Lys
以上表2中LC-MS鉴定结果显示，PT01、PF01、PF02、PF03、PF04的实测分子量与理论值基本一致，并且鉴定出PF01、PF02、PF03、PF04中PT01与偶联物之间通过Lys偶联位点连接。
实施例3：胰高血糖素样肽-2突变体的体外细胞活性测定
取HEK-293-GLP-2R细胞(HEK-293细胞购买于中国科学院典型培养物保藏委员会细胞库，GLP-2R受体基因序列由上海生博生物医药科技有限公司重组构建，重庆派金生物科技有限公司经转染筛选得HEK-293-GLP-2R细胞)测定胰高血糖素样肽-2突变体的体外活性。将实施例1中制备获得的胰高血糖样肽2突变体PT01、PT02、PT03、PT04和Teduglutide(NPS公司)分别用含1mM IBMX的稀释液将待测样品稀释到500ng/ml，以3倍倍比稀释8个梯度，再转移至含有生长状态良好的受体细胞的细胞培养孔，37℃、5％CO 2培养15分钟。加入细胞裂解液，提取细胞裂解液并用cAMP检测试剂盒(RD公司)测定cAMP的OD值，利用“Origin pro 2017”参数回归软件计算Teduglutide和胰高血糖素样肽-2突变体的EC50值，以检测其体外细胞活性。各样品体外细胞活性检测结果如表3所示。
表3：胰高血糖素样肽-2突变体的体外细胞活性
样品名称 氨基酸序列 EC50
Teduglutide HGDGSFSDEMNTILDNLAARDFINWLIQTKITD 100％
PT01 HGDGSFSDEMNTILDNLAARDFINWLIQTRITDGGGGSK 125％
PT02 HAibDGSFSDEMNTILDNLAARDFINWLIQTRITDGGGGSK 91％
PT03 HGDGSFSDEMNTILDNLAARDFINWLIQTRITDGGGGSGGGGSK 118％
PT04 HGDGSFSDEMNTILDNLAARDFINWLIQTHITDGGGGSK 109％
由表3结果可知，以Teduglutide体外细胞活性作为标准，PT01、PT03和PT04的体外细胞活性是Teduglutide的105％-130％。PT02的体外细胞活性与Teduglutide相当。
图6显示了Teduglutide、PT01、PT02、PT03和PT04的EC50曲线，曲线显示的活性趋势与表3的活性数据表现的趋势基本一致。
实施例4：胰高血糖素样肽-2突变体偶联物冻干粉制剂的制备
PF01制剂：配制磷酸盐缓冲溶液，加入His保护剂和甘露醇赋形剂搅拌溶解。磷酸盐浓度为35mmol/L，His浓度为50mmol/L，甘露醇含量为3％。称量100mg实施例2制备的PF01原料于100ml洁净烧杯，加入含有His和甘露醇的磷酸盐溶液搅拌溶解，用HCl或NaOH调解溶液pH至7.4±0.2。测定蛋白含量，无菌过滤后分装至西林瓶，低温冷冻干燥获得PF01制剂，轧盖贴签置于8℃以下保存备用。
PF04制剂的制备方式参考PF01制剂的制备方式(PF04由实施例2制备方法制得)。
实施例5：PF01、PF04促小肠增生实验
将Teduglutide(购买于NPS公司)、PF01制剂、PF04制剂和安慰剂用于小鼠实验，评价PF01促进小肠增生的效果。取40只未成年的昆明小鼠，体重10-20g，平均分成3组，每组10只。设置对照组每天皮下施用安慰剂，连续给药9d，Ted组按每天一次1mg/Kg皮下给药，连续给药9d，PF01组和PF04组按每三天给药一次3mg/Kg，一共给药3次。所有实验动物分别在给药前称量体重，给药后第10d称量体重、采血、测定小肠长度和小肠重量。
表4：PF01、PF04促进未成年小鼠小肠增长的实验结果
Figure PCTCN2022112935-appb-000006
由表4可知，PF01组和PF04在小鼠体重、血清瓜氨酸和小肠长度、小肠重量指标上均优于Teduglutide组，其中PF01最优，3个给药组均优于对照组。
实施例6：PF01和PF04在5-FU致肠道疾病上的药效学研究
将Teduglutide(购买于NPS公司)、PF01、PF04和安慰剂用于大鼠实验，评价PF01和PF04在5-FU引起的大鼠腹泻和肠黏膜损伤模型上的疗效。取40只成年的SD大鼠，体重200-300g，平均分成4组，每组10只。设置Model组每天皮下施用安慰剂，连续给药9d，Ted组按每天一次1mg/Kg皮下给药，连续给药9d，PF01组和PF04组按每三天给药一次3mg/Kg，一共给药3次。所有大鼠分别在第4-6d按每天一次50mg/Kg腹腔注射5-FU，第一次给予5-FU后每天记录大鼠腹泻情况和体重变化，在第10d分别称量体重、采血、取空肠做病例切片。体重统计数据如表5所示，病例切片如图8所示。
腹泻情况按照分数进行标识，0分：不腹泻，1分：轻度腹泻，肛门上有粪便粘附，2分：中度腹泻，后腿和尾巴有粪便粘附，3分：重度腹泻，前腿和腹部有粪便粘附，4分：死亡。根据实验记录，统计结果如表5所示。
表5：PF01和PF04在大鼠模型中的药效评价
Figure PCTCN2022112935-appb-000007
表6：各组腹泻情况得分统计
Figure PCTCN2022112935-appb-000008
表6中分子表示腹泻得分，分母表示腹泻动物数量
表7 各组血清瓜氨酸含量
Figure PCTCN2022112935-appb-000009
Figure PCTCN2022112935-appb-000010
图7显示了Teduglutide、PF01与PF04在肠道化疗损伤动物模型中CIT的变化。化疗前、化疗后48h及化疗后144h的变化趋势是一致的：PF01组瓜氨酸含量>PF04组>Ted组>Model组，化疗后96h，PF01组瓜氨酸含量与PF04组和Ted组基本一致，均高于Model组。
上述实验结果显示，在使用5-FU建立的肠道化疗损伤动物模型中，随着造模和给药进程，PF01与PF04均能明显减少由造模引起的体重降低、腹泻、血清瓜氨酸降低，并在造模后加快模型大鼠的体重和血清瓜氨酸水平的恢复。以上药效学作用均显著优于Ted组，其中PF01最优。检测结果与病例切片(图8)结果对应，病例切片从绒毛高度和隐窝细胞数量上判断，PF01组>PF04组>Ted组>对照组。
通过大鼠体重、腹泻、血清瓜氨酸含量、病例切片等指标判断，PF01在预防或治疗由5-FU药物引起的肠道疾病时的疗效均优于Ted，且PF01给药频率更低。说明PF01在预防或治疗因化疗药物引起的腹泻和肠粘膜损伤等肠道疾病时具显著的治疗效果。
实施例7：PF01在吲哚美辛致肠道疾病上的药效学研究
将Teduglutide(购买于NPS公司)、PF01和安慰剂用于大鼠实验，评价PF01在吲哚美辛引起的大鼠腹泻和肠黏膜损伤模型上的疗效。取40只成年的SD大鼠，体重200-300g，平均分成4组，每组10只。设置模型组、Ted组、PF1低剂量组、PF1高剂量组。模型组每天皮下给予等体积的PBS，连续给药12d，Ted组按每天一次0.6mg/Kg皮下给药，连续给药12d，PF01低剂量组按每三天给药一次0.6mg/Kg，一共给药4次，PF01高剂量组按每三天给药一次2.0mg/Kg，一共给药4次。所有组别大鼠分别在第4-5d按每天一次7mg/Kg单次腹腔注射吲哚美辛，首次给予吲哚美辛后每天记录大鼠体重变化，在第12d分别称量体重、采血、取空肠组织，测定血清中瓜氨酸含量，测定血清中α-酸性蛋白含量，测定小肠组织上清液中α-酸性蛋白含量。体重变化如图9所示，血清瓜氨酸含量统计结果如图10所示，血清α-酸性蛋白(α1-AGP)含量统计结果如图11所示，小肠组织上清液中α-酸性蛋白含量统计结果如图12所示。
实验结果显示，在使用吲哚美辛建立的肠道炎症动物模型中，随着造模和给药进程，PF01能够明显缓解由造模引起的体重降低、肠道组织及血清中α-酸性蛋白含量升高、血清瓜氨酸降低，并在造模后加快模型大鼠的体重和血清瓜氨酸水平的恢复。以上药效学作用均显著优于Ted组。
通过大鼠体重、血清瓜氨酸含量、血清α-酸性蛋白含量、小肠组织上清液中α-酸性蛋白含量指标判断，PF01在预防或治疗由吲哚美辛药物引起的肠道疾病时的疗效均优于Ted，且PF01给药频率更低。说明PF01在治疗药物引起的溃疡性肠炎等肠道疾病时具显著的治疗效果。
实施例8：PF01与PF04在大鼠体内的药代动力学研究
取10只成年的SD大鼠，体重200-300g，平均分成2组，每组5只。分别按照3mg/Kg单次皮下给药PF01和PF04。所有大鼠分别在给药前、给药后8h、24h、48h、72h、96h采集血清样本，用ELISA法测定血药浓度。
PF01与PF04在大鼠体内的Tmax基本一致，PF01在大鼠体内的T 1/2为20.28h，PF04在大鼠体内的T 1/2为17.63h，PF01在大鼠体内的Cmax为67.48μg/ml，PF04在大鼠体内的Cmax为57.28μg/ml。
实施例9：PF01在食蟹猴体内的药代动力学研究
取4只成年食蟹猴，雌雄各半。通过皮下注射的方式按0.05mg/Kg剂量单次给药PF01。动物于给药前、给药后4h、8h、12h、24h、48h、72h、96h、120h、192h、288h的时间点采集血样，采用LC-MS/MS方法检测样本中PF01的浓度，使用代谢动力学数据分析软件WinNonlin对血浆浓度数据进行分析，图13显示了PF01在食蟹猴体内药代动力学药时曲线。利用非房室模型法(NCA)计算t 1/2、Cmax、Tmax、AUC、Cl、Vd等。
实验结果可知，PF01在食蟹猴体内的T 1/2、Cmax、Tmax平均值分别为30.92h、458.91ng/ml、12h。Cl平均值为2.27ml/h/kg，Vd平均值为101.13ml/kg，AUC为21.71h·μg/ml。从T 1/2判断，PF01药物的体内半衰期比天然hGLP-2延长约100倍。本公开提供的胰高血糖素样肽-2突变体偶联物大大提高了胰高血糖素样肽-2在动物体内的半衰期。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本公开的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本公开的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本公开的限制，本领域的普通技术人员在本公开的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (27)

  1. 一种胰高血糖素样肽-2突变体，其中，与野生型胰高血糖素样肽-2的氨基酸序列相比，所述胰高血糖素样肽-2突变体的氨基酸序列中第30位氨基酸为非赖氨酸的碱性氨基酸，
    其中，所述野生型胰高血糖素样肽-2的氨基酸序列如SEQ ID NO:9所示。
  2. 根据权利要求1所述的胰高血糖素样肽-2突变体，其中，所述胰高血糖素样肽-2突变体具有p.Lys30Arg或p.Lys30His突变。
  3. 根据权利要求1或2所述的胰高血糖素样肽-2突变体，其中，所述胰高血糖素样肽-2突变体进一步具有p.Ala2Gly或p.Ala2Aib突变。
  4. 根据权利要求1～3中任一项所述的胰高血糖素样肽-2突变体，其中，所述胰高血糖素样肽-2突变体的氨基酸序列为：HX 1DGSFSDEMNTILDNLAARDFINWLIQTX 2ITD，
    其中，X 1为Gly或Aib，X 2为Arg或His。
  5. 一种胰高血糖素样肽-2突变体衍生物，包括权利要求1～4中任一项所述的胰高血糖素样肽-2突变体、延伸氨基酸序列以及赖氨酸Lys，所述胰高血糖素样肽-2突变体的羧基端与所述延伸氨基酸序列的氨基端相连，所述延伸氨基酸序列的羧基端与所述赖氨酸相连。
  6. 根据权利要求5所述的胰高血糖素样肽-2突变体衍生物，其中，所述延伸氨基酸序列选自(G m1S n1) x1、(S n2G m2) x2、(G m3S n3G m4) x3和(S n4G m5S n5) x4中的至少之一，
    其中，m1表示甘氨酸的数量，n1表示丝氨酸的数量，x1表示(G m1S n1)肽段的重复次数，m1、n1为1～4之间的任意整数，x1为1～3之间的任意整数，m1+n1＝5，优选地，m1＝4，n1＝1，x1＝1或2；
    任选地，m2表示甘氨酸的数量，n2表示丝氨酸的数量，x2表示(S n2G m2)肽段的重复次数，m2、n2为1～4之间的任意整数，x2为1～3之间的任意整数，m2+n2＝5，优选地，x2＝1；
    任选地，m3、m4表示甘氨酸的数量，n3表示丝氨酸的数量，x3表示(G m3S n3G m4)肽段的重复次数，m3、m4、n3为1～3之间的任意整数，x3为1～3之间的任意整数，m3+m4+n3＝5；
    任选地，m5表示甘氨酸的数量，n4、n5表示丝氨酸的数量，x4表示(S n4G m5S n5)肽段的重复次数，m5、n4、n5为1～3之间的任意整数，x4为1～3之间的任意整数，m5+n4+n5＝5。
  7. 根据权利要求5或6所述的胰高血糖素样肽-2突变体衍生物，其中，所述胰高血糖素样肽-2突变体衍生物的氨基酸序列为：HX 1DGSFSDEMNTILDNLAARDFINWLIQTX 2ITDX 3K，
    其中，X 1为Gly或Aib，X 2为Arg或His，X 3为(G m1S n1) x1，且m1表示甘氨酸的数量，n1表示丝氨酸的数量，x1表示(G m1S n1)肽段的重复次数，m1、n1为1～4之间的任意整数，x1为1～3之间的任意整数，m1+n1＝5，优选地，m1＝4，n1＝1，x1＝1或2。
  8. 根据权利要求5～7中任一项所述的胰高血糖素样肽-2突变体衍生物，其中，所述胰高血糖素样肽-2突变体衍生物的氨基酸序列如SEQ ID NO:1-4所示。
  9. 一种核酸分子，编码权利要求1～4中任一项所述的胰高血糖素样肽-2突变体或权利要求5～8中任一项所述的胰高血糖素样肽-2突变体衍生物。
  10. 一种表达载体，包含权利要求9所述的核酸分子。
  11. 一种宿主细胞，包含权利要求9所述的核酸分子或权利要求10所述的表达载体或表达权利要求1～4中任一项所述的胰高血糖素样肽-2突变体或权利要求5～8中任一项所述的胰高血糖素样肽-2突变体衍生物。
  12. 制备权利要求1～4中任一项所述的胰高血糖素样肽-2突变体或权利要求5～8中任一项所述的胰高血糖素样肽-2突变体衍生物的方法，其中，所述方法包括：
    在适于表达所述胰高血糖素样肽-2突变体或所述胰高血糖素样肽-2突变体衍生物的条件下培养权利要求11所述的宿主细胞，以便生产所述胰高血糖素样肽-2突变体或所述胰高血糖素样肽-2突变体衍生物。
  13. 一种胰高血糖素样肽-2突变体偶联物或其药学上可接受的盐，包括权利要求5～8中任一项所述的胰高血糖素样肽-2突变体衍生物以及偶联物，
    其中，所述胰高血糖素样肽-2突变体衍生物的羧基端的Lys与所述偶联物通过酰胺键相连。
  14. 根据权利要求13所述的胰高血糖素样肽-2突变体偶联物或其药学上可接受的盐，其中，所述偶联物选自脂肪酸偶联物和聚乙二醇的至少之一。
  15. 根据权利要求14所述的胰高血糖素样肽-2突变体偶联物或其药学上可接受的盐，其中，所述脂肪酸偶联物包括脂肪链和连接物，所述脂肪链和连接物相连接，其中，所述脂肪链结构通式为HOOC-(CH 2) a-COOH，a为12～24的整数，优选地，a为16或18；
    任选地，所述连接物结构通式为(17-氨基-10-氧代-3,6,12,15-四氧杂-9-氮杂十七烷酸) b-(γGlu) c，b为1或2，c为1或2，优选地，b为1，c为1；
    任选地，所述脂肪链和所述连接物通过酰胺键相连。
  16. 根据权利要求14或15所述的胰高血糖素样肽-2突变体偶联物或其药学上可接受的盐，其中，所述聚乙二醇的分子量为5KDa～40KDa，
    任选地，所述聚乙二醇为直链或支链结构，优选为直链结构，
    任选地，所述聚乙二醇为具有活化基团的聚乙二醇，所述活化基团选自N-羟基琥珀酰亚胺或酰氯。
  17. 权利要求1～4中任一项所述的胰高血糖素样肽-2突变体、权利要求5～8中任一项所述的胰高血糖素样肽-2突变体衍生物、权利要求13～16中任一项所述的胰高血糖素样肽-2突变体偶联物或其药学上可接受的盐在制备药物中的用途，其中，所述药物用于治疗和/或预防肠道相关疾病。
  18. 根据权利要求17所述的用途，其中，所述肠道相关疾病包括选自短肠综合征、化疗或放疗引起的肠粘膜损伤、溃疡性肠炎、慢性肠炎和非炎症性肠损伤的至少之一。
  19. 一种药物组合物，包括权利要求1～4中任一项所述的胰高血糖素样肽-2突变体和/或权利要求5～8中任一项所述的胰高血糖素样肽-2突变体衍生物和/或权利要求13～16中任一项所述的胰高血糖素样肽-2突变体偶联物或其药学上可接受的盐。
  20. 根据权利要求19所述的药物组合物，其中，所述药物组合物进一步包括缓冲盐、赋形剂、保护剂中的至少之一，
    任选地，所述缓冲盐选自醋酸盐、磷酸盐、硼酸盐、碳酸盐中的至少之一，优选为磷酸盐，
    任选地，所述赋形剂选自甘露醇、蔗糖、麦芽糖、海藻糖中的至少之一，优选为甘露醇和/或海藻糖，
    任选地，所述保护剂包括但不限于His、Gly、Ala、Arg，优选为His。
  21. 根据权利要求19或20所述的药物组合物，其中，进一步包括其他治疗或预防肠道相关疾病的药物或癌症化疗和/或放疗药物。
  22. 根据权利要求19～21中任一项所述的药物组合物，其中，所述药物组合物的剂型选自注射液、丸剂、冻干粉、片剂、胶囊或颗粒。
  23. 一种药物单剂型，含有0.1-10mg的权利要求1～4中任一项所述的胰高血糖素样肽-2突变体、权利要求5～8中任一项所述的胰高血糖素样肽-2突变体衍生物、权利要求13～16中任一项所述的胰高血糖素样肽-2突变体偶联物或其药学上可接受的盐。
  24. 一种治疗和/或预防肠道相关疾病的方法，包括向患有或疑似患有肠道相关疾病的受试者施用以下中的至少之一：
    权利要求1～4中任一项所述的胰高血糖素样肽-2突变体；
    权利要求5～8中任一项所述的胰高血糖素样肽-2突变体衍生物；
    权利要求9所述的核酸分子；
    权利要求10所述的表达载体；
    权利要求11所述的宿主细胞；
    权利要求13～16中任一项所述的胰高血糖素样肽-2突变体偶联物或其药学上可接受的盐；
    权利要求19～22所述的药物组合物；
    权利要求23所述的药物单剂型。
  25. 根据权利要求24所述的方法，其中，所述肠道相关疾病包括选自短肠综合征、化疗或放疗引起的肠粘膜损伤、溃疡性肠炎、慢性肠炎和非炎症性肠损伤的至少之一。
  26. 权利要求1～4中任一项所述的胰高血糖素样肽-2突变体、权利要求5～8中任一项所述的胰高血糖素样肽-2突变体衍生物、权利要求9所述的核酸分子、权利要求10所述的表达载体、权利要求11所述的宿主细胞、权利要求13～16中任一项所述的胰高血糖素样肽-2突变体偶联物或其药学上可接受的盐、权利要求19～22所述的药物组合物、权利要求23所述的药物单剂型在治疗和/或预防肠道相关疾病中的用途。
  27. 根据权利要求25所述的用途，其中，所述肠道相关疾病包括选自短肠综合征、化疗或放疗引起的肠粘膜损伤、溃疡性肠炎、慢性肠炎和非炎症性肠损伤的至少之一。
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