WO2010099746A1 - 胰高血糖素样肽-2类似物及其制备方法和用途 - Google Patents

胰高血糖素样肽-2类似物及其制备方法和用途 Download PDF

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WO2010099746A1
WO2010099746A1 PCT/CN2010/070850 CN2010070850W WO2010099746A1 WO 2010099746 A1 WO2010099746 A1 WO 2010099746A1 CN 2010070850 W CN2010070850 W CN 2010070850W WO 2010099746 A1 WO2010099746 A1 WO 2010099746A1
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asp
ile
peptide
thr
leu
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王亚里
孙长安
王瑞军
姜涛
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连云港恒邦医药科技有限公司
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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
    • A61K38/26—Glucagons
    • 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
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/08—Drugs for disorders of the alimentary tract or the digestive system for nausea, cinetosis or vertigo; Antiemetics
    • 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
    • A61P1/12—Antidiarrhoeals
    • 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
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • 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
    • C07K14/605—Glucagons

Definitions

  • the present invention relates to Glucagon Like Peptide-2 (GLP-2) analogs and their preparation for the prevention and/or treatment of diseases associated with gastrointestinal mucosal damage and for the reduction of chemotherapy and radiation therapy Use in drugs for intestinal side effects.
  • GLP-2 Glucagon Like Peptide-2
  • GLP-2 is a proglucagon-derived peptide (PGDP), which is one of the products of proglucagon (PG) degradation by prohormone convertase (PC).
  • PGDP proglucagon-derived peptide
  • PC prohormone convertase
  • GLP-2 is synthesized and released by intestinal L endocrine cells and is regulated by feeding, neurological and endocrine factors. It is most important to stimulate the secretion of L endocrine cells by eating foods containing carbohydrates and fats [Rocca AS.
  • GLP-2 Monounsaturated fatty acid diets improve glycemic tolerance through increased secretion of glucagon-like peptide- 1. Endocrinology, 2001, 142: 1148-55].
  • the concentration of GLP-2 in the blood circulation increased most significantly within 15 minutes and 1 hour after a normal adult meal.
  • the main form of GLP-2 present in intestinal mucosal tissues and blood circulation is intact GLP-2, GLP-2 (1-33).
  • the biological half-life of GLP-2 ( 1-33 ) in human blood circulation is about 7 minutes. Its metabolism is mainly hydrolyzed by renal excretion and dipeptidyl peptidase-4 (DPP-4) on the intestinal brush border.
  • DPP-4 dipeptidyl peptidase-4
  • GLP-2 The first two residues at the amino terminus form inactive GLP-2 (2-33).
  • the rats fed the GLP-2 analogue lacking the DDP-4 action site showed significantly better intestinal epithelial growth than the rats fed the native GLP-2.
  • the clearance of GLP-2 in the blood of rats with bilateral nephrectomy was significantly lower than that of unremoved kidney rats.
  • GLP-2 (2-33) has recently been shown to be a competitive antagonist of the GLP-2 receptor (GLP-2R), which inhibits GLP-2 ( 1-33 ) and nutrient-induced intestinal mucosal growth [Shin ED] , Estall JL, Izzo A, Drucker DJ, Brubaker PL.
  • the effects of GLP-2 on the intestine include: specific stimulation of intestinal mucosal growth, enhancement of regeneration after intestinal mucosal injury; inhibition of intestinal mucosal epithelial cells and crypt cells; enhancement of intestinal epithelial basal glucose transporter -2 (glucose transporter-2, GLUT2) expression and transport of glucose, promote intestinal digestion and absorption; inhibit gastrointestinal motility and gastric acid secretion; enhance intestinal mucosal barrier function; increase intestinal blood supply.
  • the effect of GLP-2 on the intestinal mucosa is essentially independent of age and gender, and intravenous, intramuscular, subcutaneous or Intraperitoneal injection has a significant effect [Burrin DG, Petersen Y, Stoll ⁇ , et al. Glucagon-like peptide 2: a nutrient-responsive gut growth factor. J Nutr, 2001, 131: 709-712].
  • the GLP-2 receptor (GLP-2R) is a member of the type B glucagon-trypsin-like G protein-coupled receptor superfamily and its expression is highly tissue specific. Immunohistochemistry and in situ hybridization have confirmed that GLP-2R is expressed on human intestinal endocrine cells, murine intestinal neurons, and subepithelial myofibroblasts of rats, mice, and marmosets [Yusta B, Huang L, Munroe D , et al. Enteroendocrine localization of GLP-2 receptor expression in humans and rodents. Gastroenterology, 119:744-55; Bjerknes M, Cheng H. Modulation of specific intestinal epithelial progenitors by enteric neurons. Proc Natl Acad Sci.2001,98: 12497-502]. The lack of expression of GLP-2R on intestinal cells suggests that GLP-2R may be indirect in promoting proliferation and cytoprotection.
  • GLP-2 Signaling of GLP-2 in cells is achieved by a specific GLP-2R-mediated cyclic adenosine monophosphate (cAMP) pathway.
  • GLP-2 activates adenylate cyclase, which increases intracellular cAMP, which activates protein kinase A (PKA), and then PKA may promote the genes of cAMP response elements through intracellular Ca2+ and/or inositol triphosphate pathways. Transcription.
  • GLP-2 phosphorylates two isoforms of ERK-1, a mitogen-activated protein kinase (MAPK), in human intestinal mucosal epithelial cells Caco-2 cells cultured in vitro by phosphorylation-type extracellular signal-regulated kinase (MEK)
  • MEK extracellular signal-regulated kinase
  • the threonine and tyrosine residues of ERK-2 increase their activity, thereby significantly promoting Caco-2 cell proliferation.
  • This effect can be specifically tyrosine protein kinase inhibitor (Genistein), phosphatidylinositol 3-kinase (PB-K) inhibitor (LY294002), mitogen-activated protein kinase (MAPK) inhibitor (PD098059) ) Block.
  • GLP-2R signaling inhibits cycloheximide-induced apoptosis in BHK-GLP-2R cells, which inhibits caspase-specific protease-3 (ca S p aS e-3) Enzyme activity, which reduces the division of poly ADP ribose polymerase.
  • caspase-specific protease-3 ca S p aS e-3 Enzyme activity, which reduces the division of poly ADP ribose polymerase.
  • PKA inhibitor H289 GLP-2 reduced cycloheximide-induced caspase-3 division.
  • GLP was present in the presence of two kinase inhibitors, the phosphatidylinositol-3-kinase (PI3-K) inhibitor LY294002 and the mitogen-activated protein kinase (MAPK) inhibitor PD98054.
  • PI3-K phosphatidylinositol-3-kinase
  • MAPK mitogen-activated protein kinase
  • -2 also increases cell viability [Yusta B, Estall J, Drucker DJ. Glucagon-like peptide-2 recepter activation engages bad and glycogen synthase kinase-3 in a protei kinase A-dependent manner and ensures apoptosis following inhibition of phosphatidylinositol 3 -kinase.
  • GLP-2 has a nutrient-absorbing effect, but whether it reduces intestinal damage, The role of promoting intestinal repair remains to be confirmed by further clinical trials.
  • the adverse effects of GLP-2 have not been found during the treatment, it is considered that the biological effects of GLP-2 are found in the study of animal glucagon-induced tumors, and the subcutaneous injection of glucagon in nude mice It is shown that GLP-2 significantly promotes intestinal mucosal growth, so it is difficult to determine whether GLP-2 stimulates the growth of intestinal tumors.
  • GLP-2 For the treatment of short bowel syndrome, the efficacy of GLP-2 requires a large-scale randomized controlled trial. The most appropriate dose of GLP-2 treatment, the route of administration, the course of treatment, and the timing of application require more in-depth research. It is believed that the special intestinal protective effect of GLP-2 has important clinical application value in the prevention and treatment of malabsorption, inflammatory bowel disease, stress gastrointestinal mucosal injury and other diseases.
  • GLP-2 is secreted as a peptide with the following 33 amino acids: His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala -Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp. GLP-2 is rapidly inactivated in vivo by the enzyme DPPIV at the 2-position alanine (Ala) at the nitrogen end to form GLP-2 (3-33). In addition to renal clearance, this rapid enzymatic degradation mechanism of GLP-2 (1-33) results in a half-life of the peptide in the human blood circulation of about 7 minutes.
  • GLP-2 analogs and their medical uses are disclosed in U.S. Patent Nos. 6,184,208, 5,789,379 and 6,184,201. The analogs are all obtained by substitution and or deletion of human GLP-2.
  • DaCambra et al. (Biochemistry 2000, 39, 8888-8894) describe structurally determined clusters of GLP-2 activity. Examples of such determinants are Phe6 and Thr5, which are believed to be critical for GLP-2 receptor binding and activation.
  • GLP-2 analogue [Gly2] GLP-2 is disclosed in WO 97/39031 by Drucker et al.
  • the alanine at position 2 is substituted with glycine to render the peptide resistant to DPPIV cleavage. Substitution of alanine is shown to increase the stability and potency of the peptide.
  • This patent application describes how to use GLP-2 analogues to combat inflammation and damage associated with intestinal epithelial mucosa. These diseases include large-scale small bowel resection, inflammatory bowel disease, chemotherapy-induced mucositis, and ischemic injury.
  • WO 02/066511 describes GLP-2 analogues with extended half-life in vivo and their use as medicaments for the treatment of gastrointestinal disorders such as inflammatory bowel disease.
  • WO 01/14779 describes the use of h [Gly2] GLP-2 as a pretreatment for inhibiting chemotherapy-induced apoptosis and promoting cell survival. Summary of the invention
  • GLP-2 analogue represented by the following formula (I), and pharmaceutically acceptable salts thereof and derivatives thereof, which include long-chain fatty acids and/or polyethylene glycols and/or Or other length
  • Z1, Z2, R1 or R2 is absent, or is a natural or unnatural amino acid, or is a peptide sequence of 2 to 33 amino acid units consisting of natural and/or unnatural amino acids, and XI, X2, X3, X10, XII, X15, X16, X24 or X25 are natural or unnatural amino acids.
  • XI is His or D-His.
  • X2 is D-Ala or
  • X10 is Nle or Met.
  • X15 is Glu or Asp.
  • X3 is Glu or Asp, and X3 forms a peptide bond with Gly as an ⁇ carboxyl group or a ⁇ carboxyl group.
  • the present invention more preferably a GLP-2 analogue having the following sequence:
  • the present invention also relates to a process for producing a GLP-2 analogue according to the present invention, characterized in that the GLP-2 analogue is prepared by solid state synthesis and purified by reverse phase liquid chromatography.
  • the invention further relates to a GLP-2 analogue pharmaceutical composition of the invention comprising a therapeutically effective amount comprising a pharmaceutically acceptable carrier and/or a cancer chemotherapeutic agent.
  • the dosage form of the composition is preferably an injection or a sustained release agent.
  • the present invention also relates to the use of the pharmaceutical composition for the preparation of a medicament for preventing and/or treating a disorder associated with gastrointestinal mucosal damage
  • a disorder associated with gastrointestinal mucosal damage wherein the gastrointestinal mucosal injury-related disorder is selected from the group consisting of a peptic ulcer, a malabsorption syndrome, Short bowel syndrome, ileocecal valve syndrome, inflammatory bowel disease, abdominal spleen diarrhea, tropical spiro diarrhea, hypogammaglobulinemia spiro diarrhea, enteritis, localized enteritis, ulcer Gastrointestinal, gastrointestinal mucosal damage caused by osteoporosis and / or malnutrition, gastrointestinal side effects of chemotherapy or radiation therapy, preferably diarrhea, abdominal pain, vomiting, radiation gastroenteritis, infectious stomach Gastrointestinal mucosal damage caused by enteritis or post-infection gastroenteritis, toxic agents or chemotherapeutic agents or DPP-IV (dipeptidyl peptidase-IV) mediated
  • the invention further relates to the use of the GLP-2 analogue for the preparation of a medicament for the prevention and/or treatment of a gastrointestinal mucosal injury-related disorder, wherein the gastrointestinal mucosal injury-related disorder is selected from the group consisting of a peptic ulcer, a malabsorption syndrome, and a short Intestinal syndrome, ileocecal valve syndrome, inflammatory bowel disease, abdominal spleen diarrhea, tropical spiro diarrhea, hypogammaglobulinemia spiro diarrhea, enteritis, localized enteritis, ulcerative colon Inflammation, gastrointestinal mucosal damage caused by osteoporosis and / or malnutrition, gastrointestinal side effects of chemotherapy or radiation therapy, preferably diarrhea, abdominal cramps, vomiting, radiation gastroenteritis, infectious gastroenteritis Or gastrointestinal mucosal damage caused by post-infection gastroenteritis, or a poison or chemotherapeutic agent or DPP-IV (dipeptidyl peptidase-IV) mediated condition.
  • the present invention relates to a novel GLP-2 analogue.
  • the amino acid at one or more positions in the sequence of the GLP-2 analogue of the present invention is replaced as compared to the well-known GLP-2, such that the GLP-2 analogue of the present invention has an improvement in in vivo biological activity. And or the characteristics of improved chemical stability.
  • the GLP-2 analogue of the present invention comprises the GLP-2 sequence in the 1st, 2nd, 3rd, 10th, 11th, 15th, 16th, 24th or 25th position. Replacement and/or replacement combinations at one or more locations.
  • the present invention provides GLP-2 analogs with improved chemical stability and or biological activity.
  • the present invention provides a GLP-2 analogue represented by the formula I or a pharmaceutically acceptable salt thereof or a derivative thereof:
  • the GLP-2 analogs of the present invention also have improved chemical stability, such as stability against acid hydrolysis, oxidation, and deamidation.
  • Substitution of the X3 position enhances the stability of the GLP-2 analog of the present invention for acid hydrolysis
  • substitution of the X10 position enhances the oxidative stability of the GLP-2 analog of the present invention.
  • substitution of one or more of the XI I, X16 and X24 positions enhances the stability of the GLP-2 analogs of the invention against deamidation.
  • the GLP-2 analogs of the present invention may exhibit enhanced resistance to degradation in acidic solutions, to deamidation and/or to oxidative degradation relative to Gly2-GLP-2.
  • GLP-2 analogs extend their half-life in vivo by long-chain fatty acids and/or polyethylene glycol or other long-acting modifications. Polyethylene glycol modification can effectively mask the analog parent, reduce the recognition of the analog by the in vivo immune system and the protease in the body, thereby reducing the immunogenicity of the analog and the probability of hydrolysis by the protease in the body, thereby prolonging the similarity of GLP-2. The time the object circulates in the body.
  • the invention in another aspect, relates to a composition
  • a composition comprising a GLP-2 analogue of the invention or a salt thereof or a derivative thereof, admixed with a carrier.
  • the composition is a pharmaceutically acceptable composition, the carrier being a pharmaceutically acceptable or pharmaceutically acceptable carrier.
  • the GLP-2 peptide analog of the present invention may be a pharmaceutically acceptable acid addition salt of the GLP-2 analogue of the present invention.
  • the invention relates to a GLP-2 analogue of the invention or a salt thereof for use in therapy.
  • the invention relates to the use of a GLP-2 analogue or a salt thereof or a derivative thereof for the manufacture of a medicament for the treatment and/or prevention of a gastrointestinal related disorder, such as treatment of impaired intestinal function, bone Neonates with plaque or DPP-IV (dipeptidyl peptidase-IV) mediated disorders.
  • a gastrointestinal related disorder such as treatment of impaired intestinal function, bone Neonates with plaque or DPP-IV (dipeptidyl peptidase-IV) mediated disorders.
  • stomach and bowel-related disorders are selected from the group consisting of peptic ulcers, gastritis, malabsorption syndrome, short bowel syndrome, ileocecal valve syndrome, inflammatory bowel disease, abdominal spiro diarrhea, tropical Sprague Diarrhea, hypogammaglobulinemia type Spirafe diarrhea, enteritis, localized enteritis, ulcerative colitis, diarrhea-related irritable bowel syndrome, small bowel injury or short bowel syndrome.
  • GLP-2 analogs of the invention include radiation gastroenteritis, infectious gastroenteritis or post-infection gastroenteritis, and due to toxic agents or other chemistry Diagnosis of small bowel damage caused by therapeutic agents. This may require simultaneous or post-administration of GLP-2 analogues with chemotherapy or radiation therapy to reduce side effects of chemotherapy or radiation therapy such as diarrhea, abdominal cramps or vomiting, and reduced chemotherapy or radiation therapy. Intestinal epithelial structure and functional impairment.
  • the invention further relates to a therapeutic kit comprising a cancer chemotherapeutic drug, a GLP-2 analogue of the invention, and instructions for use.
  • a therapeutic kit in particular, it comprises a cancer chemotherapy drug as a therapeutic agent, a GLP-2 analogue of the present invention, and instructions for use, and each of the drugs or analogs described above is optionally A pharmaceutically acceptable carrier combination.
  • the therapeutic agents can be packaged separately for separate use or can be provided in the same composition.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a cancer chemotherapeutic agent in combination with a pharmaceutically acceptable carrier and a GLP-2 analogue of the invention.
  • Patients with an increased risk of gastrointestinal mucosal formation may desire to select a compound to reduce side effects or eliminate the risk of side effects. For example, when choosing a drug for colon cancer formation in a patient who has colon cancer formation or is at risk of developing colon tumor formation, choose a drug that is more selective for the small intestine than the colon than a non-selective drug or for colon. Drugs that are higher than the small intestine may be more appropriate.
  • the invention also relates to GLP-2 analogues for use in the preparation of a therapeutic and/or preventive nutrient Use in poor drugs.
  • the invention relates to the use of a therapeutically effective amount of a GLP-2 analog of the invention, or a salt thereof, or a derivative thereof, for use in a medicament for treating a gastrointestinal related disorder in a patient.
  • the invention in another aspect, relates to a method of treating or preventing a side effect of chemotherapy or radiation therapy in a patient in need thereof, the method comprising administering a therapeutically effective amount of a GLP-2 analogue of the invention or a salt thereof Or their derivatives.
  • the invention provides a method of treating and preventing malnutrition in a patient in need thereof, the method comprising using a therapeutically effective amount of a GLP-2 analogue, or a salt thereof, or a derivative thereof, as defined herein .
  • the GLP-2 analog peptide of the present invention is synthesized by a solid phase synthesis technique well known to those skilled in the art, and the basic principle is: first, the hydroxyl group of the hydroxy terminal amino acid of the peptide chain to be synthesized is covalently bonded to the same insoluble structure.
  • the polymer resin solid phase carrier
  • the amino acid bound to the solid phase carrier is used as an amino component to deprotect the amino group and react with an excess of the activated carboxyl component to lengthen the peptide chain.
  • the intermediate control of the condensation and deprotection reaction steps is a method for detecting ninhydrin, that is, when there is a free amino group on the resin peptide chain, it is blue by the ninhydrin reagent, and does not develop color when there is no free amino group. (The ninhydrin reagent itself is yellow).
  • GLP-2R is a receptor coupled to Gs protein, and when the receptor binds to an agonist, it causes an increase in intracellular cAMP concentration.
  • HEK293 cells were co-transfected with luciferase reporter gene regulated by GLP-2R and cAMP response elements, and luciferase expression was increased when the compound binds to the receptor and activates the receptor.
  • the activation of GLP-2R by the compound can be obtained by detecting the activity of luciferase.
  • the sequence of the positive compound Teduglutide is:
  • Fmoc-Asp(OtBu)-Wang resin (0.5 mmol/g) 40 g pOmmol) was placed in a 2 L bubbling bottle, and 400 mL of DMF swelling resin was added for 30 minutes, and the DMF solution was taken out;
  • X11 is Asn
  • X15 is Asp
  • X16 is Asn
  • X24 is Asn
  • X25 is Trp.
  • the preparation method was the same as in Example 1.
  • XI is D-His
  • X2 is Gly
  • X3 is Glu
  • X10 is Met
  • Xl l It is Asn
  • X15 is Asp
  • X16 is Asn
  • X24 is Asn
  • X25 is Trp.
  • the preparation method was the same as in Example 1.
  • the preparation method was the same as in Example 1.
  • GLP-2R is a receptor coupled to a Gs protein, and when the receptor binds to an agonist, it causes an increase in intracellular cAMP concentration.
  • luciferase reporter plasmids regulated by GLP-2R and cAMP response elements were co-transfected into HEK293 cells, and luciferase expression was increased when GLP-2 analogs bind to the receptor and activate the receptor.
  • the activation of GLP-2R by GLP-2 analogues can be obtained by detecting luciferase activity.
  • Table 1 Table 1 Molecular weight, mass and final concentration of each GLP-2 analogue for GLP-2R agonistic activity assay
  • the GLP-2R and pCRE-Luc expression plasmids were introduced into HEK293 cells by electroporation, and the transfected cells were seeded into 96-well plates at a density of 40,000/well/ ⁇ and incubated at 37 °C for 24 hours.
  • GLP-2 analogs of the invention (3 replicates per concentration) or positive control GLP-2 were added at a concentration gradient and incubated for 5 hours at 37 °C.
  • Solvent DMSO was the negative control. 50 ⁇ l of the medium was taken out per well, 50 ⁇ l of luciferase substrate was added, and the mixture was shaken for 10 minutes. The 80 ⁇ l reaction solution was taken out and added to a white 96-well plate and detected on an Invision plate reader.
  • the EC50 value, 95% confidence limit and maximum response rate of the GLP-2 analogue are shown in Table 2 below.
  • both compounds 1 and 2 showed better GLP receptor agonistic activity.

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Description

胰高血糖素样肽 -2类似物及其制备方法和用途 技术领域
本发明涉及胰高血糖素样肽 -2 (Glucagon Like Peptide -2, GLP-2) 类似物及 其在制备用于预防和 /或治疗胃肠粘膜损伤相关病症以及减轻化学疗法和放射疗 法的胃肠道副作用的药物中的用途。 背景技术
GLP-2属于胰高血糖素原衍生肽类 （proglucagon-derived peptide ， PGDP) ， 是胰高血糖素原（proglucagon, PG)被激素原转化酶（prohormone convertase , PC) 降解的产物之一,是由 33个氨基酸残基组成的单链多肽， 分子量 3900道尔顿， 其氨基酸序列在哺乳动物中具有高度保守性 [Drucker DJ. Glucagon-like peptide 2. J Clin Endocrinol Metab, 2001,86:1758-1774]。 GLP-2由肠道 L内分泌细胞合成和 释放， 并受摄食、神经和内分泌等因素调节， 以进食含碳水化合物和脂肪的食物 对 L 内分泌细胞的分泌活动的剌激最为重要 [Rocca AS. LaGreca J, Kalitsky J,Brubaker PL. Monounsaturated fatty acid diets improve glycemic tolerance through increased secretion of glucagon-like peptide- 1. Endocrinology,2001 , 142:1148-55]。正 常成人餐后 15分钟内和 1小时后两个时期，血循环中 GLP-2的浓度升高最显著。 GLP-2在肠粘膜组织及血液循环中存在的主要形式是完整的 GLP-2, 即 GLP-2 ( 1-33 )。 GLP-2 ( 1-33 )在人体血循环中的生物半衰期约 7分钟， 其代谢主要通 过肾脏排泄和肠道刷状缘上的二酰肽肽酶 4 (dipeptidyl peptidase-4 , DPP-4)水解 其氨基末端前两个残基， 形成无活性的 GLP-2 (2-33 )。 词喂缺少 DDP-4作用位 点的 GLP-2类似物的大鼠，其肠上皮生长情况明显好于词喂天然 GLP-2的大鼠。 双侧肾脏切除的大鼠血中 GLP-2 的清除率明显低于未切除肾脏大鼠。 然而， GLP-2 (2-33 ) 最近被证实是 GLP-2受体 （GLP-2R) 的竞争性拮抗剂， 能抑制 GLP-2 ( 1-33 ) 和营养诱导的肠粘膜生长 [Shin ED, Estall JL, Izzo A, Drucker DJ, Brubaker PL. Mucosal adaptation to enteral nutrients is dependent on the physiologic actions of glucagons-like peptide-2 in mice. Gastroenterology, 2005,128:1340-53]。以 上说明 GLP-2的生物学活性受 DDP-4水解， 其降解产物拮抗和肾脏清除三者的 调节。
GLP-2对肠道的作用包括： 特异性地剌激肠粘膜的生长， 增强肠粘膜损伤后 的再生； 抑制肠粘膜上皮细胞和隐窝细胞的凋亡； 增强肠上皮细胞基底侧葡萄糖 转运蛋白 -2 (glucose transporter-2, GLUT2)的表达和对葡萄糖的转运， 促进肠消 化和吸收功能；抑制胃肠动力和胃酸分泌；增强肠粘膜屏障功能；增加肠道血供。 GLP-2对肠粘膜的作用基本上与年龄和性别无关， 并且静注、肌注、皮下注射或 腹腔注射都有明显效果 [Burrin DG, Petersen Y, Stoll Β, et al. Glucagon-like peptide 2: a nutrient-responsive gut growth factor. J Nutr,2001,131:709-712]。
GLP-2受体（GLP-2R)是 B型胰高血糖素-胰泌素样 G蛋白偶联受体超家族 成员， 其表达具有高度组织特异性。 免疫组织化学和原位杂交技术已经证实， GLP-2R在人类肠内分泌细胞、 鼠肠神经元以及大鼠、 小鼠、 狨猴的上皮下肌纤 维母细胞上表达 [Yusta B, Huang L, Munroe D, et al. Enteroendocrine localization of GLP-2 receptor expression in humans and rodents. Gastroenterology, 119:744-55； Bjerknes M, Cheng H. Modulation of specific intestinal epithelial progenitors by enteric neurons. Proc Natl Acad Sci.2001,98:12497-502]。 肠细胞上缺乏 GLP-2R的 表达， 说明 GLP-2R促进增生和细胞保护作用可能是间接的。
GLP-2 在细胞内的信号传导是通过特异性 GLP-2R 介导的环磷酸腺苷 (cAMP) 通路实现的。 GLP-2激活腺苷酸环化酶， 弓 I起细胞内 cAMP增加， 后 者活化蛋白激酶 A(PKA)，然后 PKA可能通过细胞内 Ca2+和 /或三磷酸肌醇通路 促进 cAMP反应元件的基因转录。 GLP-2可通过磷酸化型细胞外信号调节激酶 (MEK) 磷酸化体外培养的人肠黏膜上皮细胞 Caco-2细胞内丝裂原活化蛋白激 酶 （MAPK) 的两个异构体 ERK-1和 ERK-2的苏氨酸和酪氨酸残基， 使其活性 增加， 从而显著促进 Caco-2细胞增殖。 该作用能分别被特异性的酪氨酸蛋白激 酶抑制剂 （Genistein)、 磷脂酰肌醇 3-激酶 （PB-K) 抑制剂 （LY294002)、 丝裂 原活化蛋白激酶 （MAPK) 抑制剂 （PD098059) 阻断。 另一方面， GLP-2R信号 激活可抑制放线菌酮诱导的 BHK-GLP-2R细胞凋亡， 这与其抑制半胱氨酸天冬 氨酸特异性蛋白酶 -3(caSpaSe-3)酶活性， 减少多聚 ADP核糖聚合酶分裂相关。 并 且不论 PKA抑制剂 H289存在与否， GLP-2都能降低放线菌酮诱发的 caspase-3 的分裂。 放线菌酮给药后， 在两种激酶抑制剂即磷脂酰肌醇 -3-激酶 (PI3-K)抑制 剂 LY294002 和丝裂原活化蛋白激酶 (MAPK)抑制剂 PD98054 存在的条件下， GLP-2也能增加细胞存活率 [Yusta B, Estall J, Drucker DJ. Glucagon-like peptide-2 recepter activation engages bad and glycogen synthase kinase-3 in a protei kinase A-dependent manner and prevents apoptosis following inhibition of phosphatidylinositol 3-kinase. J Biol Chem,2002,277:24896-24906； Walsh NA, Yusta B, DaCambra MP, et al. Glucagon-like peptide-2 recepter activation in the rat intestinal mucosa. Endocrinology,2003, 144:4385-4392], 说明 GLP-2的胞内信号传 导不完全依赖 PI3-K和 MAPK途径。 由此可见， GLP-2肠道作用机制所涉及的 信号传导通道很复杂既包括 cAMP 依赖的蛋白激酶途径， 也包括酪氨酸激酶途 径、 PB-K和 MAPK途径。 GLP-2作用的确切分子机制目前尚未完全阐明。
GLP-2肠保护作用的人体试验目前还处于起步阶段，局限于短肠综合征患者 的治疗性研究。 已经明确 GLP-2具有促营养吸收作用， 但是否有减轻肠损伤、 促进肠修复的作用还有待进一步临床试验证实。虽然治疗过程中尚未发现 GLP-2 的不良作用， 但考虑到 GLP-2 的生物学作用是在动物胰高血糖素诱导肿瘤的研 究中发现的， 并且为裸鼠皮下注射胰高血糖素的研究显示， GLP-2显著促进肠粘 膜生长， 因此 GLP-2是否会剌激肠道肿瘤的生长尚难定论。 就短肠综合征的治 疗而言， GLP-2的有效性需要大规模人群随机对照研究证实。 GLP-2治疗的最适 剂量、 给药途径、 疗程和应用时机等都需要更深入的研究。 相信 GLP-2特殊的 肠保护作用在防治吸收不良、炎性肠病、应激性胃肠粘膜损伤等疾病方面具有重 要的临床应用价值。
GLP-2 以 具 有 以 下 33 个 氨 基 酸 的 肽 的 形 式 分 泌 ： His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala- Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp。 GLP-2在体内迅速地被 酶 DPPIV在氮端的 2位丙氨酸 (Ala)处切割形成 GLP-2(3-33)而失活。除肾清除外， GLP-2(l-33)的这种快速酶降解机制导致该肽在人体血循环中的半衰期为约 7分 钟。
Drucker等在 US5,994,500中描述了 GLP-2拮抗剂及其对胃肠组织生长的影 响， 其提出将所述拮抗剂配制成药物用于治疗增生或诱导发育不全。 美国 5,994,500通过突变例如替换和缺失来改变哺乳动物 GLP-2的结构。
US6,184,208、 US5,789,379和 US6,184,201公开了 GLP-2类似物及其医药用 途。 所述类似物全是通过人 GLP-2的替换和或缺失得到的。
DaCambra等 （Biochemistry2000,39,8888-8894)描述了 GLP-2活性的结构决 定簇。这样的决定簇的实例是 Phe6和 Thr5， 所述 Phe6和 Thr5据信对 GLP-2受 体结合和活化是至关重要的。
Drucker等在 WO97/39031中公开了 GLP-2类似物 [Gly2] GLP-2。 其中 2 位的丙氨酸被甘氨酸取代， 以使所述肽抵抗 DPPIV切割。 显示丙氨酸的取代提 高所述肽的稳定性和效能。 该专利申请描述了如何使用 GLP-2类似物对抗肠上 皮粘膜炎症和损伤相关的疾病。这些疾病包括大规模小肠切除、 炎性肠病、化学 疗法诱发的粘膜炎以及缺血性损伤。
WO02/066511描述了体内半衰期延长的 GLP-2类似物及其作为治疗胃肠病 症如炎性肠病的药物的用途。
WO01/14779描述了 h [Gly2] GLP-2用作抑制化学疗法诱导的凋亡和促进 细胞存活的预处理的药物的用途。 发明内容
本发明的目的在于提供一种以下通式 （ I ) 代表的 GLP-2类似物， 及其可 药用盐及它们的衍生物， 该衍生物包括经长链脂肪酸和 /或聚乙二醇和 /或其他长 效化修饰所产生的 GLP-2类似物的衍生物， 或 GLP-2类似物的可药用盐的衍生 物：
1-Z1-X1 -X2-X3-Gly-Ser-Phe-Ser-Asp-Glu-X10-X 11 -Thr-Ile-Leu-X 15-X16-L eu-Ala-Ala-Arg-Asp-Phe-Ile-X24-X25-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-(Z2)-R2
( I )，
其中 Zl、 Z2、 Rl或 R2为不存在， 或为天然或非天然氨基酸， 或为由天然 和 /或非天然氨基酸组成的具有 2至 33个氨基酸单位的肽序列， 而且 XI、 X2、 X3、 X10、 XII、 X15、 X16、 X24或 X25为天然或非天然氨基酸。
优选的是， 本发明所述的通式 （ I ) 代表的 GLP-2类似物中， XI是 His或 D-His。
优选的是， 本发明所述的通式 I代表的 GLP-2类似物中， X2是 D-Ala或
Gly。
优选的是，本发明所述的通式 I代表的 GLP-2类似物中， X10为 Nle或 Met。 优选的是，本发明所述的通式 I代表的 GLP-2类似物中， X15为 Glu或 Asp。 优选的是，本发明所述的通式 I代表的 GLP-2类似物中， X3是 Glu或 Asp， 并且 X3以 α羧基或者 β羧基与 Gly形成肽键。
具体而言， 本发明更优选具有下列序列的 GLP-2类似物：
D-His-D-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Nle-Asn-Thr-Ile-Leu-Glu-Asn-Leu -Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp。
His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Glu-Asn-Leu-Ala- Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp。
His-Gly-Glu-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Glu-Asn-Leu-Ala- Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln- Thr-Lys-Ile-Thr-Asp。
D-His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu- Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp。
D-His-Gly-Glu-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-A la-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp。
根据本发明的记载， 本发明还涉及一种本发明所述的 GLP-2类似物的制备 方法， 其特征在于该 GLP-2类似物采用固态合成法制备并用反相液相色谱法纯 化。
本发明进一步涉及一种包括治疗有效剂量的本发明所述的 GLP-2类似物药 物组合物， 其包括药学上可接受的载体和 /或者癌症化学疗法药物。
所述组合物的剂型优选为注射剂或者缓释剂。
本发明还涉及该药物组合物在制备用于预防和 /或治疗胃肠粘膜损伤相关病 症药物中的用途，其中胃肠粘膜损伤相关病症选自消化道溃疡、吸收不良综合征、 短肠综合征、回盲瓣综合征症、炎性肠病、腹型斯泼卢腹泻、热带型斯泼卢腹泻、 低丙种球蛋白血症型斯泼卢腹泻、 肠炎、局限性肠炎、 溃疡性结肠炎、 胃肠粘膜 损伤导致的骨质疏松症和 /或营养不良症、化学疗法或放射疗法的胃肠道副作用， 优选腹泻、腹部痛性痉挛、呕吐，放射性胃肠炎、传染性胃肠炎或感染后胃肠炎、 毒剂或者化学治疗剂引起的胃肠粘膜损伤或 DPP-IV (二肽基肽酶 -IV)介导的病 症。
本发明进一步涉及所述 GLP-2类似物在制备用于预防和 /或治疗胃肠粘膜损 伤相关病症药物中的用途，其中胃肠粘膜损伤相关病症选自消化道溃疡、吸收不 良综合征、 短肠综合征、 回盲瓣综合征、 炎性肠病、 腹型斯泼卢腹泻、 热带型斯 泼卢腹泻、低丙种球蛋白血症型斯泼卢腹泻、肠炎、局限性肠炎、溃疡性结肠炎、 胃肠粘膜损伤导致的骨质疏松症和 /或营养不良症、 化学疗法或放射疗法的胃肠 道副作用， 优选腹泻、 腹部痛性痉挛、 呕吐， 放射性胃肠炎、 传染性胃肠炎或感 染后胃肠炎、或者毒剂或者化学治疗剂引起的胃肠粘膜损伤或 DPP-IV (二肽基肽 酶 -IV)介导的病症。
由此可见， 本发明涉及的是一种新的 GLP-2类似物。与公知的 GLP-2相比， 本发明所述的 GLP-2类似物的序列中一个或多个位置的氨基酸被替换， 这样使 得本发明所述的 GLP-2类似物具有体内生物活性的改善和或化学稳定性改善的 特点。 具体而言， 优选地， 本发明所述的 GLP-2类似物包含 GLP-2序列 1位、 2 位、 3位、 10位、 11位、 15位、 16位、 24位或 25位中的一个或多个位置上的 替换和 /或替换组合。 本发明提供具有改善的化学稳定性和或生物活性的 GLP-2 类似物。
因此，本发明提供通式 I代表的 GLP-2类似物或其可药用盐或它们的衍生物:
1-Z1-X1 -X2-X3-Gly-Ser-Phe-Ser-Asp-Glu-X10-X 11 -Thr-Ile-Leu-X 15-X16-L eu-Ala-Ala-Arg-Asp-Phe-Ile-X24-X25-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-(Z2)-R2 (通 式 1)， 其中 Zl、 Z2,R1或 R2为不存在， 或为天然或非天然氨基酸， 或为由天然 或非天然氨基酸组成的 3至 20个氨基酸单位的肽序列，而且 XI、 X2、 X3、 X10、 XI I、 X15、 X16、 X24或 X25为天然或非天然氨基酸。
本发明所述的 GLP-2类似物还具有提高的化学稳定性， 例如针对酸水解、 氧化和脱酰胺作用的稳定性。 X3位的替换可提高本发明所述的 GLP-2类似物对 酸水解的稳定性， X10位的替换可提高本发明所述的 GLP-2类似物的氧化稳定 性。 XI I、 X16和或 X24位中一个或多个位置的替换可提高本发明所述的 GLP-2 类似物针对脱酰胺作用的稳定性。因此，相对于 Gly2-GLP-2，本发明所述的 GLP-2 类似物可显示出针对酸性溶液中降解， 针对脱酰胺作用和 /或针对氧化降解的稳 定性的增强。 X15位的替换可提高用化学合成法制备本发明所述的 GLP-2类似 物的产率。 GLP-2 类似物经长链脂肪酸和 /或聚乙二醇或其他长效化修饰可延长其在生 物体内的半衰期。聚乙二醇修饰能有效地掩饰类似物母体，减少类似物被体内免 疫系统和体内蛋白酶的辩识，从而降低了类似物的免疫原性和被体内蛋白酶水解 的几率， 从而延长 GLP-2类似物在体内循环的时间。
在另一方面， 本发明涉及包含与载体混合的本发明所述的 GLP-2类似物或 其盐或它们的衍生物的组合物。在优选实施方案中，所述组合物为可药用组合物， 所述载体为药学上可接受的或者药用载体。 本发明所述的 GLP-2肽类似物可是 本发明所述的 GLP-2类似物的可药用酸加成盐。
在另一方面中， 本发明涉及用于治疗的本发明所述的 GLP-2类似物或其盐。 在另一方面中， 本发明涉及 GLP-2类似物或其盐或它们的衍生物用于制备 治疗和 /或预防胃肠相关病症的药物中的用途， 比如治疗患有肠功能受损、 骨质 疏松症或 DPP-IV (二肽基肽酶 -IV)介导的病症的新生儿。 例如， 所述胃和肠相关 病症选自消化道溃疡、 胃炎、 吸收不良综合征、 短肠综合征、 回盲瓣综合征、 炎 性肠病、腹型斯泼卢腹泻、热带型斯泼卢腹泻、低丙种球蛋白血症型斯泼卢腹泻、 肠炎、 局限性肠炎、 溃疡性结肠炎、 腹泻相关的肠易激综合征、 小肠损伤或短肠 综合征。
可用本发明所述的 GLP-2类似物治疗或可使用 GLP-2类似物进行预防或治 疗的其它病症包括放射性胃肠炎、传染性胃肠炎或感染后胃肠炎以及由于毒剂或 其它化学诊断治疗剂引起的小肠损伤。这可能需要与化学疗法或放射疗法的药物 同时或在后施用 GLP-2类似物， 以降低化学疗法或放射疗法的副作用例如腹泻、 腹部痛性痉挛或呕吐，以及降低由化学疗法或放射疗法引起的肠上皮结构和功能 损伤。
本发明还涉及一种治疗药盒， 其包含癌症化学疗法药物、 本发明所述的 GLP-2类似物以及使用说明书。
特别是， 在治疗药盒中， 其包含作为治疗剂的癌症化学疗法药物、本发明所 述的 GLP-2类似物以及使用说明书， 并且每一种所述的药物或者类似物均任选 的与可药用载体组合。所述治疗剂可以分开包装以分别使用， 或可以在同一组合 物中提供。
因此， 本发明还涉及包含与可药用载体组合的癌症化学疗法药物和本发明 GLP-2类似物的药物组合物。
胃肠粘膜瘤形成风险提高的患者可能期望选择化合物以降低副作用或消除 副作用的风险。例如， 选择用于治疗患有结肠瘤形成， 或预防有发生结肠瘤形成 风险的患者发生结肠瘤形成药物时，选择对小肠的选择性高于结肠的药物比非选 择性药物或对结肠的选择性高于小肠的药物可能更合适。
在其他方面中， 本发明还涉及 GLP-2类似物在用于制备治疗和 /或预防营养 不良的药物中的用途。
在另一方面中， 本发明涉及通过使用治疗有效量的本发明所述的 GLP-2类 似物或其盐或它们的衍生物在用于治疗患者胃肠相关病症的药物中的用途。
在另一方面中，本发明涉及治疗或预防有此需要的患者中化学疗法或放射疗 法副作用的方法， 所述方法包括使用用治疗有效量的本发明所述的 GLP-2类似 物或其盐或它们的衍生物。
在另一方面中， 本发明提供治疗和或预防有此需要的患者中营养不良的方 法， 所述方法包括使用治疗有效量的本发明定义的 GLP-2类似物或其盐或它们 的衍生物。
采用本领域技术人员公知的固相合成技术合成本发明所述的 GLP-2类似物 肽，其基本原理是： 先将所要合成肽链的羟末端氨基酸的羟基以共价键的结构同 一个不溶性的高分子树脂（固相载体）相连， 然后以此结合在固相载体上的氨基 酸作为氨基组份经过脱去氨基保护基并同过量的活化羧基组分反应， 接长肽链。 重复（缩合→洗涤→去保护→洗涤→下一轮缩合）操作， 达到所要合成的肽链长 度， 最后将肽链从树脂上裂解下来， 经过纯化等处理， 即得所要的多肽。 其中缩 合和去保护反应步骤的中间控制采取的是茚三酮检测的方法，即当树脂肽链上有 游离的氨基时， 经茚三酮试剂检测会显蓝色， 没有游离氨基时不显色（茚三酮试 剂本身为黄色）。因此在进行缩合反应完毕后，通过茚三酮检测，如果显黄色（茚 三酮试剂本身的颜色）， 则说明本步偶合完毕可以进行下一个氨基酸的偶合前的 脱保护操作， 如果显蓝色， 则证明肽链上还有些游离氨基， 需进一步的重复偶合 或改变现有的缩合剂直至树脂肽经茚三酮检测为黄色。
本发明中涉及的 GLP-2类似物的激动活性的检测原理是 GLP-2R是与 Gs蛋 白偶联的受体， 当受体与激动剂结合会导致细胞内 cAMP 浓度升高。 本实验在 HEK293细胞中共转染 GLP-2R和 cAMP反应元件调控的荧光素酶报告基因质 粒， 当化合物与受体结合并激活受体时， 荧光素酶表达就会增加。通过对荧光素 酶活性的检测即可获知化合物对 GLP-2R的激活状况。 具体实施方式
为了更详细地说明本发明，给出下列实施例。但本发明的保护范围并非限定 于此，本领域技术人员可以根据本发明提供的技术方案结合现有技术的方法， 替 换相关的氨基酸完成本发明， 得到通式 I限定出来的衍生物。
实施例 1 阳性化合物 Teduglutide的固相合成
阳性化合物 Teduglutide的序列为：
H-His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-As n-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-A sp-OH
( 1 ) 固相合成树脂的干燥及溶胀
称量真空干燥 24 小时的 Fmoc-Asp(OtBu)-Wang 树脂 （0.5mmol/g) 40g pOmmol)置于 2L鼓泡瓶中， 加入 400mL DMF溶胀树脂 30分钟， 抽掉 DMF溶 液；
(2) Fmoc-Asp(OtBu)-Wang树脂脱除 Fmoc保护基
向装有 Fmoc-Asp(OtBu)-Wang树脂的鼓泡瓶中加入 200mL 20%哌啶 /DMF 溶液， 反应 5分钟后抽出， 再加入 200mL 20%哌啶 /DMF溶液室温反应 20分钟。 反应结束后用 DMF 200mL洗涤树脂四次。
(3) Teduglutide的固相合成
①缩合 Fmoc-Thr(tBu)-OH
称量 50mmol Fmoc-Thr(tBu)-OH， 加入 125mL 0.4M HOBt/DMF溶解， 再加 入 150mL 0.4M DIC/DCM室温下搅拌反应 10分钟； 将上述溶液加入到树脂中， 室温下通入 N2反应 4小时。反应结束后抽掉反应液，依次用 DMF， IPA和 DMF 洗涤树脂。
②肽链的延长
按照 Teduglutide 中氨基酸从羧基端（C-端）到氨基端（N-端 )的顺序 (H-His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-As n-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-A sp-OH), 氨基酸和缩合试剂的用量和 FmOC-Thr(tBU OH相同， 保护氨基酸分 别是 Fmoc-Thr(tBu)-OH、 Fmoc-Ile-OH、 Fmoc-Lys(Boc)-OH、 Fmoc-Gln(Trt)-OH Fmoc-Leu-OH、 Fmoc-Trp(Boc)-OH、 Fmoc-Asn(Trt)-OH、 Fmoc-Phe-OH、 Fmoc-Asp(OtBu)-OH Fmoc-Arg(Pbf)-OH Fmoc-Ala-OH、 Fmoc-Glu(OtBu)-OH Fmoc-Met-OH、 Fmoc-Ser(tBu)-OH Fmoc-Gly-OH和 Fmoc-His(Trt)-OH， 重复缩 合 和 脱 保 护 两 步 反 应 ， 合 成 His(Trt)-Ala-Asp(OtBu)-Gly-Ser(tBu)-Phe-Ser(tBu)-Asp(OtBu)-Glu(OtBu)-Met-Asn (Trt)-Thr(tBu)-Ile-Leu-Glu(OtBu)-Asn(Trt)-Leu-Ala-Ala-Arg(Pbf)-Asp(OtBu)-Phe-Il e-Asn(Trt)-Trp(Boc)-Leu-Ile-Gln(Trt)-Thr(tBu)-Lys(Boc)-Ile-Thr(tBu)-Asp(OtBu)-W ang树脂。
③ Teduglutide树脂肽的后处理
将②所得到的树脂肽依次用 DMF， IPA和 DMF洗涤树脂， 用无水乙醚洗涤 两次后真空干燥， 得树脂肽。 ( 4 ) Teduglutide粗品肽的制备
取干燥后的树脂肽， 加入新鲜配制的 lOmL/g 树脂肽的 TFA:乙二硫醇 EDT):TIS:水 =92.5:2.5:2.5:2.51；体积比)的裂解液， 室温下反应 4小时。 反应结束 后过滤， 用 TFA洗涤树脂二次， 收集并合并滤液， 旋转蒸发至原体积的 1/3， 加 入大量冰无水乙醚析出 Teduglutide, 离心后真空干燥得白色粗品。
(5) Teduglutide的反相液相色谱制备
取粗品肽 5g溶于一定量的水中， 经 0.45μηι膜过滤后用反相高效液相色谱 (RP-HPLC)进行分离， 流动相为 A 0.1%TFA/H2O， B 0.1%TFA/乙腈。 其中， 色 谱柱为 Denali C-18柱 (；粒径 8.3μηι， 5 <30cm)， 柱温 45度， 检测波长 220nm， 流 速 120mL/分钟。 收集产物峰， 减压浓缩除去大部分乙腈后冻干得 Teduglutide成 品 1.0g， 纯度 98.5%， 收率 20.0%。
实施例 2 化合物 1的固相合成
化合物 1的序列为：
D-His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-As n-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-A sp-OH, 即在通式 (I)中， XI是 D-His， X2是 Gly， X3是 ASP， X10是 Met，
Xll是 Asn， X15是 Asp， X16是 Asn， X24是 Asn， X25是 Trp。
制备方法同实施例 1。
实施例 3 化合物 2的固相合成
化合物 2的序列为：
D-His-Gly-Glu-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn -Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-As p-OH, 即在通式 (I)中， XI是 D-His， X2是 Gly， X3是 Glu， X10是 Met, Xl l 是 Asn， X15是 Asp， X16是 Asn， X24是 Asn， X25是 Trp。
制备方法同实施例 1。
实施例 4 化合物 3的固相合成
化合物 3的序列为：
H-D-His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Glu-Asn-Leu-Ala -Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-OH, 即在通式 (I) 中， XI是 D-His， X2是 Ala， X3是 Asp， X10是 Met， Xl l是 Asn， X15是 Gh；， X16是 Asn， X24是 Asn， X25是 Trp。
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S80 .0/0T0ZN3/X3d 91^660/ΟΐΟΖ OAV 实施例 10化合物 9的固相合成
化合物 9的序列为：
H-D-His-Gly-Glu-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Glu-Asn-Leu-Ala- Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-OH， 即在通式（I) 中， XI是 D-His， X2是 Gly， X3是 Gh；， X10是 Met， XII是 Asn， X15是 Glu， X16是 Asn， X24是 Asn， X25是 Trp。
制备方法同实施例 1。
实施例 11 化合物 10的固相合成
化合物 10的序列为：
H-His-Gly-Asp*(Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Glu-Asn-Leu- Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-OH， 即在通式 (I)中， XI是 His， X2是 Gly， X3是 Asp， X10是 Met， XI I是 Asn， X15是 Gh；， X16是 Asn， X24是 Asn， X25是 Trp。 其中 X3处 Asp* : Fmoc-Asp-OtBuo 制备方法同实施例 1。
实施例 12对 GLP-2受体（GLP-2R) 的激动活性检测
GLP-2R是与 Gs蛋白偶联的受体，当受体与激动剂结合会导致细胞内 cAMP 浓度升高。本实施例在 HEK293细胞中共转染 GLP-2R和 cAMP反应元件调控的 荧光素酶报告基因质粒， 当 GLP-2类似物与受体结合并激活该受体时， 荧光素 酶表达就会增加。通过对荧光素酶活性的检测即可获得 GLP-2类似物对 GLP-2R 的激活状况。 待测各 GLP-2类似物的相关信息如下表 1所示： 表 1进行 GLP-2R激动活性检测的各 GLP-2类似物的分子量、 质量及终浓 度
Figure imgf000012_0001
用电转方法将 GLP-2R和 pCRE-Luc表达质粒导入 HEK293细胞， 转染 后的细胞以 4万个 /孔 /ΙΟΟμΙ的密度种入 96孔板， 在 37oC孵育 24小时。 加 入一定浓度梯度的本发明所述 GLP-2类似物（每个浓度为 3复孔）或阳性对 照 GLP-2, 在 37oC孵育 5小时。 溶剂 DMSO为阴性对照。 每孔取出 50μ1 培养基， 加入 50μ1荧光素酶底物， 振荡 10分钟。 取出 80μ1反应液加入到白 色的 96孔板中， 在 Invision酶标仪上检测。 所述 GLP-2类似物的 EC50值、 95 %可信限和最大反应率如下表 2所示。
表 2各 GLP-2类似物 EC50、 95 %可信限和最大反应率
Figure imgf000013_0001
其中， 化合物 1、 2均显示了较好的 GLP受体激动活性。

Claims

权利要求书：
1、 一种以下通式 （ I ) 代表的胰高血糖素样肽 -2 类似物， 及其可药用盐及它们 的衍生物， 该衍生物包括经长链脂肪酸和 /或聚乙二醇和 /或其他长效化修饰所产生的 胰高血糖素样肽 -2 类似物的衍生物， 或胰高血糖素样肽 -2 类似物的可药用盐的衍生 物：
1-Z1-X1 -X2-X3-Gly-Ser-Phe-Ser-Asp-Glu-X 10-X11 -Thr-Ile-Leu-X 15-X16-Leu-Al a-Ala-Arg-Asp-Phe-Ile-X24-X25-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-(Z2)- 2 ( I )， 其中 Zl、 Z2、 Rl或 R2为不存在， 或为天然或非天然氨基酸， 或为由天然和 /或 非天然氨基酸组成的具有 2至 33个氨基酸单位的肽序列， 而且 XI、 X2、 X3、 X10、 XI I、 X15、 X16、 X24或 X25为天然或非天然氨基酸。
2、 根据权利要求 1所述的胰高血糖素样肽 -2类似物， 其中 XI是 His或 D-His。
3、 根据权利要求 1或 2所述的胰高血糖素样肽 -2类似物， 其中 X2是 D-Ala或
Gly。
4、 根据权利要求 1或 2所述的胰高血糖素样肽 -2类似物， 其中 X10是 Nle或
Met。
5、根据权利要求 1或 2所述的胰高血糖素样肽 -2类似物，其中 X15是 Glu或 Asp。
6、根据权利要求 1或 2所述的胰高血糖素样肽 2类似物，其中 X3是 Glu或 Asp， 并且 X3以 α羧基或者 β羧基与 Gly形成肽键。
7、 根据权利要求 1所述的胰高血糖素样肽 -2类似物， 其具有下列序列：
D-His-D-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Nle-Asn-Thr-Ile-Leu-Glu-Asn-Leu-Ala-
Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp。
8、 根据权利要求 1所述的胰高血糖素样肽 -2类似物， 其具有下列序列：
His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Glu-Asn-Leu-Ala-Ala-
Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp。
9、 根据权利要求 1所述的胰高血糖素样肽 -2类似物， 其具有下列序列：
His-Gly-Glu-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Glu-Asn-Leu-Ala-Ala- Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln- Thr-Lys-Ile-Thr-Asp。
10、 根据权利要求 1所述的胰高血糖素样肽 -2类似物， 其具有下列序列： D-His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Al a-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp。
11、 根据权利要求 1所述的胰高血糖素样肽 -2类似物， 其具有下列序列： D-His-Gly-Glu-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Al a-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys- Ile-Thr-Asp。
12、 一种权利要求 1所述的胰高血糖素样肽 -2类似物的制备方法， 其特征在于该 胰高血糖素样肽 -2类似物采用固相合成法制备并用反相液相色谱法纯化。
13、一种包括治疗有效剂量的权利要求 1所述的胰高血糖素样肽 -2类似物的药物 组合物， 其包括药学上可接受的载体和 /或癌症化学疗法药物。
14、 根据权利要求 13所述的药物组合物， 其剂型为注射剂或者缓释剂。
15、一种如权利要求 13或 14所述的药物组合物在制备用于预防和 /或治疗胃肠粘 膜损伤相关病症的药物中的用途， 其中胃肠粘膜损伤相关病症选自消化道溃疡、 吸收 不良综合征、 短肠综合征、 回盲瓣综合征、 炎性肠病、 腹型斯泼卢腹泻、 热带型斯泼 卢腹泻、 低丙种球蛋白血症型斯泼卢腹泻、 肠炎、 局限性肠炎、 溃疡性结肠炎、 胃肠 粘膜损伤导致的骨质疏松症和 /或营养不良症、 化学疗法或放射疗法的胃肠道副作用， 优选腹泻、 腹部痛性痉挛、 呕吐， 放射性胃肠炎、 传染性胃肠炎或感染后胃肠炎、 毒 剂或者化学治疗剂引起的胃肠粘膜损伤， 或二肽基肽酶 -IV介导的病症。
16、一种如权利要求 1所述的胰高血糖素样肽 2类似物在制备用于预防和 /或治疗 胃肠粘膜损伤相关病症药物中的用途， 其中胃肠粘膜损伤相关病症选自消化道溃疡、 吸收不良综合征、 短肠综合征、 回盲瓣综合征、 炎性肠病、 腹型斯泼卢腹泻、 热带型 斯泼卢腹泻、 低丙种球蛋白血症型斯泼卢腹泻、 肠炎、 局限性肠炎、 溃疡性结肠炎、 胃肠粘膜损伤导致的骨质疏松症和 /或营养不良症、化学疗法或放射疗法的胃肠道副作 用， 优选腹泻、腹部痛性痉挛、 呕吐， 放射性胃肠炎、传染性胃肠炎或感染后胃肠炎、 毒剂或者化学治疗剂引起的胃肠粘膜损伤， 或二肽基肽酶 -IV介导的病症。
17、 一种治疗药盒， 其包含癌症化学疗法药物、 权利要求 1所述的胰高血糖素样 肽 -2类似物以及使用说明书。
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