WO2016090628A1 - Analogues d'oxyntomoduline (oxm), leur synthèse et utilisation - Google Patents

Analogues d'oxyntomoduline (oxm), leur synthèse et utilisation Download PDF

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WO2016090628A1
WO2016090628A1 PCT/CN2014/093681 CN2014093681W WO2016090628A1 WO 2016090628 A1 WO2016090628 A1 WO 2016090628A1 CN 2014093681 W CN2014093681 W CN 2014093681W WO 2016090628 A1 WO2016090628 A1 WO 2016090628A1
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ser
gly
lys
pro
ala
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PCT/CN2014/093681
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Chinese (zh)
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马涛
许树森
张爱红
邱红娟
车美英
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北京韩美药品有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to oxyntomodulin (OXM) analogs, their synthesis and their use.
  • OXM oxyntomodulin
  • the present invention relates to oxyntomodulin analogs and pharmaceutical compositions thereof, and to methods and uses thereof as effective drug candidates for clinical treatment of metabolic disorders such as obesity diseases.
  • Roux-en-Y gastric bypass produced better weight loss than other procedures.
  • RYGB Roux-en-Y gastric bypass
  • intestinal hormones include Peptide YY, glucagon-like peptide-1 (GLP-1) and oxyntomodulin (OXM).
  • GLP-1 has been the most studied as a new drug development target.
  • GLP-1 is a 30 or 31 amino acid polypeptide secreted by small intestinal L cells. It binds to the GLP-1 receptor in the islets to produce an incretin effect, which promotes insulin release in a glucose-dependent manner (Kreymann, et al. Lancet, 1987, 2, 1300-4). GLP-1 also inhibits glucagon secretion, slows gastric emptying and reduces food intake Ingestion (Larsen. Diabetes, 2001, 50, 2530-9; Turton. Nature, 1996, 379, 69-72; Tang-Christensen, et al. American Journal of Physiology, 1996, 271, R848-56).
  • GLP-1 may be the result of its direct interaction with the CNS, not just by slowing gastric emptying (Abbott, et al. Brain Research, 2005, 1044, 127–31).
  • GLP-1's incretin action and satiety make it the preferred target for the development of anti-diabetic and anti-obesity drugs.
  • Natural GLP-1 is rapidly degraded in vivo by the DPP-IV enzyme and therefore cannot be directly used as a drug in clinical applications (Deacon, et al. Journal of Clinical Endocrinology & Metabolism, 1995, 80, 952-957).
  • Oxytoxin is a 37 amino acid polypeptide whose sequence includes all 29 amino acids of glucagon and is called IP-1 (intervening peptide–1) at the C-terminus. Amino acid elongation (Bataille, et al. Peptides, 1981, 2, Supplement 2, 41-44). OXM is rapidly secreted after a meal, and the main physiological effects include reducing gastric acid secretion, reducing pancreatic exocrine and delaying gastric emptying (Schjoldager, et al. European Journal of Clinical Investigation, 1988, 18, 5, 499-503). In addition, OXM has the effect of reducing food intake and increasing energy consumption.
  • OXM has been shown to activate both the GLP-1 receptor and the glucagon receptor. Its inhibitory effect on food intake is likely to be achieved by binding to the GLP-1 receptor. OXM did not cause appetite suppression in GLP-1 receptor knockout mice, but was not affected in glucagon receptor knockout mice. However, there is also evidence that the physiological role of OXM may not be entirely dependent on the GLP-1 receptor. For example, OXM has a 50-fold lower affinity for GLP-1 receptor than native GLP-1, but the same molar amount of OXM and GLP-1 can produce similar food uptake inhibition (Darkin, et al. Endocrinology, 2001, 142, 10, 4244 - 4250).
  • OXM in the ventricles of rodents or direct injection of the polypeptide into the hypothalamus can reduce food intake in animals (Dakin, et al. American Journal of Physiology - Endocrinology and Metabolism, 2002, 283, 6, E1173 - E1177). Dakin et al. found that intraperitoneal injections and repeated intraventricular injections of OXM twice daily for seven consecutive days reduced body weight gain and reduced obesity. Animals administered OXM had more body weight loss than the blank group at the same food intake, and their basal body temperature and heart rate also increased. This suggests that OXM can increase energy expenditure (Dakin, et al. Endocrinology, 2004, 145, 6, 2687-2695; American Journal of Physiology - Endocrinology and Metabolism, 2002, 283, 6, E1173 - E1177).
  • OXM ulcerative colitis
  • glucagon The association of OXM activation of glucagon receptor with its weight loss activity has not been well studied.
  • the main pharmacological activity of glucagon is to promote glycogenolysis and gluconeogenesis in the state of hypoglycemia (Exton. Advances in Enzyme Regulation, 1968, 6, 391–407), so its clinical application is limited to emergency treatment of insulin injection.
  • Glucagon also has the effect of increasing lipid breakdown, increasing satiety, increasing heat production and energy expenditure (Habegger, et al. Nature Reviews Endocrinology, 2010, 6, 689-697).
  • Glucagon increases satiety and promotes energy expenditure, making it a potential target for obesity treatment, but its role in raising blood sugar and accelerating insulin resistance limits its application.
  • dual agonists with similar activation at the GLP-1 receptor and glucagon receptor are more effective than GLP-1 receptor agonists in controlling body weight, adipose tissue content, and glucose homeostasis. Have better activity. Dual agonists can also significantly increase energy expenditure and promote lipid metabolism. In particular, continuous use of this dual agonist did not result in an increase in blood glucose produced by glucagon. One reason for this may be that activation of the GLP-1 receptor counteracts the glycemic effect caused by the glucagon receptor (Day, et al. Nature Chemical Biology, 2009, 5, 749-757).
  • OXM and other GLP-1/glucagon receptor dual agonists offer a new direction in the development of anti-obesity and metabolic disease drugs.
  • Novel peptide drugs can provide finer regulation of metabolism in the body, which may have better activity and fewer side effects.
  • a first aspect of the invention relates to an OXM analog derived from a native OXM sequence having enhanced GlP-1 receptor agonistic activity and GCG receptor agonistic activity and having the amino acid sequence of Formula I below:
  • A2 is selected from the group consisting of Ala, Gly, sarcosine, Aib, d-Ala, and d-Ser;
  • A16 is selected from the group consisting of Ser, Gln, Glu, Asp, Asn, and Lys;
  • A17 is selected from the group consisting of Arg, Asn, Asp, Lys, Lys-Z1, Glu, and Gln;
  • A18 is selected from the group consisting of Arg, Ala, Aib, and N-methyl Ala;
  • A19 is selected from the group consisting of Ala and Aib;
  • A20 is selected from the group consisting of Gln, Glu, Lys, and Lys-Z2;
  • A21 is selected from the group consisting of Glu and Asp;
  • A23 is selected from the group consisting of Val and Ile;
  • A24 is selected from the group consisting of Gln, Glu, Ala, Lys-Z3, and Cys-Z4;
  • A27 is selected from the group consisting of Met, Leu, and Lys-Z5;
  • A28 is selected from the group consisting of Asn, Ala, and Lys-Z6;
  • A29 is selected from the group consisting of Thr and Gly;
  • Y is selected from Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-A38, Gly-Pro-Ser-Ser-Pro-Pro-Pro-Ser-A38 and Lys-Arg-Asn-Arg-Asn-Asn a group consisting of -Ile-Ala-A38;
  • A38 is selected from the group consisting of (Lys)n-Z7, Cys-Z8, (Glu) m- Z9 and deletions;
  • Z1 to Z9 are independently selected from the group consisting of a bridge-PEG, a bridge-biotin, and a bridge-fatty acid;
  • n is an integer selected from 1 to 6, ie 1, 2, 3, 4, 5 or 6;
  • n is an integer selected from 0 to 3, ie 0, 1, 2 or 3;
  • a linker is a peptide having 0-5 amino acid residues, ie 0, 1, 2, 3, 4 or 5 amino acid residues;
  • Y is Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-A38;
  • A38 is selected from the group consisting of (Lys)n-Z7, Cys-Z8, (Glu) m- Z9 and deletions;
  • Z7 to Z9 are independently selected from the group consisting of -(Glu) a -PEG, -(Glu) b -biotin and -(Glu) c -fatty acid and deletion;
  • n is an integer selected from 1 to 6, ie 1, 2, 3, 4, 5 or 6;
  • n is an integer selected from 0 to 3, ie 0, 1, 2 or 3;
  • a, b and c are independently selected from integers from 0 to 5, ie 1, 2, 3, 4 or 5.
  • Y is Gly-Pro-Ser-Ser-Pro-Pro-Pro-Ser-A38;
  • A38 is selected from the group consisting of -(Lys) n -Z7 and -Cys-Z8 and a deletion;
  • Z7 or Z8 is independently selected from the group consisting of -(Glu) a -PEG, -(Glu) b -biotin, -(Glu) c -fatty acid, and deletion;
  • n is an integer selected from 1 to 6, ie 1, 2, 3, 4, 5 or 6;
  • n is an integer selected from 0 to 3, ie 0, 1, 2 or 3;
  • a, b and c are independently selected from integers from 0 to 5, ie 1, 2, 3, 4 or 5.
  • Y is Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala-A38;
  • A38 is selected from the group consisting of -(Lys) n -Z7, -Cys-Z8 and deletions;
  • Z7 or Z8 is independently selected from the group consisting of -(Glu) a -PEG, -(Glu) b -biotin, -(Glu) c -fatty acid, and deletion;
  • n is an integer selected from 1 to 6, ie 1, 2, 3, 4, 5 or 6;
  • n is an integer selected from 0 to 3, ie 0, 1, 2 or 3;
  • a, b and c are independently selected from integers from 0 to 5, ie 1, 2, 3, 4 or 5.
  • A2 is Aib.
  • A24 is Cys-Z4
  • Z4 is -(Glu) a -PEG
  • a is an integer selected from 0 to 3, that is, 0, 1, 2 or 3.
  • A38 is selected from (Lys) n -Z7;
  • Z7 is selected from the group consisting of -(Glu) a -PEG, -(Glu) b -biotin, -(Glu) c -fatty acid, and deletion;
  • n is an integer selected from 1 to 6, ie 1, 2, 3, 4, 5 or 6;
  • n is an integer selected from 0 to 3, ie 0, 1, 2 or 3;
  • a, b and c are independently selected from integers from 0 to 5, ie 1, 2, 3, 4 or 5.
  • Z1 to Z9 are independently selected from the group consisting of (Glu) a -PEG and (Glu) c -fatty acid;
  • a or c is independently selected from an integer from 0 to 2, ie 0, 1 or 2, for example Glu is ⁇ -Glu.
  • Z1 to Z9 are (Glu) a -PEG; a is an integer selected from 0 to 2, for example, Glu is ⁇ -Glu.
  • Z1 to Z9 are -(Glu) c -fatty acids
  • c is an integer selected from 0 to 2, that is, 0, 1, or 2, for example, Glu is ⁇ -Glu.
  • the fatty acid in Formula I, can be selected from the group consisting of myristic acid, palmitic acid, stearic acid, and cholic acid.
  • the molecular weight of the PEG can range from 5 kDa to 40 kDa, such as 20 kDa, 30 kDa, or 40 kDa.
  • the OXM analog or a pharmaceutically acceptable salt thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 (analog 034), SEQ ID NO: 2 (analog 044), SEQ ID NO: 3 (analog 045), SEQ ID NO: 4 (analog 046), SEQ ID NO: 5 (analog 051), SEQ ID NO: 6 (analog 052), SEQ ID NO: 7 (analog 053) SEQ ID NO: 8 (analog 054), SEQ ID NO: 9 (analog 058), SEQ ID NO: 10 (analog 060), SEQ ID NO: 11 (analog 067), SEQ ID NO: 12 (analog 068), SEQ ID NO: 13 (analog 069), SEQ ID NO: 14 (analog 070), SEQ ID NO: 15 (analog 072), SEQ ID NO: 16 (analog 073), SEQ ID NO: 17 (analog 074),
  • the OXM analog or a pharmaceutically acceptable salt thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 (analog 034), SEQ ID NO: 2 (analog 044), SEQ ID NO : 4 (analog 046), SEQ ID NO: 5 (analog 051), SEQ ID NO: 6 (analog 052), SEQ ID NO: 7 (analog 053), SEQ ID NO: 9 (analog 058) ), SEQ ID NO: 11 (analog 067), SEQ ID NO: 13 (analog 069), SEQ ID NO: 15 (analog 072), SEQ ID NO: 17 (analog 074), SEQ ID NO: a group consisting of 19 (analog 082), SEQ ID NO: 21 (analog 084), or SEQ ID NO: 23 (analog 100).
  • amino acid sequence encompassed by the OXM analog or a pharmaceutically acceptable salt thereof is SEQ ID NO: 1 (analog 034), SEQ ID NO: 5 (analog 051), SEQ ID NO : 9 (analog 058), SEQ ID NO: 13 (analog 069), SEQ ID NO: 21 (analog 084) or SEQ ID NO: 23 (analog 100).
  • a second aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of an OXM analog as described above, a pharmaceutically acceptable diluent, carrier or excipient, and optionally an anti-diabetic agent
  • the anti-diabetic agent is selected from the group consisting of insulins, biguanides, sulfonylureas, rosiglitazone or pioglitazone, alpha-glucosidase inhibitors, and aminodipeptidase IV inhibitors.
  • the pharmaceutical composition is in the form of an injection or lyophilized powder.
  • an OXM analog as described above, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described above is used to treat a metabolic disease.
  • the OXM analog, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described above for use in the treatment of a metabolic disease, wherein the metabolic disease is selected from the group consisting of diabetes, obesity, and bone A group consisting of looseness.
  • a third aspect of the invention relates to an OXM analog as described above, or a pharmaceutically acceptable salt thereof, or as described above Use of a pharmaceutical composition for the preparation of a medicament for the treatment of a metabolic disease.
  • the metabolic disease is selected from the group consisting of diabetes, obesity, and osteoporosis.
  • a fourth aspect of the invention relates to a method of treating and/or preventing a metabolic disease, comprising administering to a subject in need thereof an effective amount of an OXM analog as described above or a pharmaceutically acceptable salt thereof or A pharmaceutical composition as described above.
  • a fifth aspect of the invention relates to a method for simultaneously activating GCG and GLP-1 receptors in vivo and/or in vitro comprising administering an OXM analog as described above or a pharmaceutically acceptable salt thereof or as described above Pharmaceutical composition.
  • Figure 1 shows an in vitro activity screening assay for analog 034 on CHO cells overexpressing the human GLP-1 receptor.
  • Figure 2 shows in vitro activity screening assay for analog 034 on CHO cells overexpressing human GCG receptor.
  • Figure 3 shows in vitro activity screening assay for analog 069 on CHO cells overexpressing the human GLP-1 receptor.
  • Figure 4 shows in vitro activity screening assay for analog 069 on CHO cells overexpressing the human GCG receptor.
  • the OXM analogs referred to in the present invention are engineered based on the native OXM sequence (1 - 37, SEQ ID NO: 32 (analog 013)) and are characterized by the glucagon receptor and GLP - The agonistic activity of the 1 receptor was significantly enhanced compared to the native OXM.
  • the invention also provides pharmaceutical methods for treating or preventing a metabolic disease or condition using an OXM analog, which is primarily referred to as obesity and diabetes.
  • OXM agonist activity of glucagon receptor
  • EC 50 0.7759nM
  • EC glucagon 50 0.0905 nM
  • OXM is roughly equivalent to glucagon's agonistic activity at the GLP-1 receptor, which is only one percent of native GLP-1 (Riber, et al. WO 2008152403, 2008, June, 16).
  • a novel OXM analog can be constructed by modifying the OXM (1-37) sequence by a hybridization concept, ie, a specific site.
  • the substitution of Ser with the non-natural amino acid Aib can achieve stability of DPP-IV metabolism; the amino acid residue Glu with a negative charge in the side chain is substituted at the 16th and 21st positions, respectively.
  • the amino acid residue Lys with a positive charge in the chain is substituted at positions 17 and 20, respectively, to give analogs such as analogs 060, 067, 068, 069, 070, 082 and 083). Modification of the above sites results in a mid-segment of the above analog polypeptide sequence having two salt bridges (between 16 and 20 positions, and between positions 17 and 21, respectively) to immobilize the a-helix configuration.
  • Fatty acids and hydrophilic macromolecules are linked to the polypeptide chain by covalent bonds. All of the above measures can significantly prolong the pharmacokinetic properties of the compound in vivo, such as the analogs 060, 068, 070, 083 and 085.
  • pharmaceutically acceptable carrier includes any standard pharmaceutical carrier, such as a physiological saline buffer of phosphate, water, an emulsion such as an oil/water or water/oil emulsion, and various humectants.
  • pharmaceutically acceptable salt refers to salts of the compounds which retain the biological activity of the parent, and those which are not biologically active or otherwise different forms of the compound. Many of the compounds described herein are capable of forming salts of acids and/or bases via amino and/or carboxyl groups or other similar groups.
  • Salts derived from inorganic bases include only the examples, sodium, potassium, lithium, ammonium, calcium and magnesium salts.
  • Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines.
  • Salts derived from inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like.
  • Salts derived from organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, hydroxysuccinic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid. , mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and other similar acids.
  • treatment includes the prevention of a particular disorder or condition, or the alleviation of a condition associated with a particular disorder or condition, and/or the prevention or elimination of said condition.
  • an OXM analog refers to an amount that is non-toxic but that achieves the desired therapeutic effect.
  • expected therapeutic effects include weight loss or prevention of weight gain, as measured by the level of weight loss.
  • the "effective" dose will vary with the age and condition of the individual, the management mode, and the like. Therefore, it is unlikely that an accurate “effective dose” will be determined. However, a moderate “effective” dose for each person may need to be determined by routine experimentation.
  • purified refers to the separation of an impurity or a molecule or compound in its free form, which is usually associated with a natural molecule or compound. However, the term “purification” as used herein does not only include purity, but also relative meaning.
  • purified polypeptide refers to polypeptides isolated from other compounds, including, but not limited to, nucleic acid molecules, liposomes, and carbohydrates.
  • isolated refers to the isolation of the material from its original environment.
  • a natural polynucleotide represents a living organism but is not isolated, but the same polynucleotide, from some coexisting organisms. Being picked out is separation.
  • peptide refers to a sequence of three or more, but less than 50 amino acids, which may be natural or non-natural.
  • a non-natural amino acid refers to an amino acid that is not found in vivo, but may be included in the sequences described herein.
  • the "OXM analog” referred to herein is the amino acid sequence including the analog 013 (SEQ ID NO: 32). Or any peptide of any of the analogous amino acid sequences of SEQ ID NOS: 1 to 31, which includes substitution, addition, deletion or modification of an amino acid (eg, methylation, acetylation, alkylation, panthromination, molecule Internal covalent bonds: such as formation of lactam bridges, polyethylene glycol modifications, etc.), wherein these analogs are capable of stimulating the glucagon receptor and/or the GLP-1 receptor, as described herein for cAMP in vitro activity screening test.
  • modification refers to a substitution, addition or deletion of an amino acid, including substitution or addition of any of the 20 natural amino acids.
  • any of the specific amino acid positions mentioned e.g., position 24 refers to an amino acid at a specific position in the native OXM (analog 013) or any of the analogs.
  • native GLP-1 refers to a polypeptide comprising human GLP-1 (7-36 or 7-37) sequences
  • native OXM refers to a polypeptide of human OXM sequence (1-37).
  • GLP-1 or OXM refers to native GLP-1 or native OXM, respectively.
  • substitution of an amino acid means that one amino acid residue is replaced by another amino acid residue.
  • polyethylene glycol or “PEG” refers to a polymer of ethylene oxide and water, in the form of a straight or branched chain, of the formula H(OCH 2 CH 2 ) n OH,n The minimum is equal to 9. Unless otherwise stated, the term refers to compounds having an average total molecular weight of polyethylene glycol of between 5,000 and 40,000 Daltons. "Polyethylene glycol” or “PEG” is used with a numerical suffix to indicate the average molecular weight. For example, PEG-5000 means that the average molecular weight of polyethylene glycol is 5000 Daltons.
  • polyethylene glycol modification refers to the modification of the original site of a polypeptide by attachment of polyethylene glycol.
  • a "peptide modified by polyethylene glycol” refers to a polypeptide that is covalently bonded to polyethylene glycol.
  • a peptide as referred to herein refers to a polypeptide modified at the N-terminus and the C-terminus.
  • a natural amino acid chain is in the form of a carboxyl group at the C-terminus, and the modified peptide may be in the form of an amide.
  • linker refers to a group that links a bond, a molecule, or a link to two separate entities.
  • a bridge can optimize the spatial structure of two entities or provide a variable bond that separates two entities.
  • Variable binding bonds include photolytic groups, acid labile groups, base labile groups, and enzymatic groups.
  • dimer refers to a complex composed of two units covalently bonded by a linkage. Dimers include homodimers and hybrid dimers. A homodimer includes two identical structural units, and a hybrid dimer includes two different structural units, although the two units are very similar.
  • charged amino acid refers to an amino acid having a negative charge (ie, deprotonated) or a positive charge (ie, protonated) in the side chain within the pH range of the physiological solution.
  • negatively charged amino acids include aspartic acid, glutamic acid, cysteine, homocysteine, homologous glutamic acid, while positively charged amino acids include arginine, lysine, and Histidine.
  • Charged amino acids include the charged amino acids of the 20 natural amino acids and the unnatural amino acids.
  • acidic amino acid refers to an amino acid containing a second acidic group, including, for example, a carboxylic acid group or a sulfate group.
  • GLP-1 activity refers to native GLP-1 in the EC 50 of GLP-1 receptor with a ratio of 50 EC of this polypeptide on GLP-1 receptor.
  • Glucagon activity is the ratio of EC native glucagon at the glucagon receptor over the EC 50 of this polypeptide in the glucagon receptor 50.
  • alkyl refers to a straight or branched chain hydrocarbon containing a certain number of carbon atoms.
  • alkyl groups include methyl, ethyl and n-propyl groups.
  • heteroalkyl refers to a straight or branched chain hydrocarbon containing a number of carbon atoms, wherein the structure contains at least one heteroatom in the backbone.
  • Heteroatoms suitable for use in the present invention include, but are not limited to, N, S, O.
  • cycloalkyl refers to a cyclic hydrocarbon containing a certain number of carbon atoms, for example, cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl.
  • heterocycloalkyl refers to a cyclic hydrocarbon containing a certain number of carbon atoms and one to three heteroatoms, wherein the heteroatoms are selected from the group consisting of O, N, S.
  • the heterocyclic ring is not limited to the following groups: piperidine, tetrahydrofuran, tetrahydropyran, dihydrofuran, morpholine, thiophene and the like.
  • aryl refers to a monocyclic or polycyclic aromatic group, preferably a monocyclic or bicyclic aromatic group containing a specified number of carbon atoms, such as benzene or naphthalene. Unless otherwise specified, an aryl group can be unsubstituted or substituted.
  • Alpha-aminoisobutyric acid alpha-aminoisobutyric acid
  • sarcosine as referred to herein means an analog of glycine whose amino group is modified by a methyl group.
  • the OXM analogs of the invention can be obtained by standard polypeptide solid phase synthesis methods, recombinant DNA techniques, or any other method of preparing polypeptides and fusion proteins.
  • the OXM analogs containing non-peptide moieties of the present invention can be synthesized by standard organic synthesis reactions in addition to standard peptide synthesis methods.
  • Polypeptides were prepared by standard solid phase peptide synthesis methods using an N-terminal Fmoc-protection strategy. The assembly of the peptide chain was performed manually according to the standard Fmoc method.
  • Fmoc Rink-Amide resin 1% cross-linking degree, 100-200 mesh, degree of substitution 0.34-0.44 mmol/g commercially available from Tianjin Nankai Synthetic Technology Co., Ltd. was used as a solid phase carrier.
  • the polypeptide Upon completion of the synthesis, the polypeptide is cleaved from the solid phase polymer support and all side chains are deprotected. This can be accomplished by treatment with trifluoroacetic acid (TFA) for 2 h, while 2.5% water and 2.5% 1,2-didecylethane (EDT) are added to the trifluoroacetic acid as a scavenger for the side chain protecting group.
  • TFA trifluoroacetic acid
  • EDT 1,2-didecylethane
  • Peptide-TFA mixture from resin After filtering out, most of the TFA was removed, and cold diethyl ether was added to precipitate the polypeptide. It was centrifuged, washed with diethyl ether, and then dissolved in acetonitrile buffer.
  • the crude polypeptide was analyzed by a reverse phase HPLC analytical column.
  • the crude polypeptide was purified by semi-preparative chromatography using a Vydac C4 or C8 column (2.2 x 25 cm) using a mobile phase containing 0.1% TFA. The pure product was characterized by LC-MS and then lyophilized to obtain the target polypeptide.
  • Fmoc Rink-Amide resin 0.05 mmol was placed in a 10 mL reaction vessel, and a standard Fmoc-chemical solid phase polypeptide synthesis process was carried out according to the fitted sequence, wherein DIC/HOBt was used as a condensation reagent.
  • the polypeptide-bound resin was treated with 20% piperidine in DMF to remove the N-terminal Fmoc group, followed by the excision reagent (95% TFA, 2.5% H 2 O, 2.5% EDT). ), reaction 2h.
  • the solid resin was filtered off, and the obtained filtrate was concentrated with nitrogen.
  • the polypeptide was precipitated with cold ether and centrifuged to give a crude material.
  • the crude peptide was dissolved in acetonitrile buffer and loaded onto a semi-preparative reversed phase column. Gradient elution was carried out using an HPLC system containing acetonitrile flowing relative to Waters. The appropriate fractions were characterized by LC-MS and pooled together for lyophilization. HPLC analysis showed that the purity of the resulting product was greater than 90%, and ESI-MS showed the ion signal of the target polypeptide.
  • Fmoc Rink-Amide resin 0.05 mmol was placed in a 10 mL reaction vessel, and a standard Fmoc-chemical solid phase polypeptide synthesis process was carried out in sequence, wherein DIC/HOBt was used as a condensation reagent.
  • the polypeptide-attached resin was treated with Pd(PPh 3 ) 4 for 1 h under nitrogen to remove the Alloc/OAll protecting group, and a lactam bond was formed under the action of the coupling reagent PyBOP/DIEA (17). Between bit and 21). It was then treated with a solution containing 20% piperidine in DMF access polypeptide resin to remove the N- terminal Fmoc group removal agent was added (95% TFA, 2.5% H 2 O, 2.5% EDT), the reaction 2h. The solid resin was filtered off, and the obtained filtrate was concentrated with nitrogen. The polypeptide was precipitated with cold ether and centrifuged to give a crude material.
  • the crude peptide was dissolved in acetonitrile buffer and loaded onto a semi-preparative reversed phase column. Gradient elution was carried out using an HPLC system containing acetonitrile flowing relative to Waters. The appropriate fractions were characterized by LC-MS and pooled together for lyophilization. HPLC analysis showed that the purity of the resulting product was greater than 90%, and ESI-MS showed the ion signal of the target polypeptide.
  • the analog containing 058 containing Cys was dissolved in phosphate buffer ( ⁇ 10 mg/mL), an equivalent of maleimide-activated methoxy reagent containing methoxy reagent was added, stirred at room temperature, and analyzed by analytical HPLC. monitor. After 10-24 h of reaction, the reaction mixture was acidified, loaded onto a semi-preparative chromatograph, and purified by gradient eluting with acetonitrile-purified HPLC system. The appropriate fractions are combined and lyophilized to give the desired PEGylated polypeptide.
  • Fmoc Rink-Amide resin 0.05 mmol was placed in a 10 mL reaction vessel, and a standard Fmoc-chemical solid phase polypeptide synthesis process was carried out in sequence, wherein DIC/HOBt was used as a condensation reagent.
  • the polypeptide-attached resin was treated with Pd(PPh 3 ) 4 for 1 h under nitrogen to remove the Alloc protecting group, and the fatty acid was added to condense with the ⁇ -amino group of Lys at position 17. It was then treated with a solution containing 20% piperidine in DMF access polypeptide resin to remove the N- terminal Fmoc group removal agent was added (95% TFA, 2.5% H 2 O, 2.5% EDT), the reaction 2h. The solid resin was filtered off, and the obtained filtrate was concentrated with nitrogen. The polypeptide was precipitated with cold ether and centrifuged to give a crude material.
  • the crude peptide was dissolved in acetonitrile buffer and loaded onto a semi-preparative reversed phase column. Gradient elution was carried out using an HPLC system containing acetonitrile flowing relative to Waters. The appropriate fractions were characterized by LC-MS and pooled together for lyophilization. HPLC analysis showed that the purity of the resulting product was greater than 90%, and ESI-MS showed the ion signal of the target polypeptide.
  • the synthesized OXM analogs were all analyzed by HPLC and MS.
  • Cell culture medium ⁇ MEM (Gibco, 12561-056), plus 10% FBS (Gibco, 10099), 1 mg/mL G418 (Invitrogen, 10031035), 10 nM MTX (Sigma, M4010);
  • cAMP assay kit "cAMP Fluorescent Assay kit", Molecular Devices, R8089;
  • Ultra-clean workbench ESCO, SVE-4A1;
  • KRBG Sigma, M4892 (add 15 mM NaHCO 3 when configured);
  • Microplate oscillator TAITEC, M.BR-022UP;
  • This experiment uses a genetically engineered, highly expressed human GLP-1 receptor (hGLP-1R) or human GCG receptor (hGCGR) CHO cell line.
  • hGLP-1R highly expressed human GLP-1 receptor
  • hGCGR human GCG receptor
  • KRBG containing various concentrations of compound: 1000 nM---0, 10-fold gradient dilution
  • cell culture plate was taken out, and 50 ⁇ L of cell lysate (Molecular Devices-R7097, supplied with the kit) was added to each well, followed by shaking into a microplate shaker for 10 minutes to fully lyse the cells;
  • polypeptide sequences of the OXM analogs of the present invention are listed below.

Abstract

Cette invention concerne des analogues d'oxyntomoduline (OXM) ayant une activité d'antagoniste des récepteurs GLP-1 et une activité d'antagoniste des récepteurs GCG. Des compositions pharmaceutiques comprenant les analogues d'OXM, et leur utilisation sont en outre décrites.
PCT/CN2014/093681 2014-12-12 2014-12-12 Analogues d'oxyntomoduline (oxm), leur synthèse et utilisation WO2016090628A1 (fr)

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EP3670529A4 (fr) * 2017-08-16 2021-11-03 Dong-A St Co., Ltd. Analogue peptidique d'oxyntomoduline acylée
WO2023207107A1 (fr) * 2022-04-29 2023-11-02 苏州星洲生物科技有限公司 Co-agoniste des récepteurs glp-1/gcg, composition pharmaceutique le comprenant et son utilisation

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EP3670529A4 (fr) * 2017-08-16 2021-11-03 Dong-A St Co., Ltd. Analogue peptidique d'oxyntomoduline acylée
EP4079757A1 (fr) * 2017-08-16 2022-10-26 Dong-A St Co., Ltd. Analogue peptidique d'oxyntomoduline acylée
WO2023207107A1 (fr) * 2022-04-29 2023-11-02 苏州星洲生物科技有限公司 Co-agoniste des récepteurs glp-1/gcg, composition pharmaceutique le comprenant et son utilisation

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