WO2023227133A1 - 人胰淀素类似物、其衍生物及用途 - Google Patents

人胰淀素类似物、其衍生物及用途 Download PDF

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WO2023227133A1
WO2023227133A1 PCT/CN2023/096811 CN2023096811W WO2023227133A1 WO 2023227133 A1 WO2023227133 A1 WO 2023227133A1 CN 2023096811 W CN2023096811 W CN 2023096811W WO 2023227133 A1 WO2023227133 A1 WO 2023227133A1
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Prior art keywords
polypeptide
derivative
pharmaceutically acceptable
acceptable salt
amino acid
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PCT/CN2023/096811
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English (en)
French (fr)
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李岩
潘海
郝素娟
李昭英
李标
邹海霞
吴心乐
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杭州先为达生物科技股份有限公司
北京先为达生物科技有限公司
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Publication of WO2023227133A1 publication Critical patent/WO2023227133A1/zh

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • 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

Definitions

  • the present application relates to the field of biomedicine or biopharmaceutical technology, and more specifically to human amylin analogs, derivatives and their use in the treatment and/or prevention of amylin or metabolism-related diseases.
  • Amylin also known as islet amyloid polypeptide (IAPP), is a member of the calcitonin gene-related peptide (CGRP) family. It exerts physiological functions by activating corresponding receptors, regulating blood sugar, controlling food intake, It has many functions such as regulating metabolism.
  • CGRP calcitonin gene-related peptide
  • amylin is prone to fibrillation and the formation of amyloid deposits under neutral or alkaline conditions.
  • amylin has a short retention time in the blood, with a half-life of only about 13 minutes, making it difficult to use, which limits its use in the treatment of diseases. Therefore, the development of amylin analogs with better stability, longer half-life, and more obvious therapeutic effects is of great significance for the treatment of metabolic diseases such as diabetes and obesity.
  • Pramlintide also known as Symlin
  • Symlin is an amylin analogue that improves stability in solution by replacing amino acid residues 25, 28, and 29 with Pro. It has been approved for marketing. It is used to treat diabetes, but the half-life of pramlintide is only about 48 minutes, requiring frequent injections to maintain its efficacy. It is easy to aggregate and precipitate under neutral pH conditions, which limits the combined use of pramlintide with other therapeutic drugs.
  • WO2012162542A1 discloses a derivative of an amylin polypeptide analogue, which is substituted by lysine and connected to polyethylene glycol, thereby prolonging the action time of amylin or its analogues in the body, but the PEGylated The affinity of the derivatives for the amylin receptor is significantly reduced.
  • Li-Mei Yan et al. Proc Natl Acad Sci, 2006, 103(7):2046 ⁇ 2051
  • disclosed an amylin derivative IAPP-GI which improves The solubility of amylin is improved, but the activity is reduced compared with wild type.
  • Cagrilintide is a long-acting amylin derivative developed by Novo Nordisk and is currently in Phase II clinical stage.
  • amylin analogs/derivatives in the prior art have improvements in solubility, stability, half-life, activity, etc. compared with amylin, they still have insufficient in vivo efficacy and insufficient drug stability. and other technical problems, therefore, further improvements to amylin derivatives are still needed.
  • This application provides a new amylin analog, as well as derivatives of the analog, preparation methods, pharmaceutical compositions and products containing the polypeptide or derivatives thereof, and their use in preventing and/or treating diseases.
  • the application provides a new amylin analog or a pharmaceutically acceptable salt thereof, the analog comprising X 1 CNTA TCATQ RLAX 14 F LX 17 HX 19 X 20 X 21 X 22 X 23 X 24 X 25 ILX 28
  • X 1 is selected From K, D, Dab, Dap, E, H, Har, Orn, R or delete
  • X 14 is selected from N, Aad, Dab, Dap, E or D
  • X 17 is selected from V, Orn or R
  • X 19 is selected X 20 is selected from S or K
  • X 21 is selected from N or K
  • X 22 is selected from N or K
  • X 23 is selected from F
  • X 24 is selected from G, R, H , P, D, N, S, K, ⁇ K, Dap, Dab, Orn, Cit, Sar, Trx or AOC
  • X 25 is selected from K, D, Dab, Dap, E, H, Har
  • the amylin analog includes the amino acid sequence shown in X 1 CNTA TCATQ RLAX 14 FLX 17 HSS NNX 23 X 24 X 25 ILX 28
  • the amino acid sequence includes one or more amino acid mutations shown at position 1, position 14, position 17, position 23, position 24, position 25, position 28, position 29, and position 37,
  • X 1 is selected from K, D, Dab, Dap, E, H, Har, Orn, R or deleted
  • X 14 is selected from N, Aad, Dab, Dap, E or D
  • X 17 is selected from V
  • Orn Or R X 23 is selected from F, L or K
  • X 24 is selected from G, R, H, P, D, N, S, K, ⁇ K, Dap, Dab, Orn, Cit, Sar, Trx or AOC
  • X 25 is selected from A, K or P
  • X 28 is selected from S or P
  • X 29 is selected from S or P
  • X 37 is selected from Y, 3-Am-Pro, Aze, cis-Hyp, P, Pip, ⁇ MePro or trans -Hyp;
  • X 1 is selected from K or R
  • X 14 is selected from N
  • X 17 is selected from V
  • X 23 is selected from F
  • X 24 is selected from From G, R, H, P, D, N, S, K, ⁇ K, Dap, Dab, Orn, Cit, Sar, Trx or AOC
  • X 25 is selected from A, K or P
  • X 28 is selected from S or P
  • X 29 is selected from S or P
  • X 37 is selected from Y, 3-Am-Pro, Aze, cis-Hyp, P, Pip, ⁇ MePro or trans-Hyp;
  • X 1 is selected from K, D, Dab, Dap, E, H, Har, Orn, R or deleted
  • X 14 Selected from N Aad, Dab, Dap, E or D
  • X 17 is selected from V
  • Orn or R X 23 is selected from F
  • X 24 is selected from G
  • R or K X 25 is selected from A
  • K or P X 28 is selected from S or P
  • X 29 is selected from S or P
  • X 37 is selected from Y, 3-Am-Pro, Aze, cis-Hyp, P, Pip, ⁇ MePro or trans-Hyp;
  • X 1 is selected from K or R
  • X 14 is selected from N
  • X 17 is selected from V
  • X 23 is selected from F
  • X 24 is selected from From G
  • X 25 is selected from A
  • X 28 is selected from S or P
  • X 29 is selected from S or P
  • X 37 is selected from Y, 3-Am-Pro, Aze, cis-Hyp , P, Pip, ⁇ MePro or trans-Hyp;
  • X 1 is selected from K, D, Dab, Dap, E, H, Har, Orn, R or deleted
  • X 14 is selected from N, Aad, Dab, Dap, E or D
  • X 17 is selected from V
  • Orn Or R X 23 is selected from F, L or K
  • X 24 is selected from G, R, H, P, D, N, S, K, ⁇ K, Dap, Dab, Orn, Cit, Sar, Trx or AOC
  • X 25 is selected from A, K or P
  • X 28 is selected from S or P
  • X 29 is selected from S or P
  • X 37 is P or trans-Hyp;
  • X 1 is selected from K or R
  • X 14 is selected from N
  • X 17 is selected from V
  • Orn or R X 23 is selected from F
  • R or K X 25 is selected from A
  • X 29 is selected from S or P
  • X 37 is P or trans-Hyp;
  • X 1 is selected from K or R
  • X 14 is selected from N or D
  • X 17 is selected from V or R
  • X 23 is selected from F or L
  • X 24 is selected from G
  • X 25 is selected from A or P
  • X 28 is selected from S or P
  • X 29 is selected from S or P
  • X 37 is selected from Y, P or trans-Hyp;
  • X 1 is K
  • X 14 is selected from N or D
  • X 17 is selected from V or R
  • X 23 is selected from F or L
  • X 24 is selected from G
  • X 25 is selected from A or P
  • X 28 is selected from S or P
  • X 29 is selected from S or P
  • X 37 is selected from Y, P or trans-Hyp;
  • X 1 is R
  • X 14 is selected from N or D
  • X 17 is selected from V or R
  • X 23 is selected from F or L
  • X 24 is selected from G
  • X 25 is selected from A or P
  • X 28 is selected from S or P
  • X 29 is selected from S or P
  • X 37 is selected from Y, P or trans-Hyp;
  • X1 is selected from K or R
  • X14 is D
  • X17 is R
  • X23 is L
  • X25 is P
  • X24 is selected from G
  • X28 is P
  • X29 is P
  • X 37 is P or trans-Hyp
  • X1 is K
  • X14 is D
  • X17 is R
  • X23 is L
  • X24 is selected from G or K
  • X25 is P
  • X28 is P
  • X29 is P
  • X37 is P or trans-Hyp
  • X is K , X is D , X is R, X is L, X is K, X is P , X is P , X is P , X is P or trans -Hyp.
  • the present application provides a new derivative of amylin analogues or pharmaceuticals thereof
  • the above acceptable salt, the derivative of the amylin analog has one or more amino acid modifications, and the derivative of the analog includes the polypeptide sequence of the amylin analog described above.
  • polypeptide analogs or derivatives thereof described in the present application comprise the amino acid sequences described in SEQ ID NO. 2-104 in Table 1.
  • the derivative sequence of the polypeptide analog described in the present application includes 1-10 amino acid modifications compared to the wild-type polypeptide sequence, preferably, includes 1-8 amino acid modifications, further preferably, includes 1 - 4 amino acid modifications, more preferably, including 2 amino acid modifications, most preferably, including 1 amino acid modification.
  • the amino acid modifications of the derivative sequence of the polypeptide analog compared to the wild-type polypeptide sequence are in natural amino acids or unnatural amino acids, including but not limited to glutamic acid, aspartic acid, lysine
  • a functional group that can be used for coupling is introduced on the side chain of acid, glutamine or asparagine.
  • the amino acid modification is on the side chain of lysine.
  • the lysine is located on the polypeptide derivative.
  • the modified side chain is selected from Alpha-amino or Epsilon-amino side chains of lysine, preferably Epsilon amino side chain.
  • the coupled functional group includes an extension part and a linker connecting the extension part and the modified amino acid, wherein the linker may be composed of one or more linkers, and the connection order of the linkers is arbitrary, as long as it can be achieved Linking extensions and modified amino acids are understood to be forms within the scope of this application.
  • the single linker may be of the following structure:
  • n 0, 1, 2 or 3
  • n 1, 2 or 3
  • s is any integer from 0 to 6
  • p is any integer from 1 to 8.
  • the connector is:
  • the joint is:
  • p is any integer from 1 to 5
  • p is 1 and the linker is ⁇ Glu.
  • the extension is:
  • x is any integer from 4 to 38;
  • the extension part is where x is any integer from 4-38.
  • the amino acid sequence of the polypeptide analog includes the amino acid sequence represented by formula (I): X 1 CNTATCATQRLAX 14 FLX 17 HX 19 X 20 X 21 X 22 X 23 X 24 X 25 ILX 28 X 29 TNVGSX 35 TX 37 formula (I),
  • X 1 is selected from K or R
  • X 14 is selected from N
  • X 17 is selected from V
  • X 23 is selected from F
  • X 24 is selected from G, R, H, P, D, N, S, K, ⁇ K, Dap, Dab, Orn, Cit, Sar, Trx or AOC
  • X 25 is selected from A, K or P
  • X 28 is selected from S or P
  • X 29 is selected from S or P
  • X 37 is selected from Y, 3-Am-Pro, Aze, cis-Hyp, P, Pip, ⁇ MePro or trans-Hyp; the linker is:
  • the amino acid sequence of the polypeptide analog includes the amino acid sequence represented by formula (I): X 1 CNTATCATQRLAX 14 FLX 17 HX 19 X 20 X 21 X 22 X 23 X 24 X 25 ILX 28 X 29 TNVGSX 35 TX 37 formula (I),
  • X 1 is selected from K, D, Dab, Dap, E, H, Har, Orn, R or deleted;
  • X 14 is selected from N, Aad, Dab, Dap, E or D;
  • R is selected from F, L or K,
  • X 24 is selected from G, R or K,
  • X 25 is selected from A, K or P,
  • X 28 is selected from S or P,
  • X 29 is selected from S Or P,
  • X 37 is selected from Y, 3-Am-Pro, Aze, cis-Hyp, P, Pip, ⁇ MePro or trans-Hyp; the linker is:
  • the amino acid sequence of the polypeptide analog includes the amino acid sequence represented by formula (I): X 1 CNTATCATQRLAX 14 FLX 17 HX 19 X 20 X 21 X 22 X 23 X 24 X 25 ILX 28 X 29 TNVGSX 35 TX 37 formula (I),
  • X 1 is selected from K or R
  • X 14 is selected from N
  • X 17 is selected from V
  • X 23 is selected from F
  • X 24 is selected from G
  • X 25 is selected from A
  • X 28 is selected from S or P
  • X 29 is selected from S or P
  • X 37 is selected from Y, 3-Am-Pro, Aze, cis-Hyp, P, Pip, ⁇ MePro or trans-Hyp
  • X 1 is selected from K or R
  • X 14 is selected from N or D
  • X 17 is selected from V or R
  • X 23 is selected from F or L
  • X 24 is selected from G or K
  • X 25 is selected from A or P
  • X 28 is selected from S or P
  • X 29 is selected from S or P
  • X 37 is selected from Y or trans-Hyp
  • the amino acid sequence of the polypeptide analog includes the amino acid sequence represented by formula (I): X 1 CNTATCATQRLAX 14 FLX 17 HX 19 X 20 X 21 X 22 X 23 X 24 X 25 ILX 28 X 29 TNVGSX 35 TX 37 formula (I),
  • X 1 is K
  • X 14 is selected from N or D
  • X 17 is selected from V or R
  • X 23 is selected from F or L
  • X 24 is selected from G or K
  • X 25 is selected from A or P
  • X 28 is selected from From S or P
  • X 29 is selected from S or P
  • X 37 is selected from Y or trans-Hyp; the linker is:
  • the amino acid sequence of the polypeptide analog includes the amino acid sequence represented by formula (I): X 1 CNTATCATQRLAX 14 FLX 17 HX 19 X 20 X 21 X 22 X 23 X 24 X 25 ILX 28 X 29 TNVGSX 35 TX 37 formula (I),
  • X 1 is R
  • X 14 is selected from N or D
  • X 17 is selected from V or R
  • X 23 is selected from F or L
  • X 24 is selected from G or K
  • X 25 is selected from A or P
  • X 28 is selected From S or P
  • X 29 is selected from S or P
  • X 37 is selected from Y or trans-Hyp
  • the linker is:
  • the amino acid sequence of the polypeptide analog includes the amino acid sequence represented by formula (I): X 1 CNTATCATQRLAX 14 FLX 17 HX 19 X 20 X 21 X 22 X 23 X 24 X 25 ILX 28 X 29 TNVGSX 35 TX 37 formula (I),
  • X1 is selected from K or R
  • X14 is D
  • X17 is R
  • X23 is L
  • X25 is P
  • X24 is selected from G or K
  • X28 is P
  • X29 is P
  • X37 is trans-Hyp
  • the connector is:
  • the amino acid sequence of the polypeptide analog includes the amino acid sequence represented by formula (I): X 1 CNTATCATQRLAX 14 FLX 17 HX 19 X 20 X 21 X 22 X 23 X 24 X 25 ILX 28 X 29 TNVGSX 35 TX 37 formula (I),
  • X1 is K
  • X14 is D
  • X17 is R
  • X23 is L
  • X24 is selected from G or K
  • X25 is P
  • X28 is P
  • X29 is P
  • X37 is trans- Hyp
  • the connector is:
  • the amino acid sequence of the polypeptide analog includes the amino acid sequence represented by formula (I): X 1 CNTATCATQRLAX 14 FLX 17 HX 19 X 20 X 21 X 22 X 23 X 24 X 25 ILX 28 X 29 TNVGSX 35 TX 37 formula (I),
  • X1 is K
  • X14 is D
  • X17 is R
  • X23 is L
  • X24 is K
  • X25 is P
  • X28 is P
  • X29 is P
  • X37 is trans-Hyp
  • the amino acid sequence of the polypeptide analog includes the amino acid sequence represented by formula (I): X 1 CNTATCATQRLAX 14 FLX 17 HX 19 X 20 X 21 X 22 X 23 X 24 X 25 ILX 28 X 29 TNVGSX 35 TX 37 formula (I),
  • X 1 is selected from K, D, Dab, Dap, E, H, Har, Orn, R or deleted
  • X 14 is selected from N
  • X 17 is selected from V
  • X 24 is selected from G, R, H, P, D, N, S, K, ⁇ K, Dap, Dab, Orn, Cit, Sar, Trx or AOC
  • X 25 is selected from A, K or P
  • X 28 is selected from S or P
  • X 29 is selected from S or P
  • X 37 is P or trans-Hyp
  • the linker is:
  • the amino acid sequence of the polypeptide analog includes the amino acid sequence represented by formula (I): X 1 CNTATCATQRLAX 14 FLX 17 HX 19 X 20 X 21 X 22 X 23 X 24 X 25 ILX 28 X 29 TNVGSX 35 TX 37 formula (I),
  • X 1 is selected from K or R
  • X 14 is selected from N
  • X 17 is selected from V
  • Orn or R X 23 is selected from F
  • R or K X 25 is selected from A
  • X 29 is selected from S or P
  • X 37 is P or trans-Hyp; the linker is:
  • the derivatives of the polypeptide analogs of the present application are selected from the following compounds shown in Table 2, wherein PD065 is a Cagrilintide molecule.
  • Compounds PD001-PD198 represent polypeptide derivatives obtained by modifying the amino acid sequence with fatty acid-containing side chains.
  • the fatty acid-containing side chains are used to modify amino acids in this application.
  • Functional groups that can be used for coupling are introduced into the amino acid side chains.
  • PD001 means that the sequence RCNTATCATQRLAEFLRHSSKNLGPILPPTNVGSNTP is modified with a functional group that can be used for coupling, that is, the side chain (2*AEEA+ ⁇ Glu+C20diacid) Derivatives obtained after amino acid K at position 21 of -NH 2 .
  • the C16-C22 fatty diacid moiety is located outside the fatty acid-containing side chain used for modification relative to the amino acid sequence, at the distal end of the connection between the side chain and the amino acid sequence.
  • the derivatives of the polypeptide analogs of the present application are preferably selected from the following derivatives:
  • the derivative of the polypeptide analog of the present application is compound PD069, and the structure of PD069 is as follows,
  • the derivative of the polypeptide analog of the present application is compound PD070, and the structure of PD070 is as follows,
  • the derivative of the polypeptide analog of the present application is compound PD071, and the structure of PD071 is as follows,
  • the derivative of the polypeptide analog of the present application is compound PD072, and the structure of PD072 is as follows,
  • One aspect of the present application also relates to a method for preparing the new amylin analog or derivative thereof.
  • the novel amylin analogs or derivatives thereof can be prepared by conventional methods known to those skilled in the art.
  • polypeptide molecules can be prepared through chemical synthesis methods or biological fermentation methods, or a combination of both.
  • the polypeptide gene sequence can be synthesized, and the corresponding polypeptide gene with the tag can be inserted into the plasmid through conventional genetic engineering methods to construct an expression plasmid.
  • the expression plasmid can be transferred into competent cells to construct expression cells.
  • the expression cells containing the target polypeptide gene are fermented under suitable culture conditions. The fermentation products are collected and purified to finally obtain the target polypeptide.
  • the target polypeptide can be chemically modified. The method modified the amino acid side chain of the target polypeptide and purified the derivatives of the polypeptide analogues.
  • the present application also relates to a medicament or pharmaceutical composition
  • a medicament or pharmaceutical composition comprising the above-mentioned amylin analogs or derivatives thereof and pharmaceutically acceptable salts of the above-mentioned substances.
  • the pharmaceutical composition described in the present application also includes one or more oral delivery agents, and the oral delivery agent is preferably N-[8-(2-hydroxybenzoyl)amino]octanoic acid (NAC ) salt, more preferably PNAC, potassium N-[8-(2-hydroxybenzoyl)amino]octanoate.
  • NAC N-[8-(2-hydroxybenzoyl)amino]octanoic acid
  • PNAC potassium N-[8-(2-hydroxybenzoyl)amino]octanoate.
  • the NAC salt or PNAC salt of the oral delivery agent described in this application should be understood as any crystal form that can meet the oral delivery form of the composition of this application, such as that described in CN112661663B.
  • NAC salt The structure of the NAC salt is shown in formula (I):
  • the pharmaceutical composition described in the present application also includes another or more pharmacologically active substances.
  • the composition described in the present application further includes a pharmaceutically acceptable auxiliary material.
  • the pharmaceutically acceptable auxiliary material includes but is not limited to one or more of the following: pharmaceutically acceptable auxiliary materials Acceptable carriers, stabilizers, dispersants, co-solvents, plasticizers, etc. Typically, these substances are non-toxic, inert and pharmaceutically acceptable inactive substances.
  • the present application provides the use of the above-mentioned amylin analogs or derivatives and pharmaceutical compositions of the present application in the preparation of drugs for preventing and/or treating diseases.
  • the use includes the preparation of Use in drugs for preventing and/or treating amylin receptor-related diseases, further preferably, the use includes preparing drugs for preventing and/or treating lipodystrophy, blood glucose metabolism disorders, cardiovascular diseases, brain system Disease, psychiatric disease or neurological disease, including but not limited to abnormal weight, hyperlipidemia, hypertension, arteriosclerosis, fatty liver, dyslipidemia, cirrhosis, coronary heart disease, angina pectoris, myocardial infarction, inflammatory bowel disease, Indigestion and gastrointestinal ulcers, hyperglycemia, diabetes, impaired glucose tolerance, syndrome Abnormal weight, including but not limited to obesity or being overweight.
  • the present application provides a method of preventing and/or treating a disease, the method comprising administering to a subject in need thereof an amylin analog or derivative thereof, a medicament or a medicament Composition, the disease is an amylin receptor-related disease, preferably, lipodystrophy, glycemic metabolism disorder, cardiovascular disease, brain system disease, psychiatric system disease or neurological disease, including but not limited to abnormal weight, high blood pressure Lipemia, hypertension, arteriosclerosis, fatty liver, dyslipidemia, liver cirrhosis, coronary heart disease, angina pectoris, myocardial infarction, inflammatory bowel disease, dyspepsia and gastrointestinal ulcer, hyperglycemia, diabetes, impaired glucose tolerance, Syndrome
  • an amylin receptor-related disease preferably, lipodystrophy, glycemic metabolism disorder, cardiovascular disease, brain system disease, psychiatric system disease or neurological disease, including but not limited to abnormal weight, high blood pressure Lipemia, hypertension, arteriosclerosis,
  • amylin analogs or derivatives, drugs or pharmaceutical compositions described herein can also be administered in combination with one or more pharmacologically active substances, including but not limited to body weight regulators. , anti-obesity agent, lipid metabolism regulator, blood sugar regulator, hypertension treatment agent, cardiovascular regulator, brain system disease, mental system disease or nervous system regulator.
  • the pharmacologically active substance is selected from: GLP -1 receptor agonists (including but not limited to GLP-1, GLP-1 analogs, GLP-1 derivatives), GIP receptor agonists (including but not limited to GIP, GIP analogs, GIP derivatives), insulin Receptor agonists (including but not limited to insulin, insulin analogs, insulin derivatives).
  • polypeptide analogs or derivatives thereof provided by this application have good agonism for insulin amyloid peptide receptors, good stability, and high activity, and can be used to treat and prevent diseases related to insulin amyloid peptide receptors. Such as abnormal weight, especially obesity and overweight.
  • Figure 1 shows the stability experimental results of compounds PD065, PD069, PD070, PD071, and PD072 at 40 degrees Celsius on days 0, 7, and 15, respectively.
  • Figure 2A shows the time-varying trend of the weight loss effects of different derivatives in SD rats.
  • Figure 2B shows the effect of different derivatives on food intake in SD rats over time.
  • Figure 2C shows the body weight change rate of SD rats treated with different derivatives in Group A at 48 hours.
  • Figure 2D shows the body weight change rate of SD rats treated with different derivatives in Group B at 48 hours.
  • Figure 3A shows the time-varying trend of the weight loss effects of compounds PD065 and PD069 in SD rats at different concentrations.
  • Figure 3B shows the change trend of the weight loss effect of compounds PD065 and PD070 at different concentrations in SD rats over time.
  • Figure 3C shows the time-varying trend of the weight-reducing effects of compounds PD065 and PD071 in SD rats at different concentrations.
  • Figure 3D shows the change trend of the weight loss effect of compounds PD065 and PD072 at different concentrations in SD rats over time.
  • Figure 4A shows the effect of compounds PD065 and PD071 on food intake at different concentrations in SD rats over time.
  • Figure 4B shows the time-varying trend of the effect of each derivative treatment group on the food intake of SD rats at different concentrations.
  • Figure 5A shows the time-varying trend of the weight-reducing effects of compounds PD065 and PD187 in SD rats.
  • Figure 5B shows the time-varying trend of the weight-reducing effect of the corresponding compound in SD rats.
  • Figure 5C shows the time-varying trend of the effects of corresponding compounds on food intake in SD rats.
  • Figure 6 shows the pharmacokinetics of each polypeptide after oral administration of oral delivery compositions containing different polypeptide derivatives in beagle dogs.
  • AA Amino Acid, amino acid
  • Boc t-Butyloxy carbonyl, tert-butoxycarbonyl
  • DCM dichloromethane, dichloromethane
  • DMF N, N-Dimethyl formamide, dimethylformamide
  • DIEA N, N-Diisopropylethylamine, N, N-diisopropylethylamine
  • EDT 1,2-Ethanedithiol, ethanedithiol
  • Fmoc 9-fluorenylmethyloxycarbonyl, 9-fluorenylmethoxycarbonyl
  • OtBu tert-butyl ester group
  • Pbf 2 , 2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl chloride, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl
  • Pip Piperidine, TBTU: O -(Benzotriazol-
  • Amylin or “Amylin polypeptide” or “amylin” or “islet amyloid polypeptide” as used herein refers to having Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu -Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr sequence or a variant/analogue, fragment or fusion protein of the above sequence that substantially retains the agonistic effect of the amylin receptor.
  • Analog refers to a specific polypeptide molecule in which at least one amino acid residue has been replaced by another amino acid residue, and/or in which at least one amino acid residue has been deleted, and/or in which at least one amino acid residue has been added. to the N-terminus or C-terminus, and/or in which at least one amino acid residue is inserted between any two amino acids.
  • polypeptide names such as “Amylin”, and the terms “polypeptide,” “analog,” and “variant” are interchangeable.
  • Derivatives refer to products obtained by modifying functional groups (such as amino acid residues) of biological macromolecules (such as polypeptides and proteins) with certain compounds or molecules.
  • the modifications include, but are not limited to, acylation, amidation, esterification, and thioesterification.
  • compositions according to the present application may be administered by any suitable route, for example, they may be administered orally, nasally, intradermally, subcutaneously, intramuscularly or intravenously.
  • the "lipodystrophy” related diseases described in this application are a general term for related diseases caused by fat metabolism disorders. Common ones include weight abnormalities, such as obesity, and also include, for example: hyperlipidemia, hypertension, atherosclerosis, Fatty liver, cirrhosis, coronary heart disease, angina pectoris, myocardial infarction, inflammatory bowel disease, indigestion and gastrointestinal ulcers, etc.
  • “Abnormal body weight” as used in this application means that in the same evaluation system, the weight evaluation index of the subject is outside the normal average range of the population. In certain embodiments, the weight abnormality is obesity. In certain embodiments, “weight loss” refers to overweight or obesity.
  • the solid-phase organic synthesis method is adopted, using Fmoc-protected amino acid strategy and SPPS solid-phase synthesis technology to complete peptide synthesis, cleavage, oxidation, and purification to obtain the target product.
  • amino acid coupling sequence is as follows:
  • the lysis reagent was filtered out and precipitated with 5 times the liquid volume of glacial ether.
  • the precipitate was filtered out and washed 3 times with 3 times the liquid volume of glacial ether, and then dried under reduced pressure at room temperature to obtain a solid crude product.
  • Polypeptide oxidation Grind the crude product finely, prepare purified water, slowly add the ground crude product while stirring, and add acetonitrile aqueous solution dropwise. After the crude product is added and completely dissolved, add iodomethyl alcohol solution and stir for half an hour.
  • Purification and freeze-drying filter the above-mentioned oxidized liquid with a 0.45um microporous membrane; use C-18 column packing to prepare the column for crude product purification, separate and purify using appropriate gradients at room temperature, collect the target product, analyze and detect, and classify .
  • the purity requirement is ⁇ 90%. Collect unqualified target substances, separate and purify them again with appropriate gradients, and collect qualified liquid peaks.
  • the above-mentioned qualified liquid samples are freeze-dried under reduced pressure to obtain powdered refined peptide freeze-dried powder.
  • the purpose of this experiment is to detect the activity or potency of amylin derivatives on human amylin receptors in vitro using luciferase assay.
  • CHO-K1/Ga15/AMY3 cells were transfected with a plasmid containing multiple copies of the cAMP response element (CRE)-driven luciferase expression cassette (the calcitonin receptor and receptor-activating modified peptide RAMP have been constructed , purchased from GenScript), in F12 medium containing Zeocin 200ug/mL, puromycin (2ug/mL), hygromycin (100ug/mL) and G418 (400ug/mL) Culture to obtain amylin receptor/CRE-luc cell lines with stable transfection.
  • CRE cAMP response element
  • the lyophilized powder obtained in Example 1 was dissolved in 20mM phosphate buffer with pH 7.0, and Dilute the derivative sample with 10% FBS in F12 culture medium to obtain an initial concentration of 100nM.
  • the derivative sample is gradually diluted with F12 culture medium containing 10% FBS to obtain a concentration of 7 with a 10-fold difference between 100nM and 10 -5 nM.
  • the lyophilized powder of the derivatives of the polypeptide analog prepared in Example 1 the buffer solution was mixed at a mass-to-volume ratio of 0.2 mg/ml, and the polypeptide was dissolved in phosphate buffer (PB) 20 mM (pH 7.0), and stored in a -20°C environment Store the sample overnight, take out the sample and wait until it returns to room temperature to remelt.
  • PB phosphate buffer
  • the changes in the properties of the polypeptide after dissolution, freezing and re-thawing were observed and recorded, and the following liquid chromatography method was used to detect the concentration of the polypeptide before and after freezing and thawing:
  • Sample preparation Weigh a certain mass of polypeptide and dissolve it in 20mM PB, pH7.4 buffer. If the solubility is not good, add a small amount of 0.1M NaOH to adjust the pH to 8.0 to completely dissolve it, and control its concentration between 0.5-2mg/ml. between. Take the sample to be tested, centrifuge the sample at 10,000 rpm, 4°C, and 3 minutes, and transfer the centrifugal supernatant to the liquid phase injection bottle.
  • freeze-thaw yield concentration of polypeptide after freeze-thaw/concentration of polypeptide before freeze-thaw.
  • the thermal stability of the polypeptide derivatives of the present application is basically equivalent to it, and surprisingly, compound PD070 exhibits an extremely low percentage decrease in protein concentration.
  • SD Male Stawley rats with a body weight of 200-250g were selected for this experimental study.
  • rats arrive for at least 10-14 days to allow them to adapt to the experimental environment.
  • rats were housed under a reversed light/dark phase (meaning the lights were off during the day and on at night) for two weeks upon arrival, and were housed individually during the first week of arrival to ensure accurate and high-quality data.
  • rats had free access to food and water throughout the acclimation and experimental periods.
  • the derivative test is divided into experimental group A: A1, A2, A3, A4, A5, A6, and group B: B1, B2, B3, B4, B5.
  • Each derivative test group consists of 5-8 rats, administered subcutaneously.
  • Rats in each group were given derivatives or vehicle at a dose of 30 nmol/kg. Record the administration time of each group. After dosing, rats were returned to their home cages where they had access to food and water. Food consumption and rat body weight changes were recorded every 24 hours by online recording or manual recording.
  • SD rats obtained in the same manner as in Example 5 were used for this experimental study.
  • the derivative test is divided into experimental group C: C1-C13, and group D: D1-D7. There are 5-8 rats in each derivative test group.
  • the dosage regimen is as described in Table 11 and Table 12. Each group is administered subcutaneously. Group rats were treated with derivatives or vehicle at doses of 10 nmol/kg, 20 nmol/kg or 30 nmol/kg. Record the administration time of each group. After dosing, rats were returned to their home cages where they had access to food and water. Food consumption and rat body weight changes were recorded every 24 hours by online recording or manual recording.
  • polypeptide derivative molecules of the present application have better weight loss effects than PD065 (Cagrilintide) under the same conditions.
  • the derivative test is divided into experimental group E: testing the weight loss effect of compounds PD065 and PD187, and experimental group F, testing compounds PD065, PD071, PD140, PD144, PD145, PD146 and PD147.
  • Each derivative test group has 5-8 animals. Rats in each group were administered subcutaneously with a dose of 30 nmol/kg of the derivative or vehicle. Record the administration time of each group. After administration, rats were returned to their home cages, which were then placed with food and water available to them. Food consumption and rat body weight changes were recorded every 24 hours by online recording or manual recording.
  • the results of experimental group E are shown in Figure 5A
  • the results of experimental group F are shown in Figures 5B and 5C.
  • PD071 SEQ ID NO.3 was used as the peptide skeleton.
  • the results showed that when different fatty acid linkers were used, such as 2AEEA+C20 diacid (PD187), ⁇ Asp+C20 diacid (PD144) , ⁇ Asp+C20 diacid (PD145), Trx+C20 diacid (PD146) and Inp+C20 diacid (PD147), their activities are all better than PD065.
  • Example 8 Preparation of polypeptide derivative tablets containing fatty acid side chains
  • Polypeptide derivatives containing fatty acid side chains were formulated into oral tablets containing the same amount of the oral delivery agent PNAC.
  • the PNAC preparation method is as follows:
  • N-[8-(2-hydroxybenzoyl)amino]octanoic acid NAC was prepared with reference to the method in Example 1 of international patent application WO2008/028859.
  • the system was heated to 50°C, and the prepared 50% potassium hydroxide solution (1304.0g, 1.0eq) was added dropwise to the system. After the dripping, the system turned into a clear and transparent yellow solution, and the reaction was kept at 50°C for 1 hour.
  • the reaction solution was concentrated in batches at 40°C to obtain a crude product with a light orange color.
  • the preparation method of the polypeptide derivative tablets containing fatty acid side chains is as follows: sieve the polypeptide derivative and PNAC, mix them evenly with the excipients, and directly compress the tablets.
  • Example 9 Study on pharmacokinetic (PK) properties of polypeptide derivative tablets containing fatty acid side chains
  • PD065, PD187, PD183 polypeptide derivatives containing fatty acid side chains
  • Dosing Day 1 collect the whole blood of animals before dosing (-10min) and 2h, 4h, and 8h after dosing and prepare plasma (sodium heparin is used as an anticoagulant); on Dosing Day 2-Day 4, collect animals Before administration (-10min) and 2h and 4h after administration, whole blood was collected and plasma was prepared (sodium heparin was used as an anticoagulant); on administration Day 5, animals were collected before administration (-10min) and 2h and 4h after administration. Whole blood was collected and plasma was prepared at 4h, 8h, 24h, 48h, and 72h (sodium heparin was used as an anticoagulant). Plasma was stored at -80°C and used for subsequent analysis.
  • the charge-to-mass ratio is the ratio of the electrical charge of a charged particle to its mass.
  • the ion pair charge-to-mass ratio selected for PD065 is 1102.9/1074.5
  • the ion pair charge-to-mass ratio selected for PD187 is 1153/1120.9
  • the ion pair charge-to-mass ratio selected for PD183 is 945.6/919.8.

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Abstract

本申请涉及一种多肽类似物或其衍生物及其制备方法和应用，尤其是涉及一种胰岛素淀粉样多肽受体的激动性多肽类似物或其衍生物。本申请提供的多肽类似物或其衍生物对胰岛素淀粉样多肽受体具有良好的激动性，同时具有稳定性好，活性高等特点，可用于治疗、预防与胰岛素淀粉样多肽受体相关的疾病，如体重异常，尤其是在肥胖、体重过高。

Description

人胰淀素类似物、其衍生物及用途 技术领域
本申请涉及生物医学或生物制药技术领域，更具体地涉及人胰淀素类似物、衍生物及其用于治疗和/或预防与胰淀素或代谢相关的疾病。
背景技术
胰淀素(Amylin)，又称为胰岛淀粉样多肽(IAPP)，是降钙素基因相关肽(CGRP)家族的一员，其通过激活相应受体发挥生理功能，具有调节血糖、控制摄食、调节代谢等多方面的作用，然而胰淀素在中性或碱性条件下容易纤维化而形成淀粉样沉淀。同时，胰淀素在血液中存留时间短，半衰期仅约13分钟，难以用限制了其用于疾病的治疗。因此，开发出稳定性更好、半衰期更长、治疗效果更明显的胰淀素类似物对糖尿病、肥胖等代谢疾病的治疗具有重要的意义。
普兰林肽(Pramlintide，又称为Symlin)是一种胰淀素类似物，通过将25、28、29的氨基酸残基替换为Pro，提高了在溶液中的稳定性，目前已经批准上市用于治疗糖尿病，但是普兰林肽的半衰期仅约48分钟，需要频繁注射才能维持药效，在中性pH条件下容易聚集沉淀，限制了普兰林肽与其他治疗药物的联合使用。WO2012162542A1公开了一种胰淀素多肽类似物的衍生物，通过赖氨酸进行取代并与聚乙二醇连接，从而延长胰淀素或其类似物在体内的作用时间，但聚乙二醇化的衍生物对胰淀素受体的亲和力明显下降。Li-Mei Yan等(Proc Natl Acad Sci，2006，103(7):2046～2051)公开了一种胰淀素衍生物IAPP-GI，通过对24-Gly和26-Ile甲基化修饰，改善了胰淀素的溶解性，但是与野生型相比活性有所下降。Cagrilintide是由Novo Nordisk公司开发的长效胰淀素衍生物，目前处于II期临床阶段。
虽然现有技术中的胰淀素类似物/衍生物与胰淀素相比，在溶解性、稳定性、半衰期、活性等方面有所改进，但是仍然存在体内药效不够高、药物稳定性不足等技术问题，因此，仍然需要对胰淀素衍生物进行进一步的改进。
发明内容
本申请提供一种新的胰淀素类似物、以及该类似物的衍生物、制备方法、包含该多肽或其衍生物的药物组合物、制品以及他们在预防和/或治疗疾病中的用途。
在一个方面中，本申请提供了一种新的胰淀素类似物或其药学上可接受的盐，该类似物包含X1CNTA TCATQ RLAX14F LX17HX19X20X21X22X23X24X25ILX28X29T NVGSX35TX37[式(I)]所示的氨基酸序列，该氨基酸序列包含第1位、第14位、第17位、第19位、第20位、第21位、第22位、第23位、第24位、第25位、第28位、第29位、第35位、第37位所示的一个或多个氨基酸突变，优选地，X1选自K、D、Dab、Dap、E、H、Har、Orn、R或删除，X14选自N、Aad、Dab、Dap、E或D，X17选自V、Orn或R，X19选自S或K，X20选自选自S或K，X21选自N或K，X22选自N或K，X23选自F、L或K，X24选自G、R、H、P、D、N、S、K、αK、Dap、Dab、Orn、Cit、Sar、Trx或AOC，X25选自A、K或P，X28选自S或P，X29选自S或P，X35选自N或G，X37选自Y、3-Am-Pro、Aze、cis-Hyp、P、Pip、αMePro或trans-Hyp。
在一些优选方案中，所述胰淀素类似物包含X1CNTA TCATQ RLAX14FLX17HSS NNX23X24X25ILX28X29T NVGSN TX37[式(II)]所示的氨基酸序列，该氨基酸序列包含第1位、第14位、第17位、第23位、第24位、第25位、第28位、第29位、第37位所示的一个或多个氨基酸突变，
优选地，X1选自K、D、Dab、Dap、E、H、Har、Orn、R或删除，X14选自N、Aad、Dab、Dap、E或D，X17选自V、Orn或R，X23选自F、L或K，X24选自G、R、H、P、D、N、S、K、αK、Dap、Dab、Orn、Cit、Sar、Trx或AOC，X25选自A、K或P，X28选自S或P，X29选自S或P，X37选自Y、3-Am-Pro、Aze、cis-Hyp、P、Pip、αMePro或trans-Hyp；
优选地，X1选自K或R，X14选自N、Aad、Dab、Dap、E或D，X17选自V、Orn或R，X23选自F、L或K，X24选自G、R、H、P、D、N、S、K、αK、Dap、Dab、Orn、Cit、Sar、Trx或AOC，X25选自A、K或P，X28选自S或P，X29选自S或P，X37选自Y、3-Am-Pro、Aze、cis-Hyp、P、Pip、αMePro或trans-Hyp；
优选地，X1选自K、D、Dab、Dap、E、H、Har、Orn、R或删除，X14 选自N、Aad、Dab、Dap、E或D，X17选自V、Orn或R，X23选自F、L或K，X24选自G、R或K，X25选自A、K或P，X28选自S或P，X29选自S或P，X37选自Y、3-Am-Pro、Aze、cis-Hyp、P、Pip、αMePro或trans-Hyp；
优选地，X1选自K或R，X14选自N、Aad、Dab、Dap、E或D，X17选自V、Orn或R，X23选自F、L或K，X24选自G、R或K，X25选自A、K或P，X28选自S或P，X29选自S或P，X37选自Y、3-Am-Pro、Aze、cis-Hyp、P、Pip、αMePro或trans-Hyp；
优选地，X1选自K、D、Dab、Dap、E、H、Har、Orn、R或删除，X14选自N、Aad、Dab、Dap、E或D，X17选自V、Orn或R，X23选自F、L或K，X24选自G、R、H、P、D、N、S、K、αK、Dap、Dab、Orn、Cit、Sar、Trx或AOC，X25选自A、K或P，X28选自S或P，X29选自S或P，X37是P或trans-Hyp；
优选地，X1选自K或R，X14选自N、Aad、Dab、Dap、E或D，X17选自V、Orn或R，X23选自F、L或K，X24选自G、R或K，X25选自A、K或P，X28选自S或P，X29选自S或P，X37是P或trans-Hyp；
特别优选地，X1选自K或R，X14选自N或D，X17选自V或R，X23选自F或L，X24选自G、R或K，X25选自A或P，X28选自S或P，X29选自S或P，X37选自Y、P或trans-Hyp；
进一步优选地，X1是K，X14选自N或D，X17选自V或R，X23选自F或L，X24选自G、R或K，X25选自A或P，X28选自S或P，X29选自S或P，X37选自Y、P或trans-Hyp；
进一步优选地，X1是R，X14选自N或D，X17选自V或R，X23选自F或L，X24选自G、R或K，X25选自A或P，X28选自S或P，X29选自S或P，X37选自Y、P或trans-Hyp；
进一步优选地，X1选自K或R，X14是D，X17是R，X23是L，X25是P，X24选自G、R或K，X28是P，X29是P，X37是P或trans-Hyp；
更优选地，X1是K，X14是D，X17是R，X23是L，X24选自G或K，X25是P，X28是P，X29是P，X37是P或trans-Hyp；
最优选地，X1是K，X14是D，X17是R，X23是L，X24是K，X25是P，X28是P，X29是P，X37是P或trans-Hyp。
在一个方面中，本申请提供了一种新的胰淀素类似物的衍生物或其药学 上可接受的盐，所述胰淀素类似物的衍生物具有一个或多个氨基酸修饰，该类似物的衍生物包含上文所述胰淀素类似物的多肽序列。
在一些实施方式中，本申请所述的多肽类似物或其衍生物包含表1中SEQ ID NO.2-104所述氨基酸序列。
表1


在一些实施方式中，本申请所述的多肽类似物的衍生物序列相比于野生型多肽序列包括1-10个氨基酸修饰，优选地，包括1-8个氨基酸修饰，进一步优选地，包括1-4个氨基酸修饰，更优选地，包括2个氨基酸修饰，最优选地，包括1个氨基酸修饰。
在一些实施方式中，所述多肽类似物的衍生物序列相比于野生型多肽序列的氨基酸修饰是在天然氨基酸或非天然氨基酸，包括但不限于在谷氨酸、天冬氨酸、赖氨酸、谷氨酰胺或天冬酰胺的侧链上引入可用于偶联的官能团，优选地，所述氨基酸修饰是在赖氨酸的侧链上，优选地，所述赖氨酸位于多肽衍生物的第一位，被修饰的侧链选自赖氨酸的Alpha-氨基或Epsilon-氨基侧链，优选为Epsilon氨基侧链。
在一些实施方式中，所述偶联的官能团包括延长部分及连接延长部分与修饰氨基酸的接头，其中，所述接头可以是一个或多个接头组成，接头的连接顺序是任意的，只要能够实现连接延长部分与修饰氨基酸均应理解为本申请范围内的形式。
在优选实施方案中，所述单个接头可以是如下结构：
其中，m是0、1、2或3；n是1、2或3；s是0-6的任意整数；p是1-8的任意整数。
优选地，接头为：
其中m是1或2；n是1或2；p是1-5的任意整数。
更优选地，接头为：
其中，p是1-5的任意整数，
优选地，p为1，所述接头为γGlu。
在优选实施方案中，所述延长部分为：
其中x是4-38的任意整数；
优选地，所述延长部分为其中x是4-38的任意整数。
优选地，所述多肽类似物的氨基酸序列包含式(I)所示的氨基酸序列：
X1CNTATCATQRLAX14FLX17HX19X20X21X22X23X24X25ILX28X29TNVGSX
35TX37   式(I)，
其中，X1选自K或R，X14选自N、Aad、Dab、Dap、E或D，X17选自V、Orn或R，X23选自F、L或K，X24选自G、R、H、P、D、N、S、K、αK、Dap、Dab、Orn、Cit、Sar、Trx或AOC，X25选自A、K或P，X28选自S或P，X29选自S或P，X37选自Y、3-Am-Pro、Aze、cis-Hyp、P、Pip、αMePro或trans-Hyp；所述接头为：
其中m是1或2；n是1或2；p是1-5的任意整数；所述延长部分为其中x是4-38的任意整数。
优选地，所述多肽类似物的氨基酸序列包含式(I)所示的氨基酸序列：
X1CNTATCATQRLAX14FLX17HX19X20X21X22X23X24X25ILX28X29TNVGSX
35TX37   式(I)，
其中，X1选自K、D、Dab、Dap、E、H、Har、Orn、R或删除，X14选自N、Aad、Dab、Dap、E或D，X17选自V、Orn或R，X23选自F、L或K，X24选自G、R或K，X25选自A、K或P，X28选自S或P，X29选自S 或P，X37选自Y、3-Am-Pro、Aze、cis-Hyp、P、Pip、αMePro或trans-Hyp；所述接头为：
其中m是1或2；n是1或2；p是1-5的任意整数；所述延长部分为其中x是4-38的任意整数。
优选地，所述多肽类似物的氨基酸序列包含式(I)所示的氨基酸序列：
X1CNTATCATQRLAX14FLX17HX19X20X21X22X23X24X25ILX28X29TNVGSX
35TX37   式(I)，
其中，X1选自K或R，X14选自N、Aad、Dab、Dap、E或D，X17选自V、Orn或R，X23选自F、L或K，X24选自G、R或K，X25选自A、K或P，X28选自S或P，X29选自S或P，X37选自Y、3-Am-Pro、Aze、cis-Hyp、P、Pip、αMePro或trans-Hyp；特别优选地，X1选自K或R，X14选自N或D，X17选自V或R，X23选自F或L，X24选自G或K，X25选自A或P，X28选自S或P，X29选自S或P，X37选自Y或trans-Hyp；所述接头为：
其中m是1或2；n是1或2；p是1-5的任意整数；所述延长部分为其中x是4-38的任意整数。
优选地，所述多肽类似物的氨基酸序列包含式(I)所示的氨基酸序列：
X1CNTATCATQRLAX14FLX17HX19X20X21X22X23X24X25ILX28X29TNVGSX
35TX37   式(I)，
其中，X1是K，X14选自N或D，X17选自V或R，X23选自F或L，X24选自G或K，X25选自A或P，X28选自S或P，X29选自S或P，X37选自Y或trans-Hyp；所述接头为：
其中m是1或2；n是1或2；p是1-5的任意整数；所述延长部分为其中x是4-38的任意整数。
优选地，所述多肽类似物的氨基酸序列包含式(I)所示的氨基酸序列：
X1CNTATCATQRLAX14FLX17HX19X20X21X22X23X24X25ILX28X29TNVGSX
35TX37   式(I)，
其中，X1是R，X14选自N或D，X17选自V或R，X23选自F或L，X24选自G或K，X25选自A或P，X28选自S或P，X29选自S或P，X37选自Y或trans-Hyp；所述接头为：
其中m是1或2；n是1或2；p是1-5的任意整数；所述延长部分为其中x是4-38的任意整数。
优选地，所述多肽类似物的氨基酸序列包含式(I)所示的氨基酸序列：
X1CNTATCATQRLAX14FLX17HX19X20X21X22X23X24X25ILX28X29TNVGSX
35TX37   式(I)，
其中，X1选自K或R，X14是D，X17是R，X23是L，X25是P，X24选自G或K，X28是P，X29是P，X37是trans-Hyp；所述接头为：
其中m是1或2；n是1或2；p是1-5的任意整数；所述延长部分为其中x是4-38的任意整数。
优选地，所述多肽类似物的氨基酸序列包含式(I)所示的氨基酸序列：
X1CNTATCATQRLAX14FLX17HX19X20X21X22X23X24X25ILX28X29TNVGSX
35TX37   式(I)，
其中，X1是K，X14是D，X17是R，X23是L，X24选自G或K，X25是P，X28是P，X29是P，X37是trans-Hyp；所述接头为：
其中m是1或2；n是1或2；p是1-5的任意整数；所述延长部分为其中x是4-38的任意整数。
优选地，所述多肽类似物的氨基酸序列包含式(I)所示的氨基酸序列：
X1CNTATCATQRLAX14FLX17HX19X20X21X22X23X24X25ILX28X29TNVGSX
35TX37   式(I)，
其中，X1是K，X14是D，X17是R，X23是L，X24是K，X25是P，X28是P，X29是P，X37是trans-Hyp；所述接头为：
其中m是1或2；n是1或2；p是1-5的任意整数；所述延长部分为其中x是4-38的任意整数。
优选地，所述多肽类似物的氨基酸序列包含式(I)所示的氨基酸序列：
X1CNTATCATQRLAX14FLX17HX19X20X21X22X23X24X25ILX28X29TNVGSX
35TX37   式(I)，
X1选自K、D、Dab、Dap、E、H、Har、Orn、R或删除，X14选自N、Aad、Dab、Dap、E或D，X17选自V、Orn或R，X23选自F、L或K，X24选自G、R、H、P、D、N、S、K、αK、Dap、Dab、Orn、Cit、Sar、Trx或AOC，X25选自A、K或P，X28选自S或P，X29选自S或P，X37是P或trans-Hyp；所述接头为：
其中m是1或2；n是1或2；p是1-5的任意整数；所述延长部分为其中x是4-38的任意整数。
优选地，所述多肽类似物的氨基酸序列包含式(I)所示的氨基酸序列：
X1CNTATCATQRLAX14FLX17HX19X20X21X22X23X24X25ILX28X29TNVGSX
35TX37   式(I)，
X1选自K或R，X14选自N、Aad、Dab、Dap、E或D，X17选自V、Orn或R，X23选自F、L或K，X24选自G、R或K，X25选自A、K或P，X28选自S或P，X29选自S或P，X37是P或trans-Hyp；所述接头为：
其中m是1或2；n是1或2；p是1-5的任意整数；所述延长部分为其中x是4-38的任意整数。
在一些实施方式中，本申请的多肽类似物的衍生物选自表2所示的下列化合物，其中，PD065为Cagrilintide分子。
表2.







化合物PD001-PD198表示用含脂肪酸的侧链修饰氨基酸序列后得到的多肽衍生物，所述含脂肪酸的侧链即本申请中的用于修饰氨基酸的在氨基酸侧链上引入可用于偶联的官能团，如用表2中的对应含脂肪酸的侧链修饰对应多肽序列后得到的衍生物，例如，PD001表示用可用于偶联的官能团，即侧链(2*AEEA+γGlu+C20diacid)修饰序列RCNTATCATQRLAEFLRHSSKNLGPILPPTNVGSNTP-NH2的第21位的氨基酸K后得到的衍生物。通常情况下，C16-C22脂肪二酸部分相对于氨基酸序列位于用于修饰的含脂肪酸侧链的外侧，处于侧链与氨基酸序列连接的远端。
在一些实施方式中，本申请的多肽类似物的衍生物优选选自以下衍生物：
PD069、PD070、PD071、PD072、PD140、PD158、PD160、PD161、PD144、PD145、PD146、PD147、PD187。
在一些实施方式中，本申请的多肽类似物的衍生物是化合物PD069，PD069的结构如下所示，
在一些实施方式中，本申请的多肽类似物的衍生物是化合物PD070，PD070的结构如下所示，
在一些实施方式中，本申请的多肽类似物的衍生物是化合物PD071，PD071的结构如下所示，
在一些实施方式中，本申请的多肽类似物的衍生物是化合物PD072，PD072的结构如下所示，
本申请一个方面还涉及所述新的胰淀素类似物或其衍生物的制备方法。
在一些实施方式中，所述新的胰淀素类似物或其衍生物的制备可以通过本领域技术人员已知的常规方法制备。通常情况下，多肽分子可以通过化学合成方法或生物发酵方法，或两者结合制备获得。例如，多肽合成过程中可以通过多肽基因序列合成，并将带有标签的对应多肽基因通过基因工程常规方法插入质粒中从而构建表达质粒，将表达质粒转入感受态细胞从而构建表达细胞，经筛选后含有目标多肽基因的表达细胞在适宜培养条件下发酵，收集发酵产物并通过纯化方法最终获得目标多肽，进一步地，可通过化学修饰 方法对目标多肽氨基酸侧链进行修饰，并纯化获得多肽类似物的衍生物。
在一个方面中，本申请还涉及包含上述胰淀素类似物或其衍生物及上述物质的药学上可接受的盐的药物或药物组合物。
在一些实施方式中，本申请所述药物组合物，还包括一种或多种口服递送剂，所述口服递送剂优选为N-[8-(2-羟基苯甲酰基)氨基]辛酸(NAC)盐，进一步优选为PNAC，即N-[8-(2-羟基苯甲酰基)氨基]辛酸钾。本申请所述的口服递送剂的NAC盐或PNAC盐应理解为可以满足本申请组合物口服递送形式的任意晶体形式，如CN112661663B所述的。
所述PNAC盐的结构如式(II)所示：
所述NAC盐的结构如式(I)所示：
在一些实施方式中，本申请所述药物组合物，还包括另一种或多种具有药理学活性物质。
在一些实施方式中，本申请所述组合物进一步还包括一种药学上可接受的辅料，优选地，所述药学上可接受的辅料包括但不限于以下中的一种或多种：药学上可接受的载体、稳定剂、分散剂、助溶剂、塑形剂等。通常，这些物质是无毒的、惰性的和药学上可接受的无活性物质。
在一个方面中，本申请提供了本申请上述胰淀素类似物或其衍生物、药物组合物在制备用于预防和/或治疗疾病药物中的用途，优选地，所述用途包括在制备用于预防和/或治疗胰淀素受体相关疾病药物中的用途，进一步优选地，所述的用途包括在制备用于预防和/或治疗脂肪代谢障碍、血糖代谢障碍、心血管疾病、脑系统疾病、精神系统疾病或神经系统疾病，包括但不限于体重异常、高脂血症、高血压、动脉硬化、脂肪肝、血脂障碍、肝硬化、冠心病、心绞痛、心肌梗死、炎性肠病、消化不良和胃肠道溃疡，高血糖症、糖尿病、葡萄糖耐量降低、X综合征、认知障碍、中风，优选地，所述疾病是体重异常或血糖代谢障碍，进一步优选地，所述疾病是体重异常，包括但不限于肥胖或体重过高。
在一个方面中，本申请提供了一种预防和/或治疗疾病的方法，所述方法包括向有需要的受试者施用本申请所述的胰淀素类似物或其衍生物、药物或药物组合物，所述疾病是胰淀素受体相关疾病，优选地，脂肪代谢障碍、血糖代谢障碍、心血管疾病、脑系统疾病、精神系统疾病或神经系统疾病，包括但不限于体重异常、高脂血症、高血压、动脉硬化、脂肪肝、血脂障碍、肝硬化、冠心病、心绞痛、心肌梗死、炎性肠病、消化不良和胃肠道溃疡、高血糖症、糖尿病、葡萄糖耐量降低、X综合征、认知障碍、中风，进一步优选地，所述疾病是体重异常或血糖代谢障碍，更优选地，所述疾病是体重异常，包括但不限于肥胖或体重过高。
本申请所述的胰淀素类似物或其衍生物、药物或药物组合物还可以与另一种或多种具有药理学活性物质组合施用，所述药理学活性物质包括但不限于体重调节剂、抗肥胖剂、脂代谢调节剂、血糖调节剂、高血压治疗剂、心血管调节剂、脑系统疾病、精神系统疾病或神经系统调节剂，优选地，所述药理学活性物质选自：GLP-1受体激动剂(包括但不限于GLP-1、GLP-1类似物、GLP-1衍生物)、GIP受体激动剂(包括但不限于GIP、GIP类似物、GIP衍生物)、胰岛素受体激动剂(包括但不限于胰岛素、胰岛素类似物、胰岛素衍生物)。
发明效果
本申请提供的多肽类似物或其衍生物对胰岛素淀粉样多肽受体具有良好的激动性，同时具有稳定性好，活性高等特点，可用于治疗、预防与胰岛素淀粉样多肽受体相关的疾病，如体重异常，尤其是在肥胖、体重过高。
附图说明
附图用于更好地理解本申请，不构成对本申请的不当限定。其中：
图1示出了化合物PD065、PD069、PD070、PD071、PD072分别在40摄氏度下第0天、7天、15天的稳定性实验结果。
图2A示出了不同衍生物在SD大鼠中减重效果随时间变化趋势。
图2B示出了不同衍生物在SD大鼠中对食物摄入的影响随时间变化趋势。
图2C示出了第48小时A组中不同衍生物处理组SD大鼠体重变化率。
图2D示出了第48小时B组中不同衍生物处理组SD大鼠体重变化率。
图3A示出了SD大鼠中不同浓度下化合物PD065和PD069减重效果随时间变化趋势。
图3B示出了SD大鼠中不同浓度下化合物PD065和PD070减重效果随时间变化趋势。
图3C示出了SD大鼠中不同浓度下化合物PD065和PD071减重效果随时间变化趋势。
图3D示出了SD大鼠中不同浓度下化合物PD065和PD072减重效果随时间变化趋势。
图4A示出了SD大鼠中不同浓度下化合物PD065和PD071对食物摄入的影响随时间变化趋势。
图4B示出了不同浓度下各衍生物处理组对SD大鼠食物摄入影响随时间变化的趋势。
图5A示出了SD大鼠中化合物PD065和PD187减重效果随时间变化趋势。
图5B示出了SD大鼠中相应化合物减重效果随时间变化趋势。
图5C示出了SD大鼠中相应化合物对食物摄入的影响随时间变化趋势。
图6示出了在比格犬中口服分别含有不同多肽衍生物的口服递送组合物后，各多肽的药代动力学情况。
具体实施方式
以下对本申请的示范性实施例做出说明，其中包括本申请实施例的各种细节以助于理解，应当将它们认为仅仅是示范性的。因此，本领域普通技术人员应当认识到，可以对这里描述的实施例做出各种改变和修改，而不会背离本申请的范围和精神。同样，为了清楚和简明，以下的描述中省略了对公知功能和结构的描述。
缩写
本申请实施例中所用的一些缩写词对应名称或结构形式如下表3所示：
表3.

AA：Amino Acid，氨基酸；Boc：t-Butyloxy carbonyl，叔丁氧羰基；DCM：dichloromethane，二氯甲烷；DMF：N，N-Dimethyl formamide，二甲基甲酰胺；DIEA：N，N-Diisopropylethylamine，N，N-二异丙基乙胺；EDT：1，2-Ethanedithiol，乙二硫醇；Fmoc：9-fluorenylmethyloxycarbonyl，9-芴基甲氧基羰基；OtBu：叔丁基酯基；Pbf：2，2，4，6，7-Pentamethyldihydrobenzofuran-5-sulfonyl chloride，2，2，4，6，7-五甲基二氢苯并呋喃-5-磺酰基；Pip：Piperidine，哌啶；TBTU：O-(Benzotriazol-l-yl)-N，N，N，N，-tetramethyluronium Tetrafluoroborate，O-苯并三氮唑-N，N，N'，N'-四甲基脲四氟硼酸酯；tBu：tertiary butyl，叔丁基；TFA：Trifluoroacetic acid，三氟乙酸；TIS：Triisopropylsilane，三异丙基硅烷；Trt：Triphenylmethyl，三苯基甲基。
定义
本文所述“Amylin”或“Amylin多肽”或“胰淀素”或“胰岛淀粉样多肽”是指具有Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr序列或基本保持了胰淀素受体激动作用的以上序列的变体/类似物、片段或融合蛋白。
本文所述“类似物”是指特定多肽分子的至少一个氨基酸残基已经被另一个氨基酸残基置换，且/或者其中至少一个氨基酸残基已经缺失，且/或者其中至少一个氨基酸残基被添加到N端或C端，且/或者其中至少一个氨基酸残基被插入到任意两个氨基酸之间所形成的化合物。
除非上下文中另有明确说明，否则本申请中特定多肽名称，例如“Amylin”，及术语“多肽”、“类似物”、“变体”可互相替换。
本文所述“衍生物”是指生物大分子(如多肽、蛋白)的功能基团(如氨基酸残基)被某些化合物或分子修饰后所得到的产物。所述修饰包括但不限于酰基化、酰胺化、酯化、硫酯化。
根据本申请的药物组合物可通过任何适宜的途径施用，例如可口服、鼻、皮内、皮下、肌内或静脉内施用。
本申请所述的“脂肪代谢障碍”相关疾病为脂肪代谢紊乱导致的相关疾病的总称，常见的有体重异常，例如肥胖症，还包括例如：高脂血症、高血压、动脉粥样硬化、脂肪肝、肝硬化、冠心病、心绞痛、心肌梗死、炎性肠病、消化不良和胃肠道溃疡等。
本申请所述的“体重异常”指在相同评价体系中，所述对象体重评价指标在其所述种群正常平均范围以外。在某些实施方式中，所述体重异常是指肥胖。在某些实施方式中，所述“减重”指超重或减少肥胖。
实施例
参照以下实施例可以更好地理解本申请。但是，应理解，以下实施例仅用于举例说明目的，而不应被理解为以任何方式限制本申请的保护范围。如未特别指明之处，可根据本领域技术人员所熟悉的《分子克隆实验指南》、《细胞实验指南》等实验手册以及CFDA的试验指引等所列方法来实施。其中，所用的试剂原料均为市售品，可以通过公开渠道购买获得。
实施例1胰淀素衍生物的制备
采用固相有机合成法，利用Fmoc-保护氨基酸策略，SPPS固相合成技术，完成多肽合成，裂解，氧化，纯化即得目标产物。
以化合物PD065(Cagrilintide)为例，其结构式如下：
合成过程如下：
固相合成：用Fmoc-Linker MBHA Resin S＝0.32mmol/g，采用Fmoc/tBu工艺，按照上述序列，以表4方法从C端向N端(从右到左)依次缩合氨基酸连接。
表4.合成程序列表
氨基酸偶联顺序如下所示：
A-01Fmoc-Pro-OH，A-02Fmoc-Thr(tBu)-OH，A-03Fmoc-Asn(Trt)-OH，A-04Fmoc-Ser(tBu)-OH，A-05Fmoc-Gly-OH，A-06Fmoc-Val-OH，A-07Fmoc-Asn(Trt)-OH，A-08Fmoc-Thr(tBu)-OH，A-09Fmoc-Pro-OH，A-10Fmoc-Pro-OH，A-11Fmoc-Leu-OH，A-12Fmoc-Ile-OH，A-13Fmoc-Pro-OH，A-14Fmoc-Gly-OH，A-15Fmoc-Phe-OH，A-16Fmoc-Asn(Trt)-OH，A-17Fmoc-Asn(Trt)-OH，A-18Fmoc-Ser(tBu)-OH，A-19Fmoc-Ser(tBu)-OH，A-20Fmoc-His(Trt)-OH，A-21Fmoc-Arg(Pbf)-OH，A-22Fmoc-Leu-OH，A-23Fmoc-Phe-OH，A-24Fmoc-Glu(OtBu)-OH，A-25Fmoc-Ala-OH，A-26Fmoc-Leu-OH，A-27Fmoc-Arg(Pbf)-OH，A-28Fmoc-Gln(Trt)-OH，A-29Fmoc-Thr(tBu)-OH，A-30Fmoc-Ala-OH，A-31Fmoc-Cys(Trt)-OH，A-32Fmoc-Thr(tBu)-OH，A-33Fmoc-Ala-OH，A-34Fmoc-Thr(tBu)-OH，A-35Fmoc-Asn(Trt)-OH，A-36Fmoc-Cys(Trt)-OH，A-37Fmoc-Lys(Boc)-OH，A-38Fmoc-Glu-otbu，A-39C20diacid；最后形成此多肽衍生物树脂，多肽衍 生物树脂(肽树脂)洗涤转移出干燥至恒重，待裂解。
肽树脂裂解：
裂解试剂的配制：按体积为1g肽树脂比10ml±2ml计算裂解试剂用量，TFA:H2O:EDT:TIS＝95:1:2:2。依次将所需裂解试剂H2O，TFA，EDT，TIS加于裂解反应瓶，裂解试剂温度控制在0～10℃；裂解试剂在搅拌下加入肽树脂中，待体系温度稳定后；再温控在25～30℃搅拌反应2.5小时。将裂解试剂滤出，采用5倍液体积量的冰乙醚将其沉淀，滤出沉淀物并采用3倍液体积量的冰乙醚洗涤3次后，室温减压干燥，得固体粗品。
多肽氧化：将粗品研细，准备纯化水，在搅拌下缓慢加入研细粗品，同时滴加乙腈水溶液，待粗品加完并溶解完全后，加入碘甲醇溶液搅拌半小时。
纯化冻干：将上述氧化后液体，用0.45um的微孔滤膜过滤；粗品纯化采用C-18柱填料制备柱，在常温下用合适梯度进行分离纯化，收集目标产物，分析检测，归类。纯度要求≥90％，将不合格目标物收集，用合适梯度再次对其进行分离纯化，收集合格液体峰。将上述合格的液体样品减压冷冻干燥，得到粉末状精制多肽冻干粉。
采用与所述化合物PD065(Cagrilintide)类似方法及步骤制备表2所述化合物。
实施例2胰淀素衍生物体外效力检测
本实验目的在于采用荧光素酶测定法在体外检测胰淀素衍生物对人胰淀素受体的活力或效力。
2.1构建胰淀素受体/CRE-luc细胞系
使用标准方法，用含有多拷贝cAMP应答元件(CRE)驱动的荧光素酶表达框的质粒转染CHO-K1/Ga15/AMY3细胞(降钙素受体与受体激活修饰性肽RAMP已构建完成，购自金斯瑞)，在含有吉欧霉素(Zeocin)200ug/mL，嘌呤霉素(2ug/mL)、潮霉素(100ug/mL)和G418(400ug/mL)的F12培养基中培养，以获得具有稳定转染的胰淀素受体/CRE-luc细胞系。
2.2胰淀素荧光素酶测定
白色96孔板中每孔加入50μL生长培养基(含10％FBS的F12培养基)，将稳定转染的胰淀素/CRE-luc CHO细胞以约20000个细胞/孔的密度接种到白色96孔培养板中，以进行活性测定。
将实施例1所得冻干粉用pH7.0的20mM磷酸缓冲液溶解，并用 10％FBS的F12培养基稀释获得初始浓度100nM的衍生物样品，用含10％FBS的F12培养基梯度稀释衍生物样品，以获得100nM到10-5nM之间浓度依此相差10倍的7个浓度的样品，每孔中加入相应浓度的样品测定液50μL，在37℃和5％CO2条件下孵育24小时，进一步加入Luciferase每孔100μL，孵育3分钟，最后在SpectraMax L(Molecular Devices)上以SoftMax Pro 7.0.3 GxP软件测定发光情况，并通过荧光值绘制标准曲线，计算EC50。结果如表5所示。
表5.
表5结果显示，本申请的多肽衍生物对人胰淀素受体均具有良好的活力。
实施例3衍生物冻融稳定性
实施例1中制备得到的多肽类似物的衍生物冻干粉:缓冲溶液按0.2mg/ml的质量体积比混合，用磷酸缓冲液(PB)20mM(pH 7.0)溶解多肽，于-20℃环境保存样品，过夜保存，取出样品待其恢复至室温复融。在实验过程中，分别观察并记录多肽溶解、和冻存复融后的性状变化，并用如下液相色谱方法检测多肽冻融前后的浓度：
材料和设备：高效液相色谱仪(Agilent 1200)、超纯水(18.2MΩ自制超纯水)、乙腈(HPLC级)、三氟乙酸(HPLC级)；反相色谱柱：Sepax Bio-C4 4.6*100mm 5μm
样品准备 称取一定质量的多肽，用20mM PB，pH7.4缓冲液溶解，如溶解度不好，可加入少量0.1M NaOH调节pH至8.0使之完全溶解，控制其浓度在0.5-2mg/ml之间。取待测样品，将样品于10000rpm、4℃、3min条件下离心，取离心上清液至液相进样瓶。
色谱条件：流速：1.0ml/min；自动进样器温度：15℃；柱温：15℃；检测波长：280nm；流动相A：100％H2O+0.05％TFA；流动相B：100％CAN。洗脱梯度见下表：
表6液相色谱洗脱梯度列表
利用如下公式计算其冻融收率：冻融收率＝冻融后多肽的浓度/冻融前多肽的浓度。相关结果表7。
表7


如表7所示，本申请绝大部分多肽衍生物有良好的冻融稳定性和收率。
实施例4衍生物40℃稳定性研究
将化合物PD065、PD069、PD070、PD071、PD072按1.34mg/ml与磷酸二氢钠(1.42mg/ml)混合，溶解于超纯水中，用盐酸/氢氧化钠调节pH至7.4左右，之后在超净工作台中用0.22μm无菌滤器过滤溶液至已灭菌西林瓶中。西林瓶压盖后置于40℃稳定性试验箱中。
测前，将样品于10000rpm、4℃、3min条件下离心，取离心上清液至液相进样瓶。采用实施例3中液相方法进行检测。结果如表8和图1所示
表8

如表8结果所示，相比于PD065的热稳定性变化，本申请多肽衍生物热稳定性基本与之相当，并且令人意外的是，化合物PD070展现出了极低的蛋白浓度下降百分比。
实施例5衍生物的减重药效研究
选择体重为200-250g的SD(Sprague Dawley)大鼠用于本实验研究。实验开始前，大鼠到达至少10-14天，使其适应实验环境。在这一期间，大鼠到达后在反转的光照/黑暗阶段(意思是光照在白天期间关闭并且在夜间打)下饲养两周，且到达的第一周单独饲养，以确保数据准确和高的测试灵敏度，在整个适应期和实验期内大鼠自由获取食物和水。衍生物测试分为实验A组：A1、A2、A3、A4、A5、A6，和B组：B1、B2、B3、B4、B5，各衍生物测试组5-8只大鼠，经皮下给予各组大鼠30nmol/kg剂量的衍生物或溶媒。记录每组的给药时间。给药后，将大鼠放回它们的居住笼，然后在其中它们可以获取食物和水。通过在线记录或手动记录，每24小时记录食物消耗和大鼠体重变化。
各组SD鼠体重及摄食变化情况如图2A-2D及表9-表10所示。
表9
表10
实验结果显示，相比于PD065减重效果，本申请的多肽衍生物显示出更 高的减重能力，甚至可达到PD065效果的2倍以上。
实施例6衍生物的用量对减重效果的研究
使用与实施例5相同方法获得的SD鼠用于本实验研究。衍生物测试分为实验C组：C1-C13，和D组：D1-D7，各衍生物测试组5-8只大鼠，如表11和表12所述给药方案，经皮下分别给予各组大鼠10nmol/kg，20nmol/kg或30nmol/kg剂量的衍生物或溶媒。记录每组的给药时间。给药后，将大鼠放回它们的居住笼，然后在其中它们可以获取食物和水。通过在线记录或手动记录，每24小时记录食物消耗和大鼠体重变化。
各组SD鼠体重变化及累积摄食量变化情况如图3A-3D、4A-4B及表11和表12所示。
表11
表12
从实验结果来看，本申请多肽衍生物分子相比PD065(Cagrilintide)在相同条件下，具有更优的减重效果。
实施例7.衍生物脂肪酸侧链研究
使用与实施例5相同方法获得的SD鼠用于本实验研究。衍生物测试分为实验E组：测试化合物PD065和PD187的减重效果，和实验F组，测试化合物PD065、PD071、PD140、PD144、PD145、PD146和PD147，各衍生物测试组5-8只大鼠，经皮下给予各组大鼠30nmol/kg剂量的衍生物或溶媒。记录每组的给药时间。给药后，将大鼠放回它们的居住笼，然后在其中放置它们可以获取的食物和水。通过在线记录或手动记录，每24小时记录食物消耗和大鼠体重变化。实验E组结果如图5A所示，实验F组结果如图5B和5C所示。
动物实验结果发现，当N端为R(PD140)时，活性好于PD065。
为进一步评估脂肪酸侧链对多肽活性的作用，以PD071(SEQ ID NO.3)为多肽骨架，结果显示，使用不同脂肪酸linker时，如2AEEA+C20 diacid(PD187)、αAsp+C20 diacid(PD144)、βAsp+C20 diacid(PD145)、Trx+C20 diacid(PD146)和Inp+C20 diacid(PD147)，其活性均好于PD065。
实施例8：含脂肪酸侧链的多肽衍生物片剂的制备
将含脂肪酸侧链的多肽衍生物制作为口服片剂，该片剂包含相同含量的口服递送剂PNAC。所述PNAC制备方法如下：
参照国际专利申请WO2008/028859实施例1中的方法制备N-[8-(2-羟基苯甲酰基)氨基]辛酸NAC。向50L反应釜中加入异丙醇(22070.0ml，4.0vol)，开启搅拌，加入NAC(5518g，1.0eq)。将体系升温至50℃，向体系中滴加配制好的50％氢氧化钾溶液(1304.0g，1.0eq)。滴毕，体系变为澄清透明的黄色溶液，保温50℃反应1h。将反应液40℃下分批浓缩，得到粗品的颜色为浅橙黄色。
将粗品合批加入异丙醇(19310.0ml，3.5vol)进行打浆，打浆1h。对体系进行抽滤，滤饼以异丙醇(2760.0ml，0.5vol)进行淋洗。将滤饼转移至真空干燥箱中，干燥体系氮气平压，60℃条件下干燥16h，再次转移至真空干燥箱在100℃条件下，干燥24h。干燥完成，共得到产品4.52kg，收率72.8％，性状为类白色粉末状固体，即PNAC。
所述含脂肪酸侧链的多肽衍生物片剂中主要成分的含量如表13所示：
表13：本申请制备的多肽衍生物片剂的示例

所述含脂肪酸侧链的多肽衍生物片剂的制备方法如下：将多肽衍生物与PNAC过筛，与辅料混合均匀直接压片。
实施例9：含脂肪酸侧链的多肽衍生物片剂药代动力学(PK)特性研究
10-15月龄雄性Beagle犬(9-12kg)连续5天，每天一次口服给予实施例4制备的口服片剂，施用的含脂肪酸侧链的多肽衍生物(i.e.PD065、PD187、PD183)的剂量分别为7mg/只(n＝5)。首次口服给药的日期记为Day 1，末次口服给药的日期记为Day 5。给药Day 1时，收集动物给药前(-10min)及给药后2h、4h、8h时全血并制备血浆(肝素钠作为抗凝剂)；给药Day 2-Day 4时，收集动物给药前(-10min)及给药后2h、4h时全血并制备血浆(肝素钠作为抗凝剂)；给药Day 5时，收集动物给药前(-10min)及给药后2h、4h、8h、24h、48h、72h时全血并制备血浆(肝素钠作为抗凝剂)。血浆存储于-80℃并用于后续分析。利用LC-MS/MS(Waters ACQUITY I Class Premier UPLC tandem with Sciex 6500+QQQ)分析血浆中药物的含量。实验数据由GraphPad Prism9.3.1绘制。分别施用所述三种多肽衍生物片剂后，在所述时间点分别测得的所述三种多肽衍生物(i.e.PD065、PD187、PD183)的血浆浓度结果如图6所示。
荷质比是带电粒子的电量与其质量之比。其中PD065选用的离子对荷质比是1102.9/1074.5、PD187选用的离子对荷质比是1153/1120.9、PD183选用的离子对荷质比是945.6/919.8。
由图6所示结果可见：虽然化合物PD187及PD183均具有更优的减重药效，但在相同递送环境下，相比于含有化合物PD065的片剂组，口服利用度未见提高；令人意外的是，化合物PD183分子在递送剂PNAC的片剂中显示出了特别的协同效果，展现极高的口服生物利用效果。
尽管以上结合对本申请的实施方案进行了描述，但本申请并不局限于上述的具体实施方案和应用领域，上述的具体实施方案仅仅是示意性的、指导性的，而不是限制性的。本领域的普通技术人员在本说明书的启示下和在不脱离本申请权利要求所保护的范围的情况下，还可以做出很多种的形式，这些均属于本申请保护之列。

Claims (26)

  1. 一种胰淀素多肽类似物或其衍生物及上述物质的药学上可接受的盐，其中，包含式(I)所示的氨基酸序列：
    X1CNTATCATQRLAX14FLX17HX19X20X21X22X23X24X25ILX28X29TNVGSX
    35TX37
    式(I)，
    其中，
    X1选自K、D、Dab、Dap、E、H、Har、Orn、R或删除,
    X14选自N、Aad、Dab、Dap、E或D，
    X17选自V、Orn或R，
    X19选自S或K，
    X20选自S或K,
    X21选自N或K，
    X22选自N或K，
    X23选自F、L或K，
    X24选自G、R、H、P、D、N、S、K、αK、Dap、Dab、Orn、Cit、Sar、Trx或AOC，
    X25选自A、K或P，
    X28选自S或P，
    X29选自S或P，
    X35选自N或G，
    X37选自Y、3-Am-Pro、Aze、cis-Hyp、P、Pip、αMePro或trans-Hyp。
  2. 根据权利要求1所述的多肽类似物或其衍生物及上述物质的药学上可接受的盐，其中，包含式(II)所示的氨基酸序列：
    X1CNTATCATQRLAX14FLX17HSSNNX23X24X25ILX28X29TNVGSNTX37
    式(II)
    其中，
    X1选自K或R，
    X14选自N或D，
    X17选自V或R，
    X23选自F或L，
    X24选自G、R或K，
    X25选自A或P，
    X28选自S或P，
    X29选自S或P，
    X37选自Y、P或trans-Hyp。
  3. 根据权利要求2所述的多肽类似物或其衍生物及上述物质的药学上可接受的盐，其中所述X14是D，X17是R，X23是L，X25是P，X28是P，X29是P，X37是P或trans-Hyp。
  4. 根据权利要求3所述的多肽类似物或衍生物或其药学上可接受的盐，其中X24是K或R；优选地，所述X1是K。
  5. 根据权利要求1或2所述的多肽类似物或其衍生物及上述物质的药学上可接受的盐，其包含选自SEQ ID NO.2-104的氨基酸序列。
  6. 根据权利要求1-5任一项所述的多肽类似物或多肽衍生物或其药学上可接受的盐，其中所述多肽衍生物或其药学上可接受的盐包括1-10个氨基酸修饰。
  7. 根据权利要求6所述的多肽衍生物或其药学上可接受的盐，其中所述修饰位于1位K的Epsilon氨基上。
  8. 根据权利要求6或7所述的多肽衍生物或其药学上可接受的盐，所述氨基酸修饰是在氨基酸侧链上引入可用于偶联的官能团。
  9. 根据权利要求8所述的多肽衍生物或其药学上可接受的盐，所述可用于偶联的官能团包括延长部分及连接延长部分与修饰氨基酸的接头。
  10. 根据权利要求9所述的多肽衍生物或其药学上可接受的盐，所述延长部分选自
    或
    其中x是4-38的任意整数；
    优选地，
    所述延长部分选自：
    HOOC(CH2)14CO-、HOOC(CH2)15CO-、HOOC(CH2)16CO-、HOOC(CH2)17CO-、HOOC(CH2)18CO-、HOOC(CH2)19CO-、HOOC(CH2)20CO-、HOOC(CH2)21CO-和HOOC(CH2)22CO-。
  11. 根据权利要求9或10所述的多肽衍生物或其药学上可接受的盐，所述接头选自：


    其中，m是0、1、2或3；n是1、2或3；s是0-6的任意整数；p是1-8的任意整数；
    优选地，所述接头选自：

    其中，m是1，n是1或2，p是1-5的任意整数。
  12. 根据权利要求11所述多肽衍生物或其药学上可接受的盐，选自：PD069、PD070、PD071、PD072、PD140、PD158、PD160、PD161、PD144、PD145、PD146、PD147或PD187。
  13. 根据权利要求12所述的多肽衍生物或其药学上可接受的盐是
  14. 根据权利要求12所述的多肽衍生物或其药学上可接受的盐是
  15. 根据权利要求12所述的多肽衍生物或其药学上可接受的盐是
  16. 根据权利要求12所述的多肽衍生物或其药学上可接受的盐是
  17. 一种制备权利要求1-16中任一项所述的多肽类似物或其衍生物或上述物质的药学上可接受的盐的方法。
  18. 权利要求1-16中任一项所述的多肽类似物或其衍生物或上述物质的药学上可接受的盐在制备用于预防或治疗疾病药物中的用途。
  19. 根据权利要求18所述的用途，所述疾病是胰淀素受体相关疾病。
  20. 根据权利要求19所述的用途，所述胰淀素受体相关疾病选自脂肪代谢障碍，血糖代谢障碍，心血管疾病，脑、精神或神经系统疾病。
  21. 一种治疗或/和预防疾病的方法，所述方法包括向有需要的受试者施用权利要求1-16中任一项所述多肽类似物或其衍生物，及上述物质的药学上可接受的盐、药物或药物组合物。
  22. 根据权利要求21所述的方法，其中所述疾病选自：脂肪代谢障碍、血糖代谢障碍、心血管疾病、脑、精神或神经系统疾病。
  23. 一种药物组合物，包括权利要求1-16中任一项所述的多肽类似物或其衍生物及上述物质的药学上可接受的盐。
  24. 根据权利要求23所述的药物组合物，还包括一种或多种口服递送剂；优选地，所述口服递送剂为N-[8-(2-羟基苯甲酰基)氨基]辛酸(NAC)盐；进一步优选为PNAC。
  25. 根据权利要求23或24所述的药物组合物，还包括另一种或多种具有药理学活性物质。
  26. 根据权利要求25所述的药物组合物，其中所述具有药理学活性物质选自：体重调节剂、抗肥胖剂、脂代谢调节剂、血糖调节剂、高血压治疗剂、心血管调节剂、脑系统调节剂、精神系统调节剂或神经系统调节剂。
PCT/CN2023/096811 2022-05-27 2023-05-29 人胰淀素类似物、其衍生物及用途 WO2023227133A1 (zh)

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CN101413009A (zh) * 2008-10-22 2009-04-22 广东暨大基因药物工程研究中心有限公司 一种改构的人淀粉样多肽突变体－普兰林肽(pramlintide)的制备方法
CN103596972A (zh) * 2011-06-10 2014-02-19 诺沃—诺迪斯克有限公司 多肽
WO2015040182A2 (en) * 2013-09-19 2015-03-26 Zealand Pharma A/S Amylin analogues
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CN101413009A (zh) * 2008-10-22 2009-04-22 广东暨大基因药物工程研究中心有限公司 一种改构的人淀粉样多肽突变体－普兰林肽(pramlintide)的制备方法
CN103596972A (zh) * 2011-06-10 2014-02-19 诺沃—诺迪斯克有限公司 多肽
WO2015040182A2 (en) * 2013-09-19 2015-03-26 Zealand Pharma A/S Amylin analogues
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