WO2020228610A1 - 多肽衍生物及其制备方法 - Google Patents

多肽衍生物及其制备方法 Download PDF

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Publication number
WO2020228610A1
WO2020228610A1 PCT/CN2020/089217 CN2020089217W WO2020228610A1 WO 2020228610 A1 WO2020228610 A1 WO 2020228610A1 CN 2020089217 W CN2020089217 W CN 2020089217W WO 2020228610 A1 WO2020228610 A1 WO 2020228610A1
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Prior art keywords
polypeptide
insulin
group
lysine
glp
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PCT/CN2020/089217
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English (en)
French (fr)
Inventor
查若鹏
张振山
吴松
刘慧玲
陈卫
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宁波鲲鹏生物科技有限公司
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Priority to CN202080035118.1A priority Critical patent/CN113811614A/zh
Priority to AU2020276560A priority patent/AU2020276560A1/en
Priority to EP20806421.2A priority patent/EP3967759A4/en
Priority to JP2021567053A priority patent/JP7453698B2/ja
Priority to US17/610,606 priority patent/US20220211857A1/en
Priority to CA3139826A priority patent/CA3139826A1/en
Priority to BR112021022629A priority patent/BR112021022629A2/pt
Publication of WO2020228610A1 publication Critical patent/WO2020228610A1/zh

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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
    • A61P3/08—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • 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
    • C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y601/00—Ligases forming carbon-oxygen bonds (6.1)
    • C12Y601/01—Ligases forming aminoacyl-tRNA and related compounds (6.1.1)
    • C12Y601/01026—Pyrrolysine-tRNAPyl ligase (6.1.1.26)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention belongs to the field of biomedicine. Specifically, the invention relates to a polypeptide derivative and a preparation method thereof.
  • peptide/protein drugs are eliminated in the body mainly through degradation, excretion, and receptor-mediated endocytosis.
  • Polypeptide factors with a molecular weight of less than 20kDa are easily filtered by the glomerulus during metabolism; when they pass through the renal tubules, they are partially degraded by the proteases and excreted in the urine, so the half-life is short.
  • GLP-1 as an example, its biological half-life in the body is generally 20 minutes.
  • frequent high-dose medication is required. Long-term frequent injections not only increase the patient’s pain and treatment costs, but also easily cause a series of serious side effects. .
  • the half-life of the protein or polypeptide in the blood can be prolonged by modifying the protein or polypeptide by fatty acid modification, and then through the non-covalent bond between the fatty acid and albumin.
  • the purpose of the present invention is to provide a new and longer-acting polypeptide derivative.
  • polypeptide derivative in the first aspect of the present invention, includes:
  • n 14 An integer from to 16.
  • the group Y is a group selected from the following group:
  • the polypeptide is selected from the group consisting of insulin, GLP-1, PTH, or a combination thereof.
  • the polypeptide derivative is selected from the group consisting of insulin derivatives, GLP-1 derivatives, PTH derivatives, or a combination thereof.
  • the A chain of the insulin has a sequence shown in SEQ ID NO.: 1 or 2.
  • the B chain of the insulin has a sequence as shown in SEQ ID NO.: 3, 4, 5 or 6.
  • the GLP-1 has a sequence as shown in any one of SEQ ID NO.: 7-9.
  • the PTH has a sequence shown in SEQ ID NO.:10.
  • the structure of the polypeptide derivative is as follows, wherein Is insulin, GLP-1 or PTH:
  • n is an integer of 14 to 16.
  • polypeptide derivative is selected from the group consisting of Is insulin, GLP-1 or PTH:
  • polypeptide derivative is selected from the group consisting of L0-GFA16-polypeptide, L2-GFA16-polypeptide, L3-GFA16-polypeptide, L4-GFA16-polypeptide, L5-GFA16-polypeptide, or L6- GFA16-polypeptide.
  • polypeptide derivative L0-GFA16-polypeptide is as follows, wherein Is a peptide:
  • polypeptide derivative L2-GFA16-polypeptide is as follows, wherein Is a peptide:
  • polypeptide derivative L3-GFA16-polypeptide is as follows, wherein Is a peptide:
  • polypeptide derivative L4-GFA16-polypeptide is as follows, wherein Is a peptide:
  • polypeptide derivative L5-GFA16-polypeptide is as follows, wherein Is a peptide:
  • polypeptide derivative L6-GFA16-polypeptide is as follows:
  • the insulin includes insulin A chain and B chain.
  • the insulin includes human insulin or animal insulin, preferably, the insulin is human insulin.
  • the animal insulin includes porcine insulin and bovine insulin.
  • the A chain and the B chain of the insulin further include one or more disulfide bonds.
  • the insulin includes natural insulin, an insulin precursor, or a variant of insulin.
  • the insulin derivative includes an insulin precursor and the modification group L.
  • the A chain of the insulin has a sequence shown in SEQ ID NO.: 1 or 2.
  • the B chain of the insulin has a sequence as shown in SEQ ID NO.: 3, 4, 5 or 6.
  • the A chain of the insulin has a sequence as shown in SEQ ID NO.:1
  • the B chain of the insulin has a sequence as shown in SEQ ID NO.: 3, 5 or 6.
  • the A chain of the insulin has a sequence as shown in SEQ ID NO.: 2
  • the B chain of the insulin has a sequence as shown in SEQ ID NO.: 4 or 6.
  • the modification group L is covalently linked to the lysine (K) site.
  • the modification group L is covalently linked to the ⁇ -amino group of the lysine (K).
  • the insulin comprises a PK, DKT, PKT or KPT motif, and the modification group L is connected to the lysine (K) site in the motif.
  • the insulin comprises a TPK, TKP or TDK motif, and the modification group L is connected to the lysine (K) site in the motif.
  • the insulin comprises a YTPK, YTDKT, YTPKT or YTKPT motif, and the modification group L is connected to the lysine (K) site in the motif.
  • the modification group L is connected to the 28th or 29th lysine (K) of the B chain.
  • the modification group L is covalently linked to the 29th lysine (K) in the sequence shown in SEQ ID NO.: 3, 4 or 6.
  • the B chain of the insulin has the sequence shown in SEQ ID NO.: 3, 4, or 6, and the modification group L is connected to the sequence shown in SEQ ID NO.: 3, 4, or 6.
  • the 29th lysine (K) in the sequence is shown.
  • the B chain of the insulin has the sequence shown in SEQ ID NO.: 5, and the modification group L is connected to the 28th lysine in the sequence shown in SEQ ID NO.: 5 Acid (K).
  • the insulin derivative is selected from the group consisting of L0-GFA16-insulin, L2-GFA16-insulin, L3-GFA16-insulin, L4-GFA16-insulin, L5-GFA16-insulin, or L6- GFA16-insulin.
  • the GLP-1 derivative includes a GLP-1 analog and the modification group L.
  • the GLP-1 has a sequence as shown in any one of SEQ ID NO.: 7-9.
  • the modification group L is covalently linked to the lysine (K) site.
  • the modification group L is covalently linked to the ⁇ -amino group of the lysine (K).
  • the GLP-1 contains an AKE motif, and the modification group L is connected to the lysine (K) site in the motif.
  • the GLP-1 contains an AAKEF motif, and the modification group L is connected to the lysine (K) site in the motif.
  • the modification group L is connected to the lysine (K) at position 20 or 26 of the chain.
  • the GLP-1 derivative is selected from the following group: L0-GFA16-GLP-1, L2-GFA16-GLP-1, L3-GFA16-GLP-1, L4-GFA16-GLP-1 , L5-GFA16-GLP-1, or L6-GFA16-GLP-1.
  • the PTH derivative includes a PTH analog and the modification group L.
  • the PTH has a sequence shown in SEQ ID NO.:10.
  • the modification group L is covalently linked to the lysine (K) site.
  • the modification group L is covalently linked to the ⁇ -amino group of the lysine (K).
  • the PTH comprises an RKR motif, and the modification group L is connected to the lysine (K) site in the motif.
  • the PTH comprises the LRKRL motif, and the modification group L is connected to the lysine (K) site in the motif.
  • the modification group L is connected to the lysine (K) at position 26 of the PTH.
  • the PTH derivative is selected from the group consisting of L0-GFA16-PTH, L2-GFA16-PTH, L3-GFA16-PTH, L4-GFA16-PTH, L5-GFA16-PTH, or L6- GFA16-PTH.
  • the second aspect of the present invention provides a pharmaceutical composition comprising the polypeptide derivative of the first aspect of the present invention and a pharmaceutically acceptable carrier.
  • the third aspect of the present invention provides the use of the polypeptide derivative as described in the first aspect of the present invention for the preparation of prevention and/or treatment of osteoporosis, diabetes, hyperglycemia and other beneficial effects of lowering blood sugar Drugs or preparations for diseases.
  • the fourth aspect of the present invention provides a method for preparing a polypeptide derivative, the method comprising the steps:
  • polypeptide derivatives include:
  • Modification group L said modification group L is connected to the lysine site of said polypeptide, and said modification group L is group X, which is the same as defined in the first aspect of the present invention ;
  • the polypeptide is selected from the group consisting of insulin, GLP-1, PTH, or a combination thereof.
  • the polypeptide derivative is selected from the group consisting of insulin derivatives, GLP-1 derivatives, PTH derivatives, or a combination thereof.
  • the fifth aspect of the present invention provides a method for preparing a polypeptide derivative, the method comprising the steps:
  • a, b, c, d, e, and f are each independently selected from an integer from 0 to 10; n is an integer from 14 to 16.
  • the polypeptide is selected from the group consisting of insulin, GLP-1, PTH, or a combination thereof.
  • the polypeptide derivative is selected from the group consisting of insulin derivatives, GLP-1 derivatives, PTH derivatives, or a combination thereof.
  • the sixth aspect of the present invention provides a method for preparing a polypeptide derivative, the method comprising the steps:
  • a, b, c, d, e, and f are each independently selected from an integer from 0 to 10; n is an integer from 14 to 16.
  • the polypeptide is selected from the group consisting of insulin, GLP-1, PTH, or a combination thereof.
  • the polypeptide derivative is selected from the group consisting of insulin derivatives, GLP-1 derivatives, PTH derivatives, or a combination thereof.
  • an intermediate is provided, and the intermediate includes:
  • polypeptide wherein the polypeptide is insulin, GLP-1 or PTH;
  • the wavy line indicates the connection position to the lysine site, and m is an integer of 0-8.
  • the intermediate has a structure shown in formula IV, wherein Is insulin, GLP-1 or PTH,
  • the intermediate is used to prepare the polypeptide derivative described in the first aspect of the present invention.
  • the present invention also provides the use of the intermediate described in the seventh aspect of the present invention, which is used to prepare the polypeptide derivative described in the first aspect of the present invention.
  • the inventors obtained a polypeptide derivative through extensive and in-depth research.
  • the experimental results show that the polypeptide derivative has a significantly prolonged half-life while maintaining biological activity.
  • the present invention also provides the pharmaceutical use of the polypeptide derivative, and its functions in treating or preventing diabetes, promoting bone cell formation and the like. On this basis, the inventor completed the present invention.
  • the ideal effect of long-acting insulin is to rebuild basic insulin secretion in diabetic patients through as few insulin injections as possible.
  • Chemical modification is one of the ways to obtain long-acting insulin.
  • the structure of chemical modifiers must be stable, non-toxic, non-antigenic and have a suitable molecular weight.
  • insulin can maintain its biological activity while prolonging its half-life and reducing its antigenicity.
  • the modified insulin derivative of the present invention is a polymer compound with good biocompatibility, is non-toxic to the human body, and the water solubility of the drug is increased. And it can reduce the glomerular clearance rate, increase the half-life of the drug circulating in the body, and obtain long-term effects.
  • the insulin, GLP-1, PTH protein and fatty acid acyl compounds containing butynoxycarbonyl-lysine of the present invention are connected by a click reaction to obtain a series of GLP-1 and PTH derivatives with significantly prolonged half-life.
  • the term “about” means that the value can vary from the recited value by no more than 1%.
  • the expression “about 100” includes all values between 99 and 101 (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the term "containing” or “including (including)” can be open, semi-closed, and closed. In other words, the term also includes “substantially consisting of” or “consisting of”.
  • GLP-1 is a glucose-dependent intestinal lowering polypeptide hormone. GLP-1 stimulates insulin secretion without hypoglycemia. This glucose-dependent insulin secretion promoting properties avoids the risk of hypoglycemia that often exists in the treatment of diabetes. These physiological functions make the development of GLP-1 as a type 2 diabetes treatment drug has broad prospects. GLP-1 usually acts on the receptor GLP-1 receptor (GLP-1R) on the pancreatic ⁇ cell membrane to promote the secretion of insulin.
  • GLP-1R GLP-1 receptor
  • natural GLP-1 has many advantages in the treatment of diabetes, it is rapidly degraded by Diyl Peptidase IV (DPP-IV) in the body.
  • DPP-IV Diyl Peptidase IV
  • natural GLP-1 will be rapidly filtered and metabolized by the kidneys, so we need to modify the natural GLP-1 in order to find GLP-1 analogues that can resist the degradation of DPP-IV and avoid rapid metabolism by the kidneys. .
  • GLP-1 has a blood sugar-dependent incretin secretion effect; prevents pancreatic ⁇ -cell degeneration, stimulates ⁇ -cell proliferation and differentiation; induces the transcription of pre-insulin genes, promotes pre-insulin biosynthesis; increases insulin sensitivity; Increase the secretion of somatostatin and inhibit the production of insulin and glucagon (this effect is also blood sugar dependent).
  • PTH Parathyroid hormone
  • Parathyroid hormone is a single-chain polypeptide protein containing 84 amino acids secreted by the parathyroid glands. It is one of the most important peptide hormones that regulate calcium and phosphorus metabolism and bone turnover.
  • the physiological function of hPTH is mainly to promote the osteogenesis of bone cells, stimulate the kidney's reabsorption of calcium, the secretion of phosphorus and bone reconstruction.
  • the main disadvantage of PTH is that hPTH molecule does not contain cysteine and is very unstable in the body.
  • PTHI has a small molecular weight and is easily filtered by the glomerulus, so its half-life in the body is short. The half-life of subcutaneous administration or intramuscular injection is generally about 12 hours.
  • the invention contains butynyloxycarbonyl-lysine-containing polypeptides (such as insulin, PTH, GLP-1) and fatty acid acyl compounds connected by click reaction to obtain a series of polypeptide derivatives with significantly prolonged half-life.
  • polypeptides such as insulin, PTH, GLP-1
  • fatty acid acyl compounds connected by click reaction to obtain a series of polypeptide derivatives with significantly prolonged half-life.
  • the fatty acid acyl compound of the present invention is a fatty acid acyl compound with 14-18 carbons, and the structural formula is the compound of formula V or formula VIII:
  • a, b, c, d, e, and f are each independently selected from an integer from 0 to 10; n is an integer from 14 to 16.
  • the fatty acid acyl compound is selected from the group consisting of L0-GFA, L2-GFA, L3-GFA, L4-GFA, L5-GFA, or L6-GFA, wherein n is an integer from 14 to 16 .
  • the fatty acid acyl compound is selected from the group consisting of L0-GFA16, L2-GFA16, L3-GFA16, L4-GFA16, L5-GFA16, or L6-GFA16.
  • polypeptide analog As used herein, the terms “polypeptide analog”, “polypeptide derivative”, and “derivative of the present invention” are used interchangeably, and all refer to the polypeptide derivative described in the first aspect of the present invention.
  • the present invention also provides a polypeptide derivative as described in the first aspect of the present invention.
  • polypeptide derivatives include:
  • n 14 An integer from to 16.
  • polypeptide derivatives include insulin derivatives, GLP-1 derivatives, PTH derivatives, or a combination thereof.
  • the A chain of the insulin has a sequence shown in SEQ ID NO.: 1 or 2.
  • the B chain of the insulin has a sequence as shown in SEQ ID NO.: 3, 4, 5 or 6.
  • the GLP-1 has a sequence as shown in any one of SEQ ID NO.: 7-9.
  • HXEGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO.: 9) (where X is 2-aminoisobutyric acid (Aib))
  • the PTH has a sequence shown in SEQ ID NO.:10.
  • insulin derivatives include insulin, insulin precursors and insulin variants.
  • the insulin variant is different from any naturally occurring insulin, but can still perform a similar effect to human insulin in a way of blood sugar control in the human body.
  • the amino acid sequence of insulin can be changed, thereby changing its absorption, distribution, metabolism and secretion properties.
  • Improvements include insulin analogues that are more easily absorbed by the injection site, and therefore act faster than subcutaneously injected natural insulin, and are designed to supply the level of insulin required for mealtimes (meal insulin); and those that are between 8 hours and 24 hours In between, slow-release insulin analogs are designed to provide a basal level of insulin (basal insulin) during the day, especially at night.
  • Fast-acting insulin analogs include insulin lispro (Lilly) and insulin aspart (Novo Nordisk), while long-acting insulin analogs include NPH insulin, insulin glulisine (Sanofi-Aventis), and insulin detemir (Novo Nordisk) ) And insulin glargine (Sanofi-Aventis).
  • the term "variant" includes any variant in which (a) one or more amino acid residues are replaced by a naturally or non-naturally occurring amino acid residue; (b) two or more amino acid residues The order is reversed; (c) (a) and (b) are both present; (d) there is a spacer group between any two amino acid residues; (e) one or more amino acid residues are in the form of peptoid; (f ) The (NCC) backbone of one or more amino acid residues of the peptide is modified, or any combination of (a) to (f).
  • the variant is one of (a), (b) or (c).
  • one or two amino acid residues are replaced by one or more other amino acid residues. Still more preferably, one amino acid residue is replaced by another amino acid residue. Preferably, the substitution is homologous.
  • Homologous substitutions may occur (the substitutions and substitutions used herein refer to the exchange of existing amino acid residues with optional residues), that is, homosexual substitutions, such as basic substitutions, acid substitutions, acid substitutions, and polar substitutions. Sex etc.
  • Non-homologous substitutions may also occur, that is, one residue is replaced by another, or alternatively includes unnatural amino acids such as ornithine, ornithine diaminobutyrate, norleucine ornithine, pyridine
  • amino acid, thienylalanine, naphthylalanine and phenylglycine More than one amino acid residue can be modified at the same time.
  • amino acids are classified according to the following categories: basic: H, K, R; acidic: D, E; non-polar: A, F, G, I, L, M, P, V, W; polar : C, N, Q, S, T, Y.
  • suitable spacer groups that can be inserted between any two amino acid residues of the carrier portion include: alkyl groups such as methyl, ethyl, or Propyl.
  • alkyl groups such as methyl, ethyl, or Propyl.
  • Type (f) modification can be performed by the method described in International Publication PCT/GB99/01855.
  • Amino acid variants preferably of type (a) or (b) preferably occur independently at any position. As mentioned above, more than one homologous or non-homologous replacement may occur simultaneously. Other variants can be obtained by reversing the sequence of some amino acid residues within the sequence.
  • the replacement amino acid residue is selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine Acid, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.
  • polypeptide derivatives of the present invention may exist in the form of salts or esters, especially pharmaceutically acceptable salts or esters.
  • the pharmaceutically acceptable salts of the compounds of the present invention include suitable acid addition or base salts thereof.
  • suitable pharmaceutically acceptable salts see the review by Berge et al. J Pharm Sci, 66, 1-19 (1977). It can be combined with strong inorganic acids, such as mineral acids (such as sulfuric acid, phosphoric acid, or hydrohalic acid); and strong organic carboxylic acids, such as unsubstituted or substituted (such as halogen substituted) alkanoic carboxylic acids with 1 to 4 carbon atoms (such as acetic acid); and saturated or unsaturated dicarboxylic acids, such as oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid or terephthalic acid; and hydroxycarboxylic acids, such as ascorbic acid, Glycolic acid, lactic acid, malic acid, tartaric acid or citric acid; with amino acids, such as aspartic acid or glutamic acid; with benzoic acid; or
  • the present invention also includes the form of solvates of the derivatives of the present invention.
  • the terms used in the claims include these forms.
  • the present invention also relates to various crystal forms, polymorphs and (anhydrous) hydrated forms of the analogs of the present invention.
  • the pharmaceutical industry has established a way that chemical compounds can be separated in any such form by slightly changing the purification method and/or separation method of the solvent used in the synthetic preparation of the compound.
  • the invention also includes the derivatives of the invention in the form of prodrugs.
  • prodrugs are generally derivatives of the invention in which one or more suitable groups have been modified so that the modification can be reversed when administered in a human or mammalian subject.
  • a second agent together with such a prodrug to effect the reversal in vivo
  • such reversal is usually performed by enzymes naturally present in the subject. Examples of such modifications include esters (such as any of those described above), where the reversal can be performed by an esterase. Other such systems are well known to those skilled in the art.
  • the insulin derivatives and GLP-1 derivatives of the present invention have a more durable, stable and long-term blood sugar lowering effect, and their half-life is significantly prolonged.
  • the insulin derivatives and GLP-1 derivatives of the present invention will not cause hypoglycemia, and can reduce the number of injections, with little side effects.
  • the PTH derivative of the present invention has a longer-lasting and stable drug effect, and its half-life is significantly prolonged.
  • the PTH derivative of the present invention can reduce the number of injections and has less side effects.
  • the preparation method of the present invention has few by-products, high yield, low cost, simple process, and is suitable for large-scale production. No need for cyanogen bromide cracking, oxidative sulfite hydrolysis and related purification steps. There is no need to use high concentrations of mercaptans or hydrophobic adsorption resins. There are few purification steps and low production cost.
  • Example 1 Synthesis of recombinant insulin protein containing butynyloxycarbonyl-lysine
  • a DNA fragment encoding a butynyloxycarbonyl-lysine recombinant insulin protein containing the amino acid sequence of SEQ ID NO.: 11 was chemically synthesized, wherein the coding sequence of lysine (K) at position 80 was replaced by TAG( Encoding lysine derivatives).
  • the DNA fragment encoding the complete amino acid sequence of SEQ ID NO.: 11 was then cloned into the modified pBAD-HisA vector.
  • the resulting plasmid is used for the expression of recombinant insulin protein with the structure of butynyloxycarbonyl-lysine.
  • the plasmid and enzyme plasmid pEvol-pylRs-pylT were transformed into E.
  • the transformant was cultured on LB agar medium containing 25 ⁇ g/mL kanamycin and 17 ⁇ g/mL chloramphenicol at 37° C. overnight. A single colony was picked and cultured overnight in an LB liquid medium containing 25 ⁇ g/mL kanamycin and 17 ⁇ g/mL chloramphenicol on a constant temperature shaker at 37° C. and 220 rpm. Then, the overnight culture was inoculated into 100 ml TB liquid medium containing 25 ⁇ g/mL kanamycin and 17 ⁇ g/mL chloramphenicol, and cultured at 37° C. until the OD 600 was 2-4.
  • K at position 80 is a lysine covalently linked to a butynyloxycarbonyl group.
  • the fusion protein is expressed in the form of insoluble "inclusion bodies".
  • crush the E. coli cells with a high-pressure homogenizer remove cell debris and soluble E. coli host protein by a 5000g centrifugation method, wash the inclusion bodies with a solution containing Tween 80, EDTA, and NaCl, and then wash the inclusion bodies with pure water 1-2 times.
  • the washed inclusion bodies are dissolved in 7.5M urea with a pH of 10.5-11.5 and containing 2-10mM ⁇ -mercaptoethanol, so that the concentration of the total protein after dissolution is 10-25mg/ml.
  • the insulin protein with a terminal alkyne is introduced.
  • the alkyne reacts with the azide to form a 1,2,3-triazole ring to form a crosslink.
  • the solution can be diluted to the proper volume or protein concentration.
  • Example 1 the compound IV prepared in Example 1 was subjected to click reaction with L3-GFA16, L4-GFA16, L5-GFA16, and L6-GFA16, respectively, and the structure of the product obtained was as follows.
  • Example 4 Study on the pharmacokinetics of insulin derivatives of the present invention in rats
  • the experiment was divided into a control product group and a test product group.
  • Recombinant human insulin and L6-GFA16-insulin were injected subcutaneously, respectively, at a dose of 0.45 mg/kg, in a single administration.
  • Animals in each group were collected blood at 15min, 30min, 1h, 2h, 3h, 5h, 7h, 12h, 24h after administration.
  • LC-MS/MS analysis method was used to detect the content of different insulin analogs.
  • the kinetic data analysis software WinNonlin 7.0 was used to calculate the plasma concentration data
  • NCA non-compartmental model method
  • a DNA fragment encoding the GLP-1 protein of butynyloxycarbonyl-lysine containing the amino acid sequence of SEQ ID NO.: 12 was chemically synthesized, wherein the coding sequence of lysine (K) at position 70 was replaced by TAG (Encoding lysine derivative).
  • the DNA fragment encoding the complete amino acid sequence of SEQ ID NO.: 12 was then cloned into the modified pBAD-HisA vector.
  • the resulting plasmid is used to express the recombinant GLP-1 protein with the structure of butynoxycarbonyl-lysine.
  • the plasmid and enzyme plasmid pEvol-pylRs-pylT were transformed into E. coli strain Top10 together.
  • the transformant was cultured on LB agar medium containing 25 ⁇ g/mL kanamycin and 17 ⁇ g/mL chloramphenicol at 37° C. overnight. A single colony was picked and cultured overnight in an LB liquid medium containing 25 ⁇ g/mL kanamycin and 17 ⁇ g/mL chloramphenicol on a constant temperature shaker at 37° C. and 220 rpm. Then, the overnight culture was inoculated into 100 ml TB liquid medium containing 25 ⁇ g/mL kanamycin and 17 ⁇ g/mL chloramphenicol, and cultured at 37° C. until the OD 600 was 2-4.
  • K at position 70 is a lysine covalently attached to a butynyloxycarbonyl group.
  • the fusion protein is expressed in the form of insoluble "inclusion bodies".
  • the E. coli cells were crushed with a high-pressure homogenizer, cell debris and soluble E. coli host proteins were removed by a 5000g centrifugation method, the inclusion bodies were washed with a solution containing Tween 80, EDTA, and NaCl, and then the inclusions were washed with pure water Body 1-2 times.
  • the washed inclusion bodies are dissolved in 7.5M urea with a pH of 10.5-11.5 and containing 2-10mM ⁇ -mercaptoethanol, so that the concentration of the total protein after dissolution is 10-25mg/ml.
  • the sample Dilute the sample 5-10 times, maintain 4-8°C, and perform routine folding for 14-30 hours under the condition of 10.5-11.7.
  • the fusion protein is separated and purified with a weak anion filler under pH 9.0 conditions, and the electrophoresis purity of the target protein reaches 80%.
  • the high-salt eluted sample was desalted and maintained at a pH of about 8.0-9.0 at 25°C, and digested with enterokinase for about 10-20 hours.
  • the reverse-phase HPLC analysis showed that the digestion step produced The rate is higher than 90%.
  • the GLP-1 analog obtained after enterokinase cleavage was named butynoxycarbonyl-lysine-GLP-1. After enzyme digestion, it is purified by hydrophobic filler to extract butynoxycarbonyl-lysine-GLP-1, and the electrophoresis purity reaches 90%.
  • GLP-1 protein with terminal alkyne is introduced. Using the principle of "click chemistry", the alkyne reacts with azide to form a 1,2,3-triazole ring to form a crosslink. Add 4 ⁇ L of copper sulfate (50 ⁇ M) to a clean 1.5ml centrifuge tube, and then add 3 ⁇ L of BTTAA (300 ⁇ M) and 10 ⁇ L of the IV compound prepared in Example 5 (N-(butynyloxycarbonyl)-lysine GLP) in sequence -1 protein) (about 5 ⁇ M). At this point, the solution can be diluted to the proper volume or protein concentration.
  • Example 7 Synthesis of L2-GFA16-GLP-1, L3-GFA16-GLP-1, L4-GFA16-GLP-1, L5-GFA16-GLP-1, L6-GFA16-GLP-1 (n is 14)
  • Example 5 the compound IV prepared in Example 5 and L3-GFA16, L4-GFA16, L5-GFA16, and L6-GFA16 were respectively subjected to click reaction to obtain the following product structures.
  • the experiment was divided into a reference product group and a test product group.
  • GLP-1 and L6-GFA16-GLP-1 were injected intravenously, at a dose of 0.5 mg/kg, in a single dose.
  • Animals in each group were collected blood at 15min, 30min, 1h, 2h, 3h, 5h, 7h, 12h, 24h after administration.
  • LC-MS/MS analysis method was used to detect the content of different insulin analogs.
  • the kinetic data analysis software WinNonlin 7.0 was used to calculate the plasma concentration data, and the non-compartmental model method (NCA) was used to calculate the pharmacokinetic parameters (Table 2).
  • the DNA fragment encoding the butynyloxycarbonyl-lysine PTH protein containing the amino acid sequence of SEQ ID NO.: 13 was chemically synthesized, wherein the coding sequence of Lysine (K) at position 76 was replaced with TAG (coding Lysine derivatives).
  • the DNA fragment encoding the complete amino acid sequence of SEQ ID NO.: 13 was then cloned into the modified pBAD-HisA vector.
  • the resulting plasmid is used to express the recombinant PTH protein with the structure of butynoxycarbonyl-lysine.
  • the plasmid and enzyme plasmid pEvol-pylRs-pylT were transformed into E. coli strain Top10 together.
  • the transformant was cultured on LB agar medium containing 25 ⁇ g/mL kanamycin and 17 ⁇ g/mL chloramphenicol at 37° C. overnight. A single colony was picked and cultured overnight in an LB liquid medium containing 25 ⁇ g/mL kanamycin and 17 ⁇ g/mL chloramphenicol on a constant temperature shaker at 37° C. and 220 rpm. Then, the overnight culture was inoculated into 100 ml TB liquid medium containing 25 ⁇ g/mL kanamycin and 17 ⁇ g/mL chloramphenicol, and cultured at 37° C. until the OD 600 was 2-4.
  • K at position 76 is a lysine covalently attached to a butynyloxycarbonyl group.
  • the fusion protein is expressed in the form of insoluble "inclusion bodies".
  • the E. coli cells were crushed with a high-pressure homogenizer, cell debris and soluble E. coli host proteins were removed by a 5000g centrifugation method, the inclusion bodies were washed with a solution containing Tween 80, EDTA, and NaCl, and then the inclusions were washed with pure water Body 1-2 times.
  • the washed inclusion bodies are dissolved in 7.5M urea with a pH of 10.5-11.5 and containing 2-10mM ⁇ -mercaptoethanol, so that the concentration of the total protein after dissolution is 10-25mg/ml.
  • the fusion protein is separated and purified with a weak anion filler under pH 9.0 conditions, and the electrophoresis purity of the target protein reaches 80%.
  • the high-salt eluted sample was desalted and maintained at a pH of about 8.0-9.0 at 25°C, and digested with enterokinase for about 10-20 hours.
  • the reverse-phase HPLC analysis showed that the digestion step produced The rate is higher than 90%.
  • the PTH analogue obtained after enterokinase cleavage was named butynoxycarbonyl-lysine-PTH. After enzyme digestion, it is purified by hydrophobic filler to extract butynyloxycarbonyl-lysine-PTH, and the electrophoresis purity reaches 90%.
  • the method was the same as in Examples 6 and 7.
  • the PTH protein containing N-(butynyloxycarbonyl)-lysine was substituted for the GLP-1 protein containing N-(butynyloxycarbonyl)-lysine to synthesize L0-GFA16 -PTH, L2-GFA16-PTH, L3-GFA16-PTH, L4-GFA16-PTH, L5-GFA16-PTH and L6-GFA16-PTH.
  • the experiment was divided into a reference product group and a test product group.
  • PTH and L6-GFA16-PTH were injected subcutaneously, at a dose of 8.6 ⁇ g/kg, in a single dose.
  • Animals in each group were collected blood at the time points of 0 min, 5 min, 15 min, 30 min, 1 h, 2 h, and 4 h after administration.
  • LC-MS/MS analysis method was used to detect the content of different insulin analogs.
  • the kinetic data analysis software WinNonlin 7.0 was used to calculate the plasma concentration data, and the non-compartmental model method (NCA) was used to calculate the pharmacokinetic parameters (Table 3). It can be seen from Table 3 that the peak time in L6-GFA16-PTH animals was prolonged to about 1.6h, the drug exposure time in the body was prolonged, and the exposure increased.
  • the difference is that the L0-GFA16-PTH, L2-GFA16-PTH, L3-GFA16-PTH, L4-GFA16-PTH, L5-GFA16-PTH of the present invention are used.
  • the PTH derivatives of the present invention can significantly extend the half-life while maintaining biological activity.

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Abstract

提供了多肽衍生物及其制备方法。具体地，提供了一种多肽衍生物。实验结果表明，所述多肽衍生物在保持生物活性的同时半衰期显著延长。还公开了所述多肽衍生物的制备方法，及其在治疗中的作用。

Description

多肽衍生物及其制备方法 技术领域
本发明属于生物医药领域，具体地说，本发明涉及多肽衍生物及其制备方法。
背景技术
药代动力学研究表明多肽/蛋白类药物主要通过降解、排泄、以及受体介导的内吞等作用在体内被清除。其中分子量小于20kDa的多肽因子在代谢过程中易被肾小球滤过；通过肾小管时多肽因子又被其中的蛋白酶部分降解并从尿中排出，因而半衰期短。以GLP-1为例，其体内生物半衰期一般在20min,为了达到的治疗效果，需要频繁的大剂量用药，长期的频繁注射不仅增加了病人的痛苦和治疗费用，易引发一系列严重的毒副作用。长效多肽/蛋白类药物的开发已成为对第一代基因工程多肽/蛋白药物进行二次开发的重要方向。目前延长蛋白药物半衰期的主要基于增大蛋白药物的分子量，减少肾小球滤过率减少异源蛋白的免疫原性，从而减少其体内清除率；持续缓慢释放维持药物浓度，延长药物作用时间等二个方面考虑。常用技术有：制备缓释制剂、构建突变体、化学修饰以及基因融合等。
因此，本发明通过将蛋白或者多肽进行脂肪酸化修饰，再通过脂肪酸与白蛋白的非共价键结合可以使蛋白或者多肽在血液中的半衰期延长。
发明内容
本发明的目的是提供一种新的、更长效的多肽衍生物。
本发明的第一方面，提供了一种多肽衍生物，所述多肽衍生物包括：
(a)多肽；和
(b)修饰基团L，所述修饰基团L连接于所述多肽的赖氨酸位点，且所述修饰基团L为式I所示的基团，
Figure PCTCN2020089217-appb-000001
其中，波浪线表示与所述赖氨酸位点的连接位置，m为0-8的整数；a、b、c、d、e、f各自独立的选自0至10的整数；n为14至16的整数。
在另一优选例中，所述基团Y为选自下组的基团：
Figure PCTCN2020089217-appb-000002
在另一优选例中，所述多肽选自下组：胰岛素、GLP-1、PTH、或其组合。
在另一优选例中，所述多肽衍生物选自下组：胰岛素衍生物、GLP-1衍生物、PTH衍生物、或其组合。
在另一优选例中，所述胰岛素的A链具有如SEQ ID NO.:1或2所示的序列。
在另一优选例中，所述胰岛素的B链具有如SEQ ID NO.:3、4、5或6所示的序列。
在另一优选例中，所述GLP-1具有如SEQ ID NO.:7-9中任一所示的序列。
在另一优选例中，所述PTH具有如SEQ ID NO.:10所示的序列。
在另一优选例中，所述多肽衍生物的结构如下所示，其中
Figure PCTCN2020089217-appb-000003
为胰岛素、GLP-1或PTH：
Figure PCTCN2020089217-appb-000004
其中，m为0-8的整数；a、b、c、d、e、f各自独立的选自0至10的整数；n为14至16的整数。
在另一优选例中，所述多肽衍生物选自下组，其中
Figure PCTCN2020089217-appb-000005
为胰岛素、GLP-1或PTH：
Figure PCTCN2020089217-appb-000006
在另一优选例中，所述多肽衍生物选自下组：L0-GFA16-多肽、L2-GFA16-多肽、L3-GFA16-多肽、L4-GFA16-多肽、L5-GFA16-多肽、或L6-GFA16-多肽。
在另一优选例中，所述多肽衍生物L0-GFA16-多肽的结构如下，其中
Figure PCTCN2020089217-appb-000007
为多肽：
Figure PCTCN2020089217-appb-000008
在另一优选例中，所述多肽衍生物L2-GFA16-多肽的结构如下，其中
Figure PCTCN2020089217-appb-000009
为多肽：
Figure PCTCN2020089217-appb-000010
在另一优选例中，所述多肽衍生物L3-GFA16-多肽的结构如下，其中
Figure PCTCN2020089217-appb-000011
为多肽：
Figure PCTCN2020089217-appb-000012
在另一优选例中，所述多肽衍生物L4-GFA16-多肽的结构如下，其中
Figure PCTCN2020089217-appb-000013
为多肽：
Figure PCTCN2020089217-appb-000014
在另一优选例中，所述多肽衍生物L5-GFA16-多肽的结构如下，其中
Figure PCTCN2020089217-appb-000015
为多肽：
Figure PCTCN2020089217-appb-000016
在另一优选例中，所述多肽衍生物L6-GFA16-多肽的结构如下：
Figure PCTCN2020089217-appb-000017
在另一优选例中，所述胰岛素包括胰岛素A链和B链。
在另一优选例中，所述胰岛素包括人胰岛素或动物胰岛素，较佳地，所述胰岛素为人胰岛素。
在另一优选例中，所述动物胰岛素包括猪胰岛素、牛胰岛素。
在另一优选例中，所述胰岛素的A链和B链之间还包括一个或多个二硫键。
在另一优选例中，所述胰岛素包括天然胰岛素、胰岛素前体、或胰岛素的变体。
在另一优选例中，所述胰岛素衍生物包括胰岛素前体和所述修饰基团L。
在另一优选例中，所述胰岛素的A链具有如SEQ ID NO.:1或2所示的序列。
在另一优选例中，所述胰岛素的B链具有如SEQ ID NO.:3、4、5或6所示的序列。
在另一优选例中，所述胰岛素的A链具有如SEQ ID NO.:1所示的序列，所述胰岛素的B链具有如SEQ ID NO.:3、5或6所示的序列。
在另一优选例中，所述胰岛素的A链具有如SEQ ID NO.:2所示的序列，所述胰岛素的B链具有如SEQ ID NO.:4或6所示的序列。
在另一优选例中，所述修饰基团L与所述赖氨酸(K)位点共价连接。
在另一优选例中，所述修饰基团L与所述赖氨酸(K)的ε-氨基共价连接。
在另一优选例中，所述胰岛素包含PK、DKT、PKT或KPT基序，并且所述修饰基团L连接于所述基序中的赖氨酸(K)位点。
在另一优选例中，所述胰岛素包含TPK、TKP或TDK基序，并且所述修饰基团L连接于所述基序中的赖氨酸(K)位点。
在另一优选例中，所述胰岛素包含YTPK、YTDKT、YTPKT或YTKPT基序，并且所述修饰基团L连接于所述基序中的赖氨酸(K)位点。
在另一优选例中，所述修饰基团L连接于所述B链的第28位或第29位赖氨酸(K)。
在另一优选例中，所述修饰基团L与对应于SEQ ID NO.:3、4或6所示序列中第29位赖氨酸(K)共价连接。
在另一优选例中，所述胰岛素的B链具有如SEQ ID NO.:3、4或6所示的序列，且所述修饰基团L连接于SEQ ID NO.:3、4或6所示序列中第29位赖氨酸(K)。
在另一优选例中，所述胰岛素的B链具有如SEQ ID NO.:5所示的序列，且所述修饰基团L连接于SEQ ID NO.:5所示序列中第28位赖氨酸(K)。
在另一优选例中，所述胰岛素衍生物选自下组：L0-GFA16-胰岛素、L2-GFA16-胰岛素、L3-GFA16-胰岛素、L4-GFA16-胰岛素、L5-GFA16-胰岛素、或L6-GFA16-胰岛素。
在另一优选例中，所述GLP-1衍生物包括GLP-1类似物和所述修饰基团L。
在另一优选例中，所述GLP-1具有如SEQ ID NO.:7-9中任一所示的序列。
在另一优选例中，所述修饰基团L与所述赖氨酸(K)位点共价连接。
在另一优选例中，所述修饰基团L与所述赖氨酸(K)的ε-氨基共价连接。
在另一优选例中，所述GLP-1包含AKE基序，并且所述修饰基团L连接于所述基序中的赖氨酸(K)位点。
在另一优选例中，所述GLP-1包含AAKEF基序，并且所述修饰基团L连接于所述基序中的赖氨酸(K)位点。
在另一优选例中，所述修饰基团L连接于所述链的第20位或第26位赖氨酸(K)。
在另一优选例中，所述GLP-1衍生物选自下组：L0-GFA16-GLP-1、L2-GFA16-GLP-1、L3-GFA16-GLP-1、L4-GFA16-GLP-1、L5-GFA16-GLP-1、或L6-GFA16-GLP-1。
在另一优选例中，所述PTH衍生物包括PTH类似物和所述修饰基团L。
在另一优选例中，所述PTH具有如SEQ ID NO.:10所示的序列。
在另一优选例中，所述修饰基团L与所述赖氨酸(K)位点共价连接。
在另一优选例中，所述修饰基团L与所述赖氨酸(K)的ε-氨基共价连接。
在另一优选例中，所述PTH包含RKR基序，并且所述修饰基团L连接于所述基序中的赖氨酸(K)位点。
在另一优选例中，所述PTH包含LRKRL基序，并且所述修饰基团L连接于所述基序中的赖氨酸(K)位点。
在另一优选例中，所述修饰基团L连接于所述PTH的第26位赖氨酸(K)。
在另一优选例中，所述PTH衍生物选自下组：L0-GFA16-PTH、L2-GFA16-PTH、L3-GFA16-PTH、L4-GFA16-PTH、L5-GFA16-PTH、或L6-GFA16-PTH。
本发明的第二方面，提供了一种药物组合物，所述药物组合物包含本发明第一方面所述的多肽衍生物，以及药学上可接受的载体。
本发明的第三方面，提供了如本发明第一方面所述的多肽衍生物的用途，用于制备预防和/或治疗骨质疏松症、糖尿病、高血糖症及其他降低血糖可获益的疾病的药物或制剂。
本发明的第四方面，提供了一种多肽衍生物的制备方法，所述方法包括步骤：
(1)在X基团-赖氨酸、吡咯赖氨酰基-tRNA合成酶及其同源关联tRNA的存在下，培养含有胰岛素编码序列的菌株，其中，所述编码序列中，多肽中的赖氨酸的编码序列替换为TAG(编码赖氨酸衍生物)，从而产生多肽衍生物，其中所述多肽衍生物包括：
(a)多肽链；和
(b)修饰基团L，所述修饰基团L连接于所述多肽的赖氨酸位点，且所述修饰基团L为基团X，基团X同本发明第一方面中所定义；和任选地
(2)从发酵产物中分离所述的多肽衍生物。
在另一优选例中，所述多肽选自下组：胰岛素、GLP-1、PTH、或其组合。
在另一优选例中，所述多肽衍生物选自下组：胰岛素衍生物、GLP-1衍生物、PTH衍生物、或其组合。
本发明的第五方面，提供了一种多肽衍生物的制备方法，所述方法包括步骤：
(1)在式III化合物、吡咯赖氨酰基-tRNA合成酶及其同源关联tRNA的存在下，
培养含有多肽编码序列的菌株，其中，所述编码序列中，多肽中的赖氨酸的编码序列替换为TAG(编码赖氨酸衍生物)，从而得到式IV化合物；和
Figure PCTCN2020089217-appb-000018
(2)在惰性溶剂中，将式IV化合物与式V化合物进行反应，从而得到多肽衍生物，
Figure PCTCN2020089217-appb-000019
式V中，a、b、c、d、e、f各自独立的选自0至10的整数；n为14至16的整数。
在另一优选例中，所述多肽选自下组：胰岛素、GLP-1、PTH、或其组合。
在另一优选例中，所述多肽衍生物选自下组：胰岛素衍生物、GLP-1衍生物、PTH衍生物、或其组合。
本发明的第六方面，提供了一种多肽衍生物的制备方法，所述方法包括步骤：
(1)在式VI化合物、吡咯赖氨酰基-tRNA合成酶及其同源关联tRNA的存在下，
培养含有多肽编码序列的菌株，其中，所述编码序列中多肽中的赖氨酸的编码序列替换为TAG(编码赖氨酸衍生物)，从而得到式VII化合物；和
Figure PCTCN2020089217-appb-000020
(2)在惰性溶剂中，将式VII化合物与式VIII化合物进行反应，从而得到多肽衍生物，
Figure PCTCN2020089217-appb-000021
式VIII中a、b、c、d、e、f各自独立的选自0至10的整数；n为14至16的整数。
在另一优选例中，所述多肽选自下组：胰岛素、GLP-1、PTH、或其组合。
在另一优选例中，所述多肽衍生物选自下组：胰岛素衍生物、GLP-1衍生物、PTH衍生物、或其组合。
本发明的第七方面，提供了一种中间体，所述中间体包括：
(a)多肽，其中所述多肽为胰岛素、GLP-1或PTH；和
(b)修饰基团L，所述修饰基团L连接于所述多肽的赖氨酸位点，且所述修饰基团L为式A所示的基团，
Figure PCTCN2020089217-appb-000022
其中，波浪线表示与所述赖氨酸位点的连接位置，m为0-8的整数。
在另一优选例中，所述中间体具有如式IV所示的结构，其中
Figure PCTCN2020089217-appb-000023
为胰岛素、GLP-1或PTH，
Figure PCTCN2020089217-appb-000024
在另一优选例中，所述中间体用于制备本发明第一方面所述的多肽衍生物。
本发明还提供了本发明第七方面所述的中间体的用途，所述中间体用于制备本发明第一方面所述的多肽衍生物。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
具体实施方式
本发明人通过广泛而深入的研究，获得一种多肽衍生物，实验结果表明，所述多肽衍生物在保持生物活性的同时，具有显著延长的半衰期。本发明还提供了所述多肽衍生物的制药用途，及其在治疗或预防糖尿病、促进骨细胞的成骨等作用。在此基础上，发明人完成了本发明。
长效胰岛素的理想效果是通过尽可能少的胰岛素注射次数，在糖尿病患者体内重建胰岛素基础分泌。化学修饰是获得长效胰岛素的途径之一，化学修饰剂的结构必须稳定、无毒性、无抗原性并具有合适大小的分子量。经过本发明特定的化学修饰，可使胰岛素在保持生物活性的同时，半衰期延长，抗原性降低。本发明经过修饰的胰岛素衍生物是一种具有较好生物相容性的高分子化合物，对人体无毒性，药物的水溶性增加。并且可以降低肾小球对其的清除速率，增加药物在体内循环的半衰期，从而获得长效。本发明的含有丁炔氧羰基-赖氨酸的胰岛素、GLP-1、PTH蛋白与脂肪酸酰基化合物通过点击反应连接，得到一系列半衰期显著延长的GLP-1、PTH衍生物。
术语
在描述本发明之前，应当理解本发明不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且不意图是限制性的，本发明的范围将仅由所附的权利要求书限制。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。
如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
GLP-1
GLP-1是一种葡萄糖依赖性肠降多肽激素，GLP-1刺激胰岛素分泌而不出现低血糖，这种葡萄糖依赖性的促胰岛素分泌特性，避免了糖尿病治疗中常存在的低血糖症的危险，这些生理功能使开发GLP-1作为一种2型糖尿病治疗药物具有广阔的前景。GLP-1通常作用于胰岛β细胞膜上的受体GLP-1受体(GLP-1R)，促进胰岛素素的分泌。然而，虽然天然的GLP-1在治疗糖尿病上有诸多优点，但它在体内却会被二基肽酶IV(DPP-IV)快速降解。另外天然的GLP-1会被肾脏快速滤过代谢，所以这需要我们对天然的GLP-1进行改造，以期望寻找到能够抵抗DPP-IV降解，避免肾脏快速滤过代谢的GLP-1类似物。
GLP-1具有血糖依赖性的肠促胰岛素分泌作用；阻止胰腺β-细胞退化，刺激β-细胞的增殖和分化；诱导前胰岛素基因的转录，促进前胰岛素的生物合成；增加胰岛素的敏感性；增加生长抑素分泌，抑制胰岛素高血糖素的产生(此作用也是血糖依赖性)。
甲状旁腺激素(PTH)
甲状旁腺激素(PTH)是由甲状旁腺分泌的含84个氨基酸的单链多肽蛋白质，它是调节钙、磷代谢及骨转换最为重要的肽类激素之一。hPTH的生理功能主要是促进骨细胞的成骨作用，刺激肾对钙的重吸收、磷的分泌和骨的重建。PTH目前的缺点主要是hPTH分子不含半胱氨酸，在体内很不稳定。PTHI分子质量较小，易被肾小球滤过，因而在体内的半衰期较短，皮下给药或肌肉注射的半衰期一般在12h左右。为了达到治疗效果，一般需要频繁大剂量用药(每天皮下注射一次，连续几个月)，但如此频繁的用药方式再加上较长的治疗周期使得病人较难承受，而且临床上会造成头疼、呕吐、发烧等不良反应，病人的顺应性差。所以目前急需有PTH的长效制剂或者类似物，通过对甲状旁腺激素加以修饰，以延长其半衰期。
脂肪酸酰基化合物
本发明含有丁炔氧羰基-赖氨酸的多肽(如胰岛素、PTH、GLP-1)与脂肪酸酰基化合物通过点击反应连接，得到一系列半衰期显著延长的多肽衍生物。
本发明脂肪酸酰基化合物为14-18个碳的脂肪酸酰基化合物，结构式如下式V或式VIII化合物：
Figure PCTCN2020089217-appb-000025
其中：a、b、c、d、e、f各自独立的选自0至10的整数；n为14至16的整数。
在另一优选例中，所述脂肪酸酰基化合物选自下组：L0-GFA、L2-GFA、L3-GFA、L4-GFA、L5-GFA、或L6-GFA，其中n为14至16的整数。
Figure PCTCN2020089217-appb-000026
Figure PCTCN2020089217-appb-000027
在另一优选例中，所述脂肪酸酰基化合物选自下组：L0-GFA16、L2-GFA16、L3-GFA16、L4-GFA16、L5-GFA16、或L6-GFA16。
Figure PCTCN2020089217-appb-000028
Figure PCTCN2020089217-appb-000029
多肽衍生物
如本文所用，术语“多肽类似物”、“多肽衍生物”、“本发明衍生物”可互换使用，均指本发明第一方面所述的多肽衍生物。
本发明还提供了一种多肽衍生物，所述多肽衍生物如本发明第一方面所述。
具体地，所述多肽衍生物包括：
(a)多肽链；和
(b)修饰基团L，所述修饰基团L连接于所述多肽链的赖氨酸位点，且所述修饰基团L为式I所示的基团，
Figure PCTCN2020089217-appb-000030
其中，波浪线表示与所述赖氨酸位点的连接位置，m为0-8的整数；a、b、c、d、e、f各自独立的选自0至10的整数；n为14至16的整数。
在另一优选例中，所述多肽衍生物包括胰岛素衍生物、GLP-1衍生物、PTH衍生物、或其组合。
在另一优选例中，所述胰岛素的A链具有如SEQ ID NO.:1或2所示的序列。
在另一优选例中，所述胰岛素的B链具有如SEQ ID NO.:3、4、5或6所示的序列。
GIVEQCCTSICSLYQLENYCN(SEQ ID NO.:1)
GIVEQCCTSICSLYQLENYCG(SEQ ID NO.:2)
FVNQHLCGSHLVEALYLVCGERGFFYTPKT(SEQ ID NO.:3)
FVNQHLCGSHLVEALYLVCGERGFFYTPK(SEQ ID NO.:4)
FVNQHLCGSHLVEALYLVCGERGFFYTKPT(SEQ ID NO.:5)
FVNQHLCGSHLVEALYLVCGERGFFYTDKT(SEQ ID NO.:6)
在另一优选例中，所述GLP-1具有如SEQ ID NO.:7-9中任一所示的序列。
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG(SEQ ID NO.:7)
HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG(SEQ ID NO.:8)
HXEGTFTSDVSSYLEGQAAKEFIAWLVRGRG(SEQ ID NO.:9)(其中X为2-氨基异丁酸(Aib))
在另一优选例中，所述PTH具有如SEQ ID NO.:10所示的序列。
SVSEIQLMHNLGRHLNSMERVEWLRKRLQDVHNF(SEQ ID NO.:10)
在本发明中，胰岛素衍生物包括胰岛素、胰岛素前体和胰岛素的变体。所述胰岛素的变体不同于任何天然存在的胰岛素，但仍然可以在人体内以血糖控制的方式执行与人胰岛素类似的作用。通过基本(underlying)DNA的遗传工程，可以改变胰岛素的氨基酸序列，从而改变其吸收、分布、代谢和分泌特性。改良包括更容易被注射位点吸收的胰岛素类似物，因此比皮下注射的天然胰岛素作用更快，旨在供应进餐时间所需胰岛素的药物水平(餐时胰岛素)；而那些在8小时至24小时之间缓慢释放的胰岛素类似物，旨在在白天特别是在夜间提供基础水平的胰岛素(基础胰岛素)。速效胰岛素类似物包括赖脯胰岛素(礼来公司)和门冬胰岛素(Novo Nordisk公司)，而长效胰岛素类似物包括NPH胰岛素、赖谷胰岛素(Sanofi-Aventis公司)、地特胰岛素(Novo Nordisk公司)和甘精胰岛素(赛诺菲-安万特公司)。
如本文所用，术语“变体”包括任意变体，其中(a)一个或多个氨基酸残基被一个天然或非天然存在的氨基酸残基置换；(b)两个或多个氨基酸残基的顺序颠倒；(c)(a)和(b)都同时存在；(d)任意两个氨基酸残基之间存在间隔基团；(e)一个或多个氨基酸残基为类肽形式；(f)肽的一个或多个氨基酸残基的(N-C-C)主链被修饰，或(a)至(f)的任意组合。优选地，所述变体为(a)、(b)或(c)之一。
更优选地，一个或两个氨基酸残基被一个或多个其它的氨基酸残基置换。还更优选地，一个氨基酸残基被另一个氨基酸残基置换。优选地，所述置换是同源性的。
可能发生同源置换(本文使用的置换和替代均是指现存的氨基酸残基与可选的残基的互换)，即同性置换，如碱性置换碱性、酸性置换酸性、极性置换极性等。也可能发生非同源置换，即一种残基置换为另一种、或可选地包括非天然氨基酸，如鸟氨酸、二氨基丁酸鸟氨酸、正亮氨酸鸟氨酸、吡啶基丙氨酸、噻吩基丙氨酸、萘基丙氨酸和苯甘氨酸，下 面列出了更详细的列表。同时可以有多于一个氨基酸残基被修饰。如本文所用，根据以下类别对氨基酸分类：碱性：H、K、R；酸性：D、E；非极性：A、F、G、I、L、M、P、V、W；极性：C、N、Q、S、T、Y。
除了氨基酸间隔基团(如甘氨酸或β-丙氨酸残基)，可以插入到载体部分的任意两个氨基酸残基之间的合适的间隔基团包括：烷基，如甲基、乙基或丙基。本领域技术人员能够理解另一种变体形式，(e)型，包括以类肽形式存在的一个或多个氨基酸残基。为了避免疑问，本文使用“类肽形式”来表示α-C取代基位于所述残基的N原子而非α-C上的变体氨基酸残基。制备类肽形式的肽是本领域已知的，例如SimonRJ等，PNAS(1992)89(20),9367-9371和Horwell DC,Trends Biotechnol.(1995)13(4),132-134。(f)型修饰可由国际公开PCT/GB99/01855中描述的方法进行。氨基酸变体(优选为(a)或(b)型)优选独立地发生在任意位置。如上所述，可能同时发生多于一个的同源或非同源置换。通过在序列内反转一些氨基酸残基的序列可得到其它变体。在一个实施方案中，替换氨基酸残基选自丙氨酸、精氨酸、天冬酰胺、天冬氨酸、半胱氨酸、谷氨酸、谷氨酰胺、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸和缬氨酸。
本发明的多肽衍生物可以盐或酯的形式存在，特别是可药用的盐或酯。
本发明的化合物的可药用的盐包括其合适的酸加成盐或碱盐。合适的可药用的盐可参见Berge等人的综述J Pharm Sci,66,1-19(1977)。可与强无机酸，如矿物酸(例如硫酸、磷酸或氢卤酸)；与强有机羧酸，如未取代或取代(如被卤素取代)的具有1至4个碳原子的烷羧酸(例如乙酸)；与饱和或不饱和二羧酸，例如草酸、丙二酸、丁二酸、马来酸、富马酸、邻苯二甲酸或对苯二酸；与羟基羧酸，例如抗坏血酸、乙醇酸、乳酸、苹果酸、酒石酸或柠檬酸；与氨基酸，例如天冬氨酸或谷氨酸；与苯酸；或与有机磺酸，如未取代或取代(例如被卤素取代)的(C1-C4)-烷基-或芳基-磺酸(例如甲磺酸或对甲苯磺酸)形成盐。
本发明也包括本发明衍生物的溶剂化物的形式。权利要求中所用的术语包括这些形式。本发明还涉及本发明类似物的各种晶体形式、多晶形物和(无水型)含水型。药学产业已经建立了这样的方式，化学化合物可以任意这样的形式通过略微改变在化合物的合成制备中所用的溶剂的纯化方法和/或分离方法被分离。
本发明还包括前体药物形式的本发明的衍生物。这种前体药物通常是本发明衍生物，其中一个或多个合适的基团经修饰从而在人或哺乳动物受试者中施用时该修饰能够逆转。尽管也可与这样的前体药物一起施用第二种试剂从而在体内进行所述逆转，但是这样的逆转通常是由该受试者体内天然存在的酶进行的。这样的修饰的例子包括酯(例如上述的任意那些)，其中所述逆转可由酯酶进行。其它这样的体系是本领域技术人员所熟知的。
与现有技术相比，本发明的有益效果为：
(1)相比现有技术，本发明胰岛素衍生物、GLP-1衍生物药效更持久、平稳、长期降血糖，其半衰期显著延长。
(2)本发明胰岛素衍生物、GLP-1衍生物不会造成低血糖，并且可以减少注射次数，副作用小。
(3)相比现有技术，本发明PTH衍生物药效更持久、平稳，其半衰期显著延长。
(4)本发明PTH衍生物可以减少注射次数，副作用小。
(5)本发明制备方法副产物少，收率高，成本低，工艺简单，适合大规模生产。无需溴化氰裂解、氧化亚硫酸盐解以及相关纯化步骤。无需使用高浓度的硫醇或疏水性吸附树脂。纯化步骤少，生产成本低。
下面结合具体实施例，进一步详陈本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
实施例1：含有丁炔氧羰基-赖氨酸的重组胰岛素蛋白的合成
利用化学方法合成编码含有SEQ ID NO.:11的氨基酸序列的丁炔氧羰基-赖氨酸的重组胰岛素蛋白的DNA片段，其中，第80位赖氨酸(K)的编码序列替换为TAG(编码赖氨酸衍生物)。然后将编码SEQ ID NO.:11的完整氨基酸序列的DNA片段克隆到修饰的pBAD-HisA载体中。所得质粒则用于结构为丁炔氧羰基-赖氨酸的重组胰岛素蛋白的表达。将该质粒和酶质粒pEvol-pylRs-pylT一起转化到大肠杆菌菌株Top10中。将转化液在含有25μg/mL的卡那霉素和17μg/mL的氯霉素的LB琼脂培养基上，37℃培养过夜。挑取单菌落，在含有25μg/mL的卡那霉素和17μg/mL的氯霉素的LB液体培养基中，37℃、220rpm恒温摇床培养过夜。然后，将所述过夜培养物接种到含有25μg/mL的卡那霉素和17μg/mL的氯霉素的100ml TB液体培养基中，并于37℃培养，直到OD 600为2-4。然后，向所述培养基中加入25％的阿拉伯糖溶液至0.25％的终浓度，并加入0.1M的丁炔氧羰基-赖氨酸溶液至5mM的终浓度，以诱导所述融合蛋白的表达。将所述培养液继续培养16-20h，然后离心(10000rpm，5min，4℃)收集。
SEQ ID NO.:11的氨基酸序列：
MVSKGEELFTGVTYKTRAEVKFEGDDDDDKTLVNRIELKGIDFENLYFQGRFVNQHLCGSHLVEALYLVCGERGFFYTPKTRGIVEQCCTSICSLYQLENYCN，其中第80位K为共价连接有丁炔氧羰基的赖氨酸。
融合蛋白是以不可溶的“包涵体”形式表达。为了释放包涵体，以高压均质机 破碎大肠杆菌细胞，通过5000g离心法去除细胞碎片和可溶性大肠杆菌宿主蛋白，以含有吐温80、EDTA、NaCl溶液洗涤包涵体，再以纯水洗涤包涵体1-2次。将洗涤后的包涵体溶于pH 10.5-11.5并含有2-10mMβ-巯基乙醇的7.5M脲中，使溶解后总蛋白的浓度为10-25mg/ml。将样品稀释5-10倍，维持4-8℃，pH值在10.5-11.7的条件下进行常规折叠14-30小时。于18-25℃下，pH值维持在8.0-9.5，用胰蛋白酶和羧肽酶B进行酶切10-20小时，然后加入0.45M的硫酸铵终止酶切反应。反相HPLC分析结果表明，该酶切步骤的产率高于90％。胰蛋白酶与羧肽酶B酶切后获得的胰岛素类似物被命名为丁炔氧羰基-赖氨酸-人胰岛素。膜过滤澄清样品，以0.45M硫酸铵作为缓冲液A，纯水作为缓冲液B，经疏水层析初纯化，获得丁炔氧羰基-赖氨酸-人胰岛素粗提取液，电泳纯度达90％。后经过聚合物反相填料及C8反相填料纯化，最终获得纯度高于99％的丁炔氧羰基-赖氨酸-人胰岛素。
Figure PCTCN2020089217-appb-000031
实施例2：L0-GFA16-胰岛素的合成(n为14)
由于脂肪酸酰基化合物带有叠氮基团，引入带有端炔的胰岛素蛋白，利用“点击化学”反应原理，炔与叠氮反应生成1，2，3-三唑环，形成交连。向1.5ml干净的离心管中加入4μL硫酸铜(50μM)，随后按顺序加入3μL BTTAA(300μM)和10μL实施例1中制备的IV化合物(N-(丁炔氧羰基)-赖氨酸的人胰岛素蛋白)(约5μM)。此时，可以将溶液稀释到恰当的体积或蛋白质浓度。向该溶液中加入1μL的L0-GFA16的(1mM)，和2μL抗坏血酸钠(2.5mM)引发反应。在室温下大约1小时后，添加5μL的SDS-PAGE样品缓冲液，加热至100℃保持10min，通过12％的SDS-PAGE分析。该凝胶回收，通过凝胶成像、荧光分析，然后用考马斯亮蓝染色。
Figure PCTCN2020089217-appb-000032
实施例3：L2-GFA16-胰岛素、L3-GFA16-胰岛素、L4-GFA16-胰岛素、L5-GFA16- 胰岛素、L6-GFA16-胰岛素的合成(n为14)
向1.5ml干净的离心管中加入4μL硫酸铜(50μM)，随后按顺序加入3μL BTTAA(300μM)和10μL实施例1中制备的N-(丁炔氧羰基)-赖氨酸的人胰岛素蛋白(约5μM)。此时，可能需要加水，将溶液稀释到恰当的体积或蛋白质浓度。向该溶液中加入1μL的L0-GFA16的(1mM)，和2μL抗坏血酸钠(2.5mM)引发反应。在室温下大约1小时后，添加5μL的SDS-PAGE样品缓冲液，加热至100℃保持10min，通过12％的SDS-PAGE分析。该凝胶回收，通过凝胶成像、荧光分析，然后用考马斯亮蓝染色。
Figure PCTCN2020089217-appb-000033
类似地，将实施例1中制备的IV化合物与L3-GFA16、L4-GFA16、L5-GFA16、L6-GFA16分别进行点击反应所得产物结构如下。
Figure PCTCN2020089217-appb-000034
实施例4：本发明胰岛素衍生物在大鼠体内的药物代谢动力学研究
实验分为对照品组与供试品组，分别皮下注射重组人胰岛素、L6-GFA16-胰岛素，给药剂量均为0.45mg/kg，单次给药。各组动物均在给药后在15min、30min、1h、2h、3h、5h、7h、12h、24h时间点采集血液。采用LC-MS/MS分析方法检测不同胰岛素类似物的含量。使用代谢动力学数据分析软件WinNonlin 7.0对血浆浓度数据进行统计，利用非房室模型法(NCA)计算药代参数(表1)。由表1可知，药物在各组动物中达峰时间相近，均为0.5-1小时，L6-GFA16-胰岛素的半衰期为重组人胰岛素的3-4倍，药物在体内暴露时间延长，暴露量增加。
表1 各组动物体内的主要药代动力学参数
Figure PCTCN2020089217-appb-000035
重复上述实验，不同的是采用本发明所述的L0-GFA16-胰岛素、L2-GFA16-胰岛素、L3-GFA16-胰岛素、L4-GFA16-胰岛素、L5-GFA16-胰岛素。结果发现，本发明所述的胰岛素衍生物在保持生物活性的同时，半衰期均显著延长。
实施例5：含有丁炔氧羰基-赖氨酸的GLP-1蛋白的合成
利用化学方法合成编码含有SEQ ID NO.:12的氨基酸序列的丁炔氧羰基-赖氨酸的GLP-1蛋白的DNA片段，其中，第70位赖氨酸(K)的编码序列替换为TAG(编码赖氨酸衍生物)。然后将编码SEQ ID NO.:12的完整氨基酸序列的DNA片段克隆到修饰的pBAD-HisA载体中。所得质粒则用于结构为丁炔氧羰基-赖氨酸的重组GLP-1蛋白的表达。将该质粒和酶质粒pEvol-pylRs-pylT一起转化到大肠杆菌菌株Top10中。将转化液在含有25μg/mL的卡那霉素和17μg/mL的氯霉素的LB琼脂培养基上，37℃培养过夜。挑取单菌落，在含有25μg/mL的卡那霉素和17μg/mL的氯霉素的LB液体培养基中，37℃、220rpm恒温摇床培养过夜。然后，将所述过夜培养物接种到含有25μg/mL的卡那霉素和17μg/mL的氯霉素的100ml TB液体培养基中，并于37℃培养，直到OD 600为2-4。然后，向所述培养基中加入25％的阿拉伯糖溶液至0.25％的终浓度，并加入0.1M的丁炔氧羰基-赖氨酸溶液至5mM的终浓度，以诱导所述融合蛋白的表达。将所述培养液继续培养16-20h，然后离心(10000rpm，5min，4℃)收集。
SEQ ID NO.:12的氨基酸序列：
MVSKGEELFTGVTYKTRAEVKFEGDTLVNRIELKGIDFENLYFQGDDDDKHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG，其中第70位K为共价连接有丁炔氧羰基的赖氨酸。
融合蛋白是以不可溶的“包涵体”形式表达。为了释放包涵体，以高压均质机破碎大肠杆菌细胞，通过5000g离心法去除细胞碎片和可溶性大肠杆菌宿主蛋白，以含有吐温80、EDTA、NaCl溶液洗涤包涵体后，再以纯水洗涤包涵体1-2次。将洗涤后的包涵体溶于pH 10.5-11.5并含有2-10mMβ-巯基乙醇的7.5M脲中，使溶解后总蛋白的浓度为10-25mg/ml。将样品稀释5-10倍，维持4-8℃，pH值在10.5-11.7的条件下进行常规折叠14-30小时。复性液澄清处理后，在pH9.0条件下，以弱阴离子填料分离纯化融合蛋白，目的蛋白的电泳纯度达80％。高盐洗脱样品，经过脱盐处理后，在25℃下，pH值维持在约8.0-9.0，以肠激酶进行酶切约10-20小时，反相HPLC分析结果表明，该酶切步骤的产率高于90％。肠激酶裂解后获得的GLP-1类似物被命名为丁炔氧羰基-赖氨酸-GLP-1。酶切后经疏水填料纯化，提取丁炔氧羰基-赖氨酸-GLP-1，电泳纯度达90％。
Figure PCTCN2020089217-appb-000036
实施例6：L0-GFA16-GLP-1的合成(n为14)
由于脂肪酸酰基化合物带有叠氮基团，引入带有端炔的GLP-1蛋白，利用“点击化学”反应原理，炔与叠氮反应生成1，2，3-三唑环，形成交连。向1.5ml干净的离心管中加入4μL硫酸铜(50μM)，随后按顺序加入3μL BTTAA(300μM)和10μL实施例5中制备的IV化合物(N-(丁炔氧羰基)-赖氨酸的GLP-1蛋白)(约5μM)。此时，可以将溶液稀释到恰当的体积或蛋白质浓度。向该溶液中加入1μL的L0-GFA16的(1mM)，和2μL抗坏血酸钠(2.5mM)引发反应。在室温下大约1小时后，添加5μL的SDS-PAGE样品缓冲液，加热至100℃保持10min，通过12％的SDS-PAGE分析。该凝胶回收，通过凝胶成像、荧光分析，然后用考马斯亮蓝染色。
Figure PCTCN2020089217-appb-000037
实施例7：L2-GFA16-GLP-1、L3-GFA16-GLP-1、L4-GFA16-GLP-1、L5-GFA16-GLP-1、L6-GFA16-GLP-1的合成(n为14)
向1.5ml干净的离心管中加入4μL硫酸铜(50μM)，随后按顺序加入3μL BTTAA(300μM)和10μL实施例5中制备的N-(丁炔氧羰基)-赖氨酸的GLP-1蛋白(约5μM)。此时，可能需要加水，将溶液稀释到恰当的体积或蛋白质浓度。向该溶液中加入1μL的L0-GFA16的(1mM)，和2μL抗坏血酸钠(2.5mM)引发反应。在室温下大约1小时后，添加5μL的SDS-PAGE样品缓冲液，加热至100℃保持10min，通过12％的SDS-PAGE分析。该凝胶回收，通过凝胶成像、荧光分析，然后用考马斯亮蓝染色。
Figure PCTCN2020089217-appb-000038
类似地，将实施例5中制备的IV化合物与L3-GFA16、L4-GFA16、L5-GFA16、L6-GFA16分别进行点击反应所得产物结构如下。
Figure PCTCN2020089217-appb-000039
实施例8：本发明GLP-1衍生物在大鼠体内的药物代谢动力学研究
实验分为对照品组与供试品组，分别静脉注射GLP-1、L6-GFA16-GLP-1，给药剂量均为0.5mg/kg，单次给药。各组动物均在给药后在15min、30min、1h、2h、3h、5h、7h、12h、24h时间点采集血液。采用LC-MS/MS分析方法检测不同胰岛素类似物的含量。使用代谢动力学数据分析软件WinNonlin 7.0对血浆浓度数据进行统计，利用非房室模型法(NCA)计算药代参数(表2)。
由表2可知，药物在各组动物中达峰时间相近，均为4.8min左右，L6-GFA16-GLP-1在体内暴露时间延长，暴露量显著增加。
表2 各组动物体内的主要药代动力学参数
Figure PCTCN2020089217-appb-000040
重复上述实验，不同的是采用本发明所述的L0-GFA16-GLP-1、L2-GFA16-GLP-1、L3-GFA1-GLP-1、L4-GFA16-GLP-1、L5-GFA16-GLP-1。结果发现，本发明所述的GLP-1衍生物在保持生物活性的同时，半衰期均显著延长。
实施例9：含有丁炔氧羰基-赖氨酸的PTH蛋白的合成
利用化学方法合成编码含有SEQ ID NO.:13的氨基酸序列的丁炔氧羰基-赖氨酸的PTH蛋白的DNA片段，其中，第76位赖氨酸(K)的编码序列替换为TAG(编码赖氨酸衍生物)。然后将编码SEQ ID NO.:13的完整氨基酸序列的DNA片段克隆到修饰的pBAD-HisA载体中。所得质粒则用于结构为丁炔氧羰基-赖氨酸的重组PTH蛋白的表达。将该质粒和酶质粒pEvol-pylRs-pylT一起转化到大肠杆菌菌株Top10中。将转化液在含有25μg/mL的卡那霉素和17μg/mL的氯霉素的LB琼脂培养基上，37℃培养过夜。挑取单菌落，在含有25μg/mL的卡那霉素和17μg/mL的氯霉素的LB液体培养基中，37℃、220rpm恒温摇床培养过夜。然后，将所述过夜培养物接种到含有25μg/mL的卡那霉素和17μg/mL的氯霉素的100ml TB液体培养基中，并于37℃培养，直到OD 600为2-4。然后，向所述培养基中加入25％的阿拉伯糖溶液至0.25％的终浓度，并加入0.1M的丁炔氧羰基-赖氨酸溶液至5mM的终浓度，以诱导所述融合蛋白的表达。将所述培养液继续培养16-20h，然后离心(10000rpm，5min，4℃)收集。
SEQ ID NO.:13的氨基酸序列：
MVSKGEELFTGVTYKTRAEVKFEGDTLVNRIELKGIDFENLYFQGDDDDKSVSEIQLMHNLGRHLNSMERVEW LRKRLQDVHNF，其中第76位K为共价连接有丁炔氧羰基的赖氨酸。
融合蛋白是以不可溶的“包涵体”形式表达。为了释放包涵体，以高压均质机破碎大肠杆菌细胞，通过5000g离心法去除细胞碎片和可溶性大肠杆菌宿主蛋白，以含有吐温80、EDTA、NaCl溶液洗涤包涵体后，再以纯水洗涤包涵体1-2次。将洗涤后的包涵体溶于pH 10.5-11.5并含有2-10mMβ-巯基乙醇的7.5M脲中，使溶解后总蛋白的浓度为10-25mg/ml。将样品稀释5-10倍，维持4-8℃，pH值在10.5-11.7条件下进行常规折叠14-30小时。复性液澄清处理后，在pH9.0条件下，以弱阴离子填料分离纯化融合蛋白，目的蛋白的电泳纯度达80％。高盐洗脱样品，经过脱盐处理后，在25℃下，pH值维持在约8.0-9.0，以肠激酶进行酶切约10-20小时，反相HPLC分析结果表明，该酶切步骤的产率高于90％。肠激酶裂解后获得的PTH类似物被命名为丁炔氧羰基-赖氨酸-PTH。酶切后经疏水填料纯化，提取丁炔氧羰基-赖氨酸-PTH，电泳纯度达90％。
Figure PCTCN2020089217-appb-000041
方法同实施例6和7，用含有N-(丁炔氧羰基)-赖氨酸的PTH蛋白替换含有N-(丁炔氧羰基)-赖氨酸的GLP-1蛋白，合成了L0-GFA16-PTH、L2-GFA16-PTH、L3-GFA16-PTH、L4-GFA16-PTH、L5-GFA16-PTH和L6-GFA16-PTH。
实施例10：本发明PTH-1衍生物在大鼠体内的药物代谢动力学研究
实验分为对照品组与供试品组，分别皮下注射PTH、L6-GFA16-PTH，给药剂量均为8.6μg/kg，单次给药。各组动物均在给药后在0min、5min、15min、30min、1h、2h、4h时间点采集血液。采用LC-MS/MS分析方法检测不同胰岛素类似物的含量。使用代谢动力学数据分析软件WinNonlin 7.0对血浆浓度数据进行统计，利用非房室模型法(NCA)计算药代参数(表3)。由表3可知，L6-GFA16-PTH动物中达峰时间延长至1.6h左右，药物在体内暴露时间延长，暴露量增加。
表3 各组动物体内的主要药代动力学参数
Figure PCTCN2020089217-appb-000042
重复上述实验，不同的是采用本发明所述的L0-GFA16-PTH、L2-GFA16-PTH、L3-GFA16-PTH、L4-GFA16-PTH、L5-GFA16-PTH。结果发现，本发明所述的PTH衍生物在保持生物活性的同时，半衰期均显著延长。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种多肽衍生物，其特征在于，所述多肽衍生物包括：
    (a)多肽；和
    (b)修饰基团L，所述修饰基团L连接于所述多肽的赖氨酸位点，且所述修饰基团L为式I所示的基团，
    Figure PCTCN2020089217-appb-100001
    其中，波浪线表示与所述赖氨酸位点的连接位置，m为0-8的整数；a、b、c、d、e、f各自独立的选自0至10的整数；n为14至16的整数。
  2. 如权利要求1所述的多肽衍生物，其特征在于，所述基团Y为选自下组的基团：
    Figure PCTCN2020089217-appb-100002
  3. 如权利要求1所述的多肽衍生物，其特征在于，所述多肽选自下组：胰岛素、GLP-1、 PTH、或其组合。
  4. 如权利要求1所述的多肽衍生物，其特征在于，所述胰岛素的A链具有如SEQ ID NO.:1或2所示的序列；和/或
    所述胰岛素的B链具有如SEQ ID NO.:3、4、5或6所示的序列；和/或
    所述GLP-1具有如SEQ ID NO.:7-9中任一所示的序列；和/或
    所述PTH具有如SEQ ID NO.:10所示的序列。
  5. 一种药物组合物，其特征在于，所述药物组合物包含权利要求1所述的多肽衍生物，以及药学上可接受的载体。
  6. 如权利要求1所述的多肽衍生物的用途，其特征在于，用于制备预防和/或治疗骨质疏松症、糖尿病、高血糖症及其他降低血糖可获益的疾病的药物或制剂。
  7. 一种多肽衍生物的制备方法，其特征在于，所述方法包括步骤：
    (1)在X基团-赖氨酸、吡咯赖氨酰基-tRNA合成酶及其同源关联tRNA的存在下，培养含有胰岛素编码序列的菌株，其中，所述编码序列中，多肽中的赖氨酸位点的编码序列为TAG，从而产生多肽衍生物，其中所述多肽衍生物包括：
    (a)多肽链；和
    (b)修饰基团L，所述修饰基团L连接于所述多肽的赖氨酸位点，且所述修饰基团L为基团X，基团X同权利要求1中所定义；和任选地
    (2)从发酵产物中分离所述的多肽衍生物。
  8. 一种多肽衍生物的制备方法，其特征在于，所述方法包括步骤：
    (1)在式III化合物、吡咯赖氨酰基-tRNA合成酶及其同源关联tRNA的存在下，培养含有多肽编码序列的菌株，其中，所述编码序列中，多肽中的赖氨酸位点的编码序列为TAG，从而得到式IV化合物；和
    Figure PCTCN2020089217-appb-100003
    (2)在惰性溶剂中，将式IV化合物与式V化合物进行反应，从而得到多肽衍生物，
    Figure PCTCN2020089217-appb-100004
    式V中，a、b、c、d、e、f各自独立的选自0至10的整数；n为14至16的整数。
  9. 一种多肽衍生物的制备方法，其特征在于，所述方法包括步骤：
    (1)在式VI化合物、吡咯赖氨酰基-tRNA合成酶及其同源关联tRNA的存在下，
    培养含有多肽编码序列的菌株，其中，所述编码序列中多肽中的赖氨酸位点的编码序列为TAG，从而得到式VII化合物；和
    Figure PCTCN2020089217-appb-100005
    (2)在惰性溶剂中，将式VII化合物与式VIII化合物进行反应，从而得到多肽衍生物，
    Figure PCTCN2020089217-appb-100006
    式VIII中a、b、c、d、e、f各自独立的选自0至10的整数；n为14至16的整数。
  10. 一种中间体，其特征在于，所述中间体包括：
    (a)多肽，其中所述多肽为胰岛素、GLP-1或PTH；和
    (b)修饰基团L，所述修饰基团L连接于所述多肽的赖氨酸位点，且所述修饰基团L为式A所示的基团，
    Figure PCTCN2020089217-appb-100007
    其中，波浪线表示与所述赖氨酸位点的连接位置，m为0-8的整数。
PCT/CN2020/089217 2019-05-10 2020-05-08 多肽衍生物及其制备方法 WO2020228610A1 (zh)

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