WO2021160185A1 - Glp-1r激活剂的分子改构及其二聚体在治疗代谢病中的应用 - Google Patents

Glp-1r激活剂的分子改构及其二聚体在治疗代谢病中的应用 Download PDF

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WO2021160185A1
WO2021160185A1 PCT/CN2021/078057 CN2021078057W WO2021160185A1 WO 2021160185 A1 WO2021160185 A1 WO 2021160185A1 CN 2021078057 W CN2021078057 W CN 2021078057W WO 2021160185 A1 WO2021160185 A1 WO 2021160185A1
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peptide
glp
glu
fatty
acid
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French (fr)
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唐松山
张旭东
罗群
杨莉
谭宏梅
唐婧晅
游娟
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南京枫璟生物医药科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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 medical biology, and specifically relates to the molecular modification of a GLP-1R activator-like peptide and the application of its homodimer in the treatment of metabolic diseases.
  • Exendin-4 is an incretin analog isolated from the saliva of Heloderma suspectum. It has 39 amino acids and has 53% sequence homology with GLP-1.
  • GIP is a 42 amino acid gastrointestinal regulatory peptide, which has the functions of regulating the body's glucose metabolism, promoting the release of insulin from pancreatic ⁇ cells and reducing body weight.
  • GLP-1 is an incretin-like peptide of 30 amino acid residues, which is released by intestinal L cells during nutrient intake. Exendin-4 and GLP-1 are two GLP-1R activators discovered so far.
  • Exenatide, Polyethylene Clycol Loxenatide and Lixisenatide are allosteric molecules based on the amino acid sequence of the active polypeptide Exendin-4, and they have been clinically marketed.
  • Liraglutide, Albiglutide, Dulaglutide, Semaglutide and Taspoglutide are allosteric analogues based on the amino acid sequence of the active polypeptide GLP-1, which are produced by chemical synthesis or genetic recombination-chemical synthesis combined methods.
  • Tirzepatide is a synthetic molecule based on the GIP-Exendin-4 bifunctional receptor activator. It was developed by Lily and has completed phase II clinical trials.
  • HPA hypothalamic-pituitary-adrenal axis
  • HTPA hypothalamic-pituitary-adrenal axis
  • stimulation of the HPA axis by GLP-1R activators leads to an increase in corticosterone, leading to abnormal heart rate. Therefore, it is still necessary to: 1 When activating GIP receptors and/or GLP-1 receptors, simultaneously antagonize the activation of glucagon and GLP-2 receptors; 2 It is necessary to activate GIP receptors and/or GLP-1 receptors.
  • 3GLP-1R activator still needs to be optimized, because the current long-acting activator is in the specific activity (unit In terms of the quality of hypoglycemic effect), dosage, weight loss and side effects, it has been proved to be less effective than natural GLP-1 or Exendin-4 molecules.
  • scientific and technological progress is needed to provide longer-acting hypoglycemic activators and overcome toxicity.
  • the purpose of the present invention is to provide a GLP-1R activator-like peptide.
  • the GLP-1R activator-like peptide of the present invention performs molecular allosteric respectively on Exendin-4, GIP-Exendin-4 and GLP-1.
  • a single Cys ⁇ Ser substitution was used in the monomer peptide to study the position of cysteine in the peptide chain.
  • the modification of fatty acid or fatty acid substituents on the side chain ⁇ -amino group of lysine (K) in the peptide chain is allosteric, and different fatty acids or fatty acid substituents have different effects on the activity of GLP-1R activator-like peptides. .
  • the present invention also provides homodimers formed by similar peptides of the aforementioned GLP-1R activator. It is found that the dimers of the H-type structure formed by cysteine at different positions (intramolecular single Cys ⁇ Ser substitution) produce different activities. , The longest continuous hypoglycemic time can be up to 22 days, which is a significant increase compared with the current clinical drugs that are used once every 1-7 days.
  • the technical solution adopted by the present invention is: a glucagon-like peptide-1 receptor (GLP-1R) activator-like peptide, and the GLP-1R activator-like peptide is Exendin-4, GIP -The sequence of the Exendin-4 chimeric peptide or GLP-1 is allosterically modified and a fatty acid chain on the lysine epsilon-amino group is modified.
  • GLP-1R glucagon-like peptide-1 receptor
  • the GLP-1R activator-like peptide of the present invention is based on the amino acid sequence of Exendin-4, GIP-Exendin-4 or GLP-1 peptide as the main chain, replacing Ser on the main chain with Cys, and in the main chain amino acid sequence Contains only one Cys; the side chain is a fatty acid chain of the ⁇ -amino group of one of the lysines of the main chain.
  • the specific sequence of the GLP-1R activator-like peptide is any one of the following:
  • X 2 or X 13 is L- ⁇ -glycine or L- ⁇ -alanine or ⁇ -aminoisobutyric acid ( ⁇ Aib);
  • X 12 or X 20 or X 27 or X 28 is lysine, or fine Amino acid, or glutamyl fatty acid [ ⁇ -Glu(N- ⁇ -fatty acid)] or glutamyl fatty diacid [ ⁇ -Glu(N- ⁇ -fatty diacid)] modified lysine on the side chain ⁇ -amino group Acid, or [2 ⁇ AEEAC- ⁇ -Glu-(N- ⁇ -fatty diacid)] modified lysine on the side chain ⁇ -amino group;
  • X 38 is PS (HN 2 ) or SKKKKKK (HN 2 ).
  • the capital single letter is the abbreviation of L- ⁇ -amino acid or the symbol of amino acid substitution, the Arabic numerals are the sequence of amino acid residues, and NH 2 represents the N-terminal or C
  • the GLP-1R activator is similar to a peptide, when X 12 or X 20 or X 27 or X 28 is a side chain ⁇ -amino glutamyl fatty acid [ ⁇ -Glu(N- ⁇ -fatty acid)] or valley When aminoacyl fatty diacid [ ⁇ -Glu(N- ⁇ -fatty diacid)] modified lysine, its structure is as shown in chemical formula 1.
  • X 12 or X 20 or X 27 or X 28 is the side chain ⁇ -
  • the amino group is [2 ⁇ AEEAC- ⁇ -Glu-(N- ⁇ -fatty diacid)] modified lysine, its structure is shown in Chemical Formula 2.
  • the present invention also provides a hypoglycemic-like peptide homodimer, said dimer is connected by disulfide bonds formed by cysteine between the same monomers described in any one of claims 1-3. It constitutes an H-type GLP-1R activator-like peptide homodimer.
  • amino acid sequence of the dimer is any one of the following:
  • X 2 or X 13 is L- ⁇ -glycine or L- ⁇ -alanine or ⁇ -aminoisobutyric acid ( ⁇ Aib);
  • X 12 or X 20 or X 27 or X 28 is lysine, or fine Amino acid, or glutamyl fatty acid [ ⁇ -Glu(N- ⁇ -fatty acid)] or glutamyl fatty diacid [ ⁇ -Glu(N- ⁇ -fatty diacid)] modified lysine on the side chain ⁇ -amino group Acid, or [2 ⁇ AEEAC- ⁇ -Glu-(N- ⁇ -fatty diacid)] modified lysine on the side chain ⁇ -amino group;
  • X 38 is PS (HN 2 ) or SKKKKKK (HN 2 );
  • " indicates the disulfide bond formed between two cysteines.
  • X 12 or X 20 or X 27 or X 28 is glutamyl fatty acid [ ⁇ -Glu(N- ⁇ -fatty acid)] or glutamyl fatty diacid [ ⁇ -Glu( N- ⁇ -fatty diacid)] modified lysine
  • its structure is as shown in chemical formula 1
  • X 12 or X 20 or X 27 or X 28 is the side chain ⁇ -amino group [2 ⁇ AEEAC- ⁇ -Glu -(N- ⁇ -fatty diacid) modified lysine, its structure is as shown in chemical formula 2.
  • the present invention also provides the use of the GLP-1R activator-like peptide or the homodimer in the preparation of drugs for the treatment of metabolic syndrome.
  • the metabolic syndrome disorder includes hyperglycemia, diabetes and obesity.
  • the present invention also provides a medicament for the treatment of metabolic syndrome.
  • the above-mentioned GLP-1R activator-like peptide or homodimer and the pharmaceutically acceptable salt thereof are used as active ingredients.
  • the H-type GLP-1R activator analog homodimer of the present invention can significantly increase the corresponding monomer activator or be approved by the FDA or SFDA when the hypoglycemic intensity is not lower than the corresponding monomer peptide.
  • Approved GLP-1R activator clinical drugs have a hypoglycemic effect of about 2-3 times.
  • the provided GLP-1R activator analog homodimer maintains its activity in the body for up to 22 days, which is better than that of positive drugs. Lixinaglutide (the effect is maintained for 2 days) is significantly prolonged.
  • the structure of the new dimer changes significantly, which greatly facilitates its clinical application and market promotion.
  • Figure 1 is a schematic diagram of the blood glucose test results of a single OGTT.
  • Figure 2 is a statistical analysis diagram of body weight when 2G21 treats the T2D model.
  • Figure 3 is a statistical analysis diagram of blood glucose in the T2D model of 2G21 treatment.
  • Figure 4 is a statistical analysis diagram of glycosylated hemoglobin in 2G21 treatment T2D model.
  • Figure 5 is a statistical analysis diagram of insulin in the T2D model of 2G21 treatment.
  • Figure 6 is a statistical analysis diagram of alanine aminotransferase in 2G21 treatment T2D model.
  • Figure 7 is a statistical analysis diagram of pancreatic amylase in 2G21 treatment T2D model.
  • Resin swelling Put amino resin (amino resin for C-terminal amidation sequence) (purchased from Tianjin Nankai Synthetic Technology Co., Ltd.) into the reaction pot, add 15ml of dichloromethane (DCM, Dikma Technologies Inc.) /g resin, shake for 30min. SYMPHONY 12-channel peptide synthesizer (SYMPHONY model, software Version.201, Protein Technologies Inc.).
  • Method a three times the amount of protected amino acids and three times the amount of 2-(7-azobenzotriazole)-tetramethylurea hexafluorophosphate (HBTU, Suzhou Tianma Pharmaceutical Group Fine Chemicals Co., Ltd.), Dissolve them with as little DMF as possible and add them to the reaction pot. Immediately add ten times the amount of N-methylmorpholine (NMM, Suzhou Tianma Pharmaceutical Group Fine Chemical Co., Ltd.). The reaction is 30 minutes, and the test is negative.
  • HBTU 2-(7-azobenzotriazole)-tetramethylurea hexafluorophosphate
  • NMM N-methylmorpholine
  • Method b Three times the amount of protected amino acid FMOC-amino acid and three times the amount of 1-hydroxybenzotriazole (HOBt, Suzhou Tianma Pharmaceutical Group Fine Chemicals Co., Ltd.), both dissolved with as little DMF as possible, added to the reaction tube, and added immediately Three times the amount of N,N'-diisopropylcarbodiimide (DIC). Reaction for 30min. The test was negative.
  • Wash resin DMF (10ml/g) wash once, methanol (10ml/g) wash twice, DMF (10ml/g) wash twice.
  • Cut peptides from resin prepare cutting fluid (10ml/g): TFA 94% (JTBaker Chemical Company), water 2.5%, ethanedithiol (EDT, Sigma-Aldrich Chemistry) 2.5% and triisopropylsilane (TIS, Sigma- Aldrich Chemistry) 1%. Cutting time: 120min.
  • Drying and washing Blow dry the lysate with nitrogen as much as possible, wash it with ether six times, and then evaporate to dryness at room temperature.
  • Purify the peptides by HPLC dissolve the crude peptides in pure water or add a small amount of acetonitrile, and purify according to the following conditions: high performance liquid chromatography (analytical type; software Class-VP.Sevial System; manufacturer Japan SHIMADZU) and Venusi MRC-ODS C18 chromatographic column (30 ⁇ 250mm, Tianjin Bonna-Agela Technologies).
  • Mobile phase A solution 0.1% trifluoroacetic acid aqueous solution
  • mobile phase B 0.1% trifluoroacetic acid + 99.9% acetonitrile solution (purchased by Acetonitrile Fisher Scientific).
  • Flow rate 1.0ml/min, loading volume 30 ⁇ l, detection wavelength 220nm.
  • Elution procedure 0 ⁇ 5min: 90% solution A+10% solution B; 5 ⁇ 30min: 90% solution A/10% solution B ⁇ 20% solution A/80% solution B.
  • the purified effective solution is lyophilized (Freezone Plus 6 model of lyophilizer, L ABCONCO manufacturer), and the finished product is obtained.
  • MS method to identify the molecular weight of peptides Take the peptides with qualified purity and dissolve them in water, add 5% acetic acid + 8% acetonitrile + 87 water to dissolve the test electrospray ionization mass spectrometry to determine the molecular weight, see our authorized patent (Chinese patent ZL201410612382.3).
  • dimer peptides For the dissolved dimer peptide or the above centrifugal supernatant, it is separated and identified by Sephadex G-25 chromatography (under a 2 ⁇ 60cm G-25 column and natural flow rate, NaCl-PB solution is used as the flow item, dimer The component is the first peak, and the residual impurity component is the subsequent peak). Dimer peptides can be identified by peptide PAGE electrophoresis or mass spectrometry without sulfhydryl reducing agent. For specific methods, please refer to our authorized patent (Chinese Patent ZL201410612382.3).
  • GLP-1R activator-like peptide monomers and their dimers were synthesized by our laboratory or commissioned commercial companies. The inventors confirmed their structures through HPLC purity, ESI or laser flight mass spectrometry and cysteine oxidation.
  • the GLP-1R activator monomer synthesized in the present invention is shown in Table 1, and the amino acid sequence of the homodimer peptide is shown in Table 2.
  • Table 1 The amino acid sequence of the synthetic GLP-1R activator similar peptide monomer of the present invention and its continuous hypoglycemic activity in a single injection of the same dose
  • Tirzepatide in the table is a chimera peptide of GIP-Exendin-4 peptide; Lixisenatide is also an allosteric to Exendin 4; CFA (carbon fatty acid) or CFDA (carbon fatty acid) is carbon fatty acid or carbodiacid; K[N- ⁇ -( ⁇ -Glu-N- ⁇ -CFA or CFDA)], K[N- ⁇ -(2 ⁇ AEEAC- ⁇ -Glu-N- ⁇ -CFDA)] represents the side chain of lysine K
  • the fatty acyl or fatty diacid monoacyl glutamyl modification of the ⁇ -amino group has the specific structure shown in Formula 1 or 2.
  • mice After subcutaneous injection of drugs or monomers (dissolved with normal saline pH 6.5, normal saline for the blank control) or dimer peptide (dissolved with NaCl-PB solution pH 8.0, the blank control generally uses NaCl-PB solution) After 30 minutes, the mice were given 5% glucose solution on time, and the blood glucose level of the mouse tail was measured accurately after the administration.
  • the blood glucose meter and blood glucose test strips are products of Bayer HeathCare LLC. Take the average blood glucose of each group as the criterion: when the average blood glucose of each group's OGTT is higher than the average blood glucose of the blank control group twice in a row, the measurement is stopped.
  • mice Thirty minutes after a single dose, the tail blood of mice was taken to determine blood glucose before (0 min) and 10, 20, 40, 60, and 120 min after a single oral glucose dose.
  • the blank control group was treated with normal saline ( Figure 1). The results showed that at 10 minutes, compared with Lixisenatide and G21 groups, there was a significant increase in glucose in the 2G33 group (P ⁇ 0.05). At 20 minutes, compared with the blank control group, Lixisenatide and G21 groups showed a significant decrease (P ⁇ 0.05 or 0.01). Compared with Lixisenatide and G21 group, 2G21 group showed a significant increase in glucose (P ⁇ 0.05).
  • Lixisenatide, G21 and G33 groups showed a significant decrease (P ⁇ 0.05 or 0.01).
  • the 2G33 group showed a significant decrease or increase in glucose (P ⁇ 0.05).
  • the G21 group showed a significant decrease (P ⁇ 0.05);
  • the 2G21 or 2G33 group showed a significant increase in glucose (P ⁇ 0.05). The results showed that in the same time, the blood glucose level of the dimer was significantly higher than that of the monomer peptide, indicating that the absorption of the dimer was significantly delayed.
  • OGTT OGTT
  • mice were gavaged with 5% glucose solution on time, and measured accurately 35 minutes after gavage Rat tail blood sugar level.
  • Lixisenatide positive drugs lasted for 2 days for lowering blood sugar, G9 series maintained 2-9 days, G10 series 2-11 days, G11 series 2-11 and G12 series 7-9 days.
  • Dimer 2G9 series are maintained for 4-21 days, 2G10 series are maintained for 4-22 days, 2G11 series are maintained for 6-21 days, and 2G12 series are maintained for 16-20 days.
  • Each monomer group (shown in Table 1) is approximately 1/2 the duration of its corresponding dimer group (shown in Table 2). It is found by comparison that a disulfide bond is formed at the 11th or 12th position and a dimer peptide modified with a lysine ⁇ -amino side chain fatty acid at the 20th position (20-carbon fatty acid or aliphatic diacid is the best). Weight loss, its hypoglycemic duration also significantly increased. The comparison shows that the duration of the 11th peptide in the 2G9 series, the 20th and 21st in the 2G10 series, and the 33rd and 34th dimer peptides in the 2G11 series is the longest 21-22 days.
  • the 21st peptide (2G21) in the 2G10 series is an allosteric form of Exendin 4, which has high homology with the sequence of Lixisenatide positive control drug and the same side chain fatty acid modification. Therefore, the 2G21 peptide was selected for the treatment of type II diabetes (T2D) in vivo and follow-up experiment of. The mOGTT experiment needs to be implemented accurately, otherwise the results may be skipped.
  • CFA carbon fatty acid
  • CFDA carbon fatty diacid
  • K[N- ⁇ -( ⁇ -Glu-N- ⁇ -CFA or CFDA)] represents the fatty acyl or fatty diacid monoacyl glutamyl modification of the ⁇ -amino group of the K side chain, the specific structure is shown in formula 1 or 2.
  • the dimer is connected by the disulfide bond formed between the cysteines in the monomer peptide to form an H-type dimer; where "" means that the disulfide is formed between the two cysteines between the monomers. key.
  • T2D Type II Diabetes
  • the NaCl-PB group was a blank control and Placebo was a T2D model control. They were injected with NaCl-PB solution.
  • All T2D model groups were fed a 60kcal% high-fat diet (D12492, Changzhou Shuyishutwo Biotechnology Co., Ltd., Changzhou, China) until the end of the experiment, and the blank control group maintained a standard diet until the end of the experiment.
  • Diabetes model establishment method after 4 weeks of high-fat feeding mice, 75mg/kg streptozotocin (STZ, American Sigma Chemical Company) was injected intraperitoneally, 3 days later, 50mg/kg dose of STZ was re-injected intraperitoneally, 3 weeks later Mice with blood glucose equal to or greater than 11 mM are considered diabetic mice. These diabetic groups will undergo a 35-day treatment study on the basis of a high-fat diet.
  • Peptide solubility For Lys side chain containing fatty acid modified structure, monomer peptides that do not contain Aib amino acids show a suspended state in water, and the corresponding homodimer peptide is completely dissolved in water; for Lys side chain containing fatty acid The modified structure, monomeric peptides containing Aib amino acids or/and C-terminal amidation structures show complete dissolution in water, while their corresponding homodimer peptides dissolve poorly in water. For those without Lys side chain fatty acid modification, both the monomer peptide and the dimer are completely dissolved in water.
  • All the dimer peptides were dissolved and injected with NaCl-PB (pH 8.0), and the homodimer 2G21 of low, medium and high doses were dissolved in NaCl-PB solution [Na 2 HPO 4 buffered physiological saline solution. (pH8.0)] for animal injection.
  • the monomer peptide was dissolved in a physiological saline solution and injected (around pH 6.5).
  • Dosing concentration setting Our preliminary experiments show that a single subcutaneous injection of 0.624nmol/100 ⁇ l of Lixisenatide peptide can easily observe the time-effect relationship of OGTT for multiple days. Therefore, in all glucose tolerance experiments, normal Kunming mice were injected with a single dose of 0.624nmol/100 ⁇ l of Lixisenatide or monomeric peptides or dimer peptides subcutaneously into the hips, and blood was collected at 9 o'clock every morning to measure blood glucose and weighed. For OGTT, the time of administration, gavage and blood sampling for each animal needs to be accurate to the second. In T2D animal experiments, Lixisenatide was selected as the positive control and its administration method (subcutaneous administration).
  • 0.624nmol/100 ⁇ l of Lixisenatide can induce the postprandial blood glucose level of T2D diabetes model (postprandial blood glucose up to 20mM) to 8-11mM. At this cut-off value, the effect-dose relationship between the positive drug Lixisenatide and GLP-1R dimer is easily observed.
  • the T2D model mice were injected subcutaneously into the buttocks of each 100 ⁇ l dose within 30 minutes, and the blood glucose level of the experimental mice was measured every five days. The entire test was completed within 40 minutes.
  • the high, medium and low doses of the dimer 2G21 peptide were 1.873, 0.624, 0.208 nmol/100 ⁇ L, and the positive drug Lixisenatide dose was 0.624 nmol/100 ⁇ l (the raw material was synthesized by a commercial company), which was injected once a day until the end of the 35-day experiment.
  • Body weight change after T2D treatment Before administration, there was no significant difference in body weight in the T2D model group. After the experiment, compared with the NaCl-PB group, Placebo group, Lixisenatide group, L-2G21 group (low dose), M-2G21 group (medium dose) showed a significant increase in body weight (P ⁇ 0.05 or 0.001), but H-2G21 There is no difference between the group (high dose) and the normal group, indicating that the treatment is effective. Compared with the Placebo group, the weight loss of Lixisenatide group and H-2G21 group was significantly (P ⁇ 0.05). The body weight of each 2G21 group decreased in a dose-dependent manner, and the M-2G21 group and Lixisenatide group showed similar changes (Figure 2).
  • the homodimer series developed by us can significantly increase the efficacy time.
  • the dimer sequence shows the most promising application prospects for rodent T2D models, such as the longest lasting blood sugar lowering effect and weight reduction effect.
  • the structure-activity relationship shows that the dimer without fatty acid modification has the best solubility in water, and the fatty acid modified peptide contains the Aib amino acid structure dimer, and even has the C-terminal amidation structure, which is slightly less soluble in water. It is an important data for the study of pharmaceutical preparations, and it is also the fundamental reason for the different duration of hypoglycemic activity of different structure GLP-1R activators in this study—different spatial conformations form different physical and chemical properties, resulting in different hypoglycemic durations.
  • a single dose of the same dose, a single OGTT experiment results show that due to the slow absorption of the dimer, a longer hypoglycemic effect is produced.
  • a single dose of the same dose, multiple OGTT experiment results show that the longer duration effect involves changes in the dimer's second amino acid, disulfide bond position, symmetric fatty acid or fatty diacid modified Lys and C-terminal amidation, etc. Constellation related.
  • Table 2 shows that the longest active dimer structure contains 2 ⁇ Aib, 11 or 12 Cys-Cys disulfide bonds, symmetric 20 fatty acyl or fatty diacid monoacyl-L- ⁇ -glutamyl- 20 lysine Or 20-carbon fatty diacid acyl monoacyl- ⁇ -Glu-2 ⁇ AEEAC- 20 Lys and C-terminal amidation structure.
  • the 2G21 group has a significant reduction in the HbA 1c or fasting blood glucose (FPG) value of the diabetes model, and has a significant hypoglycemic effect.
  • FPG fasting blood glucose
  • the same molar concentration of 2G21 peptide and Lixisenatide has an effect on PPG (postprandial blood glucose) or FPG, HbA 1c. There is a similar reduction effect.
  • the body weight of 2G21 group decreased in a dose-dependent manner, and the weight loss of M-2G21 group and Lixisenatide group was consistent.
  • the better weight loss dimer peptide structure involves 11 or 12 Cys-Cys disulfide bonds, symmetrical 20 Lys [2 ⁇ AEEAC- ⁇ -Glu-20 carbon fatty acid] modification and C-terminal amidation.
  • 2G21 reduces alanine aminotransferase (ALT) in a dose-dependent manner, showing that the drug has a strong protective effect on the liver.
  • ALT alanine aminotransferase
  • the dimeric peptide of the present invention can induce more insulin release, thereby producing a better hypoglycemic effect.

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Abstract

提供了GLP-1R Exendin-4、GIP-Exendin 4嵌合多肽、GLP-1的分子变构体，该变构体通过半胱氨酸形成的二硫键连接而成二聚体，可治疗代谢综合征，包括糖尿病和肥胖症治疗。

Description

GLP-1R激活剂的分子改构及其二聚体在治疗代谢病中的应用 技术领域
本发明属于医药生物领域，具体涉及GLP-1R激活剂类似肽的分子改构及其同源二聚体在治疗代谢病中的应用。
背景技术
Exendin-4是从Heloderma suspectum唾液中分离出的肠促胰岛素类似物，有39氨基酸，与GLP-1有53％的序列同源性。GIP是一个42氨基酸的胃肠调节肽，它具有调节机体糖代谢、促进胰岛β细胞释放胰岛素和减低体重功能。GLP-1是一种30氨基酸残基的肠促胰岛素类似肽，在营养摄入时由肠L细胞释放。Exendin-4和GLP-1是目前发现的两个GLP-1R激活剂。基于这三种调节糖代谢的活性多肽氨基酸序列，经过近十年的结构显著性改变，每次变构诞生了获得美国FDA或中国SFDA上市或临床批准的降糖GLP-1R激活剂，例如每日一次给药Liraglutide(2011年上市)和Lixisenatide(2016年上市)、每日两次给药Exenatide(2010年上市)和每周一次给药Polyethylene Clycol Loxenatide(2019年上市)，Albiglutide(2014年上市)，Dulaglutide(2014年上市)，Semaglutide(2017年上市)，Taspoglutide(2015年临床失败)和Tirzepatide(2018年II期临床)。Exenatide、Polyethylene Clycol Loxenatide和Lixisenatide是基于活性多肽Exendin-4的氨基酸序列的变构分子，它们已经完成临床入市。Liraglutide、Albiglutide、Dulaglutide、Semaglutide和Taspoglutide是基于活性多肽GLP-1的氨基酸序列变构类似物，它们通过化学合成或基因重组-化学合成联合方法生产。Tirzepatide是基于GIP-Exendin-4双功能受体激活剂的合成分子，它由Lily开发，目前已经完成II期临床。
大部分GLP-1R激活剂治疗时会出现恶心和呕吐现象。由于下丘脑-垂体-肾上腺轴(The hypothalamic–pituitary–adrenal axis，HPA或者HTPA)是生理性应激反应的一部分，GLP-1R激活剂刺激HPA轴导致皮质酮增加，导致出现部分心率异常。因此，仍然需要：①在激活GIP受体和/或GLP-1受体时，同时拮抗胰高血糖素和GLP-2受体的激活；②需要通过激活GIP受体和/或GLP-1受体效应，来提供体重减轻、拮抗DPP-4和其他形式的降解机制，同时维持较低的免疫原性；③GLP-1R激活剂仍然需要进行优化，因为目前的长效激活剂在比活性(单位质量的降糖效果)、给药剂量、体重降低和副反应方面，已证明都不如天然GLP-1或 Exendin-4分子有效，同时需要科技进步提供更长效降糖激活剂和克服毒性。
发明内容
本发明的目的在于提供一种GLP-1R激活剂类似肽。本发明的GLP-1R激活剂类似肽通过对Exendin-4、GIP-Exendin-4和GLP-1分别进行分子变构。一方面，在单体肽内部以单Cys→Ser替换，对半胱氨酸在肽链中的位置进行了研究。另一方面，对肽链中赖氨酸(K)侧链ε-氨基上的脂肪酸或脂肪酸取代基修饰进行变构，不同脂肪酸或脂肪酸取代基对GLP-1R激活剂类似肽的活性产生不同影响。本发明还提供了由前述GLP-1R激活剂类似肽形成的同源二聚体，发现不同位置半光氨酸形成的H型结构的二聚体(分子内单Cys→Ser取代)产生不同活性，持续降糖时间最长可达22天，比目前1-7天一次的临床药品增加明显。
为实现上述目的，本发明采取的技术方案为：一种胰高血糖素样肽-1受体(GLP-1R)激活剂类似肽，所述GLP-1R激活剂类似肽为Exendin-4、GIP-Exendin-4嵌合肽或GLP-1的序列经变构并且一个赖氨酸ε-氨基上脂肪酸链修饰而成。本发明的GLP-1R激活剂类似肽，是以Exendin-4、GIP-Exendin-4或GLP-1肽的氨基酸序列为主链，将主链上的Ser替换为Cys，且主链氨基酸序列中仅含有一个Cys；侧链是主链的其中一个赖氨酸的ε-氨基的脂肪酸链。
优选地，所述GLP-1R激活剂类似肽的具体序列为以下任意一种：
(1)(HN 2)H-X 2-EGTFTCDLS-X 12-QMEEEAV-X 20-LFIEWL-X 27-NGGPSSGAPP-X 38或；
(2)(HN 2)H-X 2-EGTFTSDLC-X 12-QMEEEAV-X 20-LFIEWL-X 27-NGGPSSGAPP-X 38或；
(3)(HN 2)H-X 2-EGTFTSDLS-X 12-QMEEEAV-X 20-LFIEWL-X 27-NGGPCSGAPP-X 38或；
(4)(HN 2)H-X 2-EGTFTSDLS-X 12-QMEEEAV-X 20-LFIEWL-X 27-NGGPSCGAPP-X 38或；
(5)(HN 2)Y-X 2-EGTFTCDYSI-X 13-LDKIAQ-X 20-AFVQWLIAGGPSSGAPP-X 38或；
(6)(HN 2)Y-X 2-EGTFTSDYCI-X 13-LDKIAQ-X 20-AFVQWLIAGGPSSGAPP-X 38或；
(7)(HN 2)Y-X 2-EGTFTSDYSI-X 13-LDKIAQ-X 20-AFVQWLIAGGPCSGAPP-X 38或；
(8)(HN 2)Y-X 2-EGTFTSDYSI-X 13-LDKIAQ-X 20-AFVQWLIAGGPSCGAPP-X 38或；
(9)(HN 2)H-X 2-EGTFTSDVSCYLEGQAA-X 20-EFIAWLV-X 28-GRG(NH 2)；
其中，X 2或X 13为L-α-甘氨酸或L-α-丙氨酸或α-氨基异丁酸(αAib)；X 12或X 20或X 27或X 28为赖氨酸，或精氨酸，或侧链ε-氨基上谷氨酰脂肪酸[γ-Glu(N-α-fatty acid)]或谷氨酰脂肪二酸[γ-Glu(N-α-fatty diacid)]修饰的赖氨酸，或侧链ε-氨基上[2×AEEAC-γ-Glu-(N-α-脂肪二酸)]修饰的赖氨酸；X 38为PS(HN 2)或SKKKKKK(HN 2)。所述大写单字母为L-ɑ-氨基酸的缩写或氨基酸取代符号，阿拉伯数字为氨基酸残基排列顺序，NH 2代表N末端或者C端酰胺基结构。
优选地，所述GLP-1R激活剂类似肽，当X 12或X 20或X 27或X 28为侧链ε-氨基上谷氨酰脂肪酸[γ-Glu(N-α-fatty acid)]或谷氨酰脂肪二酸[γ-Glu(N-α-fatty diacid)]修饰的赖氨酸时，其结构如化学式1所示；当X 12或X 20或X 27或X 28为侧链ε-氨基上[2×AEEAC-γ-Glu-(N-α-脂肪二酸)]修饰的赖氨酸时，其结构如化学式2所示。
Figure PCTCN2021078057-appb-000001
化学式1；
Figure PCTCN2021078057-appb-000002
化学式2。
本发明还提供一种降糖类似肽同源二聚体，所述二聚体由权利要求1-3任一项所述的相同单体之间通过半胱氨酸形成的二硫键连接而成，构成H型GLP-1R激活剂类似肽同源二聚 体。
优选地，所述二聚体的氨基酸序列为以下中的任意一种：
Figure PCTCN2021078057-appb-000003
其中，X 2或X 13为L-α-甘氨酸或L-α-丙氨酸或α-氨基异丁酸(αAib)；X 12或X 20或X 27或X 28为赖氨酸，或精氨酸，或侧链ε-氨基上谷氨酰脂肪酸[γ-Glu(N-α-fatty acid)]或谷氨酰脂肪二酸[γ-Glu(N-α-fatty diacid)]修饰的赖氨酸，或侧链ε-氨基上[2×AEEAC-γ-Glu-(N-α-脂肪二酸)]修饰的赖氨酸；X 38为PS(HN 2)或SKKKKKK(HN 2)；“|”表示两个半胱氨酸之间形成的二硫键。
优选地，当X 12或X 20或X 27或X 28为侧链ε-氨基上谷氨酰脂肪酸[γ-Glu(N-α-fatty acid)]或谷氨酰脂肪二酸[γ-Glu(N-α-fatty diacid)]修饰的赖氨酸时，其结构如化学式1所示；当X 12或X 20或X 27或X 28为侧链ε-氨基上[2×AEEAC-γ-Glu-(N-α-脂肪二酸]修饰的赖氨酸时，其结构如化学式2所示。
本发明还提供所述的GLP-1R激活剂类似肽或所述的同源二聚体在制备治疗代谢综合征病症的药物中的用途。优选地，所述代谢综合征病症包括高血糖、糖尿病和肥胖症。
本发明还提供一种治疗代谢综合征的病症的药物，所述药物如上所述的GLP-1R激活剂类似肽或同源二聚体和其药学上可接受的盐作为活性成分。
本发明的有益效果：本发明的H型GLP-1R激活剂类似物同源二聚体在降糖强度不低于对应单体肽的情况下，显著增加对应单体激活剂或被FDA或SFDA批准的GLP-1R激活剂临床药物的降糖作用时间达2-3倍左右，所提供的GLP-1R激活剂类似物同源二聚体在体内的活性维持时间长达22天，较阳性药Lixinaglutide(药效维持2天)明显延长。相比临床GLP-1R激活剂，新型二聚体结构变化非常明显，极大便利了其临床应用和市场推广。
附图说明
图1为单一OGTT的血糖测试结果示意图。
图2为2G21治疗T2D模型时体重统计分析图。
图3为2G21治疗T2D模型中的血糖统计分析图。
图4为2G21治疗T2D模型中的糖化血红蛋白统计分析图。
图5为2G21治疗T2D模型中的胰岛素统计分析图。
图6为2G21治疗T2D模型中的谷丙转氨酶统计分析图。
图7为2G21治疗T2D模型中的胰淀粉酶统计分析图。
具体实施方式
为了更加简洁明了的展示本发明的技术方案、目的和优点，下面结合具体实施例及其附图对本发明做进一步的详细描述。
实施例1 单体肽和二聚体的制备
一、单体肽固相化学合成过程：手工固相多肽合成操作步骤。
1、树脂溶涨：将氨基树脂(C末端酰胺化序列用氨基树脂)(购自天津市南开合成科技有限公司)，放入反应锅中，加二氯甲烷(DCM，Dikma Technologies Inc.)15ml/g树脂，振荡30min。SYMPHONY型12通道多肽合成仪(SYMPHONY型号，软件Version.201，Protein Technologies Inc.)。
2、接第一个氨基酸：通过沙芯抽滤除去溶剂，加入3倍摩尔的C端第一个Fmoc-氨基酸(所有Fmoc-氨基酸由苏州天马医药集团精细化学品有限公司提供)，再加入10倍摩尔量的4-二甲氨基吡啶(DMAP)和N,N'-二环己基碳二亚胺(DCC)，最后加入二甲基甲酰胺(DMF)(购自Dikma Technologies Inc.)溶解，振荡30min。用醋酸酐封闭。
3、脱保护：去掉DMF，加20％哌啶-DMF溶液(15ml/g)，5min，过滤去掉溶剂，再加20％哌啶-DMF溶液(15ml/g)，15min。哌啶由国药集团上海化学试剂公司提供。
4、检测：抽掉溶剂。取十几粒树脂，用乙醇洗三次，加入茚三酮、KCN和苯酚溶液各一滴，105-110℃加热5min，变深蓝色为阳性反应。
5、洗树脂：依次DMF(10ml/g)洗两次，甲醇(10ml/g)洗两次，DMF(10ml/g)洗两次。
6、缩合：根据具体合成条件，以下方法可以在多肽合成中单独或混搭使用：
方法a：三倍量的保护氨基酸和三倍量的2-(7-偶氮苯并三氮唑)-四甲基脲六氟磷酸酯(HBTU，苏州天马医药集团精细化学品有限公司)，均用尽量少DMF溶解，加入反应锅中。立刻加入十倍量的N-甲基吗啉(NMM，苏州天马医药集团精细化工有限公司).反应30min， 检测呈阴性。
方法b：三倍量的保护氨基酸FMOC-氨基酸和三倍量1-羟基苯并三唑(HOBt，苏州天马医药集团精细化学品有限公司)，均用尽量少DMF溶解，加入反应管，立刻加入三倍量的N,N'-二异丙基碳二亚胺(DIC).反应30min.，检测呈阴性。
7、洗树脂：依次DMF(10ml/g)洗一次，甲醇(10ml/g)洗两次，DMF(10ml/g)洗两次。
8、重复2至6步操作，如表1中氨基酸没有侧链修饰的GLP-1R激活肽，或者具有侧链修饰的GLP-1R激活肽所示，从右到左依次连接相应氨基酸。带有K 12或K 20或K 27或K 28修饰的，按照如下9方法合成。
9、合成K{N-ε-[γ-Glu-(N-α-脂肪酸或脂肪二酸)]}：加入10ml 2％水合肼反应30min去除Fmoc-Lys(Dde)-OH的保护基Dde，侧链氨基裸露，用DMF和甲醇交替洗涤六次，茚三酮检测为蓝色。称取550mg的Fmoc-Glu-OTBU，HOBT 250mg，用DMF溶解，加入0.3ml的DIC，混匀，加入到反应器中与赖氨酸侧链氨基反应1h，抽干，DMF洗涤4次，茚三酮检测为无色。向反应器中加入5ml 20％哌啶DMF溶液反应20min，去除Fmoc-Glu-OTBU的氨基保护集团Fmoc,用DMF和甲醇交替洗涤六次，茚三酮检测为蓝色。称取300mg脂肪酸或脂肪二酸，HOBT 250mg，用DMF溶解，并且加入0.3ml的DIC，混匀，加入到反应器中反应1h，抽干，DMF洗涤4次，茚三酮检测为无色，用甲醇洗涤2次抽干。
合成K{N-ε-[2×AEEAC-γ-Glu-(N-α-脂肪二酸)]}：在Dde-Lys(fmoc)脱fmoc基团后，加入2mM的Fmoc-AEEAC-OH和2mM六氟磷酸苯并三唑-1-氧基三吡咯烷基磷(PyBOP)，45mM的HOBt，用DMF溶解，冰水浴下加入0.375mM的N,N'-二异丙基乙胺(DIPEA)活化3min，加入反应柱反应2h，以茚三酮法检测判断实验终点。反应结束，20％哌啶-DMF溶液(15ml/g)脱除Fmoc，DMF洗涤6次。同样方法再次偶联Fmoc-AEEAC-OH、Fmoc-Glu-OtBu和脂肪二酸链基团。用含2％水合肼反应30min去除序列赖氨酸保护基Dde，经过步骤8接在赖氨酰侧链ε氨基上。
10、将缩合完成的多肽经过DMF(10ml/g)两次，DCM(10ml/g)两次，DMF(10ml/g)两次，抽干10min。茚三酮检测阴性。
11、脱除肽链最后N端氨基酸的FMOC保护基，检测呈阳性，溶液抽干备用。
12、按照下列方法洗树脂，依次DMF(10ml/g)两次，甲醇(10ml/g)两次，DMF(10ml/g)两次，DCM(10ml/g)两次，10min抽干。
13、从树脂上切割多肽：配制切割液(10毫升/g)：TFA 94％(J.T.Baker Chemical Company)、水2.5％、ethanedithiol(EDT,Sigma-Aldrich Chemistry)2.5％和triisopropylsilane(TIS,Sigma-Aldrich Chemistry)1％。切割时间：120min。
14、吹干洗涤：将裂解液用氮气尽量吹干，用乙醚洗六次，然后常温挥干。
15、用如下方法HPLC纯化多肽、鉴定和置于-20℃避光保存。
二、检验方法如下：
1、用HPLC纯化多肽：将粗肽用纯水或者加少量乙腈溶解，按照下列条件纯化：高效液相色谱仪(分析型；软件Class-VP.Sevial System；厂商日本SHIMADZU)和Venusi MRC-ODS C18色谱柱(30×250mm，天津Bonna-Agela Technologies)。流动相A液：0.1％三氟醋酸水溶液，流动相B液：0.1％三氟乙酸+99.9％乙腈溶液(乙腈Fisher Scientific公司购买)。流速：1.0ml/min，上样体积30μl，检测波长220nm。洗脱程序：0～5min：90％A液+10％B液；5～30min：90％A液/10％B液→20％A液/80％B液。
2、最后将纯化后的有效溶液冻干(冻干机Freezone Plus 6型号，L ABCONCO厂商)，既得到成品。
3、鉴定：分别取少量的成品多肽，做HPLC分析其纯度：高效液相色谱仪(厂商日本SHIMADZU)和Venusi MRC-ODS C18色谱柱(4.6x150mm，天津Bonna-Agela Technologies)。流动相A液：0.1％三氟乙酸水溶液，流动相B液：99.9％乙腈+0.1％三氟乙酸溶液，流速：1.0ml/min，上样体积10μl，检测波长220nm。洗脱程序：0～5min：100％A液；5～30min：100％A液→20％A液/80％B液。要求测定纯度大于95％。具体方法参见我们授权专利(中国专利ZL201410612382.3)。
MS法鉴定多肽分子量：取纯度合格的多肽加入水溶解，加入5％乙酸+8％乙腈+87水溶解测试电喷雾离子化质谱测定分子量，具体方法参见我们授权专利(中国专利 ZL201410612382.3)。
4、将粉末状的多肽，密封包装，-20℃避光保存。
三、二聚体的形成：将浓度为1mg/ml的肽链内部带有单半胱氨酸的单体肽，在pH＝9.5磷酸氢二钠水溶液中，37℃保温过夜，形成同源二聚体肽。对于溶解性略差的二聚体肽，每分钟4000转离心20分钟，取沉淀为纯二聚体肽，沉淀用NaCl-PB溶液(用磷酸氢二钠调节生理盐水的pH＝8.0)溶解使用。对于溶解的二聚体肽或上述离心上清通过Sephadex G-25层析分离和鉴定(在2×60cm G-25层析柱和自然流速下，用NaCl-PB溶液为流动项，二聚体组份为第一峰，残余杂质组份为后续峰)。二聚体肽可以通过无巯基还原剂的肽PAGE电泳或质谱加以鉴定，具体方法参见我们授权专利(中国专利ZL201410612382.3)。
四、GLP-1R激活剂类似肽单体及其二聚体由本研究室或委托商业公司合成，发明人通过HPLC纯度、ESI或激光飞行质谱和半胱氨酸氧化确认其结构。本发明合成的GLP-1R激活剂单体如表1所示，同源二聚体肽的氨基酸序列如表2所示。
表1：本发明合成的GLP-1R激活剂类似肽单体的氨基酸序列和其相同剂量单次注射的持续降糖活性
Figure PCTCN2021078057-appb-000004
Figure PCTCN2021078057-appb-000005
注：表中Tirzepatide为GIP-Exendin-4肽的嵌合体肽；Lixisenatide也是对Exendin 4的变构；CFA(carbon fatty acid)或CFDA(carbon fatty diacid)为碳脂肪酸或碳脂肪二酸；所述K[N-ε-(γ-Glu-N-α-CFA或CFDA)]、K[N-ε-(2×AEEAC-γ-Glu-N-α-CFDA)]表示赖氨酸K侧链ε-氨基的脂肪酰或脂肪二酸单酰基谷氨酰修饰，具体结构如式1或2所示。
实施例2 本发明的GLP-1R激活剂降糖效果的持久性研究
1、实验方法：在广东省动物中心购买正常KM小鼠用于糖耐量测定(OGTT)：正常昆明小鼠的糖耐量测定用于筛选药物的降血糖活性和持久性。根据无差别的空腹血糖，雄性昆明小鼠(5周龄)被分成多组(NaCl-PB组、Lixisenatide组、单体G9-G12系列和二聚体2G9-2G12系列组)(n＝6)。在经过两轮14小时进食—10小时禁食的适应期后，KM小鼠在每次10小时的禁食后立即进行糖耐量测定。背部皮下注射药物或单体(用生理盐水pH6.5溶解，其空白对照使用生理盐水)或二聚体肽(用NaCl-PB溶液pH8.0溶解，其空白对照一般使用NaCl-PB溶液)后30min，小鼠准时灌胃5％葡萄糖溶液，准确在灌胃后测定鼠尾血糖值。血糖仪和血糖试纸为Bayer HeathCare LLC公司产品。以各组平均血糖值为判断标准：当各组OGTT平均血糖值连续两次高过同日空白对照组平均血糖值时，测定停止，期间低于空白组血糖持续 时间为药效持续时间。
2、实验结果
2.1口服葡萄糖耐受性试验
单次给药后30分，准时在单次口服葡萄糖前(0min)和后10、20、40、60、120min取小鼠尾部血测定血糖，空白对照组使用生理盐水(图1)。结果显示：在10min，与Lixisenatide和G21组比较，2G33组出现显著性葡萄糖增加(P<0.05)。在20min，与空白对照组比较，Lixisenatide和G21组出现显著性降低(P<0.05或0.01)。与Lixisenatide和G21组比较，2G21组出现显著性葡萄糖增加(P<0.05)。在40min，与空白对照组比较，Lixisenatide、G21和G33组出现显著性降低(P<0.05或0.01)。与空白对照、Lixisenatide或G33组比较，2G33组出现显著性葡萄糖降低或增加(P<0.05)。在60min，与空白对照组比较，G21组出现显著性降低(P<0.05)；与Lixisenatide和G21组比较，2G21或2G33组出现显著性葡萄糖增加(P<0.05)。结果显示，在相同时间内，二聚体血糖值明显高过单体肽血糖值，显示二聚体明显延迟吸收。
在单次给药后，持续多天多次OGTT试验(mOGTT)：背部皮下注射药物或单体或二聚体肽后30min，小鼠准时灌胃5％葡萄糖溶液，准确在灌胃后35min测定鼠尾血糖值。以血糖平均值为判断标准，Lixisenatide阳性药降糖持续时间2天、G9系列维持2-9天、G10系列2-11天、G11系列2-11和G12系列7-9天。二聚体2G9系列维持4-21天，2G10系列维持4-22天，2G11系列维持6-21天，2G12系列维持16-20天。各单体组(表1所示)约为其对应二聚体组(表2所示)的1/2持续时间。比较发现，在第11或12位形成二硫键同时在第20位有赖氨酸ε-氨基侧链脂肪酸修饰的二聚体肽(其中以20碳脂肪酸或脂肪二酸为最好)，有明显体重降低，其降糖持续时间也明显增加。比较发现，2G9系列中第11肽，2G10系列中第20和21，2G11系列中第33和34二聚体肽持续时间最长达21-22天。2G10系列中第21肽(2G21)为Exendin 4变构形式，和Lixisenatide阳性对照药的序列有高同源性和相同的侧链脂肪酸修饰，因此选择2G21肽进行体内II型糖尿病(T2D)的治疗以及后续的实验。mOGTT实验需要精准实施，否则结果可能有跳揺。
表2 GLP-1R激活剂二聚体序列及其相同剂量单次皮下注射降糖活性持续时间
Figure PCTCN2021078057-appb-000006
Figure PCTCN2021078057-appb-000007
Figure PCTCN2021078057-appb-000008
注：表中CFA(carbon fatty acid)或CFDA(carbon fatty diacid)为碳脂肪酸或碳脂肪二酸；所述K[N-ε-(γ-Glu-N-α-CFA或CFDA)]、K[N-ε-(2×AEEAC-γ-Glu-N-α-CFDA)]表示K侧链ε-氨基的脂肪酰或脂肪二酸单酰基谷氨酰修饰，具体结构如式1或2所示。所述二聚体由单体肽中的半胱氨酸之间形成的二硫键连接，构成H型二聚体；其中“”表示单体之间两个半光氨酸之间形成二硫键。
实施例3 二聚体对II型糖尿病模型治疗效果
一、构建II型糖尿病(T2D)小鼠模型
将C57Bl6/J小鼠放置于标准饮食的SPF级别环境中，自由饮水。所有的实验操作按照实验动物伦理与使用制度指导原则。按照标准饮食饲养天后，将5周龄的C57B16/J雄性小鼠分为6组：NaCl-PB组、Placebo组(模型对照组)、Lixisenatide组、低中高剂量二聚体肽2G21组。NaCl-PB组为空白对照和Placebo为T2D模型对照，它们注射NaCl-PB溶液。所有T2D模型组喂60kcal％的高脂饮食(D12492,常州鼠一鼠二生物技术有限公司，常州，中国)，直到实验结束，空白对照组保持标准饮食直到实验结束。糖尿病模型的建立方法：高脂喂养小鼠4周后，腹腔注射75mg/kg链脲佐菌素(STZ，美国西格玛化学公司)，3天后用50mg/kg剂量的STZ重新腹腔注射，3周后血糖等于或大于11mM的小鼠视为糖尿病小鼠。这些糖尿病组在高脂饮食的基础上，再进行35天的治疗研究。
二、对II型糖尿病治疗效果
肽的溶解度：对于Lys侧链含有脂肪酸修饰结构的，不含Aib氨基酸组成的单体肽在水中显示悬浮状态，而其对应的同源二聚体肽在水中完全溶解；对于Lys侧链含有脂肪酸修饰结构的，含有Aib氨基酸或/和有C端酰胺化结构的单体肽在水中显示完全溶解，而其对应的同源二聚体肽在水中溶解差。对于没有Lys侧链脂肪酸修饰的，单体肽和二聚体都完全溶解于水。所有的二聚体肽分别用NaCl-PB(pH8.0)溶解注射，低、中、高不同剂量的同源二聚体2G21分别溶于NaCl-PB溶液[Na 2HPO 4缓冲的生理盐水溶液(pH8.0)]中进行动物注射。单体肽溶解在生理盐水溶液注射(pH6.5左右)。
给药浓度设置：我们预实验显示，单次皮下注射0.624nmol/100μl的Lixisenatide肽，其多天多次OGTT的时效关系很容易观察到。所以，在所有的糖耐量实验中，正常昆明小鼠臀部皮下注射单次剂量为0.624nmol/100μl的Lixisenatide或单体肽或二聚体肽，每天早上9点剪尾采血测量血糖并称重。对于OGTT，每个动物的给药、灌胃和取血测糖时间需要精确到秒。T2D动物实验选择Lixisenatide为阳性对照和其给药方式(皮下给药)。
0.624nmol/100μl的Lixisenatide可诱导T2D糖尿病模型(餐后血糖达20mM)的餐后血糖值达到8-11mM。在该临界值，阳性药Lixisenatide与GLP-1R二聚体的效应-剂量关系很容易被观察到。在T2D治疗研究中，在30min内，按每只100μl剂量臀部皮下注射T2D模型鼠，每五天测量实验鼠血糖值，整个测试在40min内完成。二聚体2G21肽高中低剂量分别为1.873、0.624、0.208nmol/100μL，阳性药物Lixisenatide剂量为0.624nmol/100μl(原料药由商业公司合成)，每天注射一次直至35天实验结束。
1、T2D治疗后的体重变化：给药前，T2D模型组体重无显著性差异。实验结束后，与NaCl-PB组比较，Placebo组、Lixisenatide组、L-2G21组(低剂量)、M-2G21组(中剂量)显示体重明显增加(P<0.05或0.001)，但H-2G21组(高剂量)与正常组没有差异，显示治疗有效。与Placebo组相比，Lixisenatide组和H-2G21组的体重下降明显(P<0.05)。各2G21组体重呈剂量依赖性下降，M-2G21组与Lixisenatide组呈相似变化(图2)。
2、T2D治疗中的降血糖作用：与NaCl-PB组相比，Placebo、Lixisenatide、L-和M-2G21组有显著性高的空腹血糖值(图3)(P<0.05或0.001)，或Placebo、Lixisenatide、L-和M-、H-2G21组有显著性高糖化血红蛋白(HbA 1c)(图4)(P<0.001)，显示T2D模型是成功的。与Placebo组相比，Lixisenatide和H-2G21组空腹血糖显著性降低(P＜0.05或0.01)，或Lixisenatide、M-2G21和H-2G21组HbA 1c显著性降低(P＜0.05)。与Lixisenatide组比较，L-2G21组空腹血糖明显升高(P＜0.05)。注射肽后，血糖水平成剂量依赖性下降，随给药次数越多，效果越好。M-2G21组的血糖变化与Lixisenatide组相似。HbA 1c和血糖值在T2D治疗中产生类似的变化。
3、T2D治疗中的血液生化指标检测：在T2D治疗实验后，Placebo、Lixisenatide或L-2G21组出现空腹胰岛素水平显著低于NaCl-PB组(P<0.01或0.001)。各2G21组空腹胰岛素呈剂量依赖性增加，与Lixisenatide组和L-2G21组比较，M-2G21组胰岛素含量增加2-3倍(P<0.05或0.01)。与Placebo、Lixisenatide组或L-2G21组比较，H-2G21组胰岛素含量增加2-4倍(P<0.05 或0.001)(图5)，显示等摩尔浓度的二聚体肽诱导2-3倍胰岛素分泌。2G21组谷丙转氨酶(ALT)呈剂量依赖性下降，H-2G21组ALT水平均低于NaCl-PB或Placebo、L-2G21组(P<0.05)(图6)。各2G21组血清淀粉酶呈剂量依赖性下降，但与空白对照或Placebo、Lixisenatide没有统计学差异(P>0.05)(图7)。
从上述实施例中，可以得出以下结论：我们发展的同源二聚体系列可以明显增加药效时间。研究表明，二聚体序列对啮齿类动物T2D模型表现出最有希望应用前景，如持续时间最长的降血糖效应和减轻体重作用。
结构-活性关系表明，没有脂肪酸修饰的二聚体在水中有最好溶解性，具有脂肪酸修饰肽含有Aib氨基酸结构二聚体，甚至带有C末端酰胺化结构，在水中溶解性略差，这些是药物制剂研究的重要数据，也是本研究不同结构GLP-1R激活剂有不同降糖活性持续时间的根本原因—不同空间构象，形成不同理化性质，产生不同降糖持续时间。
单次相同剂量给药，单次OGTT实验结果显示，由于二聚体缓慢吸收而产生较长的降血糖作用。单次相同剂量给药，多次OGTT实验结果表明，较长的持续时间效应涉及二聚体第2位氨基酸、二硫键位置、对称脂肪酸或脂肪二酸修饰的Lys和C端酰胺化等变构相关。表2中显示，最长活性的二聚体结构含有 2αAib、 11或 12Cys-Cys二硫键、对称20脂肪酰或脂肪二酸单酰基-L-γ-谷氨酰基- 20赖氨酸或20碳脂肪二酸酰单酰基-γ-Glu-2×AEEAC- 20Lys和C末端酰胺化等结构。这些修饰特点如下：(1)αAib→ 2Ala代换产生更长活性；(2)与其他脂肪酸修饰比较，Lys[2×AEEAC-γ-Glu-(N-α-20碳脂肪二酸)]修饰达到最好结果；(3)C末端酰胺化明显延长活性；(4)二聚体分子中第12或11位二硫键结构显示最好活性。单体肽活性仅是对应二聚体的1/2。
在T2D治疗实验中，2G21组对糖尿病模型HbA 1c或空腹血糖(FPG)值有明显降低，有明显降血糖作用，相同摩尔浓度的2G21肽和Lixisenatide对PPG(餐后血糖)或FPG、HbA 1c有相似的降低作用。
2G21组体重呈剂量依赖性下降，M-2G21组与Lixisenatide组在体重减低方面一致。较好的体重降低的二聚体肽结构涉及 11或 12Cys-Cys二硫键、对称 20Lys[2×AEEAC-γ-Glu-20碳脂肪酸]修饰和C末端酰胺化等结构。
2G21使谷丙转氨酶(ALT)呈剂量依赖式降低，显示药物对肝脏有着很强的保护作用。
在T2D治疗实验中，2G21组胰岛素呈剂量依赖性增加，与Placebo、Lixisenatide和L-2G21比较，M-或H-2G21组诱导了2-4倍高的胰岛素水平，因而2G21具有更好的降糖作用。
综上所述，本发明的二聚体肽能诱导更多的胰岛素释放，从而产生更好的降血糖作用。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (9)

  1. 一种胰高血糖素样肽-1受体(GLP-1R)激活剂类似肽，其特征在于，所述GLP-1R激活剂类似肽为Exendin-4、GIP-Exendin-4嵌合肽或GLP-1的序列经变构并且一个赖氨酸ε-氨基上脂肪酸链修饰而成。
  2. 如权利要求1所述的GLP-1R激活剂类似肽，其特征在于，所述类似肽的具体序列为以下任意一种：
    (1)(HN 2)H-X 2-EGTFT8CDLS-X 12-QMEEEAV-X 20-LFIEWL-X 27-NGGPSSGAPP-X 38或；
    (2)(HN 2)H-X 2-EGTFTSDL11C-X 12-QMEEEAV-X 20-LFIEWL-X 27-NGGP32S33SGAPP-X 38或；
    (3)(HN 2)H-X 2-EGTFTSDLS-X 12-QMEEEAV-X 20-LFIEWL-X 27-NGGPCSGAPP-X 38或；
    (4)(HN 2)H-X 2-EGTFTSDLS-X 12-QMEEEAV-X 20-LFIEWL-X 27-NGGPSCGAPP-X 38或；
    (5)(HN 2)Y-X 2-EGTFTCDYSI-X 13-LDKIAQ-X 20-AFVQWLIAGGPSSGAPP-X 38或；
    (6)(HN 2)Y-X 2-EGTFTSDYCI-X 13-LDKIAQ-X 20-AFVQWLIAGGPSSGAPP-X 38或；
    (7)(HN 2)Y-X 2-EGTFTSDYSI-X 13-LDKIAQ-X 20-AFVQWLIAGGPCSGAPP-X 38或；
    (8)(HN 2)Y-X 2-EGTFTSDYSI-X 13-LDKIAQ-X 20-AFVQWLIAGGPSCGAPP-X 38或；
    (9)(HN 2)H-X 2-EGTFTSDVSCYLEGQAA-X 20-EFIAWLV-X 28-GRG(NH 2)；
    其中，X 2或X 13为L-α-甘氨酸或L-α-丙氨酸或α-氨基异丁酸(αAib)；X 12或X 20或X 27或X 28为赖氨酸，或精氨酸，或侧链ε-氨基上谷氨酰脂肪酸[γ-Glu(N-α-fatty acid)]或谷氨酰脂肪二酸[γ-Glu(N-α-fatty diacid)]修饰的赖氨酸，或侧链ε-氨基上[2×AEEAC-γ-Glu-(N-α-脂肪二酸)]修饰的赖氨酸；X 38为PS(HN 2)或SKKKKKK(HN 2)。
  3. 如权利要求2所述的GLP-1R激活剂类似肽，其特征在于，当X 12或X 20或X 27或X 28为侧链ε-氨基上谷氨酰脂肪酸γ-Glu(N-α-fatty acid)或谷氨酰脂肪二酸γ-Glu(N-α-fatty diacid)修饰的赖氨酸时，其结构如化学式1所示；当X 12或X 20或X 27或X 28为侧链ε-氨基上[2×AEEAC-γ-Glu-(N-α-脂肪二酸]修饰的赖氨酸时，其结构如化学式2所示：
    Figure PCTCN2021078057-appb-100001
    Figure PCTCN2021078057-appb-100002
  4. 一种降糖类似肽同源二聚体，其特征在于，所述二聚体由权利要求1-3任一项所述的相同单体之间通过半胱氨酸形成的二硫键连接而成，构成H型GLP-1R激活剂类似肽同源二聚体。
  5. 如权利要求4所述的二聚体，其特征在于，其氨基酸序列为以下中的任意一种：
    Figure PCTCN2021078057-appb-100003
    Figure PCTCN2021078057-appb-100004
    其中，X 2或X 13为L-α-甘氨酸或L-α-丙氨酸或α-氨基异丁酸(αAib)；X 12或X 20或X 27或X 28为赖氨酸，或精氨酸，或侧链ε-氨基上谷氨酰脂肪酸[γ-Glu(N-α-fatty acid)]或谷氨酰脂肪二酸[γ-Glu(N-α-fatty diacid)]修饰的赖氨酸，或侧链ε-氨基上[2×AEEAC-γ-Glu-(N-α-脂肪二酸)]修饰的赖氨酸；X 38为PS(HN 2)或SKKKKKK(HN 2)；“|”表示两个半胱氨酸之间形成的二硫键。
  6. 如权利要求5所述的所述的二聚体，其特征在于，当X 12或X 20或X 27或X 28为侧链ε-氨基上谷氨酰脂肪酸[γ-Glu(N-α-fatty acid)]或谷氨酰脂肪二酸[γ-Glu(N-α-fatty diacid)]修饰的赖氨酸时，其结构如化学式1所示；当X 12或X 20或X 27或X 28为侧链ε-氨基上[2×AEEAC-γ-Glu-(N-α-脂肪二酸]修饰的赖氨酸时，其结构如化学式2所示。
  7. 如权利要求1-3任一项所述的GLP-1R激活剂类似肽，或如权利要求4-6任一项所述的同源二聚体在制备治疗代谢综合征病症的药物中的用途。
  8. 如权利要求7所述的用途，其特征在于，所述代谢综合征病症包括高血糖、糖尿病和肥胖。
  9. 一种治疗代谢综合征病症的药物，其特征在于，所述药物以权利要求1-3任一项所述的GLP-1R激活剂类似肽，或以权利要求4-6任一项所述的同源二聚体和其药学上可接受的盐作为活性成分。
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