WO2021129779A1 - 一种新型两亲性蛋白、其制备方法及用途 - Google Patents

一种新型两亲性蛋白、其制备方法及用途 Download PDF

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WO2021129779A1
WO2021129779A1 PCT/CN2020/139216 CN2020139216W WO2021129779A1 WO 2021129779 A1 WO2021129779 A1 WO 2021129779A1 CN 2020139216 W CN2020139216 W CN 2020139216W WO 2021129779 A1 WO2021129779 A1 WO 2021129779A1
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insulin
protein
amphiphilic protein
amphiphilic
cys
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French (fr)
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李瑛�
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李瑛�
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the present invention relates to the design and construction of a new type of amphipathic protein and its application. Specifically, the present invention relates to an amphipathic protein and its preparation method and application.
  • the protein has strong amphiphilicity in solution. It can form hydrophobic and hydrophilic domains to form a stable composition with the drug through the non-covalent bond interaction between the domain and the drug. This composition can significantly improve the stability and half-life of the drug in the body.
  • Biomacromolecules are unstable and easily hydrolyzed in vivo.
  • biopharmaceuticals In recent years, the use of biological macromolecular structure modification to optimize the metabolic kinetic properties of biopharmaceuticals to extend its half-life is a frequently used method, but some biopharmaceuticals will be partially or completely inactivated during the structural modification process.
  • glucagon-like peptide-1 a large number of amino acid substitution experiments have confirmed that the easy degradation of the second amino acid Ala leads to its half-life of about 2-5 minutes, but the structural modification at this site leads to inactivation of the polypeptide function. Receptor binding.
  • Another example is the structural modification of insulin. Many sites in the insulin molecule are easily degraded by proteases, and the three pairs of disulfide bonds in the molecule are also one of its unstable factors.
  • Bio macromolecular drugs including peptide drugs or protein drugs, for example, insulin, insulin analogs.
  • Insulin analogues People use genetic engineering technology to locally modify the amino acid sequence and structure of human insulin to synthesize human insulin analogues, which generally refer to the secretion of normal insulin and are similar in structure to insulin. Of the substance.
  • Insulin and insulin analogues The term "insulin" in the present invention adopts a broad concept, and unless otherwise specified, it includes but is not limited to insulin, insulin analogues, human insulin, synthetic insulin, animal pancreas (e.g., pig pancreas) ) Peptides with lowering blood sugar effect, etc.
  • the present invention relates to a new type of amphiphilic protein with structural characteristics, which has different hydrophilic and hydrophobic domains.
  • This protein with special structural characteristics can form a composition with biological macromolecular drugs, such as insulin molecules.
  • the stability of the composition can effectively increase the serum and in vivo half-life of insulin molecules, thereby optimizing the clinical medication regimen of insulin, and achieving the purpose of reducing the number of registrations, for example, reducing to 1 injection per day, or even lower frequency.
  • An object of the present invention is to use a protein with special physical and chemical properties to form a composition with insulin, which can effectively improve the absorption, half-life and stability of the drug in the blood after oral administration.
  • One purpose of the present invention is to provide a carrier protein that can form a composition with biomacromolecule drugs in order to increase the stability of the drug against biological degradation factors and prolong the retention time of biomacromolecule drugs in the body. Its half-life in the body.
  • Another object of the present invention is to provide a carrier including the carrier protein, and its application in preparing a pharmaceutical composition.
  • Another object of the present invention is to provide a pharmaceutical composition formed by a composition of amphiphilic protein and insulin and pharmaceutically acceptable excipients (including: carrier materials, excipients, stabilizers or diluents, etc.) and ⁇ The method of preparation.
  • pharmaceutically acceptable excipients including: carrier materials, excipients, stabilizers or diluents, etc.
  • an amphiphilic protein and a pharmaceutically acceptable salt, ester, ether, amide, or mixture thereof, and the carrier polypeptide has the general formula characteristics of the amino acid residue sequence of SEQ ID NO::
  • the protein with amphipathic function in Formula 1 contains 8 cysteines and forms 4 pairs of disulfide bonds.
  • A, B, C, D, E, F, G respectively refer to amino acid fragments with certain structural characteristics, and the sequence is shown in the sequence list;
  • a 1 -A 7 in the above general formula are selected from the following fragments:
  • X refers to hydrophobic amino acids, including: A, G, V, L, I, P,
  • Y refers to hydrophilic amino acids, including: S, T, N, Q,
  • Z refers to charged amino acids, including: D, E;
  • amphiphilic protein of the present invention preferably has the following structure:
  • Amphipathic protein 1 (protein sequence 1, sequence 1):
  • Amphipathic protein 2 (protein sequence 2, sequence 2):
  • Amphiphilic protein 3 (protein sequence 3, sequence 3):
  • Amphipathic protein 4 (protein sequence 4, sequence 4):
  • amphiphilic protein of the present invention also includes pharmaceutically acceptable salts, esters, ethers, amides or mixtures thereof of the amphiphilic protein as a pharmaceutical carrier and the application in the preparation of medicines.
  • the protein molecule with the above general structural formula is prepared by a biological fermentation method, and the product is purified by hydrophobic chromatography and reversed phase chromatography.
  • a pharmaceutical composition which comprises 1) any one or more of the aforementioned amphiphilic protein and/or pharmaceutically acceptable salts, esters, ethers, amides or mixtures thereof Mixture thereof; and 2) Biomacromolecule drugs used for loading of amphiphilic proteins;
  • a pharmaceutical composition of protein sequence 1 protein and biological macromolecular drugs for example: a pharmaceutical composition of protein sequence 1 protein and biological macromolecular drugs; a mixture of protein sequence 2 protein and protein sequence 3 protein as a carrier, plus a pharmaceutical composition formed by biological macromolecular drugs; protein sequence 1 protein and protein A mixture of sequence 2 protein and protein sequence 3 protein as a carrier, plus a pharmaceutical composition formed by a biomacromolecule drug; a mixture carrier composed of a protein sequence 2 protein and an ester of a protein sequence 2 protein, plus a biomacromolecule Pharmaceutical compositions formed by drugs; etc.
  • the biomacromolecule drugs include polypeptide drugs or protein drugs, for example, insulin and insulin analogues;
  • the protein drug is insulin or an insulin analogue.
  • Said amphiphilic protein and/or any one or more of its pharmaceutically acceptable salts, esters, ethers, amides and mixtures thereof;
  • the molecular molar ratio of the amphiphilic protein to the biological macromolecular drug is 1:50-50:1; preferably, the molecular molar ratio of the amphiphilic protein to the biological macromolecular drug is 1:25 ⁇ 50:1;
  • the molecular molar ratio of the amphiphilic protein to the biological macromolecular drug is 1:15-25:1; preferably, the molecular molar ratio of the amphiphilic protein to the biological macromolecular drug is 1:10 ⁇ 15:1;
  • the molar ratio of amphiphilic protein to polypeptide or protein drug is 1:25-50:1; preferably, the molar ratio of amphiphilic protein to polypeptide or protein drug is 1:15 ⁇ 25:1;
  • the molar ratio of amphiphilic protein to insulin is 1:15-25:1; preferably, the molar ratio of amphiphilic protein to insulin is 1:10-15:1;
  • the molar ratio of amphiphilic protein to insulin/insulin analog is 1:5 to 5:1, preferably 1:2 to 4:1.
  • the pH range of the composition formed by the amphiphilic protein and other drug molecules is 4-7.8;
  • the pH range of the molecular composition of amphiphilic protein and insulin is 4-7.8; wherein, preferably, the pH range of the amphiphilic protein and insulin is 4-7.4.
  • composition of the present invention can be prepared using general techniques known in the art.
  • the amphiphilic protein and the biomacromolecule are made into a composition with different ratios, calculated on a molar basis, from 15:1 to 1:25, and the compositions obtained at different ratios have different releases. characteristic.
  • the molar ratio take an appropriate amount of the drug, dissolve it in an appropriate amount of hydrochloric acid solution and adjust the pH, add an appropriate amount of amphiphilic protein, mix with ultrasound for 1-5 minutes, or stir for 1-3 hours, or leave it overnight, the solution can be directly added with auxiliary materials It can be made into preparations, or it can be freeze-dried and then made into preparations with other excipients.
  • the amphiphilic protein and insulin are made into a composition with different ratios, from 5:1 to 1:5 on a molar basis, and the compositions obtained at the different ratios have different release characteristics.
  • the molar ratio take an appropriate amount of insulin, dissolve it in an appropriate amount of hydrochloric acid solution and adjust the pH, add an appropriate amount of amphiphilic protein, mix with ultrasound for 1-5 minutes, or stir for 1-3 hours, or leave it overnight.
  • the solution can be directly added with excipients It can be made into preparations, or it can be freeze-dried and then made into preparations with other excipients.
  • the pharmaceutical composition further includes one or more of a soluble filler, a pH adjuster, a stabilizer, water for injection, and an osmotic pressure adjuster.
  • a soluble filler is one or a combination of mannitol, low molecular dextran, sorbitol, polyethylene glycol, glucose, lactose, and galactose.
  • the pH adjuster is a non-volatile acid, or a physiologically acceptable organic or inorganic acid and base and salt, or a combination thereof; preferably, it is selected from citric acid, phosphoric acid, lactic acid, tartaric acid, hydrochloric acid, potassium hydroxide, hydrogen Sodium oxide, potassium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, ammonium carbonate, sodium bicarbonate, potassium bicarbonate, ammonium bicarbonate, and combinations thereof.
  • Stabilizers are EDTA-2Na, sodium thiosulfate, sodium metabisulfite, sodium sulfite, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, arginine, glutamic acid, polyethylene glycol 6000, polyethylene glycol 4000, ten One or a combination of sodium dialkylsulfate or tris(hydroxymethyl)aminomethane.
  • the osmotic pressure regulator is one or a combination of sodium chloride and potassium chloride.
  • the dosage form of the pharmaceutical composition is an injection dosage form or an oral dosage form, including freeze-dried powder for injection, injection, tablets, and capsules;
  • the dosage form of the pharmaceutical composition is a lyophilized powder for injection, an injection solution; further, the dosage form of the pharmaceutical composition is a lyophilized powder for injection.
  • a preparation method of a pharmaceutical composition whose dosage form is a freeze-dried powder for injection which comprises the steps of: taking an appropriate amount of insulin and an amphiphilic protein solution, adding water-soluble fillers, stabilizers, and osmotic pressure.
  • Regeneratives, etc. add appropriate amount of water for injection, adjust the pH to 7 to dissolve, add water to dilute to a proper concentration, add 0.1-0.5% activated carbon, stir at 0-10°C for 10-20 minutes, remove the activated carbon, use microporous filtration Membrane filtration sterilization, the filtrate is divided into packaging, and the white loose mass is obtained by freeze-drying method, which is then sealed.
  • the pharmaceutical composition of the present invention can be used in the preparation of therapeutic drugs, and the treatment direction is consistent with the original treatment direction of the drug, which can generally reduce the number of administrations and improve compliance.
  • the composition of the present invention can be administered in the form of oral or subcutaneous injection.
  • the dosage varies depending on the subject to be treated, the method of administration, symptoms and other factors, the composition of the present invention is effective in a relatively wide dosage range.
  • the actual dose should be determined by the doctor according to the relevant conditions, including the physical state of the person being treated, the route of administration, age, weight, the patient’s individual response to the drug, the severity of the patient’s symptoms, etc. Therefore, the above dose range does not apply. It does not limit the scope of the present invention in any way.
  • the present invention can greatly improve the stability of the drug, prolong the concentration and duration of the drug in the body, so that the originally fast-metabolized insulin has a sustained-release function; this is beneficial to reduce the frequency of administration of diabetic patients , which greatly improves the patient's treatment compliance and treatment pain.
  • liver first-pass effect of oral drugs The metabolic characteristic of oral drugs is that after absorption from the small intestine, the drug will first reach the liver and undergo first-level metabolism. This drug metabolism feature is a loss and barrier to the effective dose of the drug .
  • oral insulin which is to reduce liver glycogen.
  • the existing marketed insulin drugs cannot solve the problem of liver glycogen, and reducing liver glycogen is of great significance for the treatment of diabetes. Therefore, the oral insulin described in the present invention can be used for the treatment of high liver glycogen in diabetic patients.
  • the pharmaceutical composition prepared by using the carrier of the present invention helps to reduce drug intake, thereby reducing medical costs.
  • Figure 1 shows the SDS-PAGE profile of protein fermentation and expression
  • Figure 2 shows a liquid phase diagram of amphiphilic protein 2
  • Figure 3 shows an experiment of improving insulin serum stability by a combination of insulin and amphiphilic protein 3
  • Figure 4 shows an experiment of improving the stability of insulin in rats by the combination of insulin and amphiphilic protein 4;
  • Figure 5 shows the hypoglycemic function experiment of the composition of amphiphilic protein 1 and insulin
  • Figure 6 shows the hypoglycemic function experiment of amphiphilic protein 2 and insulin composition
  • Figure 7 shows the hypoglycemic function experiment of amphiphilic protein 3 and insulin composition
  • Figure 8 shows the hypoglycemic function experiment of the composition of amphiphilic protein 5 and insulin
  • Figure 9 shows the long-acting blood glucose control experiment of the combination of insulin and amphiphilic protein
  • the amphiphilic protein 1 was constructed and fermented to express. After the plasmid is transformed into E. coli, a stepwise amplification fermentation process is carried out, and IPTG induction is carried out when the OD reaches the range of 45-60. After induction, the dissolved oxygen is controlled at 20%-40%, and the residual sugar of the medium is less than 0.54g/L. Induce to OD90-120 for routine operations such as collecting bacterial cells, crushing and centrifuging inclusion bodies. After the inclusion is resuspended, a hydrophobic chromatography column is used for crude purification, and then a C8 reverse phase column is used for purification, and the final purity of the product is >95%. The molecular weight of the product was confirmed by SDS-PAGE ( Figure 1: The arrow in the figure refers to the amphiphilic protein 1).
  • the plasmid construction and fermentation expression of amphiphilic protein 2 were carried out according to the common methods in the industry. After the plasmid is transformed into E. coli, a stepwise amplification fermentation process is carried out, and IPTG induction is carried out when the OD reaches the range of 45-60. After induction, the dissolved oxygen is controlled at 20%-40%, and the residual sugar of the medium is less than 0.54g/L. Induced to OD90-120, carry out routine operations such as collecting bacterial cells, crushing and centrifuging inclusion bodies. After the inclusion is resuspended, a hydrophobic chromatography column is used for crude purification, and then a C8 reverse phase column is used for purification, and the final purity of the product is >95%. The purity of the product was confirmed by HPLC (figure 2 of the specification, the HPLC chart showed that the purity of the protein was 96.8%).
  • Example 15 The composition of amphiphilic protein 3 and insulin (molar ratio 2:1) greatly improved the serum stability of insulin
  • composition of rat serum and amphiphilic protein 4/insulin 100 ⁇ g/kg equivalent to insulin, the molar ratio of amphiphilic protein to insulin is 1:2, 2:1, and 4:1, respectively, after incubation
  • ELISA enzyme-linked immunosorbent assay
  • the operation is as follows: serum and 100mM ammonium acetate are given at room temperature for 10 minutes, and insulin EIA kit (Phoenix Pharmaceuticals, INC) is used to determine the concentration of insulin in rat plasma. The concentration of insulin is measured.
  • the test method refers to the company's instructions for the measurement of insulin concentration, and the insulin stability is evaluated based on the results.
  • Sample source Half-life (minutes) insulin 35 Amphiphilic protein 4+insulin (molar ratio 1:2) 80 Amphiphilic protein 4+insulin (molar ratio 2:1) 120 Amphiphilic protein 4+insulin (molar ratio 4:1) 150
  • Example 18 Experiments on the hypoglycemic function of the composition of amphiphilic protein 2 and insulin
  • Example 19 The hypoglycemic function experiment of the composition of amphiphilic protein 3 and insulin
  • a sample of the composition of amphiphilic protein 2 and insulin (molar ratio 1:2 and 1:4) was sampled and administered to SD rats orally every day. Rats take oral glucose (2g/kg) every day, and measure the blood glucose level every day. The results are shown in Figure 9 of the manual.
  • the composition of amphiphilic protein 2 and insulin shows oral activity, and the formation of the composition of insulin and amphiphilic protein has Long-acting blood sugar regulation The results show that oral administration of insulin alone does not have any blood glucose regulation function at all, but when insulin and amphiphilic protein 2 form a composition, it has oral activity.

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Abstract

提供了一种两亲性蛋白、其制备方法，以及基于该两亲性蛋白的载剂及由该载剂制备的药物组合物及其用途。该载剂能作为胰岛素的口服或长效给药载体，用于制备糖尿病治疗药物。

Description

一种新型两亲性蛋白、其制备方法及用途 技术领域
本发明涉及新型两亲性蛋白的设计与构建及其应用，具体而言，本发明涉及一种两亲性蛋白及其制备方法和用途，该蛋白在溶液状态下具有较强的两亲性，能够形成疏水及亲水结构域从而通过结构域与药物的非共价键相互作用与药物形成稳定的组合物。这种组合物能够显著提高药物在体内的稳定性和半衰期。
背景技术
生物大分子药物(核酸分子、蛋白质、多肽)由于其靶标结合性高，副反应小等优势近年来在临床治疗上取得了快速发展，尤其是癌症、内分泌、自身免疫性疾病甚至艾滋病等传染病。但是，局限生物大分子药物发展的主要障碍之一是生物大分子体内不稳定、易水解。
近年来，采用生物大分子结构修饰来优化生物药物的代谢动力学特性，以延长其半衰期是经常采用的方法，但是结构修饰过程中一些生物药物会部分或全部失活。例如胰高血糖样素肽-1，大量氨基酸替代实验证实其第二位氨基酸Ala的易降解导致其半衰期约2-5分钟，但是在此位点的结构修饰却导致多肽功能失活，无法与受体结合。再如胰岛素的结构修饰，胰岛素分子内多个位点容易被蛋白酶降解，而且分子中的三对二硫键也是其不稳定的因素之一。包括我们在内的许多研究者都开展了胰岛素的结构修饰，研究中发现结构修饰对胰岛素的活性影响非常巨大，我们将A链和B链间的二硫键进行酰胺键替换后，替换后胰岛素的活性完全丧失。
所以，寻找一种简便的、稳定的制剂技术将具有重要的意义。
以下是与本发明相关的一些概念的说明：
生物大分子药物：包括多肽药物或蛋白类药物，例如，胰岛素、胰岛素类似物。
胰岛素类似物：人们利用基因工程技术对人胰岛素的氨基酸序列及结构进行局部修饰，合成了人胰岛素类似物(insulin analogue)，泛指既可模拟正常胰岛素的分泌，同时在结构上与胰岛素也相似的物质。
胰岛素与胰岛素类似物：本发明所称的“胰岛素”采用广义的概念，如无特别说明，包括但不限于胰岛素、胰岛素类似物、人胰岛素、人工合成胰岛素、动物体胰脏(如：猪胰)中提取制得的具有降血糖作用的多肽类物质，等。
发明内容
本发明涉及一种新型结构特点的两亲性蛋白，这类蛋白具有不同的亲水、疏水结构域。这种具有特殊结构特征的蛋白可以与生物大分子药物，例如胰岛素分子形成组合物。组合物的稳定性能够有效提高胰岛素分子的血清及体内半衰期，从而优化胰岛素的临床用药方案，达到减少注册次数的目的，例如：减少到每天注射1次，甚至更低频次。
本发明的一个目的是使用具有特殊理化性质的蛋白与胰岛素形成组合物，能够有效提高药物口服后的吸收、在血液中的半衰期及稳定性。
本发明的一个目的是针对生物大分子类药物在体内存留时间较短，提供了一种能与生物大分子类药物形成组合物的载体蛋白，以增加药物对生物体内降解因素的稳定性，延长其在体内的半衰期。
本发明的另一个目的是提供了包括所述载体蛋白的载剂，及其在制备药物组合物中的应用。
本发明的另一个目的是提供了两亲性蛋白与胰岛素形成的组合物与药学上可接受的辅料(包括：载体材料、赋形剂、稳定剂或稀释剂等等)形成的药物组合物及其制备方法。
结合本发明的目的对本发明进一步加以描述：
在本发明的一个方面，提供了两亲性蛋白，及其药学上可接受的盐、酯、醚、酰胺或其混合物，所述载体多肽具有SEQ ID NO 0氨基酸残基序列的通式特征：
A 1-Cys-A 2-Cys-Cys-A 3-Cys-A 4-Cys-A 5-Cys-Cys-A 6-Cys-A 7     SEQ ID NO 0；
通式1中的具有两亲性功能的蛋白含有8个半胱氨酸，并形成4对二硫键。
在上述通式中，A，B，C，D，E，F，G分指具有一定结构特征的氨基酸片段，序列见序列表sequence list；
上述通式中的A 1-A 7选自下列片段：
[XY]n、[YX]n、Y[X]n、Z[XX]n；
其中，X指代疏水氨基酸，包括：A、G、V、L、I、P，
Y指代亲水氨基酸，包括：S、T、N、Q，
Z指代带有电荷的氨基酸，包括：D、E；
n＝1～20，优选n＝2～10。
进一步地，本发明两亲蛋白，优选以下结构：
两亲性蛋白1(蛋白序列1，sequence 1)：
Figure PCTCN2020139216-appb-000001
两亲性蛋白2(蛋白序列2，sequence 2)：
Figure PCTCN2020139216-appb-000002
两亲性蛋白3(蛋白序列3，sequence 3)：
Figure PCTCN2020139216-appb-000003
两亲性蛋白4(蛋白序列4，sequence 4)：
Figure PCTCN2020139216-appb-000004
本发明所述的两亲性蛋白，还包括两亲性蛋白的药学上可接受的盐、酯、醚、酰胺或其混合物作为药物载剂，在制备药物中的应用。
本发明中，具有上述结构通式的蛋白分子通过生物发酵方法进行制备，产品使用疏水层析及反相层析方法进行纯化。
在本发明的另一个方面，提供了药物组合物，其包括1)前述的两亲性蛋白和/或其药学上可接受的盐、酯、醚、酰胺或其混合物的任一种或几种其混合物；及2)用于两亲性蛋白装载的生物大分子药物；
例如：蛋白序列1蛋白与生物大分子药物的药物组合物；蛋白序列2蛋白与蛋白序列3蛋白的混合物作为载剂，再加上生物大分子药物形成的药物组合物；蛋白序列1蛋白与蛋白序列2蛋白、蛋白序列3蛋白的混合物作为载剂，再加上生物大分子药物形成的药物组合物；蛋白序列2蛋白与蛋白序列2蛋白的酯组成的混合物载剂，再加上生物大分子药物形成的药物组合物；等。
在本发明一个优选的方面，所述生物大分子药物包括多肽药物或蛋白类药物，例如，胰岛素、胰岛素类似物；
优选地，所述蛋白类药物为胰岛素或胰岛素类似物。
所述的两亲性蛋白和/或其药学上可接受的盐、酯、醚、酰胺的任一或几种其混合物；
在本发明一个优选的方面，两亲性蛋白与生物大分子药物的分子摩尔比为1∶50～50∶1；优选地，两亲性蛋白与生物大分子药物的分子摩尔比为1∶25～50∶1；
进一步地，在本发明一个优选的方面，两亲性蛋白与生物大分子药物的分子摩尔比为1∶15～25∶1；优选地，两亲性蛋白与生物大分子药物的分子摩尔比为1∶10～15∶1；
在本发明一个优选的方面，两亲性蛋白与多肽或蛋白药物的分子摩尔比为1∶25～50∶1；优选地，两亲性蛋白与多肽或蛋白药物的分子摩尔比为1∶15～25∶1；
在本发明一个优选的方面，两亲性蛋白与胰岛素的分子摩尔比为1∶15～25∶1；优选地，两亲性蛋白与胰岛素的摩尔比为1∶10～15∶1；
再优选，两亲性蛋白与胰岛素/胰岛素类似物的摩尔比为1∶5～5∶1，优选1∶2～4∶1。
在本发明一个优选的方面，两亲性蛋白与其他药物的分子(如生物类大分子药物)形成组合物的pH值范围是4-7.8；
在本发明一个优选的方面，两亲性蛋白与胰岛素的分子形成组合物的pH值范围是4-7.8；其中优选的，两亲性蛋白与胰岛素所采用的pH范围是4-7.4。
本发明所述的药物组合物可采用本领域公知的一般技术制备。
称取适量两亲性蛋白冻干粉，溶于磷酸缓冲液中，然后称取适量胰岛素冻干粉，加入上述两亲性蛋白的溶液中，混匀搅拌后，冷冻干燥，得到组合物固体粉末。
在本发明的一个实施方案中，将两亲性蛋白与生物大分子制成不同比例的组合物，以摩尔计算，从15∶1到1∶25，不同的比例得到的组合物具有不同的释放特性。按照摩尔比例，取适量的药物，溶于适量的盐酸溶液并调整pH后加入适量的两亲性蛋白，超声混合1-5分钟，或者搅拌1-3小时，或者放置过夜，溶液可以直接加辅料做成制剂，也可以冷冻干燥后再与其他辅料一起制成制剂。
在本发明的一个实施方案中，将两亲性蛋白与胰岛素制成不同比例的组合物，以摩尔计算，从5∶1到1∶5，不同的比例得到的组合物具有不同的释放特性。按照摩尔比例，取适量的胰岛素，溶于适量的盐酸溶液并调整pH后加入适量的两亲性蛋白，超声混合1-5分钟，或者搅拌1-3小时，或者放置过夜，溶液可以直接加辅料做成制剂，也可以冷冻干燥后再与其他辅料一起制成制剂。
在本发明一个优选的方面，所述药物组合物还包括可溶性填充剂、pH调节剂、稳定剂、注射用水、滲透压调节剂的一种或多种。其中，水溶性填充剂辅料为甘露醇、低分子右旋糖苷、山梨醇、聚乙二醇、葡萄糖、乳糖、半乳糖的一种或多种的组合。pH调节剂为非挥发性的酸，或者生理可接受的有机或无机酸和碱及盐，或其组合；优选地，其选自橼酸、磷酸、乳酸、酒石酸、盐酸、氢氧化钾、氢氧化钠、氢氧化钾、氢氧化铵、碳酸钠、碳酸钾、碳酸铵、碳酸氢钠、碳酸氢钾、碳酸氢铵及其组合。稳定剂为EDTA-2Na、硫代硫酸钠、焦亚硫酸钠、亚硫酸钠、磷酸氢二钾、碳酸氢钠、碳酸钠、精氨酸、谷氨酸、聚乙二醇6000、聚乙二醇4000、十二烷基硫酸钠或三羟甲基氨基甲烷的一种或几种的组合。优选焦亚硫酸钠、磷酸氢二钾、精氨酸、聚乙二醇6000、三羟甲基氨基甲烷的一种或多种的组合。渗透压调节剂，是氯化钠、氯化钾的一种或两种的组合。
在本发明一个优选的方面，所述药物组合物的剂型为注射剂型、口服剂型，包括注射用冻干粉、注射液、片剂、胶囊；
进一步地，所述药物组合物的剂型为注射用冻干粉、注射液；更进一步地，所述药物组合物的剂型为注射用冻干粉。
在本发明的另一个方面，提供了剂型为注射用冻干粉的药物组合物的制备方法，其包括步骤：取胰岛素和两亲性蛋白溶液适量，加入水溶性填充剂、稳定剂、滲透压调节剂等，加入注射用水适量，调节pH值至7使其溶解，加水稀释至适当浓度，加入0.1-0.5％活性炭，在0-10℃下搅拌10-20分钟，除去活性炭，采用微孔滤膜过滤除菌，滤液进行分装，采用冷冻干燥法，制得白色疏松块状物，封口即得。
本发明的药用组合物可以用在制备治疗药物方面，治疗的方向依据药物原来治疗方向一致，通常能够减少给药次数，提高依从性。具体地，本发明的组合物物可以口服或皮下注射剂形式给药。虽然剂量依治疗对象、给药方式、症状及其它因素而改变，本发明的组合物在相当宽的剂量范围内是有效的。实际剂量应该由医生根据有关的情况来决定，这些情况包括被治疗者的身体状态、给药途径、年龄、体重、患者对药物的个体反应，患者症状的严重程度等等，因此上述剂量范围并不是以任何方式限制本发明的范围。
本发明包括但不限于以下的优点：
1.稳定性与缓释功能：本发明可大幅改善药物的稳定性，延长药物在体内的浓度持续时间，使本来快速代谢的胰岛素产生了缓释功能；这有利于降低糖尿病人的给药频次，大幅提升病人的治疗顺应性和治疗痛苦。
2.口服药物肝部首过效应优势：口服药物的代谢特点是小肠吸收后，药物将首先达 到肝部并进行第一级代谢，这种药物代谢特点是对药物有效剂量的一种损失和壁垒。但是，对于口服胰岛素却存在特殊意义，那就是降低肝糖原。现有的上市的胰岛素类药物无法解决肝糖原问题，而降低肝糖原对糖尿病的治疗具有重要意义。所以，本发明描述的口服胰岛素可以用于糖尿病患者的高肝糖原治疗。
3.采用本发明的载剂制备的药物组合物有助于减少药物摄取，从而降低医疗成本。
附图说明
图1表示蛋白发酵表达SDS-PAGE图谱；
图2表示两亲性蛋白2液相图谱；
图3表示胰岛素与两亲性蛋白3组合物提高胰岛素血清稳定性实验；
图4表示胰岛素与两亲性蛋白4组合物提高胰岛素在大鼠体内稳定性实验；
图5表示两亲性蛋白1与胰岛素组合物的降血糖功能实验；
图6表示两亲性蛋白2与胰岛素组合物的降血糖功能实验；图7表示两亲性蛋白3与胰岛素组合物的降血糖功能实验；
图8表示两亲性蛋白5与胰岛素组合物的降血糖功能实验；
图9表示胰岛素与两亲性蛋白组合物长效血糖调控实验
具体实施方式
下面结合实施例对本发明作进一步的说明。实施例仅为解释性的内容，绝不意味着它以任何方式限制本发明的范围。
实施例1两亲性蛋白1的发酵表达
按照现有方法，对两亲性蛋白1进行质粒构建和发酵表达。将质粒转化入大肠杆菌后进行逐级放大发酵工艺，并在OD达到45-60范围内进行IPTG诱导。诱导后控制溶氧在20％-40％，培养基残糖＜0.54g/L。诱导至OD90-120进行收集菌体、破碎离心等包涵体收集常规操作。包涵体重悬后采用疏水层析柱进行粗纯，而后采用C8反相柱进行精纯，产品最终纯度＞95％。产品采用SDS-PAGE进行分子量确认(说明书附图1：图中箭头指两亲性蛋白1)。
实施例2两亲性蛋白2的制备、纯度
按照业内通用方法对两亲性蛋白2进行质粒构建和发酵表达。将质粒转化入大肠杆菌后进行逐级放大发酵工艺，并在OD达到45-60范围内进行IPTG诱导。诱导后控制溶氧在20％-40％，培养基残糖＜0.54g/L。诱导至OD90-120进行收集菌体、破碎离心等包 涵体收集常规操作。包涵体重悬后采用疏水层析柱进行粗纯，而后采用C8反相柱进行精纯，产品最终纯度＞95％。产品采用HPLC进行纯度确认(说明书附图2，HPLC图谱显示蛋白纯度为96.8％)。
实施例3两亲性蛋白1与胰岛素组合物(摩尔比1∶25)的制备
称取2.0mg两亲性蛋白1冻干粉，溶于1ml pH 5.8的磷酸缓冲液中，然后称取80.0mg的胰岛素冻干粉，加入上述两亲性蛋白的溶液中，充分混匀搅拌6小时后，冷冻干燥得到组合物固体粉末。
实施例4两亲性蛋白2与胰岛素组合物(摩尔比1∶15)的制备
称取1.0mg两亲性蛋白2冻干粉，溶于1ml pH 4.0的磷酸缓冲液中，然后称取24.0mg的胰岛素冻干粉，加入上述两亲性蛋白的溶液中，充分混匀搅拌4小时后，冷冻干燥得到组合物固体粉末。
实施例5两亲性蛋白1与胰岛素组合物(摩尔比1∶5)的制备
称取2.0mg两亲性蛋白1冻干粉，溶于1ml pH 5.0的磷酸缓冲液中，然后称取16.0mg的胰岛素冻干粉，加入上述两亲性蛋白的溶液中，充分混匀搅拌3小时后，冷冻干燥得到组合物固体粉末。
实施例6两亲性蛋白3与胰岛素组合物(摩尔比5∶1)的制备
称取10.0mg两亲性蛋白3冻干粉，溶于1ml pH 4.5的磷酸缓冲液中，然后称取1.2mg的胰岛素冻干粉，加入上述两亲性蛋白的溶液中，充分混匀搅拌0.5小时后，冷冻干燥得到组合物固体粉末。
实施例7两亲性蛋白3与胰岛素组合物(摩尔比25∶1)的制备
称取25.0mg两亲性蛋白3冻干粉，溶于1ml pH 5.8的磷酸缓冲液中，然后称取0.6mg的胰岛素冻干粉，加入上述两亲性蛋白的溶液中，充分混匀搅拌2小时后，冷冻干燥得到组合物固体粉末。
实施例8两亲性蛋白1与胰岛素组合物(摩尔比50∶1)的制备
称取50.0mg两亲性蛋白1冻干粉，溶于1ml pH 4.5的磷酸缓冲液中，然后称取0.3mg的胰岛素冻干粉，加入上述两亲性蛋白的溶液中，充分混匀搅拌6小时后，冷冻干燥得到组合物固体粉末。
实施例9两亲性蛋白2与胰岛素组合物(摩尔比1∶2)的制备
称取5.0mg两亲性蛋白2冻干粉，溶于1ml pH 4.0的磷酸缓冲液中，然后称取6mg的胰岛素冻干粉，加入上述两亲性蛋白的溶液中，充分混匀搅拌2小时后，冷冻干燥得 到组合物固体粉末。
实施例10两亲性蛋白3与胰岛素组合物(摩尔比1∶1)的制备
称取5.0mg两亲性蛋白3冻干粉，溶于1ml pH 7.8的磷酸缓冲液中，然后称取3mg的胰岛素冻干粉，加入上述两亲性蛋白的溶液中，充分混匀搅拌2小时后，冷冻干燥得到组合物固体粉末。
实施例11两亲性蛋白3与胰岛素组合物(摩尔比2∶1)的制备
称取8.0mg两亲性蛋白3冻干粉，溶于1ml pH 7.8的磷酸缓冲液中，然后称取2.4mg的胰岛素冻干粉，加入上述两亲性蛋白的溶液中，充分混匀搅拌2小时后，冷冻干燥得到组合物固体粉末。
实施例12两亲性蛋白4与胰岛素组合物(摩尔比4∶1)的制备
称取12.0mg两亲性蛋白4冻干粉，溶于1ml pH 4.5的磷酸缓冲液中，然后称取2.4mg的胰岛素冻干粉，加入上述两亲性蛋白的溶液中，充分混匀搅拌2小时后，冷冻干燥得到组合物固体粉末。
实施例13两亲性蛋白4与胰岛素组合物(摩尔比10∶1)的制备
称取10.0mg两亲性蛋白4冻干粉，溶于1ml pH 6.6的磷酸缓冲液中，然后称取0.6mg的胰岛素冻干粉，加入上述两亲性蛋白的溶液中，充分混匀搅拌1小时后，冷冻干燥得到组合物固体粉末。
实施例14两亲性蛋白4与胰岛素组合物(摩尔比15∶1)的制备
称取30.0mg两亲性蛋白4冻干粉，溶于1ml pH 4.0的磷酸缓冲液中，然后称取1.2mg的胰岛素冻干粉，加入上述两亲性蛋白的溶液中，充分混匀搅拌1小时后，冷冻干燥得到组合物固体粉末。
实施例15两亲性蛋白3与胰岛素组合物(摩尔比2∶1)大大提高了胰岛素的血清稳定性
称取20mg两亲性蛋白3冻干粉，溶于1ml pH 4.0的磷酸缓冲液中，然后称取0.6mg的胰岛素冻干粉，加入上述两亲性蛋白的溶液中，充分混匀搅拌3小时后，冷冻干燥得到组合物固体粉末。将两亲性蛋白与胰岛素组合物溶在10ml人血浆中，在37度下放置不同时间(0、0.5、1、4小时)，以胰岛素酶联免疫法测定血中胰岛素含量，计算稳定性。并以相同浓度的胰岛素在血浆中的稳定性进行比较(说明书附图3)，结果显示，胰岛素与两亲性蛋白组合物的形成大大提高了胰岛素的血清稳定性。
实施例16两亲性蛋白4与胰岛素组合物的体内半衰期实验
本实施例使用SD大鼠5组，每组10只，其得自上海SLAC动物中心。
将大鼠血清与两亲性蛋白4/胰岛素的组合物(折合胰岛素为100μg/kg，两亲性蛋白与胰岛素的摩尔比例分别为1∶2、2∶1及4∶1，分别于孵育后采用酶联免疫吸附方法(ELISA)检测大鼠血清中胰岛素的浓度，操作如下：血清与100mM的醋酸铵在室温赋予10分钟后，用胰岛素EIA试剂盒(Phoenix Pharmaceuticals，INC)对鼠血浆中的胰岛素的浓度进行测定。试验方法参照公司说明书进行胰岛素浓度测定，并根据结果评价胰岛素稳定性。
胰岛素及其组合物的体内药代动力学结果见说明书附图4和表1(两亲性蛋白4与胰岛素组合物大大增加了胰岛素的体内半衰期；结果显示本发明的胰岛素组合物在体内的半衰期较单独的胰岛素明显延长，具有长效特性，胰岛素与两亲性蛋白组合物的形成大大提高了胰岛素在大鼠体内的稳定性。经过说明书附图4计算的形成组合物后胰岛素的半衰期大大延长(表1)：
表1胰岛素及其组合物在大鼠体内的半衰期
样品来源 半衰期(分)
胰岛素 35
两亲性蛋白4+胰岛素(摩尔比1∶2) 80
两亲性蛋白4+胰岛素(摩尔比2∶1) 120
两亲性蛋白4+胰岛素(摩尔比4∶1) 150
实施例17两亲性蛋白1与胰岛素组合物的降血糖功能实验
称取10mg两亲性蛋白1融入pH7.8的磷酸盐缓冲液中，而后加入0.6mg的胰岛素粉末(摩尔比1∶1)中搅拌30分钟后可以用于口服给药。
试验中每组10只糖尿病大鼠，在葡萄糖刺激前测定其血糖值(0h)并立即进行2g/kg体重的口服葡萄糖刺激，并测定给糖后0.5小时时间点的血糖值。同时在0.5小时时间点糖尿病大鼠分别腹腔注射单独胰岛素、胰岛素与两亲性蛋白1组合物并跟踪血糖值变化，结果见说明书附图5。结果表明：虽然胰岛素单独口服给药途径使得胰岛素在肠胃中迅速降解失活所以没有药效，但是，经过本发明涉及的两亲性蛋白1保护后，组合物的存在能够保护胰岛素不受酸性环境的破坏、肠道内蛋白酶降解，并达到每天注射1次降血糖功能。
实施例18两亲性蛋白2与胰岛素组合物的降血糖功能实验
称取10mg两亲性蛋白2融入pH6.0的磷酸盐缓冲液中，而后加入1.2mg的胰岛素粉末(摩尔比1∶2)中搅拌30分钟后可以用于给药。试验中每组10只糖尿病大鼠，在葡萄糖刺激前测定其血糖值(0h)并立即进行2g/kg体重的口服葡萄糖刺激，并测定给 糖后0.5小时时间点的血糖值。同时在0.5小时时间点糖尿病大鼠分别腹腔注射单独胰岛素、胰岛素与两亲性蛋白2组合物并跟踪血糖值变化，结果见说明书附图6。结果表明：虽然胰岛素单独口服给药途径使得胰岛素在肠胃中迅速降解失活所以没有药效，但是，经过本专利涉及的两亲性蛋白2保护后，组合物的存在能够保护胰岛素不受酸性环境的破坏、肠道内蛋白酶降解，并每天注射1次达到降血糖功能。
实施例19两亲性蛋白3与胰岛素组合物的降血糖功能实验
称取10mg两亲性蛋白1融入pH4.8的磷酸盐缓冲液中，而后加入2.4mg的胰岛素粉末(摩尔比1∶4)中搅拌30分钟后可以用于口服给药。
试验中每组10只糖尿病大鼠，在葡萄糖刺激前测定其血糖值(0h)并立即进行2g/kg体重的口服葡萄糖刺激，并测定给糖后0.5小时时间点的血糖值。同时在0.5小时时间点糖尿病大鼠分别腹腔注射单独胰岛素、胰岛素与两亲性蛋白3组合物并跟踪血糖值变化，结果见说明书附图7。结果表明：虽然胰岛素单独口服给药途径使得胰岛素在肠胃中迅速降解失活所以没有药效，但是，经过本专利涉及的两亲性蛋白3保护后，组合物的存在能够保护胰岛素不受酸性环境的破坏、肠道内蛋白酶降解，并每天注射1次达到降血糖功能。
实施例20两亲性蛋白4与胰岛素组合物的降血糖功能实验
称取65mg两亲性蛋白1融入pH7.8的磷酸盐缓冲液中，而后加入0.6mg的胰岛素粉末(摩尔比4∶1)中搅拌30分钟后可以用于口服给药。
试验中每组10只糖尿病大鼠，在葡萄糖刺激前测定其血糖值(0h)并立即进行2g/kg体重的口服葡萄糖刺激，并测定给糖后0.5小时时间点的血糖值。同时在0.5小时时间点糖尿病大鼠分别腹腔注射单独胰岛素、胰岛素与两亲性蛋白5组合物并跟踪血糖值变化，结果见说明书附图8。结果表明：由于胰岛素口服给药途径使得胰岛素在肠胃中迅速降解失活所以没有药效。但是，经过本专利涉及的两亲性蛋白保护后，组合物的存在能够保护胰岛素不受酸性环境的破坏、肠道内蛋白酶降解，并达到每天注射1次降血糖功能。
实施例21两亲性蛋白2与胰岛素组合物的长期口服降血糖实验
取两亲性蛋白2与胰岛素的组合物(摩尔比1∶2和1∶4)的样品，每日口服给药SD大鼠。大鼠每天口服葡萄糖(2g/kg)，在每日测定血糖水平，结果见说明书附图9，两亲性蛋白2与胰岛素组合物显示具有口服活性，胰岛素与两亲性蛋白组合物的形成具有长效血糖调控功能。结果显示口服单独给予胰岛素完全不具有任何血糖调控功能，但是当胰岛素与 两亲性蛋白2形成组合物后具有口服活性。
Figure PCTCN2020139216-appb-000005
Figure PCTCN2020139216-appb-000006
Figure PCTCN2020139216-appb-000007
Figure PCTCN2020139216-appb-000008

Claims (10)

  1. 一类两亲性蛋白，具有如下通式结构：
    A 1-Cys-A 2-Cys-Cys-A 3-Cys-A 4-Cys-A 5-Cys-Cys-A 6-Cys-A 7
    其中，A 1-A 7选自下列片段：
    [XY]n、[YX]n、Y[X]n、Z[XX]n；
    其中，X指代疏水氨基酸，包括：A、G、V、L、I、P，
    Y指代亲水氨基酸，包括：S、T、N、Q，
    Z指代带有电荷的氨基酸，包括：D、E；
    n＝1～20，优选n＝2～10。
  2. 权利要求1所述的两亲性蛋白，选自：
    两亲性蛋白1，序列：
    AQCQQATGQV NLTCCEAGGD PAVELLTDAL VELLGLLIAV GCSLVSTVIG TSVIGSSASG CTANPCCSPI GGLIVSSIVP VNTAGVLIPN SASASANLIP GASCSV；
    两亲性蛋白2，序列：
    TALLGSCTGQ LTGALWTGAL WCCDPAGSEL LGLIGVVIGL GEAGLGGLCS PITVGTGGSA ACCSLLPPLI GGLVCSATAS VCNVNV；
    两亲性蛋白3，序列：
    ELLGLIGVVI GLGCTGALWT GGTVGCCELL GLLIAVGELL GLLIAVGCSV IGSSVIGSSV IGSCCQQATG QVNLTSPIGG LIVSCTAGVL IPNNLIPGAS CTALLGS；
    两亲性蛋白4，序列：
    TAGGLIGCSA TACCEGGLIV CTAGLIGVCT ATACCEAGVI LIGDAGLIGV EVAGLVDAAG LVICAQ。
  3. 权利要求1-2任一所述的两亲性蛋白和/或其药学上可接受的盐、酯、醚、酰胺或其混合物在制备药物载体中的应用。
  4. 药物组合物，其特征在于包括：
    1)权利要求1-2所述的两亲性蛋白和/或其药学上可接受的盐、酯、醚、酰胺的任一种或几种其混合物；
    2)生物大分子药物，包括多肽类药物、蛋白类药物。
  5. 权利要求4所述的药物组合物，其特征在于：所述的蛋白类药物可选自胰岛素、胰岛素类似物。
  6. 权利要求4所述的药物组合物，其特征在于载剂以多肽计，两亲性蛋白与生物大分子药物的摩尔比为1∶15～25∶1。
  7. 权利要求6所述的药物组合物，其特征在于载剂以多肽计，两亲性蛋白与蛋白类药物的摩尔比为1∶15～25∶1。
  8. 权利要求7所述的药物组合物，其特征在于载剂以多肽计，两亲性蛋白与胰岛素/胰岛素类似物的摩尔比为1∶15～25∶1，优选1∶10～15∶1。
  9. 权利要求8所述的药物组合物，其特征在于载剂以多肽计，两亲性蛋白与胰岛素/胰岛素类似物的摩尔比为1∶5～5∶1，优选1∶2～4∶1。
  10. 权利要求1-2所述两亲性蛋白在制备治疗糖尿病药物方面的应用。
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