Polypeptide composition using isolated BBI and KTI and use thereof

Abstract

The present invention provides a polypeptide composition for oral administration, wherein the polypeptide composition comprises a polypeptide, an isolated BBI, an isolated KTI, fish oil, and at least one absorption promoter. The isolated BBI is mixed with the isolated KTI in a certain ratio, so that an activity ratio of total trypsin inhibitory (total TI) activity to total chymotrypsin inhibitory (total CI) activity in the composition is 0.5:1 to 10:1.

Description

Polypeptide compositions using isolated BBI and KTI and their uses Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to a polypeptide composition using separated Bowman-Birk trypsin inhibitor (BBI) and Kunitz trypsin inhibitor (KTI) and uses thereof. Background Art

[0002] In the treatment of diseases, peptide drugs, with their high specificity and effectiveness, are becoming an ideal choice for treating numerous human ailments. With the advancement of biotechnology, the clinical application of peptide drugs, such as calcitonin, insulin, and thymosin, is becoming increasingly widespread. Peptide drugs are typically administered only by injection. However, injections are prone to side effects such as infection and inflammation, and patients struggle to overcome needle phobia, leading to poor compliance and reduced motivation to take medication regularly.

[0003] Chinese patent publication number CN101547702A, published on September 30, 2009, discloses an oral insulin composition, which uses a soybean trypsin inhibitor.

[0004] Soy Bean Trypsin Inhibitors (SBTI) are the main anti-nutritional factors in soybeans. Currently, Kunitz trypsin inhibitor (KTI) and Bowman-Birk trypsin inhibitor (BBI) have been isolated. KTI is a single-head inhibitor that can inhibit the activity of trypsin, while BBI is a double-head inhibitor that can inhibit the activity of trypsin and chymotrypsin at the same time.

[0005] Commercially available SBTI preparations were reported to produce highly variable results when used in pharmaceutical compositions (CN104244969A). Summary of the Invention

[0006] The applicant has discovered that by adjusting the activity ratio of BBI and KTI, it is possible to achieve smaller inter-individual differences in oral administration of the polypeptide composition.

[0007] There are two methods for extracting soybean trypsin inhibitor from soybeans. The first method involves directly extracting soybean trypsin inhibitor from soybeans (i.e., rather than separately extracting BBI and KTI from soybeans and then mixing them). The second method involves separately extracting BBI and KTI from soybeans and then mixing them. The applicants have found that oral compositions made with soybean trypsin inhibitor obtained using the first method exhibit significant individual variability in dosing. The applicants speculate that regulating the activities of BBI and KTI separately may yield better results.

[0008] Therefore, in one aspect, the present disclosure provides a polypeptide composition, wherein the polypeptide composition comprises:

[0009] Peptides, isolated BBI and isolated KTI, fish oil and at least one absorption enhancer;

[0010] The isolated BBI and the isolated KTI are mixed in a certain ratio so that the activity ratio of total trypsin inhibitory activity (total TI) to total chymotrypsin inhibitory activity (total CI) in the composition is 0.5:1 to 10:1.

[0011] In some embodiments, the present disclosure provides a polypeptide composition having an activity ratio of total trypsin inhibitory activity (total TI) to total chymotrypsin inhibitory activity (total CI) of 0.6:1 to 10:1.

[0012] In some embodiments, the present disclosure provides a polypeptide composition having an activity ratio of total trypsin inhibitory activity (total TI) to total chymotrypsin inhibitory activity (total CI) of 0.7:1 to 10:1.

[0013] In some embodiments, the present disclosure provides a polypeptide composition having an activity ratio of total trypsin inhibitory activity (total TI) to total chymotrypsin inhibitory activity (total CI) of 0.8:1 to 10:1.

[0014] In some embodiments, the present disclosure provides a polypeptide composition having an activity ratio of total trypsin inhibitory activity (total TI) to total chymotrypsin inhibitory activity (total CI) of 0.7:1 to 6:1.

[0015] In some embodiments, the present disclosure provides a polypeptide composition having an activity ratio of total trypsin inhibitory activity (total TI) to total chymotrypsin inhibitory activity (total CI) of 0.8:1 to 6:1.

[0016] In some embodiments, the present disclosure provides a polypeptide composition having an activity ratio of total trypsin inhibitory activity (total TI) to total chymotrypsin inhibitory activity (total CI) of 0.9:1 to 6:1.

[0017] In some embodiments, the present disclosure provides a polypeptide composition having an activity ratio of total trypsin inhibitory activity (total TI) to total chymotrypsin inhibitory activity (total CI) of 0.91:1 to 6:1.

[0018] In some embodiments, the present disclosure provides a polypeptide composition having an activity ratio of total trypsin inhibitory activity (total TI) to total chymotrypsin inhibitory activity (total CI) of 0.91:1 to 5.27:1.

[0019] In some embodiments, the activity ratio of total trypsin inhibitory activity (total TI) to total chymotrypsin inhibitory activity (total CI) in the polypeptide composition provided by the present disclosure is 0.91:1, 1.77:1, 2.16:1, 5.27:1, or a range between any two ratios.

[0020] In some embodiments, the present disclosure provides a polypeptide composition having an activity ratio of total trypsin inhibitory activity (total TI) to total chymotrypsin inhibitory activity (total CI) of 0.91:1 to 5.27:1, 0.91:1 to 2.16:1, 0.91:1 to 1.77:1, 1.50:1 to 5.27:1, 1.60:1 to 5.27:1, 1.77:1 to 5.27:1, 1.77:1 to 2.16:1, or 2.16:1 to 5.27:1.

[0021] There are two methods for extracting soybean trypsin inhibitor from soybeans. The first method involves directly extracting soybean trypsin inhibitor from soybeans (i.e., rather than separately extracting BBI and KTI from soybeans and then mixing them to obtain soybean trypsin inhibitor). The second method involves separately extracting BBI and KTI from soybeans and then mixing them to obtain soybean trypsin inhibitor. The applicant discovered that oral compositions made with soybean trypsin inhibitor obtained using the first method exhibit significant variability in individual dosing. The applicant discovered that separately regulating the activities of BBI and KTI can achieve better results.

[0022] In some embodiments, the activity ratio of total trypsin inhibitory activity (total TI) to total chymotrypsin inhibitory activity (total CI) in the polypeptide composition provided by the present disclosure is 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 or a range between any two ratios thereof.

[0023] In some embodiments, the isolated BBI and isolated KTI are prepared by the method described in Chinese patent application CN104244969, the entire contents of which are incorporated herein by reference.

[0024] The amino acid sequence of BBI is shown in SEQ ID NO: 1, and the number in the Uniprot database is UniprtoKB-P01055;

[0025] SEQ ID NO: 1

[0026] As used herein, KTI refers to KTI3, the amino acid sequence of KTI3 is shown in SEQ ID NO: 2, and the number of KTI3 in the Uniprot database is UniprtoKB-P01070.

[0027] SEQ ID NO:2

[0028] As used herein, the term "isolated BBI" refers to a preparation that is enriched in BBI relative to other components. In various embodiments, the BBI preparation utilized in the methods and polypeptide compositions described herein is at least 85% pure, as determined by SDS-PAGE, Brilliant Blue staining, or imager quantification. In the case of polypeptide compositions, these values ​​relate to the characteristics of BBI prior to mixing with one or more other components of the polypeptide composition. In other embodiments, the BBI preparation contains 5% or less KTI, and in other embodiments, the BBI preparation contains less than 0.1% high molecular weight impurities (in other words, high molecular weight impurities refer to substances with a molecular weight greater than 30,000).

[0029] As used herein, the term "isolated KTI" refers to a preparation that is enriched in KTI relative to other components. In various embodiments, the KTI preparation utilized in the methods and peptide compositions described herein is at least 85% pure, as determined by SDS-PAGE, brilliant blue staining, or imager quantification. In other embodiments, the protein content of the KTI preparation is greater than 85%, as measured by the BCA assay. In the case of peptide compositions, these values ​​relate to the characteristics of KTI prior to mixing with one or more other components of the peptide composition. In other embodiments, the KTI preparation contains 5% or less BBI, as determined by SDS-PAGE. In other embodiments, the KTI preparation contains less than 0.1% high molecular weight impurities (in other words, high molecular weight impurities refer to substances with a molecular weight greater than 30,000).

[0030] In other embodiments, the isolated BBI and isolated KTI can be obtained by separating them from their natural components by means known to those skilled in the art.

[0031] In some embodiments, the isolated BBI is isolated from a soy product, wherein the isolated BBI has been purified to a purity of at least 85% as measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); preferably, the BBI has been purified to a purity of at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, or at least 91%;

[0032] wherein the isolated BBI has been purified to a protein content of at least 85% by the BCA (dicarboxybicinol) assay; preferably, the BBI has been purified to a protein content of at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91% or at least 92%.

[0033] In some embodiments, the isolated BBI contains less than 0.1% of high molecular weight impurities.

[0034] The isolated KTI is isolated from a soy product, wherein the isolated KTI has been purified to a purity of at least 85% as measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); preferably, the KTI has been purified to a purity of at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or at least 95%;

[0035] Wherein, the isolated KTI has been purified to a protein content of at least 85% by BCA (dicarboxybicinol) assay; preferably, the KTI has been purified to a protein content of at least 86%, at least 87%, at least 88%, at least 89%, or at least 90%.

[0036] In some embodiments, the isolated KTI contains less than 0.1% of high molecular weight impurities.

[0037] The purity of isolated BBI and / or isolated KTI as described herein is determined prior to mixing it with one or more other components of the polypeptide composition.

[0038] In an embodiment of the present disclosure, the isolated BBI has activity in inhibiting trypsin and chymosin.

[0039] In embodiments of the present disclosure, the isolated BBI has a trypsin inhibitory activity of at least 0.5, 0.6, 0.7, 0.8 or 0.9 mg / mg; and the isolated BBI has a chymotrypsin inhibitory activity of at least 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7 or 1.8 mg / mg.

[0040] In an embodiment of the present disclosure, the isolated BBI has a trypsin inhibitory activity of 0.9 mg / mg; and the isolated BBI has a chymotrypsin inhibitory activity of 1.5 mg / mg.

[0041] In an embodiment of the present disclosure, the isolated KTI has trypsin inhibitory activity.

[0042] In embodiments of the present disclosure, the isolated KTI has a trypsin inhibitory activity of at least 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7 or 1.8 mg / mg.

[0043] In an embodiment of the present disclosure, the isolated KTI has a trypsin inhibitory activity of 1.4 mg / mg.

[0044] Unless otherwise indicated, chymotrypsin inhibitory activity referred to herein was measured using chymotrypsin with 40 BTEE units / mg chymotrypsin activity and expressed as mg of chymotrypsin inhibited per mg of test protein (mg inhibited chymotrypsin / mg test protein). BTEE refers to N-benzoyl-L-tyrosine ethyl ester (see Sigma-Aldrich product description No. B6125).

[0045] Unless otherwise indicated, trypsin inhibition activity referred to herein was measured using trypsin having an activity of 10,000 BAEE units / mg trypsin and is expressed as mg of trypsin inhibited per mg of test protein (mg inhibited trypsin / mg test protein). α -Benzoyl-L-arginine ethyl ester (see Sigma-Aldrich product number B4500).

[0046] Chymotrypsin inhibitory activity and trypsin inhibitory activity as described herein are determined prior to mixing isolated BBI and / or isolated KTI with one or more other components of the polypeptide composition.

[0047] As used herein, the term "total trypsin inhibitory activity (total TI)" refers to the combined trypsin inhibitory activity of BBI and KTI contained in the polypeptide composition.

[0048] As used herein, the term "total chymotrypsin inhibitory activity (total CI)" refers to the chymotrypsin inhibitory activity of the BBI contained in the polypeptide composition.

[0049] In some embodiments, the polypeptide compositions described herein have chymotrypsin inhibitory activity of inhibiting 0.03-0.3 mg chymotrypsin / mg of polypeptide composition, wherein the chymotrypsin has 40 BTEE units / mg chymotrypsin activity;

[0050] In some embodiments, the polypeptide compositions described herein have chymotrypsin inhibitory activity of inhibiting 0.04-0.15 mg chymotrypsin / mg of polypeptide composition, wherein the chymotrypsin has 40 BTEE units / mg chymotrypsin activity;

[0051] Specifically, the polypeptide compositions described herein have chymotrypsin inhibitory activity that inhibits at least 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.3 mg chymotrypsin / mg of polypeptide composition, and the chymotrypsin has 40 BTEE units / mg chymotrypsin activity.

[0052] In some embodiments, the polypeptide compositions described herein have trypsin inhibitory activity of inhibiting 0.05-0.4 mg trypsin / mg of polypeptide composition, wherein the trypsin has 10,000 BAEE units / mg of trypsin activity.

[0053] In some embodiments, the polypeptide compositions described herein have trypsin inhibitory activity that inhibits 0.1-0.3 mg trypsin / mg of polypeptide composition, wherein the trypsin has 10,000 BAEE units / mg of trypsin activity.

[0054] In some embodiments, the polypeptide compositions described herein inhibit 0.17-0.3 mg trypsin / mg polypeptide composition, wherein the trypsin has 10,000 BAEE units / mg trypsin activity.

[0055] In some embodiments, the polypeptide compositions described herein have trypsin inhibitory activity of inhibiting 0.06-0.17 mg trypsin / mg of polypeptide composition, wherein the trypsin has 10,000 BAEE units / mg of trypsin activity.

[0056] Specifically, the polypeptide compositions described herein have trypsin inhibitory activity that inhibits at least 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.3 mg trypsin / mg polypeptide composition.

[0057] In some embodiments, the mass ratio of the isolated BBI to the isolated KTI in the polypeptide composition is 0.1:1 to 10:1;

[0058] In some embodiments, the mass ratio of the isolated BBI to the isolated KTI in the polypeptide composition is 0.1:1 to 5:1;

[0059] In some embodiments, the mass ratio of the isolated BBI to the isolated KTI in the polypeptide composition is 0.2:1 to 3:1;

[0060] In some embodiments, the mass ratio of the isolated BBI to the isolated KTI in the polypeptide composition is 0.8:1 to 3:1;

[0061] In some embodiments, the mass ratio of the isolated BBI to the isolated KTI in the polypeptide composition is 0.2:1 to 0.8:1;

[0062] Optionally, the mass ratio of the isolated BBI to the isolated KTI is 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1 or any number in between any two ratios.

[0063] As used herein, the term "fish oil," also known as omega-3 triglycerides, is a type of oil extracted from fatty fish. It is rich in n-3 polyunsaturated fatty acids, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Fatty fish species from which fish oil can be extracted include, but are not limited to, mackerel, tuna, salmon, sturgeon, anchovies, sardines, herring, and trout.

[0064] In some embodiments, the polypeptides disclosed herein include, but are not limited to, any one or a mixture of several of insulin, glucagon, glucagon-like peptide-1 (GLP-1) receptor agonist, glucagon-like peptide-2 (GLP-2) receptor agonist, calcitonin (CT), growth hormone, somatostatin, thyrotropin-releasing hormone, parathyroid hormone, gonadotropin-releasing hormone, heparin, granulocyte colony-stimulating factor, prostaglandin, cyclosporine, interferon, vasopressin, vancomycin, erythropoietin, glutathione, and thymosin.

[0065] In some embodiments, the polypeptides disclosed herein include, but are not limited to, recombinant human insulin, insulin degludec, glucagon, exenatide, belaglutide, liraglutide, loxenatide, dulaglutide, lixisenatide, semaglutide, albiglutide, dulaglutide, teduglutide, calcitonin, salmon calcitonin, growth hormone, octreotide, thyrotropin-releasing hormone, parathyroid hormone fragment, teriparatide, leuprorelin, prorelin, gonadorelin, goserelin, buserelin, sermorelin, nafarelin, histrelin, treprostinil, Any one or a mixture of several of the following: priloprelin, tesmorelin, cortirelin, lanreotide acetate, terlipressin, antiangitide, teriparatide, abalaparatide, nesiritide, eptifibatide, lixisenatide, sifuvirtide, atosiban, Yunzhi glycopeptide, ginseng glycopeptide, osteopeptide, myosin, glutathione, mannan peptide, enfuvirtide, bradykinin, enkephalin, nosiheptide, hirudin, glatiramer acetate, aprotinin, alaninerelin, trypsinogenase, polymyxin, serrapeptase, thymosin α1, thymopentin and thymofasin.

[0066] In some embodiments, the insulin described in the present disclosure includes human insulin, proinsulin, and insulin-like compounds, wherein insulin-like compounds include insulin analogs and insulin derivatives, etc.

[0067] In some embodiments, the insulins described herein include animal insulin and human insulin.

[0068] In some embodiments, the insulins described herein include rapid-acting insulin, short-acting insulin, intermediate-acting insulin, long-acting insulin, and ultra-long-acting insulin.

[0069] In some embodiments, an insulin analog as described herein refers to an insulin in which one or more amino acids are substituted while retaining some or all of one or more of the glucose-related activities of insulin.

[0070] In some embodiments, the insulins described in the present disclosure include: (1) rapid-acting insulins, such as lispro insulin and aspart insulin; (2) short-acting insulins, such as recombinant human insulin; (3) intermediate-acting insulins, such as protamine zinc insulin (NPH); (4) long-acting insulins, such as protamine zinc insulin, glargine insulin, detemir insulin, degludec insulin, etc.; (5) ultra-long-acting insulin; (6) translocation insulin analogs; (7) insulin loss analogs; (8) derivatized insulins; (9) derivatized insulin analogs; (10) derivatized proinsulin; (11) human insulin analog complexes (e.g., hexameric complexes); (12) insulin mixtures and (13) PEG insulin.

[0071] In some embodiments, the glucagon-like peptide-1 described herein includes a short-acting GLP-1 receptor agonist and a long-acting GLP-1 receptor agonist.

[0072] In some embodiments, the glucagon-like peptide-1 described in the present disclosure includes: Exenatide (trade name: Byetta), Lixisenatide, Benaglutide (trade name: Yishengtai), Liraglutide (trade name: Novoli), Taspoglutid, Loxenatide (trade name: Fulaimei), Semaglutide, Albiglutide and Dulaglutide (trade name: Duyida), etc.

[0073] In some embodiments, the thymosin peptides disclosed herein include thymosin α1, thymosin α1, thymosin β4, or thymopentin.

[0074] In some embodiments, the polypeptide in the composition described in the present disclosure may also be replaced with other active substances, such as other small molecules with poor oral bioavailability.

[0075] In some embodiments, the molecular weight of the polypeptides of the present disclosure is 100Da to 20,000Da. In some embodiments, the molecular weight of the polypeptides of the present disclosure can be 100Da, 200Da, 300Da, 400Da, 500Da, 600Da, 700Da, 800Da, 900Da, 1000Da, 1100Da, 1200Da, 1300Da, 1400Da, 1500Da, 1600Da, 1700Da, 1800Da, 1900Da, 2000Da, 2100Da, 2200Da, 2300Da, 2400Da, 2500Da, 2600Da, 2700Da, 2800Da, 2900Da, 3000Da, 3100Da, 3200Da, 3300Da, 3400Da, 3600Da, 3700Da, 3800Da, 3900Da, 4000Da, 4100Da, 4200Da, 4300Da, 4400Da, 4500Da, 4600Da, 4700Da, 4800Da, 4900Da, 5000Da, 5100Da, 5200Da, 5300Da, 5400Da, 5500Da, 5600Da, 5700Da 400Da, 2500Da, 2600Da, 2700Da, 2800Da, 2900Da, 3000Da, 3100Da, 3200Da, 3300Da, 3400Da, 3500Da, 3600Da, 3700Da, 3800Da, 3 900Da, 4000Da, 4100Da, 4200Da, 4300Da, 4400Da, 4500Da, 4600Da, 4700Da, 4800Da, 4900Da, 5000Da, 5100Da, 5200Da, 5300Da, 54 00Da, 5500Da, 5600Da, 5700Da, 5800Da, 5900Da, 6000Da, 6100Da, 6200Da, 6300Da, 6400Da, 6500Da, 6600Da, 6700Da, 6800Da, 69 00Da, 7000Da, 7100Da, 7200Da, 7300Da, 7400Da, 7500Da, 7600Da, 7700Da, 7800Da, 7900Da, 8000Da, 8100Da, 8200Da, 8300Da, 840 000 Da, 17000 Da, 18000 Da, 19000 Da, 20000 Da, or any range therebetween.

[0076] In some embodiments, the molecular weight of the polypeptides disclosed herein is 1000Da to 20000Da, in some embodiments, the molecular weight of the polypeptides disclosed herein is 1000Da to 6150Da, in some embodiments, the molecular weight of the polypeptides disclosed herein is 1209Da to 6104Da, in some embodiments, the molecular weight of the polypeptides disclosed herein is 1000Da to 6000Da, in some embodiments, the molecular weight of the polypeptides disclosed herein is 2000Da to 6000Da.

[0077] In some embodiments, the polypeptides disclosed herein may be selected from the following polypeptides:

[0078] In some embodiments, the polypeptides disclosed herein may be naturally extracted polypeptides, polypeptides synthesized by chemical methods, or polypeptides prepared by genetic engineering.

[0079] In some embodiments, the human insulin disclosed herein may be insulin prepared by chemical synthesis, or recombinant human insulin obtained by genetic engineering.

[0080] In some embodiments, the polypeptide composition of the present disclosure is for oral administration; in some embodiments, the polypeptide composition for oral administration of the present disclosure is prepared into the following dosage forms: tablets, pills, capsules, emulsions, syrups or suspensions.

[0081] Compared with the nanosystems prepared in the prior art, in the polypeptide composition system provided by the present disclosure, the polypeptide exists in the form of a suspension, the system is uniform, and there is no sudden release phenomenon. There is no need to consider the encapsulation rate during the preparation process, and the product quality is easy to control.

[0082] In some embodiments, the polypeptide composition system provided by the present disclosure is a non-nanosystem.

[0083] In some embodiments, the polypeptide compositions provided by the present disclosure are not oil-in-water or water-in-oil systems.

[0084] In some embodiments, the absorption promoter of the present disclosure is selected from any one or more of ethylenediaminetetraacetic acid or its salts, ethylene glycol-bis(2-aminoethyl ether)-N,N,N'N'-tetraacetic acid or its salts, N-(8-(2-hydroxybenzoyl)amino)octanoic acid or its salts, salicylic acid or its salts, citric acid or its salts, bile acid or its salts, deoxycholic acid or its salts, glycocholic acid or its salts, taurocholic acid or its salts, alkyl glycosides, sodium lauryl sulfate, sodium docusate, cyclodextrin or its derivatives, chitosan or its derivatives, caprylic acid or its salts, capric acid or its salts, lauric acid or its salts, myristic acid, palmitic acid, stearic acid, fatty acid triglycerides, lecithin, choline and carnitine. In some embodiments, the absorption promoter of the present disclosure is selected from the sodium salt of ethylenediaminetetraacetic acid.

[0085] In some embodiments, the EDTA salt of the present disclosure is selected from disodium EDTA, dipotassium EDTA, trisodium EDTA, or tetrasodium EDTA.

[0086] In some embodiments, the polypeptide composition described in the present disclosure further comprises at least one emulsifier.

[0087] In some embodiments, the polypeptide composition of the present disclosure, wherein the emulsifier is selected from any one or more of an ionic surfactant, a nonionic surfactant, and a zwitterionic surfactant;

[0088] Preferably, the emulsifier is selected from any one or more of ammonium lauryl sulfate, N-dodecyl-β-D-maltoside, tridecyl-β-D-maltoside, sodium lauryl sulfate, sodium docusate, cyclodextrin or its derivatives, chitosan or its derivatives, sodium polyoxyethylene alkyl sulfate, sodium lauryl ether sulfate, dioctyl sodium sulfosuccinate, poloxamer, glyceryl monostearate, glyceryl monolaurate, sorbitan monolaurate, sorbitan monostearate, polyoxyethylene sorbitan fatty acid esters, sorbitan fatty acid esters, polyoxyethylene monopalmitate, polyoxyethylene hydrogenated castor oil, C6-C12 fatty acid triglycerides and C8-C10 fatty acid triglycerides;

[0089] Preferably, the at least one surfactant is selected from Tween, preferably, the at least one surfactant is selected from any one or more of Tween 20, Tween 40, Tween 60, Tween 65, Tween 80 and Tween 85.

[0090] In some embodiments, the present disclosure provides a polypeptide composition comprising, by mass fraction:

[0091] Peptide: 0.5-10%,

[0092] Fish oil: 3.0-50.0%,

[0093] Isolated BBI: 1.0-25.0%,

[0094] Isolated KTI: 1.0-20.0%,

[0095] Absorption enhancer: 5.0-45.0%, and

[0096] Emulsifier: 8.0-60.0%;

[0097] or

[0098] Peptide: 0.9-4.6%,

[0099] Fish oil: 5.5-34.9%,

[0100] Isolated BBI: 2.9-10.0%,

[0101] Isolated KTI: 2.5-14.3%,

[0102] Absorption enhancer: 12.9-36.1%, and

[0103] Emulsifier: 14.9-49.6%;

[0104] or

[0105] Peptide: 0.9-4.6%,

[0106] Fish oil: 11.9-34.9%,

[0107] Isolated BBI: 2.9-10.0%,

[0108] Isolated KTI: 2.5-14.3%,

[0109] Absorption enhancer: 12.9-36.1%, and

[0110] Emulsifier: 14.9-34.5%;

[0111] or

[0112] Peptide: 0.9-4.6%,

[0113] Fish oil: 34.5-34.9%,

[0114] Isolated BBI: 2.9-7.5%,

[0115] Isolated KTI: 2.5-14.3%,

[0116] Absorption enhancer: 12.9-35.6%, and

[0117] Emulsifier: 14.9-34.5%;

[0118] or

[0119] Peptide: 0.9-1.8%,

[0120] Fish oil: 11.9-34.5%,

[0121] Isolated BBI: 2.9-10.0%,

[0122] Isolated KTI: 12.6-14.3%,

[0123] Absorption enhancer: 12.9-36.1%, and

[0124] Emulsifier: 27.6-34.5%;

[0125] or

[0126] Peptide: 1.8-4.6%,

[0127] Fish oil: 11.9-34.9%,

[0128] Isolated BBI: 7.5-10.0%,

[0129] Isolated KTI: 2.5-12.6%,

[0130] Absorption enhancer: 35.6-36.1%, and

[0131] Emulsifier: 14.9-27.6%.

[0132] In some embodiments, the present disclosure provides a polypeptide composition comprising, by mass fraction:

[0133] Recombinant human insulin: 0.5-10%,

[0134] Fish oil: 3.0-50.0%,

[0135] Isolated BBI: 1.0-25.0%,

[0136] Isolated KTI: 1.0-20.0%,

[0137] Ethylenediaminetetraacetic acid or its salt: 5.0-45.0%, and

[0138] Tween: 8.0-60.0%;

[0139] or

[0140] Recombinant human insulin: 0.9-4.6%,

[0141] Fish oil: 5.5-34.9%,

[0142] Isolated BBI: 2.9-10.0%,

[0143] Isolated KTI: 2.5-14.3%,

[0144] Ethylenediaminetetraacetic acid or its salt: 12.9-36.1%, and

[0145] Tween: 14.9-49.6%;

[0146] or

[0147] Recombinant human insulin: 0.9-4.6%,

[0148] Fish oil: 11.9-34.9%,

[0149] Isolated BBI: 2.9-10.0%,

[0150] Isolated KTI: 2.5-14.3%,

[0151] Ethylenediaminetetraacetic acid or its salt: 12.9-36.1%, and

[0152] Tween: 14.9-34.5%;

[0153] or

[0154] Recombinant human insulin: 0.9-4.6%,

[0155] Fish oil: 34.5-34.9%,

[0156] Isolated BBI: 2.9-7.5%,

[0157] Isolated KTI: 2.5-14.3%,

[0158] Ethylenediaminetetraacetic acid or its salt: 12.9-35.6%, and

[0159] Tween: 14.9-34.5%;

[0160] or

[0161] Recombinant human insulin: 0.9-1.8%,

[0162] Fish oil: 11.9-34.5%,

[0163] Isolated BBI: 2.9-10.0%,

[0164] Isolated KTI: 12.6-14.3%,

[0165] Ethylenediaminetetraacetic acid or its salt: 12.9-36.1%, and

[0166] Tween: 27.6-34.5%;

[0167] or

[0168] Recombinant human insulin: 1.8-4.6%,

[0169] Fish oil: 11.9-34.9%,

[0170] Isolated BBI: 7.5-10.0%,

[0171] Isolated KTI: 2.5-12.6%,

[0172] Ethylenediaminetetraacetic acid or its salt: 35.6-36.1%, and

[0173] Tween: 14.9-27.6%.

[0174] As used herein, the term "mass fraction" refers to the mass percentage of each component in the total mass of the polypeptide composition.

[0175] For example, in some embodiments, the present disclosure provides a polypeptide composition comprising

[0176] Recombinant human insulin: 8mg,

[0177] Fish oil: 320mg,

[0178] Isolated BBI: 26.7 mg,

[0179] Isolated KTI: 133.3 mg,

[0180] Ethylenediaminetetraacetic acid or its salt: 120 mg, and

[0181] Tween 80: 320mg.

[0182] The total mass of the polypeptide composition is 928 mg, and the mass fraction of the recombinant human insulin is 0.9%.

[0183] In some embodiments, the present disclosure provides a polypeptide composition comprising in a unit dosage form:

[0184] Recombinant human insulin: 8-32mg,

[0185] Fish oil: 50-320mg,

[0186] Isolated BBI: 26.7-88.9 mg,

[0187] Isolated KTI: 17.5-133.3 mg,

[0188] Ethylenediaminetetraacetic acid or its salt: 120-320 mg, and

[0189] Tween: 105-450mg;

[0190] or

[0191] Recombinant human insulin: 8-32mg,

[0192] Fish oil: 105-320mg,

[0193] Isolated BBI: 26.7-88.9 mg,

[0194] Isolated KTI: 17.5-133.3 mg,

[0195] Ethylenediaminetetraacetic acid or its salt: 120-320 mg, and

[0196] Tween: 105-320 mg;

[0197] or

[0198] Recombinant human insulin: 8-32mg,

[0199] Fish oil: 245-320mg,

[0200] Isolated BBI: 26.7-52.5 mg,

[0201] Isolated KTI: 17.5-133.5 mg,

[0202] Ethylenediaminetetraacetic acid or its salt: 120-250 mg, and

[0203] Tween: 105-320 mg;

[0204] or

[0205] Recombinant human insulin: 8-16mg,

[0206] Fish oil: 105-320mg,

[0207] Isolated BBI: 26.7-88.9 mg,

[0208] Isolated KTI: 111.1-133.3 mg,

[0209] Ethylenediaminetetraacetic acid or its salt: 120-320 mg, and

[0210] Tween: 245-320 mg;

[0211] or

[0212] Recombinant human insulin: 8mg,

[0213] Fish oil: 50-320mg,

[0214] Isolated BBI: 26.7-37.5 mg,

[0215] Isolated KTI: 62.5-133.3 mg,

[0216] Ethylenediaminetetraacetic acid or its salt: 120-300 mg, and

[0217] Tween: 320-450mg;

[0218] or

[0219] Recombinant human insulin: 16-32mg,

[0220] Fish oil: 105-245mg,

[0221] Isolated BBI: 52.5-88.9 mg,

[0222] Isolated KTI: 17.5-111.1 mg,

[0223] Ethylenediaminetetraacetic acid or its salt: 250-320 mg, and

[0224] Tween: 105-245 mg;

[0225] or

[0226] Recombinant human insulin: 8-16mg,

[0227] Fish oil: 50-105mg,

[0228] Isolated BBI: 37.5-88.9 mg,

[0229] Isolated KTI: 62.5-111.1 mg,

[0230] Ethylenediaminetetraacetic acid or its salt: 300-320 mg, and

[0231] Tween: 245-450mg.

[0232] In some embodiments, the present disclosure provides a polypeptide composition comprising in a unit dosage form:

[0233] Recombinant human insulin: 8mg,

[0234] Fish oil: 320mg,

[0235] Isolated BBI: 26.7 mg,

[0236] Isolated KTI: 133.3 mg,

[0237] Ethylenediaminetetraacetic acid or its salt: 120 mg, and

[0238] Tween: 320 mg;

[0239] or

[0240] Recombinant human insulin: 16mg,

[0241] Fish oil: 105mg,

[0242] Isolated BBI: 88.9 mg,

[0243] Isolated KTI: 111.1 mg,

[0244] Ethylenediaminetetraacetic acid or its salt: 320 mg, and

[0245] Tween: 245mg;

[0246] or

[0247] Recombinant human insulin: 32mg,

[0248] Fish oil: 245mg,

[0249] Isolated BBI: 52.5 mg,

[0250] Isolated KTI: 17.5 mg,

[0251] Ethylenediaminetetraacetic acid or its salt: 250 mg, and

[0252] Tween: 105mg;

[0253] or

[0254] Recombinant human insulin: 8mg,

[0255] Fish oil: 50mg,

[0256] Isolated BBI: 37.5 mg,

[0257] Isolated KTI: 62.5 mg,

[0258] 300 mg of ethylenediaminetetraacetic acid or its salt, and

[0259] Tween: 450mg.

[0260] In some embodiments, the polypeptide composition described herein comprises a polypeptide whose mass fraction can be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or any range or point value therebetween.

[0261] In some embodiments, the polypeptide composition described herein comprises isolated BBI, and the mass fraction of the isolated BBI can be 1.0%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25% or any range or point value therebetween.

[0262] In some embodiments, the polypeptide composition described herein comprises isolated KTI, and the mass fraction of the isolated KTI can be 1.0%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% or any range or point value therebetween.

[0263] In some embodiments, the polypeptide composition described herein comprises an absorption promoter, and the mass fraction of the absorption promoter can be 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% or any range or point value therebetween.

[0264] In some embodiments, the polypeptide compositions of the present disclosure further comprise at least one emulsifier.

[0265] In some embodiments, the emulsifier described in the present disclosure includes any one or a combination of at least two of an ionic surfactant, a nonionic surfactant, or a zwitterionic surfactant.

[0266] In some embodiments, the emulsifier described in the present disclosure is an ionic surfactant. In some embodiments, the ionic surfactant described in the present disclosure includes but is not limited to sulfates, sulfonates, phosphates, carboxylates, ammonium lauryl sulfate, sodium lauryl sulfate (SDS), sodium polyoxyethylene alkyl sulfate, sodium lauryl ether sulfate, sodium docusate (sodium dioctyl sulfosuccinate, DSS), perfluorooctane sulfonate (PFOS), perfluorobutyl sulfonate, alkyl aryl ether phosphates and alkyl ether phosphates. Any one or a combination of at least two.

[0267] In some embodiments, the emulsifier described in the present disclosure is a nonionic surfactant. In some embodiments, the nonionic surfactant described in the present disclosure includes but is not limited to any one of Triton X-100, poloxamer, glyceryl monostearate, glyceryl monolaurate, sorbitan monolaurate, sorbitan monostearate, polyoxyethylene sorbitan fatty acid esters (i.e., Tween, such as Tween 20, Tween 40, Tween 60 and Tween 80), and sorbitan fatty acid esters (Span), or a combination of at least two thereof.

[0268] The emulsifier described in the present disclosure can also be any one or a combination of at least two of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-acylethanolamine (POPE), N-dodecyl-β-D-maltoside, tridecyl-β-D-maltoside, cyclodextrin and chitosan derivatives (e.g., protonated chitosan, trimethyl chitosan chloride, etc.).

[0269] In some embodiments, the polypeptide composition further comprises at least one emulsifier; preferably, the polypeptide composition comprises 0.1-75% of the emulsifier, preferably 1-60%, and more preferably 5-50%.

[0270] In some embodiments, the polypeptide composition described herein comprises an emulsifier, and the mass fraction of the emulsifier can be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, %, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75% or any range or point value therebetween.

[0271] In the above embodiment, the mass ratio of the isolated BBI to the isolated KTI in the polypeptide composition is 0.1:1 to 10:1; optionally, the mass ratio of the isolated BBI to the isolated KTI is 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1, or any number between any two ratios.

[0272] In an embodiment of the present disclosure, a polypeptide composition is provided, wherein the isolated BBI and the isolated KTI in the polypeptide composition are mixed in a certain ratio so that the activity ratio of total trypsin inhibitory activity (total TI) to total chymotrypsin inhibitory activity (total CI) in the composition is 0.5:1 to 10:1; preferably 0.7:1 to 10:1; preferably 0.91:1-6:1; preferably 0.91:1 to 5.27:1; and the polypeptide composition comprises, by mass fraction:

[0273] Recombinant human insulin: 0.5-10%,

[0274] Fish oil: 3.0-50.0%,

[0275] Isolated BBI: 1.0-25.0%,

[0276] Isolated KTI: 1.0-20.0%,

[0277] Ethylenediaminetetraacetic acid or its salt: 5.0-45.0%, and

[0278] Tween: 8.0-60.0%;

[0279] or

[0280] Recombinant human insulin: 0.9-4.6%,

[0281] Fish oil: 5.5-34.9%,

[0282] Isolated BBI: 2.9-10.0%,

[0283] Isolated KTI: 2.5-14.3%,

[0284] Ethylenediaminetetraacetic acid or its salt: 12.9-36.1%, and

[0285] Tween: 14.9-49.6%;

[0286] or

[0287] Recombinant human insulin: 0.9-4.6%,

[0288] Fish oil: 11.9-34.9%,

[0289] Isolated BBI: 2.9-10.0%,

[0290] Isolated KTI: 2.5-14.3%,

[0291] Ethylenediaminetetraacetic acid or its salt: 12.9-36.1%, and

[0292] Tween: 14.9-34.5%;

[0293] or

[0294] Recombinant human insulin: 0.9-4.6%,

[0295] Fish oil: 34.5-34.9%,

[0296] Isolated BBI: 2.9-7.5%,

[0297] Isolated KTI: 2.5-14.3%,

[0298] Ethylenediaminetetraacetic acid or its salt: 12.9-35.6%, and

[0299] Tween: 14.9-34.5%;

[0300] or

[0301] Recombinant human insulin: 0.9-1.8%,

[0302] Fish oil: 11.9-34.5%,

[0303] Isolated BBI: 2.9-10.0%,

[0304] Isolated KTI: 12.6-14.3%,

[0305] Ethylenediaminetetraacetic acid or its salt: 12.9-36.1%, and

[0306] Tween: 27.6-34.5%;

[0307] or

[0308] Recombinant human insulin: 1.8-4.6%,

[0309] Fish oil: 11.9-34.9%,

[0310] Isolated BBI: 7.5-10.0%,

[0311] Isolated KTI: 2.5-12.6%,

[0312] Ethylenediaminetetraacetic acid or its salt: 35.6-36.1%, and

[0313] Tween: 14.9-27.6%.

[0314] On the other hand, the present disclosure further provides a drug, which includes the polypeptide composition described above; in some embodiments, the drug of the present disclosure further includes a pharmaceutically acceptable excipient.

[0315] In some embodiments, the pharmaceutically acceptable excipients described herein include a coating.

[0316] In some embodiments, the coating of the present disclosure is selected from a gastric coating and / or an enteric coating.

[0317] In some embodiments, the coating materials disclosed herein include: hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), acrylic resin polyvinyl pyrrolidone, cellulose acetate phthalate (CAP), hydroxypropyl methylcellulose phthalate (HPMCP), hydroxypropyl methylcellulose acetate succinate (HPMCAS), and the like.

[0318] It should be noted that the primary function of coatings in the present disclosure is to release polypeptides in specific locations within the gastrointestinal tract. For example, enteric coatings can render polypeptides insoluble in gastric acid but soluble in intestinal fluid. Those skilled in the art can select coating materials based on actual needs to achieve polypeptide release in gastric or intestinal fluid within the gastrointestinal tract.

[0319] In some embodiments, the pharmaceutically acceptable excipients disclosed herein include excipients, disintegrants, binders, flavoring agents, or lubricants.

[0320] In some embodiments, the excipients disclosed herein are selected from any one or a combination of at least two of lactose, sorbitol, xylitol, mannitol, calcium hydrogen phosphate, microcrystalline cellulose, silicified microcrystalline cellulose, starch, pregelatinized starch, lactose starch complex, lactose cellulose complex, or mannitol starch complex.

[0321] In some embodiments, the disintegrant of the present disclosure is selected from any one or a combination of at least two of cross-linked carboxymethyl cellulose sodium, cross-linked polyvinyl pyrrolidone, cross-linked carboxymethyl starch sodium, or low-substituted hydroxypropyl cellulose.

[0322] In some embodiments, the binder of the present disclosure is selected from any one or a combination of at least two of colloidal microcrystalline cellulose, hydroxypropylmethylcellulose, hydroxypropyl cellulose, methylcellulose, ethylcellulose, polyvinylpyrrolidone, gelatin solution, starch slurry or sucrose solution.

[0323] In some embodiments, the lubricant disclosed herein is selected from any one or a combination of at least two of sodium stearyl fumarate, magnesium stearate, calcium stearate, micronized silica gel, or talc.

[0324] In some embodiments, the flavoring agent disclosed herein is any one or a combination of at least two of mannitol, sorbitol, aspartame, stevioside, sucralose, aspartame, neotame, stevioside, hydroxychloroisothiazolinone, or essence.

[0325] In some embodiments, the polypeptide compositions and medicaments comprising the same provided by the present disclosure are for oral administration.

[0326] In some embodiments, the polypeptide composition or medicine provided in the present disclosure includes tablets, pills, capsules, emulsions, syrups or suspensions. Preferably, the polypeptide in the capsule contents is in a suspended state.

[0327] In some embodiments, the polypeptide compositions or medicaments provided herein are anhydrous.

[0328] "Anhydrous" means the capsule contents contain less than 9% moisture. For example, the moisture content can be 8%, 7%, 6%, or 5%. Moisture is determined according to the Fischer method (Method 1) in General Chapter 0832 of the 2020 edition of the Chinese Pharmacopoeia.

[0329] In some embodiments, the polypeptide composition or medicament provided in the present disclosure is in an oral or injectable dosage form.

[0330] In some embodiments, the polypeptide composition or drug provided by the present disclosure is used in the preparation of an oral drug for treating or preventing a disease, wherein the disease is selected from hyperglycemia, type 1 diabetes, type 2 diabetes, non-insulin-dependent diabetes mellitus, juvenile maturity-onset diabetes, gestational diabetes, obesity, non-alcoholic fatty liver disease, hepatic steatosis, hepatitis, liver fibrosis, cirrhosis, liver cancer, Alzheimer's disease, Parkinson's disease and impaired glucose tolerance.

[0331] In some embodiments, the present disclosure provides a method for treating or preventing a disease, comprising orally administering a therapeutically effective amount of the polypeptide composition or the drug to a subject in need thereof, wherein the disease is selected from hyperglycemia, type 1 diabetes, type 2 diabetes, non-insulin-dependent diabetes mellitus, maturity-onset diabetes of the young, gestational diabetes, obesity, non-alcoholic fatty liver disease, hepatic steatosis, hepatitis, liver fibrosis, cirrhosis, liver cancer, Alzheimer's disease, Parkinson's disease, and impaired glucose tolerance.

[0332] In some embodiments, the present disclosure provides a method for treating or preventing a disease, comprising orally administering to a subject in need thereof a single dose of 8-50 mg of the polypeptide in the polypeptide composition, 1-3 times a day; or orally administering to a subject a single dose of 8-50 mg of the polypeptide in the drug, 1-3 times a day.

[0333] In some embodiments, the present disclosure provides a method for treating or preventing a disease, comprising orally administering to a subject in need thereof a single dose of 8 mg, 24 mg, or 50 mg of the polypeptide composition, 1, 2, or 3 times a day; or orally administering to a subject a single dose of 8 mg, 24 mg, or 50 mg of the polypeptide in any one of claims 24-25, 1 or 2 times a day. DETAILED DESCRIPTION

[0334] The present invention is described in detail below with reference to the accompanying drawings.

[0335] Example 1 Preparation of soybean trypsin inhibitor

[0336] Soybean trypsin inhibitors (SBTIs) are major anti-nutritional factors found in soybeans. They primarily include Kunitz trypsin inhibitor (KTI) and Bowman-Birk trypsin inhibitor (BBI). KTI has trypsin inhibitory activity, while BBI has both trypsin and chymotrypsin inhibitory activity.

[0337] Trypsin inhibitory activity is abbreviated as "TI" and chymotrypsin inhibitory activity is abbreviated as "CI".

[0338] The amino acid sequence of BBI is shown in SEQ ID NO: 1, and the number in the Uniprot database is UniprtoKB-P01055;

[0339] SEQ ID NO: 1

[0340] The KTI referred to herein is KTI3, the amino acid sequence of KTI3 is shown in SEQ ID NO: 2, and the number of KTI3 in the Uniprot database is UniprtoKB-P01070.

[0341] SEQ ID NO:2

[0342] BBI and KTI were prepared by referring to the SBTI preparation method described in Example 2 of Chinese Patent Application No. CN201380018355.7 and Patent Publication No. CN104244969A, which is incorporated herein by reference in its entirety.

[0343] Briefly, BBI and KTI were prepared as follows:

[0344] Preparation of SBTI intermediates:

[0345] Extraction buffer: 25 mM sodium phosphate and 100 mM sodium chloride, pH 4.5

[0346] Dialysis buffer: 10 mM sodium phosphate, pH 6.5

[0347] Mix 25 kg of soy flour with 250 L of extraction buffer at room temperature for 1 hour and adjust the pH to 4.5 with hydrochloric acid. The extraction buffer is 25 mM sodium phosphate and 100 mM sodium chloride, pH 4.5. Filter, wash the filter cake with extraction buffer, collect the filtrate, add ammonium sulfate to the filtrate, stir for 1-3 hours, centrifuge overnight, and collect the precipitate. Mix the collected precipitate with 12 L of 10 mM sodium phosphate, pH 7.6 buffer for 0.5 hour, centrifuge, and collect the filtrate and filter cake. The filter cake is repeatedly extracted three times with dialysis buffer, all filtrates are combined, dialyzed with dialysis buffer, centrifuged, and the filtrate is collected. The filtrate is lyophilized to obtain the SBTI intermediate.

[0348] Purification of SBTI intermediates:

[0349] The SBTI intermediate was dissolved in 10 mM sodium phosphate, pH 6.5 buffer and subjected to DEAE-Sepharose column chromatography. 1 kg of the suspended SBTI intermediate was loaded onto a 30 L column. The column was washed with 8 × CV of DEAE buffer: 10 mM sodium phosphate, pH 6.5. Elution was performed using a sodium chloride gradient, and the BBI solution and KTI solution were collected separately. The collected BBI and KTI solutions were desalted and concentrated by ultrafiltration to obtain BBI and KTI concentrates, respectively. The BBI concentrate was subjected to two steps of ultrafiltration through 30 kDa and 3 kDa ultrafiltration membranes, filtered through a 0.22 μm filter, and lyophilized to obtain BBI. The KTI concentrate was concentrated and desalted by a single step of ultrafiltration through 10 kDa, then filtered through a 0.22 μm filter and lyophilized to obtain KTI.

[0350] Example 2 Determination of the inhibitory activity of soybean trypsin inhibitor

[0351] The BBI and KTI obtained in Example 1 were assayed for trypsin inhibitory activity and chymotrypsin inhibitory activity. The specific assay method and principle are as follows:

[0352] Trypsin inhibition activity was measured using trypsin having a trypsin activity of 10,000 BAEE units / mg and expressed as mg of trypsin inhibited per mg of test protein.

[0353] Principle: Using BAEE as substrate, the absorbance at A253nm of the mixed reaction of substrate, trypsin and soybean trypsin inhibitor is measured at pH 7.6 and 25℃ to determine the trypsin inhibitory activity of soybean trypsin inhibitor.

[0354] BAEE Chinese name is N α -Benzoyl-L-arginine ethyl ester, English name Nα -Benzoyl-L-arginine Ethyl Ester.

[0355] The details are as follows:

[0356] Unit Definition:

[0357] Using BAEE as the reaction substrate, at pH 7.6, temperature 25°C, and a reaction volume of 3.20 mL, consuming 1 unit of BAEE will produce a ΔA253 of 0.001 per minute.

[0358] Prepare the following reagents:

[0359] a. 67 mM sodium phosphate buffer, pH 7.6, at 25°C, adjusted to pH 7.6 with 1 M NaOH at 25°C.

[0360] b. 0.25 mM Nα-benzoyl-L-arginine ethyl ester solution (BAEE), prepared using 67 mM sodium phosphate buffer.

[0361] c.1mM hydrochloric acid solution

[0362] d. Trypsin solution: Immediately before use, prepare a solution containing 1 mg of protein per mL of trypsin using cold 1 mM hydrochloric acid solution.

[0363] e. Trypsin inhibitor solution (BBI or KTI): immediately before use, prepare a solution containing 1.0 mg / mL trypsin inhibitor in cold reagent a.

[0364] Part A:

[0365] Take 6 centrifuge tubes and pipette (in mL) the following reagents into each of the 6 centrifuge tubes according to Table 1. Invert each centrifuge tube to mix evenly.

[0366] Table 1

[0367] Let stand at 25°C for at least 5 minutes and no more than 6 minutes.

[0368] Part B:

[0369] Take 7 centrifuge tubes and pipette (in mL) the following reagents into each of the 7 centrifuge tubes according to Table 2. Invert each centrifuge tube to mix evenly.

[0370] Table 2

[0371] Mix by inversion and equilibrate to 25°C. Monitor A253nm using a UV-Vis spectrophotometer until constant. Add the reagents of Table 3 to Table 2, i.e., pipette 0.10 mL of the non-inhibited group of Part A to the non-inhibited group of Table 2, pipette 0.10 mL of Sample 1 of Part A to Sample 1 of Table 2, pipette 0.10 mL of Sample 2 of Part A to Sample 2 of Table 2, pipette 0.10 mL of Sample 3 of Part A to Sample 3 of Table 2, pipette 0.10 mL of Sample 4 of Part A to Sample 4 of Table 2, and pipette 0.10 mL of Sample 5 of Part A to Sample 5 of Table 2. Immediately mix by inversion and record the 253nm increment over approximately 5 minutes. Using maximum linear rate, obtain the ΔA253nm / min value for each of the sample, blank, and non-inhibited group.

[0372] Table 3

[0373] calculate df = dilution factor

[0374] 0.001 = ΔA produced per minute by consuming 1 unit of BAEE at pH 7.6, 25°C, and a reaction volume of 3.20 mL. 253 0.10 = Volume of enzyme used (in ml) (Part B) 10.0 = Total volume measured in ml (Part A) 0.5 = Volume of trypsin solution used (in ml) (Part A)

[0375] Plot the trypsin activity (BAEE units / mg trypsin) versus mg trypsin inhibitor / RM. Trypsin inhibitor mg = (trypsin inhibitor mL) / trypsin inhibitor concentration, mg / mL)

[0376] Normalization Factor = (BAEE units of uninhibited trypsin per mg of solid / 10,000 BAEE units of trypsin per strength.) RM = reaction mixture

[0377] Chymotrypsin inhibitory activity was measured using chymotrypsin with an activity of 40 BTEE units / mg chymotrypsin and expressed as mg of chymotrypsin inhibited per mg of test protein.

[0378] Principle: Using BTEE as substrate, at T = 25℃, pH = 7.8, the absorbance of the mixed reaction product of substrate, chymotrypsin and soybean trypsin inhibitor at A256nm is measured to calculate the chymotrypsin inhibitory activity of soybean trypsin inhibitor.

[0379] BTEE Chinese name is N-benzoyl-L-tyrosine ethyl ester

[0380] Unit Definition:

[0381] Using BTEE as the reaction substrate, at pH 7.8 and temperature 25°C, 1 unit of chymosin activity will catalyze the hydrolysis of 1 μmol of BTEE per minute.

[0382] The details are as follows:

[0383] Prepare the following reagents

[0384] a. 80 mM Tris-HCl buffer, pH 7.8, 25°C, buffer I

[0385] b. 50% (v / v) methanol, prepared using purified water and methanol

[0386] c. 1.18 mM BTEE (prepare 50 mL by dissolving BTEE in reagent b)

[0387] d. 2M calcium chloride solution (dissolve calcium chloride in ultrapure water to prepare 5 mL)

[0388] e. 1 mM hydrochloric acid (prepare 50 ml of hydrochloric acid in ultrapure water)

[0389] f. 10 mM Tris-HCl buffer, pH 7.8, 25°C, buffer II (prepare 50 ml of hydrochloric acid in ultrapure water)

[0390] g. 2 mg / mL chymotrypsin solution, freshly prepared using cold reagent e and chymotrypsin

[0391] h. 1 mg / mL chymotrypsin inhibitor solution (TCI): Prepare fresh before use using cold reagent f and chymotrypsin inhibitor.

[0392] Part A:

[0393] According to Table 4, pipette (in mL) the following reagents into 6 centrifuge tubes and mix thoroughly by inversion.

[0394] Table 4

[0395] Part B:

[0396] Take 7 centrifuge tubes, pipette (in mL) the following reagents according to Table 5, and mix thoroughly by inversion.

[0397] Table 5

[0398] Mix by inversion and equilibrate to 25°C. Monitor the A256nm using a UV-Vis spectrophotometer until constant. Add the reagents in Table 6 to Table 5, i.e., pipette 0.10 mL of the non-inhibited group from Part A of Table 6 to the non-inhibited group from Table 5, pipette 0.10 mL of Sample 1 from Part A of Table 6 to Sample 1 from Table 5, pipette 0.10 mL of Sample 2 from Part A of Table 6 to Sample 2 from Table 5, pipette 0.10 mL of Sample 3 from Part A of Table 6 to Sample 3 from Table 5, pipette 0.10 mL of Sample 4 from Part A of Table 6 to Sample 4 from Table 5, and pipette 0.10 mL of Sample 5 from Part A of Table 6 to Sample 5 from Table 5. Immediately invert to mix and record the 256nm increment over approximately 5 minutes. Using maximum linear velocity, obtain the ΔA256nm / min value for each of the sample, blank, and non-inhibited solutions.

[0399] Table 6

[0400] calculate 3 = Total volume of Part B (mL) 0.5 = Volume of Chymotrypsin solution in Part A (mL) df = Dilution factor

[0401] 0.964 = molar extinction coefficient of BAEE at 256 nm;

[0402] 0.1 = Volume of chymotrypsin solution from Part A removed (mL) and used in Part B 10 = Total volume of Part A (mL)

[0403] Plot the relationship between chymotrypsin activity (BTEE units / mL) and trypsin volume (mL). Determine the intersection of the line and the abscissa to obtain the x-intercept.

[0404] Y0 = mg of chymotrypsin inhibitor per mL of chymotrypsin inhibitor solution that results in complete inhibition of chymotrypsin activity in part B of the experiment:

[0405] CT = mg of chymotrypsin per ml of chymotrypsin solution in part B of the experiment

[0406] mg of chymotrypsin inhibited by trypsin inhibitor

[0407] For example, the TI activity of KTI is 1.4 mg / mg, which means that each mg of KTI can inhibit 1.4 mg of trypsin, which has a trypsin activity of 10,000 BAEE units / mg.

[0408] The CI activity of BBI is 1.5 mg / mg, which means that each mg of BBI can inhibit 1.5 mg of chymotrypsin, which has 40 BTEE units / mg of chymotrypsin activity.

[0409] The BBI prepared in Example 1 was tested, and the results are shown in Table 7 below:

[0410] Table 7

[0411] The KTI prepared in Example 1 was tested, and the results are shown in Table 8 below:

[0412] Table 8

[0413] In the following examples, unless otherwise specified, all reagents and consumables used were purchased from conventional reagent manufacturers in the field; unless otherwise specified, all experimental methods and technical means used were conventional methods and means in the field.

[0414] For example, recombinant human insulin can be purchased from Hefei Tianmai Biotechnology Development Co., Ltd., fish oil can be purchased that meets the quality standards of omega-3 fatty acid triglycerides (English name Omega-3-acid triglycerides) in the European Pharmacopoeia EP10.8, disodium ethylenediaminetetraacetic acid can be purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and Tween 80 can be purchased from Sigma-Aldrich.

[0415] Example 3 Preparation of Oral Peptide Composition

[0416] This embodiment provides an oral polypeptide composition, wherein the mass ratio of recombinant human insulin, fish oil, soybean trypsin inhibitor, disodium EDTA and Tween 80 contained in the oral polypeptide composition is 1:40:20:15:40, wherein the mass of the recombinant human insulin is 8 mg.

[0417] Preparation method: 8 mg of recombinant human insulin, 320 mg of fish oil, 160 mg of soybean trypsin inhibitor, 120 mg of disodium edetate, and 320 mg of Tween 80 are weighed separately; 160 mg of soybean trypsin inhibitor is prepared by mixing 26.7 mg of BBI and 133.3 mg of KTI prepared in Example 1. The recombinant human insulin, BBI, KTI, and disodium edetate are then mixed, and the fish oil and Tween 80 are added and mixed thoroughly. The mixture is then placed into a gelatin capsule and coated with 40 mg of Eudragit L-100, 30 mg of talc, and 5 mg of polyethylene glycol 6000. The coating material is dissolved in appropriate amounts of dichloromethane and isopropyl alcohol to obtain a coating solution, which is sprayed onto the capsule surface to obtain an oral polypeptide composition.

[0418] The oral polypeptide composition prepared in this example contains capsules containing recombinant human insulin, fish oil, BBI, KTI, disodium EDTA, and Tween 80. The capsule contents are an anhydrous suspension. "Anhydrous" means that no water was intentionally added to the capsule contents during the preparation of the oral polypeptide composition in this example, but this does not preclude the absorption of water from the air by the various components in the capsule contents.

[0419] Calculations show that the oral polypeptide composition obtained in this example comprises 0.9% by mass of recombinant human insulin, 34.5% by mass of fish oil, 17.2% by mass of soybean trypsin inhibitor, 12.9% by mass of disodium edetate, and 34.5% by mass of Tween 80. Among these, the mass fraction of BBI is 2.9% and the mass fraction of KTI is 14.3%.

[0420] The weight ratio of BBI to KTI in each capsule is 0.2:1. The trypsin inhibitory activity of KTI used in the capsule is 1.4 mg / mg. The trypsin inhibitory activity of BBI used in the capsule is 0.9 mg / mg, and the chymotrypsin inhibitory activity of BBI is 1.5 mg / mg. The total TI / total CI activity ratio in each capsule is 5.27:1.

[0421] Example 4 Preparation of Oral Peptide Composition

[0422] This embodiment provides an oral polypeptide composition, which contains recombinant human insulin, fish oil, soybean trypsin inhibitor, disodium edetate and Tween 80, wherein the mass of the recombinant human insulin is 16 mg.

[0423] Preparation method: 16 mg of recombinant human insulin, 105 mg of fish oil, 200 mg of soybean trypsin inhibitor, 320 mg of disodium EDTA, and 245 mg of Tween 80 are weighed separately; 200 mg of soybean trypsin inhibitor is prepared by mixing 88.9 mg of BBI and 111.1 mg of KTI prepared in Example 1. The recombinant human insulin, BBI, KTI, and disodium EDTA prepared above are then mixed, and the fish oil and Tween 80 are added and mixed thoroughly. The mixture is then placed into a gelatin capsule and coated with 40 mg of Eudragit L-100, 30 mg of talc, and 5 mg of polyethylene glycol 6000. The coating material is dissolved in appropriate amounts of dichloromethane and isopropyl alcohol to obtain a coating solution, which is sprayed onto the capsule surface to obtain an oral polypeptide composition.

[0424] The oral polypeptide composition prepared in this example contains capsules containing recombinant human insulin, fish oil, BBI, KTI, disodium EDTA, and Tween 80. The capsule contents are an anhydrous suspension. "Anhydrous" means that no water was intentionally added to the capsule contents during the preparation of the oral polypeptide composition in this example, but this does not preclude the absorption of water from the air by the various components in the capsule contents.

[0425] Calculations show that the oral polypeptide composition obtained in this example contains 1.8% recombinant human insulin by mass, 11.9% fish oil by mass, 22.6% soybean trypsin inhibitor by mass, 36.1% disodium EDTA by mass, and 27.6% Tween 80 by mass. Among these, the mass fraction of BBI is 10.0% and the mass fraction of KTI is 12.6%.

[0426] The weight ratio of BBI to KTI in each capsule is 0.8:1. The trypsin inhibitory activity of KTI used in the capsule is 1.4 mg / mg. The trypsin inhibitory activity of BBI used in the capsule is 0.9 mg / mg, and the chymotrypsin inhibitory activity of BBI is 1.5 mg / mg. The total TI / total CI activity ratio in each capsule is 1.77:1.

[0427] Example 5 Preparation of Oral Peptide Composition

[0428] This embodiment provides an oral polypeptide composition, which contains recombinant human insulin, fish oil, soybean trypsin inhibitor, disodium edetate and Tween 80, wherein the mass of the recombinant human insulin is 32 mg.

[0429] Preparation method: 32 mg of recombinant human insulin, 245 mg of fish oil, 70 mg of soybean trypsin inhibitor, 250 mg of disodium EDTA, and 105 mg of Tween 80 are weighed separately; the 70 mg of soybean trypsin inhibitor is prepared by mixing 52.5 mg of BBI and 17.5 mg of KTI prepared in Example 1. The recombinant human insulin, BBI, KTI, and disodium EDTA are then mixed, and the fish oil and Tween 80 are added. The mixture is mixed thoroughly and placed into a gelatin capsule. The capsule is then coated with 40 mg of Eudragit L-100, 30 mg of talc, and 5 mg of polyethylene glycol 6000. The coating material is dissolved in appropriate amounts of dichloromethane and isopropyl alcohol to obtain a coating solution, which is sprayed onto the capsule surface to obtain an oral polypeptide composition.

[0430] The oral polypeptide composition prepared in this example contains capsules containing recombinant human insulin, fish oil, BBI, KTI, disodium EDTA, and Tween 80. The capsule contents are an anhydrous suspension. "Anhydrous" means that no water was intentionally added to the capsule contents during the preparation of the oral polypeptide composition in this example, but this does not preclude the absorption of water from the air by the various components in the capsule contents.

[0431] Calculations show that the oral polypeptide composition obtained in this example comprises 4.6% by mass of recombinant human insulin, 34.9% by mass of fish oil, 10.0% by mass of soybean trypsin inhibitor, 35.6% by mass of disodium edetate, and 14.9% by mass of Tween 80. Among these, the mass fraction of BBI is 7.5% and the mass fraction of KTI is 2.5%.

[0432] The weight ratio of BBI to KTI in each capsule is 3:1. The trypsin inhibitory activity of KTI used in the capsule is 1.4 mg / mg. The trypsin inhibitory activity of BBI used in the capsule is 0.9 mg / mg, and the chymotrypsin inhibitory activity of BBI is 1.5 mg / mg. The total TI / total CI activity ratio in each capsule is 0.91:1.

[0433] Example 6 Preparation of Oral Peptide Composition

[0434] This embodiment provides an oral polypeptide composition, which contains recombinant human insulin, fish oil, soybean trypsin inhibitor, disodium edetate and Tween 80, wherein the mass of the recombinant human insulin is 8 mg.

[0435] Preparation method: 8 mg of recombinant human insulin, 50 mg of fish oil, 100 mg of soybean trypsin inhibitor, 300 mg of disodium edetate, and 450 mg of Tween 80 are weighed separately; 100 mg of soybean trypsin inhibitor is prepared by mixing 37.5 mg of BBI and 62.5 mg of KTI prepared in Example 1. The recombinant human insulin, BBI, KTI, and disodium edetate weighed above are then mixed, and the fish oil and Tween 80 weighed above are added and mixed evenly. The mixture is then placed into a gelatin capsule and coated with 40 mg of Eudragit L-100, 30 mg of talc, and 5 mg of polyethylene glycol 6000. The coating material is dissolved in appropriate amounts of dichloromethane and isopropyl alcohol to obtain a coating solution, which is sprayed onto the capsule surface to obtain an oral polypeptide composition.

[0436] The oral polypeptide composition prepared in this example contains capsules containing recombinant human insulin, fish oil, BBI, KTI, disodium EDTA, and Tween 80. The capsule contents are an anhydrous suspension. "Anhydrous" means that no water was intentionally added to the capsule contents during the preparation of the oral polypeptide composition in this example, but this does not preclude the absorption of water from the air by the various components in the capsule contents.

[0437] Calculations show that the oral polypeptide composition obtained in this example comprises 0.9% by mass of recombinant human insulin, 5.5% by mass of fish oil, 11.0% by mass of soybean trypsin inhibitor, 33.0% by mass of disodium EDTA, and 49.6% by mass of Tween 80. Among these, the mass fraction of BBI is 4.0% and the mass fraction of KTI is 7.0%.

[0438] The weight ratio of BBI to KTI in each capsule is 0.6:1. The trypsin inhibitory activity of KTI used in the capsule is 1.4 mg / mg. The trypsin inhibitory activity of BBI used in the capsule is 0.9 mg / mg, and the chymotrypsin inhibitory activity of BBI is 1.5 mg / mg. The total TI / total CI activity ratio in each capsule is 2.16:1.

[0439] Example 7-Comparative Example

[0440] This embodiment provides an oral polypeptide composition, which contains recombinant human insulin, soybean trypsin inhibitor, disodium edetate and Tween 80, wherein the mass of the recombinant human insulin is 16 mg.

[0441] Preparation method: 16 mg of recombinant human insulin, 200 mg of soybean trypsin inhibitor, 320 mg of EDTA disodium, and 350 mg of Tween 80 are weighed separately; 200 mg of soybean trypsin inhibitor is prepared by mixing 88.9 mg of BBI and 111.1 mg of KTI prepared in Example 1. The recombinant human insulin, BBI, KTI, and EDTA disodium are then mixed, and Tween 80 is added and mixed thoroughly. The mixture is then placed into a gelatin capsule and coated with 40 mg of Eudragit L-100, 30 mg of talc, and 5 mg of polyethylene glycol 6000. The coating material is dissolved in appropriate amounts of dichloromethane and isopropyl alcohol to obtain a coating solution, which is sprayed onto the capsule surface to obtain an oral polypeptide composition.

[0442] Example 8 - Comparative Example

[0443] This embodiment provides an oral polypeptide composition, which contains recombinant human insulin, fish oil, soybean trypsin inhibitor and disodium edetate, wherein the mass of the recombinant human insulin is 16 mg.

[0444] Preparation method: 16 mg of recombinant human insulin, 350 mg of fish oil, 200 mg of soybean trypsin inhibitor, and 320 mg of EDTA disodium are weighed separately; 200 mg of soybean trypsin inhibitor is prepared by mixing 88.9 mg of BBI and 111.1 mg of KTI prepared in Example 1. The recombinant human insulin, BBI, KTI, and EDTA disodium are then mixed, and the fish oil is added and mixed thoroughly. The mixture is then placed into a gelatin capsule and coated with 40 mg of Eudragit L-100, 30 mg of talc, and 5 mg of polyethylene glycol 6000. The coating material is dissolved in appropriate amounts of dichloromethane and isopropyl alcohol to obtain a coating solution, which is sprayed onto the capsule surface to obtain an oral polypeptide composition.

[0445] Example 9 Stability Test

[0446] HPLC was used to detect the content of recombinant human insulin in the oral polypeptide compositions of Example 3, Example 4, Example 5 and Example 6 at 25°C on day 0, day 5, day 10, day 30 and day 60, respectively. Three samples of each oral polypeptide composition were tested at each time point. The results are shown in Table 9. The content in Table 9 is the average of the three samples at each time point.

[0447] Table 9. Stability data of recombinant human insulin at 25℃

[0448] "Recombinant human insulin content" refers to the percentage of the actual content of recombinant human insulin to the labeled amount. For example, the labeled amount of recombinant human insulin in an oral polypeptide composition is 8 mg. After standing, three samples are taken and tested by HPLC. The actual amount of recombinant human insulin (average value) is calculated to be 4.8 mg. The content of recombinant human insulin in the oral polypeptide composition is

[0449] HPLC conditions for the content of recombinant human insulin:

[0450] Chromatographic column: octadecylsilane bonded silica gel;

[0451] Mobile phase A: 600 mL of 0.1 M sodium sulfate buffer (pH 4.8), 100 mL of acetonitrile, and 300 mL of water;

[0452] Mobile phase B: 100 mL of 0.1 M sodium sulfate buffer (pH 4.8), 600 mL of acetonitrile, and 300 mL of water;

[0453] Flow rate: 1.0 ml / min; column temperature 35°C; detection wavelength UV 214 nm; injection volume 20 μL.

[0454] The gradient elution table is as follows:

[0455] Table 10 Gradient elution table

[0456] As shown in Table 9, when the oral polypeptide composition is stored at 25° C. for 30 days, the content of recombinant human insulin is greater than 85%; when stored for 60 days, the content of recombinant human insulin is greater than 62%.

[0457] HPLC was used to detect the content of recombinant human insulin in the oral polypeptide compositions of Example 3, Example 4, Example 5 and Example 6 at 40°C on day 0, day 5 and day 10, respectively. Three samples of each oral polypeptide composition were tested at each time point. The results are shown in Table 11. The content in Table 11 is the average of the three samples at each time point.

[0458] Table 11. Stability data of recombinant human insulin at 40℃

[0459] As shown in Table 11, when the oral polypeptide composition is stored at 40° C. for 5 days, the content of recombinant human insulin is greater than 85%; when stored for 10 days, the content of recombinant human insulin is greater than 76%.

[0460] Example 10

[0461] Dissolution test

[0462] According to the dissolution test method specified in the second method of Part 4 of the Chinese Pharmacopoeia 2020, a pH 6.8 phosphate buffer solution was used as the dissolution medium. The contents of the oral polypeptide compositions prepared in Examples 3, 4, 5, 6, 7, and 8 were poured into different dissolution cups and dissolved in the above dissolution medium at a speed of 50 revolutions per minute and a constant temperature of 37°C ± 0.5°C. The dissolution test was performed. Samples were taken at 0 min, 2 min, 5 min, 10 min, 20 min, 30 min, 45 min, and 60 min, and the recombinant human insulin content was measured by HPLC. The cumulative dissolution data are shown in Table 12.

[0463] Table 12 Cumulative dissolution data of recombinant human insulin

[0464] HPLC conditions for the content of recombinant human insulin:

[0465] Chromatographic column: octadecylsilane bonded silica gel;

[0466] Mobile phase A: 600 mL of 0.1 M sodium sulfate buffer (pH 4.8), 100 mL of acetonitrile, and 300 mL of water;

[0467] Mobile phase B: 100 mL of 0.1 M sodium sulfate buffer (pH 4.8), 600 mL of acetonitrile, and 300 mL of water;

[0468] Flow rate: 1.0 ml / min; column temperature 35°C; detection wavelength UV 214 nm; injection volume 20 μL.

[0469] The gradient elution table is as follows:

[0470] Table 13 Gradient elution table

[0471] From Table 12, we can see that

[0472] The oral polypeptide composition of Example 6, recombinant human insulin, has a cumulative dissolution rate of more than 86% in 30 minutes and is substantially completely dissolved in 45 minutes.

[0473] The oral polypeptide composition of Example 4, recombinant human insulin, was substantially completely dissolved within 20 minutes.

[0474] The oral polypeptide compositions of Example 3 and Example 5, and the recombinant human insulin, had a cumulative dissolution rate of greater than 86% in 2 minutes, and were substantially completely dissolved in 5 minutes.

[0475] Dissolution test

[0476] According to the dissolution test method specified in the second method of Part 0931 of the General Rules of the Fourth Volume of the 2020 Chinese Pharmacopoeia, using pH 6.8 phosphate buffer as the dissolution medium, the contents of the oral polypeptide compositions prepared in Examples 3, 4, 5, and 6 were poured into different dissolution cups and dissolved in the above dissolution medium at a speed of 50 rpm and a constant temperature of 37°C ± 0.5°C. Dissolution tests were performed. Samples were taken at 0 min, 2 min, 5 min, 10 min, 20 min, 30 min, 45 min, and 60 min, and the soybean trypsin inhibitor content was measured by HPLC. Cumulative dissolution data are shown in Table 14.

[0477] Table 14 Cumulative dissolution data of soybean trypsin inhibitor

[0478] HPLC conditions for soybean trypsin inhibitor content:

[0479] Mobile phase A: water-acetonitrile-trifluoroacetic acid (95 ml: 5 ml: 0.05 ml);

[0480] Mobile phase B: water-acetonitrile-trifluoroacetic acid (10 ml: 90 ml: 0.05 ml);

[0481] Column: C18;

[0482] Flow rate: 1 mL / min;

[0483] Column temperature: 40°C;

[0484] Detector: UV 214 nm;

[0485] Injection volume: 20 μL;

[0486] The gradient elution table is as follows:

[0487] Table 15 Gradient elution table

[0488] As can be seen from Table 14, the soybean trypsin inhibitor in the oral polypeptide compositions of Examples 3 and 5 dissolved faster than that in the oral polypeptide compositions of Examples 4 and 6. In the oral polypeptide composition of Example 3, the soybean trypsin inhibitor was substantially completely dissolved within 5 minutes. The soybean trypsin inhibitor in the oral polypeptide composition of Example 4 dissolved faster than that in the oral polypeptide composition of Example 6.

[0489] Example 11

[0490] Animal testing

[0491] The oral polypeptide compositions prepared in Examples 3-6 were subjected to animal testing.

[0492] (1) Experimental animals

[0493] Beagle, male, 8-10kg.

[0494] (2) Animal grouping and drug administration design

[0495] Group design: The experiment set up 4 groups, namely low-dose group 1, medium-dose group, high-dose group, and low-dose group 2;

[0496] Number of animals: 8 males in each dosing group;

[0497] Experimental methods: Two rounds of experiments were conducted, with four groups of four mice in each round; the washout period was set at 48 hours;

[0498] Grouping method: Randomly grouped according to body weight. Specific grouping information is shown in Table 16.

[0499] Table 16 Dosage and group information

[0500] Note: [1] The first digit of the animal number represents the group, the second letter represents the sex (M for male), and the third, fourth, and fifth digits represent the individual animal number;

[0501] [2] After the first round of the trial, the second round of the trial was conducted after 48 hours of washout and random grouping;

[0502] [3] Do not eat for more than 12 hours before administration.

[0503] (3) Method of administration

[0504] Low-dose group 1, medium-dose group, high-dose group, and low-dose group 2

[0505] Route of administration: Oral;

[0506] Dosing frequency: 1 dose;

[0507] Dosage quantity: 1 tablet;

[0508] (4) Detection method

[0509] Blood was collected from the forelimb vein using vacuum tubes (anticoagulated with heparin) at 0 (pre-dose), 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, and 8.0 hours, with approximately 2.5 mL of blood collected each time. After whole blood collection, cool it and centrifuge it at 4°C (3000 rpm for 15 minutes) within 2 hours to separate plasma. All samples were stored at -70°C until assayed. Human insulin concentrations in plasma were determined by LC / MS / MS.

[0510] Table 17 LC / MS / MS detection conditions

[0511] in conclusion:

[0512] The calculation results of the absolute bioavailability F of the oral administration group are shown in Table 18 below:

[0513] Table 18. Absolute bioavailability F of oral administration

[0514] As can be seen from Table 18, the absolute bioavailability of low-dose group 1 was 1.54% and the SD (standard deviation) was 0.43.

[0515] For comparison, when the same experimental scheme was used, the soybean trypsin inhibitor in the oral polypeptide composition was replaced with a soybean trypsin inhibitor directly extracted from soybeans on the market (i.e., BBI and KTI were not obtained separately from soybeans and then BBI and KTI were mixed to obtain the soybean trypsin inhibitor), and the rest remained unchanged, the F of each comparative example is shown in Table 19 below.

[0516] Table 19. Absolute bioavailability F of comparative examples

[0517] As shown in Table 19, the absolute bioavailability of the comparative low-dose group 1 was 1.68%, and the SD (standard deviation) was 0.84.

[0518] The applicant declares that the above description is merely a preferred embodiment of the present invention and is intended only to illustrate the technical solution of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are included within the scope of protection of the present invention.

Claims

1. A polypeptide composition, wherein the polypeptide composition comprises a polypeptide, isolated BBI and isolated KTI, fish oil and at least one absorption enhancer; wherein the isolated BBI and the isolated KTI are mixed in a certain ratio such that the activity ratio of the total trypsin inhibitory activity (total TI) to the total chymotrypsin inhibitory activity (total CI) in the composition is 0.5:1 to 10:1; preferably 0.7:1 to 10:1; preferably 0.91:1 - 6:1; preferably 0.91:1 to 5.27:

1.

2. The polypeptide composition according to claim 1, wherein the isolated BBI has been purified to a purity of at least 85% as measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); preferably, the BBI has been purified to a purity of at least 90%; more preferably, the BBI has been purified to a purity of at least 91%.

3. The polypeptide composition according to claim 1 or 2, wherein By the BCA (bicinchoninic acid) assay, the isolated BBI has been purified to a protein content of at least 85%; preferably, the BBI has been purified to a protein content of at least 90%; more preferably, the BBI has been purified to a protein content of at least 92%.

4. The polypeptide composition according to any one of claims 1 - 3, wherein the isolated BBI contains less than 0.1% high molecular weight impurities.

5. The polypeptide composition according to any one of claims 1 - 4, wherein the isolated BBI has a trypsin inhibitory activity of at least 0.5, 0.6, 0.7, 0.8, 0.9 mg / mg or 1.8 mg / mg; and the isolated BBI has a chymotrypsin inhibitory activity of at least 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 mg / mg or 1.8 mg / mg.

6. The polypeptide composition according to any one of claims 1-5, wherein, As measured by SDS-PAGE, the isolated KTI has been purified to a purity of at least 85%; preferably, the isolated KTI has been purified to a purity of at least 90%; more preferably, the isolated KTI has been purified to a purity of at least 95%.

7. The polypeptide composition according to any one of claims 1-6, wherein, As measured by the BCA assay, the isolated KTI has been purified to a protein content greater than 85%; preferably, the isolated KTI has been purified to a protein content greater than 90%.

8. The polypeptide composition according to any one of claims 1 - 7, wherein the isolated KTI contains less than 0.1% high molecular weight impurities.

9. The polypeptide composition according to any one of claims 1 - 8, wherein the isolated KTI has a trypsin inhibitory activity of at least 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 mg / mg or 1.8 mg / mg.

10. The polypeptide composition according to any one of claims 1-9, wherein the polypeptide composition has a chymotrypsin inhibitory activity of inhibiting 0.04-0.30 mg of chymotrypsin / mg of polypeptide composition, and the chymotrypsin has a chymotrypsin activity of 40 BTEE units / mg of chymotrypsin; preferably, wherein the polypeptide composition has a chymotrypsin inhibitory activity of inhibiting 0.04-0.15 mg of chymotrypsin / mg of polypeptide composition, and the chymotrypsin has a chymotrypsin activity of 40 BTEE units / mg of chymotrypsin.

11. The polypeptide composition according to any one of claims 1-10, wherein the polypeptide composition has a trypsin inhibitory activity of inhibiting 0.05-0.40 mg of trypsin / mg of polypeptide composition, and the trypsin has a trypsin activity of 10,000 BAEE units / mg of trypsin; preferably, wherein the polypeptide composition has a trypsin inhibitory activity of inhibiting at least 0.10-0.30 mg of trypsin / mg of polypeptide composition, and the trypsin has a trypsin activity of 10,000 BAEE units / mg of trypsin.

12. The polypeptide composition according to any one of claims 1-11, wherein the molecular weight of the polypeptide is 100 Da to 20,000 Da, preferably 1000 Da to 20,000 Da, preferably 1000 Da to 6150 Da, preferably 1209 Da to 6104 Da, preferably 1000 Da to 6000 Da, and more preferably 2000 Da to 6000 Da.

13. The polypeptide composition according to any one of claims 1-12, wherein the polypeptide is selected from any one or more of insulin, glucagon, glucagon-like peptide-1 receptor agonist, glucagon-like peptide-2 receptor agonist, calcitonin, growth hormone, somatostatin, thyrotropin-releasing hormone, parathyroid hormone, gonadotropin-releasing hormone, heparin, granulocyte colony-stimulating factor, prostaglandin, cyclosporine, interferon, vasopressin, vancomycin, erythropoietin, glutathione, and thymosin; Preferably, the polypeptide is selected from any one or more of recombinant human insulin, degludec insulin, glucagon, exenatide, beloran peptide, liraglutide, lixisenatide, dulaglutide, lisinopril peptide, semaglutide, albiglutide, dulaglutide, tildrakizumab, calcitonin, salmon calcitonin, growth hormone, octreotide, thyrotropin-releasing hormone, parathyroid hormone fragment, teriparatide, leuprorelin, protirelin, gonadorelin, goserelin, buserelin, sermorelin, nafarelin, histrelin, triptorelin, temolerein, coredorelin, lanreotide acetate, terlipressin, angiotensin peptide, teriparatide, abaloparatide, nesiritide, eptifibatide, lisinopril, cefovecin, atosiban, polysaccharide peptide, ginseng glycopeptide, osteopeptide, sarcosine peptide glycoside, glutathione, mannan peptide, enfuvirtide, bradykinin, enkephalin, nisin, hirudin, glatiramer acetate, aprotinin, alarelin, trypsinogen enzyme peptide, polymyxin, serrapeptase, thymosin α1, thymopentin, and thymalfasin.

14. The polypeptide composition according to any one of claims 1 to 13, wherein the absorption enhancer is selected from any one or more of ethylenediaminetetraacetic acid or its salts, N-(8-(2-hydroxybenzoyl)amino)octanoic acid or its salts, salicylic acid or its salts, citric acid or its salts, cholic acid or its salts, deoxycholic acid or its salts, glycocholic acid or its salts, taurocholic acid or its salts, alkyl glycoside, sodium dodecyl sulfate, sodium docusate, cyclodextrin or its derivatives, chitosan or its derivatives, octanoic acid or its salts, decanoic acid or its salts, lauric acid or its salts, myristic acid, palmitic acid, stearic acid, fatty acid triglyceride, lecithin, choline, and carnitine. Preferably, the ethylenediaminetetraacetic acid salt is selected from disodium ethylenediaminetetraacetate, dipotassium ethylenediaminetetraacetate, trisodium ethylenediaminetetraacetate, or tetrasodium ethylenediaminetetraacetate.

15. The polypeptide composition according to any one of claims 1-14, wherein the polypeptide composition further comprises at least one emulsifier.

16. The polypeptide composition according to claim 15, wherein the emulsifier is selected from any one or more of ionic surfactants, nonionic surfactants, and zwitterionic surfactants; Preferably, the emulsifier is selected from ammonium lauryl sulfate, N-dodecyl-β-D-maltoside, tridecyl-β-D-maltoside, sodium lauryl sulfate, sodium dioctyl sulfosuccinate, poloxamer, glycerol monostearate, glycerol monolaurate, sorbitan monolaurate, sorbitan monostearate, polyoxyethylene sorbitan fatty acid ester, sorbitan fatty acid ester, polyoxyethylene monopalmitate, polyoxyethylene hydrogenated castor oil, C6-C 12 triglyceride fatty acid and C8-C 10 any one or more of triglyceride fatty acids; Preferably, the at least one surfactant is selected from Tween. Preferably, the at least one surfactant is selected from any one or more of Tween 20, Tween 40, Tween 60, Tween 65, Tween 80, and Tween 85.

17. The polypeptide composition according to any one of claims 1 to 16, wherein the polypeptide composition is calculated by mass fraction Comprising: Polypeptide: 0.5-10%, Fish oil: 3.0-50.0%, Isolated BBI: 1.0-25.0%, Isolated KTI: 1.0-20.0%, Absorption enhancer: 5.0-45.0%, and Emulsifier: 8.0-60.0%; Or Polypeptide: 0.9-4.6%, Fish oil: 5.5-34.9%, Isolated BBI: 2.9-10.0%, Isolated KTI: 2.5-14.3%, Absorption enhancer: 12.9-36.1%, and Emulsifier: 14.9-49.6%; Or Polypeptide: 0.9-4.6%, Fish oil: 11.9-34.9%, Isolated BBI: 2.9 - 10.0%, Isolated KTI: 2.5 - 14.3%, Absorption enhancer: 12.9 - 36.1%, and Emulsifier: 14.9 - 34.5%; Or Polypeptide: 0.9 - 4.6%, Fish oil: 34.5 - 34.9%, Isolated BBI: 2.9 - 7.5%, Isolated KTI: 2.5 - 14.3%, Absorption enhancer: 12.9 - 35.6%, and Emulsifier: 14.9 - 34.5%; Or Polypeptide: 0.9 - 1.8%, Fish oil: 11.9 - 34.5%, Isolated BBI: 2.9 - 10.0%, Isolated KTI: 12.6 - 14.3%, Absorption enhancer: 12.9 - 36.1%, and Emulsifier: 27.6 - 34.5%; Or Polypeptide: 1.8 - 4.6%, Fish oil: 11.9 - 34.9%, Isolated BBI: 7.5 - 10.0%, Isolated KTI: 2.5 - 12.6%, Absorption enhancer: 35.6 - 36.1%, and Emulsifier: 14.9 - 27.6%.

18. The polypeptide composition according to any one of claims 1 to 17, wherein the polypeptide composition comprises, by mass fraction: Recombinant human insulin: 0.5 - 10%, Fish oil: 3.0 - 50.0%, Isolated BBI: 1.0 - 25.0%, Isolated KTI: 1.0 - 20.0%, Ethylenediaminetetraacetic acid or its salt: 5.0 - 45.0%, and Tween: 8.0 - 60.0%; Or Recombinant human insulin: 0.9 - 4.6%, Fish oil: 5.5 - 34.9%, Isolated BBI: 2.9 - 10.0%, Isolated KTI: 2.5 - 14.3%, Ethylenediaminetetraacetic acid or its salt: 12.9 - 36.1%, and Tween: 14.9 - 49.6%; Or Recombinant human insulin: 0.9 - 4.6%, Fish oil: 11.9 - 34.9%, Isolated BBI: 2.9 - 10.0%, Isolated KTI: 2.5 - 14.3%, Ethylenediaminetetraacetic acid or its salt: 12.9 - 36.1%, and Tween: 14.9 - 34.5%; Or Recombinant human insulin: 0.9 - 4.6%, Fish oil: 34.5 - 34.9%, Isolated BBI: 2.9 - 7.5%, Isolated KTI: 2.5 - 14.3%, Ethylenediaminetetraacetic acid or its salt: 12.9 - 35.6%, and Tween: 14.9 - 34.5%; Or Recombinant human insulin: 0.9 - 1.8%, Fish oil: 11.9 - 34.5%, Isolated BBI: 2.9 - 10.0%, Isolated KTI: 12.6 - 14.3%, Ethylenediaminetetraacetic acid or its salt: 12.9 - 36.1%, and Tween: 27.6 - 34.5%; Or Recombinant human insulin: 1.8 - 4.6%, Fish oil: 11.9 - 34.9%, Isolated BBI: 7.5 - 10.0%, Isolated KTI: 2.5 - 12.6%, Ethylenediaminetetraacetic acid or its salt: 35.6 - 36.1%, and Tween: 14.9 - 27.6%.

19. The polypeptide composition according to any one of claims 1 to 18, wherein the polypeptide composition in a unit dosage form comprises: Recombinant human insulin: 8 - 32 mg, Fish oil: 50 - 320 mg, Isolated BBI: 26.7 - 88.9 mg, Isolated KTI: 17.5 - 133.3 mg, Ethylenediaminetetraacetic acid or its salt: 120 - 320 mg, and Tween: 105 - 450 mg; Or Recombinant human insulin: 8 - 32 mg, Fish oil: 105 - 320 mg, Isolated BBI: 26.7 - 88.9 mg, Isolated KTI: 17.5 - 133.3 mg, Ethylenediaminetetraacetic acid or its salt: 120 - 320 mg, and Tween: 105 - 320 mg; Or Recombinant human insulin: 8 - 32 mg, Fish oil: 245 - 320 mg, Isolated BBI: 26.7 - 52.5 mg, Isolated KTI: 17.5 - 133.5 mg, Ethylenediaminetetraacetic acid or its salt: 120 - 250 mg, and Tween: 105 - 320 mg; Or Recombinant human insulin: 8 - 16 mg, Fish oil: 105 - 320 mg, Isolated BBI: 26.7 - 88.9 mg, Isolated KTI: 111.1 - 133.3 mg, Ethylenediaminetetraacetic acid or its salt: 120 - 320 mg, and Tween: 245 - 320 mg; Or Recombinant human insulin: 8 mg, Fish oil: 50 - 320 mg, Isolated BBI: 26.7 - 37.5 mg, Isolated KTI: 62.5 - 133.3 mg, Ethylenediaminetetraacetic acid or its salt: 120 - 300 mg, and Tween: 320 - 450 mg; Or Recombinant human insulin: 16 - 32 mg, Fish oil: 105 - 245 mg, Isolated BBI: 52.5 - 88.9 mg, Isolated KTI: 17.5 - 111.1 mg, Ethylenediaminetetraacetic acid or its salt: 250 - 320 mg, and Tween: 105 - 245 mg; Or Recombinant human insulin: 8 - 16 mg, Fish oil: 50 - 105 mg, Isolated BBI: 37.5 - 88.9 mg, Isolated KTI: 62.5 - 111.1 mg, Ethylenediaminetetraacetic acid or its salt: 300 - 320 mg, and Tween: 245 - 450 mg.

20. The polypeptide composition according to any one of claims 1 to 19, wherein the polypeptide composition in a unit dosage form comprises: Recombinant human insulin: 8 mg, Fish oil: 320 mg, Isolated BBI: 26.7 mg, Isolated KTI: 133.3 mg, Ethylenediaminetetraacetic acid or its salt: 120 mg, and Tween: 320 mg; Or Recombinant human insulin: 16 mg, Fish oil: 105 mg, Isolated BBI: 88.9 mg, Isolated KTI: 111.1 mg, Ethylenediaminetetraacetic acid or its salt: 320 mg, and Tween: 245 mg; Or Recombinant human insulin: 32 mg, Fish oil: 245 mg, Isolated BBI: 52.5 mg, Isolated KTI: 17.5 mg, Ethylenediaminetetraacetic acid or its salt: 250 mg, and Tween: 105 mg; or Recombinant human insulin: 8 mg, Fish oil: 50 mg, Isolated BBI: 37.5 mg, Isolated KTI: 62.5 mg, Ethylenediaminetetraacetic acid or its salt: 300 mg, and Tween: 450 mg.

21. The polypeptide composition according to any one of claims 1 to 20, wherein the isolated BBI and isolated KTI in the polypeptide composition are mixed in a certain ratio such that the activity ratio of the total trypsin inhibitory activity (total TI) to the total chymotrypsin inhibitory activity (total CI) in the composition is 0.5:1 to 10:1; preferably 0.7:1 to 10:1; preferably 0.91:1 - 6:1; preferably 0.91:1 to 5.27:1; and the polypeptide composition comprises, by mass fraction: Recombinant human insulin: 0.5 - 10%, Fish oil: 3.0 - 50.0%, Isolated BBI: 1.0 - 25.0%, Isolated KTI: 1.0 - 20.0%, Ethylenediaminetetraacetic acid or its salt: 5.0 - 45.0%, and Tween: 8.0 - 60.0%; or Recombinant human insulin: 0.9 - 4.6%, Fish oil: 5.5 - 34.9%, Isolated BBI: 2.9 - 10.0%, Isolated KTI: 2.5 - 14.3%, Ethylenediaminetetraacetic acid or its salt: 12.9 - 36.1%, and Tween: 14.9 - 49.6%; or Recombinant human insulin: 0.9 - 4.6%, Fish oil: 11.9 - 34.9%, Isolated BBI: 2.9 - 10.0%, Isolated KTI: 2.5 - 14.3%, Ethylenediaminetetraacetic acid or its salt: 12.9 - 36.1%, and Tween: 14.9 - 34.5%; or Recombinant human insulin: 0.9 - 4.6%, Fish oil: 34.5 - 34.9%, Isolated BBI: 2.9 - 7.5%, Isolated KTI: 2.5 - 14.3%, Ethylenediaminetetraacetic acid or its salt: 12.9 - 35.6%, and Tween: 14.9 - 34.5%; or Recombinant human insulin: 0.9 - 1.8%, Fish oil: 11.9 - 34.5%, Isolated BBI: 2.9 - 10.0%, Isolated KTI: 12.6 - 14.3%, Ethylenediaminetetraacetic acid or its salt: 12.9 - 36.1%, and Tween: 27.6 - 34.5%; or Recombinant human insulin: 1.8 - 4.6%, Fish oil: 11.9 - 34.9%, Isolated BBI: 7.5 - 10.0%, Isolated KTI: 2.5 - 12.6%, Ethylenediaminetetraacetic acid or its salt: 35.6 - 36.1%, and Tween: 14.9 - 27.6%.

22. The polypeptide composition according to any one of claims 1-21, wherein the mass ratio of the isolated BBI to the isolated KTI in the polypeptide composition is 0.1:1 to 5:1; preferably, the mass ratio of the isolated BBI to the isolated KTI is 0.2:1 to 3:1; optionally, the mass ratio of the isolated BBI to the isolated KTI is 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1 or any value between any two ratios.

23. The polypeptide composition according to any one of claims 16-22, wherein the mass ratio of the fish oil to Tween in the polypeptide composition is (10-70):(10-90), optionally, the mass ratio of the fish oil to Tween is (10-70):(10-70), (10-70):(10-30), 1:(0.43-9), 1:(0.43-2.33), 1:(0.43-1), 50:50 or 30:

70.

24. A drug, wherein the drug comprises the polypeptide composition according to any one of claims 1-23; Preferably, the drug further comprises a pharmaceutically acceptable excipient.

25. The drug according to claim 24, wherein the pharmaceutically acceptable excipient comprises a coating, preferably, the coating comprises a gastric-soluble coating and / or an enteric-soluble coating; Preferably, the pharmaceutically acceptable excipient comprises any one or more of hydroxypropyl cellulose, hydroxypropyl methylcellulose, acrylic resin, polyvinylpyrrolidone, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate; Preferably, the pharmaceutically acceptable excipient is selected from any one or more of excipients, disintegrants, binders, glidants, flavoring agents and lubricants; Preferably, the excipient is selected from any one or more of lactose, sorbitol, xylitol, mannitol, calcium sulfate, calcium carbonate, calcium hydrogen phosphate, microcrystalline cellulose, siliconized microcrystalline cellulose, starch, pregelatinized starch, lactose starch complex, lactose cellulose complex and mannitol starch complex; Preferably, the disintegrant is selected from any one or more of cross-linked sodium carboxymethylcellulose, cross-linked polyvinylpyrrolidone, sodium carboxymethyl starch, cross-linked sodium carboxymethyl starch and low-substituted hydroxypropyl cellulose; Preferably, the binder is selected from any one or more of starch paste, gelatin solution, sucrose solution, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose and polyvinylpyrrolidone; Preferably, the lubricant is selected from any one or more of sodium stearyl fumarate, magnesium lauryl sulfate, polyethylene glycol, magnesium dodecyl sulfate, sodium dodecyl sulfate, magnesium stearate, calcium stearate, colloidal silicon dioxide and talc; Preferably, the flavoring agent is selected from any one or more of mannitol, sorbitol, aspartame, stevioside, sucralose, aspartame, neotame, steviol, hydroxychloroquine, and essence.

26. The polypeptide composition according to any one of claims 1-23, or the drug according to claim 24 or 25, wherein the polypeptide composition or the drug exists in a dosage form selected from the following: tablets, pills, capsules, emulsions, syrups or suspensions. Preferably, the polypeptide in the capsule is in a suspended state.

27. The polypeptide composition according to any one of claims 1-23, or the drug according to claim 24 or 25, wherein the polypeptide composition or the drug is anhydrous.

28. The polypeptide composition according to any one of claims 1-23, or the drug according to claim 24 or 25, wherein the polypeptide composition or the drug is in an oral or injection dosage form.

29. Use of the polypeptide composition according to any one of claims 1-23, or the drug according to claim 24 or 25 in the preparation of an oral drug for the treatment or prevention of a disease, wherein the disease is selected from hyperglycemia, type 1 diabetes, type 2 diabetes, non-insulin-dependent diabetes, maturity-onset diabetes of the young, gestational diabetes, obesity, non-alcoholic fatty liver disease, hepatic steatosis, hepatitis, liver fibrosis, cirrhosis, liver cancer, Alzheimer's disease, Parkinson's disease, and diseases with reduced glucose tolerance.

30. A method for treating or preventing a disease, the method comprising orally administering to a subject in need a therapeutically effective amount of the polypeptide composition according to any one of claims 1-23, or the drug according to claim 24 or 25, wherein the disease is selected from hyperglycemia, type 1 diabetes, type 2 diabetes, non-insulin-dependent diabetes, maturity-onset diabetes of the young, gestational diabetes, obesity, non-alcoholic fatty liver disease, hepatic steatosis, hepatitis, liver fibrosis, cirrhosis, liver cancer, Alzheimer's disease, Parkinson's disease, and diseases with reduced glucose tolerance.

31. The method for treating or preventing a disease according to claim 30, the method comprising orally administering to a subject in need the polypeptide composition according to any one of claims 1-23; or the drug according to any one of claims 24-25 at a single dose of 8-50 mg of polypeptide, 1-3 times a day.

32. The method for treating or preventing a disease according to claim 30, the method comprising orally administering to a subject in need the polypeptide composition according to any one of claims 1-23; or the drug according to any one of claims 24-25 at a single dose of 8 mg, 24 mg or 50 mg of polypeptide, 1, 2 or 3 times a day.

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