Biologically active substances bound to a biotin moiety and compositions for oral administration containing the same

Abstract

The present invention relates to a physiologically active substance having a biotin moiety bound thereto, and to an oral composition containing the physiologically active substance. According to the present invention, the physiologically active substance having a biotin moiety bound thereto has the effect of being highly absorbable by the body when orally taken.

Description

[Technical field]

[0001] The present invention relates to a physiologically active substance bound to a biotin moiety and a composition for oral administration containing the physiologically active substance. More specifically, the present invention relates to a physiologically active substance bound to a biotin moiety, which is highly absorbable by the body when orally taken, and a composition for oral administration containing the physiologically active substance. [Background technology]

[0002] Recently, due to economic growth and accelerating progress in science and technology, diets have become more Westernized, and consumption of high-calorie and high-fat foods has increased, resulting in a rapid increase in the number of people suffering from various metabolic diseases such as diabetes and obesity.

[0003] Diabetes is a disease accompanied by various complications, and its management requires dietary restrictions that significantly reduce the quality of life of patients. Awareness of diabetes treatment and management is increasing, and there is an urgent need to develop therapeutic agents to improve or cure diabetes.

[0004] Diabetes is classified as either "Type I" diabetes, caused by an inability to produce insulin, or "Type II" diabetes, where insulin production is normal but metabolic control is impaired due to insulin resistance. Type II diabetes and obesity are risk factors for each other. Both type II diabetes and obesity are highly dangerous diseases, as they cause metabolic disease and increase the risk of atherosclerosis, the leading cause of death in diabetic patients.

[0005] Recently, glucagon derivatives have been attracting attention. Glucagon is produced by the pancreas when blood glucose levels begin to drop due to causes such as drug therapy, disease, and hormone or enzyme deficiency. Glucagon signals the liver to break down glycogen, release glucose, and raise blood glucose to normal levels. In addition to its hyperglycemic effect, glucagon has been reported to suppress appetite, activate hormone-sensitive lipase in adipocytes to promote fat breakdown, and exhibit anti-obesity effects. Glucagon-like peptide-1 (GLP-1), one of the glucagon derivatives, is a substance under development as a therapeutic agent for suppressing hyperglycemia in diabetic patients, and its functions include promoting insulin synthesis and secretion, suppressing glucagon secretion, suppressing gastric emptying, promoting glucose use, and suppressing food intake.

[0006] Exendin-4, produced from lizard venom, shares approximately 50% amino acid identity with GLP-1, and it has been reported that exendin-4 also activates the GLP-1 receptor and suppresses hyperglycemia in diabetic patients.

[0007] However, problems with peptide and protein drugs when administered orally are that they are degraded by enzymatic attack and have low intestinal membrane permeability. Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a physiologically active substance bound to a biotin moiety and having excellent absorbability in the body when orally taken, and a composition for oral administration containing the physiologically active substance. [Means for solving the problem]

[0009] In one aspect of the present invention, biologically active agents conjugated to a biotin moiety and methods for their preparation are provided.

[0010] Another aspect of the invention provides an orally administered composition comprising a biologically active agent linked to a biotin moiety.

[0011] In yet another aspect of the invention, a pharmaceutical composition is provided that includes a biologically active agent linked to a biotin moiety.

[0012] In yet another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating diabetes, the composition comprising a biologically active agent linked to a biotin moiety.

[0013] In yet another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating obesity, the composition comprising a biologically active agent linked to a biotin moiety.

[0014] In yet another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating osteoporosis, the composition comprising a biologically active agent bound to a biotin moiety.

[0015] In yet another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating fatty liver, the composition comprising a biologically active agent bound to a biotin moiety.

[0016] In yet another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating irritable bowel syndrome, the composition comprising a biologically active agent linked to a biotin moiety.

[0017] In yet another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating a neurodegenerative disease, the composition comprising a biologically active agent linked to a biotin moiety. Effect of the Invention

[0018] In accordance with one embodiment of the present invention, the bioactive agent bound to the biotin moiety may be bound to water-soluble biotin for improved bioavailability when taken orally.

[0019] Biologically active agents conjugated to a biotin moiety according to one embodiment of the present invention may protect peptides and other biologically active agents from enzymatic cleavage, ultimately facilitating the permeation of the biologically active agent across the intestinal membrane and absorption in the intestine.

[0020] In accordance with one embodiment of the present invention, a bioactive substance bound to a biotin moiety can be absorbed by active transport via the sodium-dependent multivitamin transporter by being bound to biotin (vitamin B7, a water-soluble vitamin).

[0021] According to one embodiment of the invention, the biotin moiety may be attached to a non-active region of the bioactive agent, such that the activity of the bioactive agent is not inhibited. [Brief description of the drawings]

[0022] [Figure 1] 1 is a chromatogram during purification according to embodiment 3 of the present invention. [Diagram 2] 1 shows chromatograms of the final substances of the first to third embodiments of the present invention. [Diagram 3] 1 shows MALDI-TOF mass spectra according to first to third embodiments of the present invention. [Figure 4] 1 is a graph showing blood concentrations over time for Embodiments 1 to 3 after oral administration to rats. [Diagram 5] 1 is a graph showing the change in blood glucose level after glucose was administered to each subject. [Figure 6] 1 is a graph showing the change in blood glucose level after glucose was administered to each subject. [Figure 7] 1 is a graph showing the change in blood glucose level after glucose was administered to each subject. [Figure 8] 1 is a graph showing the change in blood glucose level after glucose was administered to each subject. [Figure 9] 1 is a chromatogram during purification of embodiment 10 of the present invention. [Figure 10]1 is a chromatogram after purification of embodiment 10 of the present invention. [Figure 11] 1 is a reverse phase chromatogram of embodiment 11 of the present invention. [Figure 12] 1 shows MALDI-TOF mass spectra of embodiments 10 and 11 of the present invention. [Figure 13] 1 shows chromatograms during purification of embodiments 12 and 13 of the present invention. [Figure 14] Chromatogram after purification of embodiment 12 of the present invention. [Figure 15] Chromatogram after purification of embodiment 13 of the present invention. BEST MODE FOR CARRYING OUT THEINVENTION

[0023] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In order to enable those skilled in the art to easily practice the present invention, the following detailed description will be given of examples and embodiments of the present invention.

[0024] However, the present invention may be embodied in various different forms and is not limited to the examples and embodiments described herein. Throughout the specification of the present invention, when a certain part "comprises" a certain element, it does not exclude other elements, but means that it may further include other elements, unless otherwise specified to the contrary.

[0025] Throughout this specification, terms such as "about" and "substantially" are used as terms of degree to describe any inherent manufacturing or material tolerances, or values ​​that approximate those tolerances, and are used to prevent infringers from misappropriating the disclosure, which refers to exact or absolute values ​​that are provided to aid in the understanding of the invention. The term "step of" or "step of" as used throughout this specification does not mean "step for".

[0026] Throughout the present specification, the term "combinations thereof" as used in a Markush claim refers to a mixture or combination of at least one of the group of elements set forth in the Markush claim language, comprising at least one selected from the group of elements. Throughout the present specification, "and / or B" means "and B, or A or B."

[0027] According to one aspect of the present invention, there is provided a bioactive substance bound to a biotin moiety and a method for preparing the same. The bioactive substance bound to a biotin moiety according to one aspect of the present invention can be highly absorbed by the body when orally taken.

[0028] Generally, peptide and protein drugs are highly water-soluble, and fall into class 3 of the Biopharmaceutical Classification System (BCS), and their intestinal absorption is limited. Peptide and protein drugs are characterized by being highly hydrophilic, having a large molecular weight, being easily degraded by gastric acid with a low pH, and being attacked by enzymes such as trypsin, resulting in low intestinal absorption. Peptide and protein drugs generally have an oral bioavailability (BA) of around 0.1%, making them difficult to use as pharmaceutical compositions. To solve this problem, enteric-coated capsules are used to allow [such drugs] to pass through the stomach, but this technology has the problem of not being able to fundamentally improve the absorption of peptides and proteins.

[0029] In contrast, pharma- ceutical active agents conjugated to a biotin moiety, according to one embodiment of the invention, appear to have improved intestinal membrane permeability and improved intestinal absorption.

[0030] More specifically, the bioactive substance bound to the biotin moiety according to one embodiment of the present invention can be absorbed by active transport via the sodium-dependent multivitamin transporter by being bound to biotin (vitamin B7, a water-soluble vitamin).

[0031] As used herein, "unsubstituted or substituted" means that it may be unsubstituted or substituted. "Substituted" means having one or more substituents, where a substituent refers to a chemical moiety, such as alkylene or heteroalkylene, that is covalently bonded or fused to any atom in the parent group.

[0032] In the present invention, "halo" means fluorine, chlorine, bromine, or iodine, and the like.

[0033] As used herein, "alkyl" refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of an aliphatic or alicyclic saturated or unsaturated hydrocarbon compound, examples of which include methyl, ethyl, propyl, butyl, pentyl, hexyl, n-propyl, n-butyl, n-pentyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, and neopentyl.

[0034] As used herein, "heteroalkyl" refers to an alkyl containing at least one heteroatom, where the heteroatom is at any carbon atom in the alkyl, replacing C, CH, CH2, or CH3.

[0035] As used herein, "alkylene" refers to a divalent moiety obtained by removing a hydrogen atom from a carbon atom of an aliphatic or alicyclic saturated or unsaturated hydrocarbon compound.

[0036] As used herein, "heteroalkylene" refers to an alkylene containing at least one heteroatom.

[0037] As used herein, "aryl" refers to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound having ring atoms. For example, "C 5-10In "aryl," carbon has 5 to 10 ring atoms, and the aryl is a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound. Examples of aryl include groups derived from benzene, acenaphthene, fluorene, phenalene, acephenanthrene, and aceanthrene.

[0038] In the present invention, "heteroaryl" refers to aryl containing at least one heteroatom, such as pyridine, pyrimidine, benzothiophene, furyl, dioxalanyl, pyrrolyl, oxazolyl, pyridyl, pyridazinyl, pyrimidinyl, isobenzofuran, indole, isoindole, indolizine, indoline, isoindoline, purine, benzodioxane, quinoline, isoquinoline, quinolizine, benzoxazine, benzothiazine, pyridopyridine, quinoxaline, quinazoline, cinnoline, phthalazine, naphthyridine, pteridine, perimidine, pyridoindole, oxanthrene, phenoxathiin, phenazine, and phenoxazine.

[0039] As used herein, "arylene" refers to a divalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound having ring atoms.

[0040] As used herein, "heteroarylene" refers to an arylene containing at least one heteroatom.

[0041] As used herein, "alkenyl" refers to an alkyl having at least one carbon-carbon double bond, examples of which include vinyl (-CH=CH2), 1-propenyl (-CH=CHCH3), isopropenyl, butenyl, pentenyl, and hexenyl.

[0042] As used herein, "alkynyl" refers to an alkyl group having at least one carbon-carbon triple bond, examples of which include ethynyl and 2-propynyl.

[0043] According to one embodiment of the present invention, the biotin moiety can be represented by the general formula A below: [General formula A] [ka]

[0044] In the general formula A, X is a functional group capable of binding to a biologically active substance; Y is a spacer, Z is a linking unit, B can be represented by the following chemical formula A-1: [Chemical formula A-1] [ka] Z is the compound of formula A-1 [ka] It is linked to T is a terminal group; m is an integer from 1 to 10; n is 0 or an integer from 1 to 10, and when n=0, Y is directly bonded to B or T; p is an integer of 0 to 1.

[0045] In one embodiment of the present invention, in the general formula A, X is a functional group capable of binding to a biologically active substance, which may include, but is not limited to, a functional group capable of reacting with a thiol group, a carboxyl group, and / or an amine group, such as a maleimide group, a succinimide group, an N-hydroxysuccinimide group, an aldehyde group, or a carboxyl group.

[0046] In one embodiment of the present invention, the functional group X may maintain its structure, be cleaved, or be modified when bound to a biologically active agent.

[0047] Y is a spacer and may have a structure that is cleavable in vivo. For example, but not limited to, Y may represent a direct bond or may have a substituted or unsubstituted C 1-50 Linear alkylene, substituted or unsubstituted C 1-50 Non-linear alkylene, substituted or unsubstituted C 1-50 Straight chain heteroalkylene, substituted or unsubstituted C 1-50 Non-linear heteroalkylene, substituted or unsubstituted C 1-50 Arylene, substituted or unsubstituted C 1-50 heteroarylene, -O-, -C(O), -C(O)NR-, -C(O)O-, -S-, -NR- or -NOR-, wherein R is hydrogen, substituted or unsubstituted C 1-50 Alkyl, substituted or unsubstituted C 1-50 Aryl, or ethylene glycol repeating units (-(CH2CH2O) n - (wherein n is an integer of 1 or more and 20 or less).

[0048] Z is a linking unit capable of binding to B, and Z may include, for example, but is not limited to, an amino acid, a polypeptide, an alkylene, an amine, or a polyamidoamine structure.

[0049] The amino acids may include, but are not limited to, lysine, 5-hydroxylysine, 4-oxalysine, 4-thialysine, 4-selenalysine, 4-thiahomolysine, 5,5-dimethyllysine, 5,5-difluorolysine, trans-4-dihydrolysine, 2,6-diamino-4-hexenoic acid, cis-4-dihydrolysine, 6-N-methylysine, diaminopimelic acid, ornithine, 3-methylornithine, α-methylornithine, citrulline, homocitrulline, arginine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, ornithine, proline, serine, or threonine.

[0050] When n is 0, B or T may be directly bonded to Y (the spacer).

[0051] T is a terminal group and may be, for example, but not limited to, hydrogen or NH2.

[0052] When p is 0, B may be terminal.

[0053] According to one embodiment of the present invention, in the following general formula A, m may be an integer of 1 to 10, specifically, an integer of 1 to 8, 1 to 5, and 1 to 4.

[0054] In one aspect of the invention, X is maleimide, succinimide, N-hydroxysuccinimide, succinimidyl succinate, succinimidyl glutamate, succinimidyl methyl ester, succinimidyl pentyl ester, succinimidyl carbonate, p-nitrophenyl carbonate, aldehyde, amine, thiol, oxyamine, iodoacetamide, aminoxyl, hydrazide, hydroxy, propionate, pyridyl, alkyl halide, vinyl sulfone, carboxyl, hydrazide, halogen acetamide, C 2-5 Alkynyl, C 6-20 Aryl disulfide, C 5-20 It may be selected from the group consisting of heteroaryl disulfides, isocyanates, thioesters, iminoesters and derivatives thereof.

[0055] In one embodiment of the invention, X is maleimide, N-hydroxysuccinimide, an aldehyde, or an amine.

[0056] In one aspect of the invention, Y is absent or a substituted or unsubstituted linear or branched chain C 1-50 Alkylene, substituted or unsubstituted, straight or branched chain C 1-50 Heteroalkylene, substituted or unsubstituted C 6-50 Arylene, or substituted or unsubstituted C 6-50 Heteroarylene, when substituted, contains at least one selected from the group consisting of =O, -C(O)NH2, -OH, -COOH, -SH, =NH and -NH2.

[0057] In one aspect of the invention, Y is a substituted linear or branched C 1-50 It is heteroalkylene and contains at least one -C(O)-.

[0058] In one aspect of the invention, Y is -(C(O)) q -(CH2) r -(C(O)NH s -(CH2) r -(OCH2CH2) t -(C(O)) q -, wherein q, r, s, and t are independently selected; q and S are 0 or 1; r is an integer from 1 to 20; and t is an integer from 0 to 20.

[0059] In one aspect of the invention, Y is -(CH2) r C(O)NHNH-, where r is an integer from 1 to 20.

[0060] In one aspect of the invention, Y comprises -C(O)-.

[0061] In one aspect of the invention, Y comprises -C(O)NH-.

[0062] In one aspect of the invention, Z is any one of the following, each of which may be independently selected: A) together with X or separately from X, forms an amino acid or a derivative thereof; or B) Substituted or unsubstituted linear or non-linear C 1-50 is heteroalkylene, When substituted, it contains at least one selected from the group consisting of =O, -C(O)NH2, -OH, -COOH, -SH, =NH and -NH2.

[0063] In one aspect of the invention, Z is linked via B and -NH-.

[0064] In one aspect of the invention, Z is a hydrophilic amino acid or a derivative thereof.

[0065] Specifically, in one aspect of the present invention, Z may be selected from the group consisting of lysine, arginine, histidine, glutamine, asparagine, threonine, cysteine, serine, and derivatives thereof.

[0066] In one aspect of the invention, Z comprises at least one glycerol and at least one polyethylene glycol, or a combination thereof.

[0067] In one aspect of the invention, Z is [ka] Including, [ka] represents a binding site and has at least one [ka] is attached to the binding site, where u is an integer of 1 to 20.

[0068] In one aspect of the invention, Z is [ka] and -(CH2)3NH- is [ka] is further bound to

[0069] In one aspect of the invention, T is an amine, C 1-8 Alkyl, C 1-8 It may be selected from the group consisting of alkenyl, halo, hydroxy, thiol, sulfonic acid, carboxyl, phenyl, benzyl, aldehyde, azide, cyanate, isocyanate, thiocyanate, isothiocyanate, nitrile and phosphonic acid.

[0070] In a specific embodiment of the invention, T is an amine.

[0071] In one aspect of the invention, the biotin moiety is [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] is selected from the group consisting of:

[0072] According to one embodiment of the invention, the biotin moiety and the bioactive agent may be linked by various bonds, which may be formed by a functional group of the biotin moiety binding to a functional group of the bioactive agent, such as, but not limited to, a thiol-ether bond or an amide bond.

[0073] In one example, the bond between the biotin moiety and the biologically active substance may be formed by the method of the following reaction scheme 1. In the following reaction scheme 1, [ka] represents a biologically active agent containing a thiol group, and Scheme 1 represents the reaction of a biotin moiety containing a maleimide group with a thiol group (-SH) of a cysteine ​​residue present in the biologically active agent, according to one embodiment of the present invention. [ka] [Reaction Scheme 1]

[0074] In one example, the bond between the biotin moiety and the biologically active agent may be formed by the method of Reaction Scheme 2 below. In Reaction Scheme 2, [ka] represents a bioactive agent containing an amine group, and Scheme 2 represents the reaction of a biotin moiety containing an N-hydroxysuccinimide with an amine group (-NH2) present on the bioactive agent, according to one embodiment of the present invention. [ka] [Reaction Scheme 2]

[0075] According to one embodiment of the present invention, the bioactive substance may not be particularly limited.

[0076] In the present invention, a biologically active substance refers to a polymeric substance that can be administered to the body for a given purpose.

[0077] According to one embodiment of the invention, the bioactive agent may be a substance used in a pharmaceutical composition, for example, but not limited to, a substance used in the prevention or treatment of diabetes, obesity, osteoporosis, fatty liver, irritable bowel syndrome, or neurodegenerative disease.

[0078] According to one embodiment of the present invention, the bioactive substance may be, for example, but is not limited to, a polypeptide, a protein, a polysaccharide, or a derivative thereof.The bioactive substance may be, for example, but is not limited to, glucagon, GLP-1 (glucagon-like peptide-1), GLP-2 (glucagon-like peptide-2), GIP (glucose-dependent insulinotropic polypeptide), exendin g-4, insulin, parathyroid hormone, interferon, erythropoietin, calcitonin, serotonin, rituximab, trastuzumab, uricase, tissue plasminogen activator, thymoglobin, vaccine, heparin or heparinoid, antithrombin III, filgrastim, pramlintide acetate, exenatide, eptifibatide, antivenom, IgG, IgM, HGH, thyroxine, coagulation factor VII, coagulation factor VIII, monoclonal antibodies, glycolipids acting as therapeutic agents, and derivatives thereof.

[0079] According to one embodiment of the invention, the biotin moiety may be attached to an inactive region of the bioactive agent.

[0080] The biotin moiety may be attached to an inactive region of the bioactive agent so as not to inhibit the biological activity of the bioactive agent, i.e., to exhibit biological activity that is the same as or greater than the biological activity of the bioactive agent.

[0081] For example, but not limited to, PSA may contain exposed -SH groups in the inactive region and a biotin moiety may be attached to the -SH group. + The biotin moiety may include an exposed -NH3 +The PSA may be attached to the -NH2 group or the -NH2 group of a lysine amino acid in the inactive region, so that the activity of the polypeptide is not inhibited. In the case of a polypeptide, the PSA may be attached to the -NH3+ group, -NH2 group of a lysine amino acid in the inactive region, or to the N-terminus if the N-terminus of the polypeptide is the inactive region.

[0082] That is, according to one embodiment of the present invention, the position at which the biotin moiety is attached to the PSA may be adjusted so that the attachment avoids the site at which activity occurs.

[0083] According to one embodiment of the present invention, PSA may be a polypeptide having any one of the amino acid sequences of SEQ ID NOs: 1 to 7 below, or a derivative thereof. SEQ ID NO: 1: H(Aib)QGTFTSDYSKYLDEQAAKEFVQWLMNT SEQ ID NO: 2: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR SEQ ID NO:3: HADGSFSDEMNTILDNLAARDFINWLIQTKITD SEQ ID NO: 4: YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ SEQ ID NO: 5: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPS SEQ ID NO: 6: SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF SEQ ID NO: 7: HSQGTFTSDYSKYLDSRRAQDFVQWLMN

[0084] Furthermore, PSA may be a protein having the amino acid sequence of SEQ ID NO:15 and 16 below, or a protein having the amino acid sequence of SEQ ID NO:17 and 16, which is linked via disulfide bonds between cysteine ​​at position 6 of SEQ ID NO:15 or 17 and cysteine ​​at position 11, between cysteine ​​at position 7 of SEQ ID NO:15 or 17 and cysteine ​​at position 7 of SEQ ID NO:16, and between cysteine ​​at position 20 of SEQ ID NO:15 or 17 and cysteine ​​at position 19 of SEQ ID NO:16. SEQ ID NO: 15: GIVEQCCTSICSLEQLENYCN SEQ ID NO: 16: FVNQHLCGSHLVEALYLVCGERGFFTPKT SEQ ID NO: 17: GIVEQCCTSICSLYQLENYCN

[0085] According to one embodiment of the invention, cysteines may be substituted or inserted into the polypeptide to modulate the binding site for the biotin moiety.

[0086] As a non-limiting example, at least one of the amino acids in the inactive region of a polypeptide selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 7 above may be replaced with a cysteine ​​amino acid, or a cysteine ​​amino acid may be inserted at that position. In this case, the biotin moiety is bound to the -SH group of the cysteine ​​amino acid. Furthermore, the polypeptide into which the cysteine ​​amino acid is inserted may be a polypeptide having any one of the amino acid sequences of SEQ ID NOs: 8 to 14. SEQ ID NO: 8: H(Aib)QGTFTSDYSKYLDEQAAKEFVQWLMNTC SEQ ID NO: 9: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRC SEQ ID NO: 10: HADGSFSDEMNTILDNLAARDFINWLIQTKITDC SEQ ID NO: 11: YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQC SEQ ID NO: 12: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC SEQ ID NO: 13: SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFC SEQ ID NO: 14: HSQGTFTSDYSKYLDSRRAQDFVQWLMNTC

[0087] According to one embodiment of the present invention, the PSA having a biotin moiety may be covalently bound to at least one selected from the group consisting of peptide and non-peptide polymers, fatty acids, cholesterol, antibodies, antibody fragments, albumin and its fragments, nucleotides, fibronectin, transferrin, FcRn binding substances, saccharides, elastin, heparin, and derivatives thereof, or may form a microsphere with at least one selected from the group.

[0088] The non-peptide polymer may be selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol (PVA), polysaccharides, dextran, polyvinyl ethyl ether, PLA (polylactic acid), PLGA (polylactic-co-glycolic acid), lipid polymers, chitin, hyaluronic acid, and combinations thereof.

[0089] Another aspect of the present invention provides a method for preparing PSA conjugated to a biotin moiety, the method comprising the steps of obtaining a biotin moiety, reacting the biotin moiety with PSA, and isolating the PSA conjugated to the biotin moiety after completion of the reaction.

[0090] According to one embodiment of the present invention, in obtaining a biotin moiety, the biotin moiety can be represented by general formula A above.

[0091] According to one embodiment of the present invention, in reacting the above mixture, the reaction molar ratio of the biotin moiety to the PSA may be 0.5 or more. Specifically, the molar ratio of the biotin moiety to the PSA may be 0.5 to 5. An appropriate reaction molar ratio may be selected based on the molecular structure, molecular weight, solubility, pH of the reaction solution, reaction temperature, reaction time, etc. of the biotin moiety.

[0092] According to one embodiment of the present invention, the reaction may be carried out using a buffer solution or an organic solvent. The buffer solution or the organic solvent is not particularly limited, and may be appropriately selected from buffer solutions commonly used in the art according to the structure of the biotin moiety.

[0093] In one embodiment of the present invention, the temperature and duration of the reaction step may be adjusted accordingly depending on the characteristics of the biotin moiety and PSA used. The reaction may be carried out at room temperature, for example, but not limited to, at 4° C. for at least 3 hours, or for a shorter duration. The reaction may be correlated with the degree of reactivity of the biotin moiety used. After a suitable reaction time, the reaction may be stopped by lowering the pH of the reaction solution.

[0094] According to one embodiment of the present invention, the above reaction step may be followed by a step of removing unreacted materials. The method of removing unreacted materials may be a method commonly used in the art. The method may include, but is not limited to, removing the unreacted materials by dialysis using a suitable buffer solution, such as PBS (phosphate buffered saline).

[0095] According to one embodiment of the invention, the method may include a purification step after the isolation step, which may be performed using, but is not limited to, size exclusion chromatography, high performance liquid chromatography, or ion exchange chromatography.

[0096] Another aspect of the present invention provides a composition for oral administration, comprising PSA conjugated to a biotin moiety as described above.

[0097] According to one embodiment of the present invention, a bioactive substance bound to a biotin moiety can be absorbed by active transport via the sodium-dependent multivitamin transporter by being bound to biotin (vitamin B7, a type of water-soluble vitamin), thereby promoting intestinal membrane permeability and intestinal absorption.

[0098] Another aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned PSA bound to a biotin moiety. The use of this pharmaceutical composition can be determined according to the type of PSA. Furthermore, this pharmaceutical composition can be an oral administration composition.

[0099] According to one embodiment of the present invention, there may be provided a pharmaceutical composition for preventing or treating diabetes, the composition comprising PSA linked to a biotin moiety.

[0100] The PSA may be used for preventing or treating diabetes. For example, but not limited to, the PSA may be a polypeptide having the amino acid sequences of SEQ ID NOs: 1 to 14, a protein having the amino acid sequences of SEQ ID NOs: 15 and 16, a protein having the amino acid sequences of SEQ ID NOs: 17 and 16, or a derivative thereof.

[0101] Furthermore, the protein is linked via disulfide bonds between cysteine ​​at position 6 of SEQ ID NO: 15 or 17 and cysteine ​​at position 11, between cysteine ​​at position 7 of SEQ ID NO: 15 or 17 and cysteine ​​at position 7 of SEQ ID NO: 16, and between cysteine ​​at position 20 of SEQ ID NO: 15 or 17 and cysteine ​​at position 19 of SEQ ID NO: 16.

[0102] According to one embodiment of the present invention, there may be provided a pharmaceutical composition for preventing or treating obesity, the composition comprising PSA linked to a biotin moiety.

[0103] The PSA may be used for preventing or treating obesity. For example, but not limited to, the PSA may be a polypeptide having the amino acid sequences of SEQ ID NOs: 1 to 14, a protein having the amino acid sequences of SEQ ID NOs: 15 and 16, a protein having the amino acid sequences of SEQ ID NOs: 17 and 16, or a derivative thereof.

[0104] Furthermore, the protein is linked via disulfide bonds between cysteine ​​at position 6 of SEQ ID NO: 15 or 17 and cysteine ​​at position 11, between cysteine ​​at position 7 of SEQ ID NO: 15 or 17 and cysteine ​​at position 7 of SEQ ID NO: 16, and between cysteine ​​at position 20 of SEQ ID NO: 15 or 17 and cysteine ​​at position 19 of SEQ ID NO: 16.

[0105] According to one embodiment of the present invention, there may be provided a pharmaceutical composition for preventing or treating fatty liver, the composition comprising PSA bound to a biotin moiety.

[0106] The PSA may be used for preventing or treating fatty liver. For example, but not limited to, the PSA may be a polypeptide having the amino acid sequences of SEQ ID NOs: 1 to 14, a protein having the amino acid sequences of SEQ ID NOs: 15 and 16, a protein having the amino acid sequences of SEQ ID NOs: 17 and 16, or a derivative thereof.

[0107] Furthermore, the protein is linked via disulfide bonds between cysteine ​​at position 6 of SEQ ID NO: 15 or 17 and cysteine ​​at position 11, between cysteine ​​at position 7 of SEQ ID NO: 15 or 17 and cysteine ​​at position 7 of SEQ ID NO: 16, and between cysteine ​​at position 20 of SEQ ID NO: 15 or 17 and cysteine ​​at position 19 of SEQ ID NO: 16.

[0108] According to one embodiment of the present invention, there may be provided a pharmaceutical composition for preventing or treating irritable bowel syndrome, the composition comprising PSA bound to a biotin moiety.

[0109] The PSA may be used for preventing or treating irritable bowel syndrome. For example, but not limited to, the PSA may be a polypeptide having the amino acid sequences of SEQ ID NOs: 1 to 14, a protein having the amino acid sequences of SEQ ID NOs: 15 and 16, a protein having the amino acid sequences of SEQ ID NOs: 17 and 16, or a derivative thereof.

[0110] Furthermore, the protein is linked via disulfide bonds between cysteine ​​at position 6 of SEQ ID NO: 15 or 17 and cysteine ​​at position 11, between cysteine ​​at position 7 of SEQ ID NO: 15 or 17 and cysteine ​​at position 7 of SEQ ID NO: 16, and between cysteine ​​at position 20 of SEQ ID NO: 15 or 17 and cysteine ​​at position 19 of SEQ ID NO: 16.

[0111] According to one embodiment of the present invention, there may be provided a pharmaceutical composition for preventing or treating a neurodegenerative disease, the composition comprising PSA linked to a biotin moiety.

[0112] The PSA may be used for preventing or treating a neurodegenerative disease. For example, but not limited to, the PSA may be a polypeptide having the amino acid sequences of SEQ ID NOs: 1 to 14, a protein having the amino acid sequences of SEQ ID NOs: 15 and 16, a protein having the amino acid sequences of SEQ ID NOs: 17 and 16, or a derivative thereof.

[0113] Furthermore, the protein is linked via disulfide bonds between cysteine ​​at position 6 of SEQ ID NO: 15 or 17 and cysteine ​​at position 11, between cysteine ​​at position 7 of SEQ ID NO: 15 or 17 and cysteine ​​at position 7 of SEQ ID NO: 16, and between cysteine ​​at position 20 of SEQ ID NO: 15 or 17 and cysteine ​​at position 19 of SEQ ID NO: 16.

[0114] According to one embodiment of the present invention, pharmaceutical compositions containing as an active ingredient a biologically active agent bound to a biotin moiety may be formulated and administered in a variety of oral and parenteral dosage forms, including, but not limited to, the following:

[0115] When formulating, commonly used fillers, solubilizers, extenders, binders, wetting agents, disintegrants, surfactants, and other diluents and excipients may be used in the formulation.

[0116] Solid preparations for oral administration include tablets, pills, powders, granules and capsules, and such solid preparations may be prepared by mixing the compound with at least one excipient, such as starch, calcium carbonate, sucrose or lactose.

[0117] In addition to simple excipients, lubricants such as magnesium stearate or talc are used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions and syrups, which may contain various excipients such as wetting agents, flavoring agents, sweeteners and preservatives in addition to commonly used diluents such as water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized drugs and suppositories. As non-aqueous solvents or suspensions, propylene glycol, polyethylene glycol (PEG), vegetable-based oils such as olive oil, and injectable esters such as ethyl oleate may be used.

[0118] Additionally, calcium or vitamin D3 may be added to improve efficacy as a therapeutic agent for proliferative or autoimmune diseases.

[0119] The dosage range of the pharmaceutical composition according to one embodiment of the present invention may vary depending on body weight, age, sex, health condition, diet, administration interval, administration method, excretion rate and severity of disease, but generally, it may be administered once a day or multiple times within the effective dose range per day.Furthermore, the effective dose may be administered multiple times every week or every two weeks.

[0120] Hereinafter, the present invention will be described in detail through embodiments and experimental examples. However, the following embodiments and experimental examples are intended only to illustrate the present invention, and the scope of the present invention is not limited by the following embodiments and experimental examples. EXAMPLES

[0121] <Embodiment: Preparation of Biotin Moiety> List of Abbreviations HBTU: 3-[bis(dimethylamino)methyliumyl]-3H-benzotriazole-1-oxide hexafluorophosphate DIEA: Ethyldiisopropylamine HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate DIC: Diisopropylcarbodiimide HOBt: 1-hydroxybenzotriazole MBHA: 4-Methylbenzhydrylamine hydrochloride) Fmoc: 9-fluorenylmethoxycarbonyl DMF: Dimethylformamide SPPS: Solid Phase Peptide Synthesis HPLC: High-performance liquid chromatography LCMS: Liquid Chromatography Mass Spectrometry

[0122] Common SPPS methods In some cases, the solid phase synthesis of peptides can be improved by the use of groups that can be cleaved under acidic conditions (e.g., 2-Fmoc-oxy-4-methoxybenzyl, or d-peptides protected at the amide bond with 2,4,6-trimethoxybenzyl). The Fmoc-protected amino acid derivatives used were prepared according to the recommended standard materials, e.g., Anaspect, Bachem, Iris. These included Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH or Fmoc-Val-OH, all supplied by Biotech or Novabiochem. The N-terminal amino acid was Boc protected at the α-amino group, e.g., Fmoc-8-amino-3,6-dioxaoctanoic acid, Fmoc-tranexamic acid, Fmoc-isonipecotic acid, Fmoc-Glu-OtBu, Fmoc-Lys(Fmoc)-OH, supplied by Anaspec, Bachem, Iris Biotech or Novabiochem, were used.

[0123] Peptide synthesis using SPPS The peptides may be synthesized on Rink Amide MBHA resin using standard Fmoc chemistry with HBTU / DIEA, HATU / DIEA or DIC / HOBt as coupling reagents. Reactants and coupling reagents used during synthesis include the following combinations: [Table 1]

[0124] An exemplary protocol for a peptide synthesis process using SPSS is given below. 1) Add DMF to the reaction vessel containing Rink Amide MBHA resin and allow to swell for 2 hours (sub: 0.68mmol / g, 1.0mmol, 1.47g or 5mmol, 7.35g, sub: 0.68mmol / g). 2) Add 20% piperidine / DMF and mix for 30 minutes. 3) Remove the solvents from 1)-2) and wash with DMF (30 seconds x 5 times). 4) Add a reactant (one of the reactants numbered 1-5) and mix for 30 seconds, then add a coupling reagent (one of the coupling reagents numbered 1-5) and bubble with nitrogen for 1 hour. 5) Add 20% piperidine / DMF and mix for 30 minutes.

[0125] In the above exemplary protocols 1) to 5), the synthesis may be repeated at least once using the combination of reactants and coupling reagents No. 1 to No. 5. To remove Fmoc, [the above mixture] was treated with 20% piperidine / DMF solution for 30 minutes.

[0126] General process for peptide purification and analysis The crude peptide was dissolved in an appropriate mixture of water, TFA and ACN, purified by preparative HPLC, dried and quantified. The conditions for purification using preparative HPLC include those shown in Table 2 below. [Table 2]

[0127] After purification using preparative HPLC, the final products were characterized using analytical HPLC or LMS.

[0128] Analysis showed that the biotin moieties in Table 3 below were obtained. [Table 3] B1: [ka] B2: [ka] B3: [ka] B4: [ka] B5: [ka]

[0129] Furthermore, through peptide synthesis protocols 1) to 5) using SPSS, purification and analysis, the following biotin moieties were obtained. In Table 4, X, Y, Z and B are included in the definition of general formula A in this specification. [Table 4-1] [Table 4-2]

[0130] <Embodiment: Preparation of a physiologically active substance bound to a biotin moiety> <Embodiments 1 to 3> 1) Preparation Examples A mixture of polypeptide-biotin moiety (molar ratio=1:2) shown in Table 5 below, in which the polypeptide of SEQ ID NO:12 was mixed with the biotin moiety shown in Table 3 above (B1, B2 and B3 were used, respectively. Polypeptide of SEQ ID NO:12: B1, B2 or B3) was prepared using DMSO solvent with 0.3% triethylamine (TEA, Sigma) as the reaction solvent. The mixture was reacted for at least 30 minutes at room temperature, and the reaction was stopped by adding 1% trifluoroacetic acid in a volume equal to the volume of the mixture. [Table 5]

[0131] 2) Isolation, purification and identification The reaction products of the above embodiments 1 to 3 were isolated and purified using reverse phase high performance liquid chromatography (hereinafter referred to as "HPLC").

[0132] A SUPERSIL ODS-1 column (10×250 mm, 5 μm, LB Science, South Korea) was used as the column. The mobile phase conditions were linearly changed from 30 to 50% solvent B (acetonitrile containing 0.1% TFA) and solvent A (distilled water containing 0.1% TFA) at a flow rate of 4.7 m / min. Peaks detected between 12 and 14 min were collected by monitoring at 280 nm with a UV spectrophotometer. The organic solvent and TFA were evaporated under vacuum from the collected peaks, and the mixture was concentrated and purified using an ultracentrifuge filter with an appropriate molecular weight cutoff. The purity of the purified material was confirmed using HPLC analysis. Analysis was performed at a constant temperature around room temperature using a Gemini C18 column (4.6×250 mm, 5 μm, Phenomenex, CA, USA). The analysis was carried out using a gradient elution method with a mobile phase consisting of a mixture of trifluoroacetic acid test solution:acetonitrile (mixture ratio changed from 70:30 to 50:50 in 20 min) at a flow rate of 1 mL / min. UV absorbance was monitored at 280 nm.

[0133] FIG. 1 is a chromatogram during purification in embodiment 3.

[0134] FIG. 2 is a chromatogram of the final substance of the first to third embodiments.

[0135] In analysis using a UV detector, no peaks other than those of the polypeptide bound to the biotin moiety were observed, and the purity of embodiments 1 to 3 was confirmed to be 99% or more when expressed as a percentage of the peak area in each chromatogram.

[0136] Molecular weight confirmation The molecular weights of the final substances obtained in the first to third embodiments were measured.

[0137] The molecular weight was measured using MALDI-TOF mass spectrometry. The matrix solution used was a 50% acetonitrile solution containing 0.1% TFA saturated with CHCA (α-cyano-4-hydroxycinnamic acid). Mass spectra were examined in linear and reflectron modes to confirm the molecular weight at a final substance concentration of 0.1 mg / mL.

[0138] Fig. 3 shows the MALDI-TOF mass spectrum of the embodiments 1 to 3. Referring to Fig. 3, it was confirmed that the molecular weight of the isolated substance obtained by MALDI-TOF mass spectrometry was consistent with the theoretical molecular weight.

[0139] <Embodiments 4 to 9> Using the same methods as those of Examples 1-3, polypeptide-biotin moieties were prepared by attaching biotin moieties to the polypeptides of SEQ ID NOs: 8 and 13 as shown in Table 6 below. [Table 6]

[0140] [evaluation] Reaction rate and yield The reaction rate was obtained by comparing the HLPC peak areas of Examples 1 to 3 in HPLC analysis of the mixtures having biotin moieties based on the amount of polypeptide added in the step of obtaining the initial mixture.

[0141] The yields of the present invention were obtained by determining the amount of final purified material based on the amount of polypeptide added in the step of obtaining the initial mixture, and the results are shown in Table 7 below. [Table 7]

[0142] Measurement of oral absorption rate in embodiments 1 to 3 Pharmacokinetics were compared to ascertain the oral absorption rate.

[0143] The test drug was administered intravenously at 100 μg / kg and orally at 500 μg / kg to experimental rats (SD rats) weighing about 200 g, and serum was then collected and enzyme-linked immunosorbent assay was performed to measure the change in blood drug concentration over time. Blood samples were collected from the jugular vein. The average was calculated from the results, and a polypeptide having the amino acid sequence of SEQ ID NO: 5 was used as a control. The experimental results showed that the oral absorption rate was increased by about 257 times in embodiment 1, 270 times in embodiment 2, and about 557 times in embodiment 3 compared to the control.

[0144] Fig. 4 is a graph showing the blood concentration over time after oral administration to rats for Embodiments 1 to 3. As shown in Fig. 4, it was confirmed that Embodiments 1 to 3 have superior oral absorption compared to the control.

[0145] Measurement of blood glucose regulation ability in embodiments 1 to 3 To confirm glucose tolerance, the oral dosage forms of GLP-1 agonists, Examples 1 to 3, were orally administered to mice, and the effectiveness of regulating blood glucose was measured by intraperitoneal glucose tolerance test (IPGTT).

[0146] To measure peritoneal glucose tolerance in an animal model, 100 μl of test drug (1 μg / mouse, SEQ ID NO: 1) was orally administered to 9-week-old male mice (C57BL / 6) at -60 min, followed by intraperitoneal injection of 200 μl of glucose (2 g / kg), and blood glucose changes were observed in blood samples taken from the tail vein at -60, 0, 20, 40, 60, 90 and 120 min. In control 1, a polypeptide having the amino acid sequence of SEQ ID NO: 5 was administered subcutaneously, and in control 2, the same polypeptide was administered orally.

[0147] 5 is a graph showing the change in blood glucose level after administration of glucose to each subject. As shown in FIG. 5, it was confirmed that the first to third embodiments have glucose regulating ability.

[0148] Measurement of blood glucose regulation ability in embodiment 5 and embodiment 6 To confirm glucose tolerance, embodiments 5 and 6 were orally administered to mice, and the effectiveness of regulating blood glucose was measured by intraperitoneal glucose tolerance test.

[0149] To measure peritoneal glucose tolerance in an animal model, 100 μl of the test drug (500 μg / kg, sequence number 2) was orally administered to 9-week-old male mice (C57BL / 6) at -20 minutes, followed by intraperitoneal injection of 200 μl of glucose (2 g / kg). Blood was collected from the tail vein at -20 minutes, 0 minutes, 20 minutes, 40 minutes, 60 minutes, 90 minutes, and 120 minutes to observe the changes in blood glucose.

[0150] Figure 6 is a graph showing the change in blood glucose after glucose was administered to each subject. As shown in Figure 6, it was confirmed that the embodiments 5 and 6 have glucose regulating ability.

[0151] Measurement of oral absorption rate of embodiment 8 and embodiment 9 The oral absorption rate of the embodiments 8 and 9 was measured. The test drug was administered subcutaneously and orally to laboratory rats (SD rats) weighing about 200 g at a dose of 20 μg / kg, and then serum was isolated and enzyme-linked immunosorbent assay was performed to measure the change in blood drug concentration over time. Blood samples were collected from the jugular vein. The measurement results are shown in Table 8 below. A polypeptide having the amino acid sequence of SEQ ID NO: 6 was used as a control. [Table 8]

[0152] As shown above, a polypeptide conjugated to a biotin moiety according to one embodiment of the present invention has been shown to have a high rate of intestinal absorption.

[0153] <Embodiment 10> A polypeptide bound to a biotin moiety was prepared using biotin moiety B4 from Table 3 and a polypeptide having the amino acid sequence of SEQ ID NO:5 in Table 9 below. [Table 9]

[0154] 1) Preparation Examples A mixture (molar ratio 1:4) of the polypeptide of SEQ ID NO:5 and B4 of Table 3 above was prepared using DMSO solvent containing 0.3% triethylamine (TEA, Sigma) as a reaction solvent. The mixture was reacted at room temperature for 2 hours, and the reaction was stopped by adding 1% trifluoroacetic acid in a volume equal to the volume of the mixture.

[0155] 2) Isolation, purification and identification The reaction product of Example 10 was isolated and purified using reverse phase HPLC.

[0156] A SUPERSIL ODS-1 column (10×250 mm, 5 μm, LB Science, South Korea) was used as the column. The mobile phase conditions were linearly changed from 30 to 50% solvent B (acetonitrile containing 0.1% TFA) and solvent A (distilled water containing 0.1% TFA) at a flow rate of 4.7 m / min. The peak liquid detected at 12.7 min was collected by monitoring at 280 nm with a UV spectrophotometer. The organic solvent and TFA were evaporated from the collected peak liquid under vacuum, and the liquid was concentrated and purified using an ultracentrifuge filter with an appropriate molecular weight cutoff. The purity of the purified material was confirmed by HPLC analysis. Analysis was performed at a constant temperature around room temperature using a Gemini C18 column (4.6×250 mm, 5 μm, Phenomenex, CA, USA). The analysis was carried out using a mobile phase consisting of trifluoroacetic acid test solution:acetonitrile mixture (mixture ratio changed from 70:30 to 50:50 in 20 min) at a flow rate of 1 mL / min. UV absorbance was monitored at 280 nm.

[0157] 9 is a chromatogram during purification of embodiment 10 of the present invention. The unreacted polypeptide of SEQ ID NO: 1 was detected at 11.7 minutes, and the substance of embodiment 10 bound to the biotin moiety was detected at 12.7 minutes.

[0158] 10 is a chromatogram after purification of embodiment 10 of the present invention. One peak of the polypeptide bound to the biotin moiety was confirmed, and no other peaks were observed. In the chromatogram, the peak area was expressed as a percentage, confirming that the purity was 95% or more.

[0159] 3) Confirmation of molecular weight The molecular weight of the final material obtained in embodiment 10 was measured.

[0160] The molecular weight was measured using MALDI-TOF mass spectrometry. The matrix solution used was a 50% acetonitrile solution containing 0.1% TFA saturated with CHCA (α-cyano-4-hydroxycinnamic acid). Mass spectra were obtained in linear and reflectron modes.

[0161] <Embodiment 11> A polypeptide bound to a biotin moiety was prepared using biotin moiety B5 from Table 3 and a polypeptide having the amino acid sequence of SEQ ID NO:12 in Table 10 below. [Table 10]

[0162] 1) Preparation Examples A mixture (molar ratio 1:2) of the polypeptide of SEQ ID NO: 12 and B5 in Table 3 above was prepared using DMSO solvent containing 0.3% triethylamine (TEA, Sigma) as a reaction solvent. The mixture was reacted at room temperature for 30 minutes, and the reaction was stopped by adding 1% trifluoroacetic acid in a volume equal to the volume of the mixture.

[0163] 2) Isolation, purification and identification The reaction product of Example 10 was isolated and purified using reverse phase HPLC.

[0164] A SUPERSIL ODS-1 column (10×250 mm, 5 μm, LB Science, South Korea) was used as the column. The mobile phase conditions were linearly changed from 30 to 50% solvent B (acetonitrile containing 0.1% TFA) and solvent A (distilled water containing 0.1% TFA) at a flow rate of 4.7 m / min. The peak liquid detected at 11 to 13 minutes was collected by monitoring at 280 nm with a UV spectrophotometer. The organic solvent and TFA were evaporated from the collected peak liquid under vacuum, and the liquid was concentrated and purified using an ultracentrifugal filter with an appropriate molecular weight cutoff. The purity of the purified material was confirmed by HPLC analysis. Analysis was performed at a constant temperature around room temperature using a Gemini C18 column (4.6×250 mm, 5 μm, Phenomenex, CA, USA). The analysis was carried out using a mobile phase consisting of trifluoroacetic acid test solution:acetonitrile mixture (mixture ratio changed from 70:30 to 50:50 in 20 min) at a flow rate of 1 mL / min. UV absorbance was monitored at 280 nm.

[0165] 11 is a reverse phase chromatogram of embodiment 11 of the present invention. One peak of the polypeptide bound to the biotin moiety was confirmed, and no other peaks were observed. In the chromatogram, the peak area was expressed as a percentage, confirming that the purity was 90% or more.

[0166] 3) Confirmation of molecular weight The molecular weight of the final material obtained in Example 11 was measured.

[0167] The molecular weight was measured using MALDI-TOF mass spectrometry. The matrix solution used was a 50% acetonitrile solution containing 0.1% TFA saturated with CHCA (α-cyano-4-hydroxycinnamic acid). Mass spectra were obtained in linear and reflectron modes.

[0168] Fig. 12 is a MALDI-TOF mass spectrum of the present invention according to embodiment 10 and embodiment 11. Referring to Fig. 12, it was confirmed by MALDI-TOF mass spectrometry that the molecular weight of the isolated substance was consistent with its theoretical molecular weight.

[0169] <Embodiments 12 and 13> A polypeptide bound to a biotin moiety was prepared using biotin moiety B4 from Table 3 and polypeptides having the amino acid sequences of SEQ ID NOs: 15 and 16 in Table 11 below. [Table 11]

[0170] 1) Preparation Examples A mixture (molar ratio 1:16) of the polypeptides of SEQ ID NOs: 15 and 16 and B4 in Table 3 above was prepared using DMSO solvent containing 0.3% triethylamine (TEA, Sigma) as a reaction solvent. The mixture was reacted at room temperature for 2 hours, and the reaction was stopped by adding 30% acetonitrile / distilled water solution containing 1% trifluoroacetic acid in the same volume as the mixture.

[0171] 2) Isolation, purification and identification The reaction products of Examples 12 and 13 were isolated and purified using reverse phase HPLC.

[0172] A SUPERSIL ODS-1 column (10×250 mm, 5 μm, LB Science, South Korea) was used as the column. The mobile phase conditions were linearly changed from 30 to 40% solvent B (acetonitrile containing 0.1% TFA) and solvent A (distilled water containing 0.1% TFA) at a flow rate of 4.7 m / min. The peak liquid detected at 12.7 min was collected by monitoring at 280 nm with a UV spectrophotometer. The organic solvent and TFA were evaporated from the collected peak liquid under vacuum, and the liquid was concentrated and purified using an ultracentrifuge filter with an appropriate molecular weight cutoff. The purity of the purified material was confirmed by HPLC analysis. Analysis was performed at a constant temperature around room temperature using a Gemini C18 column (4.6×250 mm, 5 μm, Phenomenex, CA, USA). The analysis was carried out using a mobile phase consisting of trifluoroacetic acid test solution:acetonitrile mixture (mixture ratio changed from 70:30 to 50:50 in 20 min) at a flow rate of 1 mL / min. UV absorbance was monitored at 280 nm.

[0173] 13 shows chromatograms of the purification of the embodiments 12 and 13 of the present invention. The unreacted polypeptide of SEQ ID NO: 7 was detected at 12.1 minutes, the substance of the embodiment 12 bound to the biotin moiety was detected at 12.3 minutes, and the substance of the embodiment 13 was detected at 12.5 minutes.

[0174] 14 is a chromatogram after purification of embodiment 12 of the present invention. One peak of the polypeptide bound to the biotin moiety was confirmed, and no other peaks were observed. In the chromatogram, the peak area was expressed as a percentage, confirming that the purity was 95% or more.

[0175] 15 is a chromatogram after purification of embodiment 13 of the present invention. One peak of the polypeptide bound to the biotin moiety was confirmed, and no other peaks were observed. In the chromatogram, the peak area was expressed as a percentage, confirming that the purity was 95% or more.

[0176] 3) Confirmation of molecular weight The molecular weights of the final materials obtained in Examples 12 and 13 were measured.

[0177] The molecular weight was measured using MALDI-TOF mass spectrometry. The matrix solution used was a 50% acetonitrile solution containing 0.1% TFA saturated with CHCA (α-cyano-4-hydroxycinnamic acid). Mass spectra were obtained in linear and reflectron modes.

[0178] [evaluation] Measurement of blood glucose regulation ability according to embodiments 10 and 11 To confirm glucose tolerance, oral dosage forms of GLP-1 agonists, embodiments 10 and 11, were orally administered to mice and their effectiveness in regulating blood glucose was measured by intraperitoneal glucose tolerance test (IPGTT).

[0179] In order to measure peritoneal glucose tolerance in an animal model, 100 μl (10 μg / mouse, SEQ ID NO: 1) of the polypeptide bound to the biotin moiety prepared in embodiments 10 and 11 was orally administered to 9-week-old male mice (C57BL / 6) at -20 minutes, followed by intraperitoneal injection of 200 μl (2 g / kg) of glucose, and blood glucose changes were observed in blood collected from the tail vein at -20.0 minutes, 0 minutes, 20 minutes, 40 minutes, 60 minutes, 90 minutes and 120 minutes. As a control, exendin-4 consisting of the amino acid sequence represented by SEQ ID NO: 5 was orally administered.

[0180] 7 is a graph showing the change in blood glucose level after administration of glucose to each subject. As shown in FIG. 7, it was confirmed that the embodiments 10 and 11 have the ability to regulate blood glucose level.

[0181] Measurement of the biological activity of embodiments 12 and 13 The biological activity of the polypeptide was compared before and after the attachment of the biotin moiety, and further, the biological activity was compared according to the attachment position of the biotin moiety.

[0182] Specifically, biological activity was measured using the PathHunter® Insulin Bioassay Kit (DiscoverX, #93-0466Y3-00007).

[0183] PathHunter U2OS INSRb Bioassay cells were seeded in a 96-well plate and then cultured in AssayComplete™ Cell Plating Reagent5 (CP5) for 24 hours. Then, each drug was added at concentrations of 20 μl, 300, 60, 20, 6.67, 2.22, 0.74, 0.25, 0.05, and 0.01. After 3 hours of culture, 10 μl of detection reagent 1 was added and reacted for 15 minutes, and then 40 μl of detection reagent 2 was added and reacted for 60 minutes. Luminescence was then measured using a 96-well microplate reader. A protein consisting of chain A having the amino acid sequence of SEQ ID NO: 15 and chain B having the amino acid sequence of SEQ ID NO: 16 was used as a control. In the control, the protein has disulfide bonds between C6 of chain A and C11 of chain A, between C7 of chain A and C7 of chain B, and between C20 of chain A and C19 of chain B. [Table 12]

[0184] Measurement of blood glucose regulation ability according to embodiments 12 and 13 To confirm glucose tolerance, oral forms of the protein, Examples 12 and 13, were orally administered to mice and their effectiveness in regulating blood glucose was measured by an intraperitoneal glucose tolerance test.

[0185] In order to measure the glucose tolerance of the peritoneum in an animal model, 100 μl (10 μg / mouse) of the polypeptide bound to the biotin moiety prepared in embodiments 12 and 13 was orally administered to 9-week-old male mice (C57BL / 6) at -20 minutes, followed by intraperitoneal injection of 200 μl (2 g / kg) of glucose, and the blood glucose change was observed in blood collected from the tail vein at -20 minutes, 0 minutes, 20 minutes, 40 minutes, 60 minutes, 90 minutes and 120 minutes. Meanwhile, a protein consisting of chain A having the amino acid sequence of SEQ ID NO: 15 and chain B having the amino acid sequence of SEQ ID NO: 16 was used as a control.

[0186] Fig. 8 is a graph showing the change in blood glucose level after administration of glucose to each subject. As shown in Fig. 8, it was confirmed that the compositions according to the embodiments 12 and 13 have glucose regulating ability.

[0187] Those skilled in the art will recognize or ascertain through routine experimentation many equivalents to the specific embodiments of the invention set forth herein. Such equivalents are intended to be included in the appended claims. The above description of the invention is intended to be illustrative, and those skilled in the art to which the invention pertains will understand that the invention may be easily modified into other specific forms without changing the technical spirit or essential characteristics of the invention. That is, the above-described embodiments should be understood to be illustrative and non-limiting in all circumstances. For example, each element described as being combined may be implemented separately, and similarly, elements described as being separate may be implemented in a combined form. Any modifications or variations that can be inferred from the spirit and scope of the appended claims and their equivalent concepts may be interpreted as being included in the scope of the invention.

[0188] Industrial Applicability The present invention relates to a physiologically active substance bound to a biotin moiety, which has excellent absorbability in the body when orally taken, and a composition for oral administration containing the physiologically active substance, and the physiologically active substance bound to the biotin moiety can be prepared by preparing the biotin moiety using a method such as SPSS and mixing the biotin moiety with the physiologically active substance. Furthermore, the present invention has the advantage that the binding of the biotin moiety prevents the degradation of the physiologically active substance, and ultimately promotes the intestinal membrane permeation and intestinal absorption of the physiologically active substance. In certain embodiments, for example, the following items are provided: (Item 1) A bioactive agent conjugated to a biotin moiety, wherein the biotin moiety is conjugated to an inactive region of the bioactive agent. (Item 2) The biotin moiety is represented by the following general formula A: [General formula A] [ka] In the above general formula A, X is a functional group capable of binding to the biologically active substance, Y is a spacer, Z is a linking unit, B can be represented by the following chemical formula A-1: [Chemical formula A-1] [ka] The Z is represented by the formula A-1 [ka] It is linked to T is a terminal group; m is an integer from 1 to 10; n is 0 or an integer of 1 to 10, and when n=0, the Y is directly bonded to the B or the T; 2. The biologically active substance conjugated to a biotin moiety according to item 1, wherein p is an integer from 0 to 1. (Item 3) 2. The bioactive agent conjugated to a biotin moiety according to item 1, wherein the biotin moiety is conjugated to the bioactive agent selected from the group consisting of polypeptides, proteins and polysaccharides. (Item 4) 2. The bioactive agent conjugated to a biotin moiety according to claim 1, wherein the bioactive agent comprises an exposed -SH group and the biotin moiety is conjugated to the -SH group. (Item 5) The bioactive agent is an exposed -NH 3 + Group or -NH 2 group, and the biotin moiety comprises the -NH 3 + group or the -NH 2 2. A biologically active substance conjugated to a biotin moiety according to claim 1, wherein the biologically active substance is bound to a group. (Item 6) 2. The bioactive agent conjugated to a biotin moiety according to claim 1, wherein the bioactive agent comprises an N-terminus and the biotin moiety is conjugated to the N-terminus. (Item 7) 2. The bioactive substance conjugated to a biotin moiety according to item 1, wherein the bioactive substance is selected from the group consisting of glucagon, GLP-1 (glucagon-like peptide-1), GLP-2 (glucagon-like peptide-2), GIP (glucose-dependent insulinotropic polypeptide), exendin g-4, insulin, parathyroid hormone, interferon, erythropoietin, calcitonin, serotonin, rituximab, trastuzumab, uricase, tissue plasminogen activator, thymoglobulin, vaccines, heparin or heparinoids, antithrombin III, filgrastim, pramlintide acetate, exenatide, eptifibatide, antivenin, IgG, IgM, HGH, thyroxine, coagulation factor VII, coagulation factor VIII, monoclonal antibodies, glycolipids acting as therapeutic agents, and derivatives thereof. (Item 8) 2. The bioactive substance bound to a biotin moiety according to Item 1, wherein the bioactive substance is a polypeptide selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 7, a protein consisting of the amino acid sequences represented by SEQ ID NOs: 15 and 16, or a protein consisting of the amino acid sequences represented by SEQ ID NOs: 17 and 16. (Item 9) 9. The biologically active substance bound to a biotin moiety according to item 8, wherein the protein having the amino acid sequence of SEQ ID NO: 15 and SEQ ID NO: 16, or SEQ ID NO: 17 and SEQ ID NO: 16 is bound via disulfide bonds between the cysteine ​​at position 6 and the cysteine ​​at position 11 of SEQ ID NO: 15 or SEQ ID NO: 17, between the cysteine ​​at position 7 of SEQ ID NO: 15 or SEQ ID NO: 17 and the cysteine ​​at position 7 of SEQ ID NO: 16, and between the cysteine ​​at position 20 of SEQ ID NO: 15 or SEQ ID NO: 17 and the cysteine ​​at position 19 of SEQ ID NO: 16. (Item 10) The biotin moiety is an -NH + 3 group, -NH 2 7. A biologically active substance conjugated to a biotin moiety according to claim 5 or 6, wherein the biologically active substance is conjugated to a biotin group or to the N-terminus. (Item 11) 2. The bioactive substance conjugated to a biotin moiety according to item 1, wherein the bioactive substance is a polypeptide, in which at least one of the amino acid sequences of an inactive region of the polypeptide selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 7 is substituted with a cysteine ​​amino acid or a cysteine ​​amino acid is inserted into the at least one position, and the biotin moiety is bound to an -SH group of the cysteine ​​amino acid. (Item 12) 11. The physiologically active substance bound to a biotin moiety according to item 10, wherein the physiologically active substance is a polypeptide selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 8 to 14. (Item 13) X is maleimide, succinimide, N-hydroxysuccinimide, succinimidyl succinate, succinimidyl glutamate, succinimidyl methyl ester, succinimidyl pentyl ester, succinimidyl carbonate, p-nitrophenyl carbonate, aldehyde, amine, thiol, oxyamine, iodoacetamide, aminoxyl, hydrazide, hydroxy, propionate, pyridyl, alkyl halide, vinyl sulfone, carboxyl, hydrazide, halogenated acetamide, C 2-5 Alkynyl, C 6-20 Aryl disulfide, C 5-20 3. The biologically active substance conjugated to the biotin moiety according to item 2, which is selected from the group consisting of heteroaryl disulfides, isocyanates, thioesters, iminoesters and derivatives thereof. (Item 14) 3. The biologically active substance conjugated to a biotin moiety according to item 2, wherein X is maleimide, N-hydroxysuccinimide, an aldehyde or an amine. (Item 15) The Y is absent or is a substituted or unsubstituted linear or branched chain C 1-50 Alkylene, substituted or unsubstituted, straight or branched chain C 1-50 Heteroalkylene, substituted or unsubstituted C 6-50 Arylene, or substituted or unsubstituted C 6-50 is heteroarylene, If substituted, =O, -C(O)NH 2 , -OH, -COOH, -SH, =NH and -NH 2 3. The bioactive substance conjugated to a biotin moiety according to claim 2, comprising at least one selected from the group consisting of: (Item 16) The Y is a substituted linear or branched C 1-503. The biologically active agent conjugated to a biotin moiety according to item 2, which is heteroalkylene and contains at least one -C(O)-. (Item 17) The Y is -(C(O)) q -(CH 2 ) r -(C(O)NH s -(CH 2 ) r -(OCH 2 CH 2 ) t -(C(O)) q - and wherein q, r, s, and t are independently selected; The q and the S are 0 or 1, The r is an integer of 1 to 20, 3. A physiologically active substance bound to a biotin moiety according to item 2, wherein the t is an integer of 0 to 20. (Item 18) The Y is -(CH 2 ) r 3. The biologically active substance bound to a biotin moiety according to item 2, wherein r is C(O)NHNH-, and r is an integer of 1 to 20. (Item 19) 3. The biologically active agent conjugated to a biotin moiety according to claim 2, wherein Y comprises -C(O)-. (Item 20) 3. The biologically active agent conjugated to a biotin moiety according to claim 2, wherein Y comprises -C(O)NH-. (Item 21) 3. The biologically active agent conjugated to a biotin moiety according to claim 2, wherein Z is any one of the following, each of which may be independently selected: A) together with X or separately from X, forms an amino acid or derivative; or B) Substituted or unsubstituted linear or non-linear C 1-50 Heteroalkylene, when substituted, is ═O, —C(O)NH 2 , -OH, -COOH, -SH, =NH and -NH 2 The compound includes at least one selected from the group consisting of: (Item 22) 3. The biologically active substance conjugated to a biotin moiety according to item 2, wherein Z is linked to B via -NH-. (Item 23) 3. The biologically active substance conjugated to a biotin moiety according to item 2, wherein Z is a hydrophilic amino acid or a derivative thereof. (Item 24) 3. The biologically active substance conjugated to a biotin moiety according to item 2, wherein Z is selected from the group consisting of lysine, arginine, histidine, glutamine, asparagine, threonine, cysteine, serine and derivatives thereof. (Item 25) 3. The biologically active agent conjugated to a biotin moiety according to claim 2, wherein Z comprises at least one glycerol and at least one polyethylene glycol, or a combination thereof. (Item 26) The Z is

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Claims

1. a bioactive agent bound to a biotin moiety, said biotin moiety being bound to an inactive region of said bioactive agent and not inhibiting the biological activity of said bioactive agent; The biologically active substance has an exposed -SH group, an exposed -NH ３ ＋ group or -NH ２ a polypeptide, protein, thyroxine, glycolipid or non-peptide polymer comprising a group, The biotin moiety is represented by the following general formula A: [General formula A] 【Chemistry 19】 X is maleimide, succinimide, N-hydroxysuccinimide, succinimidyl succinate, succinimidyl glutamate, succinimidyl methyl ester, succinimidyl pentyl ester, succinimidyl carbonate, p-nitrophenyl carbonate, aldehyde, amine, thiol, oxyamine, iodoacetamide, aminoxyl, hydrazide, hydroxy, propionate, pyridyl, alkyl halide, vinyl sulfone, carboxyl, hydrazide, halogen acetamide, C ２－５ Alkynyl, C ６－２０ Aryl disulfide, C ５－２０ a functional group capable of binding to said biologically active substance selected from the group consisting of heteroaryl disulfides, isocyanates, thioesters, and iminoesters; Y is absent or a substituted or unsubstituted linear or branched chain C １－５０ Alkylene, substituted or unsubstituted straight or branched chain C １－５０ Heteroalkylene, when substituted, is ═O, —C(O)NH ２ , -OH, -COOH, -SH, =NH and -NH ２ At least one selected from the group consisting of: Z is any one independently selected from the following: (i) when Y is absent, together with X or independently of X, forms lysine, arginine, histidine, glutamic acid, glutamine, asparagine, aspartic acid, threonine, cysteine ​​or serine; or (ii) 【Chemical 22】 wherein 【Chemistry 23】 represents a binding site, and one or more 【Chemistry 24】 is attached to said attachment site, where u is an integer from 1 to 20; B is represented by the following chemical formula A-1, [Chemical formula A-1] 【Chemistry 20】 The Z is represented by the formula A-1 【Chemistry 21】 It is linked to T is an amine, C １－８ Alkyl, C １－８ an end group selected from the group consisting of alkenyl, halo, hydroxy, thiol, sulfonic acid, carboxyl, phenyl, benzyl, aldehyde, azide, cyanate, isocyanate, thiocyanate, isothiocyanate, nitrile, and phosphonic acid; m is an integer from 2 to 10; n is an integer from 1 to 10; The physiologically active substance, wherein p is an integer of 0 to 1.

2. 2. The bioactive agent conjugated to a biotin moiety of claim 1, wherein the biotin moiety is conjugated to a polypeptide.

3. 2. The bioactive agent conjugated to a biotin moiety of claim 1, wherein the bioactive agent is a polypeptide, protein or non-peptide polymer that contains an exposed -SH group, and the biotin moiety is conjugated to the -SH group.

4. The biologically active substance is an exposed -NH ３ ＋ group or -NH ２ a polypeptide, protein or non-peptide polymer comprising a -NH group, ３ ＋ group or the -NH ２ 2. The biologically active agent conjugated to a biotin moiety according to claim 1, which is conjugated to a group.

5. The biologically active substance is a compound having an exposed -NH ２ 2. The biologically active agent conjugated to a biotin moiety of claim 1, wherein the biologically active agent is a polypeptide or protein comprising a group, and the biotin moiety is attached to the N-terminus.

6. 2. The bioactive substance conjugated to a biotin moiety of claim 1, wherein the bioactive substance is selected from the group consisting of glucagon, GLP-1 (glucagon-like peptide-1), GLP-2 (glucagon-like peptide-2), GIP (glucose-dependent insulinotropic polypeptide), exendin-4, insulin, parathyroid hormone, interferon, erythropoietin, calcitonin, serotonin, rituximab, trastuzumab, uricase, tissue plasminogen activator, thymoglobulin, vaccine, heparin, antithrombin III, filgrastim, pramlintide acetate, exenatide, eptifibatide, antivenin, IgG, IgM, HGH, thyroxine, coagulation factor VII, coagulation factor VIII, monoclonal antibodies, and glycolipids.

7. 2. The bioactive agent conjugated to a biotin moiety of claim 1, wherein the bioactive agent is a polypeptide selected from the group consisting of the amino acid sequences represented by SEQ ID NO:1 to SEQ ID NO:

7.

8. 2. The bioactive agent conjugated to a biotin moiety of claim 1, wherein the bioactive agent is a protein composed of two amino acid chains represented by SEQ ID NO:15 and SEQ ID NO:16, respectively.

9. 2. The bioactive substance bound to a biotin moiety of claim 1, wherein the bioactive substance is a protein composed of two amino acid chains represented by SEQ ID NO:15 and SEQ ID NO:16, respectively, and linked via disulfide bonds between cysteine ​​at position 6 of SEQ ID NO:15 and cysteine ​​at position 11 of SEQ ID NO:15, between cysteine ​​at position 7 of SEQ ID NO:15 and cysteine ​​at position 7 of SEQ ID NO:16, and between cysteine ​​at position 20 of SEQ ID NO:15 and cysteine ​​at position 19 of SEQ ID NO:

16.

10. 2. The bioactive agent conjugated to a biotin moiety of claim 1, wherein the bioactive agent is a protein composed of two amino acid chains represented by SEQ ID NO:17 and SEQ ID NO:16, respectively.

11. 2. The bioactive substance bound to a biotin moiety according to claim 1, wherein the bioactive substance is a protein composed of two amino acid chains represented by SEQ ID NO:17 and SEQ ID NO:16, respectively, and linked via disulfide bonds between cysteine ​​at position 6 of SEQ ID NO:17 and cysteine ​​at position 11 of SEQ ID NO:17, between cysteine ​​at position 7 of SEQ ID NO:17 and cysteine ​​at position 7 of SEQ ID NO:16, and between cysteine ​​at position 20 of SEQ ID NO:17 and cysteine ​​at position 19 of SEQ ID NO:

16.

12. The biotin moiety is an —NH of a lysine amino acid of the polypeptide or protein. ＋ ３ group, -NH ２ 6. A biologically active substance bound to a biotin moiety, as claimed in claim 4 or claim 5, which is bound to a group.

13. 2. The bioactive agent conjugated to a biotin moiety according to claim 1, wherein the bioactive agent is a polypeptide in which at least one of the amino acids of an inactive region of the polypeptide selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 1-7 is replaced by a cysteine ​​amino acid or a cysteine ​​amino acid is inserted at said at least one position, and the biotin moiety is conjugated to the -SH group of the cysteine ​​amino acid.

14. 2. The bioactive agent conjugated to a biotin moiety of claim 1, wherein the bioactive agent is a polypeptide selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 8-14.

15. 2. The biologically active agent conjugated to a biotin moiety of claim 1, wherein X is maleimide, N-hydroxysuccinimide, an aldehyde, or an amine.

16. 2. The biologically active agent conjugated to a biotin moiety of claim 1, wherein Z is linked to B via --NH--.

17. 2. The biologically active agent conjugated to a biotin moiety of claim 1, wherein Z comprises at least one glycerol and at least one polyethylene glycol.

18. - (CH ２ ) ３ NH- is 【Chemistry 25】 2. The biologically active agent conjugated to a biotin moiety of claim 1, further conjugated to:

19. 2. The biologically active agent conjugated to a biotin moiety of claim 1, wherein T is an amine.

20. The biotin moiety is 【Chemistry 26-13】 【Chemistry 26-2】 【Chemistry 26-3】 【Chemistry 26-4】 【Chemistry 26-5】 【Chemistry 26-6】 【Chemistry 26-7】 【Chemistry 26-8】 【Chemistry 26-9】 【Chemistry 26-10】 【Chemistry 26-11】 【Chemistry 26-12】 2. The bioactive agent conjugated to a biotin moiety of claim 1, selected from the group consisting of:

21. The biotin moiety is 【Chemistry 35】 and the biologically active substance is a polypeptide having the amino acid sequence of SEQ ID NO: 12; A biologically active agent conjugated to a biotin moiety, wherein said biotin moiety is attached to the thiol group or the N-terminus of the cysteine ​​amino acid at position 40 of SEQ ID NO:

12.

22. The biotin moiety is 【Chemical 36】 and the biologically active substance is a polypeptide having the amino acid sequence of SEQ ID NO: 12; A biologically active agent conjugated to a biotin moiety, wherein said biotin moiety is attached to the thiol group or the N-terminus of the cysteine ​​amino acid at position 40 of SEQ ID NO:

12.

23. The biotin moiety is 【Chemical 37】 and the biologically active substance is a polypeptide having the amino acid sequence of SEQ ID NO: 12; A biologically active agent conjugated to a biotin moiety, wherein said biotin moiety is attached to the thiol group or the N-terminus of the cysteine ​​amino acid at position 40 of SEQ ID NO:

12.

24. The biotin moiety is 【Chemical Formula 38】 and the biologically active substance is a polypeptide having the amino acid sequence of SEQ ID NO: 12; A biologically active agent conjugated to a biotin moiety, wherein said biotin moiety is attached to the thiol group or the N-terminus of the cysteine ​​amino acid at position 40 of SEQ ID NO:

12.

25. 25. An orally administrable composition in a mammal, comprising a biologically active agent attached to a biotin moiety according to any one of claims 1 to 24, and one or more excipients.

26. a) a biologically active substance conjugated to a biotin moiety according to any one of claims 1 to 24; b) instructions for administering the bioactive agent to a human; and Kit including:

27. A composition comprising a biologically active agent attached to a biotin moiety according to any one of claims 1 to 24 for treating a disease in humans.

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