Physiological active materials conjugate with biotin moiety, fatty acid moiety or a combination thereof

Abstract

The present invention relates to a bioactive substance conjugate in which a biotin moiety, a fatty acid moiety, or a combination thereof is combined with a bioactive substance, and more specifically, to a bioactive substance conjugate in which a biotin moiety and a fatty acid moiety are combined. As the biotin moiety and the fatty acid moiety according to the present invention are combined, the bioactive substance has an excellent oral absorption rate in the body and excellent pharmacokinetic effects.

Description

Technology Field

[0001] The present invention relates to a bioactive conjugate comprising a novel biotin moiety, a fatty acid moiety, or a combination thereof, and more specifically, to a bioactive conjugate comprising a biotin moiety, a fatty acid moiety, or a combination thereof having excellent oral absorption rate in the body and excellent pharmacokinetics, and more specifically, to a bioactive conjugate comprising a biotin moiety and a fatty acid moiety. Background Technology

[0003] For a drug to function effectively, high bioavailability must be ensured. Bioavailability refers to the extent to which a drug is utilized at the target site after administration, and this level varies depending on the method of administration and the target environment. Drugs can be lost or degraded during the process of delivery from the site of administration to the target, and this varies depending on the method of administration.

[0004] Generally, drug delivery for therapeutic agents containing proteins, polypeptides, etc., is divided into parenteral and oral administration methods. Parenteral administration methods include intravenous injection, intramuscular injection, subcutaneous injection, and sublingual administration, while oral administration refers to the ingestion of drugs by mouth. Most therapeutic agents, such as proteins and polypeptides, are administered via parenteral methods considering bioavailability, target environment, and delivery process, and parenteral administration is known to produce direct and rapid effects. However, parenteral administration can cause patient pain or discomfort, and side effects such as injection infection or air embolism may occur depending on the route. In contrast, oral administration has the advantage of being convenient and providing sustained effects because it involves direct administration through the mouth. Accordingly, many pharmaceutical companies have continued to attempt to administer therapeutic agents via oral methods; however, oral administration presents a problem in that it requires resistance to acidic environments and enzymatic degradation as the drug passes through the digestive system. In particular, proteins and peptides are known to have a low bioavailability of about 0.1% when administered orally.

[0005] To address these issues associated with oral administration, attempts are being made to enhance drug delivery by manufacturing separate formulations using surfactants and absorption enhancers, or by methods such as miniaturizing drug particles and controlling the frequency of administration. In this regard, major pharmaceutical companies are developing oral insulin and oral GLP-1 analogs, and various other research and development efforts are underway for the oral administration of products such as oral interferon alpha. However, peptide and protein drugs are difficult to administer orally, and while various attempts are being made to resolve this, no definitive solution has been found to date. The problem to be solved

[0007] The object of the present invention is to provide a bioactive conjugate comprising a biotin moiety, a fatty acid moiety, or a combination thereof, which exhibits excellent oral absorption in the body and excellent pharmacokinetic effects. More specifically, the invention provides a bioactive conjugate comprising a biotin moiety and a fatty acid moiety. means of solving the problem

[0009] One aspect of the present invention provides a bioactive substance conjugate combined with a biotin moiety, a fatty acid moiety, or a combination thereof, and a method for preparing the same.

[0010] Another aspect of the present invention provides a pharmaceutical composition comprising a bioactive substance conjugate combined with a biotin moiety, a fatty acid moiety, or a combination thereof.

[0011] Another aspect of the present invention provides a composition for oral administration comprising a bioactive substance conjugate combined with a biotin moiety, a fatty acid moiety, or a combination thereof.

[0012] One specific aspect of the present invention provides a bioactive conjugate comprising a biotin moiety and a fatty acid moiety, and a method for preparing the same.

[0013] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of diabetes, obesity, fatty liver disease, irritable bowel syndrome, neurodegenerative disease, bone disease, osteoporosis, human growth hormone deficiency, anticancer or non-alcoholic fatty liver disease, comprising a bioactive substance conjugate combined with a biotin moiety, a fatty acid moiety, or a combination thereof. Effects of the invention

[0015] A bioactive substance conjugate combined with a biotin moiety, a fatty acid moiety, or a combination thereof according to one embodiment of the present invention has excellent effects due to the combination of water-soluble biotin. Specifically, it has improved oral absorption rate, improved pharmacokinetic effects, protection against degradation of the bioactive substance by enzymes, promotion of intestinal membrane permeation of the bioactive substance, or active transport absorption effects through a sodium-dependent multivitamin transporter.

[0016] In addition, a bioactive conjugate comprising a biotin moiety and a fatty acid moiety according to one embodiment of the present invention exhibits superior effects in terms of each of the above effects compared to a conjugate comprising only a biotin moiety or a fatty acid moiety. Brief explanation of the drawing

[0018] FIG. 1 is a purified chromatogram of conjugates 14 to 17 according to one embodiment of the present invention. FIG. 2 is a purified chromatogram of conjugates 18 to 19 according to one embodiment of the present invention. FIG. 3 is a chromatogram of the final material of conjugates 20 and 24 according to one embodiment of the present invention. Figure 4 is a figure showing changes in blood glucose after glucose administration of conjugates 20 and 24 according to one embodiment of the present invention. FIG. 5 is a chromatogram of the final material of conjugates 51 to 52 according to one embodiment of the present invention. FIG. 6 is a purified chromatogram of conjugates 53 to 54 according to one embodiment of the present invention. FIG. 7 is a figure showing changes in blood glucose after administering glucose of conjugates 53 and 54 according to one embodiment of the present invention. FIG. 8 is a purified chromatogram of conjugates 55 to 59 according to one embodiment of the present invention. FIG. 9 is a chromatogram of the final material of conjugate 56 according to one embodiment of the present invention. FIG. 10 is a figure showing the change in body weight after 2 weeks of subcutaneous administration of conjugates 58 to 59 according to one embodiment of the present invention. FIG. 11 is a purified chromatogram of conjugates 60 to 64 according to one embodiment of the present invention. FIG. 12 is a figure showing the feed intake after oral administration of conjugates 33, 36, 39, 42, 61, 62, 63 and 64 according to one embodiment of the present invention. FIG. 13 is a purified chromatogram of conjugate 66 according to one embodiment of the present invention. FIG. 14 is a figure showing the blood glucose control ability after oral administration of conjugates 65 and 66 according to one embodiment of the present invention. FIG. 15 is a purified chromatogram of conjugate 69 according to one embodiment of the present invention. FIG. 16 is a figure showing the intracellular accumulation of conjugates 68 and 69 according to one embodiment of the present invention. Specific details for implementing the invention

[0019] Hereinafter, embodiments and examples of the present invention are described in detail so that a person skilled in the art to which the present invention pertains can easily practice the present invention.

[0020] However, the present invention may be implemented in various different forms and is not limited to the embodiments and examples described herein. Throughout the specification of the present invention, when a part is described as “comprising” a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0021] Throughout the specification of this invention, terms of degree such as “about,” “substantially,” etc., are used to mean at or near the stated value when inherent manufacturing and material tolerances are presented in the stated meaning, and are used to prevent unscrupulous infringers from unfairly exploiting the disclosure in which precise or absolute values ​​are mentioned to aid in understanding the invention. Throughout the specification of this invention, terms of degree such as “step” or “step of” do not mean “step for”.

[0022] Throughout the specification of the present invention, the term “combination thereof” included in a Markush-style expression means one or more mixtures or combinations selected from the group consisting of the components described in the Markush-style expression, and means including one or more selected from the group consisting of said components. Throughout the specification of the present invention, the description “and / or B” means “and B, or A or B”.

[0024] One aspect of the present invention provides a bioactive substance conjugate combined with a biotin moiety, a fatty acid moiety, or a combination thereof, and a method for preparing the same. A specific aspect of the present invention provides a bioactive substance conjugate combined with a biotin moiety and a fatty acid moiety, and a method for preparing the same.

[0025] Generally, peptide and protein drugs belong to BCS (Biopharmaceutical Classification System) Class 3, which is characterized by high water solubility and restricted absorption sites in the gastrointestinal tract. Peptide and protein drugs possess high hydrophilicity and large molecular weight; they can be degraded by gastric acid with a low pH and are attacked by enzymes such as trypsin, resulting in low intestinal absorption rates. Generally, the oral bioavailability (BA) of peptide and protein drugs is around 0.1%, making it difficult to use them in pharmaceutical compositions. To address these issues, technology utilizing enteric capsules to pass through the stomach is employed, but it has limitations in that it cannot fundamentally improve the absorption rate of peptides and proteins.

[0026] In contrast, a bioactive substance conjugate combined with a biotin moiety, a fatty acid moiety, or a combination thereof according to one embodiment of the present invention can increase intestinal membrane permeability, thereby promoting absorption in the intestine.

[0027] In addition, a bioactive conjugate combined with a biotin moiety, a fatty acid moiety, or a combination thereof according to one embodiment of the present invention may exhibit excellent pharmacokinetic effects.

[0028] In addition, a bioactive substance conjugate combined with a biotin moiety, a fatty acid moiety, or a combination thereof according to one embodiment of the present invention can prevent the degradation of bioactive substances, such as peptides, by enzymes, and ultimately, the bioactive substances can penetrate the intestinal membrane and promote absorption in the intestine.

[0029] In addition, a bioactive conjugate combined with a biotin moiety, a fatty acid moiety, or a combination thereof according to one embodiment of the present invention can be absorbed by active transport through a sodium-dependent multivitamin transporter by being combined with biotin, which is a type of water-soluble vitamin.

[0030] In addition, a biotin moiety, a fatty acid moiety, or a combination thereof according to one embodiment of the present invention may be bound to an active or inactive site of a physiologically active substance, and accordingly, may not inhibit the activity of the physiologically active substance.

[0032] In the present invention, "unsubstituted or substituted" means that it can be unsubstituted or substituted, and "substituted" means having one or more substituents, wherein the substituents refer to a chemical portion that is covalently bonded or fused with any atom of a main group such as alkylene, heteroalkylene, etc.

[0033] In the present invention, "halo" means fluorine, chlorine, bromine, iodine, etc.

[0034] In the present invention, "alkyl" refers to a monovalent portion obtained by removing a hydrogen atom from a carbon atom of an aliphatic or alicyclic, saturated or unsaturated hydrocarbon compound, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, n-propyl, n-butyl, n-pentyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl, etc.

[0035] In the present invention, “heteroalkyl” means an alkyl containing one or more heteroatoms, wherein the heteroatom is positioned at any one carbon atom of the alkyl, thereby replacing C, CH, CH2, or CH3.

[0036] In the present invention, "alkylene" refers to a divalent portion obtained by removing a hydrogen atom from a carbon atom of an aliphatic or alicyclic, saturated or unsaturated hydrocarbon compound.

[0037] In the present invention, “alkenylene” refers to a monovalent portion obtained by removing a hydrogen atom from a carbon atom of an aliphatic or alicyclic, saturated or unsaturated hydrocarbon compound.

[0038] In the present invention, “heteroalkylene” means an alkylene comprising one or more heteroatoms.

[0039] In the present invention, "aryl" refers to a monovalent portion obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound having a ring atom. For example, "C 5-10 "Aryl" refers to a monovalent portion obtained by removing hydrogen atoms from the aromatic ring atoms of an aromatic compound, having 5 to 10 ring atoms of carbon. Examples of aryls include groups derived from benzene, acenaphthene, fluorene, phenalene, acephenanthrene, and aceanthrene.

[0040] In the present invention, "heteroaryl" is an aryl comprising one or more heteroatoms, such as pyridine, pyrimidine, benzothiophene, furyl, dioxalanyl, pyrrolyl, oxazolyl, pyridyl, pyridazinyl, pyrimidinyl, isobenzofuran, indole, isoindole, indolizine, indoline, isoindolin, purine, benzodioxane, quinoline, isoquinoline, quinolizine, benzoxazine, benzodiazine, pyridopyridine, quinoxaline, quinazolin, cinnoline, phthalazine, naphthiridine, pteridine, ferrimidine, pyridoindole, oxantrene, phenoxatin, phenazine, phenoxazine, etc.

[0041] In the present invention, “arylene” refers to a divalent portion obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound having a ring atom.

[0042] In the present invention, “heteroarylene” means arylene containing one or more heteroatoms.

[0043] In the present invention, "alkenyl" is an alkyl having one or more carbon-carbon double bonds, such as vinyl (-CH=CH2), 1-propenyl (-CH=CHCH3), isopropenyl, butenyl, pentenyl, hexenyl, etc.

[0044] In the present invention, "alkynyl" is an alkyl group having one or more carbon-carbon triple bonds, examples of which include ethinyl and 2-propynyl.

[0045] In the present invention, where part of the general formula is defined as a specific compound, it includes a form in which the said compound is combined with other components.

[0047] According to one embodiment of the present invention, the biotin moiety can be represented by the following general formula A.

[0048] [General Formula A]

[0049]

[0050] In the above general formula A,

[0051] X is a functional group capable of binding to physiologically active substances, and

[0052] Y is a spacer, and

[0053] Z is a binding unit, and

[0054] B can be represented by the following chemical formula A-1, and

[0055] [Chemical Formula A-1]

[0056]

[0057] Z is of chemical formula A-1. Connected to,

[0058] T is the terminal stage, and

[0059] m is an integer from 1 to 10, and

[0060] n is an integer of 0 or 1 to 10, and when n=0, Y is directly coupled to B or T, and

[0061] p is an integer of 0 or 1.

[0063] According to one embodiment of the present invention, in the general formula A, X is a functional group capable of being combined with a physiologically active substance. Although not limited thereto, the functional group may include, for example, maleimide, succinimide, N-hydroxysuccinimide, aldehyde, carboxyl, carboxyl ester, succinimidyl ester, tetrafluorophenyl, -O-tetrafluorophenyl (TFP, 2,3,5,6-tetrafluorophenyl), tetrafluorophenyl ester, pentafluorophenyl (PFP), pentafluorophenyl ester, -O-benzotriazole, benzotriazole, sulfotetrafluorophenyl (STP), sulfodichlorophenyl (SDP), nitrophenol, nitrophenyl carbonate (NPC), etc.

[0064] In one embodiment of the present invention, the structure of the functional group X may be maintained, detached, or modified when combined with a physiologically active substance.

[0065] The above Y may be a structure that acts as a spacer and possesses cleavage properties within the body. Although not limited thereto, for example, Y may include a direct bond, or substituted or unsubstituted alkylene, -O-, -C(O)NR-, -C(O)O-, or -C(O)-, -NR-, -NOR-, etc. More specifically, Y may include a direct bond, or substituted or unsubstituted C within the structure of Y. 1-50 Linear alkylene, substituted or unsubstituted C 1-50 Non-linear alkylene, substituted or unsubstituted C 1-50 Linear heteroalkylene, substituted or unsubstituted C 1-50 Non-linear heteroalkylene, substituted or unsubstituted C 1-50 Aryllene, substituted or unsubstituted C 1-50 It may include one or more from the group consisting of heteroarylene, -O-, -C(O), -C(O)NR-, -C(O)O-, -S-, -NR-, or -NOR-, wherein R is hydrogen, substituted or unsubstituted C 1-50Alkyl, substituted, or unsubstituted C 1-50 Aryl, or ethylene glycol repeating unit (-(CH2CH2O) n -, n can be an integer between 1 and 20 inclusive.

[0066] The above Z is a binding unit that can be combined with B, and, although not limited thereto, may include, for example, an amino acid, polypeptide, alkylene, amine, or polyamidoamine structure.

[0067] In addition, but not limited to, for example, the above amino acids may include lysine, 5-hydroxylysine, 4-oxalysine, 4-thialysine, 4-selenalysine, 4-thiahomolysine, 5,5-dimethyllysine, 5,5-difluorolysine, trans-4-dehydrolysine, 2,6-diamino-4-hexinoic acid, cis-4-dehydrolysine, 6-N-methyllysine, diminopimelic acid, ornithine, 3-methylornithine, α-methylornithine, citrulline, homocitrulline, arginine, aspartate, asparagine, glutamate, glutamine, histidine, ornithine, proline, serine, or threonine.

[0068] In the present invention, when n is 0, B or T can be directly coupled with X or Y (spacer).

[0069] In the present invention, the above T is a terminal group, which is not limited thereto but may be, for example, hydrogen or NH2.

[0070] In the present invention, when p is 0, B may be a terminal.

[0071] In the present invention, the XY can together form a bioactive substance binding site.

[0072] According to one embodiment of the present invention, in the general formula A, m may be an integer from 1 to 10, specifically an integer from 1 to 8, from 1 to 5, or from 1 to 4.

[0074] In one aspect of the present invention, X is maleimide, succinimide, N-hydroxysuccinimide, succinimidyl succinate, succinimidyl glutarate, succinimidyl methyl ester, succinimidyl pentyl ester, succinimidyl carbonate, p-nitrophenyl carbonate, aldehyde, amine, thiol, oxyamine, iodoacetamide, aminoxyl, hydrazide, hydroxy, propionate, pyridyl, alkyl halide, vinylsulfone, carboxyl, hydrazide, halogen acetamide, C 2-5 Alkinyl, C 6-20 Aryl disulfide, C 5-20 It can be selected from the group consisting of heteroaryl disulfides, isocyanates, thioesters, iminoesters, and derivatives thereof.

[0075] In one embodiment of the present invention, X is maleimide, N-hydroxysuccinimide, succinimidyl carbonate, p-nitrophenyl carbonate, thiol, aminoxyl, aldehyde, or amine.

[0076] In one embodiment of the present invention, X is maleimide, N-hydroxysuccinimide, aldehyde, or amine.

[0078] In one aspect of the present invention, Y is a linear or branched C that is absent, substituted, or unsubstituted. 1-50 Alkylene, substituted or unsubstituted linear or branched C 1-50 Heteroalkylene, substituted or unsubstituted C 6-50 Aryllene, or substituted or unsubstituted C 6-50 It is a heteroarylene and, when substituted, comprises one or more selected from the group consisting of =O, -C(O)NH2, -OH, -COOH, -SH, =NH and -NH2.

[0079] In one embodiment of the present invention, Y comprises -C(O)-. In one embodiment of the present invention, Y comprises -C(O)NH-.

[0080] In one aspect of the present invention, the Y is a substituted linear or branched C 1-50 It is a heteroalkylene and contains at least one -C(O)-.

[0081] In one embodiment of the present invention, the Y is -(C(O)) q -(CH2) r -(C(O)NH) s -(CH2) r -(OCH2CH2) t -(C(O)) q - and, where q, r, s, and t are selected independently, q and s are 0 or 1, r is an integer from 1 to 20, and t is an integer from 0 to 20.

[0082] In one embodiment of the present invention, the Y is -(CH2) r C(O)NHNH-, where r is an integer from 1 to 20.

[0083] In one aspect of the present invention, the Y is -C(O)-(OCH2CH2) u -NH- is included as a repeating unit, where u is an integer from 1 to 20.

[0084] In one aspect of the present invention, the Y is -C(O)-(OCH2CH2) u -NH- is included as a repeating unit, where u is an integer from 2 to 4.

[0085] In one embodiment of the present invention, Y comprises amino acids as part of its composition.

[0086] In one specific embodiment of the present invention, Y comprises glutamic acid, glutamine, glycine, isoleucine, or lysine as part of its composition, wherein each amino acid may exist in a combined form.

[0087] In one specific embodiment of the present invention, the Y comprises glutamic acid or lysine as part of its composition.

[0088] In one embodiment of the present invention, Y comprises fatty acids as part of its composition.

[0089] In one specific embodiment of the present invention, the Y is C 12-24 It contains fatty acids, where the fatty acids exist in a bound form.

[0090] In one embodiment of the present invention, Y is a direct connection.

[0092] In one embodiment of the present invention, Z is any one of the following and can be selected independently.

[0093] a) to form an amino acid or a derivative thereof, together with or apart from X;

[0094] b) Substituted or unsubstituted linear or branched C 1-50 It is a heteroalkylene, and

[0095] In the case of substitution here, it includes one or more selected from the group consisting of =O, -C(O)NH2, -OH, -COOH, -SH, =NH, and -NH2.

[0096] In one embodiment of the present invention, Z is connected to B through -NH-.

[0097] In one embodiment of the present invention, Z is a hydrophilic amino acid or a derivative thereof.

[0098] In one specific embodiment of the present invention, Z may be selected from the group consisting of lysine, arginine, histidine, glutamine, asparagine, threonine, cysteine, serine, and derivatives thereof.

[0099] In one embodiment of the present invention, Z comprises one or more glycerols, one or more polyethylene glycols, or a combination thereof.

[0101] In one aspect of the present invention, the Z is Includes, represents a joint site, and on one or more of the said joint sites One or more are combined, where u is an integer from 1 to 20.

[0102] In one aspect of the present invention, the Z is Includes, -(CH2)3NH- is further bonded to it.

[0104] In one aspect of the present invention, T is an amine, C 1-8 Alkyl, C 1-8 It can 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.

[0105] In one specific embodiment of the present invention, T is an amine.

[0107] In one aspect of the present invention, the biotin moiety is

[0108] , , , , , , , , , , , , , , , and It is selected from a group composed of

[0110] According to one embodiment of the present invention, the fatty acid moiety can be represented by the following general formula B.

[0111] [General Formula B]

[0112] X'-Y'-W

[0113] In the above formula,

[0114] X' is a functional group capable of binding to the above-mentioned physiologically active substance, and

[0115] Y' is a spacer, and

[0116] W is a fatty acid.

[0118] In this specification, fatty acids include carboxylic acids having long aliphatic chains that are saturated or unsaturated, but are not limited thereto, examples of which include caprylic acid, lauric acid, palmitic acid, stearic acid, arachidic acid, cerotic acid, myristoleic acid, palmitoleic acid, oleic acid, linoleic acid, alpha-linolenic acid, etc., which are types of saturated fatty acids.

[0119] According to one embodiment of the present invention, in the general formula B, X' is a functional group capable of binding to a physiologically active substance. Here, X' is identical to X in the general formula A. Accordingly, in one embodiment of the present invention, the structure of the functional group X' may be maintained, detached, or modified when binding to a physiologically active substance.

[0120] According to one embodiment of the present invention, in the general formula B, W may correspond to a fatty acid. Here, the fatty acid includes simple fatty acids and includes all modifiable forms of fatty acids, such as modifications, additions, deletions, etc.

[0122] In one aspect of the present invention, Y' is identical to Y in the general formula A. Accordingly, in one embodiment of the present invention, the spacer Y' corresponds to a direct bond, or C that is substituted or unsubstituted within the structure of Y. 1-50Linear alkylene, substituted or unsubstituted C 1-50 Non-linear alkylene, substituted or unsubstituted C 1-50 Linear heteroalkylene, substituted or unsubstituted C 1-50 Non-linear heteroalkylene, substituted or unsubstituted C 1-50 Aryllene, substituted or unsubstituted C 1-50 It may include one or more from the group consisting of 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 unit (-(CH2CH2O) n -, n can be an integer between 1 and 20 inclusive.

[0123] In one aspect of the present invention, the W is a substituted or unsubstituted linear or branched C 1-60 Alkylene, substituted or unsubstituted linear or branched C 1-60 Alkenylene, substituted or unsubstituted linear or branched C 1-60 Heteroalkylene, or substituted or unsubstituted linear or branched C 1-60 It is a heteroalkenylene, and when substituted, it may be substituted with one or more selected from the group consisting of =O, -C(O)NH2, -OH, -COOH, -SH, =NH, -NH2, and halo.

[0124] In one aspect of the present invention, the W is one or more substituted C 12-24 Alkylene or one or more substituted C 36-48 It is a heteroalkylene and may include =O or -COOH when substituted.

[0125] In one specific embodiment of the present invention, the one or more substituted Ws are substituted or unsubstituted Cs. 12-24 It is a saturated fatty acid and contains -COOH when substituted.

[0127] In one embodiment of the present invention, the fatty acid moiety may have the chemical formula of the following general formula B1.

[0128] [General Formula B1]

[0129] X'1-Y'-C(O)-F1

[0130] In the above formula,

[0131] X'1 is maleimide, N-hydroxysuccinimide, aldehyde, amine, tetrafluorophenyl ester, or nitrophenol, and

[0132] Y' is a spacer, and

[0133] F1 is C 6-28 It is a substituted or unsubstituted linear or branched alkylene, or a substituted or unsubstituted linear or branched heteroalkylene.

[0135] According to one embodiment of the present invention, in the general formula B1, X'1 may have the same meaning as X in the general formulas A and B. Accordingly, in one embodiment of the present invention, the structure of the functional group X'1 may be maintained, detached, or modified when combined with a physiologically active substance.

[0136] In addition, in the above general formula B1, Y' may have the same meaning as Y in the above general formulas A and B.

[0137] In one aspect of the present invention, Y' is a substituted or unsubstituted C 6-50 It is a linear or branched heteroalkylene, and when substituted, it comprises one or more selected from the group consisting of =O, -C(O)NH2, -OH, -COOH, -SH, =NH and -NH2.

[0138] In one embodiment of the present invention, the Y' may include -(CH2CH2O)- as a repeating unit.

[0139] In one aspect of the present invention, the Y' is -C(O)-(OCH2CH2) u -NH- is included as a repeating unit, where u is an integer from 1 to 20.

[0140] In one specific embodiment of the present invention, the Y' is -C(O)-(OCH2CH2) u -NH- is included as a repeating unit, where u is an integer from 2 to 4.

[0141] In one aspect of the present invention, Y' comprises an amino acid or a derivative thereof as part of its composition.

[0142] In one specific embodiment of the present invention, the Y' comprises glutamic acid, glutamine, glycine, isoleucine, or lysine as part of its composition, wherein each amino acid may exist in a combined form.

[0143] In one specific embodiment of the present invention, the Y' comprises glutamic acid or lysine as part of its composition.

[0145] In one aspect of the present invention, F1 is a substituted or unsubstituted C 10-28 It can be a linear or branched alkylene.

[0146] In one specific embodiment of the present invention, the one or more substituted Ws are substituted or unsubstituted Cs. 12-24 It is a saturated fatty acid and contains -COOH when substituted.

[0147] In one specific embodiment of the present invention, the F1 is -(CH2) v -COOH, and v is an integer from 10 to 20.

[0148] In one specific embodiment of the present invention, the F1 is -C(O)-(CH2) v -COOH, and v is an integer from 10 to 20.

[0150] In one specific embodiment of the present invention, the fatty acid moiety is , , , , , , , , , , , , , , and It can be selected from a group consisting of.

[0152] According to one embodiment of the present invention, a biotin moiety and / or a fatty acid moiety and a bioactive substance may be formed by various bonds. The functional group of the biotin moiety or the fatty acid moiety may be formed by bonding with the functional group of the bioactive substance, and may be formed by, for example, a thiol-ether bond or an amide bond, although this is not limited thereto.

[0153] In one embodiment, the binding of the biotin moiety and the bioactive substance can be formed by a manner similar to Reaction Scheme 1 below. In Reaction Scheme 1 below, represents a bioactive substance containing a thiol group, and represents a reaction between a biotin moiety containing maleimide according to one embodiment of the present invention and the thiol group (-SH) of a cysteine ​​residue present in the bioactive substance.

[0154]

[0155] [Reaction Equation 1]

[0157] In one embodiment, the binding of the biotin moiety and the bioactive substance can be formed by a manner similar to Reaction Scheme 2 below. In Reaction Scheme 2 below, represents a bioactive substance containing an amine group, and represents a reaction between a biotin moiety containing N-hydroxysuccinimide according to one embodiment of the present invention and an amine group (-NH2) present in the bioactive substance.

[0158]

[0159] [Reaction Equation 2]

[0161] According to one embodiment of the present invention, the physiologically active substance may not be particularly limited.

[0162] In the present invention, a physiologically active substance refers to a substance that can be administered into the body for a specific purpose and causes a physiological or biochemical reaction within the body.

[0163] According to one embodiment of the present invention, the bioactive substance may be a substance used in a pharmaceutical composition. For example, it may be a substance used for the prevention or treatment of diabetes, obesity, fatty liver disease, irritable bowel syndrome, neurodegenerative disease, bone disease, osteoporosis, human growth hormone deficiency, anticancer or non-alcoholic fatty liver disease. This is exemplary, and since the indications may vary depending on the type of bioactive substance, it is not limited to the above indications.

[0164] According to one embodiment of the present invention, the physiologically active substance may be, for example, a polypeptide or a non-peptide polymer, but is not limited thereto. For example, it may be a polypeptide, a protein, a polysaccharide, or a derivative thereof, but is not limited thereto. But is not limited thereto, for example, the above-mentioned physiologically active substance may be 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, amylin, serotonin, rituximab, trastuzumab, uricase, tissue plasminogen activator, thymoglobin, vaccine, heparin or heparin analog, antithrombin III, filgrastim, pramlintide acetate, exenatide, eptifibatide, antivenin, IgG, IgM, HGH, thyroxine, blood coagulation factors VII and VIII, glycolipids acting as therapeutic agents, and derivatives thereof. there is.

[0166] According to one embodiment of the present invention, a biotin moiety can be bound to a physiologically active substance.

[0167] Biotin moiety can bind to a bioactive substance without inhibiting the biological activity of the bioactive substance, and accordingly, can have the same biological activity as the bioactive substance or enhanced biological activity.

[0168] Although not limited thereto, for example, the above-mentioned bioactive substance may contain an exposed -SH group, and a biotin moiety may be bound to the -SH group. In addition, the above-mentioned bioactive substance may contain an exposed -NH3 + Containing a group or -NH2 group, the exposed -NH3 +A biotin moiety can be bound to the group or -NH2 group.

[0169] According to one embodiment of the present invention, the biotin moiety can be bound by controlling the binding site with the physiologically active substance to avoid the site exhibiting activity.

[0170] In addition, according to one embodiment of the present invention, the fatty acid moiety can be directly bound to a physiologically active substance. Furthermore, a portion of the fatty acid moiety may be common to the biotin moiety. For example, in one aspect of the following examples, biotin moietyes B35 and B36 commonly include a fatty acid portion that is part of the fatty acid moiety. However, this is merely an example and is not limited thereto.

[0171] In addition, according to one embodiment of the present invention, the fatty acid moiety may be bound to a physiologically active substance and may be bound to a site different from the site to which the biotin moiety is bound.

[0172] In addition, the fatty acid moiety can be bound to the active or inactive site of a physiologically active substance in the same way as the biotin moiety, and can exhibit the same characteristics as the above.

[0173] According to one embodiment of the present invention, both the biotin moiety and the fatty acid moiety can be combined with a physiologically active substance, and a physiologically active substance conjugate in which both the biotin moiety and the fatty acid moiety are combined may exhibit superior oral absorption rate, pharmacokinetics, inhibition of enzymatic degradation, and intestinal membrane permeability compared to a conjugate in which only the biotin moiety or the fatty acid moiety is combined.

[0175] According to one embodiment of the present invention, the physiologically active substance may be glucagon, calcitonin, GLP-1, GLP-2, GIP, exendin-4, parathyroid hormone, insulin, amylin, human growth hormone, or a derivative thereof.

[0176] According to one embodiment of the present invention, the physiologically active substance may be a polypeptide having an amino acid sequence of any one of the following SEQ ID NOs 1 to 7 or a derivative thereof. Specifically, the physiologically active substances of the following SEQ ID NOs 1 to 7 each refer to a glucagon derivative (SEQ ID NO 1), GLP-1 (SEQ ID NO 2), GLP-2 (SEQ ID NO 3), GIP (SEQ ID NO 4), excendin-4 (SEQ ID NO 5), parathyroid hormone (SEQ ID NO 6), and glucagon (SEQ ID NO 7).

[0178] Sequence No. 1: H(Aib)QGTFTSDYSKYLDEQAAKEFVQWLMNT

[0179] Sequence No. 2: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR

[0180] Sequence No. 3 : HADGSFSDEMNTILDNLAARDFINWLIQTKITD

[0181] Sequence No. 4: YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ

[0182] Sequence No. 5: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS

[0183] Sequence No. 6: SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF

[0184] Sequence No. 7: HSQGTFTSDYSKYLDSRRAQDFVQWLMNT

[0186] In addition, the above physiologically active substance may be a protein having the amino acid sequences of SEQ ID NOs 15 and 16 below or a protein having the amino acid sequences of SEQ ID NOs 17 and 16.

[0187] Additionally, the above-mentioned bioactive substance may be a protein having the amino acid sequences of SEQ ID NOs. 15 and 16 below or a protein having the amino acid sequences of SEQ ID NOs. 17 and 16, wherein the protein is bound by disulfide bonds between the 6th and 11th cysteines of SEQ ID NO. 15 or 17, the 7th cysteine ​​of SEQ ID NO. 15 or 17 and the 7th cysteine ​​of SEQ ID NO. 16, and the 20th cysteine ​​of SEQ ID NO. 15 or 17 and the 19th cysteine ​​of SEQ ID NO. 16. Specifically, the protein having the amino acid sequences of SEQ ID NOs. 15 and 16 below or the bioactive substance having the amino acid sequences of SEQ ID NOs. 17 and 16 refers to insulin (SEQ ID NO. 15 (insulin A chain derivative) and 16 (insulin B chain) / SEQ ID NO. 17 (insulin A chain) and 16 (insulin B chain)).

[0189] Sequence No. 15 GIVEQCCTSICSLEQLENYCN

[0190] Sequence No. 16: FVNQHLCGSHLVEALYLVCGERGFFYTPKT

[0191] Sequence No. 17: GIVEQCCTSICSLYQLENYCN

[0193] According to one embodiment of the present invention, cysteine ​​can be substituted or inserted into a polypeptide to control the binding site with a biotin moiety.

[0194] Although not limited thereto, for example, one or more of the amino acids of a polypeptide selected from the group consisting of amino acid sequences represented by SEQ ID NOs 1 to 7 may be substituted or inserted with a cysteine ​​amino acid. In this case, a biotin moiety is attached to the -SH group of the cysteine ​​amino acid.

[0195] In addition, one or more of the amino acids of the polypeptide selected from the group consisting of the above amino acid sequence may be substituted or inserted with a lysine amino acid. At this time, a biotin moiety is attached to the -NH2 group of the lysine amino acid.

[0196] In addition, the polypeptide having the inserted cysteine ​​amino acid may be a polypeptide having any one of the amino acid sequences of SEQ ID NOs 8 to 14 below. Specifically, the physiologically active substances of SEQ ID NOs 8 to 14 below refer to physiologically active substances in which the cysteine ​​amino acid is substituted or inserted from the physiologically active substances of SEQ ID NOs 1 to 7 (for example, the physiologically active substance of SEQ ID NO. 8 is in which one or more of the amino acids of the physiologically active substance of SEQ ID NO. 1 are substituted with cysteine).

[0198] Sequence No. 8: H(Aib)QGTFTSDYSKYLDEQAAKEFVQWLMNTC

[0199] Sequence No. 9: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRC

[0200] Sequence No. 10: HADGSFSDEMNTILDNLAARDFINWLIQTKITDC

[0201] Sequence No. 11:

[0202] YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQC

[0203] Sequence No. 12: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC

[0204] Sequence No. 13: SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFC

[0205] Sequence No. 14: HSQGTFTSDYSKYLDSRRAQDFVQWLMNTC

[0207] According to one embodiment of the present invention, a portion of the polypeptide can be substituted to control the binding site with the biotin moiety.

[0208] In addition, according to one embodiment of the present invention, a lysine amino acid may be substituted or inserted into the polypeptide to control the binding site with the biotin moiety.

[0209] Although not limited thereto, for example, one or more of the amino acids of the amino acid sequence represented by SEQ ID NO. 5 may be substituted or inserted with a lysine amino acid.

[0210] In addition, although not limited thereto, for example, one or more of the amino acids of the amino acid sequence represented by SEQ ID NO. 5 may be substituted with 2-aminoisobutyric acid (2-Aminoisobutyric acid, Aib), and a lysine amino acid may be inserted. At this time, a biotin moiety is attached to the -NH2 group of the lysine amino acid.

[0211] In addition, the polypeptide in which a portion of the above polypeptide is substituted, or in which a lysine amino acid is substituted or inserted, may be a polypeptide having any one of the amino acid sequences of SEQ ID NOs 18 to 21 below. Specifically, the physiologically active substances of SEQ ID NOs 18 to 21 below refer to exendin-4 derivatives in which a portion of the amino acid is substituted or inserted from the physiologically active substance of SEQ ID NO. 5.

[0213] Sequence No. 18: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK

[0214] Sequence No. 19:

[0215] HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSKKK

[0216] Sequence No. 20:

[0217] H(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK

[0218] Sequence No. 21:

[0219] H(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSKKK

[0221] According to one embodiment of the present invention, the physiologically active substance may be a polypeptide having the amino acid sequence of SEQ ID NO. 22 below or a derivative thereof. The physiologically active substance of SEQ ID NO. 22 below refers to amylin.

[0223] Sequence No. 22: KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY

[0225] According to one embodiment of the present invention, a portion of the amino acid sequence represented by SEQ ID NO. 22 may be substituted or inserted to control the binding site with the biotin moiety.

[0226] Although not limited thereto, for example, one or more of the amino acids of the amino acid sequence represented by SEQ ID NO. 22 may be substituted with proline, aspartic acid, or arginine amino acids. Additionally, although not limited thereto, for example, one or more of the amino acids of the amino acid sequence represented by SEQ ID NO. 22 may be substituted or inserted with lysine amino acids.

[0227] In addition, the polypeptide having a portion of the amino acid represented by SEQ ID NO. 22 substituted or inserted may be a polypeptide having any one of the amino acid sequences of SEQ ID NOs 23 to 31 below. Specifically, the physiologically active substances of SEQ ID NOs 23 to 31 below refer to amylin derivatives having a portion of the amino acid substituted or inserted from the physiologically active substance of SEQ ID NO. 22.

[0229] Sequence No. 23: KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTYK

[0230] Sequence No. 24: KCNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTY

[0231] Sequence No. 25: KCNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTYK

[0232] Sequence No. 26: KCNTATCATQRLADFLRHSSPNFGAIPSSTNVGSRTY

[0233] Sequence No. 27: KCNTATCATQRLADFLRHSSPNFGAIPSSTNVGSRTYK

[0234] Sequence No. 28: KCNTATCATQRLADFLRHSSNNFGAIPSSTNVGSRTY

[0235] Sequence No. 29: KCNTATCATQRLADFLRHSSNNFGAIPSSTNVGSRTYK

[0236] Sequence No. 30: RCNTATCATQRLADFLRHSSNNFGAIPSSTNVGSKTY

[0237] Sequence No. 31: RCNTATCATQRLADFLRHSSNNFGAIPSSTNVGSKTYK

[0239] According to one embodiment of the present invention, the physiologically active substance may be a polypeptide having the amino acid sequence of SEQ ID NO. 32 below or a derivative thereof. Specifically, the physiologically active substance of SEQ ID NO. 32 below refers to an exendin-4 derivative.

[0241] Sequence No. 32: H(Aib)QGTFTSDKSKYLDERAAQDFVQWLLDGGPSSGAPPPS

[0243] According to one embodiment of the present invention, a portion of the amino acid sequence of SEQ ID NO. 32 may be deleted, substituted, or inserted to control the binding site with the biotin moiety.

[0244] Although not limited thereto, for example, one or more of the amino acids of the amino acid sequence represented by SEQ ID NO. 32 may be substituted with methionine, lysine, isoleucine, tryptophan, glycine, or an amino acid. Additionally, although not limited thereto, for example, one or more of the amino acids of the amino acid sequence represented by SEQ ID NO. 32 may be deleted.

[0245] In addition, the polypeptide having a portion of the amino acids represented by SEQ ID NO. 32 deleted, substituted, or inserted may be a polypeptide having any one of the amino acid sequences of SEQ ID NOs 33 to 37 below. Specifically, the physiologically active substances of SEQ ID NOs 33 to 37 below refer to exendin-4 derivatives obtained by deleting, substituting, or inserting a portion of the amino acids from the physiologically active substance of SEQ ID NO. 32.

[0247] Sequence No. 33: H(Aib)QGTFTSDKSKYLDERAAQDFVQWLMDGGPSSGAPPPS

[0248] Sequence No. 34: H(Aib)QGTFTSDKSKYLDKIAAQDFVQWLIDGGPSSGAPPPS

[0249] Sequence No. 35: H(Aib)QGTFTSDKSWYLDKIAAQDFVQWLLGGGPSSGAPPPS

[0250] Sequence No. 36: H(Aib)QGTFTSDKSWYLDERAAQDFVQWLMGGGPSSGAPPPS

[0251] Sequence No. 37: H(Aib)QGTFTSDKSKWLDKIAAQDFVQWLIGGGPSSGAPPPS

[0253] According to one embodiment of the present invention, one or more of the amino acids of the amino acid sequence represented by SEQ ID NO 12 may be substituted with 2-aminoisobutyric acid (Aib).

[0254] According to one embodiment of the present invention, a polypeptide having one or more amino acids of the amino acid sequence represented by SEQ ID NO. 12 substituted with 2-aminoisobutyric acid (Aib) and one or more amino acids substituted with Des-amino-His(h) may be a polypeptide having the amino acid sequences of SEQ ID NOs 38 to 39 below. Specifically, the physiologically active substance of SEQ ID NO. 38 or 39 below is an exendin-4 derivative in which an amino acid is substituted from the physiologically active substance of SEQ ID NO. 12. More specifically, the physiologically active substance of the amino acid sequence of SEQ ID NO. 39 below is a physiologically active substance in which one or more amino acids are substituted with Des-amino-His(h).

[0256] Sequence No. 38: H(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC

[0257] Sequence No. 39: h(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC

[0259] According to one embodiment of the present invention, one or more of the amino acids of the amino acid sequence represented by SEQ ID NO 8 may be substituted with lysine (Lys) or arginine (Arg).

[0260] A polypeptide in which one or more of the amino acids of the amino acid sequence represented by SEQ ID NO. 8 are substituted with lysine or arginine may be a polypeptide having the amino acid sequences of SEQ ID NOs 40 to 41 below. Specifically, the physiologically active substance of SEQ ID NO. 40 or 41 below refers to a glucagon derivative in which a portion of the amino acids of the physiologically active substance of SEQ ID NO. 8 are substituted.

[0262] Sequence No. 40: H(Aib)QGTFTSDYSKYLDEQAAKEFVQWLMNTK

[0263] Sequence No. 41: H(Aib)QGTFTSDYSKYLDEKRAKEFVQWLMNTC

[0265] According to one embodiment of the present invention, the physiologically active substance may be a polypeptide having the amino acid sequence of SEQ ID NO. 42 below or a derivative thereof. Specifically, the physiologically active substance of SEQ ID NO. 42 below refers to a human growth hormone derivative.

[0267] Sequence No. 42:

[0268] MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKISFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF

[0270] According to one embodiment of the present invention, a bioactive conjugate having a biotin moiety may covalently bond with one or more selected from the group consisting of peptides and non-peptide polymers, fatty acids, cholesterol, antibodies, antibody fragments, albumin and fragments thereof, nucleotides, fibronectin, transferrin, FcRn conjugated substances, saccharides, elastin, heparin and derivatives thereof, or form microspheres.

[0271] The above-mentioned 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, polyvinylethyl ether, PLA (polylactic acid), PLGA (polylactic-glycolic acid), lipid polymers, chitin, hyaluronic acid, and combinations thereof.

[0273] Another aspect of the present invention provides a method for preparing a bioactive substance combined with a biotin moiety, comprising the steps of: obtaining a biotin moiety; reacting the biotin moiety with a bioactive substance; and separating the bioactive substance combined with the biotin moiety after the reaction is completed.

[0274] In addition, another aspect of the present invention provides a method for preparing a conjugate of a bioactive substance combined with a biotin moiety and a fatty acid moiety, comprising the steps of: obtaining a biotin moiety; reacting the biotin moiety with a bioactive substance; reacting the biotin moiety with a fatty acid moiety after the reaction between the biotin moiety and the bioactive substance is completed; and separating the bioactive substance combined with the biotin moiety and the fatty acid moiety after the reaction is completed.

[0276] According to one embodiment of the present invention, in the step of obtaining the biotin moiety, the biotin moiety may be represented by the general formula A.

[0277] According to one embodiment of the present invention, the fatty acid moiety may be represented by the general formula B.

[0278] According to one embodiment of the present invention, in the step of obtaining the mixture, the reaction molar ratio of the biotin moiety to the physiologically active substance may be 0.5 or higher. Specifically, the reaction molar ratio of the biotin moiety to the physiologically active substance may be 0.5 to 30. An appropriate reaction molar ratio may be selected by considering the molecular structure, molecular weight, solubility, pH of the reaction solution, reaction temperature, reaction time, etc. of the biotin moiety.

[0279] According to one embodiment of the present invention, in the step of obtaining the mixture, the reaction molar ratio of the fatty acid moiety to the physiologically active substance may be 0.5 or higher. Specifically, the reaction molar ratio of the fatty acid moiety to the physiologically active substance may be 0.5 to 20. An appropriate reaction molar ratio may be selected by considering the molecular structure, molecular weight, solubility, pH of the reaction solution, reaction temperature, reaction time, etc. of the fatty acid moiety.

[0280] In addition, depending on the form of the bioactive substance, the reaction molar ratio of the biotin moiety to the bioactive substance in the step of obtaining the mixture may be 20 or more. Specifically, the reaction molar ratio of the biotin moiety to the bioactive substance may be 20 to 25.

[0281] In addition, depending on the form of the bioactive substance, the reaction molar ratio of the fatty acid moiety to the bioactive substance in the step of obtaining the mixture may be 6 or more. Specifically, the reaction molar ratio of the fatty acid moiety to the bioactive substance may be 6 to 12.

[0283] 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 organic solvent is not subject to any particular limitations, and a buffer solution commonly used in the art may be appropriately selected depending on the structure of the biotin moiety and the fatty acid moiety.

[0284] In one embodiment of the present invention, the temperature and time of the reaction step may be appropriately controlled according to the characteristics of the biotin moiety, fatty acid moiety, and bioactive substance used. Although not limited thereto, for example, it may be performed at 4°C for 3 hours or more, or at room temperature for a shorter time. This may be related to the degree of reactivity of the biotin moiety, fatty acid moiety, or combination thereof used. After a suitable reaction time has elapsed, the reaction may be stopped by lowering the pH of the reaction solution.

[0285] According to one embodiment of the present invention, a step of removing unreacted material may be performed after the reaction step. The method of removing unreacted material may be carried out by a method commonly used in the art. Although not limited thereto, it may be removed by dialysis, for example, using a suitable buffer solution, such as PBS (phosphate buffered saline).

[0286] According to one embodiment of the present invention, a purification step may be included after the separation step. The separation and purification steps may be performed using size exclusion chromatography, reverse-phase high-performance liquid chromatography, ion exchange chromatography, etc., but are not limited thereto.

[0288] Another aspect of the present invention is to provide a pharmaceutical composition comprising a bioactive substance conjugate combined with the biotin moiety, fatty acid moiety, or a combination thereof described above.

[0289] Another specific aspect of the present invention is to provide a pharmaceutical composition comprising a bioactive substance conjugate combined with the biotin moiety and fatty acid moiety described above.

[0290] Here, the use of the pharmaceutical composition may be determined according to the type of physiologically active substance. Additionally, the pharmaceutical composition may be a composition for oral administration.

[0291] According to one embodiment of the present invention, a pharmaceutical composition for the prevention or treatment of diabetes, obesity, fatty liver disease, irritable bowel syndrome, neurodegenerative disease, bone disease, osteoporosis, human growth hormone deficiency, anticancer or non-alcoholic fatty liver disease may be provided, comprising a bioactive substance conjugate combined with a biotin moiety, a fatty acid moiety, or a combination thereof.

[0292] According to one embodiment of the present invention, when the bioactive substance is GLP-1, GLP-2, GIP, insulin, amylin, or a derivative thereof, the conjugate may be used for the prevention or treatment of diabetes. Specifically, a conjugate comprising the bioactive substance of SEQ ID NO. 12 may be used for the prevention or treatment of diabetes. However, this is exemplary and is not limited thereto.

[0293] In addition, according to one embodiment of the present invention, when the bioactive substance is parathyroid hormone or a derivative thereof, the conjugate may be used for the prevention or treatment of bone disease. Specifically, a conjugate comprising the bioactive substance of SEQ ID NO. 6 may be used for the prevention or treatment of bone disease. However, this is exemplary and is not limited thereto.

[0294] According to one embodiment of the present invention, when the bioactive substance is hGH or a derivative thereof, the conjugate may be used for the prevention or treatment of human growth hormone deficiency. Specifically, a conjugate comprising the bioactive substance of SEQ ID NO. 42 may be used for the prevention or treatment of human growth hormone deficiency. However, this is exemplary and is not limited thereto.

[0296] Another aspect of the present invention is to provide a composition for oral administration comprising a bioactive substance conjugate combined with the biotin moiety, fatty acid moiety, or a combination thereof described above.

[0297] Another specific aspect of the present invention is to provide a composition for oral administration comprising a bioactive substance conjugate combined with the biotin moiety and fatty acid moiety described above.

[0298] A bioactive conjugate combined with a biotin moiety, a fatty acid moiety, or a combination thereof according to one embodiment of the present invention is combined with biotin, a type of water-soluble vitamin, so that it can be absorbed by active transport through a sodium-dependent multivitamin transporter, thereby permeating the intestinal membrane and promoting absorption in the intestine. More specifically, excellent effects are exhibited as both the biotin moiety and the fatty acid moiety are combined.

[0300] According to one embodiment of the present invention, a pharmaceutical composition containing a bioactive substance combined with the biotin moiety as an active ingredient may be formulated and administered in various oral or parenteral forms, but is not limited thereto.

[0301] In addition, according to one embodiment of the present invention, a pharmaceutical composition containing a bioactive conjugate combined with the biotin moiety, fatty acid moiety, or a combination thereof as an active ingredient may be formulated and administered in various oral or parenteral forms, but is not limited thereto.

[0302] When formulating, it can be prepared using diluents or excipients such as commonly used fillers, solubilizing aids, extenders, binders, wetting agents, disintegrants, surfactants, and absorption promoters.

[0303] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms may be prepared by mixing at least one excipient with the compound, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc.

[0304] In addition, lubricants such as magnesium styrate and talc are also used in addition to simple excipients. Liquid formulations for oral administration include suspensions, oral liquids, emulsions, and syrups; in addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as humectants, sweeteners, flavorings, and preservatives, may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Propylene glycol, polyethylene glycol (PEG), vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspension solvents.

[0305] In addition, calcium or vitamin D3 may be added to enhance efficacy as a treatment for proliferative or autoimmune diseases.

[0306] The dosage of a pharmaceutical composition according to one embodiment of the present invention may vary depending on the patient's weight, age, gender, health condition, diet, time of administration, method of administration, excretion rate, and severity of the disease, but generally, it may be administered once or in multiple doses per day within the range of an effective daily dosage. In addition, an effective dosage may be administered several times over one to two weeks.

[0308] The present invention will be explained in detail below through examples and experimental examples. However, the following examples and experimental examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples and experimental examples.

[0310] [Example]

[0311] Biotin Moisture Manufacturing

[0312] List of abbreviations

[0313] HBTU: 3-[Bis(dimethylamino)methyliumyl]-3H-benzotriazol-1-oxide hexafluorophosphate

[0314] DIEA: Ethyldiisopropylamine

[0315] HATU: 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate

[0316] DIC: Diisopropylcarbodiimide

[0317] HOBt:: 1-Hydroxybenzotriazole

[0318] MBHA: 4-Methylbenzhydrylamine hydrochloride

[0319] Fmoc: 9-Fluorenylmethyloxycarbonyl (9-Fluorenylmethoxycarbonyl)

[0320] DMF: Dimethylformamide

[0321] SPPS: Solid-phase peptide synthesis

[0322] HPLC: High-performance liquid chromatography

[0323] LCMS: Liquid Chromatography Mass Spectrometry

[0325] General SPPS method

[0326] In some cases, solid-phase synthesis of peptides can be improved by using a dipeptide protected in an amide bond with a group that can be cleaved under acidic conditions, for example, 2-Fmoc-oxy-4-methoxybenzyl or 2,4,6-trimethoxybenzyl. The Fmoc-protected amino acid derivatives used were the recommended standards: e.g., 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 supplied from Anaspec, Bachem, Iris Biotech, or Novabiochem Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, or Fmoc-Val-OH, etc. The N-terminal amino acid was Boc protected at the alpha-amino group. For example, Fmoc-8-amino-3,6-dioxoctanic acid, Fmoc-tranexamic acid, Fmoc-isonifekotic acid, Fmoc-Glu-OtBu, Fmoc-Lys(Fmoc)-OH supplied from Anaspec, Bachem, Iris Biotech, or Novabiochem were used.

[0328] Peptide synthesis using SPPS

[0329] Peptides can be synthesized in link amide MBHA resins using general Fmoc chemistry with HBTU / DIEA, HATU / DIEA, or DIC / HOBt as coupling reagents. The reactants and coupling reagents used in the synthesis include the following combinations.

[0330] # reactants coupling reagent 1 Fmoc-Lys (Biotin)-OH (1.5 eq ) HBTU (1.42 eq) and DIEA (3.0 eq) 2 Fmoc-Lys (Biotin)-OH (2.0 eq ) HBTU (1.9 eq) and DIEA (4.0 eq ) 3 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) propanoic acid (3.0 eq ) DIC (3.0 eq) and HOBt (6.0 eq) 4 2,2-dimethyl-4-oxo-3,7,10,13,16,19,22-heptaoxapentacosan-25-oic acid (2.0 eq ) HATU (1.9 eq) and DIEA (4.0 eq) 5 Fmoc-21-amino-4,7,10,13,16,19-hexaoxaheneicosanoic acid (2.0 eq ) HATU (1.9 eq) and DIEA (4.0 eq)

[0331] An exemplary protocol for the peptide synthesis process using SPPS includes the following: 1) Add DMF to a container containing link-amide MBHA resin and expand for 2 hours (sub: 0.68 mmol / g, 1.0 mmol, 1.47 g or 5 mmol, 7.35 g, sub: 0.68 mmol / g). 2) Add 20% piperidine / DMF and mix for 30 minutes. 3) Remove the solvent from steps 1) and 2), then wash with DMF (30 seconds x 5 times). 4) Add a reactant (one of reactants #1 to #5) and mix for 30 seconds, then add the coupling reagent corresponding to the reactant (one of coupling reagents #1 to #5) and perform nitrogen bubbling for 1 hour. 5) Add 20% piperidine / DMF and mix for 30 minutes.

[0332] The exemplary protocols of 1) to 5) above allow for repeated synthesis using the combination of reactants and coupling reagents of #1 to #5 one or more times. To remove Fmoc, a 20% piperidine / DMF solution was treated for 30 minutes.

[0334] General process for peptide purification and analysis

[0335] Unpurified peptides were dissolved in a suitable mixture of water, TFA, and ACN, purified using preparative HPLC, dried, and quantified. The purification conditions using preparative HPLC include those shown in Table 2 below.

[0336] Purification conditions menstruum ACN / H2O equipment SHIMADZU LC-8A, or Gilson GX-281 Mobile Phase A: H2O (0.075% TFA in H2O) B: CH3CN Gradient 15-35%-60 min. Retention time: 42 min, or 20-50%-60 min. Retention time: 45 min, or 5-35%-60 min. Retention time: 50 min Column Luna25*200mm, C18, 10um, 110A+Gemin150*30mm, C18, 5um, 110A, or Luna50*25mm, C18, 10um, 100Å+Gemini®50*50mm, C8, 5um, 110Å Flow Rate 80 mL / Min or 20 mL / Min Wavelength 220 / 254 nm Oven Temp. Room temperature

[0337] After purification using preparative HPLC, the characteristics of the final product were analyzed using analytical HPLC or LC-MS.

[0338] As a result of the analysis, a biotin moiety according to Table 3 below was obtained.

[0339] Biotin Moiety designation B1 N-Biotinoyl-N'-(6-maleiidohexanoyl)hydrazide B2 3-Maleimidopropionate-Lys(Biotin)-Lys(Biotin)-CONH2 B3 3-Maleimidopropionate-Lys(Biotin)-Lys(Biotin)-Lys(Biotin)-CONH2 B4 propionate-N-hydroxysuccinimide ester- PEG-Lys(Biotin)-Lys(Biotin)-Lys(Biotin)-CONH2 B5 3-Maleimidopropionate-PEG-Lys(Biotin)-Lys(Biotin)-Lys(Biotin)-CONH2

[0340] B1: ,

[0341] B2: ,

[0342] B3: ,

[0343] B4: ,

[0344] B5:

[0345] In addition, the following biotin moiety B6 to B37 were obtained through the protocols, purification, and analysis of the peptide synthesis using SPPS1) to 5). In Table 4 below, X, Y, Z, and B are included in the definition of General Formula A of this specification.

[0346] Biotin Moiety X Y Z Number of B (Biotin) B6 Aldehyde propane Lysine 2 B7 Maleimide butyrate Glycerol and PEG 2 B8 Maleimide butyrate Glycerol and PEG 2 B9 N-hydroxysuccinimide butyrate Lysine 2 B10 N-hydroxysuccinimide glutarate Glycerol and PEG 2 B11 Maleimide PEG12 Lysine 3 B12 N-hydroxysuccinimide PEG12 Lysine 3 B13 amine - Lysine 3 B14 Aldehyde pentane Lysine 2 B15 Maleimide adipate Glycerol and PEG 2 B16 Maleimide suberate Glycerol and PEG 2 B17 Maleimide sebacate Glycerol and PEG 2 B18 N-hydroxysuccinimide adipate Glycerol and PEG 2 B19 N-hydroxysuccinimide suberate Lysine 4 B20 N-hydroxysuccinimide sebacate Lysine 4 B21 N-hydroxysuccinimide PEG6 Glycerol and PEG 2 B22 Succinimidyl carbonate PEG6 Lysine 2 B23 Succinimidyl carbonate PEG12 Lysine 3 B24 Succinimidyl carbonate pentane Lysine 3 B25 Succinimidyl carbonate hexane Lysine 3 B26 p-nitrophenyl carbonate PEG6 Lysine 3 B27 p-nitrophenyl carbonate PEG12 Lysine 4 B28 p-nitrophenyl carbonate propane Glycerol and PEG 2 B29 p-nitrophenyl carbonate pentane Glycerol and PEG 2 B30 amine - Glycerol and PEG 2 B31 thiol butyrate Lysine 2 B32 thiol glutarate Lysine 3 B33 aminoxy PEG6 Lysine 3 B34 iodoacetamide PEG6 Lysine 3 B35 Maleimide EG2-EG2-Glu-C18 Lysine 3 B36 Maleimide EG2-EG2-Glu-C18 Lysine 3 B37 Amine Lys-EG2 Lysine 3 B38 N-hydroxysuccinimide - - 1

[0347] B35:

[0348] B36:

[0349] B37:

[0350] B38:

[0353] <지방산 모이어티>

[0354] For the fatty acid moiety, materials manufactured by methods known in the art or commercially obtained may be used.

[0355] The fatty acid moiety used was the fatty acid moiety according to Table 5 below.

[0356] 지방산 모이어티 명칭 F1 C16-NHS F2 C16-MAL F3 C18-NHS F4 C18-MAL F5 C16-Glu-NHS F6 C16-Glu-MAL F7 C18-Glu-NHS F8 C18-Glu-MAL F9 C18-Glu-EG2-NHS F10 C18-Glu-EG2-MAL F11 C18-Glu-EG2-EG2-NHS F12 C18-Glu-EG2-EG2-MAL F13 C20-Glu-EG2-EG2-NHS F14 C20-Glu-EG2-EG2-MAL F15 C18-Glu-EG2-EG2-TFP F16 C18-Glu-EG2-EG2-NPC

[0357] F1:

[0358] F2:

[0360] F3:

[0362] F4:

[0364] F5:

[0366] F6:

[0368] F7:

[0370] F8:

[0371] F9:

[0373] F10:

[0375] F11:

[0376] F12:

[0378] F13:

[0380] F14:

[0381] F15:

[0382] F16:

[0384] Polypeptide

[0385] Polypeptides may be materials manufactured by methods known in the art or commercially obtained. In the present invention, the sequences of polypeptides combined with biotin moiety, fatty acid moiety, or combinations thereof are as shown in Table 6 below.

[0386] polypeptide Sequence number amino acid sequence P1 1 H(Aib)QGTFTSDYSKYLDEQAAKEFVQWLMNT P2 2 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR P3 3 HADGSFSDEMNTILDNLAARDFINWLIQTKITD P4 4 YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ P5 5 HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS P6 6 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF P7 7 HSQGTFTSDYSKYLDSRRAQDFVQWLMNT P8 8 H(Aib)QGTFTSDYSKYLDEQAAKEFVQWLMNTC P9 9 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRC P10 10 HADGSFSDEMNTILDNLAARDFINWLIQTKITDC P11 11 YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQC P12 12 HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC P13 13 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFC P14 14 HSQGTFTSDYSKYLDSRRAQDFVQWLMNTC P15 15 GIVEQCCTSICSLEQLENYCN P16 16 FVNQHLCGSHLVEALYLVCGERGFFYTPKT P17 17 GIVEQCCTSICSLYQLENYCN P18 18 HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK P19 19 HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSKKK P20 20 H(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK P21 21 H(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSKKK P22 22 KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY P23 23 KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTYK P24 24 KCNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTY P25 25 KCNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTYK P26 26 KCNTATCATQRLADFLRHSSPNFGAIPSSTNVGSRTY P27 27 KCNTATCATQRLADFLRHSSPNFGAIPSSTNVGSRTYK P28 28 KCNTATCATQRLADFLRHSSNNFGAIPSSTNVGSRTY P29 29 KCNTATCATQRLADFLRHSSNNFGAIPSSTNVGSRTYK P30 30 RCNTATCATQRLADFLRHSSNNFGAIPSSTNVGSKTY P31 31 RCNTATCATQRLADFLRHSSNNFGAIPSSTNVGSKTYK P32 32 H(Aib)QGTFTSDKSKYLDERAAQDFVQWLLDGGPSSGAPPPS P33 33 H(Aib)QGTFTSDKSKYLDERAAQDFVQWLMDGGPSSGAPPPS P34 34 H(Aib)QGTFTSDKSKYLDKIAAQDFVQWLIDGGPSSGAPPPS P35 35 H(Aib)QGTFTSDKSWYLDKIAAQDFVQWLLGGGPSSGAPPPS P36 36 H(Aib)QGTFTSDKSWYLDERAAQDFVQWLMGGGPSSGAPPPS P37 37 H(Aib)QGTFTSDKSKWLDKIAAQDFVQWLIGGGPSSGAPPPS P38 38 H(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC P39 39 h(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC P40 40 H(Aib)QGTFTSDYSKYLDEQAAKEFVQWLMNTK P41 41 H(Aib)QGTFTSDYSKYLDEKRAKEFVQWLMNTC P42 42 MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKISFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF

[0388] <실시예: 비오틴 모이어티, 지방산 모이어티 또는 이의 조합과 결합된 생리활성 물질 제조>

[0389] [제조예]

[0390] The reaction solvent was a DMSO solution to which 0.3% triethylamine (TEA, Sigma) was added, and the polypeptide of Table 6 and the biotin moiety of Tables 3 and 4 were reacted in a molar ratio of 1:X at room temperature for at least 30 minutes. Subsequently, a mixture was prepared by adding the fatty acid moiety of Table 5 to the polypeptide-biotin moiety mixture in a molar ratio of 1:Y, and the mixture was reacted at room temperature for at least 90 minutes. The reaction was stopped by adding a 1% trifluoroacetic acid solution in a volume equal to the volume of the mixture.

[0391] [Separation, Purification, and Verification]

[0392] The above reaction products were separated and purified using reverse-phase high-performance liquid chromatography. A SUPERSIL ODS-1 column (10x250 mm, 5 μm, LB Science, South Korea) was used, and the mobile phase conditions were linearly varied using 30-50% solvent B (acetonitrile with 0.1% TFA) and solvent A (distilled water with 0.1% TFA) while maintaining a flow rate of 4.7 mL / min. Peaks detected between 10 and 20 minutes were collected by monitoring at 280 nm using a UV absorbance spectrometer. The collected peaks were concentrated and purified using an ultracentrifugal filter with an appropriate molecular weight cutoff after volatilizing the organic solvent and TFA under vacuum. The purity of the purified material was confirmed by HPLC analysis. Analysis was performed using a Gemini C18 column (4.6 x 250 mm, 5 μm; Phenomenex, CA, USA) at a constant temperature near room temperature. The analysis was carried out by the gradient elution method at a flow rate of 1 mL / min using a mobile phase consisting of a trifluoroacetic acid solution:acetonitrile mixture (the mixing ratio was varied). UV absorbance was observed at 280 nm.

[0393] [Check Molecular Weight]

[0394] The molecular weight of the above reaction product was measured.

[0395] Molecular weight was confirmed using MALDI-TOF mass spectrometry. As the matrix solution, a solution of CHCA (α-Cyano-4-hydroxycinnamic acid) saturated in 50% acetonitrile containing 0.1% TFA was used. Mass spectra were analyzed in linear and positive modes, and the molecular weight was determined at a final substance concentration of 0.1 mg / mL. Through this, it was confirmed that the molecular weights of the isolated substances matched the theoretical molecular weights.

[0397] <Example: Polypeptide combined with a biotin moiety, a fatty acid moiety, or a combination thereof>

[0398] The biotin moiety, fatty acid moiety, and polypeptide used the materials described above, and the method of binding the biotin moiety, fatty acid moiety, or a combination thereof with the polypeptide was performed by a method known in the art or by the method of the above example.

[0399] Polypeptides to which a biotin moiety, a fatty acid moiety, or a combination thereof is combined are shown in Table 7 below. (Here, molecular weight refers to the molecular weight measured by molecular weight measurement or the theoretical molecular weight.)

[0400] Conjugate (sequence number) polypeptide Biotin Moiety fatty acid moiety Molecular weight (g / mol) Sequence number order number Connection location number Connection location 1 12 HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC B1 C40 - - 4744.5 2 12 HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC B2 C40 - - 5167.1 3 12 HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC B3 C40 - - 5521.6 4 8 H(Aib)QGTFTSDYSKYLDEQAAKEFVQWLMNTC B1 C30 - - 3963.5 5 8 H(Aib)QGTFTSDYSKYLDEQAAKEFVQWLMNTC B2 C30 - - 4386.1 6 8 H(Aib)QGTFTSDYSKYLDEQAAKEFVQWLMNTC B3 C30 - - 4740.6 7 13 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFC B1 C35 - - 4675.4 8 13 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFC B2 C35 - - 5098.0 9 13 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFC B3 C35 - - 5452.5 10 5 HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS B4 K27 - - 5613.5 11 12 HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC B5 C40 - - 5857.0 12 15 GIVEQCCTSICSLEQLENYCN(A print) B4 K29 of B chain - - 7200.9 16 FVNQHLCGSHLVEALYLVCGERGFFYTPKT(B-print) - - 13 15 GIVEQCCTSICSLEQLENYCN(A print) B4 F1 / K29 of B chain - - 8627.8 16 FVNQHLCGSHLVEALYLVCGERGFFYTPKT(B-print) - - 14 12 HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC B3 C40 F1 K27 5759.4 15 12 HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC B38 K12, K27 F2 C40 5120.9 16 12 HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC B3 C40 F11 K27 6237.2 17 12 HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC B38 K12, K27 F12 C40 5598.6 18 12 HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC B35 C40 - - 6208.4 19 12 HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC B36 C40 - - 6208.4 20 18 HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK B37 K40 - - 5565.4 21 19 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSKKK B K39, K40, K41 - - 5251.1 22 19 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSKKK B K39, K40, K41 F11 K27 6241.6 23 19 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSKKK B K39, K40, K41 F11 K12 6241.6 24 20 H(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK B37 K40 - - 5278.1 25 21 H(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSKKK B K39, K40, K41 - - 5279.0 26 21 H(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSKKK B K39, K40, K41 F11 K27 6241.6 27 21 H(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSKKK B K39, K40, K41 F11 K12 5759.0 28 8 H(Aib)QGTFTSDYSKYLDEQAAKEFVQWLMNTC B2 K12 C30 F6 5259.0 29 8 H(Aib)QGTFTSDYSKYLDEQAAKEFVQWLMNTC B5 C30 K12 F5 4800.0 30 22 KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY B38 K1 - - 4129.6 31 22 KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY B39 K1 - - 5842.7 32 23 KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTYK B2 K38 F11 K1 5627.5 33 24 KCNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTY B38 K1 - - 4175.7 34 24 KCNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTY B39 K1 - - 5888.8 35 25 KCNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTYK B2 K38 F11 K1 5673.6 36 26 KCNTATCATQRLADFLRHSSPNFGAIPSSTNVGSRTY B38 K1 - - 4196.7 37 26 KCNTATCATQRLADFLRHSSPNFGAIPSSTNVGSRTY B39 K1 - - 5909.8 38 27 KCNTATCATQRLADFLRHSSPNFGAIPSSTNVGSRTYK B2 K38 F11 K1 5694.5 39 28 KCNTATCATQRLADFLRHSSNNFGAIPSSTNVGSRTY B38 K1 - - 4213.7 40 28 KCNTATCATQRLADFLRHSSNNFGAIPSSTNVGSRTY B39 K1 - - 5926.8 41 29 KCNTATCATQRLADFLRHSSNNFGAIPSSTNVGSRTYK B2 K38 F11 K1 5711.5 42 30 RCNTATCATQRLADFLRHSSNNFGAIPSSTNVGSKTY B38 K35 - - 4213.7 43 30 RCNTATCATQRLADFLRHSSNNFGAIPSSTNVGSKTY B39 K35 - - 5926.8 44 31 RCNTATCATQRLADFLRHSSNNFGAIPSSTNVGSKTYK B2 K35 F11 K38 5711.5 45 32 H(Aib)QGTFTSDKSKYLDERAAQDFVQWLLDGGPSSGAPPPS B38 K12 - - 6112.9 46 33 H(Aib)QGTFTSDKSKYLDERAAQDFVQWLMDGGPSSGAPPPS B39 K12 - - 6131.0 47 34 H(Aib)QGTFTSDKSKYLDKIAAQDFVQWLIDGGPSSGAPPPS B38 K10 - - 6069.0 48 35 H(Aib)QGTFTSDKSWYLDKIAAQDFVQWLLGGGPSSGAPPPS B39 K10 - - 6069.0 49 36 H(Aib)QGTFTSDKSWYLDERAAQDFVQWLMGGGPSSGAPPPS B3 K12 F11 K40 6142.0 50 37 H(Aib)QGTFTSDKSKWLDKIAAQDFVQWLIGGGPSSGAPPPS B3 K12 F12 C40 6220.2 51 20 H(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK B37 K40 F11 K27 6309.3 52 20 H(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK B38 K12, K27 F11 K40 5511.3 53 38 H(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC B38 K12, K27 F12 C40 5626.5 54 39 h(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC B38 K12, K27 F12 C40 5597.4 55 8 H(Aib)QGTFTSDYSKYLDEQAAKEFVQWLMNTC B38 K12 F12 C30 4591.3 56 40 H(Aib)QGTFTSDYSKYLDEQAAKEFVQWLMNTK B38 K12 F11 K30 4475.1 57 41 H(Aib)QGTFTSDYSKYLDEKRAKEFVQWLMNTC B38 K12 F12 C30 4647.6 58 8 H(Aib)QGTFTSDYSKYLDEQAAKEFVQWLMNTC B1 C30 F16 K12 4679.4 59 8 H(Aib)QGTFTSDYSKYLDEQAAKEFVQWLMNTC B38 K12 F14 C30 4619.4 60 22 KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY B38 K1 F11 K1 4845.5 61 24 KCNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTY B38 K1 F11 K1 4891.6 62 26 KCNTATCATQRLADFLRHSSPNFGAIPSSTNVGSRTY B38 K1 F11 K1 4912.6 63 28 KCNTATCATQRLADFLRHSSNNFGAIPSSTNVGSRTY B38 K1 F11 K1 4929.6 64 30 RCNTATCATQRLADFLRHSSNNFGAIPSSTNVGSKTY B38 K35 F11 K1 4929.6 65 15 GIVEQCCTSICSLEQLENYCN(A쇄) B38 B쇄의 K29 - - 6000.3 16 FVNQHLCGSHLVEALYLVCGERGFFYTPKT(B쇄) 66 15 GIVEQCCTSICSLEQLENYCN(A쇄) B38 B쇄의 K29 F11 B쇄의 F1 6716.2 16 FVNQHLCGSHLVEALYLVCGERGFFYTPKT(B쇄) 67 13 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF B38 K13, K26, K27 - - 4796.7 68 42 MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKISFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF B38 Lys random - - 69 42 MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKISFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF B38 Lys random F16 Lys random 70 5 HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS B38 K12, K27 - - 71 12 HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC - - F1 C40 72 12 HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC - - F12 C40

[0401] (In the table above, B represents Native biotin.)

[0403] Conjugates 14 to 17

[0404] 1) Preparation Example

[0405] The composite of Table 8 below was prepared by the following method.

[0406] A mixture was prepared using a DMSO solution containing 0.3% triethylamine (TEA, Sigma) as the reaction solvent, with the polypeptide of SEQ ID NO. 12 listed in Table 6 and the biotin moiety listed in Tables 3 and 4 in a molar ratio of 1:2, and reacted at room temperature for at least 30 minutes. Additionally, a mixture was prepared by adding the fatty acid moiety listed in Table 5 to the polypeptide-biotin moiety mixture in a molar ratio of 1:1 to 1:2, and reacted at room temperature for at least 90 minutes. The reaction was stopped by adding a 1% trifluoroacetic acid solution in a volume equal to the volume of the mixture.

[0407] composite polypeptide Biotin Moiety fatty acid moisture g / mol Sequence number order number Connection location number Connection location 14 12 HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC B3 C40 F1 K27 5759.4 15 12 HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC B1 K12, K27 F2 C40 5120.9 16 12 HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC B3 C40 F11 K27 6237.2 17 12 HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC B1 K12, K27 F12 C40 5598.6

[0408] 2) Separation, purification, and purity verification

[0409] The reaction products of the above conjugates 14 to 17 were separated and purified by reverse-phase high performance liquid chromatography (hereinafter “).

[0410] A SUPERSIL ODS-1 column (10 x 250 mm, 5 μm, LB Science, South Korea) was used, and the mobile phase conditions were linearly varied with Conjugate 14: 30–80%, Conjugate 15: 40–70%, Conjugate 16: 30–60%, and Conjugate 17: 40–60% while maintaining a flow rate of 4.7 ml / min using Solvent A (distilled water with 0.1% TFA) and Solvent B (acetonitrile with 0.1% TFA). Subsequently, monitoring was performed at 280 nm using a UV absorbance spectrometer between 10 and 17 minutes. Peaks detected at (Conjugate 14: 15 min, Conjugate 15: 16 min, Conjugate 16: 16 min, Conjugate 17: 11 min) were collected. The collected peaks were concentrated and purified using an ultracentrifugal filter with an appropriate molecular weight cutoff after volatilizing the organic solvent and TFA under vacuum. The purity of the purified material was confirmed by HPLC analysis. Analysis was performed using a Gemini C18 column (4.6 x 250 mm, 5 μm; Phenomenex, CA, USA) at a constant temperature near room temperature. The analysis was carried out by the gradient elution method at a flow rate of 1 mL / min using a mobile phase consisting of a trifluoroacetic acid solution:acetonitrile mixture (mixing ratio was adjusted from 70:30 to 20:80 after 20 minutes). UV absorbance was observed at 280 nm.

[0411] As a result of the measurement, the purified chromatograms of conjugates 14 to 17 are shown in Fig. 1. In addition, as a result of analysis with a UV detector, no peaks other than polypeptides bound to biotin moiety and fatty acid moiety were observed, and when the area of ​​the peaks generated from each chromatogram was expressed as a percentage, it was confirmed that the purity of conjugates 14 to 17 was 95% or higher.

[0413] 3) Check molecular weight

[0414] The molecular weight of the final material obtained from the above conjugates 14 to 17 was measured.

[0415] The molecular weight was determined by MALDI-TOF mass spectrometry. As the matrix solution, a solution of CHCA (α-Cyano-4-hydroxycinnamic acid) saturated in 50% acetonitrile containing 0.1% TFA was used. Mass spectra were analyzed in linear and positive modes, and the molecular weight was determined at a final substance concentration of 0.1 mg / mL. The analysis results are shown in Table 8 above.

[0418] Conjugates 18 to 19

[0419] 1) Preparation Example

[0420] The composite of Table 9 below was prepared by the following method.

[0421] The reaction solvent was a DMSO solution to which 0.3% triethylamine (TEA, Sigma) was added, and a mixture was prepared by mixing the polypeptide of SEQ ID NO. 12 listed in Table 6 and the biotin moiety of Tables 3 and 4 in a molar ratio of 1:2 and reacted at room temperature for at least 30 minutes. The reaction was stopped by adding a 1% trifluoroacetic acid solution in a volume equal to the volume of the mixture.

[0422] composite polypeptide Biotin Moiety fatty acid moisture g / mol Sequence number order number Connection location number Connection location 18 12 HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC B35 C40 - - 6208.4 19 12 HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC B36 C40 - - 6208.4

[0423] 2) Separation, purification, and purity verification

[0424] The reaction products of the above conjugates 18 to 19 were separated and purified by reverse-phase high-performance liquid chromatography.

[0425] A SUPERSIL ODS-1 column (10 x 250 mm, 5 μm, LB Science, South Korea) was used, and the mobile phase conditions were linearly varied from 30–80% using solvent A (distilled water with 0.1% TFA) and solvent B (acetonitrile with 0.1% TFA) while maintaining a flow rate of 4.7 ml / min. Peaks detected between 12 and 13 minutes were collected by monitoring at 280 nm using a UV absorbance spectrometer. The collected peaks were concentrated and purified using an ultracentrifugal filter with an appropriate molecular weight cutoff after volatilizing the organic solvent and TFA under vacuum. The purity of the purified material was confirmed by HPLC analysis. Analysis was performed using a Gemini C18 column (4.6 x 250 mm, 5 μm; Phenomenex, CA, USA) at a constant temperature near room temperature. Analysis was performed by the gradient elution method at a flow rate of 1 mL / min using a mobile phase consisting of a trifluoroacetic acid solution:acetonitrile mixture (mixing ratio adjusted from 70:30 to 20:80 after 20 minutes). UV absorbance was observed at 280 nm.

[0426] As a result of the measurement, the purified chromatograms of conjugates 18 to 19 are shown in Fig. 2. In addition, as a result of analysis with a UV detector, no peaks other than polypeptides bound to biotin moiety and fatty acid moiety were observed, and when the area of ​​the peaks generated from each chromatogram was expressed as a percentage, it was confirmed that the purity of conjugates 18 to 19 was 95% or higher.

[0428] 3) Check molecular weight

[0429] The molecular weight of the final material obtained from the above conjugates 18 to 19 was measured.

[0430] The molecular weight was determined by MALDI-TOF mass spectrometry. As the matrix solution, a solution of CHCA (α-Cyano-4-hydroxycinnamic acid) saturated in 50% acetonitrile containing 0.1% TFA was used. Mass spectra were analyzed in linear and positive modes, and the molecular weight was determined at a final substance concentration of 0.1 mg / mL. The analysis results are shown in Table 9.

[0433] Conjugates 20 and 24

[0434] 1) Preparation Example

[0435] Conjugates 20 and 24 were synthesized from the conjugates of Table 10 below by the above general SPPS method.

[0436] composite polypeptide Biotin Moiety fatty acid moisture g / mol Sequence number order Sequence number Connection location Sequence number Connection location 20 18 HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK B37 K40 - - 5565.4 24 20 H(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK B37 K40 - - 5278.1

[0437] 2) Purity verification

[0438] The purity of the purified materials of the above conjugates 20 and 24 was confirmed by HPLC analysis. Analysis was performed using a Gemini C18 column (4.6 x 250 mm, 5 μm; Phenomenex, CA, USA) at a constant temperature near room temperature. The analysis was carried out by the gradient elution method at a flow rate of 1 mL / min using a mobile phase consisting of a trifluoroacetic acid solution:acetonitrile mixture (mixing ratio adjusted from 70:30 to 50:50 after 20 minutes). UV absorbance was observed at 280 nm.

[0439] As a result of the measurement, the purified chromatograms of conjugates 20 and 24 are shown in Fig. 3. In addition, as a result of analysis with a UV detector, no peaks other than the polypeptides bound to the biotin moiety and fatty acid moiety were observed, and when the area of ​​the peaks generated from each chromatogram was expressed as a percentage, it was confirmed that the purity of conjugates 20 and 24 was 95% or higher.

[0441] 3) Check molecular weight

[0442] The molecular weight of the final material obtained from the above conjugates 20 and 24 was measured.

[0443] The molecular weight was determined by MALDI-TOF mass spectrometry. As the matrix solution, a solution of CHCA (α-Cyano-4-hydroxycinnamic acid) saturated in 50% acetonitrile containing 0.1% TFA was used. Mass spectra were analyzed in linear and positive modes, and the molecular weight was determined at a final substance concentration of 0.1 mg / mL. The analysis results are shown in Table 10.

[0446] Conjugates 51 to 54

[0447] 1) Preparation Example

[0448] - Conjugates 51 to 54 were synthesized using the standard Fmoc manufacturing method below, as shown in Table 12.

[0449] The term "standard Fmoc manufacturing method" means that it is performed by the following method.

[0450] Add DMF to a container holding Fmoc Rink amide AM resin and allow it to swell for 2 hours. After washing with DMF, add 20% piperidine / DMF and mix for 30 minutes. After washing with DMF, mix with the Fmoc-amino acid solution for 30 seconds, add activation buffer, and administer nitrogen gas for 1 hour. Repeat the above process sequentially until the desired peptide sequence is completed, wherein the Fmoc-amino acid reagent contains the substances listed in Table 11 below.

[0451] # Materials Coupling reagents 1 Fmoc-Lys(Alloc)-OH (2.0 eq ) HATU (1.95 eq) and DIEA (4.0 eq ) 2 Fmoc-Ser(tBu)-OH (3.0 eq ) HBTU (2.85 eq) and DIEA (6.0 eq ) 3 Fmoc-Pro-OH (3.0 eq ) HBTU (2.85 eq) and DIEA (6.0 eq ) 4 Fmoc-Pro-OH (3.0 eq ) HBTU (2.85 eq) and DIEA (6.0 eq ) 5 Fmoc-Pro-OH (3.0 eq ) HBTU (2.85 eq) and DIEA (6.0 eq ) 6 Fmoc-Ala-OH (3.0 eq ) HBTU (2.85 eq) and DIEA (6.0 eq ) 7 Fmoc-Gly-OH (3.0 eq ) HBTU (2.85 eq) and DIEA (6.0 eq ) 8 Fmoc-Ser(tBu)-OH (3.0 eq ) HBTU (2.85 eq) and DIEA (6.0 eq ) 9 Fmoc-Ser(tBu)-OH (3.0 eq ) HBTU (2.85 eq) and DIEA (6.0 eq ) 10 Fmoc-Pro-OH (3.0 eq ) HOBt (3.00 eq) and DIC (3.0 eq ) 11 Fmoc-Gly-OH (3.0 eq ) HBTU (2.85 eq) and DIEA (6.0 eq ) 12 Fmoc-Gly-OH (3.0 eq ) HBTU (2.85 eq) and DIEA (6.0 eq ) 13 Fmoc-Asn(Trt)-OH (3.0 eq ) HBTU (2.85 eq) and DIEA (6.0 eq ) 14 Fmoc-Lys(Dde)-OH (3.0 eq ) HBTU (2.85 eq) and DIEA (6.0 eq ) 15 Fmoc-Leu-OH (3.0 eq ) HBTU (2.85 eq) and DIEA (6.0 eq ) 16 Fmoc-Trp(Boc)-OH (3.0 eq ) HBTU (2.85 eq) and DIEA (6.0 eq ) 17 Fmoc-Glu(OtBu)-OH (3.0 eq ) HBTU (2.85 eq) and DIEA (6.0 eq ) 18 Fmoc-Ile-OH (3.0 eq ) HBTU (2.85 eq) and DIEA (6.0 eq ) 19 Fmoc-Phe-OH (3.0 eq ) HBTU (2.85 eq) and DIEA (6.0 eq ) 20 Fmoc-Leu-OH (3.0 eq ) HBTU (2.85 eq) and DIEA (6.0 eq ) 21 Fmoc-Arg-OH (3.0 eq ) HBTU (2.85 eq) and DIEA (6.0 eq ) 22 Fmoc-Val-OH (6.0 eq ) HATU (5.70 eq) and DIEA (12.0 eq ) 23 Fmoc-Ala-OH (6.0 eq ) HBTU (5.70 eq) and DIEA (12.0 eq ) 24 Fmoc-Glu(OtBu)-OH (6.0 eq ) HBTU (5.70 eq) and DIEA (12.0 eq ) 25 Fmoc-Glu(OtBu)-OH (6.0 eq ) HBTU (5.70 eq) and DIEA (12.0 eq ) 26 Fmoc-Glu(OtBu)-OH (6.0 eq ) HBTU (5.70 eq) and DIEA (12.0 eq ) 27 Fmoc-Met-OH (6.0 eq ) HATU (5.70 eq) and DIEA (12.0 eq ) 28 Fmoc-Gln(Trt)-OH (6.0 eq ) HATU (5.70 eq) and DIEA (12.0 eq ) 29 Fmoc-Lys(Dde)-OH (3.0 eq ) HATU (2.85 eq) and DIEA (6.0 eq ) 30 Fmoc-Ser(tBu)-OH (6.0 eq ) HATU (5.70 eq) and DIEA (12.0 eq ) 31 Fmoc-Leu-OH (6.0 eq ) HATU (5.70 eq) and DIEA (12.0 eq ) 32 Fmoc-Asp(OtBu)-OH (6.0 eq ) HOBt (6.00 eq) and DIC (6.0 eq ) 33 Fmoc-Ser(tBu)-OH (6.0 eq ) HATU (5.70 eq) and DIEA (12.0 eq ) 34 Fmoc-Thr(tBu)-OH (6.0 eq ) HATU (5.70 eq) and DIEA (12.0 eq ) 35 Fmoc-Phe-OH (6.0 eq ) HATU (5.70 eq) and DIEA (12.0 eq ) 36 Fmoc-Thr(tBu)-OH (6.0 eq ) HATU (5.70 eq) and DIEA (12.0 eq ) 37 Fmoc-Gly-OH (9.0 eq ) HATU (8.55 eq) and DIEA (18.0 eq ) 38 Fmoc-Glu(OtBu)-OH (9.0 eq ) HATU (8.55 eq) and DIEA (18.0 eq ) 39 Fmoc-Aib-OH (3.0 eq ) HATU (2.85 eq) and DIEA (6.0 eq ) 40 Fmoc-His(Trt)-OH (6.0 eq ) HATU (5.70 eq) and DIEA (12.0 eq ) 41 (Boc)2O (5.0 eq ) DIEA (10.0 eq ) De-Alloc PhSiH3 (10 eq ) and Pd(PPh3)4(0.1 eq ) 42 Fmoc-AEEA-OH (3.0 eq ) HATU (2.85 eq) and DIEA (6.0 eq ) 43 Fmoc-AEEA-OH (3.0 eq ) HATU (2.85 eq) and DIEA (6.0 eq ) 44 Fmoc-γ(3.0 eq ) HATU (2.85 eq) and DIEA (6.0 eq ) 45 C18DA-tBu (3.0 eq ) HATU (2.85 eq) and DIEA (6.0 eq ) De-Dde 3% Hydrazine hydrate 46 Biotin-OSu (4.0 eq ) DIEA (12.0 eq )

[0452] - Conjugates 53 to 54 were prepared by the following method. Using a DMSO solution containing 0.3% triethylamine (TEA, Sigma) as the reaction solvent, a mixture was prepared by mixing the polypeptides of SEQ ID NOs 39 to 40 listed in Table 6 and the biotin moiety listed in Tables 3 to 4 in a molar ratio of 1:2 and reacted at room temperature for at least 10 minutes. A mixture was prepared by mixing the polypeptide-biotin moiety with the fatty acid moiety listed in Table 5 in a molar ratio of 1:3 and reacted at room temperature for at least 90 minutes. The reaction was stopped by adding a 1% trifluoroacetic acid solution in a volume equal to the volume of the mixture.

[0454] composite polypeptide Biotin Moiety fatty acid moisture g / mol Sequence number order Sequence number Connection location Sequence number Connection location 51 20 H(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK B37 K40 F11 K27 6309.3 52 20 H(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK B38 K12, K27 F11 K40 5511.3 53 38 H(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC B38 K12, K27 F12 C40 5626.5 54 39 h(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC B38 K12, K27 F12 C40 5597.4

[0455] 2) Separation, purification, and purity verification

[0456] The reaction products of the above conjugates 51 to 54 were separated and purified by reverse-phase high-performance liquid chromatography.

[0457] A Gemini C18 column (10 x 250 mm, 5 μm; Phonomenex, CA, USA) was used, and the mobile phase conditions were linearly varied from 40–60% using solvent A (distilled water with 0.1% TFA) and solvent B (acetonitrile with 0.1% TFA) while maintaining a flow rate of 4.7 ml / min. Peaks detected between 11 and 13 minutes (conjugate 53: 12 minutes, conjugate 54: 13 minutes) were collected by monitoring at 280 nm using a UV absorbance spectrometer. The collected peaks were concentrated and purified using an ultracentrifugal filter with an appropriate molecular weight cutoff after volatilizing the organic solvent and TFA under vacuum. The purity of the purified material was confirmed by HPLC analysis. Analysis was performed using a Gemini C18 column (4.6 x 250 mm, 5 μm; Phenomenex, CA, USA) at a constant temperature near room temperature. The analysis was carried out by the gradient elution method at a flow rate of 1 mL / min using a mobile phase consisting of a trifluoroacetic acid solution:acetonitrile mixture (mixing ratio adjusted from 70:30 to 20:80 after 20 minutes). UV absorbance was observed at 280 nm.

[0459] As a result of the measurement, the chromatograms for the final substances of conjugates 51 to 52 are shown in Fig. 5, and the purified chromatograms of conjugates 53 to 54 are shown in Fig. 6. In addition, as a result of analysis with a UV detector, no peaks other than polypeptides bound to biotin moiety and fatty acid moiety were observed, and when the area of ​​the peaks generated from each chromatogram was expressed as a percentage, it was confirmed that the purity of conjugates 51 to 54 was 95% or higher.

[0461] 3) Check molecular weight

[0462] The molecular weight of the final material obtained from the above conjugates 51 to 54 was measured.

[0463] The molecular weight was determined by MALDI-TOF mass spectrometry. As the matrix solution, a solution of CHCA (α-Cyano-4-hydroxycinnamic acid) saturated in 50% acetonitrile containing 0.1% TFA was used. Mass spectra were analyzed in linear and positive modes, and the molecular weight was determined at a final substance concentration of 0.1 mg / mL. The analysis results are shown in Table 12.

[0465] Conjugates 55 to 59

[0466] - Conjugate 56 was synthesized using the standard Fmoc manufacturing method used for conjugates 51 to 54.

[0467] - Conjugates 55 and 57 to 59 were prepared by the following method. Using a DMSO solution containing 0.3% triethylamine (TEA, Sigma) as the reaction solvent, a mixture was prepared by mixing the polypeptides of SEQ ID NO. 8, SEQ ID NO. 40, and SEQ ID NO. 41 listed in Table 6 and the biotin moiety listed in Tables 3 and 4 in a molar ratio of 1:2 and reacted at room temperature for at least 10 minutes. A mixture was prepared by mixing the polypeptide-biotin moiety with the fatty acid moiety listed in Table 5 in a molar ratio of 1:1 or 1:2 and reacted at room temperature for at least 90 minutes. The reaction was stopped by adding a 1% trifluoroacetic acid solution in a volume equal to the volume of the mixture.

[0468] composite polypeptide Biotin Moiety fatty acid moisture g / mol Sequence number order Sequence number Connection location Sequence number Connection location 55 8 H(Aib)QGTFTSDYSKYLDEQAAKEFVQWLMNTC B38 K12 F12 C30 4591.3 56 40 H(Aib)QGTFTSDYSKYLDEQAAKEFVQWLMNTK B38 K12 F11 K30 4475.1 57 41 H(Aib)QGTFTSDYSKYLDEKRAKEFVQWLMNTC B38 K12 F12 C30 4647.6 58 8 H(Aib)QGTFTSDYSKYLDEQAAKEFVQWLMNTC B1 C30 F11 K12 4679.4 59 8 H(Aib)QGTFTSDYSKYLDEQAAKEFVQWLMNTC B38 K12 F14 C30 4619.4

[0469] 2) Separation, purification, and purity verification

[0470] The reaction products of the above conjugates 55 to 59 were separated and purified by reverse-phase high-performance liquid chromatography.

[0471] A Gemini C18 column (10 x 250 mm, 5 μm; Phonomenex, CA, USA) was used, and the mobile phase conditions were linearly varied with solvent A (distilled water with 0.1% TFA) and solvent B (acetonitrile with 0.1% TFA) at a flow rate of 4.7 ml / min, with Conjugates 55 and 57 at 40-60% and Conjugates 58 to 59 at 40-70%. Peaks detected between 10 and 15 minutes (Conjugate 55: 13 min, Conjugate 57: 10 min, Conjugate 58: 13 min, Conjugate 59: 15 min) were collected by monitoring at 280 nm using a UV absorbance spectrometer. The collected peaks were concentrated and purified using an ultracentrifugal filter with an appropriate molecular weight cutoff after volatilizing the organic solvent and TFA under vacuum. The purity of the purified material was confirmed by HPLC analysis. Analysis was performed at a constant temperature (35°C) using a Gemini C18 column (4.6 x 250 mm, 5 μm; Phenomenex, CA, USA). The analysis was carried out by the gradient elution method at a flow rate of 1 mL / min using a mobile phase consisting of a trifluoroacetic acid solution:acetonitrile mixture (mixing ratio adjusted from 60:40 to 30:70 after 20 minutes). UV absorbance was observed at 280 nm.

[0472] As a result of measurement, the purification chromatograms of conjugate 55 and conjugates 57 to 59 are shown in Fig. 8, and the final substance chromatogram of conjugate 56 is shown in Fig. 9. In addition, as a result of analysis with a UV detector, no peaks other than polypeptides bound to biotin moiety and fatty acid moiety were observed, and when the area of ​​the peaks generated from each chromatogram was expressed as a percentage, it was confirmed that the purity of conjugates 55 to 59 was 95% or higher.

[0474] 3) Check molecular weight

[0475] The molecular weight of the final material obtained from the above conjugates 55 to 59 was measured.

[0476] The molecular weight was determined by MALDI-TOF mass spectrometry. As the matrix solution, a solution of CHCA (α-Cyano-4-hydroxycinnamic acid) saturated in 50% acetonitrile containing 0.1% TFA was used. Mass spectra were analyzed in linear and positive modes, and the molecular weight was determined at a final substance concentration of 0.1 mg / mL. The analysis results are shown in Table 13.

[0478] Conjugate 60 to 64

[0479] 1) Preparation Example

[0480] The composite of Table 14 below was manufactured by the following method.

[0481] A mixture was prepared using a DMSO solution containing 0.3% triethylamine (TEA, Sigma) as the reaction solvent, in which polypeptides of SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, and SEQ ID NO. 30 listed in Table 6 and biotin moiety listed in Tables 3 and 4 were reacted at room temperature for at least 30 minutes. A mixture was prepared by adding a fatty acid moiety listed in Table 5 to the polypeptide-biotin moiety mixture in a molar ratio of 1:2 and reacted at room temperature for at least 120 minutes. The reaction was stopped by adding a 1% trifluoroacetic acid solution in a volume equal to the volume of the mixture.

[0482] composite polypeptide Biotin Moiety fatty acid moisture g / mol Sequence number order Sequence number Connection location Sequence number Connection location 60 22 KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY B38 K1 F11 K1 4845.5 61 24 KCNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTY B38 K1 F11 K1 4891.6 62 26 KCNTATCATQRLADFLRHSSPNFGAIPSSTNVGSRTY B38 K1 F11 K1 4912.6 63 28 KCNTATCATQRLADFLRHSSNNFGAIPSSTNVGSRTY B38 K1 F11 K1 4929.6 64 30 RCNTATCATQRLADFLRHSSNNFGAIPSSTNVGSKTY B38 K35 F11 K1 4929.6

[0483] 2) Separation, purification, and purity verification

[0484] The reaction products of the above conjugates 60 to 64 were separated and purified by reverse-phase high-performance liquid chromatography.

[0485] A Gemini C18 column (10 x 250 mm, 5 μm; Phonomenex, CA, USA) was used, and the mobile phase conditions were linearly varied with solvent A (distilled water with 0.1% TFA) and solvent B (acetonitrile with 0.1% TFA) at a flow rate of 4.7 ml / min, ranging from 30-60% for Examples 18 to 20 and 25-50% for Examples 21 to 22. Peaks detected between 5 and 18 minutes were collected by monitoring at 280 nm using a UV absorbance spectrometer (Example 18: 20 min, Example 19: 15 min, Example 20: 13 min, Example 21: 17 min, Example 15 min). The collected peaks were concentrated and purified using an ultracentrifugal filter with an appropriate molecular weight cutoff after volatilizing the organic solvent and TFA under vacuum. The purity of the purified material was confirmed by HPLC analysis. Analysis was performed using a Gemini C18 column (4.6 x 250 mm, 5 μm; Phenomenex, CA, USA) at a constant temperature (35°C). The analysis was carried out by the gradient elution method at a flow rate of 1 mL / min using a mobile phase consisting of a trifluoroacetic acid solution:acetonitrile mixture (mixing ratio adjusted from 75:25 to 50:50 after 20 minutes). UV absorbance was observed at 280 nm.

[0486] As a result of the measurement, the purified chromatograms of conjugates 60 to 64 are as shown in Fig. 11.

[0487] As a result of analysis with a UV detector, no peaks other than the polypeptides bound to the biotin moiety and fatty acid moiety were observed, and when the area of ​​the peaks generated from each chromatogram was expressed as a percentage, it was confirmed that the purity of conjugates 60 to 64 was 95% or higher.

[0489] 3) Check molecular weight

[0490] The molecular weight of the final material obtained from the above conjugates 60 to 64 was measured.

[0491] The molecular weight was determined by MALDI-TOF mass spectrometry. As the matrix solution, a solution of CHCA (α-Cyano-4-hydroxycinnamic acid) saturated in 50% acetonitrile containing 0.1% TFA was used. Mass spectra were analyzed in linear and positive modes, and the molecular weight was determined at a final substance concentration of 0.1 mg / mL. The analysis results are shown in Table 14.

[0493] Conjugate 66

[0494] 1) Preparation Example

[0495] The composite of Table 15 below was prepared by the following method.

[0496] The reaction solvent was a DMSO solution to which 0.3% triethylamine (TEA, Sigma) was added. A polypeptide bonded by disulfide bonds between the 6th and 11th cysteines of SEQ ID NO. 15 of Table 6, the 7th cysteine ​​of SEQ ID NO. 15 and the 7th cysteine ​​of SEQ ID NO. 16, and the 20th cysteine ​​of SEQ ID NO. 15 and the 19th cysteine ​​of SEQ ID NO. 16 was prepared by adding the biotin moiety listed in Tables 3 and 4 in a molar ratio of 1:1 and reacted at room temperature for at least 30 minutes. A mixture was prepared by adding the fatty acid moiety listed in Table 5 in a molar ratio of 1:2 to the polypeptide-biotin moiety mixture and reacted at room temperature for at least 120 minutes. The reaction was stopped by adding a 1% trifluoroacetic acid solution in a volume equal to the volume of the mixture.

[0497] composite polypeptide Biotin Moiety fatty acid moisture g / mol Sequence number order Sequence number Connection location Sequence number Connection location 66 15 GIVEQCCTSICSLEQLENYCN(A print) B38 K29 of B chain F11 F1 of chain B 6716.2 16 FVNQHLCGSHLVEALYLVCGERGFFYTPKT(B-print)

[0498] 2) Separation, purification, and purity verification

[0499] The reaction product of the above conjugate 66 was separated and purified by reverse-phase high-performance liquid chromatography. A Gemini C18 column (10 x 250 mm, 5 μm; Phonomenex, CA, USA) was used, and the mobile phase conditions were linearly varied from 35–45% using solvent A (distilled water with 0.1% TFA) and solvent B (acetonitrile with 0.1% TFA) while maintaining a flow rate of 4.7 ml / min. The peak detected between 13 and 14 minutes was collected by monitoring at 280 nm using a UV absorbance spectrometer. The collected peak was concentrated and purified using an ultracentrifugal filter with an appropriate molecular weight cutoff after volatilizing the organic solvent and TFA under vacuum. The purity of the purified material was confirmed by HPLC analysis. Analysis was performed using a Gemini C18 column (4.6 x 250 mm, 5 μm; Phenomenex, CA, USA) at a constant temperature (35℃). The analysis was carried out by the gradient elution method at a flow rate of 1 mL / min using a mobile phase consisting of a trifluoroacetic acid solution:acetonitrile mixture (mixing ratio adjusted from 70:30 to 40:60 after 20 minutes). UV absorbance was observed at 280 nm.

[0500] As a result of the measurement, the purified chromatogram of conjugate 66 is shown in Fig. 13. In addition, as a result of analysis with a UV detector, no peaks other than the polypeptide bound to the biotin moiety and fatty acid moiety were observed, and when the area of ​​the peaks generated from each chromatogram was expressed as a percentage, it was confirmed that the purity of conjugate 66 was 95% or higher.

[0502] 3) Check molecular weight

[0503] The molecular weight of the final substance obtained from the above conjugate 66 was measured.

[0504] The molecular weight was determined by MALDI-TOF mass spectrometry. As the matrix solution, a solution of CHCA (α-Cyano-4-hydroxycinnamic acid) saturated in 50% acetonitrile containing 0.1% TFA was used. Mass spectra were analyzed in linear and positive modes, and the molecular weight was determined at a final substance concentration of 0.1 mg / mL. The analysis results are shown in Table 15.

[0506] Conjugate 69

[0507] 1) Preparation Example

[0508] The composite of Table 16 below was prepared by the following method.

[0509] The polypeptide of SEQ ID No. 42 in Table 6 above was dissolved in a pH 7.8 phosphate buffer, and the biotin moiety was prepared by dissolving it in DMSO. The reaction was carried out with a volume ratio of 90:10 for each substance. The reaction was carried out at room temperature for at least 60 minutes with a molar ratio of 1:22. A mixture was prepared by adding the fatty acid moiety listed in Table 5 above to the polypeptide-biotin moiety mixture at a molar ratio of 1:9, and the mixture was carried out at room temperature for at least 60 minutes. The reaction was inhibited by lowering the storage temperature of the reactants to 4 degrees Celsius, after which the products were immediately purified.

[0510] composite polypeptide Biotin Moiety fatty acid moisture g / mol Sequence number order Sequence number Connection location Sequence number Connection location 69 42 MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKISFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF B38 Lys random F16 Lys random

[0511] 2) Separation, purification, and purity verification

[0512] The reaction product of the above conjugate 69 was confirmed to have undergone a reaction and produced through reverse-phase chromatography analysis, and purification was carried out using an ultracentrifugal filter with an appropriate molecular weight cutoff.

[0513] As a result of the measurement, the purified chromatogram of Conjugate 69 is shown in Fig. 15. In addition, analysis using a UV detector revealed that no peaks other than the polypeptide bound to the biotin moiety and fatty acid moiety were observed, and when the peak area from each chromatogram was expressed as a percentage, it was confirmed that the purity of Conjugate 69 was over 95%.

[0515] 3) Check molecular weight

[0516] The molecular weight of the final substance obtained from the above conjugate 69 was measured.

[0517] Molecular weight was confirmed by MALDI-TOF mass spectrometry. As the matrix solution, a solution of CHCA (α-Cyano-4-hydroxycinnamic acid) saturated in 50% acetonitrile containing 0.1% TFA was used. Mass spectra were analyzed in linear and positive modes, and the molecular weight was determined at a final substance concentration of 0.1 mg / mL. The average number of biotin moiety bonds was calculated based on the average molecular weight before and after the reaction. The average number of biotin bonds in conjugate 69 was found to be 10–20. The analysis results are shown in Table 16.

[0519] Experimental Example. In vitro Activity test

[0520] Confirmation of GLP-1 receptor efficacy

[0521] Conjugate 3, 14 to 20

[0522] of the above-mentioned composites 3, 14 to 20 in vitro Activity was measured.

[0523] First, to confirm the efficacy against the GLP-1 receptor, CHO-K1 cells expressing human GLP-1 receptors were purchased from Eurofins and used, with 7 x 10 cells per well in a 96-well plate. 3Cells were seeded and cultured in a CO2 incubator at 37°C. After 24 hours, the culture medium was removed from each well, and 30 μl of 0.5 mM IBMX was added for 15 minutes. Subsequently, 0.001–1000 nM of each example and Exendin-4 were added, followed by incubation in a CO2 incubator at 37°C for 30 minutes. Afterward, the production of cAMP (Luciferase activity) was measured using the HitHunter® cAMP assay kit to determine the EC of the GLP-1 receptor. 50 The values ​​were calculated. The measurement results are shown in Table 17.

[0524] division Active (nM) Exendin-4 1.991 Conjugate 3 0.301 Conjugate 14 1.155 Conjugate 15 0.307 Conjugate 16 0.882 Conjugate 17 1.109 Conjugate 18 0.347 Conjugate 19 0.237 Assembly 20 NA

[0525] Absorption rate measurement

[0526] Conjugate 3, 14 to 20

[0527] Pharmacological behavior was compared to confirm the absorption rate of the drug.

[0528] Blood samples were collected after administering samples at doses of 100 and 500 μg / kg, respectively, into the duodenum of the small intestine of experimental rats (SD rats) weighing approximately 200 g. Each sample was dissolved in 10 mM PBS (pH 7.4) containing 0.02% polysorbate 80 according to the dosage, and then 68 mg / kg of NaCDC and 34 mg / kg of PG were added, mixed at 1,000 rpm for 20 minutes, and administered. Changes in blood drug concentration over time were measured using the enzyme-linked immunosorbent assay (ELISA). Blood samples were collected from the jugular vein. The results were calculated as average values, and the results are shown in Table 18.

[0529] division Half-life (time) Cmax(ng / mL) AUC(hr*ng / mL) Bioavailability (%) Conjugate 3 - 34 ± 24 9 ± 3 2.4 Conjugate 14 1.6 ± 0.3 65 ± 16 146 ± 50 - Conjugate 16 5.3 ± 2.1 1204 ± 275 8919 ± 7009 17.4 Conjugate 17 7.9 ± 1.8 1961 ± 752 11879 ± 3709 5.2 Conjugate 18 0.9 ± 0.2 4736 ± 2534 4411 ± 1593 - Conjugate 19 1.4 ± 0.4 693 ± 249 880 ± 368 -

[0530] After intravenously administering the above conjugates 15 to 17 to experimental rats (SD Rat), the pharmacological behavior was confirmed. The experimental results are shown in Table 19 below.

[0531] test substance Half-life (hr) AUClast (hr*ng / mL) composite Dosage (mg / kg) 15 0.154 1.1±0.1 2223.1±81.9 16 0.187 6.5±0.7 7439.9±508.0 17 0.168 6.5±1.1 29612.4±2495.4

[0533] Measurement of blood glucose control ability

[0534] Conjugates 3, 17 and 20

[0535] To confirm glucose tolerance, an oral GLP-1 agonist formulation was orally administered to mice, and the blood glucose control efficacy was measured through an intraperitoneal glucose tolerance test (IPGTT).

[0536] To measure intraperitoneal glucose tolerance in an animal model, 100 µl of the sample (10 µg / mouse, based on SEQ ID NO. 1) was orally administered to 9-week-old male mice (C57BL / 6) at -60 minutes, followed by the intraperitoneal injection of 200 µl of glucose (2 g / kg), and changes in blood glucose levels were observed in blood collected from the tail vein at -60, 0, 20, 40, 60, 90, and 120 minutes. Subcutaneous administration of the polypeptide having the amino acid sequence of SEQ ID NO. 5 was designated as Control 1, and oral administration was designated as Control 2.

[0537] division AUC (%) Vehicle 100 Conjugate 3 56.7±6.8 Conjugate 17 68.8±9.1 Assembly 20 53.0±7.0

[0538] Conjugates 20 and 24

[0539] After orally administering the above conjugates 20 and 24 to mice, the blood glucose regulating efficacy was measured through a glucose tolerance test.

[0540] The measurement results are as shown in Figure 4, and it was confirmed that the blood glucose levels in the groups administered conjugates 20 and 24 were significantly lower compared to the untreated group.

[0542] Conjugates 53 to 54

[0543] The blood glucose regulating efficacy of the above conjugates 53 to 54 was measured through a glucose tolerance test. After orally administering the conjugates to mice, glucose was administered intraperitoneally 6 hours later, and changes in blood glucose were measured.

[0544] The measurement results are as shown in Figure 7, and it was confirmed that the blood glucose levels in the groups administered conjugates 53 to 54 were significantly lower compared to the untreated group.

[0546] Conjugates 65 and 66

[0547] After orally administering the above conjugates 65 and 66 to mice, the blood glucose regulating efficacy was measured through a glucose tolerance test.

[0548] To measure intraperitoneal glucose tolerance in an animal model, the sample was orally administered to 8-week-old male mice (C57BL / 6) at -20 minutes, followed by intraperitoneal injection of glucose (2 g / kg), and changes in blood glucose levels were observed in blood collected from the tail vein at -20, 0, 20, 40, 60, 90, and 120 minutes. The control group was orally administered a polypeptide linked by disulfide bonds between the 6th and 11th cysteine ​​of SEQ ID NO. 15, the 7th cysteine ​​of SEQ ID NO. 15 and the 7th cysteine ​​of SEQ ID NO. 16, and the 20th cysteine ​​of SEQ ID NO. 15 and the 19th cysteine ​​of SEQ ID NO. 16.

[0549] The measurement results are as shown in Figure 14. As shown in Figure 14, the blood glucose level of the conjugate administration group was significantly reduced compared to the untreated group.

[0551] Measurement of Caco-2 cell membrane permeability

[0552] Conjugates 17, 51, 72, and 73

[0553] The above conjugate 51 was dissolved in Hanks Balanced Salt Solution (HBSS), and the Caco-2 cell membrane permeability was measured. First, to form a Caco-2 cell monolayer, 1.5 x 10⁶ cells were placed in each well of a 12-transwell plate. 5Cells were seeded and cultured at 37°C under CO2 conditions for 3 to 4 weeks. During the first week, the culture medium was changed every 2 days, and thereafter, it was changed every 3 days. Cells 3 to 4 weeks after seeding were used for the experiment. To verify cell monolayer formation, TEER values ​​and Lucifer yellow values ​​were measured, and only cell monolayers with a TEER value of 300 Ω·cm² or higher and a Lucifer yellow transmittance of 3% or less were used for the test. For each test, the transwells were washed with transport media (HBSS) and incubated in a 37°C CO2 incubator for 1 hour. Subsequently, 200 μL of the prepared drug and permeability marker were added to the apical side, and 1 mL of drug-free transport media was added to the basolateral side. The samples were then incubated in a 37°C CO2 incubator for 2 hours. After 2 hours, 1 mL samples were taken from each basolateral side, and the drug permeability coefficient (Papp value) was measured using the enzyme-linked immunosorbent assay (ELISA). The Papp values ​​were calculated as follows, and the analysis results are shown in Table 21. (At this time, each conjugate used was prepared in the same manner as in the above preparation example.)

[0555] [ Papp(10 -6 , cm / s) = (dCr / dt) x Vr / (A x C0) ]

[0556] (*dCr-concentration of permeated sample, dt-drug treatment time, Vr-basolateral volume, A-transwell area, CO-initial drug concentration)

[0557] test substance Permeability Coefficient (x 10 7 cm / s) Fold test substance Volume (μg / mL) Polypeptide Sequence No. 5 214.5 0.01 1 Assembly 70 232.0 1.10 110 Conjugate 72 234.8 0.99 99 Conjugate 17 280.0 1.55 155 Conjugate 52 275.6 1.33 133

[0559] Conjugates 68 and 69

[0560] The above conjugates 68 and 69 were dissolved in Hanks Balanced Salt Solution (HBSS), and the intracellular accumulation of Caco-2 cells was measured. First, to determine the intracellular accumulation, Caco-2 cells were placed in a 96-well plate with 7 x 10⁶ cells per well. 4 The cells were dispensed into individual wells and incubated in a CO2 incubator at 37°C. After 24 hours, the culture medium was removed from each well, washed with HBSS, and 100 μl of the prepared drug was added to each well. The cells were then incubated in a CO2 incubator at 37°C. After 8 minutes, each well was washed with PBS, treated with 100 μl of 10% formalin, and reacted at room temperature. After 10 minutes, each well was washed with PBS, treated with 100 μl of 0.1% TRITON X-100, and reacted at room temperature. After 10 minutes, each well was washed with PBS and blocked with 1% BSA for 1 hour. Subsequently, the cells were treated with HRP Anti-Growth Hormone antibody (1:1000). After 1 hour, each well was washed with PBST, and Ultra TMB substrate solution was added. After 10 minutes, 2N HCl stop solution was applied to each well, and the absorbance was measured at 450 nM to calculate the intracellular accumulation of each substance. The results were based on hGH as 100%, and the measurement results are shown in Table 22 and Figure 16. (In this case, the conjugate used was prepared by the same method as in the above preparation example.)

[0561] test substance Relative Uptake (%) test substance Volume (μg / mL) polypeptide sequence number 42 0.66 100 Conjugate 68 0.69 110 Conjugate 69 0.74 707( P <0.001)

[0563] Measurement of body weight changes and feed intake

[0564] Conjugates 58 and 59

[0565] Changes in body weight were measured after subcutaneously administering the above conjugates 58 and 59 to obese model mice for 2 weeks.

[0566] As shown in Figure 10, it was confirmed that body weight decreased significantly when conjugates 58 and 59 were administered compared to the untreated group.

[0568] Concatenations 33, 36, 39, 42 and 61 to 64

[0569] After orally administering the polypeptide, conjugates 61 to 64, and conjugates 33, 36, 39, and 42 to mice, body weight loss and feed intake were measured for 24 hours. (At this time, each conjugate was prepared in the same manner as in the above preparation example.)

[0570] As a result of the measurements, body weight loss is shown in Table 23 below, and feed intake is shown in Figure 12. Compared to the untreated group, the single polypeptide administration group showed body weight loss and a decrease in feed intake; however, the conjugate administration group showed superior effects in body weight loss and a decrease in feed intake compared to the single polypeptide administration group.

[0571] division Weight loss (%) compared to the control group polypeptide sequence number 24 -2.63±1.06 polypeptide sequence number 26 -3.13±1.05 polypeptide sequence number 28 -1.11±0.84 polypeptide sequence number 30 -1.17±0.76 Conjugate 33 -4.07±1.30 Conjugate 36 -4.53±0.86 Conjugate 39 -1.99±0.94 Conjugate 42 -2.60±0.62 Conjugate 61 -2.96±4.22 Conjugate 62 -3.32±1.31 Conjugate 63 -2.41±0.78 Assembly 64 -3.38±1.26

[0573] A person skilled in the art will be able to recognize or identify numerous equivalents to the specific embodiments of the invention described herein using only routine experiment. Such equivalents are intended to be included in the following claims. The foregoing description of the present invention is for illustrative purposes only, and a person skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single unit may be implemented in a distributed manner, and components described as distributed may likewise be implemented in a combined form. All modifications or variations derived from the meaning and scope of the claims set forth below and the concept of equivalents thereof may be interpreted as being included in the scope of the present invention.

Claims

Claim 1 As a bioactive polypeptide, said polypeptide comprises an amino acid sequence selected from the group consisting of: H(Aib)QGTFTSDKSKYLDERAAQDFVQWLLDGGPSSGAPPPS (SEQ ID NO. 32), wherein one or more amino acids in the amino acid sequence of SEQ ID NO. 32 are deleted, inserted, or substituted; or HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK (SEQ ID NO. 18); or HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSKKK (SEQ ID NO. 19); or H(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK (SEQ ID NO. 20); or H(Aib)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSKKK (SEQ ID NO. 21). Claim 2 A physiologically active conjugate in which the polypeptide of claim 1 is combined with one or more biotin moietyes, fatty acid moietyes, or one or more biotin moietyes and fatty acid moietyes. Claim 3 In paragraph 2, the bioactive conjugate is represented by the following general formula A, wherein the biotin moiety is a bioactive conjugate: [General Formula A] In the above general formula A, X is a functional group capable of binding to the physiologically active substance, Y is a spacer, Z is a binding unit, and B is represented by the following chemical formula A-1, [Chemical Formula A-1] Z is of chemical formula A-1 It is connected to, T is a terminal, m is an integer from 1 to 10, n is an integer from 0 or 1 to 10, and when n=0, Y is directly coupled to B or T, and p is an integer from 0 or 1. Claim 4 In paragraph 3, the above X is maleimide, succinimide, N-hydroxysuccinimide, succimidyl succinate, succimidyl glutarate, succimidyl methyl ester, succimidyl pentyl ester, succimidyl carbonate, p-nitrophenyl carbonate, aldehyde, amine, thiol, oxyamine, iodoacetamide, aminoxyl, hydrazide, hydroxy, propionate, pyridyl, alkyl halide, vinylsulfone, carboxyl, hydrazide, halogen acetamide, C 2-5 Alkinyl, C 6-20 Aryl disulfide, C 5-20 A bioactive conjugate selected from the group consisting of heteroaryl disulfide, isocyanate, thioester, iminoester, and derivatives thereof. Claim 5 In paragraph 3, the above Y is a linear or branched C that is absent, substituted, or unsubstituted. 1-50 Alkylene, substituted or unsubstituted linear or branched C 1-50 Heteroalkylene, substituted or unsubstituted C 6-50 Aryllene, or substituted or unsubstituted C 6-50 A bioactive conjugate that is heteroarylene and, when substituted, comprises one or more selected from the group consisting of =O, -C(O)NH2, -OH, -COOH, -SH, =NH and -NH2. Claim 6 In paragraph 3, the above Y is -C(O)-(OCH2CH2) u A bioactive conjugate comprising -NH- as a repeating unit, wherein u is an integer from 1 to 20. Claim 7 In paragraph 3, the above Y is a physiologically active conjugate comprising glutamic acid, glutamine, glycine, isoleucine, or lysine as part of its composition. Claim 8 In paragraph 3, the above-mentioned Z is any one of the following and each is independently selected: a physiologically active conjugate that forms an amino acid or a derivative thereof together with or separately from X; or B) a substituted or unsubstituted linear or branched C 1-50 It is a heteroalkylene, wherein, where substituted, it comprises one or more selected from the group consisting of =O, -C(O)NH2, -OH, -COOH, -SH, =NH and -NH2. Claim 9 In paragraph 3, the above T is an amine, C 1-8 Alkyl, C 1-8 A bioactive conjugate 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. Claim 10 In paragraph 2, the biotin moiety is, , , , , , , , , , , , , , , , and A bioactive conjugate selected from a group consisting of Claim 11 A bioactive substance conjugate according to paragraph 2, wherein the fatty acid moiety is bound to a bioactive substance and the biotin moiety is bound to a site different from the site of the bioactive substance to which it is bound. Claim 12 In paragraph 2, the above-mentioned fatty acid moiety is represented by the following general formula B, a physiologically active conjugate: [General Formula B] X'-Y'-W In the above formula, X' is a functional group capable of binding to the physiologically active substance, Y' is a spacer, and W is a fatty acid. Claim 13 In paragraph 12, the above X' is maleimide, succinimide, N-hydroxysuccinimide, succimidyl succinate, succimidyl glutarate, succimidyl methyl ester, succimidyl pentyl ester, succimidyl carbonate, p-nitrophenyl carbonate, aldehyde, amine, thiol, oxyamine, iodoacetamide, aminoxyl, hydrazide, hydroxy, propionate, pyridyl, alkyl halide, vinylsulfone, carboxyl, hydrazide, halogen acetamide, C 2-5 Alkinyl, C 6-20 Aryl disulfide, C 5-20 A physiologically active conjugate selected from the group consisting of heteroaryl disulfide, isocyanate, thioester, iminoester, tetrafluorophenyl ester, nitrophenyl carbonate, nitrophenyl, and derivatives thereof. Claim 14 In Clause 12, the above W is a substituted or unsubstituted linear or branched C 1-60 Alkylene, substituted or unsubstituted linear or branched C 1-60 Alkenylene, substituted or unsubstituted linear or branched C 1-60 Heteroalkylene, or substituted or unsubstituted linear or branched C 1-60 A bioactive conjugate that is heteroalkenylene, and when substituted, is substituted with one or more selected from the group consisting of =O, -C(O)NH2, -OH, -COOH, -SH, =NH, -NH2, and halo. Claim 15 In paragraph 2, the physiologically active conjugate, wherein the fatty acid moiety is represented by the chemical formula of the following general formula B1: [General Formula B1] X' 1 -Y'-C(O)-F 1 X'1 is maleimide, N-hydroxysuccinimide, aldehyde, amine, tetrafluorophenyl ester, or nitrophenol, Y' is a spacer, and F1 is C 6-28 It is a substituted or unsubstituted linear or branched alkylene, or a substituted or unsubstituted linear or branched heteroalkylene. Claim 16 In paragraph 12 or 15, the above Y' corresponds to a direct bond, or C substituted or unsubstituted within the structure of Y. 1-50 Linear alkylene, substituted or unsubstituted C 1-50 Non-linear alkylene, substituted or unsubstituted C 1-50 Linear heteroalkylene, substituted or unsubstituted C 1-50 Non-linear heteroalkylene, substituted or unsubstituted C 1-50 Aryllene, substituted or unsubstituted C 1-50 It comprises one or more groups consisting of 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 unit (-(CH2CH2O) n -, n is an integer between 1 and 20 inclusive, a physiologically active conjugate. Claim 17 In paragraph 12 or 15, the above Y' is -C(O)-(OCH2CH2) u A bioactive conjugate comprising -NH- as a repeating unit, wherein u is an integer from 1 to 20. Claim 18 In claim 12 or 15, the above Y' is a bioactive conjugate comprising glutamic acid, glutamine, glycine, isoleucine, or lysine as part of its composition. Claim 19 In claim 1, the fatty acid moiety is , , , , , , , , , , , , , , and A bioactive conjugate selected from a group consisting of Claim 20 A pharmaceutical composition for the prevention or treatment of diabetes, obesity, fatty liver disease, irritable bowel syndrome, neurodegenerative disease, bone disease, osteoporosis, human growth hormone deficiency, anticancer or non-alcoholic fatty liver disease, comprising a physiologically active substance conjugate according to any one of claims 1 to 19. Claim 21 A composition for oral administration comprising a physiologically active substance conjugate according to any one of claims 1 to 19.

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