KR20140103544A

KR20140103544A - Effective Ingredient Delivery System for Prolonging In-vivo Half-Life of an Effective Ingredient

Info

Publication number: KR20140103544A
Application number: KR1020130017045A
Authority: KR
Inventors: 김상재
Original assignee: 주식회사 카엘젬백스; 김상재
Priority date: 2013-02-18
Filing date: 2013-02-18
Publication date: 2014-08-27

Abstract

Disclosed is an active ingredient delivery system for extending an in-vivo half-line of an active ingredient. The active ingredient delivery system comprises carrier peptide and the conjugate of the active ingredient. The conjugate has a serum half-line which is extended compared to the peculiar serum half-line of the active ingredient, the carrier peptide is a peptide bonded to albumin in blood in order to extend the serum half-line of the active ingredient. The carrier peptide is a peptide including sequence number 1, a peptide having a sequence homogeny of 80% or more with the peptide sequence, or a fragment of the same. The peptide having a sequence homogeny of 80% or more and the fragment may have a serum albumin bonding function of the sequence number 1 peptide. Using the techniques disclosed in the present invention, an in-vivo half-line of an active ingredient can be extended, and the administered amount and the number of administrations of the active ingredient can be reduced, so that the effects of the active ingredient can be improved.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an effective ingredient delivery system for prolonging in vivo half life of an active ingredient,

In this specification, contents concerning an active ingredient delivery system for extending the in vivo half-life of an active ingredient are described.

The time for which the efficacy of the active ingredients administered in vivo is maintained in the body is very important. The longer the holding time, the lower the dosage and the number of administration. One of the ways to increase the in vivo efficacy maintenance time of the active ingredients may be to control the time that the active ingredient remains in the body, i.e., the half-life.

In recent years, bio-derived molecules have been widely used for the diagnosis and treatment of diseases, and representative examples are peptides-based proteins classified into antibodies, various hormones and enzymes according to their functions.

In order for such a biologically active protein to be effectively used as a therapeutic drug, it is very important to control the time, i.e., half-life, of staying in the body when administered to the body. In particular, the limitations of the potential therapeutic effects of these proteins quickly disappear in the body. As a result, there is a problem that the number of injections increases in order for the therapeutic drug to maintain a stable level in the body. Therefore, one of the most important factors for the development of a successful protein drug is the development of a method to prolong the half-life of the drug. When the half-life is increased, there is a great advantage that the level of drug exposure can be reduced and the time spent in the body can be increased to ultimately reduce the amount of drug administered.

Telomere is a genetic material that is repeatedly present at the end of a chromosome, and is known to prevent damage to the chromosome or its binding to other chromosomes. As the cell divides, the length of the telomere is getting shorter. When there is more than a certain number of cell divisions, the telomere becomes very short, and the cell stops dividing and dies. On the other hand, it is known that lengthening the telomeres prolongs the life of the cells. For example, it is known that the cancer cells secrete an enzyme called telomerase and prevent the shortening of the telomeres.

Korean Patent Publication No. 10-2007-0119622 Korean Patent Publication No. 10-2007-0024206 European Patent Publication No. 1362597 European Patent Publication No. 0841396

In one aspect of the present invention, the object of the present invention is to prolong the in vivo half-life of the active ingredient.

In another aspect of the present invention, the object of the present invention is to reduce the dose and the number of administration of the active ingredient.

An effective component delivery system for extending the in vivo half-life of an active ingredient according to one aspect of the present invention comprises a carrier peptide and a conjugate of an active ingredient, wherein the conjugate has an intrinsic serum of the active ingredient Wherein the carrier peptide is a peptide that binds to albumin in blood and that prolongs the serum half-life of the active ingredient, wherein the carrier peptide comprises a peptide comprising SEQ ID NO: 1, a peptide comprising the peptide Or a fragment thereof having a sequence homology of 80% or more with the sequence, wherein the peptide and the fragment having 80% or more of the sequence homology can have the serum albumin binding ability of the peptide of SEQ ID NO: 1.

In an effective component delivery system for extending the in vivo half-life of an active ingredient according to an aspect of the present invention, the fragment may be a fragment composed of three or more amino acids.

In an effective ingredient delivery system for extending the in vivo half life of an active ingredient according to one aspect of the present invention, the transportation peptide may be a peptide composed of 30 amino acids or less.

A composition according to one aspect of the present invention is a composition comprising an active ingredient having an extended half-life, wherein the composition comprises a carrier peptide and a conjugate of an active ingredient, Wherein the carrier peptide is a peptide that binds to albumin in the blood and is a peptide that prolongs the serum half-life of the active ingredient, wherein the carrier peptide has the amino acid sequence of SEQ ID NO: 1 A peptide having a homology of 80% or more with the above peptide sequence, or a fragment thereof, and the peptide or fragment having 80% or more homology with the sequence may have a serum albumin binding ability of the peptide of SEQ ID NO: 1 .

In the composition according to one aspect of the present invention, the fragment may be a fragment composed of three or more amino acids.

In a composition according to one aspect of the invention, the carrying peptide may be a peptide consisting of up to 30 amino acids.

A method according to one aspect of the invention is a method for prolonging the duration of an active ingredient comprising administering to a subject in need thereof a carrier peptide and a conjugate of an active ingredient, Wherein the conjugate has an extended serum half-life compared to the intrinsic serum half-life of the active ingredient, wherein the transport peptide is a peptide that binds to albumin in the blood and is a peptide that prolongs the serum half-life of the active ingredient, Wherein the peptide or fragment has a sequence homology of at least 80% with the peptide sequence, or a fragment thereof, wherein the peptide or fragment having at least 80% sequence homology has a serum albumin binding ability of the peptide of SEQ ID NO: 1 Lt; / RTI >

In the method according to one aspect of the present invention, the fragment may be a fragment composed of three or more amino acids.

In the method according to one aspect of the invention, the carrying peptide may be a peptide consisting of up to 30 amino acids.

Using the techniques disclosed herein, the in vivo half-life of an active ingredient can be extended. This can reduce the dosage of the active ingredient and the frequency of administration. Also, the effect of the active ingredient can be increased.

Figure 1 is a schematic representation of a primer sequence for the conjugate production of pep1, a carrier peptide, and Green Fluorescent Protein (GFP).
Figure 2 is a schematic representation of a vector for the conjugation of pep1, a carrier peptide, and Green Fluorescent Protein (GFP).
FIG. 3 shows the results of SDS-PAGE electrophoresis of binding proteins after immunizing the cell contents of Mycobacterium tuberculosis with the pep1-GFP conjugate attached to a resin.
FIG. 4 shows the result of SDS-PAGE electrophoresis of bound proteins after immunizing the human sera after attaching the pep1-GFP conjugate to the resin.
Figure 5 is toxicity experiment data for pep1.

Serum albumin is the most abundant protein in blood and plays an important role as a delivery medium for fatty acids and drugs (cf. Advanced Protein Chemistry, 37, 161-245, 1985). The albumin synthesized in the liver not only can increase the half-life of the body through interaction with the FcRn receptor, a major histocompatibility (MHC class I) molecule (see Protein Engineering, Design & Selection 23 : 11, 827-834, 2010) and are known to help maintain homeostasis (cf. The Lancet, December 16, 1989). Therefore, the peptide binding to the albumin becomes capable of increasing the blood half-life of the active ingredient bound to the peptide.

"Carrier peptide" as used herein refers to a peptide that binds to an active ingredient and carries the active ingredient in vivo. In one aspect of the present invention, the carrying peptide is a peptide comprising SEQ ID NO: 1, a peptide having 80% or more sequence homology with the peptide sequence, or a fragment thereof, wherein the peptide having the sequence homology of 80% May have the serum albumin binding ability of the peptide of SEQ ID NO: 1.

The peptides of SEQ ID NO: 1 are shown in Table 1 below. SEQ ID NO: 2 is the sequence of human telomerase full-length protein. SEQ ID NO: 1 is a peptide derived from telomerase and consisting of 16 amino acids. The "name" in Table 1 below is what the peptides are named for. In another aspect of the present invention, the peptide of SEQ ID NO: 1 includes a "synthetic peptide" synthesized by selecting peptides at corresponding positions among the peptides contained in the telomerase.

SEQ ID NO: name Telomerase phase location order Length One. pep1 [611-626] EARPALLTSRLRFIPK 16 aa 2. Telomerase [1-1132] MPRAPRCRAVRSLLRSHYREVLPLATFVRR LGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTW KMSVRDCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQ VNSLQTV CTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD 1132 aa

One aspect of the present invention provides a polynucleotide encoding a peptide comprising SEQ ID NO: 1, or a fragment thereof, or a peptide having 80% or more sequence homology with the above peptide sequence. The polynucleotide can be used to mass produce a peptide comprising SEQ ID NO: 1 or a peptide thereof or a peptide having 80% or more sequence homology with the peptide sequence. For example, a vector containing a polynucleotide encoding the peptide can be cultured in a host cell to mass-produce the peptide.

The peptides disclosed herein may comprise peptides having greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99% sequence homology. Also, the peptide disclosed in the present specification can be prepared by reacting a peptide or a fragment thereof comprising SEQ ID NO: 1 with one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, Or 7 or more amino acid altered peptides.

In one aspect of the invention, the amino acid change is of a property that causes the physicochemical properties of the peptide to change. For example, amino acid changes such as improving the thermal stability of the peptide, altering the substrate specificity, changing the optimum pH, etc. can be performed.

As used herein, the term "amino acid" includes D-isomers and modified amino acids as well as the 22 standard amino acids that are naturally incorporated into the peptide. Accordingly, in one aspect of the present invention, the peptide may be a peptide comprising a D-amino acid. In another aspect of the present invention, the peptide may include post-translationally modified non-standard amino acids and the like. Examples of post-translational modifications include phosphorylation, glycosylation, acylation (including, for example, acetylation, myristoylation and palmitoylation), alkylation ), Carboxylation, hydroxylation, glycation, biotinylation, ubiquitinylation, changes in chemical properties (such as, for example, beta-depleted deamidation , Deamidation) and structural changes (e.g., formation of a disulfide bridge). Also included are amino acid changes such as changes in amino acids, such as changes in amino groups, carboxy groups or side chains, caused by chemical reactions that take place during binding with crosslinkers to form peptide conjugates.

The peptides disclosed herein may be wild type peptides identified and isolated from natural sources. Alternatively, the peptides disclosed herein may be artificial variants, including amino acid sequences in which one or more amino acids are substituted, deleted and / or inserted, as compared to peptides that are fragments of SEQ ID NO: 1. Amino acid changes in wild-type polypeptides as well as in artificial variants include conservative amino acid substitutions that do not significantly affect folding and / or activity of the protein. Examples of conservative substitutions include, but are not limited to, basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, valine and methionine) Tryptophan and tyrosine), and small amino acids (glycine, alanine, serine and threonine). In general, amino acid substitutions that do not alter specific activity are known in the art. The most commonly occurring interactions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly, and vice versa. Other examples of conservative substitutions are shown in the following table.

Original amino acid Exemplary residue replacement Preferred residue substitution Ala (A) val; leu; with Val Arg (R) lys; gln; asn Lys Asn (N) gln; feeling; asp, lys; arg Gln Asp (D) glu; asn Glu Cys (C) ser; ala Ser Gln (Q) asn; glu Asn Glu (E) asp; gln Asp Gly (G) ala Ala His (H) asn; gln; lys; arg Arg Ile (I) leu; val; met; ala; phe; norleucine Leu Leu (L) norleucine; with; val; met; ala; phe Ile Lys (K) arg; gln; asn Arg Met (M) leu; phe; with Leu Phe (F) leu; val; with; ala; tyr Tyr Pro (P) ala Ala Ser (S) thr Thr Thr (T) ser Ser Trp (W) tyr; phe Tyr Tyr (Y) trp; phe; thr; ser Phe Val (V) with; leu; met; phe; ala; norleucine Leu

Substantial variations in the biological properties of the peptide include (a) the structure of the polypeptide backbone within the substitution region, e.g., their effect in maintaining the sheet or helical conformation, (b) the charge of the molecule at the target site Or their effect in maintaining hydrophobicity, or (c) their effect in maintaining the bulk of the side chain is significantly different. The natural residues are grouped into the following groups based on their usual side chain properties:

(1) hydrophobicity: norleucine, met, ala, val, leu, ile;

(2) Neutral hydrophilic: cys, ser, thr;

(3) Acid: asp, glu;

(4) Basicity: asn, gln, his, lys, arg;

(5) Residues affecting chain orientation: gly, pro; And

(6) Aromatic: trp, tyr, phe.

Non-conservative substitutions will be made by exchanging one member of these members for another. Any cysteine residue not associated with maintaining the proper stereostructure of the peptide can generally be replaced with a serine to enhance the oxidative stability of the molecule and prevent strange cross-linking. Conversely, cysteine bond (s) can be added to the peptide to improve its stability

Other types of amino acid variants of peptides are those in which the glycosylation pattern of the antibody is altered. The term change refers to the deletion of one or more carbohydrate moieties found in the peptide and / or the addition of one or more glycosylation sites that are not present in the peptide.

Glycosylation of peptides is typically N-linked or O-linked. N-linked refers to a carbohydrate moiety attached to the side chain of an asparagine moiety. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatically attaching carbohydrate moieties to asparagine side chains. Thus, the presence of one of these tripeptide sequences in the polypeptide creates a potential glycosylation site. O-linked glycosylation refers to attaching one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine May be used.

Addition of the glycosylation site to the peptide is conveniently accomplished by varying the amino acid sequence to contain one or more of the above-mentioned tripeptide sequences (in the case of N-linked glycosylation sites). Such changes may be made by adding one or more serine or threonine residues to the sequence of the original antibody or by substituting these residues (for O-linked glycosylation sites).

One aspect of the present invention provides a conjugate in which a peptide comprising SEQ ID NO: 1, or a peptide thereof, or a peptide having a sequence homology of 80% or more with the peptide sequence and the target substance to be delivered are conjugated with each other.

As used herein, the term "active ingredient" is intended to encompass all substances that bind carrier peptides, including, for example, any substance that desires to increase serum half-life, in particular drugs, cosmetics or active substances of health food, Nanoparticles, biological agents, viruses, contrasting substances or other chemicals, which may be exemplified by proteins, nucleic acids, peptides, minerals, and glucose, But is not limited to. In one aspect of the present invention, the active ingredient peptide may be a cytokine, an antibody, an antibody fragment, a therapeutic enzyme, a soluble receptor, or a ligand.

In one aspect of the present invention, the active ingredient may be at least one selected from proteins, nucleic acids, peptides, lipids, glycolipids, minerals, sugars, contrast agents, drugs and chemical compounds. In one aspect of the present invention, the active ingredient may be a peptide. As used herein, the term "drug" is a broad concept that includes materials for alleviating, preventing, treating or diagnosing diseases, wounds or certain symptoms.

In one aspect of the present invention, the protein or peptide as an active ingredient to be delivered includes, but is not limited to, one or more of hormones, hormone analogs, enzymes, enzyme inhibitors, signal transduction proteins (or peptides), antibodies and vaccines. In one aspect of the invention, the nucleic acid can be a naturally occurring or artificial DNA or RNA molecule, and can be single-stranded or double-stranded. The nucleic acid molecule may be one or more, it may be a nucleic acid molecule of the same type (e.g. having the same nucleotide sequence), or it may be a nucleic acid molecule of another type. But are not limited to, DNA, cDNA, decoy DNA, RNA, siRNA, miRNA, shRNA, stRNA, snoRNA, snRNA, PNA, antisense oligomer, plasmid and other modified nucleic acids It is not. In one aspect of the invention, the virus may comprise a whole virus or a viral core comprising a nucleic acid of the virus. In one aspect of the invention, a chemical is a broad concept including a chemical capable of acting as a drug, including natural or synthetic chemicals.

In one aspect of the invention, the active ingredient may be in particular a protein or a peptide. Types of proteins or peptides include cytokines, chemokines, lymphokines, ligands, receptors, hormones, apoptosis-inducing polypeptides, enzymes, antibodies and antibody fragments, and growth factors. Examples of receptors include TNF type I receptor, IL-1 receptor type II, IL-1 receptor antagonist, IL-4 receptor, and any soluble receptor chemically or genetically modified. Examples of enzymes include activated protein C, factor VII, collagenase, agarcidase-beta; Dornase-alpha; Alteplase; Pegylated apataginase; Asparaginease, and the like. (G-CSF), macrophage colony stimulating factor (M-CSF), colony stimulating factor (CSF), interferon beta (IFN-?), Interferon (IFN-gamma), interferon gamma inducible factor I (IGIF), transforming growth factor beta (TGF- beta), RANTES (regulated upon activation, expressed normal T-cells and possibly secreted), macrophage inflammatory protein (EGF), vascular endothelial growth factor (VEGF), and vascular endothelial growth factor (VEGF), such as MIP-1-alpha and MIP-1- Neurotrophin-2 (NT-2), neurotrophin-3 (NT-3), fibroblast growth factor (FGF), neurotrophic factor (BGF) ), Neurotrophin-4 (NT-4), neurotrophin-5 (NT-5), collagen cell-derived neurotrophic factor (GDNF), filamentous neurotrophic factor (CNTF) Eri As can be given erythropoietin (EPO), insulin and soluble glycoproteins, such as the glycoprotein gp120 and gp160. The gp120 glycoprotein is the human immunodeficiency virus (HIV) envelope protein, and the gp160 glycoprotein is a known precursor of the gp120 glycoprotein

In one aspect of the present invention, the half-life of the active ingredient is increased relative to the native half-life through binding with the carrier peptide.

The efficacies may be for the treatment of diseases such as Parkinson's disease, cancer, and heart disease from a disease standpoint. Also, peptides that are active ingredients include multiple infarction; Sjogren's syndrome; Sarcoidosis; Insulin dependent diabetes mellitus; Autoimmune thyroiditis; Arthritis (such as osteoarthritis, rheumatoid arthritis, reactive arthritis, and psoriatic arthritis); Ankylosing spondylitis; And for the treatment of autoimmune diseases such as scleroderma. Peptides that are also active ingredients can be used to treat acute and chronic inflammatory diseases, to promote growth, to promote wound healing, and to prevent rejection of cells, tissue EH after transplantation of organs have.

In one aspect of the invention, the drug delivered into the cell by the carrier peptide may further comprise a drug delivery vehicle such as a liposome, micelle, nanoparticle, magnetic particle or quantum dot.

As used herein, the term " imaging material "is a broad concept that encompasses all materials used for imaging in vivo structures or fluids in medical imaging. Suitable contrast agents include, but are not limited to, radiopaque contrast agents, paramagnetic contrast agents, superparamagnetic contrast agents, CT (computed tomography) contrast agents, and other contrasting agents . For example, a radiation non-transmucosal contrast material (for X-ray imaging) is an inorganic iodine compound and an organic iodine compound (for example, diatrizoart), a radiation non-transmissive metal and salts thereof Etc.) and other radiation non-transmissive compounds (for example, calcium salts, barium salts such as barium sulphate, tantalum and tantalum). Suitable paramagnetic contrast materials (for MR imaging) include gadolinium diethylene triaminepentaacetic acid (Gd-DTPA) and derivatives thereof and other gadolinium, manganese, iron, dysprosium, copper, Such as europium, erbium, chromium, nickel and cobalt complexes such as 1,4,7,10-tetraazacyclododecane-N, N ', N ", N" (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclododecane-N, -N ', N "-triacetic acid (D03A) N, N ', N' '- tetraacetic acid (TETA), hydroxy-N, N', N '' - triacetic acid (NOTA), 1,4,8,10-tetraazacyclotetradecane- Benzyl ethylene-diamine diacetic acid (HBED) and the like. Suitable superparamagnetic contrast media (for MR imaging) include magnetite, super-paramagnetic iron oxide (SPIO), ultrasmall superparamagnetic iron oxide (USPIO), and monocrystalline iron oxide. do. Other suitable contrast agents are iodinated and non-iodinated, ionic and non-ionic CT contrast agents, and contrast agents such as spin-labels, or other diagnostically effective agents.

Other examples of contrast agents include, but are not limited to, beta-galactosidase, green fluorescent protein, blue fluorescent protein, luciferase, etc., and when expressed in cells, a marker gene encoding an easily detectable protein . Various labels such as radionuclide, fluorine, enzyme, enzyme substrate, enzyme cofactor, enzyme inhibitor, ligand (especially, hapten), etc. can be used.

In one embodiment of the present invention, the contrast material may be a ferrocenecarboxylic acid of formula (2). The structure of the ferrocene is shown in Formula (1).

[Chemical Formula 1]

(2)

In one embodiment of the invention, the conjugate of the carrier peptide and the contrast agent can be ferrocenecarboxylic-pepl of the formula (3).

(3)

In one aspect of the invention. The peptide or composition may be fused with one or more detectable labels. The label may be a compound that directly or indirectly generates a detectable compound or signal in a chemical, physical, or enzymatic reaction. Labeling and subsequent detection can be carried out by methods known in the art (see, for example, Sambrook, J., and Russel, DW (2001); and Lottspeich, F., and Zorbas H. (1998) Bioanalytik, Spektrum Akademischer Verlag, Heidelberg / Berlin, Germany). The label includes, but is not limited to, a fluorescent label, an enzyme label, a chromogenic label, a luminescent label, a radiolabel, a hapten, a biotin, a metal complex, a metal and a colloidal gold. All these types of labels are well known in the art and are commercially available from a variety of suppliers.

In one aspect of the invention, the cargo can be bound directly to the peptide. In another aspect of the invention, the carrier can be conjugated to the peptide through several binding methods, including covalent or noncovalent bonding. The subject to be delivered can, for example, bind to the N-terminus or C-terminus of the peptide according to one aspect of the present invention. For example, the peptide can be attached to the peptide via a covalent bond that binds the carrier to an amine of a disulfide bond, or an alpha amine or a C-terminal lysine (K) residue of the peptide N-terminal glutamate (E) The conveying object can be combined. Alternatively, the peptides and the carrier can be bound to each other through non-covalent bonding in which the peptide and the carrier can be encapsulated in the form of a capsule, for example.

In another aspect of the invention, the peptide and the carrier can be coupled via a linker. For example, a linker of Hynic (6-hydrazinopyridine-3-carboxylic acid) with an amine of a peptide N-terminal glutamate (alpha-amine) or a C- After introducing the same linker, the carrier can be bound to the peptide by binding the carrier to the linker.

In another aspect of the present invention, when the object to be transfected is DNA or RNA, a SH group (thiol group) is introduced into the peptide, a maleimide group is introduced into DNA or RNA, and then the SH group and DNA Or by binding the maleimide group of the RNA to the peptide.

In another aspect of the present invention, when the object to be transfected is a protein or a peptide, a DNA expressing a transporter is bound to a DNA expressing the transporter, and then the DNA is expressed to express the transporter peptide as a fusion protein of the transporter and the peptide And the object to be conveyed can be combined. Specific examples of the binding by the fusion protein are as follows: when a primer for producing a fusion protein is produced, a nucleotide encoding a carrier peptide is attached in front of a nucleotide expressing a carrier, and the obtained nucleotide is then reacted with a restriction enzyme The pET vector is inserted into the example vector and BL-21 (DE3) is transformed into an exemplary cell and expressed. At this time, an expression inducing agent such as IPTG (isopropyl-1-thio- [ beta] -D-galactopyranoside) may be treated to effectively express the fusion protein. Thereafter, the fusion protein expressed through a method such as His tag purification can be purified, dialyzed with PBS, and concentrated by centrifugation at 2,000 to 4,000 rpm, for example, at 5,000 to 4,000 rpm in a kit .

In one aspect of the invention, the carrier peptide can be conjugated to a dye or a fluorescent material, specifically fluorescein isothiocyanate (FITC) or green fluorescence protein (GFP). In one aspect of the invention, FITC can bind to the amino group (NH ₃ ⁺ ) of the N-terminal or C-terminal Lys of the carrier peptide. If the peptide lacks Lys at the end, the peptide and FITC can be bound by a linker containing Lys.

A peptide comprising the sequence of SEQ ID NO: 1 disclosed herein, or a peptide thereof, or a peptide having 80% or more sequence homology with the peptide sequence, is bound to the cargo at a molar ratio of 1: 1 But it is also possible to combine them at other molar ratios. For example, the molar ratio of CPP: transport target may be 2: 1 or more. Specifically, it may be 2: 1 or more, 3: 1 or more, 4: 1 or more, 5: 1 or more, 6: 1 or more, 7: 1 or more, 8: 1 or more, 9: 1 or more or 10: This means that the carrier peptide of a plurality of molecules can bind to one carrier molecule. The plurality of carrying peptide molecules may be connected to each other in series or in parallel. Linked in series means that they are bonded to each other at terminal amino acid sites of the carrier peptide, and when they are connected in parallel, they are bonded to each other at a portion other than the terminal amino acid of the carrier peptide. Conversely, the molar ratio of the carrier peptide to the carrier may be 1: 2 or more. This means that a plurality of carrier molecules can bind to one carrier peptide molecule. For example, the molar ratio of carrier peptide: migrate may be 1: 2. Specifically, it may be 1: 2 or more, 1: 3 or more, 1: 4 or more, 1: 5 or more, 1: 6 or more, 1: 7 or more, 1: 8 or more, 1: 9 or more or 1:10 or more.

Since the peptide bound to the contrast material can easily grasp its migration pathway, the carrier peptide according to one aspect of the present invention can be utilized for cell imaging or intracellular drug delivery pathway tracing.

One aspect of the present invention provides a use for increasing the in vivo half-life of one or more active ingredients of a peptide comprising SEQ ID NO: 1 or a peptide thereof or a peptide having 80% or more sequence homology with the peptide sequence .

An aspect of the present invention relates to a pharmaceutical composition comprising a drug; And a peptide comprising SEQ ID NO: 1, or fragments thereof, or a peptide having a sequence homology of at least 80% with the peptide sequence, to a subject, .

An aspect of the present invention is a method for detecting a peptide comprising an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence thereof, comprising applying to a subject a peptide comprising SEQ ID NO: 1 or a peptide thereof or a peptide having the sequence homology of at least 80% Provides a method for tracing an applied drug delivery pathway of a subject capable of tracing a drug delivery pathway.

An aspect of the present invention relates to a composition comprising a peptide comprising SEQ ID NO: 1 or a peptide thereof or a conjugate of a drug to be delivered and a peptide having a sequence homology of at least 80% with the peptide sequence; And an instruction that discloses one or more of a dose, an administration route, a frequency of administration, and an indication of the composition, wherein the drug has an extended in vivo maintenance time.

An aspect of the present invention relates to a pharmaceutical composition comprising an active ingredient; And a peptide of SEQ ID NO: 1 or fragments thereof, or a peptide having 80% or more sequence homology with the above peptide sequence. Another aspect of the present invention provides a pharmaceutical, cosmetic or food composition comprising a peptide comprising SEQ ID NO: 1 or a peptide thereof, or a conjugate of an active ingredient with a peptide having a sequence homology of at least 80% with said peptide sequence do.

One aspect of the present invention is a pharmaceutical composition comprising a peptide comprising SEQ ID NO: 1 or a peptide thereof, or a conjugate of an active ingredient with a peptide having a sequence homology of at least 80% with the peptide sequence, Cosmetics or food compositions that exhibit good shelf-life.

In another embodiment, the present invention provides a nucleic acid molecule encoding the polypeptide, wherein the nucleotide sequence thereof has, for example, GAA GCG CGC CCG GCG CTG CTG ACC AGC CGC CTG CGC TTT ATT CCG AAA sequence (SEQ ID NO: 5). The nucleic acid molecule may be introduced into the host cell according to techniques known to those skilled in the art. For example, calcium phosphate method, liposome, electroporation, transformation by contacting the virus with a cell, or micro-injection into a cell directly. The host cell may be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell such as a yeast cell, or a prokaryotic cell such as a bacterial cell. Suitable prokaryotic hosts for transformation include, for example, species belonging to the genus Escherichia coli, Bacillus subtilis, Salmonella typhimurium, Pseudomonas, Streptomyces, and Mike bacteria.

The vector comprising the nucleic acid molecule is generally a recombinant expression vector, which contains a source of replication and a selectable marker (e. G., Dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or e. Tetracycline or ampicillin resistance in E. coli, or S. cerevisiae TRP1 gene), and promoters that regulate transcription of the protein coat sequence. Useful expression vectors that may be used include, for example, SV40 and derivatives of pcDNA and known vectors such as col E1, pCR1, pBR322, pMal-C2, pET, pGEX (Smith, et al., Gene 67: 31-40 Phage DNA such as plasmids such as the bacterial plasmid, pMB9 and its derivative RP4, phage DNA such as numerous derivatives of phage I such as NM989, and phage DNA of M13 and filament type single strand; Yeast plasmids such as phage DNA or vectors derived from a combination of phage DNA and modified plasmids for use in expression control sequences. Mammalian expression vectors include the origin of replication, appropriate promoters and enhancers. It may also contain essential ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and 5 'flanking non-promoter sequences. The mammalian expression vector may be a vector comprising an inducible promoter such as a dihydrofolate reductase promoter, any expression vector comprising a DHFR expression cassette may be a DHFR / methotrexate co-amplification vector such as pED (Randal J, Kaufman, 1991, Randal J. Kaufman, Current Protocols in Molycular Biology, 16, 12 (1991)). Or glutamine synthetase / methionine sulfoximine co-amplification vector, such as pEE14 (Celltech). Such as pREP4 (Invitrogen), pCEP4 (Invitrogen), pMEP4 (Invitrogen), pREP8 (Invitrogen), pREP9 (Invitrogen), and the like, which direct episomal expression under the control of Epstein Barr virus (EBV) or nuclear antigen pEBVHis (Invitrogen) may be used. Selectable mammalian expression vectors include Rc / CMV (Invitrogen), pRc / RSV (Invitrogen), and the like. The vaccinia virus mammalian expression vectors that can be used in the present invention include pSC11, pMJ601, pTKgptF1S, and the like.

Yeast expression systems that may be used in the present invention include non-fusion pYES2 vector (Invitrogen), fusion pYESHisA, B, C (Invitrogen), pRS vector, and the like.

The vector may be introduced into various cell mammals, particularly human-derived cells, as well as bacteria, yeast, fungi, insects, nematodes and plant cells. Examples of suitable cells include VERO cells, HELA cells, e.g. CCL2, a CHO cell line such as ATCC No. CCL61, COS cells such as COS-7 cells and ATCC No. 7 cells. CRL 1650 cells, W138, BHK, HepG2, 3T3, such as ATCC No. CRL6361, A549, PC12, K562 cells, 293 cells, Sf9 cells, e.g. CRL1711 and Cv1 cells, such as ATCC No. 1. CCL70 and so on.

Other suitable cells that may be used in the present invention include prokaryotic host cell strains such as E. coli (such as DH5-a strain), Bacillus subtilis, Salmonella typhimurium, or strains belonging to the genus Pseudomonas, Streptomyces and Staphylococcus .

The composition according to one aspect of the present invention comprises a peptide comprising SEQ ID NO: 1 or a peptide thereof, or a peptide having a sequence homology of not less than 80% with the peptide sequence in an amount of from 0.1 μg / mg to 1 mg / / Mg to 0.5 mg / mg, more specifically from 10 μg / mg to 0.1 mg / mg. When it is included in the above range, it is not only suitable for exhibiting the intended effect of the present invention but also can satisfy both the stability and safety of the composition, and may be suitably included in the above range in terms of cost effectiveness.

The composition according to one aspect of the present invention can be applied to all animals including humans, dogs, chickens, pigs, cattle, sheep, guinea pigs or monkeys.

The pharmaceutical composition according to one aspect of the present invention can be administered orally, rectally, transdermally, intravenously, intramuscularly, intraperitoneally, intramuscularly, intradermally or subcutaneously.

Formulations for oral administration may be, but are not limited to, tablets, pills, soft or hard capsules, granules, powders, solutions or emulsions. Formulations for parenteral administration may be, but are not limited to, injections, drops, lozenges, ointments, gels, creams, suspensions, emulsions, suppositories, patches or spraying agents.

The pharmaceutical composition according to one aspect of the present invention may contain additives such as a diluent, an excipient, a lubricant, a binder, a disintegrant, a buffer, a dispersant, a surfactant, a colorant, a fragrance or a sweetener as necessary. The pharmaceutical composition according to one aspect of the present invention can be prepared by a conventional method in the art.

The effective ingredients of the pharmaceutical composition according to one aspect of the present invention will vary depending on the age, sex, weight, pathological condition and severity of the subject to be administered, route of administration, or judgment of the prescriber. Determination of the amount of application based on these factors is within the level of ordinary skill in the art and its daily dose is, for example, from 0.1 [mu] g / kg / day to 1 g / kg / day, Kg / day, more specifically, 10 μg / kg / day to 1 mg / kg / day, and more particularly 50 μg / kg / day to 100 μg / kg / day. The pharmaceutical composition according to one aspect of the present invention may be administered once to three times a day, but is not limited thereto.

The cosmetic composition according to one aspect of the present invention may be provided in all formulations suitable for topical application. For example, it may be provided as a solution, an emulsion obtained by dispersing an oil phase in an aqueous phase, an emulsion obtained by dispersing an oil phase in water, a suspension, a solid, a gel, a powder, a paste, a foam or an aerosol. Such formulations may be prepared according to conventional methods in the art.

The cosmetic composition according to one aspect of the present invention may contain other ingredients, which may be synergistic to the main effect, preferably to the extent that the main effect is not impaired. In addition, the cosmetic composition according to one aspect of the present invention may further contain a moisturizing agent, an emollient agent, a surfactant, an ultraviolet absorber, an antiseptic, a bactericide, an antioxidant, a pH adjuster, an organic or inorganic pigment, a fragrance, . The compounding amount of the above components can be easily selected by those skilled in the art within the range not impairing the object and effect of the present invention, and the amount thereof is 0.01 to 5% by weight, specifically 0.01 to 3% by weight .

The formulation of the food composition according to one aspect of the present invention is not particularly limited, but can be formulated into, for example, tablets, granules, powders, liquids, solid preparations and the like. Each formulation may be blended without difficulty by a person skilled in the art according to the purpose of formulation or use, in addition to the active ingredient, and the synergistic effect may occur when the composition is applied simultaneously with other ingredients.

Determination of the dosage of the active ingredient is within the level of ordinary skill in the art and its daily dose is, for example, 1 μg / kg / day to 10 mg / kg / day, more specifically 10 μg / kg / day Day to 100 mg / kg / day, but it is not limited thereto, and it may be various factors such as the age, health condition, and complication of the subject to be administered &Lt; / RTI >

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The abbreviated term in front of a noun is not intended to limit the quantity but to indicate that there is more than one noun item mentioned. The terms " comprising ", "having ", and" containing "are to be construed as open (i.e., meaning including but not limited to).

It is important to note that the reference to a range of values is an easy way to substitute for referring individually to each distinct value within the range, and unless otherwise specified, each separate value is referred to individually Which is incorporated herein by reference. All range end values are contained within that range and can be combined independently.

All methods mentioned herein may be performed in any suitable order unless otherwise indicated or clearly contradicted by context. It is to be understood that the use of any embodiment and all of the embodiments or example language (e.g., "such as ") is for the purpose of describing the present invention only, It is not intended to be limiting. No language in the specification is to be construed as obliging any non-claimed component to be essential to the practice of the invention. Unless defined otherwise, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Preferred embodiments of the present invention include the most optimal mode known to the inventors for carrying out the present invention. Variations of the preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to appropriately utilize such variations, and the inventors expect the invention to be practiced otherwise than as described herein. Accordingly, the present invention includes equivalents and all modifications of the subject matter of the invention as recited in the appended claims, as permitted by the patent law. Moreover, any combination of the above-mentioned components within all possible variations is included in the present invention unless otherwise specified or contradicted by context. While the present invention has been particularly shown and described with reference to exemplary embodiments, those skilled in the art will readily appreciate that various changes in form and detail may be made without departing from the spirit and scope of the invention as defined by the following claims.

Example

Example 1: Synthesis of carrier peptide

The carrier peptide was prepared according to a conventional solid phase peptide synthesis method. Specifically, the peptides were synthesized by coupling one amino acid from the C-terminal through Fmoc solid phase peptide synthesis (SPPS) using ASP48S (Peptron, Inc., Daejeon, Korea). The first amino acid at the C-terminus of the peptides attached to the resin was used as follows. For example:

NH ₂ -Lys (Boc) -2-chloro-Trityl Resin

NH ₂ -Ala-2-chloro-Trityl Resin

NH ₂ -Arg (Pbf) -2-chloro-Trityl Resin

All amino acid sources used for peptide synthesis are Trt, Boc, t-Bu (t-butylester), Pbf (2,2,4,6, 7-pentamethyl dihydro-benzofuran-5-sulfonyl). For example:

Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Phe-OH, Fmoc-Arg- Fmoc-Lys (Boc) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Met-OH, Fmoc- -Asn (Trt) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ahx-OH, Trt-Mercaptoacetic acid.

As the coupling reagent, HBTU [2- (1H-Benzotriazole-1-yl) -1,1,3,3-tetramethylaminium hexafluorophosphate] / HOBt [N-Hydroxxybenzotriazole] / NMM [4-Methylmorpholine] Respectively. Fmoc removal was performed using piperidine in DMF in 20% DMF. Cleavage Cocktail (TFA (trifluoroacetic acid) / TIS (triisopropylsilane) / EDT (ethanedithiol) / H ₂ O = 92.5 / 2.5 / 2.5 / 2.5] was used to remove the synthetic peptides from the resin and to remove the protecting groups of the residues. Respectively.

Each of the peptides was synthesized by repeating the steps of reacting corresponding amino acids with a starting amino acid having an amino acid protecting group bonded thereto on a solid support, washing with a solvent, followed by deprotection. The synthesized peptide was cleaved from the resin and purified by HPLC. The synthesis was confirmed by MS and lyophilized.

A specific procedure will be described below taking Pep 1 of SEQ ID NO: 1 as an example.

1) Coupling

The protected amino acid (8 eq) and the coupling reagent HBTU (8 eq) / HOBt (8 eq) / NMM (16 eq) were dissolved in DMF and added to NH ₂ -Lys (Boc) -2-chloro-Trityl Resin , And reacted at room temperature for 2 hours and washed with DMF, MeOH and DMF in that order.

2) Fmoc deprotection

Piperidine in DMF in 20% DMF was added, and the reaction was carried out at room temperature for 5 minutes twice, followed by washing with DMF, MeOH and DMF.

3) By the reaction of 1 and 2 repeatedly peptide basic skeleton _{NH 2 -E (OtBu) -AR (} Pbf) -PALLT (tBu) -S (tBu) -R (Pbf) LR (Pbf) -FIPK (Boc) -2-chloro-Trityl Resin).

4) Cleavage: Cleavage cocktail was added to the synthesized peptide Resin to separate the peptide from Resin.

5) Cooling diethyl ether is added to the obtained mixture, and the resulting peptide is precipitated by centrifugation.

6) After purification by Prep-HPLC, molecular weight was confirmed by LC / MS and frozen to prepare powder.

Example 2: Preparation of a conjugate of carrier peptide and FITC

(1) Preparation of FITC-CPP conjugate

A conjugate comprising SEQ ID NO: 1 and fragments thereof conjugated with FITC was prepared as follows. For example, a conjugate of pep1 and FITC of SEQ ID NO: 1, namely FITC-linker-pep1, was prepared as follows.

Example 1 Peptide basic skeleton obtained in a similar manner as NH ₂ - linker -E (OtBu) -AR (Pbf) -PALLT (tBu) -S (tBu) -R (Pbf) LR (Pbf) -FIPK (Boc) - 2-chloro-Trityl Resin) was reacted with FITC. Specifically, fluorescein-5-isothiocyanate (FITC) (8 equivalents) and N, N-diisopropylethylamine (DIPEA) (16 equivalents) DMF, and the mixture was reacted at room temperature for 2 hours and washed with DMF, MeOH and DMF. As a result, FITC-linker-E (OtBu) -AR (Pbf) -PALLT (tBu) -S (tBu) -R (Pbf) LR (Pbf) -FIPK (Boc) -2-chloro-Trityl Resin was obtained. The linker is 6-aminohexanoic acid (Ahx). TFA / TIS / H ₂ O = 95 / 2.5 / 2.5 was added to the synthesized peptide Resin, and the conjugate was separated from the resin. Cooling diethyl ether was added to the resulting mixture, followed by centrifugation to precipitate the resulting conjugate. After purification by Prep-HPLC, the purity was confirmed by analytical HPLC and the molecular weight was confirmed by LC / MS. The substance obtained through confirmation of molecular weight proved to be FITC-pep1. And then freeze-dried.

(2) Preparation of CPP-FITC conjugate

Peptide as a fundamental skeleton as that of Example _{1 (NH 2 -E (OtBu)} -AR (Pbf) -PALLT (tBu) -S (tBu) -R (Pbf) LR (Pbf) -FIPK (Dde) -2-chloro -Trityl Resin). The N-term was protected with Boc to selectively introduce FITC into the C-term of the prepared peptide backbone. Di-tert-butyl dicarbonate (30 eq.) And DIPEA (30 eq.) Were dissolved in DMF and then reacted at room temperature for 2 hours, followed by washing with DMF, MeOH and DMF. As a result, Boc-E (OtBu) -AR (Pbf) -PALLT (tBu) -S (tBu) -R (Pbf) LR (Pbf) -FIPK (Dde) -2-chloro-Trityl Resin was obtained. Then, hydrazine in DMF was treated with 2% DMF to attach FITC to the residue of C-term K to remove Dde, the residue protecting group of C-term Lys. As a result, Boc-E (OtBu) -AR (Pbf) -PALLT (tBu) -S (tBu) -R (Pbf) LR (Pbf) -FIPK (NH2) -2-chloro-Trityl Resin was obtained. Then, FITC (8 eq.) And DIPEA (16 eq.) Were dissolved in DMF and added, followed by reaction at room temperature for 2 hours, followed by washing with DMF, MeOH and DMF. As a result, Boc-E (OtBu) -AR (Pbf) -PALLT (tBu) -S (tBu) -R (Pbf) LR (Pbf) -FIPK (FITC) -2-chloro-Trityl Resin was obtained. TFA / TIS / H ₂ O = 95 / 2.5 / 2.5 was added to the synthesized peptide Resin to isolate the peptide from Resin. Cooling diethyl ether was added to the resultant mixture, followed by centrifugation to precipitate the obtained peptide. After purification by Prep-HPLC, the purity was confirmed by analytical HPLC and the molecular weight was confirmed by LC / MS. The resultant substance proved to be pep1-FITC. And then lyophilized.

Example 3 Preparation of Ferrocenecarboxylic-Carrier Peptide Conjugates

(8 eq.) And coupling reagent HBTU (8 eq.) / HOBt (8 eq.) / NMM (16 eq.) In NH ₂ -Lys (Dde) -2-chloro- , And the mixture was reacted at room temperature for 2 hours and washed with DMF, MeOH and DMF in that order. Then piperidine in DMF in 20% DMF was added for Fmoc deprotection. The reaction was carried out at room temperature for 5 minutes twice, followed by washing with DMF, MeOH and DMF. (Tbu) -S (tBu) -R (Pbf) LR (Pbf) -FIPK (Dde) -2- chlorobenzene - trityl Resin). Here, hydrazine in DMF was treated with 2% DMF to remove Dde, the residue protecting group of C-terminal Lys. The coupling reagent HBTU (16 equiv.) / HOBt (16 equiv.) / NMM (32 equiv.) Was dissolved in DMF and added to the reaction mixture, For 2 hours and washed with DMF, MeOH and DMF in that order. TFA / TIS / H2O = 95 / 2.5 / 2.5 was added to the synthesized peptide Resin to isolate the peptide from Resin. Cooling diethyl ether was added to the resulting mixture, followed by centrifugation to precipitate the obtained peptide. After purification by HPLC, it was confirmed by MS and lyophilized.

Example 4: Preparation of siRNA-carrying peptide conjugates

(TBu) -S (tBu) -R (Pbf) LR (Pbf) -FIPK (Boc) -ARP (Pbf) -2-chloro-Trityl Resin). Where Ahx refers to 6-aminohexanoic acid. The resulting Trt-Mercaptoacetyl-Ahx-E (OtBu) -AR (Pbf) -PALLT (tBu) -S (tBu) -R (Pbf) LR (Pbf) -FIPK (Boc) The synthesis was confirmed by HPLC analysis and mass spectrometry.

The peptide was dissolved in maleimide-modified SiLuc (75 μmol) and PBS buffer (1 × 1 ml) supplied from Korean biona, and conjugated by reacting at room temperature for 2 hours. Conjugation occurs through the reaction of a thiol group on the peptide with a maleimide on the siRNA. The conjugation reaction was confirmed using Ellman's reagent (5,5'-dithiobis- (2-nitrobenzoic acid) or DTNB). Ellman's reagent is a chemical used to quantify the number or concentration of thiol groups in a sample.

Example 5: Preparation of carrier peptide-GFP conjugate

Conjugates of carrier peptides and Green Fluorescent Protein (GFP) (SEQ ID NO: 3, SEQ ID NO: 4) were prepared by the following method. Specifically, for example, a gene encoding the pep1 peptide (16mer) of SEQ ID NO: 1 was cloned into the pET28a (+) vector expressing GFP to induce overexpression of the pep1 peptide (16mer) Expressed in E. coli BL21 and purified.

First, as shown in FIG. 1, a primer was prepared for cloning Green Fluorescent Protein (GFP) into pET28a (+) vector (Promega). The primer contained an EcoRI cleavage site at the 5 'end and a HindIII cleavage site at the 3' end. The EcoRI site contained about 21 bp of the GFP prefix sequence and a Hind codon at the HindIII site. For the production of the GFP-carrying peptide protein gene, the 5 'side was identical to GFP but was prepared so as to include a sequence encoding a carrier peptide in front of GFP. For example, in the case of SEQ ID NO: 1 (Pep 1) peptide, it was made to include the following sequence: GAA GCG CGC CCG GCG CTG ACC AGC CGC CTG CGC TTT ATT CCG AAA (SEQ ID NO: 5). As a comparative example, "TAT GGT CGT AAA AAA CGT CGT CAA CGT CGT CGT" was prefixed to GFP to prepare TAT-GFP.

The forward primer was prepared by adding 21 GFP sequences to the pET-28a-GFP vector using the above primers and the forward primer followed by the carrier peptide coding sequence. The reverse primer was a C-terminal sequence part of GFP And PCR was carried out. PCR was carried out at 95 ° C for 5 minutes and 30 cycles at 95 ° C for 5 minutes (denaturation), 63 ° C for 30 seconds (annealing), 72 ° C for 1 minute (elongation) and 72 ° C for 7 minutes. Each primer was used at 10 pmol. Using these PCR conditions, GFP and GFP-carrying peptide DNA fragments were amplified and cloned into the EcoRI and HindIII sites of pET28a (+) vector to obtain a vector expressing GFP and GFP-carrying peptide ).

The vector was then transformed into bacteria to isolate the protein. Specifically, E. coli BL21 (DE3) (Invitrogen, Carlsbad, Calif., USA) was transformed with each of the above vectors, cultured in 5 ml LB / kanamycin medium and transferred to 100 ml medium. In this case, kanamycin was added in 1/1000 volume ratio. After incubation at 37 ° C for about 2-3 hours with stirring, the absorbance was measured to about 0.6-0.8, followed by 1 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside). After further culturing for 3-4 hours, the cells were centrifuged at 5000 rpm for 5 minutes.

As a result of the centrifugation, the expressed protein can be visually confirmed that the cell is over-expressed when it becomes green light. The protein separation kit (Prod, # 21277, Thermo Scientific, IL, USA), a His tag purification kit, was isolated according to the manufacturer's instructions.

After separation, the protein was purified by dialysis and concentrated. Specifically, dialyzed with sterilized PBS at 4 ° C. First, the dialysis bag was equilibrated in advance with PBS, and the protein solution separated from the above was taken in a 5 ml syringe and put in a dialysis bag, and dialyzed overnight at 4 ° C. with stirring. Subsequently, the protein dialyzed on BIBASPIN 20 (Prod, # VS2092, Sartorius Stedin biotech, Germany) was added to increase the concentration of the protein and to remove unnecessary substances, followed by concentration by centrifugation at 3,000 rpm at 4 ° C.

Example 6: Binding experiment of carrier peptide-GFP conjugate with serum albumin

Mycobacteria intracellulare, a Mycobacteria intracellulare, was lysed in a 7H9 culture medium (Prod. # 271310, Becton, Dickinson and Company, MD, USA) containing fetal bovine serum and then overexpressed Immunoprecipitation (IP) was performed with the conjugate and protein obtained from Example 5 above. As a result, a band of about 65 kDa was confirmed (FIG. 3). The protein bands specifically binding to the 16-mer were cut and subjected to a proteomic analysis (Maldi-TOF) to a specialized analysis institution. Furthermore, human serum was immunoprecipitated by the same method using biotin-labeled 16-mer peptides. As a result, 16-mer peptides were found to bind to albumin (65 kDa) more than the control group ).

Each of the concrete steps will be described in more detail below.

(1) Immunoprecipitation of the expression protein 16-mer and cell lysates

Mycobacteria intracellulare (Mycobacteria intracellulare) , a Mycobacterium bacterium, was lysed in a 7H9 culture medium (Prod. # 271310, Becton, Dickinson and Company, MD, USA) containing fetal bovine serum and then overexpressed And then separated as follows. Mycobacteria intracellulare was lysed in 7H9 medium containing 10% ADC (albumin dextrose and catalase, Prod. # 272352, Becton, Dickinson and Company, MD, USA). Lysing conditions are as follows. The process of stopping 0.7 seconds after 0.3 second pulse was repeated 3 times for 5 minutes to perform sonication. The sonicated sample was then centrifuged at 10,000 rpm for 15 minutes to separate the supernatant.

1 ml of HEK 293 cells (approximately 1 × 10 6) was collected and centrifuged at 1300 rpm for 5 minutes to obtain a precipitate. One ml of a lysis buffer (Prod. # 87787, Thermo Scientific, UL, USA) and a protease ingibitor complex, # 05892791001, IN, USA) were added, vortexed, and placed in ice for 30 minutes. After centrifugation at 13,000 rpm for 10 minutes in a 4 ° C centrifuge, the supernatant was taken.

6xHis-labeled cobalt resin was prepared (Prod. # 89964, Thermo Scientific, UL, USA) to attach the purified GFP-expressing protein to the resin in Example 5. 50-100 μl of resin was added to the column, and washed with 500 μl each 5 times in a washing buffer. Each of the expressed proteins purified in Example 5 was added thereto, and allowed to mix for 30 minutes to 1 hour while being rotated at 4 캜. After that, 500 μl of the expressed protein-resin polymer was washed 5 times in the washing buffer. Then, the above prepared cell lysate is added, and the mixture is allowed to stand overnight while being rotated at 4 ° C overnight. The next day, the resin was washed with a washing buffer in the same manner and 200 μl of an elution buffer was added to elute the resin. As a control, GFP Control expression protein (WT-GFP), another 11-mer expression protein (11-GFP) and ccg-GFP were tested in the same manner as above. 11mer-GFP is an 11-mer protein of 11 aa derived from HBV conjugated with GFP. ccg is the replication origin of Hepatitis B virus.

(2) Protein electrophoresis and specific binding protein analysis using SDS-PAGE

A 5X SDS sample buffer was added to 20-40 용 of the eluted protein prepared in the above (1), boiled, and centrifuged at 13,000 rpm for 1 minute. The supernatant was then loaded onto a 12% SDS-PAGE protein electrophoresis gel and electrophoresed at 100 volts for 2 hours 30 minutes. After electrophoresis, the gel was placed in distilled water for 20 minutes, and stained with a silver staining kit (PROT-S1L1, Sigma-Aldrich, St. Louis, USA). The results are shown in Fig. A dyed band of about 65 kDa (FIG. 3) was cut and subjected to analysis by a proteome analysis institution (Yonsei University Proteome Research Institute, Seoul, Korea). As a result, the protein was identified as serum albumin. From this, it can be seen that the present 16mer polypeptide binds to serum albumin.

(3) Identification of albumin binding using human serum

Blood from a healthy donor was mixed in a volume ratio of 4: 1 with phycol and centrifuged at 400 g for 30 minutes to separate the serum. Subsequently, immunoprecipitation was carried out in the same manner as in the above (1), followed by SDS-PAGE analysis. The result is shown in Fig. As a result, it was confirmed that pep1-GFP conjugate (16-mer peptide) binds more to 65kDa albumin than the control group.

Example 7: Toxicity test

(1) Preparation of cells

HeLa cell lines obtained from ATCC were cultured in MEM (Minimum Essential Medium) supplemented with 10% fetal bovine serum (Invitrogen, USA) and Earle's salts, non-essential amino acids, sodium pyruvate and 100 μg / ml penicillin and streptomycin ) Was added, and the cells were cultured in a 5% CO ₂ incubator at 37 ° C.

(2) Cell survival rate and toxicity analysis

In the meantime, the cultured cell line was divided into 96-well plates, and 10% fetal bovine serum (Invitrogen, USA), 100 μg / ml penicillin and 100 units / ml streptomycin were added to the medium. For 12 hours. After washing with PBS, the cells were starvated in MEM (Minimum Essential Medium) for 1 hour. After 20 uM of each pep1 was cultured at 37 ° C for 24 hours, cell viability and toxicity were analyzed by MTT assay. The results are shown in Fig.

&Lt; 110 > KAEL-GemVax Co., Ltd. KIM, Sangjae <120> Effective Ingredient Delivery System for Prolonging In-vivo Half-Life of an Effective Ingredient <130> 12p114kg <160> 5 <170> PatentIn version 3.2 <210> 1 <211> 16 <212> PRT <213> Homo sapiens <400> 1 Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro Lys 1 5 10 15 <210> 2 <211> 1132 <212> PRT <213> Homo sapiens <400> 2 Met Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser 1 5 10 15 His Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Val Arg Arg Leu Gly 20 25 30 Pro Gln Gly Trp Arg Leu Val Gln Arg Gly Asp Pro Ala Ala Phe Arg 35 40 45 Ala Leu Val Ala Gln Cys Leu Val Cys Val Pro Trp Asp Ala Arg Pro 50 55 60 Pro Pro Ala Ala Pro Ser Phe Arg Gln Val Ser Cys Leu Lys Glu Leu 65 70 75 80 Val Ala Arg Val Leu Gln Arg Leu Cys Glu Arg Gly Ala Lys Asn Val 85 90 95 Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro 100 105 110 Glu Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr 115 120 125 Asp Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val 130 135 140 Gly Asp Asp Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val 145 150 155 160 Leu Val Ala Pro Ser Cys Ala Tyr Gln Val Cys Gly Pro Pro Leu Tyr 165 170 175 Gln Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro His Ala Ser Gly 180 185 190 Pro Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn His Ser Val Arg 195 200 205 Glu Ala Gly Val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg 210 215 220 Gly Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg 225 230 235 240 Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gln Gly Ser Trp 245 250 255 Ala His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val 260 265 270 Val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala 275 280 285 Leu Ser Gly Thr Arg His Ser Ser Ser Val Gly Arg Gln His His 290 295 300 Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro Trp Asp Thr Pro 305 310 315 320 Cys Pro Pro Val Tyr Ala Glu Thr Lys His Phe Leu Tyr Ser Ser Gly 325 330 335 Asp Lys Glu Gln Leu Arg Pro Ser Phe Leu Leu Ser Ser Leu Arg Pro 340 345 350 Ser Leu Thr Gly Ala Arg Arg Leu Val Glu Thr Ile Phe Leu Gly Ser 355 360 365 Arg Pro Trp Met Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gln 370 375 380 Arg Tyr Trp Gln Met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His 385 390 395 400 Ala Gln Cys Pro Tyr Gly Val Leu Leu Lys Thr His Cys Pro Leu Arg 405 410 415 Ala Ala Val Thr Pro Ala Ala Gly Val Cys Ala Arg Glu Lys Pro Gln 420 425 430 Gly Ser Val Ala Ala Pro Glu Glu Glu Asp Thr Asp Pro Arg Arg Leu 435 440 445 Val Gln Leu Leu Arg Gln His Ser Ser Pro Trp Gln Val Tyr Gly Phe 450 455 460 Val Arg Ala Cys Leu Arg Arg Leu Val Pro Pro Gly Leu Trp Gly Ser 465 470 475 480 Arg His Asn Glu Arg Arg Phe Leu Arg Asn Thr Lys Lys Phe Ile Ser 485 490 495 Leu Gly Lys His Ala Lys Leu Ser Leu Gln Glu Leu Thr Trp Lys Met 500 505 510 Ser Val Arg Asp Cys Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys 515 520 525 Val Pro Ala Ala Glu His Arg Leu Arg Glu Glu Ile Leu Ala Lys Phe 530 535 540 Leu His Trp Leu Met Ser Val Tyr Val Val Glu Leu Leu Arg Ser Phe 545 550 555 560 Phe Tyr Val Thr Glu Thr Thr Phe Gln Lys Asn Arg Leu Phe Phe Tyr 565 570 575 Arg Lys Ser Val Trp Ser Lys Leu Gln Ser Ile Gly Ile Arg Gln His 580 585 590 Leu Lys Arg Val Gln Leu Arg Glu Leu Ser Glu Ala Glu Val Arg Gln 595 600 605 His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile 610 615 620 Pro Lys Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val 625 630 635 640 Gly Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser 645 650 655 Arg Val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg 660 665 670 Pro Gly Leu Leu Gly Ala Ser Val Leu Gly Leu Asp Asp Ile His Arg 675 680 685 Ala Trp Arg Thr Phe Val Leu Arg Val Arg Ala Gln Asp Pro Pro Pro 690 695 700 Glu Leu Tyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr Asp Thr Ile 705 710 715 720 Pro Gln Asp Arg Leu Thr Glu Val Ile Ala Ser Ile Ile Lys Pro Gln 725 730 735 Asn Thr Tyr Cys Val Arg Arg Tyr Ala Val Val Gln Lys Ala Ala His 740 745 750 Gly His Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp 755 760 765 Leu Gln Pro Tyr Met Arg Gln Phe Val Ala His Leu Gln Glu Thr Ser 770 775 780 Pro Leu Arg Asp Ala Val Valle Glu Gln Ser Ser Ser Leu Asn Glu 785 790 795 800 Ala Ser Ser Gly Leu Phe Asp Val Phe Leu Arg Phe Met Cys His His 805 810 815 Ala Val Arg Ile Arg Gly Lys Ser Tyr Val Gln Cys Gln Gly Ile Pro 820 825 830 Gln Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu Cys Tyr Gly Asp 835 840 845 Met Glu Asn Lys Leu Phe Ala Gly Ile Arg Arg Asp Gly Leu Leu Leu 850 855 860 Arg Leu Val Asp Asp Phe Leu Leu Val Thr Pro His Leu Thr His Ala 865 870 875 880 Lys Thr Phe Leu Arg Thr Leu Val Arg Gly Val Pro Glu Tyr Gly Cys 885 890 895 Val Val Asn Leu Arg Lys Thr Val Val Asn Phe Pro Val Glu Asp Glu 900 905 910 Ala Leu Gly Gly Thr Ala Phe Val Gln Met Pro Ala His Gly Leu Phe 915 920 925 Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Gln Ser 930 935 940 Asp Tyr Ser Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr Phe 945 950 955 960 Asn Arg Gly Phe Lys Ala Gly Arg Asn Met Arg Arg Lys Leu Phe Gly 965 970 975 Val Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gln Val Asn 980 985 990 Ser Leu Gln Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu Gln 995 1000 1005 Ala Tyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe His Gln Gln 1010 1015 1020 Val Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser Asp Thr Ala 1025 1030 1035 1040 Ser Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly Met Ser Leu 1045 1050 1055 Gly Ala Lys Gly Ala Gly Pro Leu Pro Ser Glu Ala Val Gln Trp 1060 1065 1070 Leu Cys His Gln Ala Phe Leu Leu Lys Leu Thr Arg His Arg Val Thr 1075 1080 1085 Tyr Val Pro Leu Leu Gly Ser Leu Arg Thr Ala Gln Thr Gln Leu Ser 1090 1095 1100 Arg Lys Leu Pro Gly Thr Thr Leu Thr Ala Leu Glu Ala Ala Ala Asn 1105 1110 1115 1120 Pro Ala Leu Pro Ser Asp Phe Lys Thr Ile Leu Asp 1125 1130 <210> 3 <211> 239 <212> PRT <213> Artificial Sequence <220> <223> Green Fluorescent Protein <400> 3 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 225 230 235 <210> 4 <211> 720 <212> DNA <213> Artificial Sequence <220> <223> Green Fluorescent Protein <400> 4 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720 720 <210> 5 <211> 48 <212> DNA <213> Homo sapiens <400> 5 gaagcgcgcc cggcgctgct gaccagccgc ctgcgcttta ttccgaaa 48

Claims

An effective component delivery system for extending the in vivo half-life of an active ingredient,
Wherein the active ingredient delivery system comprises a carrier peptide and a conjugate of an active ingredient,
Wherein the conjugate has an extended serum half-life compared to the intrinsic serum half-life of the active ingredient,
The carrier peptide is a peptide that binds to albumin in the blood and is a peptide that prolongs the serum half-life of the active ingredient,
The carrier peptide may be,
A peptide comprising SEQ ID NO: 1, a peptide having 80% or more sequence homology with the above peptide sequence, or a fragment thereof,
Wherein the peptide and the fragment having 80% or more of sequence homology have a serum albumin binding ability of the peptide of SEQ ID NO: 1.

The method according to claim 1,
Wherein said fragment is a fragment consisting of three or more amino acids.

The method according to claim 1,
Wherein said carrier peptide is a peptide consisting of no more than 30 amino acids.

The method according to claim 1,
The carrier peptide may be,
An effective ingredient delivery system for prolonging the in vivo half-life of an active ingredient, which is a peptide of SEQ ID NO: 1.

The method according to claim 1,
Wherein the active ingredient is at least one selected from a protein, a nucleic acid, a peptide, a lipid, a glycolipid, a mineral, a sugar, a nanoparticle, a biological agent, a contrast agent, drugs and a chemical compound. Active ingredient delivery system.

2. The active ingredient delivery system for the in vivo half-life extension of an active ingredient according to claim 1, wherein the carrier peptide and the active ingredient are conjugated by a covalent bond, optionally by a linker.

2. The active ingredient delivery system according to claim 1, wherein the carrier peptide and the active ingredient are conjugated by non-covalent bond.

6. The active ingredient delivery system according to claim 5, wherein the active ingredient is a protein or a peptide.

The active ingredient delivery system according to claim 8, wherein the active ingredient is a cytokine, an antibody, an antibody fragment, a therapeutic enzyme, a soluble receptor, or a ligand.

A composition comprising an active ingredient having an extended half-life,
The composition comprises a carrier peptide and a conjugate of an active ingredient,
Wherein the conjugate has an extended serum half-life compared to the intrinsic serum half-life of the active ingredient,
The carrier peptide is a peptide that binds to albumin in the blood and is a peptide that prolongs the serum half-life of the active ingredient,
The carrier peptide may be,
A peptide comprising SEQ ID NO: 1, a peptide having 80% or more sequence homology with the above peptide sequence, or a fragment thereof,
Wherein the peptide and the fragment having 80% or more of the sequence homology have the serum albumin binding ability of the peptide of SEQ ID NO: 1.

11. The method of claim 10,
Wherein said fragment is a fragment consisting of three or more amino acids.

11. The method of claim 10,
Wherein the delivery peptide is a peptide consisting of no more than 30 amino acids.

11. The method of claim 10,
The carrier peptide may be,
Wherein the peptide is SEQ ID NO: 1.

11. The method of claim 10,
Wherein the active ingredient is at least one selected from a protein, a nucleic acid, a peptide, a lipid, a glycolipid, a mineral, a sugar, a nanoparticle, a biological agent, a contrast agent, drugs and a chemical compound.

11. The composition of claim 10, wherein the carrier peptide and the active ingredient are conjugated by covalent bonds, optionally mediated by a linker.

11. The composition of claim 10, wherein the carrier peptide and the active ingredient are conjugated by noncovalent bonding.

15. The composition of claim 14, wherein the active ingredient is a protein or peptide.

18. The composition of claim 17, wherein the active ingredient is a cytokine, an antibody, an antibody fragment, a therapeutic enzyme, a soluble receptor, or a ligand.

11. The method of claim 10,
Wherein the composition is a pharmaceutical composition.

11. The method of claim 10,
Wherein the composition is a cosmetic composition.

11. The method of claim 10,
Wherein the composition is a food composition.

A method for prolonging the duration of an active ingredient,
Comprising administering to a subject in need thereof a carrier peptide and a conjugate of an active ingredient,
Wherein the conjugate has an extended serum half-life compared to the intrinsic serum half-life of the active ingredient,
The carrier peptide is a peptide that binds to albumin in the blood and is a peptide that prolongs the serum half-life of the active ingredient,
The carrier peptide may be,
A peptide comprising SEQ ID NO: 1, a peptide having 80% or more sequence homology with the above peptide sequence, or a fragment thereof,
Wherein the peptide and the fragment having 80% or more of sequence homology have a serum albumin binding ability of the peptide of SEQ ID NO: 1.

23. The method of claim 22,
Wherein said fragment is a fragment consisting of three or more amino acids.

23. The method of claim 22,
Wherein said carrier peptide is a peptide consisting of no more than 30 amino acids.

23. The method of claim 22,
Wherein the active ingredient is at least one selected from a protein, a nucleic acid, a peptide, a lipid, a glycolipid, a mineral, a sugar, a nanoparticle, a biological agent, a contrast agent, drugs and a chemical compound .

23. The method according to claim 22, wherein the carrier peptide and the active ingredient are conjugated by covalent bonds, optionally by a linker, thereby being a conjugated conjugate.

23. The method of claim 22, wherein the carrier peptide and the active ingredient are conjugates conjugated by non-covalent association.

26. The method according to claim 25, wherein the active ingredient is a protein or a peptide.

29. The method of claim 28, wherein the active ingredient is a cytokine, an antibody, an antibody fragment, a therapeutic enzyme, a soluble receptor, or a ligand.