AU2024257036A1 - Novel use of cpne7 protein-derived peptide - Google Patents

Abstract

A composition for inhibiting skin aging or treating wounds according to the present invention comprises a peptide consisting of an amino acid sequence of general formula 1: K-Y-R1-R2-R3-R4-R5-R6-R7-R8 (General formula 1), wherein, in general formula 1, R1 is arginine (R), lysine (K), or glutamine (Q); R2 is arginine (R) or glutamine (Q); R3, R4, and R5 are each arginine (R) or lysine (K); R6 is asparagine (N) or serine (S); and R7 and R8 are lysine (K) or tyrosine (Y).

Description

[DESCRIPTION] [DESCRIPTION]

[Title of the

[Title of theInvention] Invention]

NovelUse Novel UseofofPeptides PeptidesDerived Derivedfrom from CPNE7 CPNE7 Protein Protein

[Technical Field]

[Technical Field]

[0001] The

[0001] The present present invention invention relatestotoa anovel relates noveluse useofofpeptides, peptides,and andmore more specifically,toto aa specifically,

novel use novel useofofpeptides peptidesderived derived from from CPNE7 CPNE7 proteinprotein that promote that promote skin regeneration, skin regeneration, elasticity elasticity

enhancement, andmoisturizing enhancement, and moisturizing ability. ability.

[Background Art]

[Background Art]

[0002] The

[0002] The skin skin is is thelargest the largesttissue tissuethat that exists exists in in the the outermost layer of outermost layer of our our body bodyand andisis

simultaneously distributed throughout simultaneously distributed throughoutthe theentire entire body. body.Therefore, Therefore,itit is is exposed exposedtotovarious variousstimuli stimuli

including pressure, including pressure, impact, impact, friction, friction, andand others others from from the external the external environment. environment. The skin corresponds The skin corresponds

to an important tissue that protects internal organs from such external environments, regulates body to an important tissue that protects internal organs from such external environments, regulates body

temperature, and temperature, and enables enablessensation. sensation.

[0003] The

[0003] The structureofofskin structure skinisisdivided dividedinto intoepidermis, epidermis,dermis, dermis,and andsubcutaneous subcutaneous fatfat layer. layer.

The subcutaneous fat layer is composed of fat cells below the dermis and is a fat layer where excess The subcutaneous fat layer is composed of fat cells below the dermis and is a fat layer where excess

nutrients from nutrients from consumed foodareareaccumulated consumed food accumulatedin in thethe form form of of fat.ItIt is fat. is located located between the skin between the skin and and

musclesand muscles andhashas insulation insulation effects,performing effects, performing the the function function of maintaining of maintaining body temperature body temperature

through this through this mechanism, thusbeing mechanism, thus beingdistinguished distinguished from from thethe dermal dermal layer. layer. TheThe externally externally observed observed

epidermis is the epidermis is the layer layer that that directly directlycontacts contactsthe theexternal externalenvironment at the environment at the outermost part of outermost part of the the

skin skin and is mainly and is composed mainly composed of of thestratum the stratum corneum. corneum. The The stratum stratum corneum corneum functions functions as a barrier as a barrier

that protects our body, preventing even a single drop of water, bacteria, or viruses from penetrating that protects our body, preventing even a single drop of water, bacteria, or viruses from penetrating

from theoutside. from the outside.TheThe dermis dermis is layer is the the layer located located directly directly beneath beneath the epidermis the epidermis and mainlyand mainly consists consists of fiber components of fiber componentssuch such as collagen as collagen fibers fibers and andfibers, elastic elasticwith fibers, blood with blood vessels, hairvessels, hair follicles, follicles, nerves, and sweat glands existing in between. The dermis provides strength (tension) to the skin and nerves, and sweat glands existing in between. The dermis provides strength (tension) to the skin and is is an an important region important region forfor forming forming the skin's the skin's shape. shape. When collagen When collagen fibers or fibers elasticor elastic fibers fibers within thewithin the dermal layer become dermal layer becomeweak weak or or thin,wrinkles thin, wrinklesform form and and skin skin elasticitydecreases. elasticity decreases.

[0004] The

[0004] The skin skin is isboth botha aprotective protectivemembrane membrane with with various various physiological physiological functions functions and and an an

important tissue for important tissue for obtaining obtaining aesthetic aestheticappeal appealfrom fromothers. others.Therefore, Therefore,aged agedskin skinisis accompanied accompanied by by

functional functional disorders disorders in in addition addition to tovisible visiblechanges. changes.Moreover, Moreover, severe severe skin skin aging has psychological aging has psychological

effects onvitality effects on vitalityofoflife lifeand andcancan cause cause depression depression in severe in severe cases. cases. Prevention Prevention or treatment or treatment of skin of skin

aging is increasingly important in modern society. Particularly, the increase in average lifespan and aging is increasingly important in modern society. Particularly, the increase in average lifespan and

decrease decrease ininbirth birthrate ratehave have brought brought about about an increase an increase in the in the elderly elderly population. population. Korea is Korea is predicted predicted to to

reach the super-aged society standard designated by the UN by 2025, so efforts to prevent skin aging reach the super-aged society standard designated by the UN by 2025, so efforts to prevent skin aging

cannot be overlooked cannot be overlookedfrom froma asocial socialcost cost perspective. perspective.

[0005]

[0005] TheThe characteristics characteristics of aging of skin skin include, aging include, first of first all, of all, decreased decreased skin thickness. skin thickness. This This

is is particularly prominent particularly prominent in the in the epidermis, epidermis, and itand has itbeen hasreported been reported that the that the reduction reduction in epidermal in epidermal

thickness is thickness is accelerated accelerated gradually gradually after after adulthood. adulthood.The Thethickness thickness of of thethe epidermis epidermis is known is known to to

decrease by approximately decrease by approximately6.4% 6.4% every every 10 10 years years on average. on average. The The reduction reduction in epidermal in epidermal thickness thickness

can be aa direct can be direct pathological pathological cause cause of of skin skin dryness, dryness,asas moisture moistureononthetheskin skinsurface surfaceandand moisture moisture

content in content in the the stratum stratum corneum corneum within within thethe epidermis epidermis decrease, decrease, and and overall overall lipidlipid content content is also is also

knowntotodecrease known decreasebybyapproximately approximately 65%. 65%. Additionally, Additionally, as as keratinocyteproliferation keratinocyte proliferationdecreases decreaseswith with

age, age, recovery speed after recovery speed after keratin keratin removal becomessignificantly removal becomes significantlyslower slowerininthe theelderly elderly compared compared toto

youngpeople. young people.

[0006] Meanwhile,

[0006] Meanwhile, the the most most prominent prominent structural structural change change inside inside the the skinskin due due to aging to aging is the is the

flattening flattening of of the the dermo-epidermal junctionduedue dermo-epidermal junction to to thethe loss loss of of dermal dermal papillae. papillae. Flattening Flattening of of thethe

dermo-epidermal junction dermo-epidermal junction reduces reduces resistance resistance to to external external friction,making friction, makingit it vulnerable vulnerable to to wound wound 2 formation. Internally, it formation. Internally, it reduces the supply reduces the supplyofofnutrients nutrientsandand oxygen oxygen between between the dermis the dermis and and epidermis, disrupts various epidermis, disrupts variousintercellular intercellular signaling signaling systems, systems,and andcauses causes separation separation between between the the dermis andepidermis. dermis and epidermis.This Thisprocess processis is known known asasone oneofofthe the major majormechanisms mechanismsof of wrinkle wrinkle formation. formation.

[0007] The

[0007] The causes causes of skin of skin aging aging can can be divided be divided into into intrinsic intrinsic aging aging and extrinsic and extrinsic aging. aging.

Extrinsic aging mainly refers to photoaging caused by ultraviolet rays. However, according to recent Extrinsic aging mainly refers to photoaging caused by ultraviolet rays. However, according to recent

studies, it is studies, it is estimated thatvarious estimated that various extrinsic extrinsic factors factors that that promote promote skin including skin aging, aging, including ultravioletultraviolet

rays as well as tobacco smoke and pollution, are linked with intrinsic factors to promote skin aging. rays as well as tobacco smoke and pollution, are linked with intrinsic factors to promote skin aging.

Intrinsic factors that promote skin aging include genome instability, epigenetic alteration, hormonal Intrinsic factors that promote skin aging include genome instability, epigenetic alteration, hormonal

changes, or oxidative stress. It has not been clearly revealed at what point genetic level or hormonal changes, or oxidative stress. It has not been clearly revealed at what point genetic level or hormonal

changes activate and changes activate and link link these thesemechanisms. Onthe mechanisms. On theother other hand, hand, reactive reactive oxygen species (ROS) oxygen species (ROS)have have

been identified been identified as as aamajor major cause cause of of overall overallaging agingphenomena. It is phenomena. It isknown throughmany known through manystudies studiesthat that

reactive oxygen reactive species can oxygen species can cause cause changes in extracellular changes in extracellularmatrix matrix(ECM) in the (ECM) in the dermis dermis in in connection connection

with internal with internal and external factors and external factors of of the the skin. skin. Reactive oxygenspecies Reactive oxygen speciesnaturally naturallygenerated generated after after

external factorssuch external factors suchasasultraviolet ultraviolet rays rays or or cellular cellular metabolism metabolism bind bind to to cysteine cysteine locatedlocated at the enzyme at the enzyme

site site within within Receptor ProteinTyrosine Receptor Protein TyrosinePhosphatase Phosphatase (RPTP), (RPTP), inhibiting inhibiting RPTP's RPTP's ability ability to inhibit to inhibit

ReceptorTyrosine Receptor TyrosineKinase Kinase (RTK). (RTK). RTK phosphorylation RTK phosphorylation affects intracellular affects various various intracellular signaling signaling

systems andinduces systems and inducesthe theactivity activityofof Mitogen-Activated Mitogen-Activated Protein Protein Kinase Kinase (MAPK) (MAPK) and transcription and transcription

factors factors Nuclear Nuclear Factor-κB (NF-κB)andand Factor-kB (NF-kB) Activator Activator Protein-1 Protein-1 (AP-1). (AP-1). Activation Activation of of NF-κB NF-kB and and AP-1AP-1

inhibits inhibits collagen collagen production andincreases production and increasesMMP MMPgenegene transcription, transcription, contributing contributing to reducing to reducing the the

amount amount of of collagen collagen in skin in skin tissue tissue and causing and causing abnormal abnormal structural structural characteristics. characteristics.

[0008] Treatment

[0008] Treatment and and prevention prevention of skin of skin agingaging havea been have been a subject subject of consistent of consistent great great

interest interest parallel parallelto to human history, and human history, andresearch researchonon mechanisms mechanisms and treatments and treatments has continued. has continued.

However,basic However, basicresearch researchononskin skinaging agingtreatment treatmentand andprevention prevention has has not not shown shown outstanding outstanding results results

compared compared to to research research on treatments on treatments for other for other diseases. diseases. This isThis also is also related related to not finding to not finding fundamental fundamental

3 answers to the answers to the topic topic of of "cellular "cellular aging," aging," which whichisisthe thehigher-level higher-levelconcept conceptofofskin skinaging. aging.While While countless studies countless studies on on fibroblasts fibroblasts in vitro in vitro exist,exist, there there arecountless are also also countless studies studies that have that have failed to failed to connect research results connect research results to to anti-aging anti-aging measures for human measures for human skin skin tissueatatthe tissue theorganism organism level.This level. This suggests suggests that that the theeffects effectsofof systemic aging systemic ononthethe aging skin may skin bebemore may morecomplex complex than than expected, expected, meaning meaning that research that research related related to to skin skin aging aging should should be viewedfrom be viewed fromananexpanded expanded perspective perspective including including not not only the skin only the skin but but also alsosubcutaneous fat, vascular subcutaneous fat, vascularsystem, system, nervous nervous system, system, or or immune system. immune system.

[Disclosure]

[Disclosure]

[Technical

[Technical Problem] Problem]

[0009] The

[0009] The present present invention invention aims aims to to provide provide peptides peptides and and theiruses their usesthat that prevent preventskin skin aging aging

and promoteskin and promote skinelasticity elasticity by by synthesizing synthesizingType TypeI ICollagen Collagenandand Type Type III III Collagen Collagen in fibroblasts in fibroblasts

within the dermal layer of the skin. within the dermal layer of the skin.

[0010] Another

[0010] Another purpose purpose of the of the present present invention invention is to is to provide provide peptides peptides and and their their usesuses that that

prevent skin prevent skin aging aging and andpromote promoteskin skinelasticity elasticity by bysynthesizing synthesizingType TypeIVIV Collagen Collagen in the in the basement basement

membrane membrane of of thedermo-epidermal the dermo-epidermal junction junction of the of the skin. skin.

[0011] Another

[0011] Another purpose purpose of the of the present present invention invention is to is to provide provide peptides peptides and and their their usesuses that that

promoteregeneration promote regenerationofofthetheepidermal epidermal layer layer and and prevent prevent agingaging by enhancing by enhancing cell division cell division and and

differentiation differentiationthrough through increasing increasing Ki-67 expression in Ki-67 expression in keratinocytes keratinocytes within within the the epidermal epidermallayer layerofof

the skin. the skin.

[0012] Another

[0012] Another purpose purpose of the of the present present invention invention is to is to provide provide peptides peptides and and their their usesuses that that

promoteskin promote skinmoisturization moisturizationandand prevent prevent aging aging by enhancing by enhancing intercellular intercellular junction junction function function of of

keratinocytes through keratinocytes through synthesizing synthesizingFilaggrin Filaggrinand andInvolucrin Involucrinininkeratinocytes keratinocyteswithin withinthe theepidermal epidermal

layer of the layer of theskin. skin.

4

[0013] Another

[0013] Another purpose purpose of the of the present present invention invention is to is to provide provide peptides peptides and and their their usesuses that that

prevent skin prevent skin aging agingbybyinhibiting inhibitingcollagen collagen degradation degradation through through reducing reducing MMP-1 MMP-1 expression expression in in

keratinocytes and dermal cells within the epidermal and dermal layers of the skin. keratinocytes and dermal cells within the epidermal and dermal layers of the skin.

[0014] The

[0014] The purposes purposes of of thethe present present invention invention arenot are notlimited limitedtotothose thosementioned mentioned above, above, and and

other purposes other purposes notnot mentioned mentioned will will be be clearly clearly understood understood by those by those with withskill ordinary ordinary in theskill in the technical technical

field field to to which thepresent which the present invention invention belongs belongs from from the the description description below. below.

[Technical Solution]

[Technical Solution]

[0015]

[0015] ToTo solve solve thethe above above technical technical problems, problems, the present the present inventors inventors conducted conducted various various

studies and developed studies and developeda composition a composition for inhibiting for inhibiting skin skin agingaging or treating or treating woundswounds containing containing

peptides derived peptides derived from fromCPNE7 CPNE7 protein. protein.

[0016]

[0016] AsAs one one embodiment embodiment to achieve to achieve the above-mentioned the above-mentioned purposes, purposes, the present the present invention invention

provides aa composition provides compositionfor forinhibiting inhibitingskin skinaging agingorortreating treating wounds wounds comprising comprising peptides peptides derived derived

from CPNE7 from CPNE7 protein protein having having thethe amino amino acidacid sequence sequence of General of General Formula Formula 1 below. 1 below.

[0017] K-Y-R1-R2-R3-R4-R5-R6-R7-R8

[0017] K-Y-R1-R2-R3-R4-R5-R6-R7-R8 (General (General Formula Formula 1) 1)

[0018]

[0018] InInGeneral General Formula Formula 1 above, 1 above,

[0019]

[0019] R1R1 is is arginine(R), arginine (R),lysine lysine (K), (K), or or glutamine (Q); glutamine (Q);

[0020]

[0020] R2R2 is is arginine(R) arginine (R)ororglutamine glutamine(Q); (Q);

[0021] R3,

[0021] R3, R4, R4, and and R5 R5 areare each each arginine arginine (R)(R) or or lysine(K); lysine (K);

[0022]

[0022] R6R6 is is asparagine(N)(N) asparagine oror serine(S); serine (S);and and

[0023]

[0023] R7R7 andand R8 R8 areare lysine lysine (K)(K) or or tyrosine(Y). tyrosine (Y).

[0024]

[0024]

[0025] The

[0025] The peptides peptides provided provided bypresent by the the present invention invention can provide can provide excellent excellent collagen collagen

synthesis abilitywhile synthesis ability whilenotnot exhibiting exhibiting cytotoxicity. cytotoxicity.

5

[0026] The

[0026] The peptides peptides provided provided by present by the the present invention invention include include variantvariant peptides peptides having having

sequences that differ sequences that differby byone oneor ormore more amino amino acid acid residues residues from from the the amino amino acid acid sequences constituting sequences constituting

them, as long as they can exhibit skin aging inhibition or wound treatment effects including peptides them, as long as they can exhibit skin aging inhibition or wound treatment effects including peptides

derived fromCPNE7 derived from CPNE7 protein. protein.

[0027] Generally,amino

[0027] Generally, amino acid acid exchanges exchanges in proteins in proteins and polypeptides and polypeptides thatnot that do doentirely not entirely

alter the alter theactivity activityof of thethe molecule areare molecule known knownininthe art.art. the TheThe most commonly most commonly occurring exchangesare occurring exchanges are

betweenamino between amino acidacid residues residues Ala/Ser, Ala/Ser, Val/Ile, Val/Ile, Asp/Glu, Asp/Glu, Thr/Ser, Thr/Ser, Ala/Gly, Ala/Gly, Ala/Thr,Ala/Thr, Ser/Asn, Ser/Asn,

Ala/Val, Ser/Gly, Ala/Val, Ser/Gly,Tyr/Phe, Tyr/Phe, Ala/Pro, Ala/Pro, Lys/Arg, Lys/Arg, Asp/Asn, Asp/Asn, Leu/Ile, Leu/Ile, Leu/Val, Leu/Val, Ala/Glu,Ala/Glu, Asp/Gly. Asp/Gly.

Additionally, peptides Additionally, peptides withwith increased increased structural structural stability stability against against heat, pH,heat, etc., pH, etc., or skin or increased increased skin

aging inhibition or aging inhibition or wound treatmenteffects wound treatment effects by variations or by variations or modifications modifications in in amino acid sequences amino acid sequences

maybebeincluded. may included.

[0028] Forexample,

[0028] For example, glutamine, glutamine, which which is aisneutral a neutral polaramino polar amino acid acid acid acid at at position3 3ofofthe position the

peptide of peptide of SEQ SEQIDIDNO:NO: 1 provided 1 provided by the by the present present invention, invention, can can be substituted be substituted withwith basic basic amino amino

acids lysine or arginine while still exhibiting the effects of the peptides provided by the present acids lysine or arginine while still exhibiting the effects of the peptides provided by the present

invention. Arginine, invention. Arginine, which which is a is a basic basic aminoamino acid atacid at position position 4 or 4 or 5 of the5peptide of the of peptide SEQ ID of NO:SEQ 1, ID NO: 1,

can besubstituted can be substitutedwith with acidic acidic amino amino acid acid glutamine glutamine or basicoramino basicacid amino acid lysine lysine while stillwhile still exhibiting exhibiting

the effects of the peptides provided by the present invention. Lysine, which is a basic amino acid at the effects of the peptides provided by the present invention. Lysine, which is a basic amino acid at

position 6, 7, or 9 of the peptide of SEQ ID NO: 1, can be substituted with basic amino acid arginine position 6, 7, or 9 of the peptide of SEQ ID NO: 1, can be substituted with basic amino acid arginine

or or aromatic aminoacid aromatic amino acidtyrosine tyrosinewhile whilestill still exhibiting exhibiting the the effects effects of of the the peptides peptides provided bythe provided by the

present invention. present invention. Asparagine, whichisisa aneutral Asparagine, which neutral polar polar amino aminoacid acidatatposition position8 8ofofthe thepeptide peptideofof

SEQ SEQ IDID NO:NO: 1, be 1, can cansubstituted be substituted with neutral with neutral amino amino acid acid serine serine while while still still exhibiting exhibiting the effects the of effects of

the peptides provided by the present invention. Tyrosine, which is an aromatic amino acid at position the peptides provided by the present invention. Tyrosine, which is an aromatic amino acid at position

10 of the 10 of the peptide peptide of of SEQ SEQIDID NO:NO: 1, can 1, can be substituted be substituted withwith basicbasic aminoamino acid lysine acid lysine while while still still

exhibiting theeffects exhibiting the effectsofofthethepeptides peptides provided provided by theby the present present invention. invention.

6

[0029] Thus,

[0029] Thus, even even if if thethe acidicamino acidic amino acids, acids, basic basic amino amino acids, acids, or aromatic or aromatic aminoamino acids acids

constituting thepeptides constituting the peptides of of the the present present invention invention are substituted are substituted with different with different acidic acidic amino amino acids, acids,

basic amino acids, neutral amino acids, or aromatic amino acids respectively, they can still exhibit basic amino acids, neutral amino acids, or aromatic amino acids respectively, they can still exhibit

the effects of the peptides provided by the present invention. Therefore, it is obvious that variant the effects of the peptides provided by the present invention. Therefore, it is obvious that variant

peptides having peptides havingsequences sequencesthat thatdiffer differbybyoneone or or more more amino amino acid acid residues residues from from the amino the amino acid acid

sequences constituting sequences constituting the the peptides peptides of present of the the present invention invention areincluded are also also included in theofscope in the scope of peptides peptides

provided by provided bythe the present present invention. invention.

[0030] Additionally,

[0030] Additionally, even even if theifpeptides the peptides of the of the present present invention invention have have a form witha arbitrary form with arbitrary

amino acids added to their N-terminus or C-terminus, they can still exhibit the effects of the peptides amino acids added to their N-terminus or C-terminus, they can still exhibit the effects of the peptides

provided by provided bythe thepresent present invention inventionand andare aretherefore thereforeincluded includedininthe the scope scopeofofpeptides peptidesprovided providedbyby

the present the present invention. invention. As As one one example, they can example, they can be be in in aa form form with with 1 1 to to 300 300 amino acids added amino acids to the added to the

N-terminusororC-terminus N-terminus C-terminusofofthe thepeptide. peptide.AsAsanother anotherexample, example, they they cancan be be in in a form a form with with 1 to 1 to 100100

aminoacids amino acidsadded addedtotothe the N-terminus N-terminusororC-terminus C-terminusofofthe thepeptide. peptide. As Asyet yet another another example, example,they theycan can

be in be in aa form form with with 1 1 to to 24 24 amino acids added amino acids addedto to the the N-terminus orC-terminus N-terminus or C-terminusofofthe thepeptide. peptide.

[0031]

[0031] AsAs another another aspect, aspect, the the present present invention invention provides provides polynucleotides polynucleotides encoding encoding the the

above peptides. above peptides.

[0032] The

[0032] The polynucleotides polynucleotides may may have have one one or or bases more more substituted, bases substituted, deleted, deleted, inserted, inserted, or or

combinations thereof.When combinations thereof. When preparing preparing by chemically by chemically synthesizing synthesizing nucleotide nucleotide sequences, sequences, well- well-

knownsynthesis known synthesismethods methods in in thethe artart can can be be used, used, forfor example, example, methods methods described described in literature in the the literature

(Engels and Uhlmann, (Engels and Uhlmann,Angew Angew ChemChem IntEdIntEd Engl., Engl., 37:73-127, 37:73-127, 1988), 1988), and synthesis and synthesis canperformed can be be performed

using triester, using triester, phosphite, phosphite,phosphoramidite andH-phosphate phosphoramidite and H-phosphate methods, methods, PCR PCR and other and other auto-primer auto-primer

methods,oligonucleotide methods, oligonucleotidesynthesis synthesisononsolid solidsupports, supports,etc. etc. For For example, example,polynucleotides polynucleotidesencoding encoding

the peptides the peptides of of the thepresent presentinvention inventionmay may include include the the nucleotide nucleotide sequence of SEQ sequence of SEQIDIDNO: NO: 4. 4.

7

[0033]

[0033] AsAs yetyet another another aspect,the aspect, thepresent presentinvention inventionprovides providesexpression expression vectors vectors containing containing

the abovepolynucleotides, the above polynucleotides,transformants transformants containing containing the expression the expression vectors, vectors, and methods and methods for for

producingthe producing the peptides peptides using using the the transformants. transformants.

[0034] The

[0034] The term term "expression "expression vector" vector" in the in the present present invention invention means means a recombinant a recombinant vectorvector

capable capable ofof expressing expressing the the desired desired peptide peptide in the in the desired desired host host cell, andcell, andtorefers refers to aconstruct a genetic genetic construct

containing essential containing essential regulatory regulatory elements operablylinked elements operably linkedsosothat that the the gene geneinsert insert is is expressed. The expressed. The

expression vector expression vector includes includes expression expression control control elements elements such as such start as start codons, codons, stoppromoters, stop codons, codons, promoters,

operators, etc., where operators, etc., wherethethe startcodon start codon and and stop stop codoncodon are generally are generally considered considered part part of the of the nucleotide nucleotide

sequence encodingthe sequence encoding thepolypeptide polypeptide and and must must function function in in thethe individual individual when when the the genetic genetic construct construct

is is administered administered and must be and must be in in frame framewith withthe the coding codingsequence. sequence.The Thepromoter promoter of of thethevector vectormay may be be

constitutive or inducible. constitutive or inducible.

[0035] The

[0035] The term term "operably "operably linked" linked" in in thepresent the presentinvention inventionmeans means a statewhere a state where nucleicacid nucleic acid

expression control sequences expression control sequencesand andnucleic nucleic acid acid sequences sequences encoding encoding desired desired proteins proteins or are or RNA RNA are

functionally linked toto perform functionally linked performgeneral general functions. functions. For For example, example, a promoter a promoter and acid and nucleic nucleic acid

sequences encodingproteins sequences encoding proteinsor or RNARNA canoperably can be be operably linkedlinked to affect to affect expression expression of the of the coding coding

sequence. Operative sequence. Operativelinkage linkagewith withexpression expressionvectors vectorscan canbebe prepared prepared using using genetic genetic recombination recombination

techniques well techniques well known knownin in thethe art,and art, andsite-specific site-specificDNA DNA cleavage cleavage and and ligation ligation can can use enzymes use enzymes

generally known generally known in the in the art.art.

[0036] Additionally,the

[0036] Additionally, theexpression expressionvector vectormay may include include signalsequences signal sequences forsecretion for secretionofofthe the

peptide to facilitate separation of the peptide from cell culture medium. Specific initiation signals peptide to facilitate separation of the peptide from cell culture medium. Specific initiation signals

may also be necessary for efficient translation of the inserted nucleic acid sequence. These signals may also be necessary for efficient translation of the inserted nucleic acid sequence. These signals

include ATG include ATG startcodons start codonsandand adjacent adjacent sequences. sequences. In some In some cases, cases, exogenous exogenous translation translation control control

signals signals that that may include ATG may include ATG startcodons start codonsmust must be be provided. provided. These These exogenous exogenous translation translation control control

8 signals signals and and start startcodons codons may befrom may be fromvarious variousnatural naturaland andsynthetic syntheticsources. sources.Expression Expression efficiency efficiency can beincreased can be increasedby by introduction introduction of appropriate of appropriate transcriptional transcriptional or translational or translational enhancers. enhancers.

[0037] Furthermore,

[0037] Furthermore, thethe expression expression vector vector maymay additionally additionally include include protein protein tags tags thatcan that canbebe

removed using endopeptidases, if desired, to facilitate detection of the peptide. removed using endopeptidases, if desired, to facilitate detection of the peptide.

[0038] The

[0038] The term term "tag" "tag" in the in the present present invention invention meansmeans a molecule a molecule showing showing quantifiable quantifiable

activity activity or characteristics and or characteristics and may maybe be fluorescent fluorescent molecules molecules including including chemical chemical fluorescent fluorescent

materials such materials as fluorescein, such as fluorescein, polypeptide polypeptide fluorescent fluorescent materials materials such such as as fluorescent fluorescent proteins proteins(GFP) (GFP)

or related proteins; or related proteins;orormay may be epitope be epitope tags tags such such as Myc as Myc tags, tags, Flag Flag tags, tags, histidine histidine tags,tags, tags, leucine leucine tags,

IgG tags, streptavidin IgG tags, streptavidin tags, tags, etc. etc. Particularly, Particularly, when usingepitope when using epitopetags, tags,preferably preferably peptide peptide tags tags

composed composed of of 6 or 6 or more more amino amino acid acid residues, residues, more more preferably preferably composed composed of amino of 8 to 50 8 to 50 amino acid acid

residues can be used. residues can be used.

[0039] The

[0039] The term term "photodermatitis" "photodermatitis" in the in the present present invention invention may may mean mean a a condition condition where where

ultraviolet rays ultraviolet raysor orspecific specificsubstances substancescombined with sunlight combined with sunlight cause cause inflammatory inflammatoryreactions reactionsininthe the

skin. skin. One ofthe One of themajor major causes causes of skin of skin damage damage due todue to ultraviolet ultraviolet exposure exposure is oxidative is oxidative stress.stress.

Oxidative stress can Oxidative stress canaffect affectdermal dermal fibroblasts,which fibroblasts, which can promote can promote collagen collagen degradation degradation and and

inflammatory reactions.This inflammatory reactions. Thisprocess process cancan change change skinskin characteristics characteristics and and worsen worsen symptoms symptoms of of

photodermatitis. photodermatitis.

[0040] Theterm

[0040] The term"atopic "atopicdermatitis" dermatitis" ininthe thepresent presentinvention inventionisischronic chronicdermatitis dermatitis

characterized characterized byby drydry andand itchy itchy skinskin with with recurring recurring inflammation. inflammation. It istoknown It is known to be be related to related genetic to genetic

factors, factors,environmental factors, immune environmental factors, systemabnormalities, immune system abnormalities,etc. etc.Stress Stress in in dermal fibroblasts may dermal fibroblasts may

be associated be associated with with the the onset onset and andprogression progressionofofatopic atopicdermatitis. dermatitis.Oxidative Oxidativestress stressgenerates generatesfree free

radicals that radicals thatdamage cells, causing damage cells, causing inflammatory reactionsand inflammatory reactions andfunctional functionaldecline declineofof cells. cells. Dermal Dermal

fibroblasts arelocated fibroblasts are locatedin inthethe dermal dermal layerlayer ofskin of the the and skinplay andimportant play important roles in maintaining roles in maintaining skin skin

structure andfunction. structure and function. When When the function the function of dermal of dermal fibroblasts fibroblasts is impaired is impaired due tostress, due to oxidative oxidative stress, 9 the protective the protective function function of of the the skin skin may maybe be weakened. weakened. This This makesmakes themore the skin skinvulnerable more vulnerable to to external external stimuli stimuli and and can worsensymptoms can worsen symptoms of atopic of atopic dermatitis. dermatitis. TheThe resulting resulting oxidative oxidative stresscan stress can promoteinflammatory promote inflammatory reactions. reactions. Such Such inflammatory inflammatory reactions reactions are of are one onethe of major the major symptoms symptoms of of atopic dermatitis atopic dermatitis and and become become a acause causeofofdermal dermalfibroblasts fibroblastsnot notfunctioning functioningproperly. properly.Additionally, Additionally, cell cell damage damage duedue to oxidative to oxidative stress stress can worsen can worsen damage damage to tobarrier. the skin the skinWhen barrier. When the skin the is barrier skin barrier is damaged,substances damaged, substancesthat thatcause cause allergicreactions allergic reactionscan canpenetrate penetratethetheskin skinmore more easily, easily, which which can can further further worsen symptoms worsen symptoms of of atopicdermatitis. atopic dermatitis.

[0041]

[0041] InInthe thepresent presentinvention, invention,thetheexpression expression vector vector maymay include include nucleotide nucleotide sequences sequences

encoding CPNE7 encoding CPNE7 protein protein or or peptides peptides consisting consisting of of amino amino acids acids of of SEQSEQ ID NO: ID NO: 1. vectors 1. The The vectors used used

are not particularly are not particularlylimited limitedas as long long as as they they can can produce produce the peptides, the peptides, but preferably but preferably may be plasmid may be plasmid

DNA,phage DNA, phageDNA, DNA, etc., etc., more more preferablycommercially preferably commercially developed developed plasmids plasmids (pUC18, (pUC18, pBAD, pBAD,

pIDTSAMRT-AMP, pIDTSAMRT-AMP, etc.), etc.), E. E. coli-derived plasmids coli-derived plasmids (pYG601BR322, pBR325, (pYG601BR322, pBR325, pUC118, pUC118, pUC119, pUC119,

etc.), Bacillus etc.), Bacillus subtilis-derived subtilis-derivedplasmids plasmids (pUB110, pTP5, (pUB110, pTP5, etc.),yeast-derived etc.), yeast-derived plasmids plasmids (YEp13, (YEp13,

YEp24, YCp50,etc.), YEp24, YCp50, etc.), phage phageDNA DNA (Charon4A, (Charon4A, Charon21A, EMBL3, Charon21A, EMBL3, EMBL4, EMBL4, λgt10, Agt10, λgt11,AZAP, Agt11, λZAP,

etc.), animal etc.), virus vectors animal virus vectors(retrovirus, (retrovirus, adenovirus, adenovirus,vaccinia vaccinia virus, virus, etc.),insect etc.), insectvirus virus vectors vectors

(baculovirus, etc.). (baculovirus, etc.). Since the expression Since the expressionvector vectorshows shows different different protein protein expression expression levels levels and and

modifications depending on the host cell, it is preferable to select and use the most suitable host cell modifications depending on the host cell, it is preferable to select and use the most suitable host cell

for the purpose. for the purpose.

[0042] The

[0042] The transformants transformants provided provided by the by the present present invention invention cancan be be prepared prepared by introducing by introducing

the expression the vectors provided expression vectors by the provided by the present present invention into hosts invention into hosts and and transforming them, and transforming them, andcan can

be used be usedtotoproduce producethethepeptides peptides by by expressing expressing the the polynucleotides polynucleotides contained contained in theinexpression the expression

vectors. The vectors. The transformation can be transformation can be performed performedbybyvarious variousmethods methods andand is is notnot particularlylimited particularly limitedasas

long as long as ititcan canproduce produce the thepeptides, peptides,but butCaCl2 CaCl2 precipitation precipitationmethod, method, Hanahan methodthat Hanahan method thatincreases increases

efficiency efficiency by using dimethyl by using dimethylsulfoxide sulfoxide(DMSO) (DMSO) as a as a reducing reducing agent agent in the in the precipitation CaCl2 CaCl2 precipitation 10 method,electroporation, method, electroporation,calcium calcium phosphate phosphate precipitation precipitation method, method, protoplast protoplast fusion fusion method, method, agitation agitation method usingsilicon method using siliconcarbide carbidefibers, fibers,Agrobacterium-mediated Agrobacterium-mediated transformation transformation method, method,

PEG-mediated PEG-mediated transformation transformation method, method, dextran dextran sulfate, sulfate, lipofectamine, lipofectamine, andand dry/inhibition-mediated dry/inhibition-mediated

transformation methods, transformation etc. can methods, etc. be used. can be used. Additionally, Additionally, the the hosts hosts used for preparing used for preparing the the

transformants are also not particularly limited as long as they can produce the peptides, but may be transformants are also not particularly limited as long as they can produce the peptides, but may be

bacterial cells bacterial cells such such asasE.E.coli, coli,Streptomyces, Streptomyces, Salmonella Salmonella typhimurium; typhimurium; yeastsuch yeast cells cells as such as

Saccharomyces cerevisiae,Schizosaccharomyces Saccharomyces cerevisiae, Schizosaccharomyces pombe; pombe; fungal fungal cellscells suchsuch as Pichia as Pichia pastoris; pastoris; insect insect

cells cells such such as as Drosophila, Drosophila, Spodoptera Sf9cells; Spodoptera Sf9 cells; animal cells such animal cells such as as CHO, COS, CHO, COS, NSO, NSO, 293,293, bovine bovine

melanoma cells; or plant cells. melanoma cells; or plant cells.

[0043] The

[0043] The transformants transformants cancan also also be be used used in in methods methods for for producing producing peptides peptides consisting consisting of of

amino acids of amino acids of SEQ SEQIDIDNO: NO: 1 ofthe 1 of thepresent presentinvention. invention.Specifically, Specifically, methods for producing methods for producingpeptides peptides

consisting consisting of of amino acids of amino acids of SEQ SEQIDID NO: NO: 1 the 1 of of the present present invention invention maymay include include (a) (a) culturing culturing thethe

transformants to transformants to obtain obtain cultures; cultures; and and (b) (b) recovering recovering the the peptides peptides of of the the present present invention invention from from the the

cultures. cultures.

[0044] Theterm

[0044] The term"culture" "culture"ininthethepresent presentinvention inventionmeans means a method a method of growing of growing

microorganisms under microorganisms under artificiallycontrolled artificially controlledenvironmental environmental conditions. conditions. In In thethe present present invention, invention,

methodsfor methods forculturing culturing the the transformants transformantscan canbebeperformed performed using using methods methods widely widely known known in theinart. the art.

Specifically, theculture Specifically, the culture is is notnot particularly particularly limited limited as as as long long as itexpress it can can express andpeptides and produce produce peptides

consisting consisting of of amino amino acids acids of of SEQ IDNO: SEQ ID NO: 1 of 1 of thethe presentinvention, present invention,but butcan canbebecultured cultured

continuously in batch processes or fed batch or repeated fed batch processes. continuously in batch processes or fed batch or repeated fed batch processes.

[0045] The

[0045] The medium medium usedculture used for for culture mustthemeet must meet the requirements requirements of strains of specific specificinstrains in

appropriate wayswhile appropriate ways whilecontrolling controllingtemperature, temperature,pH, pH,etc. etc.under underaerobic aerobicconditions conditionsininconventional conventional

mediumcontaining medium containing appropriatecarbon appropriate carbon sources,nitrogen sources, nitrogensources, sources,amino aminoacids, acids,vitamins, vitamins,etc. etc. Carbon Carbon

sources that can sources that can be used include be used include mixed mixedsugars sugarsofofglucose glucoseandand xylose xylose as as main main carbon carbon sources, sources, and and 11 additionally sugars additionally sugars andand carbohydrates carbohydrates such assuch as sucrose, sucrose, lactose, lactose, fructose,fructose, maltose, maltose, starch, starch, cellulose, cellulose, oils oils and fatssuch and fats suchasassoybean soybean oil, oil, sunflower sunflower oil, castor oil, castor oil, coconut oil, coconut oil,acids oil, fatty fattysuch acids as such as palmitic palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, and organic acids such as acetic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, and organic acids such as acetic acid. These acid. substances can These substances canbebeused usedindividually individuallyororasasmixtures. mixtures.Nitrogen Nitrogensources sourcesthat thatcan canbebeused used include inorganic nitrogen include inorganic nitrogensources sourcessuch such as as ammonia, ammonia, ammonium ammonium sulfate, sulfate, ammoniumammonium chloride, chloride, ammonium ammonium acetate, ammonium acetate, ammonium phosphate,ammonium phosphate, ammonium carbonate, carbonate, andand ammonium ammonium nitrate; nitrate; amino amino acids acids such as glutamic such as glutamic acid, acid, methionine, methionine,glutamine glutamineand andorganic organicnitrogen nitrogen sources sources such such as as peptone, peptone,

NZ-amine,meat NZ-amine, meat extract,yeast extract, yeastextract, extract,malt maltextract, extract, corn cornsteep steepliquor, liquor, casein casein hydrolysate, hydrolysate, fish fish or or

their decomposition their products,defatted decomposition products, defatted soybean soybean cake cake or their or their decomposition decomposition products. products. These These

nitrogen sources nitrogen sources can can be be used used alone alone or or in in combination. combination. The mediummay The medium may includepotassium include potassium

dihydrogenphosphate, dihydrogen phosphate, dipotassium dipotassium hydrogen hydrogen phosphate, phosphate, and corresponding and corresponding sodium-containing sodium-containing

salts salts as as phosphorus sources. Phosphorus phosphorus sources. Phosphorus sources sources that that cancan be be used used include include potassium potassium dihydrogen dihydrogen

phosphate or phosphate or dipotassium dipotassiumhydrogen hydrogenphosphate phosphate or corresponding or corresponding sodium-containing sodium-containing salts. salts.

Additionally, inorganiccompounds Additionally, inorganic compoundssuchsuch as sodium as sodium chloride, chloride, calciumcalcium chloride, chloride, iron chloride, iron chloride,

magnesium magnesium sulfate,iron sulfate, ironsulfate, sulfate, manganese manganese sulfate,andand sulfate, calcium calcium carbonate carbonate can can be used. be used. Finally, Finally,

essential essential growth substances such growth substances suchas as amino aminoacids acidsand andvitamins vitaminscan canbebeused used in in additiontotothe addition theabove above

substances. substances.

[0046] Additionally,appropriate

[0046] Additionally, appropriateprecursors precursorscan canbebeused used inin theculture the culturemedium. medium.TheThe above- above-

mentionedraw mentioned rawmaterials materialscancan be be added added to the to the culture culture in in appropriate appropriate ways ways by batch, by batch, fed-batch, fed-batch, or or

continuous methodsduring continuous methods during thethe cultureprocess, culture process,but butare arenot notparticularly particularly limited limited thereto. thereto. The pHofof The pH

the culture the culture can can be controlled by be controlled appropriately using by appropriately using basic basic compounds compounds such such as sodium as sodium hydroxide, hydroxide,

potassiumhydroxide, potassium hydroxide,ammonia, ammonia,or or acid acid compounds compounds such such as phosphoric as phosphoric acidsulfuric acid or or sulfuric acid. acid.

[0047] Additionally,antifoaming

[0047] Additionally, antifoaming agents agents such such as as fattyacid fatty acidpolyglycol polyglycol esterscan esters canbebeused usedtoto

suppress foamformation. suppress foam formation.ToTo maintain maintain aerobic aerobic conditions, conditions, oxygen oxygen or oxygen-containing or oxygen-containing gas (e.g., gas (e.g.,

12 air) is injected into the culture. The temperature of the culture is usually 27°C to 37°C, preferably air) is injected into the culture. The temperature of the culture is usually 27°C to 37°C, preferably

30°C to 35°C. 30°C to 35°C.Culture Culturecontinues continuesuntil until the the maximum production maximum production of of thethe peptides peptides is is obtained.For obtained. Forthis this

purpose, it is usually achieved in 10 to 100 hours. purpose, it is usually achieved in 10 to 100 hours.

[0048] Furthermore,

[0048] Furthermore, thethe step step of of recovering recovering thethe peptides peptides from from thethe culture culture can can be be performed performed

by methods known in the art. Specifically, the recovery method is not particularly limited as long as by methods known in the art. Specifically, the recovery method is not particularly limited as long as

it it can can be usedfor be used forrecovery recoveryof of thethe produced produced peptides, peptides, but preferably but preferably methods methods such such as centrifugation, as centrifugation,

filtration, filtration, extraction, spraying, extraction, spraying, drying, drying, evaporation, evaporation, precipitation, precipitation, crystallization, crystallization, electrophoresis, electrophoresis,

fractional fractional dissolution dissolution(e.g., (e.g.,ammonium sulfate precipitation), ammonium sulfate precipitation), chromatography (e.g., ion chromatography (e.g., ion exchange, exchange,

affinity, hydrophobic and size exclusion), etc. can be used. affinity, hydrophobic and size exclusion), etc. can be used.

[0049] Accordingtotothe

[0049] According thepresent presentinvention, invention, pharmaceutical pharmaceutical compositions compositionsfor forwound wound

treatment containing treatment containing peptides peptides derived derived from CPNE7 from CPNE7 proteincancan protein be be usedused in form in the the form of of

pharmaceuticalcompositions pharmaceutical compositions for for treating treating skinskin diseases diseases caused caused by oxidative by oxidative stress stress in in dermal dermal

fibroblasts. fibroblasts. Such compositionscancan Such compositions be be prepared prepared by additionally by additionally including including appropriate appropriate carriers carriers

(natural or non-natural (natural or non-naturalcarriers), carriers),excipients, excipients,or or diluents diluents commonly commonly used. Specifically, used. Specifically, the the

pharmaceuticalcompositions pharmaceutical compositions can can be be formulated formulated andand used used in the in the form form of sterileinjectable of sterile injectablesolutions solutions

that can that can be be administered to areas administered to areas where skin diseases where skin diseases have been induced have been inducedaccording accordingtotoconventional conventional

methods.InInthe methods. thepresent presentinvention, invention,carriers, carriers, excipients, excipients, and diluents that and diluents that may maybebeincluded includedin inthethe

pharmaceuticalcompositions pharmaceutical compositions include include lactose, lactose, dextrose, dextrose, sucrose, sucrose, sorbitol, sorbitol, mannitol, mannitol, xylitol,xylitol,

erythritol, erythritol, maltitol, maltitol,starch, starch,acacia acacia gum, alginate, gelatin, gum, alginate, gelatin, calcium phosphate,calcium calcium phosphate, calcium silicate, silicate,

cellulose, cellulose, methyl methyl cellulose, microcrystalline cellulose, microcrystalline cellulose, cellulose, polyvinyl polyvinyl pyrrolidone, pyrrolidone,water, water,

methylhydroxybenzoate, propylhydroxybenzoate, methylhydroxybenzoate, propylhydroxybenzoate, talc,talc, magnesium magnesium stearate, stearate, mineral mineral oil, collagen, oil, collagen,

etc. etc. When formulating,they When formulating, theycan canbebeprepared prepared using using diluents diluents oror excipientssuch excipients such as as commonly commonly used used

fillers, fillers, extenders, binders, extenders, binders, wetting wetting agents, agents, disintegrants, disintegrants, surfactants, surfactants, etc. In sterile etc. In particular, particular, sterile

aqueoussolutions, aqueous solutions,non-aqueous non-aqueous solvents, solvents, suspensions, suspensions, emulsions, emulsions, freeze-dried freeze-dried preparations, preparations,

13 suppositories, suppositories, ointments (e.g., root ointments (e.g., rootcanal canalsealers, sealers,etc.) may etc.) maybebeincluded. included.As As non-aqueous solvents non-aqueous solvents and suspensions, and suspensions, propylene propylene glycol, glycol, polyethylene polyethylene glycol, vegetable glycol, vegetable oils oils such as such olive asinjectable oil, olive oil, injectable esters esters such such as as ethyl ethyl oleate, oleate,etc. etc.can canbebeused. used.As Assuppository suppository bases, bases, witepsol, witepsol, macrogol, tween61, macrogol, tween 61, cacao butter,laurin cacao butter, laurinbutter, butter,glycerogelatin, glycerogelatin, etc.etc. can can be used. be used.

[0050] The

[0050] The content content of the of the peptides peptides included included in pharmaceutical in the the pharmaceutical compositions compositions of the of the

present invention present is not invention is not particularly particularlylimited, but limited, may but maybe be included included in in amounts of 0.0001 amounts of 0.0001 to to 50% 50%byby

weight, more weight, morepreferably preferably0.01 0.01to to 20% 20%bybyweight weight based based on on thethe totalweight total weightofofthe thefinal final composition. composition.

[0051] The

[0051] The pharmaceutical pharmaceutical compositions compositions ofpresent of the the present invention invention can becan be administered administered in in

pharmaceuticallyeffective pharmaceutically effective amounts. amounts.The The term term "pharmaceutically "pharmaceutically effective effective amount" amount" in the in the present present

invention means invention means an amount an amount sufficient sufficient to or to treat treat or prevent prevent diseases diseases with a reasonable with a reasonable benefit/risk benefit/risk ratio ratio

applicable applicable to to medical medical treatment treatment or or prevention. prevention. The The effective effectivedose dose level levelcan canbe bedetermined determined according according

to factors including the severity of the disease, activity of the drug, age, weight, health, sex of the to factors including the severity of the disease, activity of the drug, age, weight, health, sex of the

patient, patient's sensitivity to the drug, administration time of the composition used in the present patient, patient's sensitivity to the drug, administration time of the composition used in the present

invention, invention, administration administration route route and excretion rate, and excretion rate, treatment treatment period, period, drugs drugs used used in in combination or combination or

simultaneously withthe simultaneously with thecomposition compositionofofthe thepresent presentinvention, invention,and andother otherfactors factorswell wellknown knownin in the the

medicalfield. medical field. The The pharmaceutical compositionsofofthe pharmaceutical compositions thepresent presentinvention inventioncan canbebeadministered administeredalone alone

or or in in combination withknown combination with known pharmaceutical pharmaceutical compositions compositions for treating for treating skin skin diseases diseases caused caused by by

oxidative stressinindermal oxidative stress dermal fibroblasts. fibroblasts. Considering Considering allabove all the the above factors, factors, it is important it is important to administer to administer

amounts that can amounts that can obtain obtain maximum maximum effects effects with with minimum minimum amounts amounts without without side effects. side effects.

[0052] The

[0052] The dosage dosage of the of the pharmaceutical pharmaceutical compositions compositions of the of the present present invention invention can be can be

determined by those skilled in the art considering the purpose of use, severity of disease, patient's determined by those skilled in the art considering the purpose of use, severity of disease, patient's

age, weight,sex, age, weight, sex,medical medical history, history, or type or the the of type of substance substance used as used activeas active ingredient. ingredient. For example, For example,

the pharmaceutical the compositions pharmaceutical compositions of of thepresent the present invention invention cancan be be administered administered at about at about 0.1 0.1 ng ng to to

about 100 mg/kg, about 100 mg/kg,preferably preferably11ngngtotoabout about1010mg/kg mg/kg peradult. per adult.The Theadministration administrationfrequency frequency of of the the 14 compositions compositions of of thethe present present invention invention is notisparticularly not particularly limited, limited, but canbut be can be administered administered once daily once daily or or divided divided into into multiple multiple doses. doses. The abovedosage The above dosagedoes doesnot notlimit limitthe thescope scopeofofthe the present present invention invention in in any any way. way.

[0053]

[0053] AsAs another another aspect,thethepresent aspect, presentinvention inventionprovides providesmethods methods forfor treatingskin treating skindiseases diseases

caused byoxidative caused by oxidativestress stressinindermal dermal fibroblasts fibroblasts comprising comprising administering administering the pharmaceutical the pharmaceutical

compositions inpharmaceutically compositions in pharmaceuticallyeffective effectiveamounts amountsto to subjects subjects other other than than humans humans in whom in whom skin skin

diseases caused by oxidative stress in dermal fibroblasts have developed. diseases caused by oxidative stress in dermal fibroblasts have developed.

[0054] The

[0054] The term term "subject" "subject" in in thethe present present invention invention cancan include include mammals mammals including including mice, mice,

livestock, etc. that livestock, etc. that require treatmentforforskin require treatment skin diseases diseases caused caused by oxidative by oxidative stressstress in dermal in dermal fibroblasts fibroblasts

without limitation, without limitation, but but excludes excludes humans fromsubjects humans from subjectsininwhom whomthethe diseases diseases have have developed. developed.

[0055] The

[0055] The administration administration route route of pharmaceutical of the the pharmaceutical compositions compositions for treating for treating skin skin

diseases caused by oxidative stress in dermal fibroblasts of the present invention can be administered diseases caused by oxidative stress in dermal fibroblasts of the present invention can be administered

through any general route as long as it can reach the target tissue. The pharmaceutical compositions through any general route as long as it can reach the target tissue. The pharmaceutical compositions

of the present of the presentinvention inventionareare notnot particularly particularly limited, limited, butbecan but can be administered administered through through routes suchroutes as such as

oral administration,dermal oral administration, dermal administration, administration, intramuscular intramuscular injection, injection, intravenous intravenous injection, injection,

subcutaneous injection, subcutaneous injection, respiratory respiratory administration, administration, rectal rectal administration, administration, topical topical administration, administration, etc. etc.

according according toto thedesired the desired purpose. purpose.

[0056] 'Oral

[0056] 'Oral administration administration (intraoral (intraoral administration)' administration)' is a method is a method of ingesting of ingesting drugs through drugs through

the mouth, the mouth,andand generally generally pills, pills, capsules, capsules, and and liquid liquid formsforms of can of drugs drugs can be be used. used. 'Dermal 'Dermal

administration' administration' isisa amethod method of applying of applying or attaching or attaching drugs drugs to to theand the skin, skin, can and can in be used beforms usedsuch in forms such

as creams,gels, as creams, gels,patches, patches, etc.etc. 'Intramuscular 'Intramuscular injection' injection' is a method is a method of injecting of injecting drugsinto drugs directly directly into

muscletissue, muscle tissue, mainly mainlyusing usingsyringes syringesand andneedles. needles.'Intravenous 'Intravenousinjection' injection' is is aa method methodofofinjecting injecting

drugs directly into drugs directly into blood vessels, using blood vessels, using syringes and needles, syringes and needles, or or venous venouscannulas. cannulas.'Subcutaneous 'Subcutaneous

injection' injection'isisa amethod method of of injecting injecting drugs drugs into into fat fat tissue tissueunder under the the skin, skin,and and can can be used for be used for drug drug 15 administration such administration such as insulin. as insulin. 'Respiratory 'Respiratory administration' administration' is aofmethod is a method of delivering delivering drugs to the drugs to the respiratory system respiratory throughthe system through themouth mouthor or nose, nose, andand cancan be used be used in forms in forms such such as inhalers, as inhalers, gases, gases, vapors, etc. 'Rectal administration' is a method of injecting drugs into the rectum, and can be used vapors, etc. 'Rectal administration' is a method of injecting drugs into the rectum, and can be used in in forms suchasasrectal forms such rectal medications, medications, rectal rectal suppositories, suppositories, etc. 'Topical etc. 'Topical administration' administration' is aofmethod of is a method applying drugs applying drugs directly directly to areas to areas requiring requiring treatment, treatment, and and can be can used be in used forms in forms such such gels, as creams, as creams, gels, patches, etc. according to disease characteristics. patches, etc. according to disease characteristics.

[0057]

[0057] AsAs another another aspect, aspect, the the present present invention invention provides provides quasi-drug quasi-drug compositions compositions for for

preventing or preventing or improving improvingskin skindiseases diseasescaused causedbyby oxidative oxidative stressinindermal stress dermal fibroblastscontaining fibroblasts containing

the above the peptides. above peptides.

[0058] The

[0058] The term term "improvement" "improvement" in theinpresent the present invention invention means means all all actions actions that at that leastat least

reduce parameters reduce parametersrelated related to to the the condition condition being being treated, treated,for forexample, example, the thedegree degree of ofsymptoms. symptoms.

[0059]

[0059] InInthe thepresent presentinvention, invention,the theimprovement improvementcancan be be interpreted interpreted as as meaning meaning all all actions actions

that administer that pharmaceuticalcompositions administer pharmaceutical compositions containing containing the the peptides peptides of the of the present present invention invention as as

active active ingredients ingredients to to subjects subjects requiring requiring treatment for skin treatment for skin diseases diseases caused causedbybyoxidative oxidativestress stressinin

dermal fibroblasts to dermal fibroblasts to promote promoteremoval removalof of reactive reactive oxygen oxygen species species in dermal in dermal fibroblasts, fibroblasts, thereby thereby

improving improving or or benefiting benefiting symptoms symptoms of skin of skin diseases diseases caused bycaused bystress oxidative oxidative stressfibroblasts. in dermal in dermal fibroblasts.

[0060] The

[0060] The term term "quasi-drug" "quasi-drug" in the in the present present invention invention means means products products with with milder milder effects effects

than pharmaceuticals than pharmaceuticalsamong among products products used used for purpose for the the purpose of diagnosing, of diagnosing, treating, treating, improving, improving,

alleviating, alleviating,treating, treating,ororpreventing preventingdiseases diseasesininhumans or animals. humans or For example, animals. For example,according according to to the the

PharmaceuticalAffairs Pharmaceutical AffairsAct, Act,quasi-drugs quasi-drugs areare those those excluding excluding products products usedpharmaceutical used for for pharmaceutical

purposes, including purposes, includingfiber fiber and andrubber rubberproducts products used used for for human human and animal and animal diseasedisease treatment treatment or or

prevention, products prevention, products that that have have mild mildeffects effects on onthe thehuman human body body or not or do do not act act directly directly andand areare notnot

instruments ormachines instruments or machines and and similar similar products, products, disinfectants disinfectants and insecticides and insecticides for preventing for preventing

infectious diseases,etc. infectious diseases, etc. 16

[0061]

[0061] InInthe thepresent present invention, invention, thethe types types or formulations or formulations of quasi-drug of quasi-drug compositions compositions

containing the peptides containing the peptides are are not not particularly particularly limited, limited, but but as as examples, maybebeointments, examples, may ointments,patches, patches,

powders, gels, sprays, tablets, or sprays, etc. powders, gels, sprays, tablets, or sprays, etc.

[0062]

[0062] AsAs another another aspect, aspect, thepresent the presentinvention invention provides provides functional functional food food compositions compositions for for

preventing or preventing or improving improvingskin skindiseases diseasescaused causedbyby oxidative oxidative stressinindermal stress dermal fibroblastscontaining fibroblasts containing

the above the peptides. above peptides.

[0063] The

[0063] The term term "food" "food" in in thethe present present invention invention includes includes meat, meat, sausages, sausages, bread, bread, chocolate, chocolate,

candies, snacks, confectionery, candies, snacks, confectionery, pizza, pizza, ramen, ramen,other othernoodles, noodles,gums, gums, dairy dairy products products including including ice ice

cream, various soups, cream, various soups, beverages, beverages,teas, teas, drinks, drinks, alcoholic alcoholic beverages, vitamincomplexes, beverages, vitamin complexes,functional functional

foods, andhealth foods, and health foods, foods, andand includes includes all foods all foods in thein the conventional conventional sense. sense.

[0064] The

[0064] The functional functional foodfood refers refers to food to food withmedical with high high medical and therapeutic and therapeutic effects effects

processed toto efficiently processed efficiently exhibit exhibit biological biological regulatory functions in regulatory functions in addition addition to to nutritional nutritional supply, supply,

whichis which is the the same termas same term as food food for for special special health health use use (FoSHU). Here,"functional" (FoSHU). Here, "functional"means meansobtaining obtaining

useful effects for health purposes such as regulating nutrients for human body structure and function useful effects for health purposes such as regulating nutrients for human body structure and function

or or physiological physiological actions. actions.Foods Foods of ofthe thepresent presentinvention inventioncan canbebe manufactured manufactured by by methods commonly methods commonly

used in used in the the art, art,and and during during manufacturing, they can manufacturing, they canbe bemanufactured manufacturedby by adding adding raw raw materials materials and and

components commonly components commonly addedadded in theinart. the Additionally, art. Additionally, the formulation the formulation of theoffoods the foods canbealso be can also

manufactured without limitation as long as it is a formulation recognized as food. Food compositions manufactured without limitation as long as it is a formulation recognized as food. Food compositions

of of the the present invention can present invention can be bemanufactured manufacturedin in various various formulations formulations and,and, unlike unlike general general drugs, drugs,

have the have the advantage advantageofofbeing beingmade made from from foodfood materials materials and and having having no side no side effects effects thatthat may may occuroccur

with long-term with long-termuse useof of drugs. drugs. They Theyhave haveexcellent excellentportability, portability, so so foods of the foods of the present present invention invention can can

be consumed be consumedasassupplements supplementsto to enhance enhance effects effects of of preventing preventing oror improving improving skin skin diseases diseases caused caused by by

oxidative stressinindermal oxidative stress dermal fibroblasts. fibroblasts.

17

[0065] Health

[0065] Health food food means means foodfood withwith more more active active health health maintenance maintenance or promotion or promotion effects effects

compared compared totogeneral generalfood, food, andand health health supplement supplement food food means means food food for for supplementation health health supplementation

purposes. Occasionally, purposes. Occasionally, the the terms terms functional functional food, food, health health food, food, and health supplement and health supplementfood foodcan canbebe

used interchangeably. used interchangeably.

[0066] Specifically,the

[0066] Specifically, thefunctional functional foods foods can canbe befoods foodsmanufactured manufacturedby by adding adding thethe peptides peptides

of the of the present present invention invention to to food food materials materials such as beverages, such as teas, spices, beverages, teas, spices,gums, gums, confectionery, confectionery, or or

manufacturedasascapsules, manufactured capsules,powders, powders, suspensions, suspensions, etc.When etc. When consumed, consumed, they they mean bringing mean bringing about about

specific specific health health effects, effects,but butunlike general unlike drugs, general they drugs, have they havethe advantage the advantageof ofbeing beingmade made from food from food

materials and materials having no and having noside side effects effects that thatmay may occur with long-term occur with long-termuse use of of drugs. drugs.

[0067] Since

[0067] Since thefood the food compositions compositions of the of the present present invention invention can can be consumed be consumed daily,daily, they they

can be very can be very usefully usefully used as they used as they can can be be expected to have expected to have high higheffects effects for for preventing preventing or or improving improving

skin diseasescaused skin diseases caused by oxidative by oxidative stress stress in dermal in dermal fibroblasts. fibroblasts.

[0068] The

[0068] The food food compositions compositions may may additionally additionally include include physiologically physiologically acceptable acceptable carriers, carriers,

and thetypes and the typesofofcarriers carriers areare notnot particularly particularly limited, limited, andcarrier and any any carrier commonly commonly used in theused in the art can art can

be used. be used.

[0069] Additionally,the

[0069] Additionally, thefood foodcompositions compositions maymay include include additional additional components components commonly commonly

used in used in food food compositions that can compositions that can improve improvesmell, smell,taste, taste, vision, vision,etc. etc.For Forexample, example,they theymay may include include

vitamins A, vitamins A, C, C, D, D,E,E,B1, B1,B2, B2,B6, B6,B12, B12, niacin,biotin, niacin, biotin,folate, folate, pantothenic acid, etc. pantothenic acid, etc. They They may also may also

include minerals such include minerals suchasaszinc zinc(Zn), (Zn),iron iron(Fe), (Fe),calcium calcium (Ca), (Ca), chromium chromium (Cr),(Cr), magnesium magnesium (Mg), (Mg),

manganese(Mn), manganese (Mn), copper copper (Cu), (Cu), chromium chromium (Cr), (Cr), etc. They etc. They mayinclude may also also include amino amino acids acids such as such as

lysine, tryptophan,cysteine, lysine, tryptophan, cysteine, valine, valine, etc.etc.

[0070] Additionally,the

[0070] Additionally, thefood foodcompositions compositions may may include include food food additives additives such such asas preservatives preservatives

(potassiumsorbate, (potassium sorbate, sodium sodium benzoate, benzoate, salicylicacid, salicylic acid,sodium sodium dehydroacetate, dehydroacetate, etc.), etc.), disinfectants disinfectants

(bleaching powderandand (bleaching powder high-grade high-grade bleaching bleaching powder, powder, sodium sodium hypochlorite, hypochlorite, etc.), antioxidants etc.), antioxidants 18

(butylhydroxyanisole(BHA), (butylhydroxyanisole (BHA), butylhydroxytoluene butylhydroxytoluene (BHT), (BHT), etc.),etc.), coloring coloring agents agents (tar dyes, (tar dyes, etc.), etc.),

color developers color developers (sodium (sodium nitrite, nitrite, sodium sodium acetate, acetate, etc.),etc.), bleaching bleaching agents agents (sodium (sodium sulfite), sulfite), seasonings seasonings

(MSG monosodium (MSG monosodium glutamate, glutamate, etc.), etc.), sweeteners sweeteners (dulcin,(dulcin, cyclamate, cyclamate, saccharin, saccharin, sodium, etc.), sodium, etc.),

fragrances (vanillin,lactones, fragrances (vanillin, lactones, etc.), etc.), leavening leavening agents agents (alum, (alum, potassium potassium hydrogen etc.), hydrogen D-tartrate, D-tartrate, etc.),

fortifiers, fortifiers,emulsifiers, emulsifiers,thickeners thickeners(starch syrup), (starch coating syrup), coatingagents, agents,gum gum bases, bases, antifoaming agents, antifoaming agents,

solvents, improvers, etc. solvents, improvers, etc. The Theadditives additivescancan be be selected selected according according to types to food food types andin used in and used

appropriate appropriate amounts. amounts.

[0071] The

[0071] The peptides peptides of of thepresent the presentinvention inventioncancanbebeadded added as as is is ororused usedtogether togetherwith withother other

foods or food foods or components,and food components, and can can be be appropriately appropriately used used according according to conventional to conventional methods. methods. The The

mixingamount mixing amountofof activeingredients active ingredientscan canbebeappropriately appropriatelydetermined determined according according to to theirpurpose their purposeofof

use (prevention, use (prevention,health, health,orortherapeutic therapeutic treatment). treatment). Generally, Generally, whenwhen manufacturing manufacturing foods orfoods or

beverages, the beverages, the food food compositions compositionsofofthe thepresent presentinvention inventioncan canbebeadded addedinin amounts amounts of of 50 50 parts parts by by

weight or weight or less, less, specifically specifically 20 20 parts parts by by weight or less weight or less relative relativetotothe thefood foodor orbeverage. beverage. However, However,

whenconsumed when consumedforfor long long periods periods forfor healthand health andhygiene hygiene purposes, purposes, they they may may contain contain amounts amounts below below

the above range, and since there are no safety issues, active ingredients can also be used in amounts the above range, and since there are no safety issues, active ingredients can also be used in amounts

above the above above the aboverange. range.

[0072]

[0072] AsAs one one example example of the of the food food compositions compositions of the of the present present invention, invention, they they cancan be used be used

as as health beveragecompositions, health beverage compositions,andand in in thiscase, this case,they theymaymay contain contain various various flavoring flavoring agents agents or or

natural carbohydrates natural as additional carbohydrates as additional components components likeconventional like conventional beverages. beverages. TheThe aforementioned aforementioned

natural carbohydrates natural maybebemonosaccharides carbohydrates may monosaccharides such such as glucose as glucose and and fructose; fructose; disaccharides disaccharides such such as as

maltose and maltose andsucrose; sucrose;polysaccharides polysaccharides such such as dextrin as dextrin and and cyclodextrin; cyclodextrin; sugarsugar alcohols alcohols such such as as

xylitol, sorbitol, erythritol, etc. Sweeteners may use natural sweeteners such as thaumatin and stevia xylitol, sorbitol, erythritol, etc. Sweeteners may use natural sweeteners such as thaumatin and stevia

extracts; synthetic sweeteners extracts; synthetic sweetenerssuch such as saccharin as saccharin and aspartame, and aspartame, etc. Theetc. The ratio ratio of natural of natural

19 carbohydrates cangenerally carbohydrates can generallybebeabout about0.01 0.01toto0.04 0.04g,g,specifically specifically about about 0.02 0.02 to to 0.03 0.03 gg per per 100 100 mL mL of the health of the healthbeverage beverage composition composition of theof the present present invention. invention.

[0073]

[0073] InInaddition additiontoto the the above, above, health health beverage compositionsmay beverage compositions maycontain containvarious variousnutrients, nutrients,

vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, salts of pectic acid, alginic acid, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, salts of pectic acid, alginic acid,

salts salts of alginic acid, of alginic acid, organic organicacids, acids,protective protectivecolloidal colloidalthickeners, thickeners,pHpH adjusters, adjusters, stabilizers, stabilizers,

preservatives, glycerin, alcohol, or carbonating agents, etc. Additionally, they may contain pulp for preservatives, glycerin, alcohol, or carbonating agents, etc. Additionally, they may contain pulp for

manufacturing natural fruit juices, fruit juice beverages, or vegetable beverages. These components manufacturing natural fruit juices, fruit juice beverages, or vegetable beverages. These components

can beused can be usedindependently independently or inor in combination. combination. The The ratio of ratio of such additives such additives is not critically is not critically important, important,

but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the

health beverage health compositionofofthe beverage composition thepresent presentinvention. invention.

[0074] Thefood

[0074] The foodcompositions compositionsof ofthethe presentinvention present inventioncancan containvarious contain variousweight weight

percentages as percentages as long long as as they they can can exhibit exhibit skin skin aging aging inhibition inhibition or or wound treatmenteffects wound treatment effects including including

peptides derived peptides derived from fromCPNE7 CPNE7 protein, protein, but specifically but specifically may may contain contain the peptides the peptides of theofpresent the present

invention in 0.00001 invention in to 100% 0.00001 to 100% bybyweight weightoror0.01 0.01toto80% 80%by by weight weight relativetotothe relative thetotal total weight of the weight of the

food composition, food composition, but but are are not limited not limited thereto. thereto.

[Advantageous Effects]

[Advantageous Effects]

[0075] The

[0075] The compositions compositions for for inhibiting inhibiting skin skin aging aging or treating or treating wounds wounds containing containing peptides peptides

derived derived from from CPNE7 proteinaccording CPNE7 protein according to to embodiments embodimentsofofthe the present present invention invention can can promote promote

synthesis of Type synthesis of TypeI ICollagen Collagenandand Type Type III Collagen III Collagen involved involved in promoting in promoting skin elasticity skin elasticity and and

preventing aging in fibroblasts within the dermal layer of the skin. preventing aging in fibroblasts within the dermal layer of the skin.

[0076] The

[0076] The compositions compositions for for inhibiting inhibiting skin skin aging aging or treating or treating wounds wounds containing containing peptides peptides

derived from derived fromCPNE7 CPNE7 protein protein according according to other to other embodiments embodiments of present of the the present invention invention can can promote promote

20 synthesis synthesis of of Type IVCollagen Type IV Collagen involved involved in in promoting promoting skinskin elasticity elasticity andand preventing preventing aging aging in the in the basementmembrane basement membrane of the of the dermo-epidermal dermo-epidermal junction junction of skin. of the the skin.

[0077] The

[0077] The compositions compositions for for inhibiting inhibiting skin skin aging aging or treating or treating wounds wounds containing containing peptides peptides

derived from derived fromCPNE7 CPNE7 protein protein according according to yet to yet other other embodiments embodiments of theofpresent the present invention invention aim toaim to

provide peptides provide peptides and and their their uses uses that that promote regeneration of promote regeneration of the the epidermal layer and epidermal layer and prevent prevent aging aging

in in keratinocytes within keratinocytes within thethe epidermal epidermal layer layer of theof the skin. skin.

[0078] The

[0078] The compositions compositions for for inhibiting inhibiting skin skin aging aging or treating or treating wounds wounds containing containing peptides peptides

derived fromCPNE7 derived from CPNE7 protein protein according according toother to yet yet other embodiments embodiments of the present of the present invention invention can can

promotesynthesis promote synthesisofofFilaggrin Filaggrin andand Involucrin Involucrin involved involved in promoting in promoting skin moisturization skin moisturization and and

preventing aging in keratinocytes within the epidermal layer of the skin. preventing aging in keratinocytes within the epidermal layer of the skin.

[0079] The

[0079] The compositions compositions for for inhibiting inhibiting skin skin aging aging or treating or treating wounds wounds containing containing peptides peptides

derived derived from CPNE7 from CPNE7 protein protein according according to to yetother yet otherembodiments embodimentsof of thethe presentinvention present inventioncan canreduce reduce

expression of MMP-1 expression of MMP-1 involved involved in preventing in preventing skinskin aging aging by inhibiting by inhibiting collagen collagen degradation degradation in the in the

epidermaland epidermal anddermal dermallayers layersofofthe the skin. skin.

[0080] The

[0080] The effectsofofthe effects thepresent presentinvention inventionarearenot notlimited limitedtotothose thosementioned mentioned above, above, and and

other effectsnot other effects notmentioned mentionedwill will be clearly be clearly understood understood by those by those with withskill ordinary ordinary in theskill in the technical technical

field field to to which thepresent which the present invention invention belongs belongs from from the the description description below. below.

[Description of Drawings]

[Description of Drawings]

[0081] Figure1 shows

[0081] Figure 1 shows the the results results of measuring of measuring the peptides the peptides of theofpresent the present invention invention at at

various various concentrations after 48 concentrations after 48 hours hours of of treatment, treatment,followed followed by by dispensing dispensing WST-8 solutioninto WST-8 solution intothe the

culture medium culture medium at aat10:1 a 10:1 ratio, ratio, light-shielding, light-shielding, reacting reacting for 2for 2 hours hours under under 37°C incubator 37°C incubator conditions, conditions,

and then and then measuring measuringatat450nm 450nm absorbance absorbance with with a microplate. a microplate.

21

[0082] Figure2 (A)

[0082] Figure 2 (A) shows shows the confirmation the confirmation of IType of Type I Collagen Collagen protein protein formation formation after after

treating the peptide (SEQ ID NO: 96) of the present invention at various concentrations for 7 days, treating the peptide (SEQ ID NO: 96) of the present invention at various concentrations for 7 days,

and Figure22(B) and Figure (B)shows showsthethe resultsofofconfirming results confirming thethe EC50 EC50 valuevalue according according to IType to Type I Collagen Collagen

protein formation protein after treating formation after treatingthe thepeptide peptide(SEQ (SEQ ID NO:96) ID NO: 96)ofofthe thepresent presentinvention inventionatat 100 100uM uM7 7

times over times over 77 days. days.

[0083] Figure3a3aisisaagraph

[0083] Figure graphcomparing comparingthethe Type Type I Collagen I Collagen protein protein expression expression levels levels forfor the the

peptides in Table 13. peptides in Table 13.

[0084] Figure3b3b

[0084] Figure shows shows a comparison a comparison of collagen of collagen deposition deposition levels levels with with the control the control groupgroup

after treating after treatingthe peptide the (SEQ peptide (SEQ ID ID NO: 96) of NO: 96) of the the present present invention invention at at aaconcentration concentration of of100 100 uM at uM at

24-hourintervals 24-hour intervals for for 77 days, days,where where (A) (A) shows the Confocal shows the ConfocalMicroscope Microscope (Type (Type I Collagen) I Collagen) analysis analysis

results and results and (B) (B) shows shows the the analysis analysis results resultsusing thethe using Confocal ConfocalMicroscope Microscope Z-Stack Imagingprogram. Z-Stack Imaging program.

[0085] Figure3c3c

[0085] Figure shows shows thethe confirmation confirmation of alpha-smooth of alpha-smooth muscle muscle actin actin (α-SMA) (-SMA) synthesis synthesis

efficacy ofthe efficacy of thepeptides peptidesof of thethe present present invention invention in (N=3). in hDFs hDFs (N=3).

[0086] Figure4 4shows

[0086] Figure shows thethe resultsofofconfirming results confirming human-derived human-derived dermal dermal fibroblast fibroblast collagen collagen

formation abilityupon formation ability upon multiple multiple treatments treatments of the of the peptides peptides of the invention. of the present present invention.

[0087] Figure

[0087] Figure 5 shows 5 shows the results the results of confirming of confirming the penetration the penetration of theofpeptides of the peptides of the present the present

invention into human-derived invention into three-dimensional human-derived three-dimensional skintissue. skin tissue.

[0088] Figure6a6ashows

[0088] Figure shows thethe resultsofofconfirming results confirmingthethe efficacyofofthe efficacy thepeptides peptidesofofthe the present present

invention in human-derived invention in three-dimensional human-derived three-dimensional skintissue. skin tissue.

[0089] Figure6b6b

[0089] Figure shows shows thethe confirmation confirmation of of peptide peptide collagen collagen formation formation ability ability in in Neoderm- Neoderm-

ED skintissue ED skin tissue (Ex (Ex vivo). vivo).

[0090] Figure7 shows

[0090] Figure 7 shows the the results results of of confirming confirming the the keratinocyte keratinocyte proliferation proliferation efficacy efficacy of of

the peptides of the present invention. the peptides of the present invention.

22

[0091] Figure8 shows

[0091] Figure 8 shows the the results results of of confirming confirming the the efficacy efficacy according according to skin to skin treatment treatment

with the peptides of the present invention. with the peptides of the present invention.

[0092] Figure9 shows

[0092] Figure 9 shows the the results results of confirming of confirming (A) reactive (A) reactive oxygenoxygen speciesspecies reduction reduction

effects effects and and (B) anti-photodamageeffects (B) anti-photodamage effectsaccording according to to treatment treatment with with thethe peptides peptides of of thethe present present

invention. invention.

[0093] Figure1010

[0093] Figure shows shows the the results results of of confirming confirming the the melanin melanin secretion secretion reduction reduction efficacy efficacy

according to concentration-dependent according to concentration-dependenttreatment treatmentwith withthe thepeptides peptidesofofthe the present present invention. invention.

[0094] Figure1111

[0094] Figure shows shows the the results results of of confirming confirming thethe migration migration efficacy efficacy of of thethe peptides peptides of of

the present the present invention invention in in keratinocytes keratinocytes and and hDFs. hDFs.

[Modes

[Modes ofofthe theInvention] Invention]

[0095] The

[0095] The purposes purposes andand effects effects of of thethe present present invention, invention, andand thethe technical technical configurations configurations

for for achieving them, will achieving them, will become becomeclear clearbybyreferring referringtotothe theembodiments embodiments described described in detail in detail below below

along with along with the the accompanying accompanying drawings. drawings. In In describing describing thethe present present invention, invention, when when it isdetermined it is determined

that detailed that detailed descriptions descriptionsof ofknown functions or known functions or configurations configurations may unnecessarilyobscure may unnecessarily obscurethe thegist gist

of the present of the presentinvention, invention, such such detailed detailed descriptions descriptions will will be be omitted. omitted. The The terms terms below described described are below are

terms defined terms definedconsidering consideringthe thefunctions functionsininthe thepresent presentinvention, invention,and andmaymay vary vary according according to to the the

intentions orpractices intentions or practicesofofusers users andand operators. operators.

[0096] However,

[0096] However, the the present present invention invention is not is not limited limited to to thethe embodiments embodiments disclosed disclosed belowbelow

but may but beimplemented may be implementedin in various various differentforms. different forms.The Thepresent presentembodiments embodiments are are provided provided onlyonly to to

makethe make thedisclosure disclosure of of the the present present invention invention complete and to complete and to completely informthose completely inform thosewith withordinary ordinary

skill skill in in the the technical fieldtotowhich technical field whichthethe present present invention invention belongs belongs of the of theofscope scope of the invention. the invention. The The

present invention present invention is is defined only by defined only bythe thescope scopeofofthe theclaims. claims.Therefore, Therefore,the thedefinition definitionshould shouldbebe

madebased made basedononthe thecontent contentthroughout throughoutthis thisspecification. specification. 23

[0097] The

[0097] The embodiments embodiments of present of the the present invention invention willwill be described be described in detail in detail below. below.

[0098] Example

[0098] Example 1. Synthesis 1. Synthesis of of Peptides Peptides Derived Derived fromfrom CPNE7 CPNE7 Protein Protein

[0099] The

[0099] The present present inventors inventors synthesized synthesized peptides peptides derived derived fromfrom CPNE7CPNE7 proteinprotein (SEQ ID(SEQ ID

NO:1)1)bybythethe9-fluorenylmethyloxycarbonyl NO: 9-fluorenylmethyloxycarbonyl (Fmoc) (Fmoc) method method and synthesized and synthesized peptides peptides of each of each

group group byby substituting substituting amino amino acidsacids of theofsynthesized the synthesized peptidespeptides (Tables 1(Tables to 12). 1 to 12).

[00100]

[00100]

[00101]

[00101] N-KYQRRKKNKY-C N-KYQRRKKNKY-C (SEQ (SEQ IDIDNO: NO:1) 1)

[00102]

[00102]

[00103]

[00103] First, Group First, 1 peptides Group 1 peptides were weresynthesized synthesizedbybysubstituting substitutingamino aminoacids acids5 5toto7 7

of of the the peptide peptide of ofSEQ IDNO: SEQ ID NO:1 1ororthe thepeptide peptideofof SEQ SEQIDID NO: NO: 1 with 1 with lysine lysine or or arginine arginine (Table (Table 1).1).

[00104]

[00104]

[00105]

[00105] [Table 1]

[Table 1]

[00106]

[00106] Group11Peptides Group Peptides

SEQ ID NO SEQ ID NO AminoAcid Amino AcidSequence Sequence(N-C) (N-C)

11 KYQRRKKNKY KYQRRKKNKY

2 2 KYQRRKRNKY KYQRRKRNKY

33 KYQRRRKNKY KYQRRRKNKY

4 4 KYQRRRRNKY KYQRRRRNKY

55 KYQRKKKNKY KYQRKKKNKY

66 KYQRKRKNKY KYQRKRKNKY

77 KYQRKKRNKY KYQRKKRNKY

24

88 KYQRKRRNKY KYQRKRRNKY

[00107]

[00107] Next, Group Next, Group2 2peptides peptideswere weresynthesized synthesized by by substituting substituting amino amino acids acids 5 to 5 to 7 7

of of the the peptide peptide of of SEQ IDNO: SEQ ID NO: 1 with 1 with lysine lysine or or arginine arginine and and substitutingamino substituting amino acid acid 8 with 8 with serine serine

(Table 2). (Table 2).

[00108]

[00108] [Table 2]

[Table 2]

[00109]

[00109] Group Group 22Peptides Peptides

SEQ ID NO SEQ ID NO AminoAcid Amino AcidSequence Sequence(N-C) (N-C)

9 9 KYQRRKKSKY KYQRRKKSKY

10 10 KYQRRKRSKY KYQRRKRSKY

11 11 KYQRRRKSKY KYQRRRKSKY

12 12 KYQRRRRSKY KYQRRRRSKY

13 13 KYQRKKKSKY KYQRKKKSKY

14 14 KYQRKRKSKY KYQRKRKSKY

15 15 KYQRKKRSKY KYQRKKRSKY

16 16 KYQRKRRSKY KYQRKRRSKY

[00110]

[00110] Next, Group Next, Group3 3peptides peptideswere weresynthesized synthesized by by substitutingamino substituting amino acids acids 5 to 5 to 7 7

of of the the peptide peptide of of SEQ IDNO: SEQ ID NO:1 1with withlysine lysineororarginine arginineand andsubstituting substituting amino aminoacid acid99with withtyrosine tyrosine

(Table 3). (Table 3).

25

[00111]

[00111] [Table 3]

[Table 3]

[00112]

[00112] Group Group 33Peptides Peptides

SEQ ID NO SEQ ID NO AminoAcid Amino AcidSequence Sequence(N-C) (N-C)

17 17 KYQRRKKNYK KYQRRKKNYK

18 18 KYQRRKRNYK KYQRRKRNYK

19 19 KYQRRRKNYK KYQRRRKNYK

20 20 KYQRRRRNYK KYQRRRRNYK

21 21 KYQRKKKNYK KYQRKKKNYK

22 22 KYQRKRKNYK KYQRKRKNYK

23 23 KYQRKKRNYK KYQRKKRNYK

24 24 KYQRKRRNYK KYQRKRRNYK

[00113]

[00113] Next, Group Next, Group4 4peptides peptideswere were synthesized synthesized by by substituting substituting amino amino acids acids 5 to 5 to 7 7

of of the the peptide of SEQ peptide of SEQIDID NO:NO: 1 with 1 with lysine lysine or arginine, or arginine, substituting substituting amino amino acid acid 8 serine, 8 with with serine,

substituting amino substituting amino acid acid 9 with 9 with tyrosine, tyrosine, and substituting and substituting amino amino acid acidlysine 10 with 10 with lysine (Table 4). (Table 4).

[00114]

[00114] [Table 4]

[Table 4]

[00115]

[00115] Group Group 44Peptides Peptides

SEQ ID NO SEQ ID NO AminoAcid Amino AcidSequence Sequence(N-C) (N-C)

25 25 KYQRRKKSYK KYQRRKKSYK

26

26 KYQRRKRSYK KYQRRKRSYK

27 27 KYQRRRKSYK KYQRRRKSYK

28 28 KYQRRRRSYK KYQRRRRSYK

29 29 KYQRKKKSYK KYQRKKKSYK

30 30 KYQRKRKSYK KYQRKRKSYK

31 31 KYQRKKRSYK KYQRKKRSYK

32 32 KYQRKRRSYK KYQRKRRSYK

[00116]

[00116] Next, Group Next, Group5 5peptides peptideswere weresynthesized synthesizedbyby substitutingamino substituting amino acid acid 3 3 ofof the the

peptide of peptide of SEQ IDNO: SEQ ID NO: 1 with 1 with arginine,substituting arginine, substitutingamino amino acid4 4with acid withglutamine, glutamine, and and substituting substituting

amino acids amino acids 5 to 5 to 7 with 7 with lysine lysine or arginine or arginine (Table (Table 5). 5).

[00117]

[00117] [Table 5]

[Table 5]

[00118]

[00118] Group Group 55Peptides Peptides

SEQ ID NO SEQ ID NO AminoAcid Amino AcidSequence Sequence(N-C) (N-C)

33 33 KYRQRKKNKY KYRQRKKNKY

34 34 KYRQRKRNKY KYRQRKRNKY

35 35 KYRQRRKNKY KYRQRRKNKY

36 36 KYRQRRRNKY KYRQRRRNKY

37 37 KYRQKKKNKY KYRQKKKNKY

27

38 KYRQKRKNKY KYRQKRKNKY

39 39 KYRQKKRNKY KYRQKKRNKY

40 40 KYRQKRRNKY KYRQKRRNKY

[00119]

[00119] Next, Group Next, Group6 6peptides peptideswere weresynthesized synthesizedbyby substitutingamino substituting amino acid acid 3 3 ofof the the

peptide of peptide of SEQ IDNO: SEQ ID NO:1 1with witharginine, arginine,substituting substituting amino acid 44 with amino acid with glutamine, glutamine, substituting substitutingamino amino

acids acids 55to to 77with withlysine lysineor or arginine, arginine, andand substituting substituting aminoamino acid 8 acid 8 with(Table with serine serine6).(Table 6).

[00120]

[00120] [Table 6]

[Table 6]

[00121]

[00121] Group Group 66Peptides Peptides

SEQ ID NO SEQ ID NO Amino AcidSequence Amino Acid Sequence(N-C) (N-C)

41 41 KYRQRKKSKY KYRQRKKSKY

42 42 KYRQRKRSKY KYRQRKRSKY

43 43 KYRQRRKSKY KYRQRRKSKY

44 44 KYRQRRRSKY KYRQRRRSKY

45 45 KYRQKKKSKY KYRQKKKSKY

46 46 KYRQKRKSKY KYRQKRKSKY

47 47 KYRQKKRSKY KYRQKKRSKY

48 48 KYRQKRRSKY KYRQKRRSKY

28

[00122]

[00122] Next, Group Next, Group7 7peptides peptideswere weresynthesized synthesizedbyby substitutingamino substituting amino acid acid 3 3 ofof the the

peptide of peptide of SEQ IDNO: SEQ ID NO:1 1with witharginine, arginine,substituting substituting amino acid 44 with amino acid with glutamine, glutamine, substituting substitutingamino amino

acids acids 55to to 77with withlysine lysineor or arginine, arginine, substituting substituting amino amino acid 9acid with 9 with tyrosine, tyrosine, and substituting and substituting amino amino

acid 10with acid 10 withlysine lysine (Table (Table 7). 7).

[00123]

[00123] [Table 7]

[Table 7]

[00124]

[00124] Group Group 77Peptides Peptides

SEQ ID NO SEQ ID NO Amino AcidSequence Amino Acid Sequence(N-C) (N-C)

49 49 KYRQRKKNYK KYRQRKKNYK

50 50 KYRQRKRNYK KYRQRKRNYK

51 51 KYRQRRKNYK KYRQRRKNYK

52 52 KYRQRRRNYK KYRQRRRNYK

53 53 KYRQKKKNYK KYRQKKKNYK

54 54 KYRQKRKNYK KYRQKRKNYK

55 55 KYRQKKRNYK KYRQKKRNYK

56 56 KYRQKRRNYK KYRQKRRNYK

[00125]

[00125] Next, Group Next, Group8 8peptides peptideswere weresynthesized synthesizedbyby substitutingamino substituting amino acid acid 3 3 ofof the the

peptide of peptide of SEQ IDNO: SEQ ID NO:1 1with witharginine, arginine,substituting substituting amino acid 44 with amino acid with glutamine, glutamine, substituting substitutingamino amino

acids acids 55to to77with withlysine lysineor or arginine, arginine, substituting substituting amino amino acid 8acid with 8serine, with serine, substituting substituting amino amino acid 9 acid 9

with tyrosine, and substituting amino acid 10 with lysine (Table 8). with tyrosine, and substituting amino acid 10 with lysine (Table 8).

[00126]

[00126] [Table 8]

[Table 8]

29

[00127]

[00127] Group Group 88Peptides Peptides

SEQ ID NO SEQ ID NO AminoAcid Amino AcidSequence Sequence(N-C) (N-C)

57 57 KYRQRKKSYK KYRQRKKSYK

58 58 KYRQRKRSYK KYRQRKRSYK

59 59 KYRQRRKSYK KYRQRRKSYK

60 60 KYRQRRRSYK KYRQRRRSYK

61 61 KYRQKKKSYK KYRQKKKSYK

62 62 KYRQKRKSYK KYRQKRKSYK

63 63 KYRQKKRSYK KYRQKKRSYK

64 64 KYRQKRRSYK KYRQKRRSYK

[00128]

[00128] Next, Group Next, Group9 9peptides peptideswere weresynthesized synthesizedbyby substitutingamino substituting amino acid acid 3 3 ofof the the

peptide of peptide of SEQ SEQIDIDNO:NO: 1 with 1 with lysine, lysine, substituting substituting amino amino acidacid 4 with 4 with glutamine, glutamine, and and substituting substituting

amino acids amino acids 5 to 5 to 7 with 7 with lysine lysine or arginine or arginine (Table (Table 9). 9).

[00129]

[00129] [Table 9]

[Table 9]

[00130]

[00130] Group Group 99Peptides Peptides

SEQ ID NO SEQ ID NO AminoAcid Amino AcidSequence Sequence(N-C) (N-C)

65 65 KYKQRKKNKY KYKQRKKNKY

66 66 KYKQRKRNKY KYKQRKRNKY

30

67 KYKQRRKNKY KYKQRRKNKY

68 68 KYKQRRRNKY KYKQRRRNKY

69 69 KYKQKKKNKY KYKQKKKNKY

70 70 KYKQKRKNKY KYKQKRKNKY

71 71 KYKQKKRNKY KYKQKKRNKY

72 72 KYKQKRRNKY KYKQKRRNKY

[00131]

[00131] Next, Group Next, Group1010peptides peptideswere weresynthesized synthesizedbybysubstituting substitutingamino aminoacid acid3 3ofofthe the

peptide of peptide of SEQ IDNO: SEQ ID NO: 1 with 1 with lysine,substituting lysine, substitutingamino aminoacid acid4 4with withglutamine, glutamine,substituting substitutingamino amino

acids acids 55to to 77with withlysine lysineor or arginine, arginine, andand substituting substituting aminoamino acid 8 acid 8 with(Table with serine serine10). (Table 10).

[00132]

[00132] [Table 10]

[Table 10]

[00133]

[00133] Group 10Peptides Group 10 Peptides

SEQ ID NO SEQ ID NO Amino AcidSequence Amino Acid Sequence(N-C) (N-C)

73 73 KYKQRKKSKY KYKQRKKSKY

74 74 KYKQRKRSKY KYKQRKRSKY

75 75 KYKQRRKSKY KYKQRRKSKY

76 76 KYKQRRRSKY KYKQRRRSKY

77 77 KYKQKKKSKY KYKQKKKSKY

78 78 KYKQKRKSKY KYKQKRKSKY

31

79 KYKQKKRSKY KYKQKKRSKY

80 80 KYKQKRRSKY KYKQKRRSKY

[00134]

[00134] Next, Group Next, Group1111peptides peptideswere weresynthesized synthesizedbybysubstituting substitutingamino aminoacid acid3 3ofofthe the

peptide of peptide of SEQ IDNO: SEQ ID NO: 1 with 1 with lysine,substituting lysine, substitutingamino aminoacid acid4 4with withglutamine, glutamine,substituting substitutingamino amino

acids acids 55to to77with withlysine lysine or or arginine, arginine, substituting substituting amino amino acid 9acid with 9 with tyrosine, tyrosine, and substituting and substituting amino amino

acid 10with acid 10 withlysine lysine (Table (Table 11).11).

[00135]

[00135] [Table 11]

[Table 11]

[00136]

[00136] Group 11Peptides Group 11 Peptides

SEQ ID NO SEQ ID NO AminoAcid Amino AcidSequence Sequence(N-C) (N-C)

81 81 KYKQRKKNYK KYKQRKKNYK

82 82 KYKQRKRNYK KYKQRKRNYK

83 83 KYKQRRKNYK KYKQRRKNYK

84 84 KYKQRRRNYK KYKQRRRNYK

85 85 KYKQKKKNYK KYKQKKKNYK

86 86 KYKQKRKNYK KYKQKRKNYK

87 87 KYKQKKRNYK KYKQKKRNYK

88 88 KYKQKRRNYK KYKQKRRNYK

32

[00137]

[00137] Finally, Group Finally, 12 peptides Group 12 peptides were weresynthesized synthesizedbybysubstituting substitutingamino aminoacid acid3 3ofof

the the peptide of SEQ peptide of SEQ IDID NO: NO: 1 with 1 with lysine, lysine, substituting substituting amino amino acidacid 4 with 4 with glutamine, glutamine, substituting substituting

amino acids amino acids 5 to 5 to 7 with 7 with lysine lysine or arginine, or arginine, substituting substituting amino amino acidserine, acid 8 with 8 withsubstituting serine, substituting amino amino

acid 9 with tyrosine, and substituting amino acid 10 with lysine (Table 12). acid 9 with tyrosine, and substituting amino acid 10 with lysine (Table 12).

[00138]

[00138] [Table 12]

[Table 12]

[00139]

[00139] Group1212Peptides Group Peptides

SEQ ID NO SEQ ID NO AminoAcid Amino AcidSequence Sequence(N-C) (N-C)

89 89 KYKQRKKSYK KYKQRKKSYK

90 90 KYKQRKRSYK KYKQRKRSYK

91 91 KYKQRRKSYK KYKQRRKSYK

92 92 KYKQRRRSYK KYKQRRRSYK

93 93 KYKQKKKSYK KYKQKKKSYK

94 94 KYKQKRKSYK KYKQKRKSYK

95 95 KYKQKKRSYK KYKQKKRSYK

96 96 KYKQKRRSYK KYKQKRRSYK

[00140]

[00140]

[00141]

[00141] Example Example 2.2.WST-8 WST-8 Assay Assay (Cellrix, (Cellrix, B1007-500) B1007-500)

[00142]

[00142] Human Human dermal dermal fibroblasts(hDFs) fibroblasts (hDFs) were were dispensed dispensed at 0.3×10⁵/well at 0.3x10/well in 96-well in 96-well

plates (using plates (using Fibroblast FibroblastGrowth Growth Medium (Lonza, Medium (Lonza, CC-4126) CC-4126) containing containing 2% FBS, 2% FBS, 0.1% Insulin, 0.1% Insulin, 0.1% 0.1%

FGF,0.1% FGF, 0.1%Gentamycin Gentamycin sulfate-Amphotericin) sulfate-Amphotericin) and and cultured cultured for for 24 24 hours hours in in an an incubator incubator at at 37°C,5%5% 37°C,

CO₂ conditions. CO2 conditions.

33

[00143]

[00143] WST-8 WST-8 analysiswaswas analysis performed performed to confirm to confirm cytotoxicity cytotoxicity of the of the peptide peptide (SEQ (SEQ

ID NO: ID NO:96) 96)inindermal dermalfibroblasts. fibroblasts.The Thepeptide peptidewas was treatedonondermal treated dermal fibroblastsatatconcentrations fibroblasts concentrations

of of 10, 10, 100 100 μM, 1, 5, µM, 1, 5, 10 10 mM respectively.After mM respectively. Aftertreating treating for for 48 48 hours at each hours at each concentration, concentration, WST-8 WST-8

solution was solution was dispensed dispensed into into the culture the culture mediummedium at a 10:1 at a 10:1 ratio, ratio, light-shielded, light-shielded, reacted for reacted 2 hours for 2 hours

under 37°C under 37°Cincubator incubatorconditions, conditions,and andthen thenmeasured measuredat at 450nm 450nm absorbance absorbance with with a microplate. a microplate.

[00144]

[00144] Figure 11 shows Figure showsthe theresults results of of measuring the peptide measuring the peptide (SEQ (SEQIDIDNO:NO: 96)96) of of thethe

present invention present invention at at various various concentrations concentrationsafter after 4848hours hoursofoftreatment, treatment,followed followed by by dispensing dispensing

WST-8 WST-8 solutioninto solution intothe theculture culturemedium mediumat at a 10:1 a 10:1 ratio,light-shielding, ratio, light-shielding, reacting reacting for for 22 hours hours under under

37°Cincubator 37°C incubatorconditions, conditions, and andthen thenmeasuring measuringatat450nm 450nm absorbance absorbance withwith a microplate. a microplate.

[00145]

[00145] The peptide The peptide (SEQ (SEQIDIDNO:NO: 96)96) of of thethe presentinvention present inventionwas was treatedonondermal treated dermal

fibroblasts at concentrations fibroblasts at concentrationsof of 10,10, 100 100 μM, µM, 1, 5, 1, 10 5, mM 10 mM respectively. respectively. After cell After 48 hours, 48 hours, cell viability viability

was quantified was quantified through throughWST-8 WST-8 analysis. analysis. As As a result, a result, thethe peptide peptide (SEQ (SEQ ID NO: ID NO: 96) 96) of ofpresent the the present

invention showednonocytotoxicity invention showed cytotoxicitytotodermal dermalfibroblasts fibroblastsup uptoto aa concentration concentration of of 55 mM mM and and showed showed

significant cytotoxicityat ata aconcentration significant cytotoxicity concentration ofmM. of 10 10 mM.

[00146]

[00146]

[00147]

[00147] Example3-1. Example 3-1.Confirmation Confirmation of of EC50 EC50 ValueValue for Type for Type I Collagen I Collagen Formation Formation

FollowingMultiple Following MultipleTreatments Treatmentsofof PeptidesininhDFs Peptides hDFs

[00148]

[00148] To confirm To confirmthe theType Type I collagen I collagen synthesisability synthesis abilityfollowing followingmultiple multiple

treatments of treatments of the the peptide peptide(SEQ (SEQ ID NO: ID NO: 96) of96) theofpresent the present invention, invention, dermal dermal fibroblasts fibroblasts were were

dispensed at 10/6 dispensed at 1×10⁵/6 wellwell plate plate in 60mm in 60mm dishes dishes and cultured and cultured forhours for 24 24 hours in anin an incubator incubator at 37°C, at 37°C,

5% CO₂.The 5% CO2. The peptide peptide (SEQ (SEQ ID NO: ID NO: 96)treated 96) was was treated at concentrations at concentrations of 1 of pM,1 10 pM, pM,10 pM, 100 pM,100 1 pM, 1

nM,1010nM, nM, nM, 1000 1000 nM,nM, 1 μM, 1 µM, 10100 10 µM, μM, 100 µM, μM, 1 mM 1 mM (total 11 (total groups)11atgroups) 24-houratintervals 24-hour intervals for 7 for 7

days. days.

34

[00149]

[00149] Subsequently, after washing Subsequently, after washingcells cellswith withPBS, PBS, proteins proteins werewere separated separated and and

changesin changes in Type TypeI ICollagen Collagenprotein proteinlevels levelswere wereconfirmed confirmed through through Western Western blot, blot, andand comparisons comparisons

were made were madethrough through normalization normalization with with β-Actin ß-Actin protein protein levels. levels.

[00150]

[00150] Figure 22 (A) Figure (A) shows showsthetheconfirmation confirmation of of Type Type I Collagen I Collagen protein protein formation formation

after treating after treatingthe thepeptide peptide(SEQ (SEQ ID ID NO: 96)ofofthe NO: 96) the present present invention invention at at various various concentrations for 77 concentrations for

days, days, and and Figure Figure 2 2 (B) (B) shows the results shows the results ofofconfirming confirming the theEC50 value according EC50 value accordingto to Type TypeII Collagen Collagen

protein formation protein after treating formation after treatingthe thepeptide peptide(SEQ (SEQ ID NO:96) ID NO: 96)ofofthe thepresent present invention inventionat at 100 100µM μM7 7

times over 7 days. Referring to Figure 2, the EC50 value for multiple treatments of the peptide (SEQ times over 7 days. Referring to Figure 2, the EC50 value for multiple treatments of the peptide (SEQ

ID NO: ID NO:96) 96)ofofthe thepresent presentinvention inventionwas wasconfirmed confirmed to to be be 8.621 8.621 pM,pM, and and it can it can be be seen seen that that Type Type I I

Collagenexpression Collagen expressionsignificantly significantly increased increased compared comparedtotothe thecontrol controlgroup. group.

[00151]

[00151]

[00152]

[00152] Example3-2. Example 3-2.Confirmation Confirmationof of Type Type I Collagen I Collagen Synthesis Synthesis Ability Ability of of Group Group 1 1

to 12 to 12 Peptides Peptides in in Human-Derived Dermal Human-Derived Dermal Fibroblasts Fibroblasts

[00153]

[00153] To confirm To confirmthe theType Type I collagensynthesis I collagen synthesisability abilityofofpeptides peptidesinineach eachgroup, group,

dermal fibroblasts were dermal fibroblasts dispensedatat 1x10/6 were dispensed 1×10⁵/6 well well plateinin60mm plate 60mm dishes dishes and and cultured cultured for for 24 hours 24 hours

in in an an incubator at 37°C, incubator at 5%CO2. 37°C, 5% CO₂.Each Each peptide peptide waswas treated treated at 100 at 100 μM cultured µM and and cultured forhours. for 24 24 hours.

After 24 After 24 hours, hours,after after washing washingcells cellswith with PBS, PBS, proteins proteins werewere separated separated and changes and changes in Typein I Type I

Collagen protein levels Collagen protein levels were confirmedthrough were confirmed throughWestern Western blot,and blot, andcomparisons comparisons were were mademade through through

normalization with β-Actin protein levels. normalization with ß-Actin protein levels.

[00154]

[00154] Table 13 Table 13 shows showsthe theresults results of of confirming changesininType confirming changes TypeI ICollagen Collagenprotein protein

levels after single levels after treatment ofof peptides single treatment peptidesfrom from Groups Groups 1 to 112to of 12 theof the present present invention invention at a at a

concentration of 100 concentration of 100 µM μMonon dermal dermal fibroblastsandand fibroblasts 24 24 hours hours later.Figure later. Figure3a3aisisa agraph graphcomparing comparing

TypeII Collagen Type Collagenprotein protein expression expressionlevels levels for for the thepeptides peptidesininTable Table13. 13.As Ascan canbe beconfirmed confirmed through through

35 this, it can be seen that the peptides of the present invention show excellent Type I collagen synthesis this, it can be seen that the peptides of the present invention show excellent Type I collagen synthesis ability. ability.

SEQ ID NO SEQ ID AminoAcid NO Amino AcidSequence Sequence Relative Protein Relative Protein Expression levels of Expression levels of Type Type II Collagen Collagen

Control Control -- 11

11 KYQRRKKNKY 1.33516 1.33516 KYQRRKKNKY

88 KYQRKRRNKY 2.31151 2.31151 KYQRKRRNKY

9 9 KYQRRKKSKY KYQRRKKSKY 1.5182 1.5182

16 16 KYQRKRRSKY KYQRKRRSKY 2.83006 2.83006

17 17 KYQRRKKNYK 1.99857 1.99857 KYQRRKKNYK

24 24 KYQRKRRNYK 2.8985 2.8985 KYQRKRRNYK

25 25 KYQRRKKSYK KYQRRKKSYK 1.88521 1.88521

32 32 KYQRKRRSYK KYQRKRRSYK 3.29178 3.29178

33 33 KYRQRKKNKY 2.03416 2.03416 KYRQRKKNKY

40 40 KYRQKRRNKY 3.50524 3.50524 KYRQKRRNKY

41 41 KYRQRKKSKY KYRQRKKSKY 1.6575 1.6575

48 48 KYRQKRRSKY KYRQKRRSKY 3.15834 3.15834

49 49 KYRQRKKNYK KYRQRKKNYK 1.27334 1.27334

56 56 KYRQKRRNYK 2.80845 2.80845 KYRQKRRNYK

36

57 KYRQRKKSYK KYRQRKKSYK 1.40067 1.40067

64 64 KYRQKRRSYK KYRQKRRSYK 3.62404 3.62404

65 65 KYKQRKKNKY 1.61994 1.61994 KYKQRKKNKY

72 72 KYKQKRRNKY 3.08218 3.08218 KYKQKRRNKY

73 73 KYKQRKKSKY KYKQRKKSKY 1.91764 1.91764

80 80 KYKQKRRSKY KYKQKRRSKY 4.68996 4.68996

81 81 KYKQRKKNYK 3.28704 3.28704 KYKQRKKNYK

88 88 KYKQKRRNYK 4.40885 4.40885 KYKQKRRNYK

89 89 KYKQRKKSYK KYKQRKKSYK 2.31899 2.31899

93 93 KYKQKKKSYK 2.63127 2.63127 KYKQKKKSYK

95 95 KYKQKKRSYK KYKQKKRSYK 3.43182 3.43182

96 96 KYKQKRRSYK KYKQKRRSYK 2.14839 2.14839

[00155]

[00155] Example 3-3. Confirmation Example 3-3. ConfirmationofofType Type I Collagen I Collagen Deposition Deposition Following Following

Multiple Administrations Multiple AdministrationsofofPeptides Peptidesinin hDFs hDFs

[00156]

[00156] To confirm To confirmthe theType Type I collagen I collagen synthesisability synthesis abilityfollowing followingmultiple multiple

administrations of the administrations of the peptide peptide (SEQ (SEQ IDIDNO: NO: 96)96) of of thethe present present invention, invention, dermal dermal fibroblasts fibroblasts were were

dispensed at 1x10/6 dispensed at 1×10⁵/6well wellplate plateinin60mm 60mm dishes dishes andand cultured cultured forfor 24 24 hours hours in in an an incubator incubator at at 37°C, 37°C,

5% CO₂.TheThe 5% CO2. peptide peptide (SEQ (SEQ ID 96) ID NO: NO:and96)Vitamin and Vitamin C (positive C (positive control)control) were were each each at treated treated a at a

concentration of 100 concentration of μMatat24-hour 100 µM 24-hourintervals intervalsfor for 77 days. days. 37

[00157]

[00157] Subsequently, after washing Subsequently, after washingcells cellswith withPBS, PBS, proteins proteins werewere separated separated and and

changesinin Type changes TypeI ICollagen Collagenprotein proteinlevels levelswere were confirmed confirmed through through Confocal Confocal Microscope Microscope (Type I(Type I

Collagen). Collagen).

[00158]

[00158] Figure 3b Figure 3b shows showsa acomparison comparisonof of collagen collagen deposition deposition levelswith levels withthethecontrol control

group aftertreating group after treatingthethepeptide peptide (SEQ (SEQ ID96)NO: ID NO: 96)present of the of the invention present invention at a concentration at a concentration of 100 of 100

μMatat24-hour µM 24-hourintervals intervalsfor for 77 days, days, where where(A) (A)shows shows thethe Confocal Confocal Microscope Microscope (Type(Type I Collagen) I Collagen)

analysis results analysis resultsand and (B) (B)shows shows the the analysis analysis results resultsusing usingthe Confocal the ConfocalMicroscope Z-StackImaging Microscope Z-Stack Imaging

program. program.

[00159]

[00159] Throughthis, Through this, it it was was confirmed that on confirmed that day 7, on day 7, the the amount of Type amount of TypeII Collagen Collagen

in in the the ECM (Extracellular Matrix) ECM (Extracellular Matrix)significantly significantly increased increased in inthe thepeptide peptide(SEQ (SEQ ID ID NO: 96)treatment NO: 96) treatment

group of the group of the present present invention invention compared compared totothe the control control group. group.

[00160]

[00160]

[00161]

[00161] Example3-4. Example 3-4.Confirmation Confirmation Test Test ofof α-SMA -SMA Synthesis Synthesis Efficacy Efficacy of Peptides of Peptides in in

hDFs hDFs

[00162]

[00162] To confirm To confirmthethepossibility possibility ofofpromoting promoting wound wound healing healing following following

administration of the administration of the peptide peptide (SEQ (SEQIDIDNO:NO: 96) 96) of the of the present present invention, invention, dermal dermal fibroblasts fibroblasts werewere

dispensed at dispensed at 1.5x105/Confocal 1.5×10⁵/ConfocalDish Dish andand cultured cultured forfor 24 24 hours hours in incubator in an an incubator at 37°C, at 37°C, 5% 5% CO2. CO₂.

Peptide (SEQ Peptide (SEQIDIDNO: NO: 96)96) andand Vitamin Vitamin C (positive C (positive control) control) at at 100100 µM μM werewere treated treated for for 48 hours. 48 hours.

[00163]

[00163] Subsequently, after washing Subsequently, after cells with washing cells with PBS, changesinin alpha-smooth PBS, changes alpha-smoothmuscle muscle

actin actin (α-SMA) protein (-SMA) protein levelswere levels were confirmed confirmed through through Confocal Confocal Microscope. Microscope.

[00164]

[00164] Alpha-smooth Alpha-smooth muscle muscle actin actin ("α-SMA") ("-SMA") is expressed is expressed in fibroblasts in fibroblasts of the of the skin skin

dermis and dermis and plays plays a role a role in in providing providing structural structural support support to skintotissue skin tissue and maintaining and maintaining skin elasticity. skin elasticity.

Whenwounds When wounds occur, occur, fibroblasts fibroblasts at at thethe wound wound sitesite express express -SMAα-SMA to contract to contract the wounded the wounded skin. skin.

This can This can reduce woundsize reduce wound sizeand andprotect protectwounded wounded skin skin from from tissuedamage tissue damage during during thethe wound wound healing healing 38 process. Therefore, process. wheninflammation Therefore, when inflammation occurs occurs in in thethe skin,-SMA skin, α-SMA increases increases at inflammatory at inflammatory sites sites to regulate to regulateinflammatory responses, so inflammatory responses, so α-SMA plays -SMA plays an an important important role role ininmaintaining maintainingand andstabilizing stabilizing skin tissue. skin tissue.

[00165]

[00165] Figure 3c Figure 3c shows shows the the confirmation confirmationofofalpha-smooth alpha-smoothmuscle muscleactin actin(α-SMA) (-SMA)

synthesis synthesis efficacy efficacy of of the thepeptide peptide(SEQ (SEQ ID NO:96) ID NO: 96)ininhDFs hDFs(N=3). (N=3). Referring Referring to to Figure Figure 3c,ititcan 3c, canbe be

seen that α-SMA seen that significantly -SMA significantly increaseseven increases even with with justa asingle just singletreatment treatmentofofpeptide peptide(SEQ (SEQID ID NO:NO:

96) at 96) at 100 100 μM, indicating wound µM, indicating woundhealing healingpromotion promotion efficacy. efficacy.

[00166]

[00166]

[00167]

[00167] Example4.4.Observation Example ObservationofofCellular CellularChanges Changes Through Through Optical Optical Microscopy Microscopy

[00168]

[00168] The collagen The collagen synthesis synthesis efficacy efficacybetween betweenVitamin VitaminC,C, known as aa known as

representative substance that increases collagen synthesis ability of dermal fibroblasts in vitro, and representative substance that increases collagen synthesis ability of dermal fibroblasts in vitro, and

the peptide the peptide (SEQ IDNO: (SEQ ID NO: 96) 96) ofof thepresent the presentinvention inventionwas wascompared compared by by applying applying to human-derived to human-derived

dermal fibroblasts. Groups dermal fibroblasts. Groupswere were divided divided into into single single application application (total(total 1 and 1 time) time) and multiple multiple

application (total application (total77times) times)groups, groups,and and peptide peptide (SEQ IDNO: (SEQ ID NO:96)96)atat100 100µMμM and and Vitamin Vitamin C at C at 100 100

μM were each treated for a total of 7 days. µM were each treated for a total of 7 days.

[00169]

[00169] Specifically, Specifically, dermal fibroblasts were dermal fibroblasts dispensedatat0.3x10/well were dispensed 0.3×10⁵/well in in 96-well 96-well

plates and plates and cultured cultured for for 24 24 hours hours in inan anincubator incubatoratat37°C, 37°C,5% 5% CO₂. CO2.

[00170]

[00170] Picro Sirius Picro Sirius Red Redstaining stainingis isoneone of of the the representative representative collagen collagen staining staining

methodsthat methods that mainly mainlytargets targets Type TypeII Collagen Collagenand andType Type IIICollagen. III Collagen.After Aftertreating treating peptide peptide (SEQ (SEQIDID

NO:96) NO: 96)ofofthe thepresent presentinvention inventionatat100 100µMμM andand Vitamin Vitamin C at C at µM 100 100forμM for a of a total total of 7 with 7 days days with

single application(total single application (total1 time) 1 time) and and multiple multiple application application (total 7(total 7 times), times), PicroRedSirius Picro Sirius Red staining staining

analysis was analysis was performed. When performed. When collagen collagen positivefor positive forPicro Picro Sirius Sirius Red wasquantified, Red was quantified, ititwas wasobserved observed

to be to be higher higher in in the the peptide peptide (SEQ ID NO: (SEQ ID NO:96) 96)treatment treatmentgroup group of of thepresent the presentinvention inventioncompared compared to to

the control the control group groupinin the thesingle singleapplication applicationgroup group(Figure (Figure 4).4). InIn themultiple the multiple application application group, group, 39 collagen positive collagen positive for for Picro PicroSirius SiriusRed Redclearly clearlyincreased increasedmuch much more in the more in the peptide peptide (SEQ IDNO: (SEQ ID NO:96) 96) treatment group treatment of the group of the present present invention invention and and the the Vitamin Vitamin C treatment group C treatment groupcompared comparedtotothe thecontrol control group, andsignificance group, and significance was was also also observed observed in the in the quantitative quantitative results 4). results (Figure (Figure 4).

[00171]

[00171]

[00172]

[00172] Example5-1. Example 5-1.Results Results of of Confirming Confirming Skin Skin Penetration Penetration of Peptides of Peptides of the of the

Present Invention Present Invention in in Human-Derived Three-Dimensional Human-Derived Three-Dimensional Skin Skin Tissue Tissue

[00173]

[00173] To confirm the possibility of skin penetration of the peptides of the present To confirm the possibility of skin penetration of the peptides of the present

invention, three-dimensionalskin invention, three-dimensional skintissue tissuecomposed composed of epidermis of epidermis and dermis and dermis was created was created using using

human-derivedkeratinocytes human-derived keratinocytes andand dermal dermal fibroblasts, fibroblasts, and and then then fluorescence-conjugated fluorescence-conjugated peptidepeptide

(SEQ (SEQ IDID NO: NO: 96)96) of the of the present present invention invention at 1atmM 1 was mMtreated. was treated. After After treatment, treatment, the position the position of of

fluorescence wascontinuously fluorescence was continuouslyobserved observed through through confocal confocal microscopy microscopy at 6, at 1, 1, 6, 12,12, andand 24 24 hours. hours.

[00174]

[00174] After 11 hour After hourofoftreatment, treatment,thethepeptide peptide (SEQ (SEQ ID96) ID NO: NO:of 96) the of the present present

invention wasobserved invention was observedininthe thestratum stratumcorneum, corneum,andand after6 6hours after hoursitit was wasobserved observedininthe theepidermal epidermal

layer layer (Figure (Figure 5). 5). It Itbegan began to to be be observed in the observed in the dermal layer from dermal layer from1212hours hoursonward, onward, andand after after 24 24

hours it hours it was was clearly clearly observed that the observed that the peptide peptide (SEQ IDNO: (SEQ ID NO:96)96)ofofthe thepresent presentinvention inventionexisted existedinin

the dermal the layer (Figure dermal layer (Figure 5). 5).Through this, ititwas Through this, wasconfirmed confirmed that thatthe thepeptide peptide(SEQ (SEQ ID ID NO: 96) of NO: 96) of the the

present invention can penetrate through the stratum corneum and epidermal layer to the dermal layer. present invention can penetrate through the stratum corneum and epidermal layer to the dermal layer.

[00175]

[00175]

[00176]

[00176] Example 5-2. Example 5-2. Confirmation Confirmation of of Skin Skin Penetration Penetration of of Peptides Peptides in in Human- Human-

DerivedThree-Dimensional Derived Three-Dimensional Skin Skin Tissue Tissue

[00177]

[00177] FITCfluorescence FITC fluorescencewas wasattached attachedtotothe thelysine lysineresidue residue of of the the peptide peptide (SEQ ID (SEQ ID

NO:96) NO: 96)ofofthe the present present invention invention to to create create Peptide-FITC usingthe Peptide-FITC using thepeptide peptide(SEQ (SEQID ID NO:NO: 96) 96) of the of the

present invention. present invention. Peptide-FITC Peptide-FITCatat1 1mMmM was was treated treated on human-derived on human-derived three-dimensional three-dimensional skin skin

40 tissue (TegoScience, tissue Neoderm-ED) (TegoScience, Neoderm-ED) and and the the skinskin penetration penetration state state of of Peptide-FITC Peptide-FITC was confirmed was confirmed through confocal through confocalmicroscopy microscopyatat1 1hour, hour,6 6hours, hours,1212hours, hours,and and2424hours. hours.

[00178]

[00178] Specifically, Specifically, to to confirm tissue and confirm tissue andcytological cytologicalchanges changeswhen when treating treating the the

peptide (SEQ peptide (SEQIDIDNO: NO: 96)96) of of thethe presentinvention present inventionininhuman-derived human-derived three-dimensional three-dimensional skinskin tissue, tissue,

Hematoxylin& & Hematoxylin Eosin Eosin (H&E) (H&E) staining staining was performed was performed on tissue on tissue sections sections 24 hours 24 hours after after treating treating the the

peptide (SEQ peptide (SEQIDIDNO: NO:96)96) of of thepresent the presentinvention inventionatat11mMmM forfor analysis. analysis.

[00179]

[00179] Normal epidermal Normal epidermal layers layers consist consist of of stratum stratum corneum, stratum lucidum, corneum, stratum lucidum,

stratum granulosum,stratum stratum granulosum, stratum spinosum, spinosum, and stratum and stratum basalebasale from from the the outside. outside. Millions Millions of new of new

keratinocytes are keratinocytes are formed formeddaily dailyininthe thebasal basallayer, layer,and andthese thesedodonotnot remain remain in one in one place place but but are are

continuouslypushed continuously pushedoutward outwardtotothe theoutermost outermostpart partofof the the epidermis. epidermis. While keratinocytes are While keratinocytes are pushed pushed

upward,these upward, thesecells cells gradually graduallychange changeinto intohard hardkeratin. keratin.Aged Aged keratinocytes keratinocytes continuously continuously fallfall off off

from the surface from the surface of of human skin,but human skin, butin in aged agedskin, skin, new newkeratinocyte keratinocyteformation formationdoes does notoccur not occur well, well,

so it so it takes takesmore more time for the time for the stratum stratum corneum corneum totofall fall off, off, causing causing the the stratum stratum corneum corneum totothicken. thicken.

Therefore, functional Therefore, functional decline decline of of keratinocytes keratinocytescauses causes more more dead keratinocytes to dead keratinocytes to accumulate, accumulate, which which

can beaadirect can be directcause causeof of fine fine wrinkles wrinkles and skin and skin roughening. roughening.

[00180]

[00180] Histological analysis results showed that in the control group, keratinocytes Histological analysis results showed that in the control group, keratinocytes

were observed were observedininthe thebasal basal layer, layer, but but the the stratum stratum spinosum, stratumgranulosum, spinosum, stratum granulosum, stratum stratum lucidum, lucidum,

and stratum corneum and stratum corneumwere were not not yetproperly yet properlyformed formed (Figure (Figure 6a).InIncontrast, 6a). contrast, in in the the peptide peptide treatment treatment

group ofthe group of thepresent presentinvention, invention,normal normal histological histological characteristicsof of characteristics thethe epidermis epidermis werewere well well

observed (Figure 6a). observed (Figure 6a). This means that This means that the the peptides peptides of of the the present present invention invention can can promote promote

keratinization through keratinization through enhancing keratinocytefunction. enhancing keratinocyte function.

[00181]

[00181]

[00182]

[00182] Example 5-3. Example 5-3. Confirmation ConfirmationofofPeptide PeptideCollagen CollagenFormation Formation Abilityin in Ability

Human-Derived Human-Derived Three-Dimensional Three-Dimensional Skin Tissue Skin Tissue (Ex (Ex vivo) vivo) 41

[00183]

[00183] The collagen The collagensynthesis synthesisefficacy efficacybetween betweenVitamin Vitamin C and C and the the peptides peptides of of the the

present invention present invention was wascompared compared by applying by applying to human-derived to human-derived dermal fibroblasts. dermal fibroblasts. To To confirm confirm

peptide collagen peptide collagen formation formationability abilityinin skin skintissue tissue of of the the peptides peptidesofofthe thepresent presentinvention, invention,three- three-

dimensional skin dimensional skin tissue tissuecomposed composed of of epidermis epidermis and and dermis dermis was created (Neoderm-ED) was created using (Neoderm-ED) using

human-derivedkeratinocytes human-derived keratinocytes andand dermal dermal fibroblasts, fibroblasts, and and thenthen Vitamin Vitamin C at µM C at 1,000 1,000 and μM the and the

peptide (SEQ peptide IDNO: (SEQ ID NO:96)96) of of thepresent the presentinvention inventionatat10 10µM, μM,100 100 μM, µM, 300300 µM,μM, 1,000 1,000 μM were µM each each were

treated twice. After treatment, collagen formation ability for negative control, positive control, and treated twice. After treatment, collagen formation ability for negative control, positive control, and

treatment groups treatment groups was wasconfirmed confirmedatat48-hour 48-hourintervals intervalsusing usingthe theMasson Masson Trichrome Trichrome Staining Staining method. method.

[00184]

[00184] Figure 6b Figure 6bshows showsthetheconfirmation confirmation of of peptide peptide collagen collagen formation formation ability ability in in

Neoderm-ED Neoderm-ED skinskin tissue tissue (Ex (Ex vivo). vivo). Confirming Confirming FigureFigure 6b, excellent 6b, excellent collagen collagen formation formation ability ability

according according toto treatment treatment withwith the peptides the peptides of theof the present present invention invention can be confirmed. can be confirmed. Specifically,Specifically, it it

can be confirmed can be confirmedthat that collagen collagen (blue) (blue) deposition deposition amount amountincreases increasesdose-dependently dose-dependentlyin in thepeptide the peptide

(SEQ IDNO: (SEQ ID NO:96)96) treatment treatment group group compared compared to the to the negative negative control control (NC (NC group). group).

[00185]

[00185]

[00186]

[00186] Example5-4. Example 5-4.Test Testfor forConfirming Confirming Peptide Peptide Skin Skin Efficacy Efficacy in in Human-Derived Human-Derived

Three-Dimensional Three-Dimensional Skin Skin Tissue Tissue

[00187]

[00187] To confirm the proliferation efficacy of keratinocytes in the basal layer of the To confirm the proliferation efficacy of keratinocytes in the basal layer of the

peptides of peptides of the the present present invention, invention, immunofluorescence staininganalysis immunofluorescence staining analysisofofKi-67, Ki-67,a arepresentative representative

cell division cell divisionand and proliferation proliferationmarker, marker,was wasperformed. performed. For For this, this,the peptide the (SEQ peptide (SEQ ID ID NO: 96) of NO: 96) of the the

present invention present invention at 1 mM at 1 mMwaswas treated treated on human-derived on human-derived three-dimensional three-dimensional skin skin tissue tissue

(TegoScience,Neoderm-ED) (TegoScience, Neoderm-ED)and and cultured cultured for for 24 24 hours hours in an in an incubator incubator at at 37°C, 37°C, 5%5% CO₂. CO2. Then, Then, eacheach

human-derivedthree-dimensional human-derived three-dimensional skin skin tissuewas tissue was fixed fixed and and frozen frozen sectioned.TheThe sectioned. sectioned sectioned tissues tissues

were stained were stained with with Hematoxylin Hematoxylin &&Eosin Eosinforforcomparative comparativeanalysis analysisusing usingoptical optical microscopy microscopy

(confocal microscopy). (confocal microscopy). 42

[00188]

[00188] Referring to Referring to Figure Figure 7, 7, it it can can be be confirmed that the confirmed that the number number ofofcells cells positive positive

for for Ki-67 in keratinocytes Ki-67 in keratinocytes within within the the basal basal layer layerincreased increased compared to the compared to the control control group 24 hours group 24 hours

after treating after treatinghuman-derived three-dimensionalskin human-derived three-dimensional skin tissue tissue with with the the peptide peptide (SEQ IDNO: (SEQ ID NO:96) 96)ofofthe the

present invention at 1 mM. Quantitative results for the ratio of Ki-67 positive cells showed that the present invention at 1 mM. Quantitative results for the ratio of Ki-67 positive cells showed that the

peptide (SEQ peptide (SEQIDIDNO: NO: 96)96) treatment treatment group group of the of the present present invention invention waswas 64.664.6 (±11.1%), (±11.1%), whichwhich was was

aa 5.3-fold increasecompared 5.3-fold increase compared to control to the the control group's group's 12.2 (±3.6)% 12.2 (±3.6)% (Figure (Figure 7). 7).beThis This can can be interpreted interpreted

as the peptides as the peptidesofofthethepresent present invention invention promoting promoting cell division cell division and proliferation and proliferation of keratinocytes. of keratinocytes.

[00189]

[00189] Example5-5. Example 5-5.Test Testfor forConfirming Confirming Peptide Peptide Skin Skin Efficacy Efficacy in in Human-Derived Human-Derived

Three-Dimensional Three-Dimensional Skin Skin Tissue Tissue

[00190]

[00190] To confirm To confirmthethe skin skin efficacy efficacy of peptides of the the peptides ofpresent of the the present invention, invention,

expression patterns of expression patterns of Type Type IV IV Collagen, MMP-1, Collagen, MMP-1, Filaggrin,and Filaggrin, andInvolucrin Involucrinwere were compared compared through through

immunofluorescence analysis immunofluorescence analysis 24 24 hours hours after after treatinghuman-derived treating human-derived three-dimensional three-dimensional skin skin tissue tissue

with the peptides of the present invention at 1 mM. with the peptides of the present invention at 1 mM.

[00191]

[00191] TypeIVIVCollagen Type Collagenisismainly mainlyexpressed expressed in in theepidermal the epidermal basement basement membrane membrane

at at the the dermo-epidermal junction.The dermo-epidermal junction. Thebasement basement membrane membrane has anatomical has anatomical functions functions of joining of joining the the

epidermis andthe epidermis and the underlying underlyingdermis dermisstructure, structure, facilitating facilitating permeation permeation of ofliquid liquidsubstances substancesbetween between

the epidermis the and dermis epidermis and dermiswhile whilesimultaneously simultaneouslyperforming performing a defensive a defensive role role controllingpermeation controlling permeation

of inflammatorycells of inflammatory cellsor ortumor tumor cells. cells. According According to recent to recent studies, studies, changes changes in the in the basement basement

membrane occur in aged skin, and it has been revealed that Type IV Collagen decreases at this time. membrane occur in aged skin, and it has been revealed that Type IV Collagen decreases at this time.

Such changescancan Such changes cause cause not not onlyonly deformation deformation of keratinocytes of keratinocytes butincreases but also also increases in MMP, in a MMP, a

collagen-degrading enzyme. collagen-degrading enzyme.

[00192]

[00192] Referring to Referring to Figure Figure 8, 8, it itcan canbe beconfirmed confirmed that thatthe thepeptide peptide(SEQ (SEQ ID NO:96) ID NO: 96)

of of the the present present invention invention reduced reduced MMP-1 expression MMP-1 expression in in thethe epidermis epidermis andand dermis dermis of human-derived of human-derived

three-dimensional skin three-dimensional skin tissue tissueand and increased increasedType Type IV Collagen expression IV Collagen expression in in the the basement basement 43 membrane.Maintaining membrane. Maintaining skinskin moisture moisture is aisbasic a basic condition condition for for maintaining maintaining healthy healthy skin,skin, and and the the stratum corneummaintains stratum corneum maintains moisture moisture through through natural natural moisturizing moisturizing factors factors (Natural (Natural Moisturizing Moisturizing

Factor) such Factor) such asas Filaggrin FilaggrinororInvolucrin, Involucrin,lipid lipid layers layers existing existing between betweenkeratinocytes, keratinocytes,andand sebum sebum

secreted secreted from sebaceousglands. from sebaceous glands.

[00193]

[00193] Filaggrin acts Filaggrin acts as asan anadhesive adhesiveconnecting connecting the theouter outermembrane of keratinocytes membrane of keratinocytes

and keratin intermediate and keratin intermediatemicrofibers, microfibers,andand Involucrin Involucrin has has important important functions functions in forming in forming and and

maintaining the function of keratinocyte cell membranes that act as physical barriers for the skin. maintaining the function of keratinocyte cell membranes that act as physical barriers for the skin.

Decreased expression of these can cause skin diseases such as psoriasis and atopic dermatitis due to Decreased expression of these can cause skin diseases such as psoriasis and atopic dermatitis due to

impaired skin impaired skin barrier barrier function. function. In regard, In this this regard, referring referring to 8,Figure to Figure it can 8, be itconfirmed can be that confirmed the that the

peptides of peptides of the the present present invention inventionincrease increasethe theexpression expression levels levels of of natural natural moisturizing moisturizing factors factors

Filaggrin and Filaggrin Involucrin in and Involucrin in the the epidermis epidermis of of human-derived human-derived three-dimensional three-dimensional skinskin tissue tissue (Figure (Figure

8). 8).

[00194]

[00194]

[00195]

[00195] Example6-1. Example 6-1.Confirmation Confirmation of Anti-photodamage of Anti-photodamage Efficacy Efficacy of Peptides of Peptides in in

Neoderm-ME Neoderm-ME Tissue Tissue

[00196]

[00196] To confirm To confirmthetheanti-photodamage anti-photodamage efficacy efficacy of peptides of the the peptides ofpresent of the the present

invention, invention, UV-B radiationwas UV-B radiation wasapplied applied to to human-derived human-derived three-dimensional three-dimensional skin skin tissue tissue composed composed

of of keratinocytes keratinocytes and and melanocytes. Subsequently,4848hours melanocytes. Subsequently, hours aftertreating after treating the the peptides peptides of of the the present present

invention at 100 invention at 100µM, μM, expression expression patterns patterns of DCFDA of DCFDA staining, staining, Filaggrin, Filaggrin, and Involucrin and Involucrin were were

compared throughimmunofluorescence compared through immunofluorescence analysis. analysis.

[00197]

[00197] Figure 99 shows Figure shows the the results results of of confirming confirming (A) (A) reactive reactive oxygen species oxygen species

reduction effects reduction effects and (B) anti-photodamage and (B) anti-photodamage effectsaccording effects according to to treatment treatment with with peptide peptide (SEQ (SEQ ID ID

NO:96), NO: 96),confirming confirmingthat thatthe thepeptide peptide(SEQ (SEQID ID NO: NO: 96) treatment 96) treatment groupgroup provides provides significant significant skin skin

damage reductioneffects damage reduction effectsaccording accordingtotoUV-B UV-B irradiation. irradiation. 44

[00198]

[00198]

[00199]

[00199] Example6-2. Example 6-2.Confirmation Confirmationof of Skin Skin Whitening Whitening Efficacy Efficacy Using Using Neoderm-ME Neoderm-ME

[00200]

[00200] To confirm To confirmthethe skin skin whitening whitening efficacy efficacy ofpeptides of the the peptides of the of the present present

invention, invention, UV-B radiationwas UV-B radiation wasapplied applied to to human-derived human-derived three-dimensional three-dimensional skin skin tissue tissue composed composed

of of keratinocytes keratinocytes and melanocytes.Subsequently, and melanocytes. Subsequently,4848hours hours aftertreating after treating the the peptides peptides of of the the present present

invention at 30 invention at 30 μM, 100µM, µM, 100 μM,300 300µM,μM, 1000 1000 µM, μM, comparisons comparisons werethrough were made made through Melanin Melanin Content Content

Assayand Assay andFontana FontanaMasson Masson Staining Staining analysis analysis methods. methods.

[00201]

[00201] Figure 10 Figure 10shows showsthetheconfirmation confirmation of melanin of melanin secretion secretion reduction reduction efficacy efficacy

according to concentration-dependent according to concentration-dependent treatment treatment with with peptide peptide (SEQ(SEQ ID96), ID NO: NO:confirming 96), confirming that that

significant significant melanin melanin secretion secretion reduction reduction occurs occurs at at concentrations concentrations above above 30 μM. 30 µM.

[00202]

[00202]

[00203]

[00203] Example7.7.Test Example Testfor forConfirming Confirming Migration Migration Efficacy Efficacy of ID of SEQ SEQNO:ID 96 NO: in 96 in

Keratinocytes and Keratinocytes andhDFs hDFs

[00204]

[00204] To confirm To confirmthe thepossibility possibility of of cell cell migration migration and andwound wound healing healing promotion promotion

following administrationofof the following administration the peptide peptide(SEQ (SEQID ID NO:NO: 96) 96) of the of the present present invention, invention, keratinocytes keratinocytes

and dermalfibroblasts and dermal fibroblasts were dispensedatat 1.5x10³/Transwell, were dispensed 1.5×10⁵/Transwell,treated treated with with peptide peptide (SEQ (SEQIDIDNO:NO: 96)96)

at at 100 100 μM, andcultured µM, and culturedfor for 48 48 hours hours in in an an incubator incubator at at 37°C, 37°C, 5% CO₂. 5% CO2.

[00205]

[00205] Subsequently, after washing Subsequently, after washing cellswith cells with PBS, PBS, Crystal Crystal violet violet staining staining was was

performedtotoconfirm performed confirmchanges changesinincell cellmigration migrationthrough throughLight LightMicroscope. Microscope.

[00206]

[00206] Figure 11 Figure 11 shows showsthe theconfirmation confirmationof of cellmigration cell migrationefficacy efficacyofofthe thepeptides peptides

of of the the present present invention invention in in keratinocytes keratinocytes and and hDFs, confirmingthat hDFs, confirming thatcell cell migration migrationisis promoted promotedbyby

the peptides the peptides of of the the present present invention inventionininhuman human dermal dermal fibroblasts fibroblasts and and keratinocytes, keratinocytes, indicating indicating

woundhealing wound healingpromotion promotion efficacy. efficacy.

[00207]

[00207] 45

[00208]

[00208] The present The presentspecification specification and anddrawings drawings have have disclosed disclosed preferred preferred

embodiments embodiments ofof thepresent the presentinvention, invention,and andalthough althoughspecific specificterms termshave havebeen beenused, used,these thesewere wereused used

only in aa general only in general sense sense to to easily easily explain explain the the technical technical content content of of the the present present invention inventionand andhelp help

understanding understanding of of thethe invention, invention, notlimit not to to limit the scope the scope of theof the present present invention. invention. It is obvious It is obvious to those to those

with ordinary with ordinaryskill skillininthe thetechnical technicalfield fieldto towhich which the the present present invention invention belongs belongs that that other other

modifications based modifications basedononthe thetechnical technical spirit spirit ofofthe thepresent presentinvention inventioncan canbe beimplemented in addition implemented in addition

to the to the embodiments disclosedherein. embodiments disclosed herein.

46

Claims (1)

1. [CLAIMS]

   [CLAIMS]

   [Claim 1]

   [Claim 1]

   A composition A compositionfor fortreating treating skin skin aging aging or or skin skin wounds comprising wounds comprising a peptideconsisting a peptide consistingofofthe the

   amino acidsequence amino acid sequenceofofGeneral GeneralFormula Formula 1 below: 1 below:

   K-Y-R1-R2-R3-R4-R5-R6-R7-R8 K-Y-R1-R2-R3-R4-R5-R6-R7-R8 (General (General Formula Formula 1) 1)

   In General In Formula1,1, General Formula

   R1 is arginine (R), lysine (K), or glutamine (Q); R1 is arginine (R), lysine (K), or glutamine (Q);

   R2 is arginine (R) or glutamine (Q); R2 is arginine (R) or glutamine (Q);

   R3, R4, R3, R4, and andR5 R5are areeach eacharginine arginine(R) (R)oror lysine lysine (K); (K);

   R6 is asparagine (N) or serine (S); and R6 is asparagine (N) or serine (S); and

   R7and R7 andR8R8are arelysine lysine(K) (K)or or tyrosine tyrosine (Y). (Y).

   [Claim 2]

   [Claim 2]

   Thecomposition The compositionaccording according toto Claim Claim 1, 1, characterizedininthat characterized thatthe the composition compositionpromotes promotes

   activity of Type I Collagen, Type III Collagen, Type IV Collagen, Ki-67, Filaggrin, or Involucrin, activity of Type I Collagen, Type III Collagen, Type IV Collagen, Ki-67, Filaggrin, or Involucrin,

   or or inhibits inhibitsactivity activityofof MMP-1. MMP-1.

   [Claim 3]

   [Claim 3]

   Thecomposition The compositionaccording according toto Claim Claim 1, 1, characterizedininthat characterized thatthe the peptide peptide is is any any one amino one amino

   acid sequence acid among sequence among SEQ SEQ ID NOs: ID NOs: 1 to 196. to 96.

   47

   [Claim 4]

   [Claim 4]

   A polynucleotide A polynucleotideencoding encodingthe thepeptide peptideofofClaim Claim1.1.

   [Claim 5]

   [Claim 5]

   Anexpression An expressionvector vectorcontaining containingthe thepolynucleotide polynucleotideofofClaim Claim4.4.

   [Claim 6]

   [Claim 6]

   The composition The compositionaccording according toto Claim Claim 1, 1, characterizedininthat characterized thatthe the composition compositioncomprises comprisesa a

   polypeptide in which the peptide is repeatedly linked. polypeptide in which the peptide is repeatedly linked.

   [Claim 7]

   [Claim 7]

   A composition A compositionfor forpreventing, preventing,improving, improving,orortreating treating skin skin aging aging through throughpromoting promoting skin skin

   regeneration, elasticity, and moisturization comprising a peptide consisting of any one amino acid regeneration, elasticity, and moisturization comprising a peptide consisting of any one amino acid

   sequence among sequence among SEQSEQ ID NOs: ID NOs: 1 to 1 to 96. 96.

   [Claim 8]

   [Claim 8]

   A quasi-drug A quasi-drugcomposition compositionfor forpreventing preventingororimproving improving skin skin aging aging through through promoting promoting skinskin

   regeneration, elasticity, and moisturization comprising a peptide consisting of any one amino acid regeneration, elasticity, and moisturization comprising a peptide consisting of any one amino acid

   sequence among sequence among SEQSEQ ID NOs: ID NOs: 1 to 1 to 96. 96.

   [Claim 9]

   [Claim 9]

   A cosmetic A cosmeticcomposition composition forpreventing for preventingororimproving improving skin skin aging aging through through promoting promoting skinskin

   regeneration, elasticity, and moisturization comprising a peptide consisting of any one amino acid regeneration, elasticity, and moisturization comprising a peptide consisting of any one amino acid

   sequence among sequence among SEQSEQ ID NOs: ID NOs: 1 to 1 to 96. 96.

   48

   [Claim 10]

   [Claim 10]

   A functional A functional food food composition compositionfor forpreventing preventingororimproving improving skinaging skin aging through through promoting promoting

   skin regeneration, skin regeneration, elasticity,andand elasticity, moisturization moisturization comprising comprising a peptide a peptide consisting consisting of any oneof any one amino amino

   acid sequence acid among sequence among SEQ SEQ ID NOs: ID NOs: 1 to 196. to 96.

   [Claim 11]

   [Claim 11]

   Theskin The skin treatment treatment composition compositionaccording accordingtotoClaim Claim7, 7, characterizedbybyadditionally characterized additionally

   comprising pharmaceutically comprising pharmaceutically acceptable acceptable carrier,carrier, excipient, excipient, or diluent. or diluent.

   [Claim 12]

   [Claim 12]

   A method A methodfor forpreventing, preventing,improving, improving,orortreating treatingskin skin aging agingthrough throughpromoting promoting skin skin

   regeneration, elasticity, and moisturization by administering the composition of Claim 7 to regeneration, elasticity, and moisturization by administering the composition of Claim 7 to

   subjects other subjects other than than aa human. human.

   [Claim 13]

   [Claim 13]

   Thecomposition The compositionaccording according toto Claim Claim 1, 1, wherein wherein thethe skin skin wounds wounds are are caused caused by trauma, by trauma,

   diabetes, orbedsores. diabetes, or bedsores.

   49

   [FIGURES]

   [FIGURES]

   [Figure 1]

   [Figure 1] 3.5

   3 (WST-8) Viability Cell (WST-8) Viability Cell

   [OD 450nm] 2.5

   2

   1.5

   1

   0.5

   0 Con 10uM 100uM 1mM 5mM 10mM Dose dependent # 96

   [Figure 2]

   [Figure 2] (A) (B)

   1.0 EC50 shift # # # *** # 0.8 # # ß-actin) to (relatie ß-actin) to (relatie *** collagen I Type collagen I Type # 100 * % control

   0.6

   0.4 50

   0.2

   0.0 0 Control WELL whool 100PMwull way WOOM writ 100nM 10HM -10 -8 -6 -4 -2 0 log(agonist)(mM)

   EC50: 8.621 X 10 mM (= 8.621 pM) Seven times (7 times/7 days)

   # 96: 100 µM treatment

   1/9 1/9

   [Figure 3a]

   [Figure 3a] 5

   4.5 Levels Expression Protein Relative Levels Expression Protein Relative 4 6-Actin] Collagen/ I

   [Type 6-Actin] Collagen/ I

   [Type 3.5

   3

   2.5

   2

   1.5

   1

   0.5

   0 Control 8 9 16 24 s2 32 EE is 48 6s 56 5> 64 65 << E< 80 81 88 89 93 95 96 " O Peptide Sequence Number

   2/9 2/9

   [Figure 3b]

   [Figure 3b] (A)

   7 days Control #96 100uM

   Type I Collagen

   5 8x10 Intensity) (Fluorescence Intensity) (Fluorescence ** deposition Collagen deposition Collagen 6x10

   4x10

   2x10

   0 Control wn OOL 96#

   (B)

   Control #96 100uM

   Coronal Plane

   17 um 9 um Saggital Plane

   3/9 3/9

   [Figure 3c]

   [Figure 3c] 24hr 24hr

   Seeding PBS Cell harvest #96 (100 uM)

   or L-Ascorbic acid (100 uM)

   F-Actin DAPI Merged -SMA 1.5x10

   Intensity) (Fluorescence Intensity) (Fluorescence **

   *** a-SMA 1*10

   Control

   5*10 ns

   0 Control L-Ascorbic #96 100 100 HM Acid 100 100 UM

   #96 100 uM 1.5x10

   Intensity) (Fluorescence Intensity) (Fluorescence F-actin 1*10

   5*10

   L-Ascorbic Acid

   100 uM 0 Control L-Ascorbic #96 100 HM Acid PM 100 UM 100 MM

   4/9 4/9

   [Figure 4]

   [Figure 4]

   Control # 96 100uM L-Ascorbic Acid 100uM

   Treatment Single Treatment Multiple Single Treatment Multiple 7 days

   ** 0.4 **

   0.35 540nm OD Red Sirius 540nm OD Red Sirius 0.3

   0.25

   0.2 *

   0.15

   0.1

   0.05

   0 Vit. C Vit. C # 96 # 96 Control 100 µm 100 µm Control 100 µm 100 µm (Single) (Single) (Single) (Multiple) (Multiple) (Multiple)

   5/9 5/9

   [Figure Figure 5] 5

   1x PBS Stratum Corneum

   Epidermis

   Dermis

   1hour 6hour 12hour 24hour

   # 96 1000 µM

   [Figure 6a]

   [Figure 6a] Control # 96 1mM

   Stratum corneum

   Stratum granulosum

   Stratum spinosum

   Basement Stratum basale membrane

   Dermis

   [Figure 6b]

   [Figure 6b] Negative Control # 96 10 uM # 96 100 uM # 96 300 uM # 96 1000 uM Vit.C 1000 uM

   #1

   #2

   #3

   6/9 6/9

   [Figure 7]

   [Figure 7]

   Control (PBS) # 96 1mM

   80.0 ***

   70.0 Ki-67 of Percentage % Ki-67 of Percentage % 60.0

   50.0

   40.0

   30.0

   20.0

   I 10.0

   0.0

   Control # 96

   [Figure 8]

   [Figure 8] Control (PBS) # 96 1mM Control (PBS) # 96 1mM Collagen IV Type Collagen IV Type Involucrin

   Filaggrin

   MMP-1

   7/9 7/9

   [Figure 9]

   [Figure 9] (A)

   Control #96 100 µM

   ROS (LSM Filter X RT

   4x101 Intensity) (Fluorescence Intensity) (Fluorescence DCFDA Assay 3x10

   2x10

   1x10 #

   0 Control #961 100 PM

   (B)

   Filaggrin Involucrin DAPI Merged

   Control

   Vit. C 100 µM Intensity) (Fluorescence Intensity) (Fluorescence 1000000 ** Anti-photodamage Anti-photodamage Filaggrin 800000 Involucrin

   600000

   400000

   #96 100 uM 200000

   0 Control Vit.C # 96 100pM

   8/9 8/9

   [Figure 10]

   [Figure 10] #96 Control 30 uM 100 uM 300 uM 1000 uM Vit. C 1 mM

   150 30

   (µg/ml) content Melanin (µg/ml) content Melanin *** (%) content Melanin (%) content Melanin ***

   100 20

   *** *** *** 50 10 ***

   0 0 # 96 (µM) 0 30 100 300 1000 # 96 (µM) 0 30 100 300 1000 Vit.C (µg/ml) 200 (= 1000 µM) Vit.C (µg/ml) - - - - - - - - - - 200 (= 1000 pM)

   [Figure 11]

   [Figure 11]

   Transwell Assay

   Control #96 100 uM Migration of HaCaT

   0.10 nm) (595 Absorbance nm) (595 Absorbance 0.08 ***

   0.06 Keratinocytes 0.04

   0.02

   0.00 Control 100 uM

   Migration of hDF

   0.10 nm) (595 Absorbance nm) (595 Absorbance 0.08 ***

   0.06 Dermal Fibroblasts 0.04

   0.02

   0.00 Control 100 uM

   9/9 9/9