Letter
Anti-WrinkleEfficacyofNeuropeptideSubstance
P-BasedHydrogelinHumanVolunteers
DaJungKim*,SeulBiLee,SongSunChangandJungsunLee
ResearchandDevelopmentInstitute,Biosolution,SeoulTechnopark,232Gongneung-ro,Nowon-gu, Seoul01811,Korea;xkssls@kaist.ac.kr(S.B.L.);kimdj1404@kaist.ac.kr(S.S.C.);xkssls@naver.com(J.L.)
* Correspondence:kimdj1404@biosolutions.co.kr
Received:2April2020;Accepted:20May2020;Published:21May2020
Abstract: ThisletterisintendedtoclarifytheclinicaleffectivenessofneuropeptidesubstanceP (SP)inreducingage-relatedskinwrinkling.Thisisthefirstclinicalstudytoevaluatetheefficacy ofthetopicalapplicationofSPonwrinklereduction.Fortheclinicalstudy,wetestedtheefficacy ofastableSPformulationknownasSP-basedhydrogelagainstskinaginginstudyparticipants. WefoundthatthisSP-basedhydrogelreducedskinagingbasedonchangesobservedinseveral cutaneousparameters.
Keywords: substanceP;hydrogel;anti-wrinkleefficacy;clinicalstudy;cosmeceutical
1.Introduction
SubstanceP(SP),asmall-sizedneuropeptideconsistingof11aminoacids,isproducedinwound areasandexhibitseffectiveskinregenerativeactivity[1].Theanti-agingeffectsofSPareattributedto itsanti-inflammatoryactivityandeffectsoncollagensynthesis.StudieshaveshownthatSPincreases typeIprocollagenlevels,correlatedwiththeregulationofmatrixmetalloproteinase(MMP)-1aswell asanti-inflammatoryactivity,thusindicatingitspotentialanti-agingefficacyinclinicalpractice[1,2]. Althoughthepotentialanti-agingactivityofSPfacilitatesitsuseincosmeceuticals,itsapplication hasbeenlimitedowingtoitslowstability[3].Inapreviousstudy,astableSPformulationknownas SP-basedhydrogelwasdeveloped[3].TopreventoxidationofSPandminimizephysicaldamagetoSP fromaninterfacialinteractioncausedbySP–surfactantassociationand/orcoatinginterfaces,sodium thiosulfate[4]andpolysorbate80[5–7]wereaddedtotheSP-basedhydrogel.Inaddition,agelling agent(hydroxyethylcellulose)wasusedtoenhancetheviscosityandSPstabilityofthehydrogel[8]. TheSP-basedhydrogeldemonstratedsuperior invitro stabilityandanti-agingefficacycomparedtoSP alone[2,3].Inparticular,theSP-basedhydrogelwasstableingrowthmediumcontainingfetalbovine serumforupto24handathightemperature(~60 °C)forupto4weeks[3].ToevaluateSPstability, weremovedaliquotsofsamplesfromthegrowthmediumorathightemperatureandthendiluted themwithphosphatebufferedsalineforanalysisofSPcontentbyenzyme-linkedimmunosorbent assay[3].Althoughpreviousreportsonthe invitro anti-agingeffectsandbetterstabilityprofileof SP-basedhydrogelindicatetheneedforclinicalstudiesscreeningtheanti-agingeffectsofSP-based hydrogel,nonehavebeenperformedtodate.
2.Methods
Theanti-wrinkleefficaciesofSP-basedhydrogelandplacebowerecomparedin24women(age, 59.5 ± 6.9years;onewomanwhodiscontinuedthestudyprotocolwasexcluded).Forthepurposeof thestudy,ampouleformulationwithSP-basedhydrogel(1 µg/mLSP)orwithoutSP-basedhydrogel (placebo,containingdistilledwaterinsteadofSP-basedhydrogel)wasprepared.Theformulation comprisedseveralingredientsincludingglycerine,dipropyleneglycol,1,2-Hexanediol,sodium
hyaluronate,tromethamine,andcarbomer(Table 1).Thestudywasperformedinaccordancewith theGuidelineforFunctionalCosmetics(KFDA11-1470000-000863-01; https://www.mfds.go.kr/brd/ m_218/view.do?seq=708;lastaccessed21May2020),whichrequiresthecomparativeevaluationof vehicleswithorwithoutactiveingredientsinatleast20patientsforstatisticalanalysis.Subsequently, 23womenwererandomizedtoreceiveeithertopicalSP-basedhydrogelontherightorleftsideof thefaceorplacebowithoutSP-basedhydrogelontheothersideofthefacetwicedailyfor8weeks. Thefollowingphotographicstandardsofwrinkleswerestudied(0,none;1,none/mild;2,mild;3, mild/moderate;4,moderate;5,moderate/severe;6,severe;and7,verysevere)atweeks0,4,and8[9]by twoindependentdermatologists.Moreover,skinroughness(R1),maximumroughness(R2),average roughness(R3),smoothnessdepth(R4),andarithmeticaverageroughness(R5)weremeasuredat8 weeksusingSkin-VisiometerSV600(Courage&Khazaka,Cologne,Germany)[10].Forstatistical analysis,SPSSsoftwareversion19.0(SPSSInc.,Chicago,USA)wasusedtodeterminethesignificanceof themeasuredvaluesbeforeusingthetestproduct.Afterrepeatedmeasurements,Analysisofvariance (ANOVA)wasusedasaparametricmethodwithaBonferronicorrection.Inaddition,Friedman testandMann–WhitneyUtestwereusedtocomparewithin-groupdataandbetween-groupdata, respectively,and P < 0.05wasconsideredsignificant.Thisstudywasapprovedbytheinstitutional reviewboardoftheEthicsCommitteeoftheP&KSkinResearchCentre,Seoul,Korea(approval no:P1801-115).
Table1. FormulationsofsubstanceP(SP)-basedhydrogelandplaceboampoule.
ComponentPlacebo(% w/w)SPHydrogel(% w/w) Water75.5–80.575.5–80.5 Glycerine5–65–6 Dipropyleneglycol8–98–9 1,2-Hexanediol2–32–3 Sodiumhyaluronate0.2–0.50.2–0.5 Tromethamine0.2–0.50.2–0.5 Carbomer0.2–0.50.2–0.5 SP-basedhydrogel (SP20 µg/mL) -5
Viscosity(25 ◦C)2000–2500cPs2000–2500cPs
3.Results
Wefoundthatafter8weeksoftreatment,theaveragescoresofwrinklegradeoftheSP-based hydrogel-treatedsidedecreasedsignificantlywhencomparedtothosebeforethetreatment(Figure 1b). Figure 1aillustratestheremarkabledecreaseinwrinklesafter8weeksofSP-basedhydrogeltreatment. Inaddition,Visiometeranalysistakenat8weekstoanalyzethefiveagingparameters(R1–R5)between placeboandSP-basedhydrogelrevealedastatisticallysignificantdecreaseinalltheseparametersfor theSP-basedhydrogel-treatedside(P-value < 0.05)(Figure 1c);however,nosignificantdecreasewas notedat4weeks(datanotshown).Nevertheless,theabovevaluesdidnotreflectthevaluespriorto placeboorSP-basedhydrogeltreatment.Toclarifythis,weanalyzedthepercentageofchangereflected inthebaselinevalues.ThechangeinRvaluesafter8weekswascalculated(Figure 1d).Thechange valuewasrepresentedasthevalueconvertedtoapercentagebysubtractingthebaselinevaluesfrom theabsolutevaluesafterplaceboorSP-basedhydrogeltreatment.AsshowninFigure 1d,therateof changeinalltheparametersoftheSP-basedhydrogel-treatedsidesignificantlyimprovedat8weeks whencomparedtothatoftheplacebo.Takentogether,theseresultsdemonstratetheremarkableeffect ofSP-basedhydrogelonwrinklereduction.
address these limitations, we are now working on a clinical study in two institutes, comparing the anti-wrinkle efficacy of SP-based hydrogel and retinol (as the positive control) for a duration of 12 weeks.
Figure1. Changesinclinicalparametersduringthestudy:(a)participantswithwrinklesat0and8weeks aftertreatmentwithplaceboandsubstanceP-basedhydrogel;(b)wrinklegradeassessedbyindependent dermatologistsat0,4,and8weeks. * indicatesasignificantdifferencefrombaseline(P-value < 0.05; Friedmantest,posthocWilcoxonsigned-ranktestwithBonferronicorrection);(c)skinreplicaanalysis resultusingtheSkin-Visiometer600after8weeksoftreatmentwithplaceboandSP-basedhydrogel. # indicatesasignificantdifferencefromplacebo(P-value < 0.05;Mann–WhitneyUtest);(d)changesin skinwrinkleparametersafter8weeks.(percentageof change = (after before)/before × 100) # indicates asignificantdifferencefromplacebo(P-value < 0.05;Mann–WhitneyUtest).
Figure 1. Changes in clinical parameters during the study: (a) participants with wrinkles at 0 and 8 weeks after treatment with placebo and substance P-based hydrogel; (b) wrinkle grade assessed by independent dermatologists at 0, 4, and 8 weeks. * indicates a significant difference from baseline (Pvalue < 0.05; Friedman test, post hoc Wilcoxon signed-rank test with Bonferroni correction); (c) skin replica analysis result using the Skin-Visiometer 600 after 8 weeks of treatment with placebo and SPbased hydrogel. # indicates a significant difference from placebo (P-value < 0.05; Mann–Whitney U test); (d) changes in skin wrinkle parameters after 8 weeks. (percentage of change = (afterbefore)/before × 100) # indicates a significant difference from placebo (P-value < 0.05; Mann–Whitney U test).
In this study, statistically significant wrinkle reduction was observed following topical application of SP-based hydrogel after 8 weeks of treatment. These findings imply that the SP-based hydrogel can be used in the cosmetic industry as a novel anti-wrinkle agent.
Toourknowledge,thisisthefirstclinicalstudyevaluatingtheefficacyofthetopicalapplication ofSPonwrinklereduction.However,furtherstudiesarerequiredtoclarifytheanti-wrinkleefficacyof SP.Indeed,ourstudyhadfewlimitationssuchas(i)smallsamplesizeandrecruitmentofparticipants fromasinglecenter,(ii)lackofpositivecontrol,and(iii)shortdurationofscreening(longerscreening durations,lastingmorethan8weeksincludingafollow-upperiodareneeded).Toaddressthese limitations,wearenowworkingonaclinicalstudyintwoinstitutes,comparingtheanti-wrinkle efficacyofSP-basedhydrogelandretinol(asthepositivecontrol)foradurationof12weeks.
Author Contributions: Conceptualization, D.J.K. and J.L.; validation, S.B.L.; formal analysis, D.J.K.; investigation, D.J.K.; data curation, D.J.K.; writing original draft preparation, D.J.K.; writing review and editing, D.J.K.; visualization, D.J.K.; supervision, D.J.K.; project administration, D.J.K., S.S.C and J.L. All authors have read and agreed to the published version of the manuscript.
Inthisstudy,statisticallysignificantwrinklereductionwasobservedfollowingtopicalapplication ofSP-basedhydrogelafter8weeksoftreatment.ThesefindingsimplythattheSP-basedhydrogelcan beusedinthecosmeticindustryasanovelanti-wrinkleagent.
Funding: This research received no external funding
Conflicts of Interest: The authors declare no conflict of interest.
References
AuthorContributions: Conceptualization,D.J.K.andJ.L.;validation,S.B.L.;formalanalysis,D.J.K.;investigation, D.J.K.;datacuration,D.J.K.;writing—originaldraftpreparation,D.J.K.;writing—reviewandediting,D.J.K.; visualization,D.J.K.;supervision,D.J.K.;projectadministration,D.J.K.,S.S.C.andJ.L.Allauthorshavereadand agreedtothepublishedversionofthemanuscript.
Funding: Thisresearchreceivednoexternalfunding.
ConflictsofInterest: Theauthorsdeclarenoconflictofinterest.
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