In Vitro ADME Properties of Two Novel Antimicrobial Peptoid-Based Compounds as Potential

15 Pages·2017·1.81 MB·English

molecules
Article
In Vitro ADME Properties of Two Novel
Antimicrobial Peptoid-Based Compounds as
Potential Agents against Canine Pyoderma
InesGreco1,2,3,* ID,BernardD.Hummel3,JaspreetVasir3,JeffreyL.Watts3,JasonKoch3,
JohannesE.Hansen1,HanneMørckNielsen4,PeterDamborg5andPaulR.Hansen1,* ID
1 DepartmentofDrugDesignandPharmacology,UniversityofCopenhagen,Universitetsparken2,
2100CopenhagenØ,Denmark;[email protected]
2 DepartmentofFoodScience,UniversityofCopenhagen,Rolighedsvej30,DK-1958Frederiksberg,Denmark
3 ZoetisInc.,333PortageSt.,Kalamazoo,MI49007,USA;[email protected](B.D.H.);
[email protected](J.V.);[email protected](J.L.W.);[email protected];(J.K.)
4 DepartmentofPharmacy,UniversityofCopenhagen,Universitetsparken2,2100CopenhagenØ,Denmark;
[email protected]
5 DepartmentofVeterinaryandAnimalSciences,UniversityofCopenhagen,Stigbøjlen4,
1870FrederiksbergC,Denmark;[email protected]
* Correspondence:[email protected](I.G.);[email protected](P.R.H.);Tel.:[phone](I.G.);
[phone](P.R.H.)
Received:17January2018;Accepted:5March2018;Published:10March2018
Abstract: Antimicrobial peptides (AMPs) hold promise as the next generation of antimicrobial
agents,butoftensufferfromrapiddegradationinvivo. ModifyingAMPswithnon-proteinogenic
residuessuchaspeptoids(oligomersofN-alkylglycines)providesthepotentialtoimprovestability.
Wehaveidentifiedtwonovelpeptoid-basedcompounds,B1andD2,whichareeffectiveagainstthe
canineskinpathogenStaphylococcuspseudintermedius,themaincauseofantibioticuseincompanion
animals. We report on their potential to treat infections topically by characterizing their release
fromformulationandinvitroADMEproperties. InvitroADMEassaysincludedskinpenetration
profiles,stabilitytoproteasesandlivermicrosomes,andplasmaproteinbinding. BothB1andD2
wereresistanttoproteasesand>98%boundtoplasmaproteins. Whilehalf-livesinlivermicrosomes
forbothwere>2h,peptoidD2showedhigherstabilitytoplasmaproteasesthanthepeptide-peptoid
hybridB1(>2versus0.5h). Bothcompoundsweresuitableforadministrationinanoil-in-water
creamformulation(50%releasein8h),anddisplayednoskinpermeation,intheabsenceorpresence
ofskinpermeabilitymodifiers. Ourresultsindicatethatthesepeptoid-baseddrugsmaybesuitable
asantimicrobialsforlocaltreatmentofcaninesuperficialpyodermaandthattheycanovercomethe
inherentlimitationsofstabilityencounteredinpeptides.
Keywords: antimicrobialpeptoids;peptidomimetics;invitroADME;topicalformulation;metabolic
stability
1. Introduction
The emergence of bacterial pathogens resistant to multiple antimicrobial agents over the past
decadesisagrowingpublichealththreat,andthereisanurgentneedfornovelantimicrobialdrugs
forhumanandveterinaryuse[1,2]. Incompanionanimals,canineskininfectionsconstitutethemain
causeofantibioticuse[3].Staphylococcuspseudintermediusistheprimarypathogenofthisdisease,asit
isisolatedfromaround90%ofcanineclinicalskinsamples[4]. Intherecentdecade,theworldwide
emergenceofmethicillin-resistantS.pseudintermedius(MRSP)[5]hasresultedininfectionsuntreatable
with antimicrobial agents licensed for systemic use in dogs. Therefore, there is an urgent need to
Molecules2018,23,630;doi:10.3390/molecules23030630 www.mdpi.com/journal/molecules
Molecules 2018, 23, x 2 of 15
Molecules2018,23,630 2of15
untreatable with antimicrobial agents licensed for systemic use in dogs. Therefore, there is an urgent
ndeeevdel otop adletevrenlaotpiv aelatenrtnimatiicvreo bainaltiamgeicnrtosbtioacl oamgebnattst htios pcoatmhobgate nt.hFisu rptahtehromgoerne., sFyusrttehmeircmaodrme,i nsiysstrtaetmioinc
aodfmanintiibstioratitciosnm oafy apnrtiobmiootitces tmheayd epvreolmopomtee nthteo fderveseilsotpamnceenat nodf rceusrirsetanntcgeu aidnedl incuersreenntc oguuriadgeelinthese
eunsceooufratogpe itchael uthsee roafp toypfiocralt trheeartampeyn ftoor ftrMeaRtmSPenitn ofef cMtiRonSsP [i6n]f.ecPtieopntsi d[6e]s. Pweipthtidaenst wimitihcr aonbtiiamliaccrtoivbiiatyl
a(ActMivPitsy) a(rAeMthPeso) bajercet othfrea poibdjleyc-tg roofw rianpgidrelsye-agrrcohwinintegr ersetsaesarthche nienxttergeesnt eraast iothneo fnaenxtti bgaecnteerriaatliso[n7 –o9]f.
aInntgibeancetrearli,aAlsM [7P–s9]a.r Iens gmeanlelr(a<l,5 A0MamPisn aorae csimdsa)l,lc (a<t5i0o naimc,ianmo apchiidpsa)t, hciactihoonsitc,d aemfepnhseippaethpitcid heossnt dateuferanlsley
pperepsteidnetsi nnatthuerainllnya ptereismenmt uinn ethsey sintenmatse [i1m0]m. uAnMe Psyssgteemnesr [a1ll0y]. sAhMowPss pgeecniefircailtlyy fsohrobwa cstpeerciaiflictaitryg efotsr,
bbaroctaedr-iaspl etcatrrguemts,a cbtriovaitdy-sapnedctlrouwme raacftfiivniittyy afonrdm loamwemr aalifafninhitoys tfocre llmsa[1m1–m1a4l]i.anA lhthoosut gchelplsr o[m11i–s1in4g].,
Athlethyoduisgphl apyrolimmiisteindgs, ttahbeilyit dyiisnpvlaiyv olibmeiitnegde satsaiblyilidtyeg irna dveivdob byepinrogt eeaasseilsy[ 1d5e]g.rIandreedce bnyt yperaortse,acsheesm [1ic5a].l
Iann drescterunct tyueraalrsm, ocdheifmicaictaiol nasnhda vsterubecetunruals emdoadsisftircaatteiogniess hfoarviem bpereonv iunsgesdt aabsi listtyraotfegpieepst ifdoers .imStprurocvtuinragl
satnaablioligtyu eosf opfeppetpidteids.e sStsruucchtuarsalp aenpatoloidgsue(ps oolfy mpeeprtsidoefsN s-usuchb satsit upteepdtoaildksy l(gploylcyimneesr)s[ 1o6f ,N17-]suhbasvteitbueteedn
ainlktryoldgluycceindetso) o[v16e,r1c7o]m heasvtea bbielietny iisnstureosd.uIcnetdro dtou cotvioenrcoofmpee psttaobidilirteys iidsusueessi.n Itnhterosdtruuccttiuonre ooff pexeipsttoinidg
roersiddeuenso ivno thdee sstigruncetdurAe Mof Pesxihstaisnga losro dbee ncoovmoe daessiugncceeds sAfuMlPasp hparso aalcsho tboecimompero av seucthceesirsfauclt aivpiptyroaanchd
tpoh iamrmpraocvoek tinheeitric acptrivoiptyer atineds p[1h5a]r.maWcohkeirneeatsice pffroorptehrtaiess b[1e5e]n. Wmhaedreeatso efafsosrets hsatsh beeesntr mucatduree t-oa catsisveistys
trhelea tsitornuschtuipreo-afcatnivtiimtyi crreolbatiaiolnpsehpitpo iodfs atnotiimmpicrroovbeiaalc tpivepittyo[id18s] ,toth iemirpAroDvMe Eac(taibvsitoyr p[1ti8o]n, ,tdhiesitrr iAbuDtMionE,
(mabestaobrpotliisomn, adnidstreixbcurteitoinon, )mpertoafbiloelihsmas nanodtb eexecnreintivoens)t ipgraotfeidle thoatsh enosat mbeeeenx tiennvte.sItnigtahteedp rteos ethnet sstaumdye,
ethxetesneta. sInp etchtes pwreerseencth satruadctye,r tizheedsef oasrpaecptesp wtiedree- pcehpatroaicdtehriyzberdi dfo(rB a1 )paenpdtidaep-pepeptotoidid( Dhy2b)rdides (iBgn1)e danads
aa npteimptiocirdo b(Dial2s) fdoerstiogpniecda laus saenatigmaiincrsotbSi.aplss efuodri ntoteprimcaeld uiusse. aTghaeinsstrt uSc.t pusreeusdoinftBer1maenddiuDs. 2Tharee ssthruocwtunreins
oFfi gBu1re a1n.dF irDst2g eanree rashtioownnfo rimn uFliagtuiorne s1o.f FBi1rsatn gdeDne2rfaotirotno pfiocramlaudlamtiionnisst roaft ioBn1h aanvde bDee2n fdoerv teoloppiceadl
aadnmdiunsiesdtrafotironte shtainvge .bTehene idnevvietlroopAedD ManEd pursoefdil efoorf tBes1tianngd. TDh2ei nint veritmros AofDdMegEr apdraotfiiolen obfy Bm1 eatnadb oDli2c
ienn zteyrmmess ,opf ldasemgraadpartoiotenin bbyi nmdeitnagboalnicd epnezrymmeeast,i opnlatshmroau pghroetexicni sbeidnddiongg saknind wpeerrmeeinavtieosnt igthartoedugihn
ethxicsissetudd dyo.g skin were investigated in this study.

Figure 1. Structure of B1 (A) and D2 (B) both isolated as trifluoacetic acid salts.
Figure1.StructureofB1(A)andD2(B)bothisolatedastrifluoaceticacidsalts.
2. Results
2. Results
2.1. MIC and Hemolytic Activity In Vitro
2.1. MICandHemolyticActivityInVitro
B1 and D2 were selected after screening of a combinatorial library of de novo designed
B1andD2wereselectedafterscreeningofacombinatoriallibraryofdenovodesignedcompounds
compounds for their activity against S. pseudintermedius and host toxicity in vitro measured as lytic
fortheiractivityagainstS.pseudintermediusandhosttoxicityinvitromeasuredaslyticactivityagainst
activity against red blood cells. B1 and D2 were designed on the basis that the majority of
redbloodcells. B1andD2weredesignedonthebasisthatthemajorityofantimicrobialpeptides
antimicrobial peptides are cationic and hydrophobic. We envisaged that short linear peptide-peptoid
arecationicandhydrophobic. Weenvisagedthatshortlinearpeptide-peptoidhybridsconsistingof
hybrids consisting of cationic lysines and hydrophobic peptoid residues would result in derivatives
cationiclysinesandhydrophobicpeptoidresidueswouldresultinderivativeswithpotentantibacterial
with potent antibacterial activity and low cytotoxicity. Minimum inhibitory concentrations against S.
activityandlowcytotoxicity. MinimuminhibitoryconcentrationsagainstS.pseudintermediuswere
pseudintermedius were 2–4 μg/mL for B1 and 1–2 μg/mL for D2, irrespective of the strain being MSSP
2–4µg/mLforB1and1–2µg/mLforD2,irrespectiveofthestrainbeingMSSPorMRSP.Preliminary
or MRSP. Preliminary data suggest that B1 and D2 also are active against a wide range of canine
data suggest that B1 and D2 also are active against a wide range of canine pathogens (data not
pathogens (data not shown, Greco et al., manuscript in preparation). The hemolytic activity of B1 and
shown,Grecoetal.,manuscriptinpreparation). ThehemolyticactivityofB1andD2asafunction
D2 as a function of their concentration is shown in Figure 2. Repeated experiments on different days
oftheirconcentrationisshowninFigure2. Repeatedexperimentsondifferentdayswithdifferent
with different human blood samples confirmed the results. The concentration of compound resulting
human blood samples confirmed the results. The concentration of compound resulting in 10 and
i5n0 %10h aenmdo 5ly0s%is hoefmeroyltyhsriosc oyft eesry(EthCrocayntedsE (CEC1)0 wanerde E64C5µ0)g /wmerLe a6n4d μ2g1/9mµLg /amndL 2f1o9r Bμ1g/amndL 1fo28r Bµg1 /amndL
10 50
128 μg/mL and >256 μg/mL for D2. In a separate experiment, performing the test with dog blood
and>256µg/mLforD2. Inaseparateexperiment,performingthetestwithdogbloodresultedin
resulted in hemolysis at 150 μM of 45% and 22% for B1 and D2, respectively.
hemolysisat150µMof45%and22%forB1andD2,respectively.
Molecules2018,23,630 3of15
Molecules 2018, 23, x 3 of 15
Molecules 2018, 23, x 3 of 15

Figure 2. Hemolytic activity of peptide-peptoid hybrid B1 and peptoid D2 in human erythrocytes as
Figure2.Hemolyticactivityofpeptide-peptoidhybridB1andpeptoidD2inhumanerythrocytesasa
a function of concentration. Mean ± SEM, n = 3.
functionofconcentration.Mean±SEM,n=3.

2.2. Stability to Proteases
2.2. StabilityFtioguPrer o2t. eHaesmesolytic activity of peptide-peptoid hybrid B1 and peptoid D2 in human erythrocytes as
The stability of B1 and D2 was tested against trypsin and chymotrypsin—proteases of
a function of concentration. Mean ± SEM, n = 3.
Tmhaemsmtaabliilainty oorifgBin1—aannddD p2rownaasset, eas mteidx aogf adiinffsetrternytp esniznyamneds cehxytrmacotetdry fprosmin —bapctreortieuamse Sstroefpmtomamycmes alian
origingr—is2ea.u2ns. .dS tBap1b riaolinntyda tsDoe ,P2 rawotmeeraeisx eisn ocfubdaitfefder esinntguelnazrlyym ine sdiefxfetrreanctt evdialfsr owmithb tarcytpesriinu, mchySmtroeptrtyopmsyinc,e sangdr iseus.
pronase at 37 °C (trypsin and pronase) and 30 °C (chymotrypsin), and the results were analyzed by
B1 and D2 Twheer estainbicluityb aotfe dB1s inangdu lDar2l ywians dteisftfeedr eangtavinisatl strwypistihn tarnydp scihny,mcohtyrympsointr—ypprsointe,asaens dofp ronase
at37c◦oCmm(ptaramyripmnsgail niaaranen aodsri gopifnr ot—hneaa nrsdees )puarlontindnags3 eR0, aP◦ m-CHix(Pc oLhfC yd mipffeoeartkernys tpa esfntieznry) m,6a eansn dedx tt2hr4aec htre.ed sT ufhrloetm spw ebpaectritdeereai-unpmaepl Syttozrieedpd thobymybyrccioedsm Bp1a ring
showed 6% degradation after 24 h of incubation with trypsin, 38% after pronase exposure, and no
areas ofgrtihseeusr.e sBu1 ltainndg DR2P w-HerPe LinCcupbaetaekds sianfgtuerlar6lya innd di2ff4erhen.t Tvhiaels pweiptht itdryep-psienp, tcohyidmohtyrybprsiidn, Ba1nds howed
degprraodnaatsieo nat a3f7te °rC α (-tcryhpysmino atrnydp psirno neaxspeo) saunrde 3(0F i°gCu (rceh y3m). oPterpyptosiidn) ,D a2n ds hthoew reedsu 0lt.2s %w edreeg arnaadlyatzieodn bayf ter
6%degradationafter24hofincubationwithtrypsin,38%afterpronaseexposure,andnodegradation
24 hco imncpuabriantgio anr ewasi tohf pthroe nraesseu,l tainngd RnPo- HdePgLrCa dpaetaikosn awftaesr 6o basnedr v2e4d h b. yT htrey ppespinti daen-dp ecphtoyimd ohtyrybprisdi nB 1(s ee
afterα-chymotrypsinexposure(Figure3). PeptoidD2showed0.2%degradationafter24hincubation
Supshpolewmeedn 6ta%r yd eMgaratedraitaiolsn, Saeftcetri o2n4 Sh3 o).f incubation with trypsin, 38% after pronase exposure, and no
withprodnegarsaed,aatinodn anfoterd αe-gcrhaydmaottiroynpswin aesxpoobssuerrev (Feidgubrye 3tr).y Ppespitnoiadn Dd2 cshhyowmeodt r0y.2p%s idneg(sraedeaStiuonp paflteemr entary
100 Pronase
Materia2ls4, hS eincctuiobnatiSo3n) .with pronase, and no degradation was observed by trypsin and chymotrypsin (see
90 Trypsin
Supplementary Materials, Section S3).
80 Chymotrypsin
100 Pronase
7090 Trypsin
n
o6080 Chymotrypsin
ita
d5070
a
rged %dation34005600
a
r
eg2040
d
% 1030
20
0
100 2 4 6 8 10 12 14 16 18 20 22 24
0 Time (h)

0 2 4 6 8 10 12 14 16 18 20 22 24
Time (h)

FigurFeig3u.rPer o3t. ePorlyotteicoldyetigcr addeagtriaodnatoiofnp eopft ipdeep-ptidepe-tpoeidp thoiydb rhidybBri1d bBy1c hbyym cohtyrmypostriynp,striny,p tsriynp,sainnd, apnrdo nase.
pronase.
Figure 3. Proteolytic degradation of peptide-peptoid hybrid B1 by chymotrypsin, trypsin, and
2.3. StabilityinPlasma
2.3. Stabpilriotnya isne . Plasma
PlasmastabilityofB1andD2wastestedasanestimationoftheirhalf-lifeincaseofsystemic
2.3. Stability in Plasma
absor ptionorintravenousadministration. Plasmastabilitywasdeterminedbyincubationofaknown
concentrationofthepeptoid/hybridofinterestwithdogplasmaupto60minat37◦Candanalyzing

theconcentrationofthepeptideinthesupernatantbyLC-MS/MSafterproteinprecipitation. Results
Molecules2018,23,630 4of15
arepresentedasaverageoftriplicatesfromasingledonor.Threepeptideswithknownstabilityprofiles
(Peptide1–3)wereusedascontrolsinthisstudy(seeTable1). B1showedsomedegradationinplasma,
withahalf-lifeof28.9minutes,whileD2showednodetectabledegradationandahalf-life>120min
(seeSupplementaryMaterials,SectionS6).
2.4. StabiltyinLiverMicrosomes
Livermicrosomalassayisusedtodeterminehepaticintrinsicclearanceofadrugandidentification
andprofileofmetabolitesacrossspecies. Evaluationofmicrosomalstabilityofcompoundsindifferent
speciesiscommonprocedureinthedrugdiscoveryprocesstoevaluatedifferentmetabolitesincase
of instability. Invitro stability of B1 and D2 in liver microsomes was investigated by incubating
B1andD2withdogandhumanmicrosomes,andanalyzingtheresultingsolutionviaLC-MS/MS
todeterminetheresidualamountofB1andD2aftermicrosomaldegradation. BothB1andD2were
rather stable upon incubation with liver microsomes, both displaying invitro half-lives >120 min
(Table1). After60min,77%ofD2and100%ofB1wererecovered.
Table1.Half-lives(minutes)ofB1andD2andcontrolpeptides1inplasmaandlivermicrosomes.
Compound Plasma(Dog) LiverMicrosomes(Dog) LiverMicrosomes(Human)
B1 28.9 >120 >120
D2 >120 >120 >120
Peptide-1 >120 - -
Peptide-2 <3 - -
Peptide-3 81.2 - -
1Peptide-1,-2,and-3arecontrolpeptideswithlong,mediumandshortplasmahalf-life,respectively.
2.5. PlasmaProteinBinding
The plasma protein binding properties of candidate drugs can influence their efficacy, safety,
anddistribution.Thefractionofpeptoidboundtoproteinsofplasmacollectedfromdogswasdetermined
usingrapidequilibriumdialysis. BothB1andD2werehighlybound(>98%)todogplasmaproteins
(Table2). Afreefractionof0.16%ofthepeptide-peptoidhybridB1and1.43%ofthepeptoidD2were
detectedviaLC-MS/MSfromdogplasma.RecoveriesforbothB1andD2were>70%.
Table2.ProteinbindingofB1,D2,andcontrolpeptideafter4hincubationinplasma.
Entry %AverageRecovery SD1 %Unbound Bound SD1
B1 75.6 20.7 0.16 99.8 0.000
D2 70.8 6.5 1.43 98.6 0.005
Control 67.1 12.6 0.27 99.7 0.001
1Mean±SD(n=4).
2.6. InVitroRelease
To identify a suitable carrier for B1 and D2, their release from three different formulations
(oil-in-watercream,20%poloxamergelandointment,seeTable3andSection4.6)wasinvestigated.
Releasewasevaluatedasfunctionoftheamountofpeptidediffusedthroughacellulosemembranein
thereceptorofaFranzcellanddetectedviaRP-HPLCfrom0to8h. Approximately50%ofthecontent
ofbothB1andD2wasreleasedfromtheoil-in-watercreamover8h(Figure4). Thereleasekinetics
indicatedzero-orderkinetics,andrateswerecomparableforthetwocompounds. Onthecontrary,
nodetectablereleaseofB1andD2wasobservedfromthe20%poloxamergelandtheointmentafter
eighthours(datanotshown).
Molecules 2018, 23, x 5 of 15
Molecucloesn2t0r1a8r,y2, 3n,6o3 0detectable release of B1 and D2 was observed from the 20% poloxamer gel and the5 of15
Molecules 2018, 23, x 5 of 15
ointment after eight hours (data not shown).
contrary, no detectable release of B1 and D2 was observed from the 20% poloxamer gel and the
ointment after eight hours (data not shown).

Figure 4. In vitro release of B1 and D2 from oil-in-water cream formulation. Mean ± SEM, n = 2.
Figure4.InvitroreleaseofB1andD2fromoil-in-watercreamformulation.Mean ±SEM,n=2.
2.7. SkinF iPgeurrme e4a.b Iinli tvyit ro release of B1 and D2 from oil-in-water cream formulation. Mean ± SEM, n = 2.
2.7. SkinPermeability
To evaluate the absorption of B1 and D2 into systemic circulation after topical administration,
2.7. Skin Permeability
Tthoeierv ianl uvaittreo tpheermabeasotiropnt ipornofiolef Bth1roaungdh Dex2ciisnetdo dsoygs tsekmini cwcairsc iunlvaetsitoignaatefdte rint odpififcearelnatd emxciinpiisetnrta tion,
theircionnvdiiTttirooo nevsp,a elwrumiatthee a atthnieod n awbpsiotrhropofiutlitoe ntth hoefr opBur1eg sahenndec exD co2ifs ienad toskd sioynsg tpesemkrmiicne cawibrcialuistlyait nimovnoe dsatiffitigeerar tt(eotdpraicnianslc duadtiofmfle)i.rn eFisnotrtr aeetaixoccnhi,p ient
condpittheieorminr sei,ant wivoiinttr host uapdneyrdm, wdeaoitntihooonr ucphtraotmhfielbee prtshr ercosouenngtahcie neexodcfi sfaeodrsm kduionlga tpsioeknrinm a wte asaubs iitilanitbvylees mtciogonadcteiefidn etirrna t(idtorinfaf neorsfe cnpute tpeotxlo)c.iidpF ifeoonrrt each
UcoPnLdCit idoentse,c twiointh o fa npden wetirtahtoiount othf e1 %p roefs etontcael coof nat esnkti nan pde ervmeerayb sialimtyp mle owdaifsi eprr e(tsreannts tchurteoel )t.i mFoers . eTahceh
permeation study, donor chambers contained formulation at suitable concentration of peptoid for
epxepremriemateionnt w stausd ryep, edaotneodr t wchicaem. Pbeerrms ceoantitoanin wedas f moremasuularetido na sa at fsuunitcatibolne ocof tnhcee ncotrnacteionntr aotfi opne pdteotiedc tfeodr
UPLC detection of penetration of 1% of total content and every sample was present three times.
iUn PthLeC d doenteocrt icohna mofb peer noeft raa Ftiroann zo fc 1e%ll aopfp toartaalt ucso.n Ate nret parnedse envteartyiv sea cmhprolem wataosg praremse ins ts thhorween t iimn eFsig. Tuhree
Theexperimentwasrepeatedtwice. Permeationwasmeasuredasafunctionoftheconcentration
5e.x Npeor,i omre nnetg wliagsib rleep, epaetremd etwatiicoen. P(<e1r%m)e oatfi Bon1 wanads mD2e aaspuprleided a sin a afunnyc otifo tnh eo fv tehhei ccloens cwenatsr adteitoenc tdeedt eacftteedr
detectedinthedonorchamberofaFranzcellapparatus. Arepresentativechromatogramisshownin
4in8 hth, es udgogneosrt icnhga pmrbacetri coafl lay F nroa nszk icne plle anpeptraartaiotuns .o Af B r1e panreds eDn2ta. tHivoew cehvreorm, tahteo gprraemse nist esdh orewsnu litns dFoig nuoret
Figurr5eu. 5lNe. ooN,u oot r,p onoretegnnleitggiailbli glpeia,b rplteeit,rimopneeiranmtgio einna tt(oi<o 1tn%he()< so1tfr% aBt1)u oamnf dBco 1Drna2e naudpmpD.l i2eda pinp alineyd oifn thaen yveohfictlhees wveahs idcleetsecwteads adfteetre cted
after4488 hh,, ssuuggggeesstitning gprparcaticctaicllayl lnyon sokisnk pinenpeetrnaetitorna toiof nB1o afnBd1 Da2n.d HDow2.evHeor,w theev perr,etshenetpedre rseesnutletsd droe nsuoltt sdo
notruruleleo ouuttp pootteennttiiaall ppaarrttitiitoionninign ginitnot toheth setrastturamtu cmornceourmne. um.

Figure 5. Skin penetration of B1 from 50% transcutol formulation detected via UV-UPLC after 24 h.

No penetration of B1 through dog skin is observed (UV-UPLC detection limit: 1 µg/L). Since B1 and
FigurDFei2g5 su.hrSoekw 5ien. dSpk sieinmn peiltearnra esttkiroiannt ipooenfn oBeft1 rBaf1tri oofrnmo pm5a t05t0%e%rnt rtirnaa nanslslcc tuuettsootell dffo ofrormrmmuulualltaaiottiinoo nndse,d toeencttleeycd rt eevpdiare vUsieaVn-UtUaVtPiv-LUeC dP aaLfttaCe rfo a2rf4 tB eh1r. 24h.
NopaeNrneoe stphreaontweiotnrn.a toiofnB o1f tBh1r othurgohugdho dgosgk sinkinis ios bosbeservrveedd ((UUVV--UUPPLLCC ddeetetecctitoino nlimlimit: i1t: µ1gµ/Lg)/. SLi)n.cSei Bn1c eanBd1 and
D2shDo2w sehdowsiemd isliamrislakri nskpine npeetnreattriaotnionp aptattetrenrni nina allll tteesstteedd ffoorrmmuulaltaitoinosn, so,nolyn lryeprreepsreenstaetnivtaet divaetad faotra Bf1o rB1
3. Disacrue ssshioownn .
areshown.
3. Discussion
3. Di scussion
There is an urgent need for novel antibacterials specifically tailored for veterinary use only.

Developingsuchagentsforveterinarymedicinewouldaddressthecurrentlackoftherapeuticoptions
withinthisfield,andreducetheuseofcriticallyimportantantibioticsapprovedforhumansinthe
treatmentofanimalinfections[19–21].
Molecules2018,23,630 6of15
AMPs act quickly and selectively at micromolar concentrations, are rarely associated with
antimicrobialresistance,andcanbestructurallyoptimizedbychemicalsynthesis[22–24]. Theirmain
limitations as therapeutics are their very short invivo half-life and high production costs.
Despite this, to date several AMPs are under commercial development [25]. De novo design
of AMPs and peptidomimetics, such as peptoids [26–30], peptide-peptoid hybrids [31–33] and
α-peptide/β-peptoids [34,35] have been successful strategies to increase the invivo stability.
The therapeutic potential of peptoids resides in their biological activity comparable to the parent
peptide,andtheirresistancetoproteolyticdegradation[36]. Despitethelargeinterestindeveloping
peptoid-based antimicrobial agents, though, only a few studies on ADME properties of peptoid
exist [37–41]. However, these are mainly limited to short peptoids—up to four residues. In this
study,wedescribeapeptide-peptoidhybrid,B1,andapeptoid,D2,thatwerehighlyactiveagainst
S.pseudintermedius(MIC2–4µg/mL).Moreover,theinvitroADMEpropertiesofthesepeptoid-based
antimicrobialswereinvestigatedinordertoassesstheirpotentialastopicaldrugmoleculestotreat
caninepyoderma.
Topicalantimicrobialtreatmentofcaninesuperficialpyodermaisfavorabletosystemictreatment,
as very high concentrations can be reached at the site of infection. Furthermore, it minimizes the
sideeffectsandresistancedevelopmentinotherbodysites,includingtheintestinaltract[6]. Creams
andointmentsarecurrentlyemployedforthetreatmentofstaphylococcalinfectionsinveterinary
medicine [42] and AMPs formulated in ointments (neomycin, bacitracin and polymyxin B [43]),
cream(pexiganan)[44]andpoloxamergel(vancomycin)[45]areknown.
To our knowledge there is no evidence in the literature of topical formulation studies of
peptoid-basedmolecules, althoughtheyhavebeenindicatedaspromisingcompoundsfortopical
application [46]. To characterize the potential of B1 and D2 for topical treatment, we evaluated
their release from three potential formulations with different hydrophilicity features (a hydrogel,
anointmentandanoil-in-watercream),andassessedtheirpermeationthroughcanineskin. Norelease
wasobservedwhenB1andD2wereformulatedinapoloxamergeloranointment. Onlyoil-in-water
creamindicatedsuitabilityfortopicaldeliveryofB1andD2.
Thepermeationacrossexcisedskinwasfoundtobenegligibleforbothcompoundsasexpected
from their physicochemical properties. The skin permeability is often the most difficult challenge
toovercomeforpotentialtopicaldrugs[47]. Drugswithamolecularweight>500Daltonarelikely
tofailpenetratingtheSC,theexternallayeroftheskin. Furthermore,thediffusionthroughskinof
positivelychargedpeptidesmaybepreventedbyassociationwithnegativelychargedconstituentsin
theskin[48]. Toassessthepermeationthroughskin,B1andD2weretestedaloneandincombination
withchemicalpenetrationenhancers(ethanolandtranscutol)modifyingphysicalpropertiesofthe
SC [49,50]. Ethanol is widely used in topical applications and has been previously reported as a
skin-penetratingagentforitseffectoflipid/proteinextractionfromtheSCandforits“pusheffect”due
toevaporation[51]. Transcutol(diethyleneglycolmonoethylether)increasesthesolubilityofdrugsin
theskinandisfrequentlyusedasatransdermalpenetrationenhancerinrecentyears[49,52]. Evenin
the presence of enhancers known to modify the physical properties of the SC [50], no permeation
wasobserved. TheseresultsoverallindicatethatB1andD2aresuitablefortopicaladministrationto
skin. ThelackofpermeationlikelypredictsnosystemicabsorptionofB1andD2followingtopical
administration invivo. Skin penetration of peptides is generally difficult: as an example, a recent
paperreportsof5000publicationsonformulationandclinicaltrialsfortheimprovementofpenetration
ofcyclosporine[53],anaturally-occurringmultipleN-methylatedcyclicpeptidewithaMWsimilar
toD2. FurtherexperimentsareneededtoevaluatetheextentofthepenetrationofB1andD2into
theskinlayers,andincaseofskinlesionscausedbypyoderma,includinginvivostudies. However,
theseinitialresultsindicatethatB1andD2aresuitableforthetreatmentofsuperficialinfections.
To characterize invitro the behavior of B1 and D2 if systemically absorbed, their metabolic
stability,hemolyticactivity,andplasmaproteinbindingwereevaluatedalongwiththeirsusceptibility
toproteolysisbytrypsin,chymotrypsinandpronase. Thepeptide-peptoidhybridB1onlyshowed
Molecules2018,23,630 7of15
6% degradation after 24 h of incubation with trypsin and 38% with pronase, while D2 was stable.
Ourresultisingoodagreementwithoneoftheearlieststudiesonstabilityofpeptoids[54].Inthatstudy,
homologousL-aminoacid,D-aminoacid,andN-substitutedglycinepeptideandpeptoidoligomers
wereincubatedwithdifferentproteasesfromeachmajorclass(carboxypeptidaseA,chymotrypsin,
elastase,papain,pepsin,trypsin). Theauthorsfoundthattheanalogueswerevirtuallyintact,whilethe
L-peptideswererapidlydegraded.
Inourstudy,thepeptoidD2wasalsostabletoplasmaproteases,whiletheinitialconcentrationof
thepeptide-peptoidhybridB1washalvedinabout30min.Resultsindicateanincreasedstabilityindog
plasmaconferredbytheintroductionofpeptoidresidues,whilethepresenceofpeptidebondsresultsin
lowerstability. Furthermore,theN-terminusofapeptidecorrelateswithitsresistancetoproteolysisin
plasma[15],andthatmaycorrespondtothedecreasedmetabolicstabilityofthepeptide-peptoidhybrid
B1aswell. Althoughthecleavagesiteisnotknownwithcertainty,thepresenceoftwoN-terminal
Lysresiduessuggeststhesiteofproteolyticsusceptibility. Stabilitytoproteasesisalsorelevantwhen
considering topical applications, due to the presence of proteolytic enzymes of bacteria and skin.
In one of the few studies addressing the invivo pharmacokinetics of peptoids, [37] compared the
distributionofatripeptoidandatetrapeptideafterIVadministration. Thepeptoidwasnotdegraded
andrapidlyexcreted,whilethedegradationproductsofthepeptideweredetectedinperipheralbody
sites[37]. Similarresultswerereportedby[41],whoreportedcompletemetabolicdegradationofa
labelledtripeptideinbloodaftertwohourscomparedtostabilityofalabelledtripeptoid.
BothB1andD2showedgoodstabilitytoinvitrodegradationbylivermicrosomes,anassaywhich
hasbeenpreviouslyreportedtocorrelatewithinvivodata[55].Half-livesforB1andD2werehigher
than 2 h, indicating a trend of low-medium clearance, compared to controls with known behavior.
ThissuggeststhatB1andD2arenotreadilysusceptibletodegradationbylivermetabolism.Furthermore,
theskincontainsmetabolizingenzymessimilartotheliver(CYPandUDP-glucuronosyltransferase)[56],
hence,theassaymayaidinpredictingstabilityaftercutaneousadministration.
Toxicityisanimportantissuewhendevelopingantimicrobialagents. Hemolysisisastandard
assayusedwhenscreeningantimicrobialpeptides. TheconcentrationofB1resultingin50%ofhuman
bloodhemolysiswascloseto256µg/mL,comparabletoapproximately100×MIC,whilehemolysis
ofD2atthehighestconcentrationtesteddidnotreach30%. Asimilartrendalsoappearedwhenthe
experimentwasrepeatedwithdogblood. Thiscouldbeexplainedbythehigherstructuralflexibility
ofpeptoidchainscomparedtopeptidesandadifferenttypeinteractionwitherythrocytemembranes.
The absence of backbone hydrogen bonds in peptoids prevents backbone-driven aggregation and
makes peptoids better membrane permeating agents [57]. Compared to B1, the peptoid D2 has a
reducednumberofhydrogendonorsduetothepeptoidbackbone,higherhydrophilicity(featuring
fivepositivecharges,seeFigure1)andahigherdegreeofrotationalflexibility.Rotationalflexibility[58]
isgenerallydirectlyproportionaltomolecularweight,anditcountsnon-ringbondstononterminal
non-hydrogen atoms. Despite the promising results, toxicity needs to be further assessed, e.g.,
bycytotoxicitytofibroblastsorotherskincells,aswellasinvivo.
Both B1 and D2 have high protein binding, as both were >98% bound to plasma proteins in
equilibriumdialysisinvitro. Plasmaproteinbindingisalsolikelytooccurwithadministrationtoa
siteofinfection. Otherantimicrobialpeptidesanddrugcandidatesarealsohighlyproteinbound[59],
especiallyapolipoproteinandserumalbumin. Despitesomeinhibitionofantimicrobialactivity[60],
thesecompoundsretainedpartoftheirantimicrobialactivityand,furthermore,werelesscytotoxicat
thesiteofaction[61].
4. MaterialsandMethods
4.1. SynthesisandCharacterizationofPeptide-PeptoidHybrid(B1)andPeptoid(D2)
HOAt (1-hydroxy-7-azabenzotriazole) and HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3-
triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate) were from GL Biochem Shanghai (Shanghai,
Molecules2018,23,630 8of15
China). The compounds 1-naphthylmethylamine, 4-methylbenzylamine, butylamine, DIPCDI (NN-
diisopropylcarbodiimide),DIEA(diisopropylamine)andTIS(triisopropylsilane)werefromSigma-Aldrich
(Copenhagen,Denmark. TentaGelSRAM(loading0.2mmol/g),TFA(trifluoroaceticacid),piperidine,
andFmoc-protectedaminoacidswerepurchasedfromIris-BiotechGmbH(Marktredwitz, Germany).
Disposable5-mLpolypropylenereactorsfittedwithaPTFEfilterwereacquiredfromThermoScientific
(Hvidovre,Denmark).
Acetic acid and DMF (dimethylformamide), DCM (dichloromethane), ACN (acetonitrile) were
purchased from VWR (Copenhagen, Denmark). All reagents and solvents were used without
further purification. The manual solid-phase synthesis of peptide-peptoid hybrid B1 and peptoid
D2 were carried out in disposable syringes on 100 mg of TentaGel S RAM (loading 0.2 mmol/g)
using Fmoc chemistry for amino acids and the submonomer approach for peptoid building blocks
as previously described [31]. Following synthesis, the product was cleaved from the resin using
TFA/H O/triisopopylsilane(95:2.5:2.5).CrudeproductswerepurifiedbypreparativeRP-HPLCuntil
2
≥97%puritywasobtained(determinedbyanalyticalRP-HPLC(WatersCorporation,Milford,MAUSA)).
TheidentityoftheproductswasverifiedbyMALDI-TOF-MS(BrukerDaltonikGmbH,Bremen,Germany)
(seesupplementaryinformation,SectionS2).
4.2. MinimumInhibitoryConcentration
The antimicrobial activity of B1 and D2 was investigated by determining their minimum
inhibitory concentration (MIC) against a methicillin-susceptible and a methicillin-resistant strain
of Staphylococcus pseudintermedius isolated from canine skin and ears at the Veterinary Diagnostic
laboratoryatUniversityofCopenhagen,Denmark. Assayswereperformedbymicrobrothdilution
of test compounds in Mueller-Hinton broth (MHB) in microtiter plates (Corning 96-well clear
polypropyleneroundbottomplates,non-treated)accordingtotheClinicalandLaboratoryStandard
Instituteguidelines[62]. Thecompoundsweretestedinconcentrationsrangingfrom1to64µg/mL
(1, 2, 4, 8, 16, 32, 64 µg/mL). Briefly, bacteria were streaked on blood agar plates and incubated
overnight at 37 ◦C. From these plates, colonies were suspended in saline solution and adjusted
bynephelometertoobtainturbidityequivalenttoa0.5McFarlandstandard. Thesuspensionwas
diluted1:100insterile0.9%(w/v)saline,and50µLofthissuspensionwastransferredtoeachwell
ofthemicrotiterplatecontaining50µLofpeptidesolutioninwater,resultinginaconcentrationof
5×105CFU/well. Themicrotiterplatewassealedandincubatedat35◦Cfor16–20h. Resultswere
readbyvisualinspection.
4.3. HemolyticActivity
EC andEC valuesrepresentconcentrationsthatresultin10and50%hemolysis,respectively.
10 50
TheEC andEC valuesweredeterminedforB1andD2,aspreviouslydescribed[63].Briefly,two-fold
10 50
serialdilutions(1to256µg/mL)wereincubatedinPBSwithasuspensionoffreshlywithdrawnhuman
redbloodcellsinPBSin96-wellv-shapedmicrotiterplatesat37◦Cfor1hcoveredwithMicrosealfoilto
avoidevaporation,andtransferredto96-wellflatbottomedmicrotiterplates.Thereleaseofhemoglobin
wasdetectedbymeasuringtheabsorbanceat414nmbyaUVspectrophotometer(VERSAmax™Tunable
MicroplateReader,MolecularDevices,SanJosé,CA,USA).PBSandmelittinwereusedasnegativeand
positivecontrols,respectively.Allhumanbloodsampleswereobtainedafterinformedconsentofthe
voluntarydonorsuponunderstandingoftheresearchpurposes,withinlocalhospitalsandaccordingto
localguidelines.
4.4. StabilityofPeptide-PeptoidHybrid(B1)andPeptoid(D2)
4.4.1. StabilitytoProteases
The effect of proteases on B1 and D2 was tested by incubation of the compounds at 37 ◦C
with three commercially-available proteases (trypsin and α-chymotrypsin from bovine pancreas,
Molecules2018,23,630 9of15
andpronasefromStreptomycesgriseus,allpurchasedfromSigmaAldrich). Solutions,concentrations,
and reaction temperatures were selected according to manufacturer’s recommendations for total
peptidedigestion. Astocksolutionof1mMtrypsinwaspreparedin1mMTrisHClbuffersolution
containing20mMCa2+. Fortheproteolyticstabilityassay, a10µg/mLtrypsinconcentrationand
1mg/mLconcentrationofB1andD2in0.1MTrisbufferwereusedtoobtaina1:100enzyme:peptide
ratio. Reactionwasconductedat37◦Cfor6h. Chymotrypsinwassolubilizedat10mg/mLin1mM
HClsolutioncontaining2mMCaCl . Aratioof1:60chymotrypsin: peptidewasused(17µg/mL
2
chymotrypsin, 1 mg/mL peptide). Chymotrypsin reaction was conducted in 100 mM Tris HCl
containing10mMCaCl ,pH7.8at30◦C.Thereactionwithpronasewasconducted,followingthe
2
enzyme producer’s recommendation, using 0.2 µM B1 and D2 and 1% pronase powder (w/w) in
50mMammoniumbicarbonatebufferatpH8at37 ◦C.Attime0, 3, and6h, 50µLaliquotswere
removedforRP-HPLCanalysis(seesupplementaryMaterials,SectionS3).
4.4.2. StabilityinPlasma
Dog plasma was collected from a single Beagle dog at the research facilities of Zoetis Animal
Health (Kalamazoo, MI, USA) and frozen immediately until use. Proprietary linear peptides with a
short and medium half-life and a cyclic peptide with a long half-life were used as controls (Table 2).
B1,D2,andcontrolsweredilutedto10µMin2%(w/v)BSAcontaining0.2%(v/v)formicacidtoprevent
non-specificbindingtomaterials.Testcompoundswereaddedtoplasmadiluted1:1withPBSbufferpH
7.4andpreheatedfor5minat37◦C.Sampleswereremovedat0,5,10,20,30,and60min.Thereactionwas
stoppedbyproteinprecipitationbyaddinga9:1(v/v)ACN:MeOHmixturefollowedbycentrifugation
at10,000rpmfor5min,andtheremainingamountofintacttestcompoundsinthesupernatantwas
quantifiedviaAPISciex4000QLC-MS/MS(Framingham,MA,USA)(coupledwithanHPLCcolumnto
filterofftheplasmaproteins—seeSupplementaryMaterials,SectionS6)uponcompletionoftheassay.
Experimentswereperformedinquadruplicate.Thehalf-lifevalueswerecalculatedonthebasisofthe
rateofsubstratedisappearance(Equation(1))andcalculatingtheinvitrohalf-lives(t1)(Equation(2)):
2
%compoundremaining=Ae−kt, (1)
wherekistherateconstantfordisappearanceofparentcompound, tistimeandAisthestarting
concentrationandiscloseto100whendataisnormalizedtothezerotimepoint:
t =0.693/k, (2)
1/2
4.4.3. StabilitytoLiverMicrosomes
EvaluationofstabilityofB1andD2totheenzymesinlivermicrosomeswasperformedwitha
roboticsystemforliquidhandling(HamiltonMicrolabSTAR,HamiltonRobotics,Reno,NV,USA).
Dog and human microsomes were obtained from XenoTech LLC (Kansas City, KS, USA) and
reconstitutedinbuffercontainingD-(L)-isocitratetrisodiumsaltand10U/mLisocitratedehydrogenase
typeIVglycerolsolution(procine).Astocksolutionof10mMsubstrate(B1,D2orcontrols,proprietary
peptides of Zoetis, Kalamazoo, MI, USA) was diluted 1:1000 in 1% (v/v) ACN before adding to
thereactionmixture. ThereactionwasinitiatedbyaddingNADPHandsubstratetoreconstituted
microsomes. Afinalconcentrationof1µMsubstratewasincubatedwith0.5mg/mLmicrosomes
in the presence of 1 mM NADP+ in 100 mM PBS with 1 mM MgCl . Samples were collected at
2
0,5,10,20,30,and60min. Thereactionwasconductedat37◦Cbeforeterminationbyadditionof
ACN,andanalysiswasconductedbyLC-MS/MS(similartothemethoddescribedabove). Controls
withnodrugandwithnomicrosomeregenerationsystem(bufferonly)wereincluded.Allsamples
were prepared in triplicate. The rate of degradation by metabolism was measured on the basis of
the difference of substrate concentration remaining at every time point. Metabolic half-lives were
calculatedasmentionedabove.
Molecules2018,23,630 10of15
4.5. PlasmaProteinBinding
TheinvitroplasmaproteinbindingpropertiesofB1andD2wereinvestigatedwiththeuseof
ThermoScientific™rapidequilibriumdialysispolypropylene96-wellplates. Plateinsertscomprised
twoside-by-sidechambersseparatedbyadialysismembrane(8KMWCO).Thechamberscontained
1100µLplasmaor350µLdialysisbuffer(PBSbufferpH7.4),respectively. Dogplasmalotwasidentical
to the one used in the plasma stability assay. Ten mM stock solutions of B1 and D2 were diluted
1:50inwater,and200µLofdilutedB1,D2,andcontrolcompound(sarolaner,>99%proteinbound)
wereaddedtothewellscontainingplasmainanautomatedrobotforliquidhandling. Plateswere
sealedandincubatedat37◦Cfor4honashaker. Eachtestwasruninquadruplicateindogplasma.
Uponcompletionoftheassay,theplasmaproteinbindingwasdeterminedbyproteinprecipitation
withACNofbothplasmaandPBS,anddruglevelswerequantifiedviaLC-MS/MSasdescribedabove.
ThepercentageofboundcompoundswasdeterminedfrompeakareasusingEquations(3)and(4):
%free=(concentrationbufferchamber/concentrationplasmachamber)×100% (3)
%bound=100%−%free (4)
4.6. FormulationPreparation
Anoil-in-watercreamandanointmentwerepreparedasdescribedbyDanishHealthandMedicines
Authority[64].Fortheoil-in-watercream,ingredientsoftheoilphase(Table3)weremeltedonawater
bathat70◦Candmixedwiththewaterphase,whichwaspreviouslybroughttoboilandcooleddown
to65–70◦C.Themixturewasstirreduntilcooled. Theointmentformulationwaspreparedbymixing
meltedVaseline(80%)withparaffinoil(20%)onawaterbathandallowedtocoolunderstirring.Thecold
methodwasusedforpreparationofthe20%(w/w)poloxamergel[65],wherethepoloxamerpowderwas
dissolvedinpurifiedwaterat4◦Candsubsequentlyleftovernightat2–8◦Ctostabilize.Allformulations
werestoredat4◦Cformaximumsevendaysbeforeuse. LowdosesofB1orD2wereloadedintothe
formulationsjustbeforeinitializingthereleaseexperimentbyfirstdissolvinganappropriateamountof
compoundin40to60µLofwater(or96%ethanolincaseoftheointment)andmixingthisinto1mLof
theformulationtoobtainafinalconcentrationof600µg/mL.Forthehydrogel,thecompoundsdissolved
inwaterweremixedwiththecoldpoloxamersolutionbeforegellingatroomtemperature.
Table3.Compositionofthetestedcompounds.
Formulation Ingredients %(w/w)
Oil-in-watercream OilPhase
Polysorbate80 0.5
Cetostearylalcohol 5
Paraffinoil 5
Glycerolmonostearate 6
WaterPhase
Methylparahydroxy-benzoate 0.1
85%glycerol 4
Sorbitol 7
Purifiedwater 72.4
Oinment
Paraffinoil 20
Vaselin 80
Hydrogel
Poloxamer407 20
Purifiedwater 80