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Theemergenceofpeptidesinthepharmaceutical business:Fromexplorationtoexploitation
ThomasUhlig a ,ThemisKyprianou a ,FilippoGiancarloMartinelli a , CarloAlbertoOppici a ,DaveHeiligers a ,DiederikHills a , XavierRibesCalvo a ,PeterVerhaert a,b,∗
a LaboratoryforAnalyticalBiotechnology&InnovativePeptideBiology,DepartmentofBiotechnology,Delft UniversityofTechnology,Delft,Netherlands b BiomedicalResearchInstitute,UniversityofHasselt,Diepenbeek,Belgium
articleinfo
Articlehistory: Availableonline29May2014
Keywords: Peptidedrugs
Pharmaceuticalindustry
Drugdiscovery
Drugdevelopment abstract
1.Introduction
Thisminireviewtouchesuponthechallengesandopportunitiespeptidesexperienceonthe tracktobecomeanapprovedpharmaceutical.
Peptideattributesoriginallyconsideredtroublesomewithrespecttodrugdevelopment maynowturnouttobemoreconvenientratherthanunfavourable.
Besidescharacteristichightargetaffinity,biologicalpeptidesoftenexhibithigherthan expectedstability.Clearlynaturalselectivepressurehasoptimisedthesebiomolecules beyondwhatcanbeanticipatedsolelyonthebasisoftheirchemicalnature.Thisconcept isgraduallyfindingitswayintothepharmaandbiotechindustry,asillustratedbyarisein medicinalpeptidepatentapplicationsanddevelopmentalwork.
©2014TheAuthors.PublishedbyElsevierB.V.onbehalfofEuropeanProteomics Association(EuPA).ThisisanopenaccessarticleundertheCCBY-NC-SAlicense (http://creativecommons.org/licenses/by-nc-sa/3.0/).
Drugdevelopmentpipelines,whichinthefirstcenturyof theindustryhavebeendominatedbysmallmolecules,are characterisedbyhighattritionrates.Theroadtomarket authorisationhasmanyobstaclesandnexttoefficacyand tolerability,newdrugcandidateshavetomeetseveralother requirements.Besidesessentialpharmacodynamics,pharmacokinetics,toxicity,andsafetyissues,alsoeconomicfactors arevital,includingproducibility,marketcompetition,intellectualproperty,andothers.Thisiswhyinatypicaldrug
developmentprocessoftoday,>90%noveldrugcandidatesfail betweentheiridentificationandbeingputonthemarket.
Aspeptidesarereadilydegradedinsidethehumanbody, whichisequippedwithroughly600molecularlydifferentproteases [37],thisclassof(bio)chemicalshaslongbeenheld ineligiblefordrugdevelopment,anddeemedwidelyinferior tosmallmolecules.Despitesuchneglect,anumberofrecent technologicalbreakthroughsandadvanceshavesparked majorinterestintheirusagebothasdiagnosticsaswellas therapeutics.Inparticular,modern-dayanalyticalmethods, whichgreatlyexcelinsensitivity,resolutionandthroughputoverthoseavailabletothetraditionalpharmaceutical
∗ Correspondingauthorat:LaboratoryforAnalyticalBiotechnology&InnovativePeptideBiology,DepartmentofBiotechnology,Delft UniversityofTechnology,Delft,Netherlands.Tel.:+31152782332.
E-mailaddress: p.d.e.m.verhaert@tudelft.nl (P.Verhaert).
http://dx.doi.org/10.1016/j.euprot.2014.05.003 2212-9685/©2014TheAuthors.PublishedbyElsevierB.V.onbehalfofEuropeanProteomicsAssociation(EuPA).Thisisanopenaccess articleundertheCCBY-NC-SAlicense(http://creativecommons.org/licenses/by-nc-sa/3.0/).
industry,facilitatethediscoveryandidentificationofawealth ofnovelpeptideswithpharmaceuticalpotential.Furthermore,presentcombinatorialchemistryprovidesthemeans tomodifythemandtocreatecompletelyartificialvariants andalternatives.Somepharmacodynamic‘weaknesses’of peptidescannotbefullyabolishedthisway,butcleverformulationsmaymaskoramendthem.Incombinationwithan in-depthstudyofthecompletebiologyofpeptidesintheir originalnaturalsources,current(bio)technologieshavethe potentialtogenerateanamplespectrumofefficaciousand safepeptidedrugs.
Here,wereviewthesteadyriseoftherapeuticpeptides whichisapparentinthepharmaceuticalandbiotechindustryoftoday.Wewillfocusonhowapeptidedrugcandidate passesthevariousphasesinthetraditionalpharmaceutical developmentpipelineandthedifferenceshereintobothsmall moleculesandthelargerbiopharmaceuticals.Withthis,we aimtorevealthatpeptidesarebyfarnot‘undrugable’,but, instead,offerimmensemedicinalpotential.
2.Concept/history
Intermsofchemicalcomplexity,peptidesfillanichebetween typicalsmallmoleculechemicalsandthelargerproteins.Just asthelatter,theyfeatureamodularstructurewithamino acidslinkedbypeptidebondsasbaseunits.Theirsizeis limited,witharbitraryboundariessetatupto100residues [20].Nonetheless,withintheselimits,peptidesexhibitmultifariousstructureswithregardtoaminoacidsequence, post-translationalmodificationsandresultantspatialshape.
Startingaboutacenturyago(WorldWarI),theadvent ofthemoderndrugeracamewithpioneeringtherapeutic compoundsliketheopiatemorphineandthecyclicpeptide penicillin,followedintheearly1920sbythe(poly)peptide insulin.Thesedrugsintroducedanewstandardindisease treatment.Althoughpeptidesthusheldtheirplaceamong theinitialtherapeuticdiscoveries [50],smallmoleculesrapidly tookpreferenceinthedrugdevelopmentindustry,primarily duetotheireaseofproduction,simplicityofadministration(asoral‘pill’)andsuperiorpharmacodynamicproperties. Meanwhile,therapidenzymaticbreakdownofpeptidesin biologicalsystemsandtheconsequentlymorechallenging administrationroutes(e.g.injectionsuchasforinsulin)led tomoreandmoreneglectofthisbiochemicalclassinthe traditionaldrugdevelopmentprocess.
Inthe21stcentury,thepharmaceuticalbusinessisexperiencingdramaticchanges.Stringentsafetyregulations, lengthycompounddevelopmentprocessesandmassive financialefforts(Vlieghe,Lisowskietal.,2010)allincurconcernthat,despitetheincreasinginvestmentintoresearchand development,medicinalinnovationisdeclining.Especially thelastdecadehasseenamajorparadigmshiftinthescope ofthepharmaceuticalsector,focusingmoreonorphanor repurposeddrugsandreducingproductioncosts,astoendure thehighexpensesassociatedwithdrugdevelopment.Fewer newdrugsmakeittothemarketandthepatentprotection ofcurrentblockbusterdrugsisdeteriorating,witharesultingdrainageofthedrugpipelines.Allthismayultimately pushthepharmaceuticalindustrytowardsanewfrontierin
moderndrugdevelopment.Freshstrategiesareneededto revivepharma’slostmomentumandweagreewithVlieghe andcoauthors(Vlieghe,Lisowskietal.,2010)thatthesector’s hope(partly)liesinpeptides.
3.Peptidediscovery
3.1.Naturalsources
Natureharboursanimpressivevarietyofbiologicallyactive peptidesexpressedinvirtuallyalllivingspeciesand,therefore, representsoneofthemostpromisingsourcesforpeptidedrug discovery(seealso www.NP2D.com).
Withinthemulticellularbody,peptidesexertdiverse biologicalroles,mostprominentlyassignalling/regulatory moleculesinabroadvarietyofphysiologicalprocesses, includingdefence,immunity,stress,growth,homeostasis, andreproduction [24].
Throughevolution,numerouspeptideshaveevolvedto exhibittheir‘natural’bioactivityoutsideoftheproducing organism.Manyofthesehavebeenisolatedandcharacterised fromtheskinoffrogsandtoads [49,55].Thesegenetically encodedcompoundshavebeenshowntoprotectanddefend theirmanufacturersagainstmanyfoes,bothpredatorsand pathogens [13].Hitherto,over300antimicrobialpeptideshave beenidentifiedfromamphibiansthatholdpromiseforfuture antibioticresearchanddevelopment [33].
Intriguingly,manyexternallyactivepeptideshaveevolved asmeansofactivepredation,especiallyinvenomousanimals suchasspiders,snailsandsnakes(see [64]).Whilethetoxicity arisesfrominterferingwithneuronaltransmission(blocking synapticsignalling,ionchannel;e.g.conotoxins)or,ingeneral, disruptingcriticalbiochemicalsignallingnetworkswithinthe prey’sbody [61],lowdosesofthesepeptidescanactuallycounteractdisturbancesfromdiversedisorders.Accordingly,toxic peptidesmayaidintreatingpain [41],neurologicalandcardiovasculardiseases,diabetesandcancer [32].Aprominent exampleisthetype2diabetesdrug Exenatide,asyntheticversionofaglucagon-likepeptide-1analoguefoundinthevenom oftheGilamonster Helodermasuspectum [7]
Asbioactivepeptidesobtainedfromnaturalsourceshave beensubjecttoaeonsofselectivepressure,theyshow considerableplussesoverartificially/chemicallyconceived peptide-likecompounds.Namely,theyexcelinstability andtargetaffinity,bothofwhichareextremelychallengingtoachieveorreproducethroughrationalpeptide design,screeningoflibrariesofrandomlycomposedpeptides orpeptidomimetics.Althoughweappreciatetheintelligenceofpeptidemedicinalchemists,andothertraditional (bio)chemistrybasedpharmacologists,webelievethatmuch isstilltobediscoveredfromthenaturalbioactivepeptides usedalloverthebiologicaltaxonomy(frommicroorganisms overplantstoanimals).Withsomanyofthesebeingusedas drugsbysomanydifferentspeciesforsomanydifferentpurposes,itisclearthatmankindcanstilllearnalotfromthe impliedbiology.Wewould,therefore,wholeheartedlysupport anadjustmentofthenameofthe‘NaturalPeptidestoDrugs’ NP2Ddiscussionforumto‘NP4D’(NaturalPeptidesforDrugs).
3.2.Peptidomics
Todate,oneonlybeginstograspthemagnitudeofpeptides occurringinnature,aconsiderableshareofwhichpotentiallyofferstherapeuticordiagnosticmerit.Therefore,more emphasisatstudyingtheseeminglyendlessvarietyofnatural peptidesandtheirbioactivities,isdefinitelyjustified.Thisis exactlywhattherecentscienceof‘Peptidomics’isallabout.
Inthepastdecade,wehaveseengreattechnological stridesinpeptidemanipulationandanalyticalassessment. Theseincludeadvancesinliquidhandlingdevices,syntheticproteinsynthesis,recombinantproteinexpression, multispectralmicro-platetechnologies,massspectrometry, liquidchromatography,cellculturemethodologies,sequencingtechnologies,imagingtoolsandhighthroughputpeptide screeningprotocols [6].Coupledtothesetechnological advances,thecommercialbiotechnologysectorissteadily growing [6],yieldingincreasednumbersofcommerciallyavailablebiochemicalassaysandhighthroughputscreeningtools. Whereasexpensivehigh-techproteomictechniqueshavepreviouslymainlybeenadoptedinbigpharmaandbiotech,we nowseethatthenewesttechnologiesarebecomingaccessibleandarebeingdevelopedprimarilyinacademiaandsmall biotech.Giventhemanifoldofmoderntools,anewgeneration ofmoderndrugdesignersisemerging,exploitingthepotentialofferedbythelatestdevelopmentsinallrelevantscientific disciplines,suchasbiology,biochemistry,genetics,transcriptomics,proteo/peptidomics,computerscience,mathematics, andmanyothers.
Asystematicapproachtoidentifybiologicallyandphysiologicallyactivepeptidesandthustheirpharmacological promiseisfundamentaltothestudyofpeptidomics.This relativelyyoungdisciplineaimstoholisticallyanalysethe spectrumofpeptidesfoundinanychosenorganism,mostlyby meansofmassspectrometry(MS),tightlylinkedtoadvancementsinbioinformaticstechnologywhichisessentialtokeep trackofandcopewiththehugedatasetsgenerated.
Evolutiontowardshigh-throughputMSanalysisinboth qualitativeidentificationandquantificationwasaidedbythe latestimprovementsinionisation(mainlyelectrospray,ESI) andinhighspeed,highsensitivityandresolutionanalysers suchasorbitrapsystems.Inaddition,theemergenceofmass spectrometryimaging(MSI)representsoneofthemostfascinatingprogressesinthisfieldrevealingthedistributionof peptidesinabiologicalsampleandconsequentiallyinferring theirpotentialbiologicalsourceandpurpose [40]
3.3.Peptidedrugcandidatescreening
Foraconventionaldrugcandidatescreening,theso-called ‘leaddiscovery’process,inwhichtraditionallylargesetsof typicallysyntheticsmallmolecularcompoundsofpredefined structurearepharmacologicallytested,itisessentialtodisposeofwell-characterisedpeptidelibrariestorunapeptide drugdiscoveryprogramme.However,also‘reversepharmacology’usingchromatographicallyfractionatedextractsfrom originallyimpurebiologicalsourcesof‘natural’peptidemixtures,isanapproachwhich,thankstothetechnological advancesinpeptidomicsofthepastyears,mayprovetobe muchmoresuccessfulcomparedtothepast.
Fig.1–Approachesforpeptideaffinityanalysis.Legend: cyanrods:specifictargetbinders,lightgreyrodsother peptides.Darklargerrodsinrightpanelrepresent bacterophages.Orangeforksrepresentspecificpeptide targets(e.g.aGPcR).Greyforksrepresentnon-specific targets.Peptideaffinitiestowardsoneorseveraltargetscan beinvestigatedbyimmobilisingeitherofbothand identifyingthebindingspecies(e.g.viadetectabletags and/orviamassspectrometry).Athirdoptionisphage display,wherepeptidesofinterestexposedonthesurface ofbacteriophagesbindtotheirtarget.TheirrespectiveDNA sequenceiscontainedwithinthephageandallows identificationofthepeptide.
Entirelyparalleltothehighthroughputscreeningof smallmoleculechemicallibraries,syntheticpeptidechemicallibrariescanbeproducedusingtoday’stechnologies. Thesecanincludenaturalpeptidesequences,supplemented withderivativesthereof,suchasnaturallyoccurringposttranslationallymodifiedandtruncatedisoforms,orentirely artificialpeptides.Indeed,peptidesofanydeliberatelychosen orrandomlyassembledsequencecanbeproducedbypurely chemicalmeansorbyrecombinantorganisms.Bothallow foranimmenseflexibilityinsequenceandpost-translational modifications(PTMs)enablingthecreationofcorrespondingly vastlibrarieswhichpotentiallycontainsubstancesoftherapeuticinterest.Weremarkherethatthechoiceofresidues isbyfarnotlimitedtoproteinogenicaminoacids,andis basicallyonlylimitedbytheimaginationandcompetence ofthesyntheticpeptidechemistand/orpeptidemolecular biologist.Indeed,whilechemicalsynthesismayintegrateany compoundabletobondwiththenascentpeptide [1],theintroductionofartificiallyexpandedgeneticcodeshaswidened therangeofrecombinantpeptidestructuresbyinsertingnonconventionalaminoacids [66,52]
Threestrategiesforscreeningartificialpeptidelibrariescan bedistinguished(Fig.1).Inthefirstapproach,peptidessynthesisedonasolidsupportarecleavedoffforactivityscreening [26].Secondly,peptidesareassessedwhilestillattachedtothe solidphaseonwhichtheyweresynthesised.Finally,phage displayisthethirdapproachwherebacteriophagesexpressingthepeptideandexposingitontheirsurfaceareanalysed fortheiraffinitytoaselectedtarget.
Besidestheformerpeptidechemistry-andmolecular biology-basedstrategiestopopulatepeptidelibrarieswhich canbescreenedinstraightforwardpharmacologytests,also reversepharmacologicalapproachesstartingfromnatural sourcesofbiologicalpeptidemixtureshavebecomeavery realisticalternative.Here,screeningsareperformedwithinitiallyuncharacterisednaturalpeptidestructuresisolatedand fractionatedfromtheirbiologicalsource.Biologistshelpto
identifythesourceswiththehighestpotential,considering thediseasetargetaimedfor.Richmixturesofbiologicalpeptidescanbefoundthroughouttheentiretaxonomy,from vertebratesoverinvertebratesandplantstomicroorganisms. Classicalexamplesarevenomanddefensiveglandsecretions whicharefoundthroughoutzoology(seee.g. www.np2d.com; www.venomics.eu).
Thecomponentsofpeptidelibrariescanbeassayedfor theirbioactivity invitro (targetaffinity,bindingkinetics,etc.) and invivo (alteredgeneexpression,cytotoxicity,etc.).Combinatorialanalysisofknownpeptidesequences(e.g.,Ala-scans) andtheextentastohowchangesintheprimarystructureaffecttheoriginalbiologicalactivity,enablestoreveal structure–functionrelationships [18].This‘deconvolution’ allowstheselectionofsequencesforpeptideswithadesired activity.Assuch,itessentiallyprovidesthefundamentfor rationaldesignofpeptideswithpredefinedbiologicaleffects.
Inallcases,peptidelibraryscreeningsareintendedto showmolecularinteractionwithadrugtargetusingdifferent visualisationtechnologiescomprisingcolorimetry,fluorescencemicroscopy,flowcytometryandothers.Classesof biomoleculeswhichhavepreviouslybeenextensivelyinvestigatedandsuccessfullymodulatedastraditionaldrugtargets includeionchannels,nuclearorG-protein-coupledreceptors, transcriptionfactorsorenzymes.
4.Large-scalepeptideproduction
Onceapeptidehasbeenselectedforfurtherdevelopmentas pharmacon,itneedstobeproducedinlargequantitieswith consistentquality,accordingtogoodmanufacturingpractice (GMP)rules.
Thestrategyforproducingapeptideislargelydetermined byitssizeandchemicalfeatures.Avarietyoftechnologies suchaschemicalsynthesis,recombinantDNAtechnologies, cell-freeexpressionsystems(invitro translation)andtransgenicplantsoranimalshavebeenadoptedforthispurpose (Fig.2).
Traditionally,theproductionofnaturalcompoundsis achievedbyusingamicrobialorfungalstrainthatunderwent aseriesofinducedmutationsandsubsequentscreenings forproductivity.Throughthisprocess,theproductionyield maytypicallyberaisedbyuptothreeordersofmagnitude. Specificallydesignedstrainswithenhancedproteinsynthesis,secretionandfoldingcapabilityprovideasolidplatform andstartingpointforreachinghighpeptideyields [34]
Large-scaleoperationsincorporatingchemicalsynthesisasthecoreproductiontechnologymaybeseenasan attractivealternativetoexistingrecombinantDNA-basedor biocatalyst-basedmethodologies [65].Chemicalsynthesiscan bedistinguishedintothreemajorcategories,namelysolution phase,solidphaseandhybridapproaches.Selectionofthe mostfavourableproductionprocessprimarilydependsonthe setobjectiveandthelimitationsthateverymethodpresents.
Themajorityofpeptidepharmaceuticalsareproducedin highvolumesusingsolutionphasechemistrywhichispreferablyemployedforsmalltomedium-sizedpeptides.Prolonged developmenttimesareamajordrawbackfortheapplication ofthistechniqueespeciallyduringearlyclinicalstudieswhen
Fig.2–Meansofproducingtherapeuticpeptides.Peptide manufacturingcanbeachievedentirelythroughchemical synthesis,eitherinthesolutionphase(topleft),coupledto asolidphase(topright)orbythecombinationofboth. Alternatively,peptidescanbeproducedbyrecombinant microorganisms(bottomleft)orbyextractionfromtheir natural(plantsoranimal)source(bottomright). rapidproductionofthedesiredsubstanceiscrucial.Nevertheless,significantadvantagesofthismethodologyarethe well-establishedisolation,characterisationandpurification protocolsoftheintermediateproducts [1].Ontheotherhand, solidphasepeptidesynthesis(SPPS)hasenabledtheproductionoflarge,complexpeptidesofpharmaceuticalgradepurity onalargescale [1,65].Finally,hybridprocessescombiningthe advantagesofbothtechniquesmayofferevengreaterpotential [1]
Irrespectiveoftheupstreamproductionmethodofchoice, downstreamprocessingisavitalstepinthemanufacturingof peptidepharmaceuticalproducts,sinceitinvolvesthecritical stepsassociatedwithproductisolationandpurification [1]
5.Peptidedrugregistration
Peptidesrepresentaspecialcaseinregulatoryaffairs,since, dependingonitspropertiesandmanufacturing,apeptideis sometimesregardedasaconventionalchemicalmedicinal product,andinothercasesasabiologicalentity.
TheUnitedStatesFoodandDrugAdministration(FDA) traditionallyhandlespeptidesasconventionaldrugs,notas biologicalproducts [20].Thisgoesalongwiththefocusof examinationonthedrugcompositionandcompoundstructureratherthanthemeansofmanufacturing.Accordingtothe FDA,theuppersizeboundaryofchemicallysynthesisedpeptidesisat100aminoacidresidues.Exceptions,however,are madewherepeptidesotherwisemeetthestatutorydefinition ofabiologicalproduct,suchasinthecaseofpeptidevaccines.
7.Peptidedrugformulation
Aspromisingaspeptidedrugcandidatesmaybe,anumberof conceptuallimitationsremainassociatedwiththetraditional imageofapeptideasatypicalmedicine.Thisperceptionis largelysubstantiatedbyLipinski’s“ruleoffive”whichsummarisestheidealpharmacokineticpropertiesofthemost successful(chemical)drugcandidatesoftheoriginalpharma industry.Thisdecreedogmatisesthatpeptidesarelesslikely topassthroughthegastrointestinal(GI)tractwallascompared tosmallmoleculesduetotheirlargersizeandcomparatively lowsolubility [65]
Manychallengespeptidesexperienceontheirwayto becominganeffectivedrugoriginateintheirphysicochemical propertieswhichtogetherresultinapoororalbioavailability.Duetotheirhydrophilicity,peptidesexhibitlimitedability tocrossphysiologicalbarriers.Inaddition,peptidesareconfrontedwithefficienthepaticandrenalclearance.Evenonce insidethesystemiccirculation,peptidestypicallyhaverather shorthalf-lifesduetoaggressivedegradationbyamultitudeof proteases [3].Theseaspects,intrinsictothechemicalnatureof peptides,havecompelledcumbersomeadministrationroutes suchasdirectinjectionofrepeateddoses,whichinturn resultedinlowpatientcompliance [3]
Asidetheclassicsubcutaneous,intramuscularandintravenousadministration,alternativerouteshavebeendeveloped,includingthemucosaltrack(nasalspray,pulmonary deliveryorsublingualdelivery),theoralroute(GItractpenetrationenhancers,proteaseinhibitorsorcarriers)andthe transdermalpath(patches; [3,65]).Forinstance,apeptidepill fororaladministrationisdeveloped(EnterisBiopharma)the coatingofwhicheffectivelyprotectstheactivesubstancefrom digestioninthestomach,allowingitsreleaseintheduodenum.Anumberofexcipientsprotectthepeptideagainst peptidasesandfacilitateitsparacellularuptakethroughthe intestinalwallintothesystemiccirculation [57]
Benefitsofthepulmonaryintakeareaverylargeavailable absorptiveandhighlypermeablesurface,extensivevascularisation,withconcomitantrapidonsetofpharmacological action,amoreuniformdistributionofthedrugproductand amoresustaineddrugreleasewhichallowsareductionof thedosingfrequency [2,28].Likeotherparenteraladministrations,thefirstpassmetabolismisavoided.Alsoa10–200times higherbioavailability(higherdrugproductplasmaconcentrations)canbeachievedbecauseofthesmallervolumesused [56,63,2].
Besidesalternativedeliverysystems,variousformulationshavebeendevisedinthepast,tryingtodealwiththe aforementionedphysicochemicallimitations,tohelpadvance promisingpeptidedrugleadsintopharmaceuticaldevelopment.Again,mostofthesemedicinalpreparationsare chemistry-andnanophysics-based.Indeed,chemicalincorporationofsugarsliketrehalose,sucrose,maltose,glucose, ofsaltslikepotassiumphosphate,sodiumcitrate,ammoniumsulphateand/orofotheragentssuchasheparininto theprospectiveformulationshavebeenfoundtoincrease thesolubilityand invivo stabilityofpeptides.Employmentof cationicandanionicsurfactantssuchascetrimideandsodium dodecylsulfate(SDS)haveshownenhancedtransportation
Fig.3–Modernformulationsfortheprotectedand optionallytargeteddeliveryoftherapeuticpeptides.Several modernformulationsofferenhancedtissuepenetration, delayeddecayoftherapeuticpeptidesandtargeteddelivery byexposingspecificreceptorligands.Liposomes(top) harbourlipophilicpeptideswithinthelipidbilayer,while hydrophilicpeptidesarestoredintheaqueouscore.Onthe otherhand,micelles(bottomleft),nanoemulsions(bottom centre)andpolymernanoparticles(bottomright) incorporatelipophilicpeptidesintotheircore.Theinsertat thetopleftliposomerepresentsatypicalcellmembrane amphiphilic(phospho)lipidwithlightgreydenotingthe polarheadgroupanddarkgreyindicatingtheapolar moieties. ofpeptidesacrossbodilymembranes.Considerableincrease inresistanceagainstproteolysisisoftenachievedthrough co-administrationofproteaseinhibitorssuchassodiumglycocholate,camostatmesilateorbacitracin.Moreover,the attachmentofpolymericmoleculessuchaspolyethylene glycol(PEG)andpolyvinylpyrrolidone(PVP)andeventhe encapsulationofthepeptideintonanocarriersareemployed forextendedbioavailability(Antosova,Mackovaetal.,2009).
Nanocarriertechnologyindeedseemsapromisinginnovationtoincreasepeptidepharmacodistributionthrough,for example,nanoparticles,liposomesandmicelles(Fig.3).These arethoughttoeffectivelycreateaclosedcarrierthatprotectstheactivecompoundfromdestabilisingexternalthreats suchapeptidases.Incombinationwithpulmonaryinhalation, nanocarriershavethebenefitofprolongeddrugreleasedue tothecombinationofpeptideprotectionbythecarrier’sshell withcarrieraccumulation [2]
Ingeneral,anidealnanocarriershouldbecomposedof inertandbiodegradablematerialandbeabletoefficiently encapsulateandprotectthepeptideagainstdegradationwhile atthesametimemaintainingproperdrugproducttargeting [28].Compositionsofpolymericformulationscanbeused totunethebiologicalbehaviourofnanocarriersbymodulatingcompoundpropertiessuchasmucoadhesiveness [8] Anexampleisthecationicpolysaccharidechitosan,theionic interactionsofwhichwiththenegativelychargedsialicacid groupsinmucinprovideexceptionalbinding.Incombination
withitsabilitytoopentightjunctions,chitosaneffectively favourstheutilisationoftheparacellularpathway [11,53,2] Hybridscanbecreatedbycombiningchitosanwithother polysaccharidesoroligosaccharidesinanattempttofurther improvethephysicalpropertiesandpharmacologicalperformanceofpeptidedrugformulations [23,28]
Othersyntheticpolymerssuchaspolylacticacid(PLA) andpoly(lactic-co-glycolic)acid(PLGA)havebeenfound viablesubstitutes.Theirconsistencyintermsofpeptidedrug productreleaseincombinationwithgoodbiodegradability guaranteesoptimalsafety.Accordingly,theFDAhasalready approvedanumberofrespectivemarketedproductsandclinicalapplications [11,2,28]
Liposomalnanocarriersarecomposedofoneormultiple phospholipidbilayers.SpecificligandsconjugatedwithmodifiedPEGmoleculesintheshelloffertargeteddelivery [30,28]. However,liposomesoftenfacevaryinginstabilitiesinbiologicalfluids [2].Nanoparticleswhichconsistofasolid(bothat ambientandbodytemperature)lipidmatrixdispersedinan aqueousphasemayofferanalternative.Onedistinguishes solidlipidnanoparticles(SLNs)andnanostructuredlipidcarriers(NLCs).WhileSLCsarerigidlystructured,NLCsconsist ofbothsolidandliquidlipidsallowingforanincreasedloadingcapacity [39,43,2].Finally,self-nanoemulsifyingsystems, i.e.isotropicmixturesofoilandsurfactantformingthermodynamicallystableoil-in-wateremulsions,mayhavepotential forenhancedoralbioavailabilityofpoorlywatersolublepeptides [28]
Besidesthesemodern(nano)technology-inspiredformulations,weherewouldliketodrawtheattentiontothewide varietyofbioactivepeptidedeliverysystemsfoundinnature. Evolutionhasdesigneddirectinjectiondevices,verymuchlike injectionneedles,suchasfangsorvenomteethinvariousreptiles,harpoon-likeradulasinsnails,orclaws.Howeveralso organismslackingtheseappeartosuccessfullyusebioactive peptidesintheirglandsecretions.Acarefulanalysisofthe compositionofpeptide-containingvenomanddefenseglands inanimalslikeamphibians,may,therefore,givehintsasto howpotentialtherapeuticpeptidescanbesuccessfullyformulated.Theexosomalreleasewhichisobservedfromvarious venomglandsinawayiscomparabletothemicellarencapsulationofpeptidesdescribedabove.Biologyalsohintsto additionaltrickstoensureeffectiveuseofpeptidebioactives. Innaturepeptidesarealmostneversecretedontheirown, butalwaysincocktails,basicallyamixtureofthreetypesof peptides.Thesenotseldomcontainpolycationicamphipatic (poly)peptides(sometimescataloguedas‘antimicrobialpeptides’(AMPs))whichcouldserveasmembranepenetrating deliverysystems.Co-secretionof‘peptidaseinhibitors’isalso astrategyseeninvariousanimaldefenseglands.Finally various(oftenpost-translational)modificationsonthetrue ‘bioactivepeptides’fitinastrategytorenderthe(active partofthemolecule)improved/prolongedstabilityaswellas tighterreceptorbinding.Theseincludesequenceelongation protectingthebioactivesitefromrapidexoproteasedigestion,C-terminalamidationand/orN-terminalringformation (pyroglutamicacidfromglutamine)forchargesuppression (reductionofhydrophilicity),disulfidebridgeformationfor increasedstructurepreservationandrigidity,andisomerisationofselectedpeptideterminalresidues(from l-into
Fig.4–Graphicalrepresentationofnumberofpeptidesin preclinicalandclinicaldevelopmentasof2013 [31].
d-aminoacid)forenhancedexopeptidaseresistance.Examplescanbefoundinthedefensivesecretionsof Phyllomedusa burmeisteri and Bombinavariegata,asdescribedelsewherein thisspecialissue [47]
8.Clinicaldevelopment
Thetherapeuticpeptidedevelopmentisgrowing.In2011 alone,therewerebetween500and600peptidesinpre-clinical phases(Fig.4; [31]).Theyear2012hasproventobeanother milestoneforthepeptidepharmaceuticalsector,with5and6 peptidesmeetingmarketapprovalrespectivelyinEuropeand intheUSA.Thiswasthehighestnumberofapprovalsever achievedfornewbiologicalentities(NBEs)inoneyear [29] whichrenderssomeoptimismtothesector.Thisoptimismis confirmedbythestatistics,astheregulatoryapprovalratefor peptidesisaround20%,versus10%forsmallmolecules [31] Inaddition,thenumberofpeptidesperyearenteringclinical trialshassteadilyincreasedfrom1in1970tocurrentlyaround 20 [31]
AsofApril2012,theclinicalpipelineforpeptidedrugswas composedof128peptidecandidates.Theseincluded40in phaseI,74inphaseIIand14inphaseIII(Fig.4).Therobustnessofthispipelineislargelyduetothenotableexpansion inthefieldofpeptidetherapeuticsduringthelate1990sand 2000s,ultimatelyleadingtothenumberofapprovalsobserved in2012.Consideringthegeneralfailurerateoftheclinical pipeline [29],thesenumbersproveverypromising.
InPhaseI,themostrepresentedindicationsarepain(more than30%),cancerandcardiovasculardiseases.InPhaseIIand III,inwhichcanceristheleadingtarget(accountingformore than15%and40%,respectively),onefinds,nexttopainand infectiousdiseases,alsoindicationsnotmuchrepresentedin thecurrentmarket,suchasdermatology,allergies,andCNS disorders [29]
8.1.Typical(GPC)receptorbindingpeptides
Oneareaofresearchthathasshownpromisingdevelopmentaretheuseofpeptidetherapeuticsintreatingtype 2diabetes(targetingtheglucagon-likepeptide1receptor). Alreadythreepeptideshavereceivedapprovalin2012,with 14workingtheirwaythroughthepipeline.Amostexcitingaspectofthesepeptidedrugcandidatesisthevarietyin drugformulationofmolecularformats(withpeptidesbeing covalentlylinkedtosmallmolecules,carbohydrates,lipids, biopolymers,polyethyleneglycolorproteins(seeabove))and
Fig.5–Trendinpatentapplicationsfortherapeutic peptidesfrom1980until2012 [14].
asfood.AnEspacenetdatabasequeryforpatentapplications belongingtocategoryA61K38foreachyearbetween1980and 2013(thedataforthepartialyear2013wereextrapolatedto thewholeyear)yieldsaninterestinggraph(Fig.5; [14]).
Approximately389,320patentapplicationswithinthepeptidefieldshavebeenpublishedintheinterval1980–2013. Startingfromtheyear1996thenumberofpatentapplicationsperyearhaveinvariably surmounted10,000,averyhigh number,reflectingaverydynamicdevelopmentofthepeptidemarket.Atthesametime,italsoshowsanapparentpeak inthenumberofapplicationsperyearreachedin2003(with 23,690newpeptide-relatedpatentapplications).
Consideringthatanaverageapprovaltimefornewdrugs is10yearsandthatthepeakinpeptidepatentapplications occurredintheinterval2000–2005,onemaypredictapeak inthenumberofpeptidesenteringthemarketasnewdrugs between2010and2015.Apparently,anunprecedentednumberofpeptideshaveinfactreceivedmarketapprovalinthe years2010–2013(seeabove).Consideringthatatypicalprotectiontimeforagrantedpatentis20years,andthatan off-patentdrugtendstoloseasignificantpartofitssalesrevenuetoprice-basedcompetitionbygenericsinthefewyears aftertheendofpatentprotection,weanticipatethatpeptidebaseddrugsmaydeliverrisingsalesrevenuesforpharmaina significantnumbersofyearstocome,reasonablybeyond 2020.
10.Thepeptidepharmamarket
Todate,around100therapeuticpeptides(mostlyinnovative syntheticones)areonthemarketintheUSA,Europeand Japan,includingthosefordiagnosticsapplications [29].The increasingnumbersofrecentlyapprovedpeptidesisaresult ofthewell-filledpipeline,asdescribedabove.Themarket appearsdominatedbythethreepeptidesGoserelin/Zoladex andLeuprolide(twogonadotropinreleasinghormoneagonists,usedinhormone-sensitivebreastandprostatecancer), andOctreotide(asomatostatinmimicusedagainstvarious tumours),whichaccountforannualsales(2011)betweenUSD 1.2and1.4billion [45,4,59],intotalaround25%oftheglobal peptidemarket [62]
Fig.6–Worldwidepeptidedrugmarketdistributionin 2011.GlobalpeptidesnetsalesamounttoUSD14.7billion (therelativelyminorcontributionofOctreotidetothe genericsmarket(USD0.15billion)wasnotconsidered).
Acleartrendoverthepastyearsisthecontinuallyexpandingcontributionofpeptidestotheworldwidepharmaceutical market.OfaglobalmarketworthUSD956billionin2011 [27], thepeptides’sharewasaboutUSD14.4billion [29,62],comprisingaround1.5%.ThemajorcontributoristheUSmarket, whichaccountsfor40%oftheglobalpeptidesales.In2011,the cancersub-marketwasthelargest,representing21%ofthe totalpeptidemarket,followedbymetabolicdisorders,gastrointestinaldiseasesandrespiratoryindications [62] with86% oftheapprovedpeptidedrugsworkingthroughtheparenteral route.Asindicatedabove,however,alternativeroutesareforeseentoexpand [29].Thetotalmarketpotentialofpeptidesmay besignificantlylarger,since,inadditiontothetherapeutic pharmaceuticalmarket,peptidesareexpectedtocontribute moreandmoreinothermarketssuchasthenutraceutical business [5]
Inthecurrentstagnatingpharmaceuticalindustry,peptidesareconsideredtohaveaddedvalue,byrepresenting apotentialsolutiontomoreefficaciousdiseasetreatment. Alreadytodaytheyappearmaturecompoundsaddressing unmetmedicalneeds,andacceleratingthepersonalised medicinemodel [10].Inaddition,peptidespromisetocombine thelowerproductioncostsofconventional(smallmolecular chemical)drugswiththehighspecificityof(thelarger)biologicalentities.
Importantly,theproportionofpeptidesinpharmaisanticipatedtoincrease,sinceitisestimatedtogrowfaster(9.4% annualgrowthin2012–2018) [62] thantheglobalindustry (3–6%annualgrowthin2012–2016) [27].Notonlyisthenumberofapprovedpeptidedrugsexpectedtogrowbutalsothe diversityoftreatedindications.
Itisremarkablethatgenericsalesalreadyaccountfora considerableportionofthepeptides’market.In2011,genericsrepresented35%ofthepeptidemarket,withthreeofthe fivetopsellingpeptidesbeinggenerics [5,62].Octreotidefor instancehasagenericversionaccountingforUSD0.15billionannualsales [9].Incomparison,genericsalesintheglobal pharmaceuticalmarketaccountforaround21%andbiosimilarsinthebiologicssub-marketforaround0.4%inthesame year [27]
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