The emergence of peptides in the pharmaceutical business: From exploration to exploitation

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Available

online

at

www.sciencedirect.com

ScienceDirect

j o ur na l h o me p a g e:h t t p : / / w w w . e l s e v i e r . c o m / l o c a t e / e u p r o t

The

emergence

of

peptides

in

the

pharmaceutical

business:

From

exploration

to

exploitation

Thomas

Uhlig

a

_,

_Themis

_Kyprianou

a

_,

_Filippo

_Giancarlo

_Martinelli

a

_,

Carlo

Alberto

Oppici

a

_,

_Dave

_Heiligers

a

_,

_Diederik

_Hills

a

_,

Xavier

Ribes

Calvo

a

_,

_Peter

_Verhaert

a,b,∗

a_Laboratory_for_Analytical_{Biotechnology}_&_Innovative_Peptide_Biology,_Department_of_{Biotechnology,}_Delft UniversityofTechnology,Delft,Netherlands

b_Biomedical_Research_Institute,_University_of_Hasselt,_Diepenbeek,_Belgium

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Availableonline29May2014

Keywords: Peptidedrugs Pharmaceuticalindustry Drugdiscovery Drugdevelopment

a

b

s

t

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a

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t

Thisminireviewtouchesuponthechallengesandopportunitiespeptidesexperienceonthe tracktobecomeanapprovedpharmaceutical.

Peptideattributesoriginallyconsideredtroublesomewithrespecttodrugdevelopment maynowturnouttobemoreconvenientratherthanunfavourable.

Besidescharacteristichightargetafﬁnity,biologicalpeptidesoftenexhibithigherthan expectedstability. Clearly naturalselective pressure hasoptimised thesebiomolecules beyondwhatcanbeanticipatedsolelyonthebasisoftheirchemicalnature.Thisconcept isgraduallyﬁndingitswayintothepharmaandbiotechindustry,asillustratedbyarisein medicinalpeptidepatentapplicationsanddevelopmentalwork.

1. Introduction

Drug development pipelines, which in the ﬁrst century of theindustry havebeen dominatedbysmall molecules,are characterised by high attrition rates. The road to market authorisationhasmany obstaclesand next toefﬁcacy and tolerability,newdrugcandidateshavetomeetseveralother requirements.Besidesessentialpharmacodynamics, pharma-cokinetics,toxicity,andsafetyissues,alsoeconomicfactors arevital, includingproducibility,marketcompetition, intel-lectualproperty, and others.This iswhy ina typicaldrug

∗ _{Corresponding}_author_at:_Laboratory_for_Analytical_{Biotechnology}_&_Innovative_Peptide_Biology,_Department_of_{Biotechnology,}_Delft

UniversityofTechnology,Delft,Netherlands.Tel.:+31152782332. E-mailaddress:p.d.e.m.verhaert@tudelft.nl(P.Verhaert).

developmentprocessoftoday,>90%noveldrugcandidatesfail betweentheiridentiﬁcationandbeingputonthemarket.

Aspeptidesarereadilydegradedinsidethehumanbody, whichisequippedwithroughly600molecularlydifferent pro-teases [37], this class of(bio)chemicalshaslong been held ineligiblefordrugdevelopment,anddeemedwidelyinferior tosmallmolecules.Despitesuchneglect,anumberofrecent technological breakthroughs and advances have sparked majorinterestintheirusagebothasdiagnosticsaswellas therapeutics.Inparticular,modern-dayanalytical methods, which greatly excel in sensitivity, resolution and through-put over those available to the traditional pharmaceutical

http://dx.doi.org/10.1016/j.euprot.2014.05.003

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industry,facilitatethediscoveryandidentificationofawealth of novel peptides with pharmaceutical potential. Further-more,presentcombinatorialchemistryprovides themeans tomodifythemand tocreatecompletely artificialvariants and alternatives. Some pharmacodynamic ‘weaknesses’ of peptidescannotbefullyabolishedthisway,butclever formu-lationsmaymaskoramend them.Incombination withan in-depthstudyofthecomplete biologyofpeptidesintheir originalnaturalsources, current (bio)technologieshavethe potentialtogenerateanamplespectrumofefficaciousand safepeptidedrugs.

Here,we reviewthe steady rise oftherapeuticpeptides whichisapparentinthepharmaceuticalandbiotech indus-tryoftoday.Wewillfocusonhowapeptidedrugcandidate passesthevariousphasesinthetraditionalpharmaceutical developmentpipelineandthedifferenceshereintobothsmall moleculesandthelargerbiopharmaceuticals.Withthis,we aimtorevealthatpeptidesarebyfarnot‘undrugable’,but, instead,offerimmensemedicinalpotential.

2. Concept/history

Intermsofchemicalcomplexity,peptidesﬁllanichebetween typicalsmallmoleculechemicalsandthelargerproteins.Just asthe latter, theyfeaturea modularstructurewith amino acids linked by peptide bonds as base units. Their size is limited,witharbitraryboundariessetatup to100residues

[20].Nonetheless,withintheselimits,peptidesexhibit mul-tifarious structures with regard to amino acid sequence, post-translationalmodiﬁcationsandresultantspatialshape.

Startingabout a century ago (World War I), the advent ofthe modern drug era camewith pioneering therapeutic compoundsliketheopiatemorphineandthecyclicpeptide penicillin, followed in the early 1920s bythe (poly)peptide insulin.These drugsintroduced anewstandard indisease treatment. Althoughpeptides thus heldtheir place among theinitialtherapeuticdiscoveries[50],smallmoleculesrapidly tookpreferenceinthedrugdevelopmentindustry,primarily due to their ease of production, simplicity of administra-tion(asoral‘pill’)andsuperiorpharmacodynamicproperties. Meanwhile, the rapidenzymatic breakdown of peptides in biological systems and the consequently morechallenging administrationroutes(e.g. injectionsuchasforinsulin)led tomoreand more neglect ofthis biochemicalclass inthe traditionaldrugdevelopmentprocess.

Inthe21stcentury,thepharmaceuticalbusinessis expe-riencing dramatic changes. Stringent safety regulations, lengthy compound development processes and massive ﬁnancialefforts(Vlieghe,Lisowskietal.,2010)allincur con-cernthat,despitetheincreasinginvestmentintoresearchand development, medicinal innovation is declining.Especially thelastdecadehasseenamajorparadigmshiftinthescope ofthe pharmaceutical sector, focusingmore on orphanor repurposeddrugsandreducingproductioncosts,astoendure thehighexpensesassociatedwithdrugdevelopment.Fewer newdrugsmakeittothemarketandthepatentprotection ofcurrent blockbusterdrugsisdeteriorating,witha result-ing drainageofthe drug pipelines.All this may ultimately pushthepharmaceuticalindustrytowardsanewfrontierin

modern drug development. Fresh strategiesare needed to revivepharma’slostmomentumandweagreewithVlieghe andcoauthors(Vlieghe,Lisowskietal.,2010)thatthesector’s hope(partly)liesinpeptides.

3. Peptide

discovery

3.1. Naturalsources

Natureharboursanimpressivevarietyofbiologicallyactive peptidesexpressedinvirtuallyalllivingspeciesand,therefore, representsoneofthemostpromisingsourcesforpeptidedrug discovery(seealsowww.NP2D.com).

Within the multicellular body, peptides exert diverse biological roles, most prominently as signalling/regulatory molecules in a broad variety of physiological processes, including defence, immunity, stress, growth, homeostasis, andreproduction[24].

Through evolution, numerous peptides have evolved to exhibit their ‘natural’ bioactivity outside of the producing organism.Manyofthesehavebeenisolatedandcharacterised from the skin offrogs and toads [49,55]. These genetically encodedcompoundshavebeenshowntoprotectanddefend theirmanufacturersagainstmanyfoes, bothpredators and 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],lowdosesofthesepeptidescanactually coun-teractdisturbancesfromdiversedisorders.Accordingly,toxic peptidesmayaidintreatingpain[41],neurologicaland car-diovasculardiseases,diabetesandcancer[32].Aprominent exampleisthetype2diabetesdrugExenatide,asynthetic ver-sionofaglucagon-likepeptide-1analoguefoundinthevenom oftheGilamonsterHelodermasuspectum[7].

Asbioactivepeptidesobtainedfromnaturalsourceshave been subject to aeons of selective pressure, they show considerable plusses over artiﬁcially/chemically conceived peptide-like compounds. Namely, they excel in stability and target afﬁnity, both of which are extremely chal-lenging to achieve or reproduce through rational peptide design,screeningoflibrariesofrandomlycomposedpeptides or peptidomimetics. Although we appreciate the intelli-gence ofpeptide medicinal chemists,and other traditional (bio)chemistrybasedpharmacologists,webelievethatmuch isstilltobediscoveredfrom thenaturalbioactivepeptides usedalloverthebiologicaltaxonomy(frommicroorganisms overplantstoanimals).Withsomanyofthesebeingusedas drugsbysomanydifferentspeciesforsomanydifferent pur-poses,itisclearthatmankindcanstilllearnalotfromthe impliedbiology.Wewould,therefore,wholeheartedlysupport anadjustmentofthenameofthe‘NaturalPeptidestoDrugs’ NP2Ddiscussionforumto‘NP4D’(NaturalPeptidesforDrugs).

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3.2. Peptidomics

Todate,oneonlybeginstograspthemagnitudeofpeptides occurring in nature, a considerable share ofwhich poten-tiallyofferstherapeuticordiagnosticmerit.Therefore,more emphasisatstudyingtheseeminglyendlessvarietyofnatural peptidesandtheirbioactivities,isdeﬁnitelyjustiﬁed.Thisis exactlywhattherecentscienceof‘Peptidomics’isallabout.

In the past decade, we have seen great technological stridesinpeptidemanipulationand analyticalassessment. These include advances in liquid handling devices, syn-thetic protein synthesis, recombinant protein expression, multispectral micro-plate technologies,mass spectrometry, liquidchromatography,cellculturemethodologies, sequenc-ingtechnologies,imagingtoolsandhighthroughputpeptide screening protocols [6]. Coupled to these technological advances, the commercial biotechnology sector is steadily growing[6],yieldingincreasednumbersofcommercially avail-ablebiochemicalassaysandhighthroughputscreeningtools. Whereasexpensivehigh-techproteomictechniqueshave pre-viouslymainlybeenadoptedinbigpharmaandbiotech,we nowseethatthenewesttechnologiesarebecoming accessi-bleandarebeingdevelopedprimarilyinacademiaandsmall biotech.Giventhemanifoldofmoderntools,anewgeneration ofmoderndrugdesignersisemerging,exploitingthe poten-tialofferedbythelatestdevelopmentsinallrelevantscientiﬁc disciplines,suchasbiology,biochemistry,genetics, transcrip-tomics,proteo/peptidomics,computerscience,mathematics, andmanyothers.

Asystematicapproachtoidentifybiologicallyand phys-iologically active peptides and thus their pharmacological promise isfundamental to the study of peptidomics. This relatively young discipline aims to holistically analyse the spectrumofpeptidesfoundinanychosenorganism,mostlyby meansofmassspectrometry(MS),tightlylinkedto advance-mentsinbioinformaticstechnologywhichisessentialtokeep trackofandcopewiththehugedatasetsgenerated.

Evolutiontowards high-throughput MS analysis in both qualitativeidentificationandquantificationwasaidedbythe latestimprovementsinionisation(mainlyelectrospray,ESI) andinhighspeed,highsensitivityandresolutionanalysers suchasorbitrapsystems.Inaddition,theemergenceofmass spectrometryimaging(MSI)representsoneofthemost fas-cinatingprogressesinthisfieldrevealingthedistributionof peptidesinabiologicalsampleandconsequentiallyinferring theirpotentialbiologicalsourceandpurpose[40].

3.3. Peptidedrugcandidatescreening

For a conventional drugcandidate screening, the so-called ‘leaddiscovery’process,inwhichtraditionally largesetsof typicallysyntheticsmallmolecularcompoundsofpredeﬁned structurearepharmacologicallytested,itisessentialto dis-poseofwell-characterisedpeptidelibrariestorunapeptide drugdiscoveryprogramme.However,also‘reverse pharma-cology’usingchromatographicallyfractionatedextractsfrom originallyimpurebiologicalsourcesof‘natural’peptide mix-tures, is an approach which, thanks to the technological 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.

Entirely parallel to the high throughput screening of small moleculechemical libraries,syntheticpeptide chem-ical libraries can be produced using today’s technologies. Thesecanincludenaturalpeptidesequences,supplemented with derivatives thereof, such as naturally occurring post-translationallymodifiedandtruncatedisoforms,orentirely artificialpeptides.Indeed,peptidesofanydeliberatelychosen orrandomlyassembledsequencecanbeproducedbypurely chemical means or by recombinant organisms. Both allow foranimmenseflexibilityinsequenceandpost-translational modifications(PTMs)enablingthecreationofcorrespondingly vastlibrarieswhichpotentiallycontainsubstancesof thera-peuticinterest.Weremarkherethat thechoiceofresidues is by far not limited to proteinogenic amino acids, and is basically only limited by the imagination and competence of the syntheticpeptide chemistand/or peptide molecular biologist.Indeed,whilechemicalsynthesismayintegrateany compoundabletobondwiththenascentpeptide[1],the intro-duction ofartificiallyexpandedgeneticcodes has widened therangeofrecombinantpeptidestructuresbyinserting non-conventionalaminoacids[66,52].

Threestrategiesforscreeningartiﬁcialpeptidelibrariescan bedistinguished(Fig.1).Intheﬁrstapproach,peptides synthe-sisedonasolidsupportarecleavedoffforactivityscreening

[26].Secondly,peptidesareassessedwhilestillattachedtothe solidphaseonwhich theywere synthesised.Finally,phage displayisthethirdapproachwherebacteriophages express-ingthepeptideandexposingitontheirsurfaceareanalysed fortheirafﬁnitytoaselectedtarget.

Besides the former peptide chemistry- and molecular biology-basedstrategiestopopulatepeptidelibrarieswhich canbescreenedinstraightforwardpharmacologytests,also reverse pharmacological approaches starting from natural sourcesofbiological peptide mixtureshavebecomea very realisticalternative.Here,screeningsareperformedwith ini-tiallyuncharacterisednaturalpeptidestructuresisolatedand fractionatedfrom their biological source.Biologists helpto

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identifythe sourceswiththehighestpotential,considering thediseasetargetaimedfor.Richmixturesofbiological pep-tides can be found throughout the entire taxonomy, from vertebratesoverinvertebratesandplantstomicroorganisms. Classicalexamplesarevenomanddefensiveglandsecretions whicharefoundthroughoutzoology(seee.g.www.np2d.com; www.venomics.eu).

Thecomponentsofpeptide librariescan beassayed for theirbioactivityinvitro(targetaffinity,bindingkinetics,etc.) andinvivo(alteredgeneexpression,cytotoxicity,etc.). Combi-natorialanalysisofknownpeptidesequences(e.g.,Ala-scans) and the extent as to how changes in the primary struc-tureaffect the originalbiological activity,enablestoreveal structure–function relationships [18]. This ‘deconvolution’ allowstheselectionofsequencesforpeptideswithadesired activity.Assuch, it essentiallyprovides thefundament for rationaldesignofpeptideswithpredefinedbiologicaleffects. In all cases, peptide library screenings are intended to showmolecularinteractionwithadrugtargetusingdifferent visualisation technologies comprising colorimetry, fluores-cence microscopy, flow cytometry and others. Classes of biomoleculeswhichhavepreviouslybeenextensively investi-gatedandsuccessfullymodulatedastraditionaldrugtargets includeionchannels,nuclearorG-protein-coupledreceptors, transcriptionfactorsorenzymes.

4. Large-scale

peptide

production

Onceapeptidehasbeenselectedforfurtherdevelopmentas pharmacon,itneedstobeproducedinlargequantitieswith consistentquality,accordingtogoodmanufacturingpractice (GMP)rules.

Thestrategyforproducingapeptideislargelydetermined byits sizeandchemicalfeatures. Avarietyoftechnologies suchaschemicalsynthesis,recombinantDNAtechnologies, cell-freeexpressionsystems(invitrotranslation)and trans-genicplantsoranimalshavebeenadoptedforthispurpose (Fig.2).

Traditionally, the production of natural compounds is achievedbyusingamicrobialorfungalstrainthatunderwent a series of induced mutations and subsequent screenings forproductivity.Through thisprocess,theproductionyield maytypicallyberaisedbyuptothreeordersofmagnitude. Speciﬁcallydesignedstrainswithenhancedprotein synthe-sis,secretionandfoldingcapabilityprovideasolidplatform andstartingpointforreachinghighpeptideyields[34].

Large-scale operations incorporating chemical synthe-sis as the core productiontechnology may be seen as an attractivealternativetoexistingrecombinantDNA-basedor biocatalyst-basedmethodologies[65].Chemicalsynthesiscan bedistinguishedintothreemajorcategories,namelysolution phase,solidphase andhybridapproaches.Selectionofthe mostfavourableproductionprocessprimarilydependsonthe setobjectiveandthelimitationsthateverymethodpresents. Themajorityofpeptidepharmaceuticalsareproducedin highvolumesusingsolutionphasechemistrywhichis prefer-ablyemployedforsmalltomedium-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. Never-theless, signiﬁcantadvantagesofthismethodology are the well-established isolation, characterisation and puriﬁcation protocolsoftheintermediateproducts[1].Ontheotherhand, solidphasepeptidesynthesis(SPPS)hasenabledthe produc-tionoflarge,complexpeptidesofpharmaceuticalgradepurity onalargescale[1,65].Finally,hybridprocessescombiningthe advantagesofbothtechniquesmayofferevengreater poten-tial[1].

Irrespectiveoftheupstreamproductionmethodofchoice, downstreamprocessingisavitalstepinthemanufacturingof peptidepharmaceuticalproducts,sinceitinvolvesthecritical stepsassociatedwithproductisolationandpuriﬁcation[1].

5. Peptide

drug

registration

Peptidesrepresentaspecialcaseinregulatoryaffairs,since, dependingonitspropertiesandmanufacturing,apeptideis sometimes regarded as aconventional chemicalmedicinal product,andinothercasesasabiologicalentity.

The United States Food and Drug Administration(FDA) traditionallyhandlespeptidesasconventionaldrugs,notas biological products [20]. This goes along with the focus of examinationonthedrugcompositionandcompound struc-tureratherthanthemeansofmanufacturing.Accordingtothe FDA,theuppersizeboundaryofchemicallysynthesised pep-tidesisat100aminoacidresidues.Exceptions,however,are madewherepeptidesotherwisemeetthestatutorydeﬁnition ofabiologicalproduct,suchasinthecaseofpeptidevaccines.

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TheEuropeanMedicinesAgency(EMA) doesnotprovide such distinction based on size. Instead, peptides are sim-plytreatedasbiologicalentities,iftheyareeitherextracted fromtheirnaturalsourcesorrecombinantlyproduced[15,16]. Chemicallysynthesisedpeptides,accordingly,aretreatedas conventionalsmallmolecularchemicalentities.Nevertheless, apeptide may beregarded assigniﬁcant therapeutic inno-vation,asforinstancehasbeenthecaseforExenatide[17]. Thisenablesthecentralisedapprovalprocedureforgaining marketingauthorisationintheentireEUatonce.

In terms of manufacturing, only one guideline specifi-callyaddressespeptides, i.e.the“GuidanceforIndustryfor the Submission ofChemistry, Manufacturing, and Controls InformationforSyntheticPeptideSubstances”fromtheFDA’s CenterforDrugEvaluationandResearch[19].Itspecifiesthat thelotreleasespecificationsshouldbesufficienttoensurethe identity,purity,strengthand/orpotencyofthepeptideandto demonstratelot-to-lotconsistency[58].

Apoignantsaga illustratingthe‘resistance’ certain pep-tide compounds face on their way to the drug market, is thisofmagainin[42].EvenaftercompletionofphaseIII,FDA in1999decidedagainstapprovalofpexiganan(a22mer lin-earpeptide analogueofmagaininoriginallyidentiﬁedfrom

Xenopuslaevis).Thereasonwasthat thetrialdidnotshow superiorefficacyoverotherantibioticsusedintheindication underinvestigation.Thefact thatmagainin, becauseofits uniquemodeofaction(polycationicpeptidewith hydropho-bic residues forming transmembrane pores in the highly negatively charged bacterial cell membranes, bleeding the microbialcelltodeath),isvirtuallyimpossibleforabacterium todevelop resistanceagainst, was nottaken into account. Whereastypically,regulatoryissuestendtoelongatethe dura-tion ofclinical trials by a significant amount of time, the FDAlaunchedtheAntibacterialDrugDevelopmentTaskForce inSeptember2012[21]inordertoincreasetheefficiencyof antibioticdevelopmentincludingtheclinicaltrialdesign. Sim-ilardevelopmentsare on-goinginEuropebythe EMA;new guidelinesarereleasedwhichconcerntheclinicalcriteriafor evaluatingantimicrobials[22,31].

Thecurrentlychangedattitutetowardsantimicrobial pep-tides is illustrated by surotomycin (developed by Cubist Pharmaceuticals).Thecompound,alipopeptidevery compa-rabletovancomycin(acurrentlyavailablecommonantibiotic;

[35]),isnowinphaseIIIagainstC.difficileinfectionsandhas beendesignated‘qualifiedinfectiousdiseaseproduct’(QIDP) sta-tusundertheFDA‘generatingantibioticincentivesnow’(GAIN) act.Thismeansthatpriorityreview,fast-trackstatus,anda fiveyearexclusivityafterlicenseareapplicable[22].

6. Peptide

pharmacodynamics

and

pharmacokinetics

Manyofthechallengespeptidesfaceinthedrugdevelopment processoccurinthepreclinicaldevelopmentphases. Preclin-icalbiologicalactivity isevaluated usinginvitroand invivo

pharmacologyassaysthatdeterminetheeffectsofaproduct (pharmacodynamics)relatedtoitsclinicalactivity.Additional important pharmacological parameters include the phar-macokinetics(PK, absorption, distribution, metabolismand

excretion;ADME)[38].Determinationofalltheseparameters tothefullextentisespeciallychallenginginthecaseof syn-theticorrecombinantpeptides(orproteins),astheyusually showpatternsdeviatingfrommoretraditionalsmallmolecule pharmaceuticals[60]whichtypicallyreachtheir(oftenintra-) cellular targets bydiffusion into all cells ofthe body.This is quite differentfrom the regular bioactivepeptide which exertsitseffectthroughbindingwithacellsurfacereceptor, afterhavingsuccessfullyovercomethe challengesinherent toreachingthegeneralcirculation(seealsobelow“Peptide drugformulation”).Herepeptideshaveaslightdisadvantage comparedtoconventional smallmoleculedrugs,whichnot seldomareselectedfortheireasycrossingofcellular mem-branes/barriers.

Pharmacologystudiesofpeptidedrugcandidatesarestill verytough,asthetargetedanalyticalidentificationand quan-tificationofpeptidedrugsubstancesfromcomplexmatrices isstillstronglylimited[12].Theactualanalyticscreeningfor peptidesinpreclinicalstudiesismainlybasedondetection viaimmunologicalassays.Althoughthesemethodologiesdo offerahighthroughput, theysuffer frommajorlimitations intermsofspecificityanddynamicrange[25].Atthesame time, the developmentofnewprotocolsand assays is cur-rentlystillextensiveandlaborious[12].Consequently,interms of reliabilityand economics, analytical techniques suitable forroutinetargetedpeptidemetabolism(‘bioanalysis’)studies stillneedtobedeveloped.

A solutionto cross this technological chasmmay come from themostrecentadvancementsinpeptidemass spec-trometry(MS),expandingthetoolboxofMStechnologies.The optimalintegrationofinnovativeinstrumentations,protocols andefficientdatatreatmenttoolsavailabletodatewillleadto dedicatedworkflowstoanalysespecificpeptidesinbiological complexmatrices[12].

Aspectslikeinternal(isotopicallylabelled)standard avail-ability for the generation of reliable calibration curves, togetherwithinstrumentalsetuparecapitalinpeptide quan-tificationandADMEstudies.Moreover,thepanoramaofdata acquisitioniscomplicatedbythediversityofmassspectrum deconvolution technologies and datamining software. Yet, severalexamplesexistofhowthemostrecentMSevolutions canbesuccessfullyappliedinthecontextofpeptide identifi-cationandquantificationfromcomplexbiologicalmatrices. Technologies like selected reaction monitoring (SRM) and highresolutionmassspectrometry(HRMS)actuallyemergeas verypromising[12],especiallyintheinvestigationofpeptide metabolism. Theseadvancements, hand-in-handwith ade-quatesamplepreparationworkflows,includinghighandultra performanceliquidchromatography–biofluidssuchasblood, cerebrospinalfluidorothers compriseverydistinctanalytic environments–mayhelpsolvethisstillmajorbottleneckin pharmaceuticalpeptidemetabolismstudies.

Finallyalsotheabovementionedmassspecimaging(MSI) technology is very promising as tool to pre-clinically map the distributionofdrugsand theirmetabolitesinthebody, replacingthecurrentautoradiography(MSIdoesnotrequirea radiolabel). WhereasMSIisalreadybeingsuccessfullyused in small molecular drug PK (see e.g.[48]), it stillneeds to be successfully demonstrated for peptide drugs and their metabolites.

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7. Peptide

drug

formulation

Aspromisingaspeptidedrugcandidatesmaybe,anumberof conceptuallimitationsremainassociatedwiththetraditional imageofapeptideasatypicalmedicine.Thisperceptionis largelysubstantiatedbyLipinski’s“ruleofﬁve”which sum-marises the ideal pharmacokinetic properties of the most successful(chemical)drugcandidatesoftheoriginalpharma industry.Thisdecreedogmatisesthatpeptidesarelesslikely topassthroughthegastrointestinal(GI)tractwallascompared tosmallmoleculesduetotheirlargersizeandcomparatively lowsolubility[65].

Many challenges peptides experience on their way to becominganeffectivedrugoriginateintheirphysicochemical propertieswhichtogetherresultinapoororal bioavailabil-ity.Duetotheirhydrophilicity,peptidesexhibitlimitedability tocrossphysiologicalbarriers.Inaddition,peptidesare con-frontedwithefﬁcienthepaticandrenalclearance.Evenonce insidethesystemiccirculation,peptidestypicallyhaverather shorthalf-lifesduetoaggressivedegradationbyamultitudeof proteases[3].Theseaspects,intrinsictothechemicalnatureof peptides,havecompelledcumbersomeadministrationroutes such as direct injection of repeated doses, which in turn resultedinlowpatientcompliance[3].

Asidetheclassicsubcutaneous,intramuscularand intra-venousadministration, alternative routeshave been devel-oped,including the mucosal track (nasal spray, pulmonary deliveryorsublingualdelivery),theoralroute(GItract pen-etrationenhancers,proteaseinhibitors orcarriers) and the transdermalpath(patches;[3,65]).Forinstance,apeptidepill fororaladministrationisdeveloped(EnterisBiopharma)the coatingofwhicheffectivelyprotectstheactivesubstancefrom digestion in the stomach, allowing its release in the duo-denum.Anumber ofexcipientsprotectthepeptideagainst peptidasesandfacilitateitsparacellularuptakethroughthe intestinalwallintothesystemiccirculation[57].

Beneﬁtsofthepulmonaryintakeareaverylargeavailable absorptive and highly permeable surface, extensive vascu-larisation,withconcomitantrapidonsetofpharmacological action,amoreuniformdistributionofthedrugproductand amoresustaineddrugrelease whichallowsareductionof thedosingfrequency[2,28].Likeotherparenteral administra-tions,theﬁrstpassmetabolismisavoided.Alsoa10–200times higherbioavailability(higherdrugproductplasma concentra-tions)canbeachievedbecauseofthesmallervolumesused

[56,63,2].

Besides alternative delivery systems, various formula-tionshavebeendevisedinthepast,tryingtodealwiththe aforementionedphysicochemicallimitations,tohelpadvance promising peptide drug leads into pharmaceutical devel-opment. Again, most of these medicinal preparations are chemistry-and nanophysics-based.Indeed,chemical incor-porationofsugarslike trehalose,sucrose,maltose,glucose, of salts like potassium phosphate, sodium citrate, ammo-niumsulphateand/orofother agentssuchasheparininto the prospective formulations have been found to increase thesolubilityandinvivostabilityofpeptides.Employmentof cationicandanionicsurfactantssuchascetrimideandsodium dodecyl sulfate(SDS)haveshown enhancedtransportation

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 in resistanceagainst proteolysis is oftenachieved through co-administrationofproteaseinhibitorssuchassodium gly-cocholate, camostat mesilate or bacitracin. Moreover, the attachment of polymeric molecules such as polyethylene glycol (PEG) and polyvinylpyrrolidone (PVP) and even the encapsulationofthepeptideintonanocarriersareemployed forextendedbioavailability(Antosova,Mackovaetal.,2009).

Nanocarriertechnologyindeedseems apromising inno-vationtoincreasepeptidepharmacodistributionthrough,for example,nanoparticles,liposomesandmicelles(Fig.3).These are thoughtto effectively create aclosed carrierthat pro-tectstheactivecompoundfromdestabilisingexternalthreats suchapeptidases.Incombinationwithpulmonaryinhalation, nanocarriershavethebeneﬁtofprolongeddrugreleasedue tothecombinationofpeptideprotectionbythecarrier’sshell withcarrieraccumulation[2].

In general,an idealnanocarriershould becomposedof inert and biodegradable materialand beable to efﬁciently encapsulateandprotectthepeptideagainstdegradationwhile at the same time maintaining properdrug product target-ing[28].Compositionsofpolymericformulationscanbeused to tune the biological behaviourof nanocarriersby modu-latingcompound propertiessuchas mucoadhesiveness[8]. Anexampleisthecationicpolysaccharidechitosan,theionic interactionsofwhichwiththenegativelychargedsialicacid groupsinmucinprovideexceptionalbinding.Incombination

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withits abilitytoopentight junctions,chitosan effectively favourstheutilisationofthe paracellularpathway[11,53,2]. Hybrids can be created by combining chitosan with other polysaccharidesoroligosaccharidesinanattempttofurther improvethephysicalpropertiesandpharmacological perfor-manceofpeptidedrugformulations[23,28].

Other synthetic polymers such as polylactic acid (PLA) and poly(lactic-co-glycolic) acid (PLGA) have been found viablesubstitutes.Theirconsistencyintermsofpeptidedrug productrelease in combination with good biodegradability guaranteesoptimalsafety.Accordingly,theFDAhasalready approvedanumberofrespectivemarketedproductsand clin-icalapplications[11,2,28].

Liposomalnanocarriersarecomposedofoneormultiple phospholipidbilayers.Specificligandsconjugatedwith mod-ifiedPEGmoleculesintheshelloffertargeteddelivery[30,28]. However,liposomesoftenfacevaryinginstabilitiesin biolog-icalfluids[2].Nanoparticleswhichconsistofasolid(bothat ambientandbodytemperature)lipidmatrixdispersedinan aqueousphasemay offeranalternative.Onedistinguishes solidlipidnanoparticles(SLNs)andnanostructuredlipid car-riers(NLCs).WhileSLCsarerigidlystructured,NLCsconsist ofbothsolidandliquidlipidsallowingforanincreased load-ingcapacity[39,43,2].Finally,self-nanoemulsifyingsystems, i.e.isotropicmixturesofoilandsurfactantforming thermo-dynamicallystableoil-in-wateremulsions,mayhavepotential forenhancedoralbioavailabilityofpoorlywatersoluble pep-tides[28].

Besidesthesemodern(nano)technology-inspired formula-tions,weherewouldliketodrawtheattentiontothewide varietyofbioactivepeptidedeliverysystemsfoundinnature. Evolutionhasdesigneddirectinjectiondevices,verymuchlike injectionneedles,suchasfangsorvenomteethinvarious rep-tiles,harpoon-likeradulasinsnails,orclaws.Howeveralso organismslackingtheseappeartosuccessfullyusebioactive peptidesintheirglandsecretions. Acarefulanalysisofthe compositionofpeptide-containingvenomanddefenseglands inanimalslikeamphibians,may,therefore,givehintsasto howpotentialtherapeuticpeptidescanbesuccessfully formu-lated.Theexosomalreleasewhichisobservedfromvarious venomglandsinawayiscomparabletothemicellar encap-sulation ofpeptides described above. Biology alsohints to additionaltrickstoensureeffectiveuseofpeptidebioactives. Innaturepeptidesarealmost neversecreted ontheirown, butalwaysincocktails,basicallyamixtureofthreetypesof peptides.Thesenotseldomcontainpolycationicamphipatic (poly)peptides(sometimescataloguedas‘antimicrobial pep-tides’(AMPs)) whichcould serveas membranepenetrating deliverysystems.Co-secretionof‘peptidaseinhibitors’isalso a strategy seen in various animal defense glands. Finally various (oftenpost-translational)modifications on thetrue ‘bioactive peptides’ fit in a strategy to render the (active partofthemolecule)improved/prolongedstabilityaswellas tighterreceptorbinding.Theseincludesequenceelongation protecting the bioactivesite from rapidexoprotease diges-tion,C-terminalamidationand/orN-terminalringformation (pyroglutamic acid from glutamine)for chargesuppression (reduction ofhydrophilicity), disulfide bridgeformation for increasedstructure preservation and rigidity, and isomeri-sation of selected peptide terminal residues (from l- into

Fig.4–Graphicalrepresentationofnumberofpeptidesin

preclinicalandclinicaldevelopmentasof2013[31].

d-aminoacid)forenhancedexopeptidaseresistance. Exam-plescanbefoundinthedefensivesecretionsofPhyllomedusa burmeisteri andBombinavariegata,asdescribedelsewherein thisspecialissue[47].

8. Clinical

development

The therapeutic peptide development is growing. In 2011 alone,therewerebetween500and600peptidesinpre-clinical phases(Fig.4;[31]).Theyear2012hasproventobeanother milestoneforthepeptidepharmaceuticalsector,with5and6 peptidesmeetingmarketapprovalrespectivelyinEuropeand intheUSA.Thiswasthe highestnumberofapprovalsever achieved fornewbiologicalentities (NBEs)inone year[29]

whichrenderssomeoptimismtothesector.Thisoptimismis conﬁrmedbythestatistics,astheregulatoryapprovalratefor peptidesisaround20%,versus10%forsmallmolecules[31]. Inaddition,thenumberofpeptidesperyearenteringclinical trialshassteadilyincreasedfrom1in1970tocurrentlyaround 20[31].

AsofApril2012,theclinicalpipelineforpeptidedrugswas composed of128peptide candidates. These included40 in phaseI,74inphaseIIand14inphaseIII(Fig.4).The robust-nessofthispipelineislargelyduetothenotableexpansion intheﬁeldofpeptidetherapeuticsduringthelate1990sand 2000s,ultimatelyleadingtothenumberofapprovalsobserved in 2012.Consideringthe generalfailurerate ofthe clinical 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

One area of research that has shown promising develop-ment are the use of peptide therapeutics in treating type 2 diabetes (targetingthe glucagon-like peptide 1receptor). Alreadythreepeptideshavereceivedapprovalin2012,with 14 working their way through the pipeline. A most excit-ingaspectofthesepeptidedrugcandidatesisthevarietyin drugformulationofmolecularformats(withpeptidesbeing covalently linkedtosmall molecules,carbohydrates,lipids, biopolymers,polyethyleneglycolorproteins(seeabove))and

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theirmechanismsofactions(includingspeciﬁccell-targeting peptidesandcell-penetrating peptides)currentlybeing elu-cidated.Thus,substantialeffortsarebeingmadetomodify molecularpropertiesofpeptidedrugleadstoimprovetheir functionality.Forexample,half-lifeextensionwasthe ratio-naleforfourpeptides(CBX129801,CVX060,LAPSExd4,PB1023) inphaseII,wherebypeptideconjugationtopolyethyleneor IgG substantially increased peptide stability in circulation fromminutestodaysorevenweeks.Improvedbiological bar-riercrossing/cell-penetration was the rationale behind the designofthreeotherpeptides(CBP501,AM111,ACT1)alsoin phaseII.Typicalamphipathicandcationicfeaturesofthese peptides are enhanced by the molecular addition of cell-penetrationpromotingsequences suchasthetranscription transactivation(TAT)sequencefromtheHIVvirus[36].

8.2. Antimicrobialpeptides

With the frightening advent of global increase of micro-bial resistance to conventional antibiotics, the search for alternativeshasbecomeofutmostimportance,andthe indus-try as well as the regulatory authorities are realising the potentialofantimicrobialpeptides(seealsoabove).

Arecentoverviewofantimicrobialpeptides currentlyin clinicalphasesby[22],includes10compounds(developedin 10differentcompaniesbothinNorthAmericaandinEurope). InthislistweseePexiganan(magainin,seeabove)reappear, albeitnolongerdevelopedbytheoriginalcompanyMagainin Pharmaceuticals,butbyGenaera,thenametheenterprisewas re-baptisedin.

8.3. Peptidesasvaccines

An entirely different sort of therapeutic peptides are the peptide vaccines. These peptides, representing inactive, non-virulent fragmentsofpathogen proteinsare becoming graduallymoremainstream.On-goingtrialsarespanningall phasesofclinicaldevelopment.Thelistofbeneﬁtsfor espe-ciallysynthetic peptidesas vaccinesincludes theirease of qualitycontrol,chemicalstabilityand theabsenceof onco-genic,toxic or infectious material[51]. Whereas notmany successeshaverecentlybeenachievedbyemployingpeptide vaccines[44],theadventofpersonalisedpeptidevaccination (PPV)couldheraldchangingtimes.Takingfactorsintoaccount suchasthehumanleucocyteantigen(HLA)systemand pre-existinghostimmunity[44],PPVmayhaveafuture,providing currentphaseIIItrialsareassuccessfulastheypromisetobe

[44,67].

8.4. Futureperspective

Someverypromisingpeptidestowatchoutforinthecoming years are now in late phase clinical trials. About half of them are intendedfor oncology,metabolic or cardiovascu-lartreatment as well as forremedying infectious diseases

[31]. Especially for illnesses requiring prolonged therapy, peptides have a competitive advantage over conventional small molecule drugs. In terms ofgeneral safety,peptides have a comparatively small toxicological footprint. Due to theirextremelyhighspeciﬁcityfortheirintendedtarget(the

peptidereceptor),incombinationwiththefactthattheyare extracellularly active (not requiring systemic diffusion and henceextremedilutionoverallcells),muchloweramounts canbeformulated.Moreoverafterpeptide receptorbinding and signal triggering, highly efﬁcient peptide catabolism through proteolyticdegradation yieldssimpleaminoacids, whicharerecycledinthebodyineverydaymetabolismsuch asproteinsynthesis.Comparedwithother smallmolecular chemicalentities,whichoftenrepresentextremechallenges tothebody’sdetoxiﬁcationmechanisms,peptidessufferfrom littleifanyaccumulationinthebody,norintheenvironment. Incontrasttovariouspoorlymetabolisingorabsorbingsmall chemicaldrugs,nosurfacewaterpollutionoccursbyresidual activesubstanceexcretionintotheenvironmentafterpeptide druguse.

It can be concluded that the pharmaceutical peptide pipeline is strong and stable, with several candidates approachingdrugapprovalstatus.Thecommercialvalueof thetherapeuticpeptidemarketiswellestablished(seebelow). However,therecentandnascentapprovalspromiseto sub-stantiallyincreasethemarketvalueofpeptidetherapeutics inthecomingyears,intheareasofdiabetes,oncology[29,31]

andbeyond.

9. Peptide

patents

Severalsourcesreportthatthenumberofpatentapplications involvingpeptide-relatedtechnologyhassigniﬁcantlygrown inthelastdecades.Arecentupdateonpatentapplicationsis available[46].

Anillustrativeexampleofapatentapplicationinvolving peptides with pharmaceutical potential is provided by the approvedpatentEP1590458B1“BradykininB2Receptor Antag-onistPeptidefromAmphibianSkin”[54].Thepatentdiscloses the sequence of a peptide (kinestatin) isolated from toad (Bombinamaxima)defensiveskinsecretion,whileclaimingthe protectionforkinestatin analogues,prodrugsincluding the peptides, fusion peptides and multimeric peptides. At the sametime,itgivesabroadindicationofthepotential thera-peuticapplicationareas,includingcardiovasculardisorders, inﬂammation, asthma, allergic rhinitis, pain, angiogenesis and thelike,glaucoma,hydrocephalus,spinalcordtrauma, spinal cordoedema, neurodegenerative diseases, including Alzheimer’sdisease.Theclaimscomprisetheapplicationof thepatentedmoleculeswhereinsaidpeptidesarepresentin or conjugatedontoaliposomeormicroparticlethat isofa suitablesizeforintravenousadministration,butthatlodges incapillarybeds,thusopeningtheroadtoovercoming poten-tialadministrationhurdles.Thispatentapplicationprovides the readerwithageneralframeworkforpatentapplication involvingpeptides,namelydisclosureofthemainaminoacid sequencemotif,examplesofanalogues,prodrugderivatives andabroaddeﬁnitionoftherapeuticareas.

Within the Cooperative Patent Classification (European Patent Office, 2013), category A61K38 includes “Medicinal preparationscontainpeptides”asasubdivision ofcategory “Preparations formedical,dental,ortoiletpurposes”A61K. ThisisnottobeconfusedwithcategoryC07K“Peptides”that isincludingapplicationofpeptideswithinseveralfields,such

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0 5000 10000 15000 20000 25000 Annual nu mber of pat ent s Year

Fig.5–Trendinpatentapplicationsfortherapeutic

peptidesfrom1980until2012[14].

asfood.AnEspacenetdatabasequeryforpatentapplications belongingtocategoryA61K38foreachyearbetween1980and 2013(thedataforthepartialyear2013wereextrapolatedto thewholeyear)yieldsaninterestinggraph(Fig.5;[14]).

Approximately389,320patentapplicationswithinthe pep-tide ﬁelds have been published in the interval 1980–2013. Startingfrom the year1996 thenumber ofpatent applica-tionsperyearhaveinvariablysurmounted10,000,averyhigh number,reﬂectingaverydynamicdevelopmentofthe pep-tidemarket.Atthesametime,italsoshowsanapparentpeak inthenumberofapplicationsperyearreachedin2003(with 23,690newpeptide-relatedpatentapplications).

Consideringthatanaverageapprovaltimefornewdrugs is10yearsandthatthepeakinpeptidepatentapplications occurredintheinterval 2000–2005,onemaypredictapeak inthenumberofpeptidesenteringthemarketasnewdrugs between2010and2015.Apparently,anunprecedented num-berofpeptideshaveinfactreceivedmarketapprovalinthe years2010–2013(seeabove).Consideringthatatypical pro-tection time fora granted patent is 20 years,and that an off-patentdrugtendstoloseasigniﬁcantpartofitssales rev-enuetoprice-basedcompetitionbygenericsinthefewyears aftertheendofpatentprotection,weanticipatethat peptide-baseddrugsmaydeliverrisingsalesrevenuesforpharmaina signiﬁcantnumbersofyearstocome,reasonablybeyond2020.

10. The

peptide

pharma

market

Todate,around100therapeuticpeptides(mostlyinnovative syntheticones) are onthe market inthe USA, Europeand Japan,includingthosefordiagnosticsapplications[29].The increasingnumbersofrecentlyapprovedpeptidesisaresult ofthe well-ﬁlled pipeline,as described above. Themarket appearsdominatedbythethreepeptidesGoserelin/Zoladex and Leuprolide (two gonadotropin releasing hormone ago-nists,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).

Acleartrendoverthepastyearsisthecontinually expand-ingcontributionofpeptidestotheworldwidepharmaceutical market.OfaglobalmarketworthUSD956billionin2011[27], thepeptides’sharewasaboutUSD14.4billion[29,62], com-prisingaround1.5%.ThemajorcontributoristheUSmarket, whichaccountsfor40%oftheglobalpeptidesales.In2011,the cancersub-marketwasthelargest,representing21%ofthe totalpeptidemarket,followedbymetabolicdisorders, gastro-intestinaldiseasesandrespiratoryindications[62]with86% oftheapprovedpeptidedrugsworkingthroughtheparenteral route.Asindicatedabove,however,alternativeroutesare fore-seentoexpand[29].Thetotalmarketpotentialofpeptidesmay be signiﬁcantlylarger,since,inaddition tothetherapeutic pharmaceuticalmarket,peptidesareexpectedtocontribute moreand moreinother marketssuchasthenutraceutical business[5].

In the current stagnatingpharmaceutical industry, pep-tides are considered to have added value, by representing a potential solutionto moreefﬁcacious disease treatment. Already today they appear mature compounds addressing unmet medical needs, and accelerating the personalised medicinemodel[10].Inaddition,peptidespromisetocombine thelowerproductioncostsofconventional(smallmolecular chemical)drugswiththehighspeciﬁcityof(thelarger) biolog-icalentities.

Importantly,theproportionofpeptidesinpharmais antic-ipatedtoincrease,sinceitisestimatedtogrowfaster(9.4% annual growth in 2012–2018) [62] than the global industry (3–6%annualgrowthin2012–2016)[27].Notonlyisthe num-berofapprovedpeptidedrugsexpectedtogrowbutalsothe diversityoftreatedindications.

Itisremarkablethatgenericsalesalready accountfora considerableportionofthepeptides’market.In2011, gener-icsrepresented35%ofthepeptidemarket,withthreeofthe ﬁvetopsellingpeptidesbeinggenerics[5,62].Octreotidefor instancehasagenericversionaccountingforUSD0.15 bil-lionannualsales[9].Incomparison,genericsalesintheglobal pharmaceuticalmarketaccountforaround21%and biosimi-larsinthebiologicssub-marketforaround0.4%inthesame year[27].

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Summarising, the peptide market today, although still dependingonafewblockbustersandmaturedrugsasobvious fromthecontributionofgenericstotheglobalsales(Fig.6), ispredictablyincreasing.Theimminentpipelinesindicatea brightfutureforpeptidepharmaca,withnumerous innova-tivepeptidesonthevergeofapproval.Thisendorsespeptides asﬁrmcandidatestocontributetothegrowthandinnovation ofthefuturepharmaceuticalindustry.

11. Conclusion

Therapeutic peptides have spent decades as niche prod-ucts,whilethe pharmaceuticalindustry focussed onsmall molecules asmedicinal agents. Given the increasing chal-lengeswiththelattercompounds,drugdevelopersareturning backtothesmallaminoacidchains.Whereaspeptideshave beendeemedunsuitableforalongtime,modernformulations andpeptidedrugdesingshaveachievedtocircumventtheir weaknessestoclearlyrevealmorethanafewadvantagesof thesemolecules. For example,the originalneedfor inject-ingpeptides like insulinisfading,withprogressively more patient-friendlyadministrationsbeingdeveloped. Addition-ally, today’s societywhich critically judges the undesirable side effects as well as environmental impact ofcandidate medicinesshouldembracethesafetyprovidedbypeptides.

Whereas,therapeuticpeptidesoriginallywere developed to replace their endogenous lack, the spectrum of avail-able candidate peptide drugs is by far not limited to the humanpeptide pool. Indeed,through the modern tools of peptidomics,bioactivepeptidesfrommultifariousorganisms are being discovered. Nature certainly still harbours a vir-tually infinite array ofpotential peptidic medications that await(human)pharmacologicalcharacterisation.Atthesame time,themethodsofpeptidesynthesishaveevolvedto per-mithighlyefficientproductionofremarkablylongandheavily modifiedcompounds.

Inthelightoftheseadvances,therecentriseofpeptide drugsisnotasurpriseatall.Atﬁrstglance,thedeclinein ther-apeuticpeptidepatentapplicationsafterapriorpeakabout 10yearsagomayseemdiscouraging,butthisdoesnot nec-essarilymeanthatthemarketisreﬂectingthistrend.Onthe contrary,alargenumberofclinicaltrialsofpeptidedrug candi-datesisconductedtodateandthemarketisgrowingsteadily. Giventhesepremises,weanticipateabrightfuturefor thera-peutic(aswellasdiagnostic)peptides.

Transparency

document

TheTransparencydocumentassociatedwiththisarticlecan befoundintheonlineversion.

Acknowledgements

Fortheirsupportandvaluableinput,wewishtothankour colleaguesparticipatingintheDelftUniversityModernDrug Developmentcourse,NeclaSenaAlikisioglu, SarojGhimire, JoseMariaGuillotdeMergelina,AlinaMiron,KeerthiPrasad Rajaprasad,PhuongVoandMartijnWapenaar.Inadditionwe

gratefullyacknowledgethemotivationbyJanineKiersandJur Thöneandtheirguidancewithinthe“BioProductDesign” Pro-fessional Doctorate inEngineeringprogramme.Dr.Dikvan HarteofAgentschapNLandMr.AntonvanGeel(Fujichrome, Tilburg)arethankedfortheirinputregardingthetherapeutic peptidepatenttopic.

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