ChristinaAvantiDepartmentofPharmaceutics

UniversityofSurabaya,Surabaya,Indonesia

Assoc. Prof. Christina Avanti, PhD

Graduated in Pharmacy at the University of Surabaya , she worked at Sandoz Biochemie Farma Indonesia and did her Master Program in

Cosmetic Formulation at the Department of Pharmaceutics AirlanggaUniversity. She did her PhD at the Department of Pharmaceutical Technology University of Groningen, The Netherlands. She worked

within the Dutch Top Institute Pharma Project entitled “Breaking The Cold Chain of Polypeptide-Based Medicine”. She is now working as a

Senior Researcher and teaching Physical Pharmacy, Formulation, Technology, and Biopharmacy at The School of Pharmacy University

of Surabaya. The overall aim of her work is to achieve a stable peptide/protein formulation to be transported, stored, and used

particularly in tropical developing countries.(Patent: (WO/2010/030180) PEPTIDE FORMULATIONS AND USES THEREOF)

Latest publication:• Stability of lysozyme in aqueous extremolyte solutions during heat

shock and accelerated thermal conditions. Plos One Journal, January 2014, Volume 9, Issue 1, e86244

• The formation of oxytocin dimers is suppressed by the zinc-aspartate-oxytocin complex. J. Pharm. Sci., 2013, DOI

10.1002/jps.23546 (early published online)• Aspartate buffer and divalent metal ions affect oxytocin in

aqueous solution and protect it from degradation. Int. J. Pharm, 2013 , 444(1-2):139-45

DeanofFacultyofPharmacyinUniversityofSurabaya

Outline

Conclusion

Strategiestoimprovepeptide

stabilityinliquid

formulations

Peptideinstabilityandthepossiblecausesof

degradation

Introduction

• Peptidesaspotentialdrugs:SuccessfulchemicalsynthesisofoxytocinbyduVigneaud in1953

• overahundredpeptidedrugcandidatesareindevelopmentforawidevarietyofdiseases

• Peptidesdifferfromproteins:smallerandtypicallylackadefinedtertiarystructure

Introduction

• Aminoacidslinkedtoeachotherbypeptidebonds• Peptidebondoccurswhennitrogenatomononeamino

acidbindstothecarboxylgroupofanotheraminoacid.

Whatispeptideandpeptidebond?

• Malavolta (2011)Peptidesaremoleculescontainingfewerthan40aminoacidresidues,whileproteinscontain50

residuesormore

• Lee(1991)Aminoacidsjoinedtogetherinchainsof50aminoacidsorlessaredefinedaspeptides,50-100aminoacidsaredefinedaspolypeptides,andover100aminoacidsaredefinedasproteins

DefinitionofPeptide

Tyr

Lys

Arg

Ser

Trp

Asp

Glu

Gln

Met

AminoAcidBuildingBlock

• Anumberofhormones,enzymes,antitumoragents,antibioticsandneurotransmittersarepeptides.

• Peptidesregulatemanyphysiologicalprocesses,actingatsomesitesasendocrineorparacrinesignalsandatothersitesasneurotransmittersorgrowthfactors.

• Nowadays,peptidesareusedastherapeuticagentsagainstdiversediseaseareassuchasneurological,endocrinological andhematologicaldisorders

PurposesofPeptides

Table1Severalpeptide-basedparenteralproductsinthemarketin2010-2013

Genericname

Tradename Supplier Dosageform Shelf-lifeandstoragetemp.

pH

ThyrotropinereleasinghormonesTRH/TRF Relefact TRH® Sanovi-

AventisLiquidinj. 2yr15-

25°C6.5

Antibioticpeptides

Daptomycin Cubicin® Novartis Powderforinj. 3yr,2°C–8°C

4-5

Teicoplanin Targocid® Sanofi-Aventis

Powderforinj. 4yr,2-8°C

Plateletaggregatesinhibitors

Eptifibatide Integrilin® GlaxoSmithKline

Liquidforinfusion

3yr,2-8°C 5.35

SomatostatinanalogOctreotideacetate

Sandostatin® Novartis Liquidinj. 3yr,2-8°C 4.2

VasopressinsandanalogsDesmopressin DDAVP® Ferring Liquidinj. 4yr,2-8°C 4-5

Octostim® Ferring Liquidinj. 4yr,2-8°C 4Minrin® Ferring Liquidinj. 4yr,2-8°C 4

Felypressin Citanest3%Octapressin®

Densply Liquidinj. 3yr,15-25°C 3.5-5.2

Genericname Tradename Supplier Dosageform Shelf-lifeandstoragetemp.

pH

Table1Severalpeptide-basedparenteralproductsinthemarketin2010-2013

Genericname

Tradename Supplier Dosageform Shelf-lifeandstoragetemp.

pH

OxytocinsOxytocin Syntocinon® Defiante Liquidinj. 4yr,2-8°C 4Carbetocin Pabal® Ferring Liquidinj. 2yr,2-8°C 3.8OxytocinantagonistAtosiban Tractocile® Ferring Liquidinj. 4yr,2-8°C 4.5

Tractocile® Ferring Liquidinj. 4yr2-8°C 4.5

Table1Severalpeptide-basedparenteralproductsinthemarketin2010-2013

Table1Severalpeptide-basedparenteralproductsinthemarketin2010-2013

Genericname

Tradename Supplier Dosageform Shelf-lifeandstoragetemp.

pH

GNRH/LHRHagonistsGoserelin Zoladex® Div Liquidinj. 3yr,<25°CGonadorelin Relefact

LH-RH®Sanofi-Aventis

Liquidinj. 15-25°C

Triptorelin Decapeptyl-CR®

Ipsen Powderandsolv.forsolforinj.

3yr,2-8°C

Nafarelin Synarel® Pfizer Liquidnasalspray

2yr,<25°C 5-7

Leuprolide Eligard® Sanofi-Aventis

Powderandsolv.forsolforinj.

2yr,2-8°C

Cetrorelix Cetrotide® Serono Powderforinj. 3yr,15-25°C

Table1Severalpeptide-basedparenteralproductsinthemarketin2010-2013

Genericname

Tradename Supplier Dosageform Shelf-lifeandstoragetemp.

pH

Non-NSAID analgesicZiconotide Prialt® Elan Liquidinj. 3yr,2-8°C 4-5CalcitoninsSalmonCalcitonin

CalcitoninSandoz®

Novartis Liquidinj. 5yr,2-8°C 3-5

Humanparathyroidhormone[hPTH(1–34)]Teriparatide Forsteo® EliLily Liquidinj. 2yr,2-8°CFusioninhibitorofHIV-1withCD4cellsEnfuvirtide Fuzeon® Roche Powderand

solv. solutionforinj.

4yr,2-8°C

ACTHandderivativesCRH/CRF/Corticorelin

CRH-Ferring®

Ferring Powderforinj. 3yr,<25°C

• Thesensitivitytoenzymaticbreakdown• Thepoorabilitytopassabsorbingmembranestypicallyresultsinapoorbioavailabilityfollowingnon-parenteral administration.

• Thelackofphysicalandchemicalstabilityleadtosignificantdegradationduringprocessingandstorageofthe(aqueous)formulations

Pharmaceuticalpeptides:Challenge

• buccal,• rectal,• vaginal,• percutaneous,• ocular,• transdermal,• nasal,• pulmonarymanyoftheseroutesofadministrationarestillunderinvestigationandtheymaybeinsufficientlyefficient,especiallywhenarapideffectisdesired.

Non-invasiveroutesforpeptidedrugadministration

Intravenousadministrationisthemostefficientwaytodeliverpeptidedrugsdirectlyintothesystemiccirculation

most peptide drugs have to be stored and transported at low temperatures

Chemicaldegradationpathways• hydrolysis,• oxidation,• deamidation .

Physicaldegradation:• adsorption• aggregation

betterunderstandingoftheunderlyingmechanismsofinstabilityofacertainpeptide todesignrationalstrategiesinthedevelopmentprocessofpharmaceuticalstooptimizethestabilityofthepeptideinthefinalformulation

probleminthedevelopmentasactive

pharmaceuticalsNeed

PeptideDegradationPathways

Peptideinstabilityandthepossiblecausesofdegradation

• Hydrolyticpathways– Chaincleavageofthepeptidebackbone– DeamidationofAsnandGlnresidues– IsomerizationofAspresidues

• Oxidationpathways– Autoxidation– Metal-catalyzedoxidation– Light-inducedoxidation

• β-eliminationreactions• Disulfideexchangereactions• Dimerization,aggregation,andprecipitation

Deamidation pathwaysofAsn residueviaA.directhydrolysisandB.succinimide mediation

SelectedoxidationreactionsofMetandHis.(A):methionineoxidationbyperoxidetomethioninesulfoxide inacidicsolution(HA=acid).(B):2-oxo-His,Asn andAsp(adaptedfromLi,et.al,1995).

Oxytocindegradationpathways

Strategiestoimprovepeptidestabilityinliquidformulations

Degradationpathway Stabilizationstrategy Aminoacidresidue(s)involved

ChemicalInstabilityAcid/basecatalyzedhydrolysis

pHbufferspeciesco-solvents

SerTrpAsn-ProAsn-Tyr

Deamidation pH3-5increasedsolventviscosity

Asn,Gln

β-elimination Bufferspeciesdivalentmetalions

Cys-Cys

Table 2 Degradation pathways and possible stabilization strategies for peptides including amino acid residues involved in the degradation.

Degradationpathway Stabilizationstrategy Aminoacidresidue(s)involved

ChemicalInstabilityOxidation pH<7

airexclusionantioxidants

Trp,Met,Cys,Tyr,His

Lightinducedoxidation Protectfromlight TrpMetalinducedoxidation Chelatingagents

PolyolsHis,Cys,Arg,Pro,Met

Disulfideexchange SurfactansPolyolsandsugarsBufferanddivalentmetalions

Cys-Cys

Table 2 Degradation pathways and possible stabilization strategies for peptides including amino acid residues involved in the degradation, continued

Degradationpathway Stabilizationstrategy Aminoacidresidue(s)involved

PhysicalInstabilityDimerizationandfurtheraggregation

Lowerconcentrationminimalmechanicalstressorganicsolventsalkylsaccharidealkylpolyglycoside

Cys-CysTyr-Tyr

Adsorption surfactanspolymers

HisArg

Table 2 Degradation pathways and possible stabilization strategies for peptides including amino acid residues involved in the degradation, continued

Conclusion

• In aqueous solutions peptides are often unstable. • Peptides have unique structures that the side chain

of nearly all of the amino acid residues are fully solvent exposed, allowing maximal contact with solvents

• the degradation rates appear to correlate with the degree of solvent exposure.

• Based on knowledge of the peptide’s structure and an understanding of the predominant degradation pathways, the strategies may be developed to achieve adequate stability of the formulation.

• The degradation pathways of peptides are mainly dependent on the amino acid sequence.

• The most prominent degradation pathways for peptides are hydrolysis, oxidation, and dimerization.

• Formulating peptides in a specific pH with a specific buffer, avoiding oxygen reactive species, and minimizing solvent exposure eliminate chemical degradation.

• Increasing solution viscosity by using sugars or polymers reduces peptide mobility and further decelerates physical degradation.

Conclusion

Instabilityisthemoststablecharacteristicof

adissolvedpeptide

ChristinaAvanti,2011

Instabilitiesareanaturalwaytoachievethermodynamicequilibrium

ChristinaAvanti,2012