Aspartate buffer and divalent metal ions affect oxytocin in aqueous solution and protect it from degradation - Ubaya Repository

InternationalJournalofPharmaceutics444 (2013) 139–145

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International

Journal

of

Pharmaceutics

j ou rn a l h o m e pag e :w w w . e l s e v i e r . c o m / l o c a t e / i j p h a r m

Aspartate

buffer

and

divalent

metal

ions

affect

oxytocin

in

aqueous

solution

and

protect

it

from

degradation

Christina

Avanti

a,b,∗,1

,

Nur

Alia

Oktaviani

c,1

,

Wouter

L.J.

Hinrichs

a

,

Henderik

W.

Frijlink

a

_,

_Frans

_A.A.

_Mulder

c,d

a_{DepartmentofPharmaceuticalTechnologyandBiopharmacy,UniversityofGroningen,AntoniusDeusinglaan1,9713AV,Groningen,TheNetherlands} b_{DepartmentofPharmaceutics,FacultyofPharmacy,UniversityofSurabaya(Ubaya),Surabaya,Indonesia}

c_{GroningenBiomolecularSciencesandBiotechnologyInstitute,UniversityofGroningen,Nijenborgh4,9747AG,Groningen,TheNetherlands} d_{DepartmentofChemistryandInterdisciplinaryNanoscienceCenteriNANO,UniversityofAarhus,Langelandsgade140,DK-8000AarhusC,Denmark}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received25October2012

Receivedinrevisedform22January2013 Accepted24January2013

Available online 1 February 2013

Keywords: Oxytocin Conformation Aspartatebuffer Aqueoussolution Divalentmetalions NMR

a

b

s

t

r

a

c

t

Oxytocinisapeptidedrugusedtoinducelaborandpreventbleedingafterchildbirth.Duetoits instabil-ity,transportandstorageofoxytocinformulationsundertropicalconditionsisproblematic.Inaprevious study,wehavefoundthatthestabilityofoxytocininaspartatebufferedformulationisimprovedbythe additionofdivalentmetalions(unpublishedresults).ThestabilizingeffectofZn2+_{wasbyfarsuperior}

comparedtothatofMg2+_{.Inaddition,itwasfoundthatstabilizationcorrelatedwellwiththeabilityof}

thedivalentmetalionstointeractwithoxytocininaspartatebuffer.Furthermore,LC–MS(MS) measure-mentsindicatedthatthecombinationofaspartatebufferandZn2+_{inparticularsuppressedintermolecular}

degradationreactionsneartheCys1,6_{disulfidebridge.Theseresultsleadtothehypothesisthatin}

aspar-tatebuffer,Zn2+_{changestheconformationofoxytocininsuchawaythattheCys}1,6_{disulfidebridge}

isshieldedfromitsenvironmenttherebysuppressingintermolecularreactionsinvolvingthisregion ofthemolecule.Toverifythishypothesis,weinvestigateheretheconformationofoxytocinin aspar-tatebufferinthepresenceofMg2+_orZn2+_{,using2DNOESY,TOCSY,}1_H–13_CHSQCand1_H–15_NHSQC

NMRspectroscopy.Almostall1_H,13_Cand15_{NresonancesofoxytocincouldbeassignedusingHSQC}

spectroscopy,withouttheneedfor13_Cor15_Nenrichment.1_H–13_Cand1_H–15_{NHSQCspectrashowed}

thataspartatebufferaloneinducesminorchangesinoxytocininD2O,withthelargestchemicalshift

changesobservedforCys1_.Zn2+_{causesmoreextensivechangesinoxytocininaqueoussolutionthan}

Mg2+_{.Ourfindingssuggestthatthecarboxylategroupofaspartateneutralizesthepositivechargeofthe}

N-terminusofCys1_{,allowingtheinteractionswithZn}2+_{tobecomemorefavorable.Theseinteractionsmay}

explaintheprotectionofthedisulfidebridgeagainstintermolecularreactionsthatleadtodimerization.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Oxytocin is a nonapeptide hormone secreted by the

pos-terior lobe of the pituitary gland, and is involved in the

control of labor and bleeding cessation after child birth

(Maughanetal.,2006).Thepeptideconsistsofnineaminoacids

(Cys–Tyr–Ile–Gln–Asn–Cys–Pro–Leu–Gly–NH2) with an internal

disulfidebridge,andanamidatedC-terminus(duVigneaudetal., 1953).Oxytocinisthepreferreddrugtopreventpostpartum

hem-orrhage and is commonly formulated in aqueous solution for

∗Correspondingauthor.Presentaddress:DepartmentofPharmaceutics,Faculty ofPharmacy,UniversityofSurabaya(Ubaya),RayaKalirungkut,Surabaya60293, Indonesia.Tel.:+62312981110;fax:+62312981111.

E-mailaddresses:c.avanti@rug.nl,cavanti@staff.ubaya.ac.id(C.Avanti). 1 _{Equallycontributedfirstauthor.}

parenteral administration (Gard et al., 2002). The instabilityof oxytocininaqueoussolutionunderharshcircumstances, partic-ularlyundertropicalconditions,presentsasignificantchallenge topharmaceuticalscientists(Haweetal.,2009).Theinstabilityof oxytocininaqueoussolutionhasbeenreportedinseveral stud-ies(Hogerzeiletal.,1993;Trisseletal.,2006).Ithasbeenfound thatthedegradationratestronglydependsonthepHofthe formu-lation,withthehigheststabilityreportedatpH4.5(Haweetal.,

2009).Severalstudieshave beenaimedattheimprovement of

thestabilityofoxytocininaqueoussolution(Avantietal.,2011; Hawe et al., 2009). The mostrecent findingis that the useof divalentmetalions,incombinationwithcertainbuffers,strongly increasesthestabilityofoxytocininaqueoussolution(Avantietal., 2012).

Previously, we have shown that Zn2+ _{in combination with}

aspartatebufferstronglystabilizesoxytocininaqueoussolutions, whereasCa2+_andMg2+_{onlyhaveminoreffects.Thestabilization}

0378-5173/$–seefrontmatter© 2013 Elsevier B.V. All rights reserved.