pharmacokinetics of diclofenac in healthy controls with wild type phenotype for cyp2c9 shows metabolism variability

Please cite this article in press as: Martín-De Saro M, et al.. Cuevas-Covarrubiasc , d , ∗ aDepartamento de Genética Médica, Hospital Materno Infantil ISSEMyM, Toluca, Mexico bDepartame

Please cite this article in press as: Martín-De Saro M, et al Pharmacokinetics of diclofenac in healthy

con-www.elsevier.es/hgmx

´

´

ORIGINAL ARTICLE

M Martín-De Saroa, O Amancio-Cassinb, H Urueta-Cuéllarc, L González-Huertac,

Q1

S Cuevas-Covarrubiasc , d , ∗

aDepartamento de Genética Médica, Hospital Materno Infantil ISSEMyM, Toluca, Mexico

bDepartamento de Farmacovigilancia, Hospital General de México, Mexico City, Mexico

cServicio de Genética, Hospital General de México, Mexico City, Mexico

dFacultad de Medicina, Universidad Nacional Autónoma de México, Mexico City, Mexico

Received1December2016;accepted1February2017

KEYWORDS

Pharmacogenomic;

Diclofenac;

CYP2C9;

Cytochromep450

Abstract

Background: CytochromeP4502C9(CYP2C9)isanimportantenzymeinthemetabolismofmany drugs,includingNSAIDs,antidepressantsandanticoagulants.In vitroandin vivostudieshave demonstratedthatCYP2C9polymorphismsmodifytheactivityoftheenzymeandsubsequently themetabolismofdifferentdrugs

Objective: Tocharacterizethediclofenacpharmacokineticsinhealthysubjectswiththewild typeformofCYP2C9genotype

Methods:Twentyfivehealthywomenwereincludedinthestudy;asingledoseofdiclofenac (50mg) was administered orally Pharmacokinetic analyses at 12 different times were per-formed.DNAwasextractedfromperipheralbloodandanalyzedthroughdirectsequencing.The CYP2C9*1/*1allelewasfoundinallsubjects.UsingtheAUC0-infparameter,weclassifiedthe

25volunteersaspoor,intermediateandextensivemetabolizer(2285.421/3217.442/4637.450, respectively) Wedetected statisticalsignificantdifferencesbetween thegroups,especially betweenpoormetabolizersversusintermediateandextensivemetabolizers

∗Correspondingauthorat:Genética,HospitalGeneraldeMéxico,Dr.Balmis148,Col.Doctores,MexicoCity,Mexico.

E-mail addresses:sergiocuevasunam@gmail.com, sergioa@servidor.unam.mx, sercuevas@yahoo.com (S Cuevas-Covarrubias).

http://dx.doi.org/10.1016/j.hgmx.2017.02.001

0185-1063/© 2017 Sociedad M´ edica del Hospital General de M´ exico Published by Masson Doyma M´ exico S.A This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

Conclusions:This data indicate that CYP2C9 is not the only enzyme responsible of the metabolismofdiclofenac,soitisimportanttoanalyzeothercytochromesandtheirvariants potentiallyinvolvedinthemetabolismofthisdrug

©2017SociedadM´edicadelHospitalGeneraldeM´exico.PublishedbyMassonDoymaM´exico S.A.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons org/licenses/by-nc-nd/4.0/)

PALABRAS CLAVE

Farmacogenómica;

Diclofenaco;

CYP2C9;

Citocromop450

Variabilidad metabólica de la farmacocinética del diclofenaco en controles de salud con fenotipo silvestre de CYP2C9

Resumen

Antecedentes: ElcitocromoP4502C9(CYP2C9)esunaenzimaimportanteenelmetabolismo

demuchosfármacos, incluyendoAINEs,antidepresivosyanticoagulantes Estudiosin vitroe

in vivohandemostradoquelospolimorfismosdelCYP2C9modificanlaactividaddelaenzima

yposteriormenteelmetabolismodediferentesfármacos

Objetivo:Caracterizar lafarmacocinéticadeldiclofenacoensujetossanosconlaforma sil-vestredelgenotipoCYP2C9

Métodos: Veinticincomujeressanasfueronincluidasenelestudio;Seadministróporvíaoral una dosis única de diclofenaco (50mg) Se realizaron análisis farmacocinéticos a 12 tiem-posdiferentes ElADNse extrajo desangreperiférica yse analizó mediantesecuenciación directa.ElaleloCYP2C9*1/*1seencontróentodoslossujetos.Utilizandoelparámetro AUC 0-inf, seclasificaron alos25 voluntarios como metabolizadorespobres, intermediosy rápidos(2285.421/3217.442/4637.450,respectivamente).Sedetectarondiferencias estadís-ticamentesignificativasentrelosgrupos,especialmenteentremetabolizadorespobresfrente

ametabolizadoresintermediosyrápidos

Conclusiones:Estos datos indican que CYP2C9 no es la única enzima responsable del metabolismodeldiclofenaco,porloqueesimportanteanalizarotroscitocromosysusvariantes potencialmenteimplicadasenelmetabolismodeestefármaco

©2017SociedadM´edicadelHospitalGeneraldeM´exico.PublicadoporMassonDoymaM´exico S.A.Esteesunart´ıculoOpenAccessbajolalicenciaCCBY-NC-ND(http://creativecommons org/licenses/by-nc-nd/4.0/)

Introduction

The interindividualvariability indrug responseis a major

causeofadverse effects.Inmany cases,thisvariabilityis

linkedtopolymorphismsofgenesencodingenzymes

respon-sibleforthebiotransformationofthesedrugs.1Carriersof

mutations in genes encoding drug metabolizing enzymes,

whentreatedwithstandarddosesofcertaindrugs,usually

have higher plasma levels.They present lower clearance

andan increase in thefrequency andseverity of adverse

reactions secondary tothe use of the drug.2 Cytochrome

P450ssuperfamily(CYP)comprisesenzymesinvolvedinthe

metabolismof endogenoussubstances and

biotransforma-tionofdrug.3

The CYP2C subfamily consists of 4 members clustered

on chromosome 10q24

(Cen-CYP2C18-CYP2C19-CYP2C9-CYP2C8 -Tel)andconstituteapproximately20%ofallP450

enzymesintheliver Theyalsoareexpressedinother

tis-suessuchaskidney,intestine,brain,heart,lungandaorta

TheCYP2Cenzymesarewellknowntometabolizejustover

20%ofalldrugs.4 -9Todate,33differentalleleshavebeen

described in CYP2C9 The most studied alleles CYP2C9 *2

(R144C) andCYP2C9 *3 (I359L) leadto a decrease in the

enzymaticactivity.6,9,10

CytochromeP4502C9(CYP2C9)isanimportantenzyme involvedinthebiotransformationofmanydrugs,including non-steroidalanti-inflammatorydrug(NSAIDs), antidepres-sants and anticoagulants.1 Diclofenac sodium is a NSAID, humansmetabolizediclofenacinagreatnumberof hydroxy-latedmetabolites,themainmetaboliteinplasmaandurine

is 4-hydroxy (OH) diclofenac whereas 3-OH diclofenac and 5-OH diclofenacare minor metabolites.11 Apart from CYP2C9,otherCYP2CenzymessuchasCYP2C8seemtobe important indiclofenacmetabolism.2 Studies in vitro and

in vivohavedemonstratedthatCYP2C9genepolymorphisms modifytheenzymaticactivityofCYP2C9andalterthe bio-transformationofvariousdrugs.7

Theaim ofthis studywastocharacterize the pharma-cokineticofdiclofenacinhealthywomenwiththewildtype formtoexcludetheexclusiveparticipationofCYP2C9inthe metabolismofdiclofenac

Topreventfromgenderbias,onlywomenwereincludedin thestudy.TwentyfiveunrelatedhealthyMexicanvolunteers [meanageof29±5yearsandbodymassindexwithinnormal parameters]wereevaluated.Medicalhistorywasdoneinall

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96

97

98 99 100 101

Please cite this article in press as: Martín-De Saro M, et al Pharmacokinetics of diclofenac in healthy

con-subjects.Volunteerswithhistoryofadversedrugeffectsand

thosewithanydrugintakewereexcluded.The25 women

volunteerswere nonsmokersand abstainedfrom

caffeine-oralcohol-containing beveragesaswellasfoodstuffsfrom

grapefruit during the course of the study The pre-study

healthcheckconsistedofphysicalexamination,laboratory

tests,includingbloodcellcountsandhepaticfunctiontests,

urineanalysisandanelectrocardiogram.Thesubjectswere

informedabouttheaimofthestudyandtheyagreedto

par-ticipate.ThestudywasapprovedbytheEthicsCommittee

oftheGeneralHospitalofMexico

To analyze the CYP2C9 gene, DNA was obtained from

peripheral leukocytes under standard techniques To

per-formPCR, oligonucleotidesweredesigned considering the

database NBCI of the CYP2C9 gene The protocol was

approvedbytheEthicsCommitteeoftheGeneralHospital

ofMexico

Analysis concentrations of plasma diclofenac

Afterafastingperiodof12h,the25volunteersweregiven

a single oral dose of 50mg diclofenac sodium (Voltaren,

Novartis,Switzerland).Bloodsamplesweretakenat0,0.33,

0.66,1,1.33,1.66,2,2.5,3,4,6and9h.Serumlevelsof

diclofenacwereassayedinthesamplesusingaspecific

liq-uidchromatography,ultrahighresolutioncoupledtotandem

mass spectrometry Mobile phase was: acetonitrile:water

(62:38 v/v) Flow rate: 0.5ml/min, Column temperature:

Table 1 Demographicdata

BMI: body mass index; DBP: diastolic blood pressure; SBP: sys-tolic blood pressure.

35◦C, autosampler temperature: 13◦C, injection volume:

5␮L,Column:AcquityUPLCC18.Themethodofacceptance criteriaforthevalidationparametersisestablishedinNOM -177 -SSA1199825,Mexico

Statistics

Descriptivestatisticalanalysiswasperformedonthesample

ofthe25volunteersforthevariablesofsex, age,weight, height,BMI,systolicanddiastolicbloodpressureandheart rate.LateranalysiswithABCvariable0 -∞wasperformed

byANOVA

Thedemographicdataof25volunteersareshowninTable1 PharmacokineticvaluesareshowninTable2togetherwith themeanandstandard deviation.Usingthevaluesof ABC

in infinite time, it was possible to make a classification

Table 2 Pharmacokineticparameters

Vol Tmax(h) Cmax(ng/ml) AUC0-t(ngh/ml) AUC0-inf(ngh/ml) t1/2(h)

Mean(SD) 1.551(0.864) 1788.733(362.315) 3214.062(1009.1) 3334.559(998.895) 1.274(1.18)

AUC: area under the curve.

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128 129 130 131

132

133 134 135 136 137

138

139 140 141 142

Table 3 AUCinfandmetabolizers.

Groups MeanABC0-inf(ngh/ml) SD

AUC: area under the curve; FM: fast metabolizer; IM:

interme-diate metabolizer; PM: poor metabolizer.

1600

PM IM FM

1400

1200

0 0.33 0.66 1 1.33 1.66

Time

2 2.5 3 4 6 9

1000

800

600

400

200

0

Figure 1 AUC valuesininfinite timefor poorintermediate

andfastmetabolizers

betweenslow(ML),intermediate(MI)andfast(MR)

metab-olizers(Table3)

AUC valuesin infinite time showedstatistically

signifi-cantdifferencesbetweengroups(p<0.05).Post hocanalysis

(Scheffé)wasconductedtodeterminedifferencesbetween

groups;the groupofpoor metabolizingshowedsignificant

differencesagainstthefastandintermediatemetabolizers

(p<0.05)(Fig.1)

The present study aimedtoevaluate theeffect ofthe

CYP2C9 wild type on the pharmacokinetics of diclofenac

in healthy Mexican volunteers Twenty five subjects with

genotype * 1/* 1 (wild type) were included in the study,

allwere females becauseit hasbeen reportedthat some

NSAIDsbiotransformationbyCYP2C9isaffecteddepending

ongender.12Withinthestudiedparameters,weestablished

theareaunderthecurvefromzerotimetoinfinity

(AUC0-inf) as the most important one, it was done because it

permits us to have a better estimate of the

transforma-tionofthe drug.Basedonthis result,quartileseparation

allowed us to classify the volunteers in 6 PM, 13 IM

and 6 FM We found statistically significant differences

(p<0.05) among the 3 groups and when we compared

the PM group against IM and FM together we detected

(p<0.05)

Studies about the biotransformation of diclofenacand

CYP2C9variants dividesubjectsinto groups depending on

theirgenotypeandcomparethepharmacokinetic

parame-ters.AnexamplewouldbethestudyofYasaretal.in2001,

whichadministered a dose of 50mg oral diclofenacto20

healthysubjects andcomparedthe valuesof Cmax, AUC,

timeandclearanceofthefollowinggroups:*1/*2,*1/*3,

*2/*2,*2/*3and*3/*3against*1/*1group;theauthors

didnotfindstatisticaldifferences.13

Our findings agree with authors like Zi et al., who refersthat theinvolvement ofCYP2C9 in the biotransfor-mationofdiclofenaccannotbeexclusiveandtheremaybe other enzymesinvolvedasCYP3A4, CYP2C19,CYP2C8 and CYP2C18.6Itisalsoimportanttoconsiderthevariationsin the PORgene encodingan enzyme involved inthe proper functioningofCYP2C919

In conclusion, the variability in the metabolism of diclofenac in the * 1/* 1 genotype of CYP2C9 indicates that CYP2C9 is not the only enzyme responsible for the metabolismofdiclofenac.Differentresponsesinthesame genotypemeanthatothercytochromescouldparticipatein the metabolismof thisdrug.It is importantto character-izeother cytochromes possiblyrelated todiclofenacsuch

asCYP2C8,CYP2C18,CYP2C19 andCYP2B614or evenPOR genepolymorphism

Protection of human and animal subjects.The authors declarethatnoexperimentswereperformedonhumansor animalsforthisstudy

Confidentiality of data.Theauthorsdeclarethattheyhave followedtheprotocolsoftheirworkcenteronthe publica-tionofpatientdata

Right to privacy and informed consent.The authors declarethatnopatientdataappearinthisarticle

Theauthorshavenoconflictofinteresttodeclare

References

1 Aguilar B, Rojas JC, Collados MT Prevalence of CYP2C9 variants

in the Mexican population Arch Med Res 2008;39:463.

2 Martínez C, Blanco G, García E, et al Farmacogenómica clínica

de CYP2C8 y CYP2C9: conceptos generales y aplicación al uso

de AINE Farmacia Hospitalaria 2006;30:240 -8.

3 Smith G, Stubbins MJ, Harries LW, et al Molecular genetics of the human cytochrome P450 monooxygenase superfamily Xeno-biotica 1999;28:1129 -65.

4 Chen Y, Goldstein JA The transcriptional regulation of the human CYP2C genes Curr Drug Metab 2009;10:567 -78.

5 Xie HG, Prasad HC, Kim RB, et al CYP2C9 allelic variants: eth-nic distribution and functional significance Adv Drug Deliv Rev 2003;54:1257 -70.

6 Zi J, Liu D, Ma P, et al Effects of CYP2C9*3 and CYP2C*13 on diclofenac metabolism and inhibition-based drug -drug interac-tions Drug Metab Pharmacokinet 2010;25:343 -50.

7 Zhou S, Zhou Z, Huang M Polymorphisms of human cytochrome P450 2C9 and the functional relevance Toxicology 2010;278:165 -88.

8 Castelán O, Hoyo C, Sandoval E, et al Allele frequency distribution of CYP2C9*2 and CYP2C9*3 polymorphisms in six Mexican populations Gene 2013, http://dx.doi.org/ 10.1016/j.gene.2013.03.128.

9 Shimamoto J, Leiri I, Urae A, et al Lack of differences in diclofenac (a substrate for CYP2C9) pharmacokinetics in healthy volunteers with respect to the single CYP2C9*3 allele Eur J Clin Pharmacol 2000;56:65 -8.

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191

192

193 194 195

196 197 198

199 200

201

202

203

204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230

Please cite this article in press as: Martín-De Saro M, et al Pharmacokinetics of diclofenac in healthy

con-10 Kirchheiner J, Seeringer A Clinical implications of

pharmacoge-netics of cytochrome P450 drug metabolizing enzymes Biochim

Biophys Acta 2007;1770:489 -94.

11 Yasar U, Eliasson E, Forslund-Bergengren C, et al The role of

CYP2C9 genotype in the metabolism of diclofenac in vivo and in

vitro Eur J Clin Pharmacol 2001;57:729 -35.

12 Scandlyn M, Stuart E, Rosengren R Sex-specific differences in CYP450 isoforms in humans Expert Opin Drug Metab Toxicol 2008;4:413 -24.

13 Wang B, Wang J, Huang S, et al Genetic polymorphism of the human cytochrome P450 2C9 gene and its clinical significance Curr Drug Metab 2009;10:781 -834.

231

232

233

234

235

236 237 238 239 240 241