Low efficacy of entecavir therapy in adefovir-refractory hepatitis B patients with prior lamivudine resistance.

(1)

Low

Low efficacy

efficacy

efficacy of

of

of entecavir

entecavir

entecavir therapy

entecavir

therapy

therapy in

in

adefovir-refractory

adefovir-refractory hepatitis

hepatitis

hepatitis B

hepatitis

B

B patients

patients

with

with prior

prior

prior lamivudine

lamivudine

lamivudine resistance

lamivudine

resistance

by

Kyung

Kyung Hyun

Hyun

Hyun Koh

Koh

Major

Major in

in

in Medicine

Medicine

Department

Department of

of

of Medical

Medical

Medical Sciences

Sciences

The

(2)

Low

Low efficacy

efficacy

efficacy of

of

of entecavir

entecavir

entecavir therapy

entecavir

therapy

therapy in

in

adefovir-refractory

adefovir-refractory hepatitis

hepatitis

hepatitis B

hepatitis

B

B patients

patients

with

with prior

prior

prior lamivudine

lamivudine

lamivudine resistance

lamivudine

resistance

by

Kyung

Kyung Hyun

Hyun

Hyun Koh

Koh

A

A Dissertation

Dissertation Submitted

Dissertation

Submitted

Submitted to

to

to The

The

The Graduate

The

Graduate School

Graduate

School

School of

of

of Ajou

Ajou

Ajou University

University

in

in Partial

Partial

Partial Fulfillment

Fulfillment

Fulfillment of

of

of the

the

the Requirements

the

Requirements for

Requirements

for

for the

the

the Degree

Degree

Degree of

Degree

of

Master

Master of

of

of Medical

Medical

Medical Sciences

Sciences

Supervised

Supervised by

by

Sung

Sung Won

Won

Won Cho,

Won

Cho,

Cho, M.D.

Cho,

M.D.

Department

Department of

of

of Medical

Medical

Medical Sciences

Sciences

The

The Graduate

Graduate

Graduate School,

School,

School, Ajou

Ajou

Ajou University

University

August,

(3)

This

This certifies

certifies

certifies that

that

that the

the

the dissertation

dissertation

of

of Kyung

Kyung

Kyung Hyun

Hyun

Hyun is

is

is approved.

approved.

SUPERVISORY

SUPERVISORY COMMITTEE

COMMITTEE

Sung

Sung Won

Sung

Won

Won Cho

Cho

Kwang

Kwang Jae

Kwang

Jae

Jae Lee

Jae

Lee

Jae

Jae Youn

Jae

Youn

Youn Cheong

Cheong

The

The Graduate

Graduate

Graduate School,

School,

School, Ajou

Ajou

Ajou University

University

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We determined the virologic response,incidence ofentecavirresistance, and evolution oflamivudine and adefovir-resistantmutants during entecavir (ETV) therapy in adefovir-refractory patients with prior lamivudine resistance.Forty adefovir-refractory chronic hepatitis B patients with prior lamivudine resistance who had received entecavir for ≥6 months were included and monitored forvirologic response and entecavirresistance.Ten percentofpatients achieved HBV DNA < 50 copies/mL by PCR after24 weeksofETV therapy,andan initialvirologicresponse(IVR)wasobserved in 12 outof40 patients(30%).HigherpretreatmentALT (p=0.039)and the presenceofthertL180M mutation(p=0.038)wereassociatedwithIVR.During a mean follow-up of11.4 months,fourpatients (10%)experienced virologic breakthrough,while ETV-resistant mutants were detected in six patients (15%).YMDD andadefovir-resistantmutantsweredetectedin57% and35% ofpatientsatbaseline,respectively.At48weeksoftherapy,96% and4% of patientshad YMDD and adefovir-resistantmutants,respectively.Thesedata suggestan early developmentofETV resistanceand low antiviralresponse during ETV therapy in adefovir-refractory patients with prior lamivudine resistance.

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ABSTRACT ···ⅰ TABLE OF CONTENTS ···ⅱ LIST OF TABLES ···ⅲ Ⅰ.INTRODUCTION···1

Ⅱ.PATIENTS AND METHODS···4

A.Analysisofvirologicalmarkers···4

B.Genotypicanalysis···5

C.Statisticalanalysis···5

Ⅲ.RESULTS ···7

Ⅳ.DISSCUTION ···16

REFERENCES···19

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Table1.Baselinecharacteristicsofpatients(n=40)···8

Table2.Virologic,serologicandbiochemicalresponsetoentecavir ···10

Table3.Baselinefactorsassociatedwithaninitialvirologicresponse····11

Table4.EvolutionofETV,LAM,andADV-resistantHBV duringETV therapyin6patientswhodevelopedETV-resistantmutant···13

Table5.Evolutionoflamivudineandadefovir-resistantmutantsduring entecavirtherapy···15

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Chronic hepatitis B virus (HBV) infection is an important health problem throughout the world,and leading frequently to cirrhosis,liver failure,and hepatocellularcarcinoma (Ganem and Prince,2004;Wrightand Lau, 1993). Nucleos(t)ide analogues have been found to suppress HBV replication and toimprovebiochemicaland histologicalstatusofhepatitisB patients(Laietal,1998;Dienstag etal,2003;Leung etal,2001).Prolonged antiviral therapy in patients with chronic HBV infection can prevent progression to cirrhosis and hepatocellular carcinoma (Liaw et al,2004); however,italso often results in the emergence ofdrug-resistantmutants andanensuingtreatmentfailure(Laietal,2003).

Prolonged lamivudine(LAM)therapy isassociated with ahigh rateof selectionforLAM-resistantHBV,atapproximately24% and70% after1and 4 years oftherapy,respectively (Laietal,2003).Mutations in the YMDD catalytic motif in the C domain of HBV polymerase (rtM204V/I) are responsible for LAM resistance (Melegariet al,1998).Although adefovir (ADV)hasshowntobeeffectiveagainstbothwild-typeandLAM-resistant HBV (Marcellinetal,2003;Schiffetal,2003),suboptimalviralresponsehas been frequently observed in LAM-resistantpatients (Fung etal,2006)and ADV-resistant mutants were found to appear more frequently in LAM-resistantpatients than in treatment-naive patients (Lee etal,2006). Combination therapy with ADV and LAM is considerable by international guidelines as the standard ofcare options forLAM-resistantpatients.The selection ofthe rtN236T orrtA181V/T mutants was associated with ADV resistance (Lee etal,2006;Hadziyannis etal,2006).Entecavir (ETV)is

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anotherdrug thatdisplays potentantiviralactivity againstwild-type HBV (Innaimo et al, 1997; Zoulim, 2006). LAM-resistant mutants exhibit an intermediatesusceptibility toETV asadministration ofahigh doseofETV is required to suppress these mutants (Levine etal,2002;Tenney etal, 2007). Althought ETV resistance seems to be rare in treatment-naive patients(Colonnoetal,2006;Gishetal,2007),itdoesemergewitharateof 6%, 15% and 51% after 1, 2 and 5 years therapy, respectively, in LAM-resistant patients (Tenney et al, 2007; Tenney et al, 2006). The emergenceofrtT184,rtS202,and rtM250mutationsisassociated with viral reboundinLAM-resistantpatients(Tenneyetal,2004).

Sequentialnucleos(t)ide analogue monotherapies increase the risk of selection of multi-drug resistant strains (Fung et al, 2006) and the development of multi-drug resistance to LAM and ADV is becoming a common problem.Combination therapy with ETV and tenofovir has been recommendedforthetreatmentofpatientswithresistancetoLAM andADV (Lok etal,2007);however,tenofovir has notyetbeen available for the treatmentofchronicHBV infection in many countries,and the ministry of Health,Welfare,and Family Affairs does not reimburse for combination therapyinKorea.

In vitro studies have shown that ETV is effective in suppressing ADV-resistantmutants[23].AlthoughETV hasbeenreportedtobeeffective in suppressing HBV DNA levels in two ADV-resistantpatients with prior LAM resistance(Fungetal,2006),theantiviraleffectofETV inthissetting has notbeen fully investigated.Furthermore,studies on the emergence of ETV resistance in ADV-refractory patients with priorLAM resistance are limited.In the present study,we determined the virologic response and

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emergenceofETV-resistantmutantsin ADV-refractory patientswith prior LAM resistance during ETV therapy, and the evolution of LAM and ADV-resistantmutantswasobserved.

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The study subjects included consecutive 40 ADV-refractory chronic hepatitis B patients with prior LAM resistance.All patients had LMV resistance documented by virologic breakthrough defined as an increase in thelevelofHBV DNA ofatleast1log10copies/mL from thelowestpoint during therapy ,genotypic analysis of rtM204 sequences,and LAM was switched to ADV monotherapy. Patients were considered to be ADV refractory if they had alimited virological response with or without documented ADV mutations while on ADV.Fourteen patients developed ADV resistance and another26 patients experienced suboptimalvirological response to ADV monotherapy.These were switched to ETV monotherapy from ADV.Patients were positive forHBsAg atleast1 yearbefore LAM therapy. None of the patient had co-infections(HCV,HIV) or other concomitantliverdiseasesuchasalcoholicliverdiseaseorautoimmuneliver disease.Allpatients had HBV DNA level> 5log10 copies/mL before ETV administration and received 1.0mg ETV oncedaily.Biochemistry and HBV DNA levels were tested before and every 3 months during ETV therapy. Serialblood samplesweretaken beforeand every 3monthsduring therapy andstoredat-70℃ untilusedforHBV molecularanalyses.Thisstudywas approved by the InstitutionalReview Board ofourinstitution,and allthe patientsgavetheirinformedconsent.

A A

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during therapy. HBsAg, HBeAg, and anti-HBe were tested with a commercialradioimmunoassay kit(AbbottLaboratories,Chicago,IL,USA). HBV DNA wasdetermined quantitatively by branched DNA (bDNA)assay (versantTM_3._0,BayerHeal_{thcareLLC Di}_agnosti_cDi_vi_si_{on,New York,USA),} which has a detection limit of 2000 copies/mL. In samples showing undetectableHBV DNA by bDNA assay,detection ofHBV DNA wasdone by the COBAS TaqManTM _{HBVtest (TaqMan test; Roche Di}_agnosti_cs, Branchburg,NJ),whichhasadetectionlimitof50copies/mL (or12IU/mL).

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We performed restriction fragment mass polymorphism (RFMP) to detectLAM-resistantmutations (rt180,rt204),Adefovir-resistantmutations (rt181,rt236),and ETV-resistantmutations (rt169,rt184,rt202,rt250 plus rt204) at baseline and every 3 months in all patients during ETV administration,as previously described [24,12].The genotypic analysis by RFMP wasconfirmedinsomepatientsbysequencinganalysis.Thisanalysis was performed using primers with the sequences 5'-TCC TAC GAC CCC TGC TCG TGT TAC-3'(nucleotide177-200)and5'-CTG TAA ATA GAC CTA TTG ATT GGA-3'(nucleotide959-982).

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Statisticaltesting was performed using SPSS version 13 (SPSS Inc., Chicago,IL).Results are reported as mean ±SD ormedian (range).HBV DNA levels were logarithmically transformed for analysis. Continuous variables were compared using the independentsample’st-test.Categorical datawerecomparedusingthePearson χ2_testorFi_sher’_{sexacttest.Factors}

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associated with a initial virologic response were analyzed by univariate analysis.A P-valueoflessthan.05wasconsideredstatisticallysignificant.

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The study population comprised 40 adefovir-refractory patients who had previously shown LAM resistance.The baseline characteristics ofthe patients studied are shown in Table 1.Thirty-five patients were men and themean agewas45±10.48years.Ten patients(25%)had cirrhosisand 36 patients (90%) were positive for HBeAg.Fourteen patients (35%) were treated with ETV at the time of virologic breakthrough due to ADV resistance,and the remaining 26 patients were treated due to suboptimal responsetoADV.Themean duration ofETV therapy was11.4±3.2months. At the commencement of ETV therapy,23 patients (57.5%) had YMDD mutants;seven with rtM204V,13 with rtM204I,and three with rtM204V/I. Nineteen patients (47.5%)had rtL180M.ADV-resistantmutants were found in14patients.

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Patients

Mean age, years (SD) 45.1 (10.48)

Male (%) 35 (87.5)

HBeAg-positive (%) 36 (90)

Mean ALT, IU/L (SD) 83.75 (155.72)

Mean AST, IU/L (SD) 84.28 (252.83)

HBV DNA, log10copies/mL(SD) 6.68 (0.93)

Mean duration of ADV prior to ENT, mos (SD) 17.13 (7.72)

Mean duration of ENT, mos (SD) 11.40 (3.21)

Cirrhosis (%) 10 (25) LAM-resistant mutation rtM204I (%) 13 (32.5) rtM204V (%) 7 (17.5) rtM204I+rtM204V (%) 3 (7.5) rt204M (wild) (%) 17 (42.5) rtL180M (%) 19 (47.5) rt180L (wild) (%) 21 (52.5) ADV-resistant mutation rtA181T (%) 6 (15) rtA181V (%) 4 (10) rtA181T + rtA236T (%) 1 (2.5) rtA181V + rtA236T (%) 3 (7.5)

rt181A + rt236A (wild) (%) 26 (65)

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Atthe startofETV therapy,allpatients had HBV DNA> 5 log10 copies/mL and 23 patients had elevated ALT levels.ETV redeuced HBV DNA levelsto undetectableby PCR (<50copies/mL)in 10 % and 12 % of patientsby week24andweek48,respectively,andinitialvirologicresponse (IVR)definedasHBV DNA < 4log10copies/mL after6monthsoftherapy wasobservedin12of40patients(30%)(Table2).PatientswhoachievedIVR had higherbaselineALT and AST levels(80vs44IU/L,p=0.039;51vs31 IU/L,p=0.036,respectively)comparedtothosewhodidnotachieveIVR.The rtL180M mutations were significantly more detected at baseline among patientswithIVR (75vs.35%,p=0.038).However,therewasnodifferencein baselineHBV DNA levels,HBeAgpositivity,presenceofYMDD mutationor ADV-resistantmutation between patients with and withoutIVR (Table3). Serum ALT levels were normalized in 13 of23 patients (56%)with high baseline ALT levelat 6 months of therapy.Among 36 HBeAg-positive patients,four(11.1%)achieved HBeAg loss(n=2)orHBeAg seroconversion (n=2)duringETV therapy(mean11.4months)(Table2).

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ETV,entecavir

* _Detecti_{on l}_i_mi_{t of COBAS TaqMan}TM_{assay i}_{s < 50 copi}_{es/mL (or 12} IU/mL). Week 12 (n=40) Week 24 (n=40) Week 36 (n=34) Week 48 (n=33) Total HBV DNA, log10 copies/mL,n(%) Undetectable by PCR* 4 (10) 4 (12.1) < 3.3 1 (2.5) 8 (20) 7 (20.6) 5 (15.2) 3.3-3.9 5 (12.5) 4 (10) 2 (5.9) 4 (12.1) 4.0-4.9 8 (20) 7 (17.5) 7 (20.6) 6 (18.2) ≥ 5.0 26 (65) 21 (52.5) 18 (52.9) 18 (54.5) HBeAg seroconversion /loss (n=36) (%) 0/1 2/1 2 (5.5) /2 (5.5) Virologic breakthrough (n=40) (%) 1 3 4 (10) Emergence of ETV resistance (n=40) (%) 1 1 4 6 (15)

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Patientswithout IVR

(n=28)

p-valu e Mean age,years(SD) 49 (11)02 43 (9) 0.102 Male (n=35) 10 25 0.627 HBV DNA,log10copies/mL

<7(n=24) 9 15 0.297

≥7(n=16) 3 13

MedianALT,IU/L 80(12-1007) 44(19-136) 0.039 MedianAST,IU/L 51(18-1594) 31(19-61) 0.036 HBeAg-positive(n=36)(%) 11(30.5) 25(69.4) 1.000 Cirrhosis(n=10)(%) 5(41.7) 5(17.9) 0.133 rtM204V/I(n=23)(%) 7(58.3) 16(57.1) 0.738 rtL180M (n=19)(%) 9(75) 10(35.7) 0.038 rtA181T/V,rtA236T (n=14)(%) 4(33.3) 10(35.7) 1.000

IVR,initialvirologic response Initialvirologicalresponse defined as HBV DNA < 4log10copies/mL after6monthsofentecavirtherapy.

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During a mean follow-up of 11.4±3.2 months,four patients (10%) experienced virologic breakthrough.ETV-resistantmutants emerged in six out of 40 patients (15%). Among the six patients with ETV-resistant mutants, four had virologic breakthrough and ETV-resistant mutants transiently appearedintwopatients.ETV-resistantmutantsemergedinone, oneandfourpatientsat6,9,and12monthsoftherapy,respectively.Among thesix patientswith ETV-resistantmutants,fourpatientshad thertS202G andtwohadthertT184L mutants(Table2,4).

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Among the six patients with ETV-resistant mutants, four had wild-type YMDD before ETV administration.YMDD mutations were found to emerge at12 weeks ofETV therapy in allfourpatients.The rtM204V mutation wasdetectedin threepatients,andthertM204Imutation in oneat the time ofemergence ofETV-resistantmutants.ADV-resistantmutants (rtA181V/T,rtA236T)were detected in three patients before ETV therapy. ADV-resistantmutantswerereplacedwith wild-typeHBV within 24weeks of therapy in allthree patients and were not detected at the time of emergenceofETV-resistantmutants(Table4).

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T T

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*_Combi_nedwi_thvi_rol_ogi_{cbreakthrough} † _notdetectabl_e,<3._3l_og

10copies/mL

ETV,entecavir;LAM,lamivudine;ADV,adefovir.

Entecavir-resistant mutations are rtS202G, rtT184L, lamivudine-resistant mutations are rtM204V/I,andadefovir-resistantmutationsarertA181T/V,rtA236T.

Pati ents Time to resistance (weeks)

HBV DNA level (log10copies/mL)andgenotypicresistance

Baseline week 12 week 24 week 36 week 48

1 24 5.7 5.0 ND† ND ND

rt202S rt202S rtS202G>rt202s rt202S not detected

rtM204V rtM204V rtM204V rtM204V not detected

rt181A,rt236A rt181A,rt236A rt181A,rt236A rt181A,rt236A not detected

2 36 > 8.0 5.2 ND 5.4 7.9

rt202S rt202S rt202S rt202S>rtS202G* rt202S<rtS202G* rt204M rtM204V rtM204V rtM204V

rtM204V

rt181A,rt236A rt181A,rt236A rt181A,rt236A rt181A,rt236A rt181A>rtA181T, rt236A

3 48 6.1 5.0 ND ND ND

rt202S rt202S rt202S rt202S rS202G

rt204M rt204M>rtM204I rt204M>rtM204I rtM204I rtM204I rtA181T,rt236A rt181A,rt236A rt181A,rt236A rt181A,rt236A rt181A,rt236A

4 48 > 8.0 6.4 6.9 6.8 7.4 rt184T rt184T rt184T rt184T rt184T>rtT184L* rt204M rtM204V>rt204 M rtM204V rtM204V rtM204V rtA181V,rtA236T rt181A>rtA181V, rt236A rt181A,rt236A r t 1 8 1 A , r t 2 3 6 A rt181A,rt236A 5 48 6.3 6.2 6.2 6.0 7.2 rt184T rt184T rt184T rt184T rtT184L* rtM204V rtM204V rtM204V rtM204V rtM204V

rt181A,rt236A rt181A,rt236A rt181A,rt236A rt181A,rt236A rt181A,rt236A

6 48 6.0 4.1 3.6 3.3 7.1 rt184T,rt202S rt202S rt202S rt202S rtS202G*>rt202S rt204M rtM204I>rt204M rtM204I>rt204M rtM204V rtM204V rt181A>rtA181V, rt236A>rtA236T

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Among the 40 patients studied,23 (57.5%) had rtM204V/Iand 19 (47.5%)hadrtL180M mutantsbeforeETV administration.RFMP analysisof the position rtM204 in patients receiving ETV therapy showed that rtM204V/I mutants were detected in 87.5% (35/40) and 96% (25/26) of patients at24 and 48 weeks oftherapy,respectively.In addition,rtL180M mutantsemerged in 62.5% (25/40)and 73.0% (19/26)ofpatientsat24 and 48weeksofETV therapy.TheseresultssuggestthatETV therapy selects for LAM-resistantmutants.ADV-resistant mutants (rtA181V/T,rtA236T) were detected in 14 of 40 patients (35%) before ETV administration. ADV-resistant mutants remained positive in five (12.5%) patients at 24 weeksandonepatient(3.8%)at48weeksofETV therapy(Table5).

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T T Taaabbbllleee 555.. E. EEvvvooollluuutttiioioonnn ooofff lllaaammimiivvvuuudddiiinnnee ae aannnddd aaadddeeeffofoovvviiirrr---rrreesessiiissstttaaannntt mt mmuuutttaaannntttsss ddduuurrriiinnngggeeennnttteeeccacaavvviiirrrttthhheeerrraaapppyyy Genotype B a s e l i n e (n=40) (%) week 12 (n=40) (%) week 24 (n=40) (%) week 36 (n=35) (%) week 48 (n=26)(%) YMDD, wild-type 17 (42.5) 5 (12.5) 5 (12.5) 3 (7.5) 1 (2.5) YIDD, YVDD 23 (57) 35 (87) 35 (87) 32 (91) 25 (96) rt181A, rt236A 26 (65) 30 (75) 35 (87.5) 33 (94) 25 (96) rtA181T/V, rtA236T 14 (35) 10 (25) 5 (12) 2 (6) 1 (4) rt180L 21 (52.5) 12 (30) 15 (37.5) 11 (31) 7 (26.9) rtL180M 19 (47) 28 (70) 25 (62) 24 (68) 19 (73)

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I

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Multi-drug resistancetoLAM andADV isbecoming prevalentdueto sequential treatment of LAM followed by ADV.ETV displays antiviral activityagainstbothLAM-resistantandADV-resistantHBV (Innaimoetal, 1997;Zoulim,2006;Vileneuve etal,2003).A previous study showed that 79% ofLAM-resistantpatientshadundetectableHBV DNA levelsbybDNA assayat24weeksofETV therapyandthatHBV DNA wasundetectableby PCR assay in 26% of patients at 48 weeks (Chang et al, 2005). A preliminary study of12 patients showed ETV administration reduced HBV DNA levels in patients with a limited virologicalresponse to adefovirbut only 33% of patients achieved HBV DNA levels of less than 3 log10 copies/mL at24weeks(Reijndersetal,2007).Theseresultssuggestalow response to ETV in patients with LAM resistance and in those with a limitedresponsetoADV.In ourstudy investigating theefficacy ofETV in ADV-refractorypatientswithpriorLAM resistance,IVR wasobservedin30 % ofpatientsandHBV DNA levelswereundetectableby PCR assay in 10 % ofpatientsafter6monthsofETV therapy.Thesefindingsdemonstrated thatthe antiviralactivity ofETV is low in ADV-refractory patients with LAM resistance.

In this study, high baseline ALT/AST levels were found to be associated with IVR on ETV in ADV-refractory patients with LAM resistance.Previous studies with LAM therapy have shown thatbaseline ALT isthemostimportantpredictorofHBeAg seroconversion (Chien etal, 1999). Thus,theresultsofthisstudyconfirmedthatnucleos(t)ideanalogues

(23)

are more effective in patients who have high pretreatment ALT levels. Among 40studysubjectswhohadhadYMDD mutationspreviously,YMDD mutations were detected in 23 subjects (57.5%)before ETV therapy.The presence of YMDD mutations was not associated with IVR on ETV. However,the presence ofthe rtL180M mutation appeared to be associated with IVR.TheroleofrtL180M asapredictorofIVR needstobevalidated infurtherstudieswithlargernumbersofpatients.

Ithad been reported thatviralrebounds due to ETV resistance were detectedin 10% ofLAM-resistantpatientsafter48weeksandan additional 9% after 96 weeks (Tenney etal,2007).In the presentstudy,virologic rebounds were observed in four(10%)outof40 ADV refractory patients with prior LAM resistance during a mean follow-up of 11.4 months, suggesting that ETV-resistant mutations develop early during therapy in thesepatients.WeobservedemergenceofETV-resistantmutantsinsix out of40patients.Amongthosesixpatients,4hadartS202G mutantand2had aT184L mutant.

Virologic rebound occurs in nucleoside-naive patients receiving ETV treatmentdue to selection ofa LAM-associated mutation (Colonno etal, 2006).We investigated the emergence ofLAM-associated mutations during ETV treatment in ADV-refractory patients who had had previous LAM resistance.Fifty-seven percentofpatientshad YMDD mutantsbeforeETV treatment,and ETV treatmentincreased the rate ofemergence ofYMDD mutantsto96% ofpatientsat48weeks.ThissuggeststhatYMDD mutants hadreappearedin almostallpatientswithinoneyearofETV therapy.ETV has been shown to be less effective againstLAM-resistantmutants than wild-type HBV (Chang et al,2005).Thus,ongoing ETV treatment may

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confer a selective advantage to YMDD mutants over wild-type HBV in patientsinfectedbyamixtureofwild-typeandLAM-resistantmutantHBV. Early emergence ofYMDD mutants,coupled with suboptimalresponse to ETV,can increase the risk ofselection for ETV-associated mutations in ADV refractorypatientswithLAM resistance.

Ontheotherhand,reversionfrom ADV-resistantmutantstowild-type HBV occurred in nine (64.2%) out of 14 patients at 24 weeks of ETV therapy and mostpatients had reverted wild-type HBV by 48 weeks of therapy.These findings suggest that ETV may suppress ADV resistant mutants more effectively than wild-type HBV and that ETV could be effective forthe treatmentofthose ADV-refractory patients with no prior exposuretoLAM.

In conclusion,only 30% ofADV-refractory patients with priorLAM resistanceachieved IVR on ETV and high pretreatmentALT leveland the presenceofrtL180M mutationwereassociatedwithIVR.Wealsofoundthat YMDD mutants reappeared in the majority ofpatients within one yearof therapy,even in the absence ofYMDD mutants before therapy.However, ETV was efficacious in suppressing the replication of ADV-resistant mutants.A suboptimalresponse to ETV,coupled with early emergence of YMDD mutants,ledtoearlyandfrequentdevelopmentofETV resistancein thesepatients.

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1.Chang TT,Gish RG,Hadziyannis SJ,Cianciara J, Rizzetto M, Schiff ER, Pastore G, Bacon BR, Poynard T, Joshi S, Klesczewski KS, Thiry A, Rose RE, Colonno RJ, Hindes RG:A dose-ranging study ofthe efficacy and

tolerability of entecavir in lamivudine-refractory chronic hepatitis B patients.Gastroenterology129:1198-1209,2005

2.Chien R-N,Liaw Y-F,AtkinsM:Pretherapy alaninetransaminaselevel as a determinant for hepatitis B e antigen seroconversion during lamivudine therapy in patients with chronic hepatitis B.Hepatology 30: 770-774,1999

3.ColonnoRJ,RoseR,BaldickCJ,LevineS,PokornowskiK,YuCF,Walsh A, Fang J, Hsu M, Mazzucco C, Eggers B, Zhang S, Plym M, KlesczewskiK,Tenney DJ:Entecavir resistance is rare in nucleoside naivepatientswithhepatitisB.Hepatology44:1656-1665,2006

4. Dienstag JL, Goldin RD, Heathcote EJ, Hann HW, Woessner M, Stephenson SL,Gardner S,Gray DF,SchiffER:Histologicaloutcome duringlong-term lamivudinetherapy.Gastroenterology124:105-117,2003

5.Fung SK,ChaeHB,FontanaRJ,Conjeevaram H,MarreroJ,Oberhelman K,Hussain M,Lok AS:Virologicresponseand resistancetoadefovirin patientswith chronichepatitisB.JHepatol44:283-290,2006

6.Ganem D,Prince AM:Hepatitis B virus infection-naturalhistory and clinicalconsequences.N EnglJMed350:1118-1129,2004

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7.GishRG,LokAS,ChangTT,deManRA,GadanoA,SollanoJ,HanKH, Chao YC, Lee SD, Harris M, Yang J, Colonno R, Brett-Smith H: Entecavir therapy up to 96 weeks in patients with HBeAg-positive chronichepatitisB.Gastroenterology133:1437-1444,2007

8.Hadziyannis SJ,Tassopoulos NC,Heathcote EJ,Chang TT,Kitis G, Rizzetto M,Marcellin P,Lim SG,Goodman Z,Ma J,Brosgart CL, Borroto-Esoda K, Arterburn S, Chuck SL: Long-term therapy with adefovir dipivoxilfor HBeAg-negative chronic hepatitis B for up to 5 years.Gastroenterology131:1743-1751,2006

9.HongSP,Kim NK,HwangSG,ChungHJ,Kim S,HanJH,Kim HT,Rim KS,Kang MS,Yoo W,Kim SO:Detection ofhepatitis B virusYMDD variantsusingmassspectrometricanalysisofoligonucleotidefragments.J Hepatol40:837-844,2004

10.InnaimoSF,SeiferM,BisacchiGS,Standring DN,ZahlerR,ColonnoRJ: Identification of a BMS-200475 as a potent and selective inhibitor of hepatitisB virus.AntimicrobAgentsChemother41:1444-1448,1997

11.LaiCL,ChienRN,Leung NW,Chang TT,GuanR,TaiDI,Ng KY,Wu PC,DentJC,BarberJ,Stephenson SL,Gray DF:A one-yeartrialof lamivudine for chronic hepatitis B. Asia Hepatitis Lamivudine Study Group.N EnglJMed339:61-68,1998

12.LaiCL,Dienstag J,SchiffE, Leung NW,AtkinsM,HuntC,Brown N, WoessnerM,BoehmeR,CondreayL:Prevalenceandclinicalcorrelatesof YMDD variants during lamivudine therapy for patients with chronic hepatitisB.ClinInfectDis36:687-696,2003

13.LeeYS,Suh DJ,Lim YS,Jung SW,Kim KM,LeeHC,Chung YH,Lee YS,Yoo W,Kim SO:Increased risk ofadefovirresistance in patients

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with lamivudine-resistantchronic hepatitis B after48 weeks ofadefovir dipivoxilmonotherapy.Hepatology43:1385-1391,2006

14.Leung NY,LaiCL,Chang TT,Guan R,LeeCM,Ng KY,Lim SG,Wu PC, Dent JC, Edmundson S, Condreay LD, Chien RN:. Extended lamivudine treatment in patients with chronic hepatitis B enhances hepatitis B e antigen seroconversion rates: results after 3 years of therapy.Hepatology33:1527-1532,2001

15.LevineS,HernandezD,Yamanaka G,Zhang S,RoseR,WeinheimerS, Colonno RJ:Efficacies ofentecaviragainstlamivudine-resistanthepatitis B virus replication and recombinant polymerase in vitro.Antimicrob AgentsChemother46:2525-2532,2002

16.Liaw YF,Sung JJY,Chow WC,FarrellG,LeeCZ,Yuen H,Tanwandee T, Tao QM, Shue K, Keene ON, Dixon JS, Gray DF, Sabbat J: Lamivudine for patients with chronic hepatitis B and advanced liver disease.N EnglJMed351:1521-1531,2004

17.LokAS,Zoulim F,LocarniniS,BartholomeuszA,Ghany MG,Pawlotsky JM,Liaw YF,MizokamiM,Kuiken C:Antiviraldrug-resistantHBV: Standardization of nomenclature and assays and recommendations for management.Hepatology46:254-265,2007

18.Marcellin P,Chang TT,Lim SG,Tong MJ,SievertW,Shiffman ML, Jeffers L,Goodman Z,Wulfsohn MS,Xiong S,Fry J,Brosgart CL: Adefovir dipivoxilfor the treatment of hepatitis B e antigen-positive chronichepatitisB.N EnglJMed348:808-816,2003

19. Melegari M, Scaglioni PP, Wands JR: Hepatitis B virus mutants associated with 3TC and famciclovir adminstration are replication defective.Hepatology27:628-633,1998

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20.ReijndersJG,deMan RA,PasSD,Schutten M,Janssen HL:Entecavir: A rescue therapy for chronic hepatitis B patients with a limited virologicalresponsetoadefovir.Hepatology46:660A,2007

21.SchiffER,LaiCL,HadziyannisS,NeuhausP,TerraultN,Colombo M, Tillmann HL,SamuelD,Zeuzem S,Lilly L,Rendina M,Villeneuve JP, LamaN,JamesC,WulfsohnMS,NaminiH,WestlandC,Xiong S,Choy GS,Van Doren S,Fry J,BrosgartCL:Adefovir dipivoxiltherapy for lamivudine-resistant hepatitis B in pre-and pos-liver transplantationpatients.Hepatology38:1419-1427,2003

22.Tenney DJ,LevineSM,RoseRE,Walsh AW,WeinheimerSP,Discotto L,Plym M,PokornowskiK,Yu CF,AngusP,AyresA,Bartholomeusz A,SievertW,Thompson G,WarnerN,LocarniniS,ColonnoRJ:Clinical emergence of entecavir resistant hepatitis B virus requires additional substitutionsin virusalready resistanttolamivudine.Antimicrob Agents Chemother48:3498-3507,2004

23.Tenney DJ,Pokornowsk:KA,Rose RE:Entecaviratfive years shows long-term maintenance of high genetic barrier to hepatitis B virus resistance.HepatolInt2:A88,2008

24.Tenney DJ,Rose RE,Baldick CJ,Levine SM,PokornowskiKA,Walsh AW,Fang J,Yu CF,Zhang S,Mazzucco CE,EggersB,Hsu M,Plym MJ, Poundstone P, Yang J, Colonno RJ: Two-year assessment of entecavir resistance in lamivudine-refractory hepatitis B virus patients reveals different clinical outcomes depending on the resistance substitutionspresent.AntimicrobAgentsChemother51:902-911,2007

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25.VileneuveJP,DurantelD,DurantelS,WestlandC,XiongS,BrosgartCL, GibbsCS,ParvazP,WerleB,TrépoC,Zoulim F:Selectionofahepatitis B virus strain resistantto adefovirin a livertransplantation patient.J Hepatol39:1085-1089,2003

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27.Zoulim F:Entecavir:A new treatmentoption forchronichepatitis B.J ClinVirol:36:8-12,2006

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국문요약

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아주대학교 대학원의학과 고 경 현 (지도교수 : 조 성 원 ) 본 연구에서는 이전에 라미부딘 및 아데포비어 내성을 보였던 환자를 엔테 카비어로 전환하여 치료하여 이후 발생된 바이러스 반응,엔테카비어 내성 및 라미부딘과 아데포비어 내성변종의 발현을 관찰하였다.대상 환자는 예전에 라 미부딘 내성을 보였던 아데포비어 내성 환자 중에서 6개월 이상 엔테카비어를 복용한 40명의 환자를 대상으로 하였으며 이들 환자에서의 바이러스 반응 및 엔 테카비어 내성을 모니터링하였다.전체 10% 환자에서 24주 동안의 엔테카비어 치료로 HBV DNA가 50 (copies/mL)이하가 되었으며 40명의 환자 중 12명 (30%)이 초기 바이러스 반응(initialvirologicresponse,IVR)을 보였다.치료전 높은 ALT수치(p=0.039)및 rtL180M 변이(p=0.038)가 초기 바이러스 반응과 연 관이 있었고 평균 11.4개월 추적 기간 동안 4명(10%)의 환자에서 바이러스 돌파 현상을 보였으며 엔테카비어 내성은 6명(15%)의 환자에서 관찰되었다.YMDD 및 아데포비어 내성 변이는 치료전 각각 57%,35%를 보였고 48주 동안의 치료 후 전체 환자의 96% 와 4%에서 YMDD 및 아데포비어 내성변이를 보였다.본 연구 결과 이전에 라미부딘 내성을 보였던 아데포비어 내성 환자에서의 엔테카 비어 치료는 낮은 항바이러스 반응 및 엔테카비어 조기 내성을 유발하는 것으로 사료된다. 핵심어 :B형 간염,엔테카비어,라미부딘,아데포비어,약제 내성