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A NTIMICROBIAL A GENTS AND C HEMOTHERAPY,Aug.2010,p.3205–3211Vol.54,No.8 0066-4804/10/$12.00doi:10.1128/AAC.01372-09

Importance of Serum Concentration of Adefovir for

Lamivudine-Adefovir Combination Therapy in

Patients with Lamivudine-Resistant

Chronic Hepatitis B?

Fukiko Mitsui,1,3Masataka Tsuge,1,2,3Takashi Kimura,1,3Shosuke Kitamura,1,3Hiromi Abe,1,3 Hiromi Saneto,1,3Tomokazu Kawaoka,1,3Daiki Miki,1,3Tsuyoshi Hatakeyama,1,3

Nobuhiko Hiraga,1,3Michio Imamura,1,3Yoshiiku Kawakami,1,3Hiroshi Aikata,1,3

Shoichi Takahashi,1,3C.Nelson Hayes,1,3Harue Igarashi,4Kentaro Morimoto,4

Masao Shimizu,4and Kazuaki Chayama1,3*

Department of Medicine and Molecular Science,Division of Frontier Medical Science,Programs for Biomedical Research, Graduate School of Biomedical Sciences,Hiroshima University,1-2-3,Kasumi,Minami-ku,Hiroshima-shi,734-8551, Japan1;Natural Science Center for Basic Research and Development,Hiroshima University,Hiroshima,Japan2;

Liver Research Project Center,Hiroshima University,Hiroshima,Japan3;and Bioanalysis Department,

Development and Medical Affairs Division,GlaxoSmithKline K.K.,4-6-15,

Sendagaya,Shibuya-ku,Tokyo,151-8566,Japan4

Received29September2009/Returned for modi?cation21February2010/Accepted1May2010

Lamivudine(LMV)-adefovir pivoxil(ADV)combination therapy suppresses the replication of LMV-

resistant hepatitis B virus(HBV),although its ef?cacy in suppressing HBV varies among patients.This

study analyzed the clinical,virological,and pharmaceutical factors that in?uence the effect of the

combination therapy.Patients negative for hepatitis B virus e antigen(HBeAg)and with low HBV DNA

titers immediately prior to the combination therapy effectively cleared serum HBV DNA(P?0.0348and

P?0.0310,respectively).The maximum concentration of ADV in serum(ADV C

max )was higher in

patients who showed HBV DNA clearance(P?0.0392),and the cumulative clearance rates of HBV DNA

were signi?cantly higher in patients with ADV C

max equal to or greater than24ng/ml(P?0.0284).HBeAg

negativity and lower HBV DNA at the start of the combination therapy and higher ADV C

max were found

to be independent factors for serum HBV DNA clearance.Serum creatinine increased signi?cantly during

the combination therapy,and the ADV C

max was higher in patients with low creatinine clearance rates.In

conclusion,higher serum concentrations of ADV are associated with a good response to therapy based on clearance of HBV DNA in serum.However,care should be taken to prevent worsening of renal function due to high ADV serum concentrations.

Hepatitis B virus(HBV)infection is a serious global health problem.The risk of chronic HBV infection in immunocom-petent adults is generally less than5%but increases signi?-cantly in young children and immunocompromised adults(15, 36).Chronically infected individuals often develop chronic hepatitis,liver cirrhosis,and hepatocellular carcinoma,and more than500,000people die every year from advanced liver diseases(6).Complete elimination of the virus is dif?cult,and patients are generally treated with interferon and nucleoside/ nucleotide analogues,which suppress viral replication and pre-vent the progression of liver disease by combating in?amma-tion(11,23,33).However,the emergence of drug-resistant viral mutants and hepatitis?are-up(breakthrough hepatitis)is a serious concern for such suppressive therapies(7,9,20,32). Lamivudine(LMV)is the?rst approved nucleoside ana-logue that terminates viral DNA synthesis by inhibiting chain elongation(31).Serum HBV DNA levels decrease soon after commencement of LMV therapy.However,long-term therapy frequently results in the emergence of drug-resistant HBV mutants(8,24).In one study,the rate of LMV resistance increased from24%in patients treated for1year to70%after 4years of treatment(21).LMV resistance is usually associated with amino acid substitutions in the YMDD motif of the viral reverse transcriptase(RT)(rtM204V/I/S)(4,5,19,26).Addi-tional substitutions,rtL180M and rtV173L,then further en-hance the mutated transcriptase activity(1,12,26).The emer-gence of resistant mutants also often results in viral breakthrough and subsequent breakthrough hepatitis(21). The nucleotide analogue adefovir dipivoxil(ADV)potently suppresses the replication of both wild-type and LMV-resistant HBV both in vitro and in vivo(17,25,27,37).LMV-ADV combination therapy is therefore recommended as a standard therapy for breakthrough hepatitis in Japan.Although both the combination therapy and ADV monotherapy are reported to be ef?cacious in patients with LMV-resistant HBV(28),the combination therapy carries a lower risk of emerging LMV-ADV double-resistant mutants(13,14,18).Recently,muta-

*Corresponding author.Mailing address:Department of Medical and Molecular Science,Division of Frontier Medical Science,Pro-grams for Biomedical Research,Graduate School of Biomedical Sci-ence,Hiroshima University,1-2-3Kasumi,Minami-ku,Hiroshima734-8551,Japan.Phone:81-82-257-5190.Fax:81-82-255-5194.E-mail: chayama@hiroshima-u.ac.jp.

?Published ahead of print on24May2010.

3205

tions conferring resistance to both LMV and ADV(through a combination of rtA181T/V and rtI233V or rtA181T/V and rtN236T)have been reported(2,30),although the incidence of these mutations remains lower than the incidence associated with monotherapy.

We recently observed that some patients on LMV-ADV combination therapy who developed LMV resistance showed a poor response to long-term combination therapy.Decrease of serum HBV DNA levels in these patients leveled off,and HBV DNA levels sometimes remained higher than4log copies/ml. The present study investigated those factors that affect the virological response to LMV-ADV combination therapy.We considered the nucleotide and amino acid sequences of HBV reverse transcriptase virological factors and the LMV/ADV concentrations pharmacological factors and then correlated the results with the clinical data of the patients.

MATERIALS AND METHODS

Patients.Between July2003and May2009,59consecutive patients with chronic hepatitis or cirrhosis due to LMV-resistant HBV infection were treated with LMV-ADV combination therapy at Hiroshima University Hospital.Of these,53patients who received the combination therapy for more than48weeks were analyzed in this study.Patients began to receive the combination therapy based on the following criteria:(i)increase in serum HBV DNA levels of?1log copy/ml in comparison with the nadir level during LMV monotherapy with or without breakthrough hepatitis,(ii)detection of mutations in the HBV RT domain related to LMV resistance by direct sequence analysis before the com-bination therapy,and(iii)serum creatinine levels of?1.5mg/dl.The study protocol conformed to the1975Declaration of Helsinki and was approved by the Hiroshima University Hospital ethics committee.Written informed consent was obtained from each patient.Patients coinfected with hepatitis C virus or human immunode?ciency virus were excluded from the study.In addition,patients were not administered drugs that affected serum concentrations of LMV and ADV. The53patients were divided into two groups according to virological re-sponse:virological responders(VR)and non-VR.Since cessation of the combi-nation therapy in LMV-resistant chronic hepatitis B patients is likely to lead to severe acute exacerbation,response to the therapy was assessed under extended combination therapy.VR were de?ned by sustained negative serum HBV DNA (?2.6log copies/ml by the Amplicor HBV Monitor test[Roche Diagnostics, Basel,Switzerland])for at least12weeks,while non-VR showed sustained positive HBV DNA tests until the?nal observation.In cases of cessation of the combination therapy,the point of discontinuation was de?ned as the?nal ob-servation point.Table1details the clinical and virological features of the two groups.

The patients were administered daily oral doses of10mg ADV and100mg LMV.Sera were collected from the patients every month during the combination therapy and stored at?80°C until they were used.Serum HBV DNA,liver function,complete blood count,and serum creatinine were measured every month.

Sequence analysis of the HBV polymerase RT domain.HBV DNA was ex-tracted from100?l of stored serum samples using the Smitest R&D(Genome Science Laboratories,Tokyo,Japan)and dissolved in20?l of sterile water.The extracted DNA was then ampli?ed by nested PCR using1?l of DNA as a template for the?rst PCR.PCR was performed in25?l of reaction mixture containing2.5mM MgCl2,0.4mM each deoxynucleoside triphosphate(dNTP), 20pmol of each primer,and1.25units of LA Taq(Takara Bio Inc.,Shiga,Japan) with the buffer supplied by the manufacturer.The?rst PCR products were diluted10-fold,and1?l was used as a template for the second PCR.The primers used in this study were S2F(nucleotides[nt]3189to3215;5?-CAGGGATCCT CAGGCCATGCAGTGGAAC-3?)and X2R1(nt1606to1625;5?-GTTCACG GTGGTCTCCATGC-3?)for the?rst PCR,and B2(nt65to84;5?-GGCTCM AGTTCMGGAACAGT-3?)(where M is A or C)and X2R1for the second PCR. The PCR protocol was as follows:initial denaturation at94°C for2min and35 cycles of denaturation at94°C for1min,annealing at58°C for1min,extension at72°C for2min,and?nal extension at72°C for7min.After ampli?cation,the ?nal PCR products were gel puri?ed with the QIAquick gel extraction kit (Qiagen,Hilden,Germany)and sequenced using the dye terminator cycle se-quencing kit(Applied Biosystems,Foster City,CA).Sequence analysis was performed on an ABI Prism3100Avant Genetic Analyzer(Applied Biosystems). Measurement of serum concentrations of LMV and ADV.Serum concentra-tions of LMV and ADV were measured at the last time of observation in39of 53patients who received the combination therapy.Blood sampling for trough values of LMV and ADV was performed at least24h after the drugs were taken. Subsequent blood sampling was performed1and2h after both of the drugs were taken for concentration measurement by liquid chromatography-tandem mass spectrometry(LC–MS-MS)analysis,using an LC-20A system(Shimadzu,Japan) and a Chromolith Performance RP-18e high-performance liquid chromatogra-phy(HPLC)column(Waters)for chromatography and an API4000system

TABLE1.Baseline characteristics of53patients who received LMV-ADV combination therapy

Characteristic a

Value

(n?39)

Non-VR

(n?14)

P value

Sex(male/female)31/88/6NS c CH/LC24/158/6NS HBV genotype C3914

At start of LMV monotherapy

Age(yr)54b(31–70)52b(27–66)NS HBV DNA(log copies/ml) 6.7b(2.6–8.5) 6.7(3.9–8.4)NS HBeAg(?/?)14/2510/40.0236 Duration of LMV monotherapy(wk)96b(0–166)69b(0–213)NS At start of LMV plus ADV combination therapy

Age(yr)56b(32–73)54b(27–69)NS BMI(kg/cm2)22.3b(15.6–27.3)22.2b(18.6–26.2)NS Breakthrough hepatitis(?/?)25/148/6NS HBV DNA(log copies/ml) 5.6b(2.6–8.7)7.2b(4.4–8.0)0.0310 HBeAg(?/?)15/2410/40.0348 ALT(IU/liter)44b(12–654)39b(18–310)NS Cr(mg/dl)0.74b(0.49–1.28)0.73b(0.45–1.05)NS CL CR(ml/min/1.73m2)114.3b(56.7–163.1)101.4b(74.9–180.7)NS Duration of combination therapy(wk)186b(68–311)168b(58–276)NS

a CH,chronic hepatitis;LC,liver cirrhosis;ALT,alanine transaminase;Cr,creatinine;?,positive;?,negative.

b Median value.

c NS,not signi?cant.

3206MITSUI ET AL.A NTIMICROB.A GENTS C HEMOTHER.

(MDS Sciex,Canada)for mass detection and analysis.The instrument was operated in electrospray–positive-ionization mode,and the signal was detected by multiple-reaction monitoring.We de?ned the highest concentration for the three time points as the maximum concentration of LMV(LMV C max)or ADV (ADV C max)in serum.The AUC0-2(the area under the drug concentration-time curve at0to2h)of LMV and ADV was calculated by the trapezoidal rule. Statistical analysis of clinical data.The background characteristics and serum concentrations were compared using the chi-square test and the Mann-Whitney U test.The cumulative probability of undetectable HBV DNA was analyzed by the Kaplan-Meier method,and differences between the curves were tested by the log rank test.P values of less than0.05were considered statistically signi?cant.

RESULTS

Effects of LMV-ADV combination therapy.Table1details the clinical characteristics of39VR and14non-VR subjects. Serum HBV DNA in39VR decreased to continuously unde-tectable levels,while serum HBV DNA in14non-VR de-creased to plateau levels but never became undetectable by the ?nal observation.A larger proportion of VR than non-VR were HBeAg negative prior to the start of LMV monotherapy. Similarly,a larger proportion of VR than non-VR patients were HBeAg negative and had lower serum HBV DNA im-mediately prior to the combination therapy.The cumulative clearance rates of HBV DNA were signi?cantly higher in

HBeAg-negative patients and in those with lower HBV DNA levels(?6.5log copies/ml)just before the combination therapy than in patients positive for HBeAg or with HBV DNA levels equal to or greater than6.5log copies/ml(Fig.1A and B).Out of25patients who were HBeAg positive immediately prior to combination therapy,none had seroconverted to anti-HBe af-ter completing the combination therapy,and none of the total 53showed viral breakthrough or breakthrough hepatitis during the combination therapy.

Genotyping of LMV-and ADV-resistant mutants.The nu-cleotide and amino acid sequences were determined for the RT domain in47of the53patients by the direct-sequencing method at the time just before HBV DNA clearance or at the nadir of HBV DNA levels after initiation of the combination therapy.Negative ampli?cation of HBV DNA because of low HBV DNA values precluded such analysis in the remaining6 patients.As shown in Table2,the amino acid substitutions rtS85A and A181T,previously reported to confer ADV resis-tance(16,40),were detected in2patients and1patient,re-spectively.The2patients with an rtS85A mutation also had YMDD motif mutations(Table2),and their HBV DNA levels decreased gradually to undetectable levels at62and177weeks after the beginning of combination therapy,respectively.In contrast,HBV levels in the patient with a unique rtA181T mutation did not decrease to undetectable levels following58 weeks of combination therapy until the patient was successfully treated with entecavir(ETV)monotherapy(reference39and data not shown).

Virological response to the combination therapy according to serum concentrations of LMV and ADV.To further explore the poor response of non-VR to therapy,drug concentration analysis was then undertaken in29VR and10non-VR,and the C

max

and AUC

0-2

values of LMV and ADV were com-

pared.ADV C

max

was signi?cantly higher in VR than in

non-VR(Fig.2A),although the difference for ADV AUC

0-2 was not statistically signi?cant(Fig.2B).The median values of ADV C

max

and ADV AUC

0-2

were24ng/ml and37ng?h/ml, respectively.The cumulative HBV DNA clearance rates were

signi?cantly higher in patients with high ADV C

max

values (?24ng/ml)(Fig.2C),and most of these patients belonged to

FIG.1.Cumulative HBV DNA clearance rates in patients treated

with lamivudine plus adefovir.Patients were assessed for HBeAg sta-

tus(A)and HBV DNA levels(B).

TABLE2.Amino acid sequence substitutions in the

HBV RT domain

Substitution a

No.of patients with substitution a VR

(n?33)

Non-VR

(n?14)

rtM204M

Alone73

?rtA181T01

rtM204V/I

Alone1910

?rtV214A/E20

?rtQ215H20

?rtV84I10

?rtS85A10

?rtS85A?rtV214E10

a Two known ADV-resistant amino acid substitutions(A181T and S85A)are

underlined.

V OL.54,2010IMPORTANCE OF ADEFOVIR CONCENTRATION3207

the VR group(Table3).However,the C

max and AUC

0-2

LMV were not signi?cantly different between the VR and non-VR groups,and there was no difference in HBV clearance

rates between patients with high or low C

max or AUC

0-2

LMV(Fig.2D and data not shown).The AUC

0-2

and C

max levels of both LMV and ADV did not correlate with the body

mass index(BMI)(data not shown).

Analysis of independent predictive factors for VR.To ana-

lyze predictive factors for achieving VR,multivariate analysis

was conducted.When factors appearing in Tables1,2,and3

were analyzed simultaneously,higher ADV C

max

and HBeAg negativity and lower HBV DNA at the start of the combination

therapy were found to be independent factors for VR(Table

4).ADV C

max

,in particular,was a strong determinant factor for VR(odds ratio,16.818;95%con?dence interval[CI],2.833

to99.836).

Renal function and serum concentrations of the drugs.

LMV and ADV are excreted from the kidney.Serum creati-

nine levels increased in17(32.1%)of53patients during the

combination therapy,while the median serum creatinine levels

increased signi?cantly from0.74mg/dl at baseline to0.86mg/dl

at the end of the observation period in53patients treated with

LMV and ADV(Fig.3A).The dose of ADV was reduced in6

(11.3%)of the53patients to5mg/day or10mg every2days,

and ADV administration was stopped in3(5.7%)patients due

to elevated serum creatinine levels(?1.5mg/dl).The HBV

DNA titers of6patients who reduced the dose of ADV never

had a?are-up after the reduction.Five of the6patients be-

longed to the VR and one to the non-VR group.Serum cre-

atinine levels returned to pretherapy values in all patients who

reduced or stopped treatment with ADV.Next,we investi-

gated whether the drug concentration was related to renal

function.The C

max

and AUC

0-2

values of LMV and ADV were compared between patients whose creatinine clearance rates

(CL

)were normal and those whose rates were low.As

shown in Fig.3B and C,both C

max

and AUC

0-2

of ADV were

signi?cantly higher in patients with CL

of?80ml/min/1.73

m2.In contrast,there was no relationship found between CL

CR and C

max

/AUC

0-2

of LMV(Fig.3D and E).

DISCUSSION

The poor response of chronic HBV infection to nucleotide/

nucleoside therapy is commonly attributed to amino acid sub-

stitutions in the RT domain of HBV polymerase.Several RT

amino acid mutations that induce resistance to ADV have

been reported,although the incidence is much lower than that

reported for LMV.The HBV polymerase RT domain substi-

FIG.2.Serum concentrations of ADV and effects of combination therapy.(A and B)Effects of the combination therapy based on ADV

max (A)and AUC0-2(B)determinations.In these box-and-whisker

plots,the lines within the boxes represent median values;the upper and lower lines of the boxes represent the25th and75th percentiles, respectively;and the upper and lower bars outside the boxes represent the90th and10th percentiles,respectively.(C and D)Cumulative clearance rates of HBV DNA by ADV C max(C)and LMV C max(D).

TABLE3.Serum concentration of ADV and ef?cacy of

LMV-ADV combination therapy

Parameter and value

No.(%)with value

P value VR Non-VR

AUC0-2(ng?h/ml)

?3715(52)3(30)0.2071?3714(48)7(70)

Total2910

max (ng/ml)

?2420(69)2(20)0.0097?249(31)8(80)

Total2910

TABLE4.Multivariate analysis of factors associated with HBV DNA clearance in LMV-ADV combination therapy

Factor a Category P value

Odds

ratio

95%CI

HBeAg1(positive)0.01701 1.475–25.129

2(negative)7.194

HBV DNA

(log copies/ml)

1(?6.5)0.04851 1.178–22.367

2(?6.5) 4.185

ADV C max

(ng/ml)

1(?24)0.00191 2.833–99.836

2(?24)16.818

a At the start of LMV-ADV combination therapy.Factors:gender,age,back-ground liver status,HBeAg,HBV DNA,ALT,Cr,RT mutation,ADV C max,and ADV AUC0-2.

3208MITSUI ET AL.A NTIMICROB.A GENTS C HEMOTHER.

tutions rtV84,rtS85,rtA181,rtV214,rtQ215,rtI233,rtN236, and rtP237are associated with ADV resistance(16,40),and rtA181and rtQ215mutations are associated with cross-resis-tance to LMV and ADV(23,35).To study the incidence and the effects of amino acid substitutions in the RT domain of HBV polymerase in patients receiving combination therapy, this study initially analyzed serum samples for amino acid se-quences in the region.We identi?ed the previously reported A181T and S85A substitutions,as well as substitutions at rt84, rt214,and rt215that might confer resistance to ADV.How-ever,all these mutations,except for A181T,were found in VR. These results are consistent with a previous report that most of these mutations confer only limited resistance to ADV ther-apy(16).In contrast,one of14patients who failed to clear HBV DNA in the present study had an apparent ADV resis-tance mutation.This unique mutation,A181T,which disrupts a stop codon in the HBs gene,is reported to be involved in resistance against both LMV and ADV(39).Therefore,it became apparent in the present series of experiments that only one of14patients responded poorly to the combination ther-

apy due to the emergence of a resistant viral clone. However,none of the remaining13patients had amino acid substitutions known to induce resistance to ADV.This is con-sistent with a recent report by Lampertico et al.(22)citing11% of patients who failed to clear serum HBV DNA despite3 years of combination therapy.In addition,none of these pa-tients had a known ADV-resistant strain of HBV.Yatsuji et al (40)also reported6of132patients with transiently?uctuating HBV DNA levels(from?2.6to3.1log copies/ml)and wild-type genotypes for rtA181and rtN236.

To further explore the poor response to combination ther-apy,the concentration of ADV was investigated with respect to the drug’s ef?cacy.Although it is noted that ADV is converted to the diphosphate derivative in hepatocytes by adenylate kinase and inhibits viral DNA polymerase(3,29), the detailed metabolic pathway remains unclear.According to experimental data from GlaxoSmithKline K.K.,when chronic hepatitis B patients were administered oral doses of 10mg ADV and100mg LMV,the ADV C

max

and AUC

0-24 were20.1?3.3ng/ml and231.5?33.7ng?h/ml,respec-tively(AUC

0-2

data not shown).The reported50%inhibi-

tory concentration(IC

)of ADV is0.36to0.39?M,equal to180.5to195.6ng/ml(38,39)and much higher than the

values obtained in this study(ADV C

max

,5.1to54.6ng/ml). This difference might come from the fact that the concen-tration of orally administered ADV should be higher in portal blood but lower in the peripheral blood.At any rate, there have been no reports detailing effective serum con-

centrations of ADV.In this study,the ADV C

max

was higher in VR,and cumulative clearance rates of HBV DNA were

higher in patients with higher ADV C

max

values.The reason for the lack of association between the ef?cacy of the com-

bination therapy and the ADV AUC

0-2

remains unclear and might be related to different absorption pro?les or meta-bolic pro?les for the drugs or lack of power due to the small number of patients analyzed.However,these results indi-cate that poor response to the combination therapy arises at least in part from a low serum concentration of ADV.Be-

cause90.9%(20/22)of patients with ADV C

max

values equal to or greater than24ng/ml could clear serum HBV DNA,it

is expected that non-VR with ADV C

max

values below24 ng/ml can achieve VR by boosting the serum level of ADV. Therefore,it might be recommended to raise the serum level of ADV to over24ng/ml in such cases.Two choices are considered for boosting the serum concentration of ADV: increasing the dose of ADV or using drugs that affect the serum concentration of ADV,such as an inhibitor of organic anion transporters(10,34).

Meanwhile,renal dysfunction sometimes occurs as a side effect of ADV,and serum creatinine levels actually increased in patients administered the combination therapy;11.3%of patients had to reduce the dose of ADV,and5.7%of patients had to discontinue ADV due to elevated serum creatinine levels.Furthermore,the serum concentration of ADV was

higher in patients with low CL

.This?nding suggests a pos-sible worsening of renal dysfunction in patients treated with ADV due to the generation of a vicious cause-effect circle(a higher ADV concentration worsens renal function).Although we did not investigate the safety range of ADV concentrations

FIG.3.(A)Comparison of serum creatinine concentrations just

before the start of the combination therapy and at the end of the

observation period.Renal function and concentrations of LMV and

ADV are shown.(B to E)The ADV C max(B),LMV C max(D),and

AUC0-2of ADV(C)and LMV(E)were compared between patients

with high(?80ml/min/1.73m2)and low(?80ml/min/1.73m2)

CL CR.In these box-and-whisker plots,the lines within the boxes

represent median values;the upper and lower lines of the boxes

represent the25th and75th percentiles,respectively;and the upper

and lower bars outside the boxes represent the90th and10th per-

centiles,respectively.

V OL.54,2010IMPORTANCE OF ADEFOVIR CONCENTRATION3209

in this study,and the upper limit of the range is not known,it is considered important that adequate and precise doses of ADV should be prescribed to patients,especially those with impaired renal function,instead of simply increasing the serum concentration of ADV.This study suggests that monitoring the serum ADV concentration would be useful to?ne tune the appropriate drug dosage.

ACKNOWLEDGMENTS

This work was carried out at the Research Center for Molecular Medicine,Faculty of Medicine,Hiroshima University,and the Analy-sis Center of Life Science,Hiroshima University.We thank Rie Akiyama,Sachi Tanaka,and Miyuki Matsushita for their excellent technical assistance and Yoshiko Nakata and Aya Furukawa for sec-retarial assistance.

This study was supported in part by a Grant-in-Aid for Scienti?c Research from the Japanese Ministry of Labor and Health and Wel-fare.

We have no con?icts of interest.

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