Ni cot i ne Met abol i c Capabi l i t y Fol l owi ng Ci garet t e Smoki ng i n Japanes e Smokers wi t h CYP2A64 /9 Genot ype

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Ni cot i ne Met abol i c Capabi l i t y Fol l owi ng Ci garet t e Smoki ng i n Japanes e Smokers wi t h CYP2A64 /9 Genot ype

Set s uko A

^KI^ZUKI

and Aki hi r o O

^HNI^SHI

Depar tment of Labor ator y Medicine, Daisan Hospital, The Jikei Univer sity School of Medicine

ABSTRACT

Objectives:To investigat

e whether smoking induced‑nicotine metabolism is reduced in Japanese intermediate metabolizers (IMs)pos sessing the cytochrome P450 (CYP)2A64/9 genotype,we compared urinary excretion of ni cotine and its metabolites(cotinine and trans‑3′

‑hydroxycotinine［3OH‑cotinine］catalyzed by CYP2A6)between IMs and extensive metabolizers (EMs). We also compared the saliva levels of nicotine and cotinine with blood levels after cigarette smoking and analyzed the kinetic disposition.

Study design:The subjects were 10 male smokers:3 IMs(CYP2A64/9)and 7 EMs(1 with CYP2A61/4,2 with 1/9,and 4 with 1/1). Af ter abstaining from smoking for 3 days,the subjects smoked 3 cigarettes,and then serial blood and saliva samples were collected at given time intervals and timed urine specimens were collect ed. Percent urinary excretion of nicotine and its metabolites and the metabolic ratio or index as an indication of in vivo CYP2A6 activity were compared between IMs and EMs.

Results:The 24‑hour urinary excretion of nicotine as a percentage of total nicotine intake was significantly higher in IMs(37.0±6.9)than in EMs( 16.3±2.4%,p＜0.05). Conversely,urinary excretion of cotinine＋ 3OH‑cotinine as a percent age of total nicotine intake was slightly but not significantly lower in IMs(63.0±6.9)than in EMs( 83.8±2.4%,p＝0.30). Metabolic clearance

(239.8±98.4 vs.959.9±264.4 ml/min,p＜0.05)and metabolic index(10.4±21.3 vs.39.3±11.7 mL/min, p＜0.05)were also lower in IMs than in EMs. Nicotine and cotinine levels in saliva were much higher than in serum (nicotine:21 to 37 times hi gher during the first 3 hours;cotinine:1.9 to 3.4 times higher up to 24 hours).

Conclusion:These results suggest that nicotine metabolism via CYP2A6 is moderately suppres- sed in Japanese subjects with the CYP2A64/9 genotype compared with that in EMs. Nicotine and cotinine appear to be secreted to a great ext ent in saliva,and the salivary level may be an excellent substitute for the serum level. (Jikeikai Med J 2007;54:11‑9)

Key words:nicotine,cotinine,cigarette smoking,CYP2A6,saliva

I

NTRODUCTION

Ni cot i ne,a maj or cons t i t uent of t obacco,r api dl y ent er s t he bl ood af t er bei ng abs or bed t hr ough t he l ung dur i ng t obacco s moki ng t o an ext ent s i mi l ar t o t hat af t er i nt r avenous admi ni s t r at i on. Ni cot i ne i s

met abol i zed pr i mar i l y by C‑oxi dat i on t o cot i ni ne,and t o a l es s er ext ent by N ‑oxi dat i on t o ni cot i ne N ‑1′ ‑ oxi de, N ‑demet hyl at i on,and N‑gl ucur oni dat i on( con- j ugat ed) . Cot i ni ne i s f ur t her hydr oxyl at ed t o t r ans ‑3′ ‑hydr oxycot i ni ne and 5′ ‑hydr oxycot i ne,N ‑ oxi dat i on t o cot i ni ne N ‑1‑oxi de,and N‑gl ucr oni da-

Received for publication,August 4,2006 秋月摂子，大西明弘

Mailing address:Akihiro O^HNI^SHI,Department of Laboratory Medicine,Daisan Hospital,The Jikei University School of Medicine, 4‑11‑1 Izumihoncho,Komae,Tokyo 201‑8601,Japan.

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t i on( conj ugat ed) . Tr ans ‑3′ ‑hydr ocot i ni ne i s f ur t her met abol i zed by O ‑gl ucur oni dat i on ( conj ugat ed) . Typi cal l y,70% t o 80% of ni cot i ne i s conver t ed t o cot i ni ne,whi ch i s t hen conver t ed t o t r ans ‑3′ ‑hydr ox- ycot i ni ne. Cyt ochr ome P450 ( CYP)2A6,a s uper f ami l y of CYPs ,i s a r es pons i bl e enzyme f or t hes e cat abol i c pat hways .

Lar ge i nt er s ubj ect var i abi l i t y i n t he ki net i c di s po- s i t i on of ni cot i ne and i t s met abol i t es af t er t obacco s moki ng has been document ed. Sever al f act or s have been pr opos ed t o expl ai n t hi s l ar ge var i abi l i t y,and t he mos t l i kel y f act or i s t he i ndi vi dual di f f er ence i n met abol i c capabi l i t y . For t he r es pons i bl e enzyme CYP2A6,s ever al mut at ed al l el es have been r epor t - ed . Among t he maj or mut at i ons whi ch decr eas e t he act i vi t y of CYP2A6 i n Japanes e,CYP2A64( del e- t i on t ype) and CYP2A69( T‑48G) have al l el e f r equenci es of 15. 8% and 22. 3%, r es pect i vel y.

Anot her maj or f act or af f ect i ng i nt er s ubj ect var i abi l - i t y i s t he di f f er ence i n ni cot i ne i nt ake af t er t obacco s moki ng. The dept h of t obacco s moke i nhal at i on, t he vol ume of each puf f ,and t he f or ce of dr awi ng woul d af f ect t he i nt ake of ni cot i ne af t er s moki ng.

Even by uni f yi ng s moki ng s t at us ,s i gni f i cant i nt r as ub- j ect var i abi l i t y i n ni cot i ne i nt ake woul d exi s t .

Cons i der i ng t he above t wo f act or s ,t he pr es ent s t udy anal yzed t he CYP2A64 and CYP2A69 mut a- t i ons i n Japanes e s moker s as a f i r s t s t ep,us i ng I nvader met hods combi ned wi t h copy number as s ay . I n t he s econd s t ep,we at t empt ed t o uni f y ni cot i ne bi oavai l abi l i t y af t er s moki ng by as s umi ng t ot al ni co- t i ne i nt ake as t he s um of 24‑hour ur i nar y excr et i ons of ni cot i ne,cot i ni ne ( unconj ugat ed:f r ee＋conj ugat - ed) , and tr ans ‑3′ ‑hydr oxycot i ni ne ( f r ee＋conj ugat - ed) . We t hen cal cul at ed on a mol ecul ar bas i s t he per cent age of ur i nar y excr et i on of ni cot i ne and t hat of ni cot i ne met abol i t es r el at i ve t o t he t ot al ur i nar y excr et i on of ni cot i ne and i t s met abol i t es and compar - ed t he r es ul t s i n i nt er medi at e met abol i zer s( I Ms )and ext ens i ve met abol i zer s( EMs ) . I n addi t i on,we al s o eval uat ed t he ni cot i ne met abol i c cl ear ance or i ndex obt ai ned f r om s er um and s al i var y dat a,r es pect i vel y,

i n t he t wo gr oups t o as s es s i ndi vi dual cat abol i c capa- bi l i t y of CYP2A6 f ol l owi ng ci gar et t e s moki ng.

Anot her ai m of pr es ent s t udy was t o eval uat e t he

degr ee of pas s age of ni cot i ne and cot i ni ne i nt o s ecr e- t i ons , s uch as s al i va, af t er t obacco s moki ng.

Smoker s of t en exper i ence a per s i s t ent bi t t er t as t e f or hour s . Many s t udi es have demons t r at ed t hat ni co-

t i ne l evel s ar e much hi gher i n s al i va t han i n bl ood . Ros e et al . have demons t r at ed t hat t he s al i var y ni cot i ne l evel may be us ed as an al t er nat i ve t o t he bl ood ni cot i ne l evel dur i ng ni cot i ne s ki n pat ch admi n- i s t r at i on. Tenneggi et al . have al s o s ugges t ed t hat s al i var y meas ur ement s ar e a us ef ul mar ker of bl ood l evel s . Thes e f i ndi ngs s ugges t t hat s al i va col l ect i on i s advant ageous ,es peci al l y when whi ch bl ood col l ec- t i on i s i mpr act i cal . Mor eover ,i f bl ood l evel s cannot be meas ur ed becaus e of l ower s ens i t i vi t y,meas ur e- ment of s al i var y l evel s i s mor e pr act i cal becaus e t hey ar e pr obabl y hi gher t han bl ood l evel s . I n t he pr es ent s t udy,we meas ur ed and compar ed s er i al s er um and s al i va concent r at i ons of ni cot i ne and cot i ni ne s i mul t a- neous l y over t i me f ol l owi ng t obacco s moki ng.

MATERIALS AND METHODS

Subjects

Ten heal t hy men( 28 t o 48 year s of age;mean±

SD,41±7 year s;52 t o 82 kg i n wei ght;mean wei ght , 72±10 kg)who wer e r egul ar ci gar et t e s moker s and had mi l d t o heavy s moki ng habi t s( 2 t o 35 ci gar et t es per day)wer e s t udi ed. The s ubj ect s r ef r ai ned f r om cons umpt i on of cof f ee and caf f ei ne‑cont ai ni ng f oods and bever ages and gr apef r ui t j ui ce f or 5 days bef or e and dur i ng t he s t udy per i od. Ci gar et t e s moki ng was pr ohi bi t ed f or 3 days bef or e and dur i ng t he s t udy per i od. None of t he s ubj ect s wer e t aki ng any medi - cat i on. At 9:00 AM on t he s t udy day t he s ubj ect s s moked t hr ee ci gar et t es( Long Peace,1. 9 mg ni cot i ne, 21 mg t ar ,Japan Tobacco I nc. ,Tokyo,Japan)wi t hi n 30 mi nut es . To meas ur e ni cot i ne and ni cot i ne met abol i t e l evel s i n bi ol ogi cal f l ui ds ,bl ood s ampl e wer e col l ect ed f r om t he ant ecubi t al vei n bef or e and 1,

2,3,6,9,12,and 24 hour s af t er s moki ng. Si mul t ane- ous l y,2 ml of s al i va was col l ect ed wi t hout s t i mul at i on af t er t he s ubj ect s had r i ns ed and gar gl ed at l eas t t wi ce. To accur at el y det er mi ne s al i var y ni cot i ne l evel s ,r epeat ed r i ns i ng and gar gl i ng wer e r equi r ed af t er s moki ng and bef or e each col l ect i on t o avoi d

S.A^KI^ZUKI ,et al. Vol.54,No.1 12

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cont ami nat i on di r ect l y by ci gar et t e s moke or by r es i d- ual s al i va f r om pr evi ous s ampl i ng. Ur i ne s ampl es wer e col l ect ed bef or e s moki ng( s pot t ed ur i ne)and f or t he per i ods 0 t o 6,6 t o 12. and 12 t o 24 hour s af t er s moki ng. Sampl es of s er um,s al i va,and ur i ne wer e s t or ed at−20° C unt i l anal yzed.

The s t udy pr ot ocol was appr oved by our uni ver - s i t yʼ s et hi cs commi t t ee,and wr i t t en i nf or med cons ent was obt ai ned f r om al l s ubj ect s .

Genetic analysis of CYP2A6

CYP2A6 9 (T ‑ 48G) Genotyping Assay :We anal yzed a s i ngl e nucl eot i de pol ymor phi s m ( SNP) CYP2A6 9 T‑48G wi t h t he PCR‑I nvader as s ay ( Thi r d Wave Technol ogi es ,Madi s on,WI ,USA).

Genomi c DNA was ext r act ed f r om EDTA‑t r eat ed venous bl ood us i ng t he pr ocedur e of Kunkel et al ..

Pol ymer as e chai n r eact i on( PCR)of genomi c DNA was per f or med us i ng a modi f i cat i on of t he met hod of Pi t ar que et al .. The r egi on cont ai ni ng t he pol ymor - phi c s i t e CYP2A6 9 T‑48G was ampl i f i ed wi t h PCR us i ng a pai r of f or war d and r ever s e ol i gonucl eot i de pr i mer s 9/S1 and ex1R( Tabl e 1) ,i n a 25‑μL mi xt ur e of 10 mM Tr i s ‑HCl( pH 8. 3) ,50 mM KCl ,1. 5 mM MgCl,and 0. 001% gel at i n cont ai ni ng 200μM of dNTP,0. 5μM of each pr i mer ,and 1. 25 U of Am-

pl i Taq Gol d DNA pol ymer as e( Appl i ed Bi os ys t ems , Fos t er Ci t y,CA,USA) . Af t er an i ni t i al denat ur at i on s t ep of 10 mi nut es at 95° C,35 cycl es of ampl i f i cat i on ( 0. 5 mi nut e at 94° C f or denat ur at i on;0. 5 mi nut e at 65° C f or anneal i ng;1 mi nut e at 72° C f or ext ens i on) wer e car r i ed out wi t h a t her mal cycl er( GeneAmp 9700,Appl i ed Bi os ys t ems ) ,f ol l owed by a f i nal ext en-

s i on per i od of 7 mi nut es at 72° C.

The s i gnal pr obes and t he I nvader ol i gonu- cl eot i des f or det ect i on of t he CYP2A6 9 T‑48G pol ymor phi s m was des i gned us i ng t he I nvader Cr eat or s of t war e pr ogr am ( Thi r d Wave Technol ogi es )( Tabl e 2) . I nvader r eact i ons wer e per f or med us i ng 384‑wel l pl at es wi t h r eagent s cont ai ni ng Cl eavas e XI and bot h FAM dye and Redmond Red( RED)dye( Epoch Bi os - ci ences ,Redmond,WA,USA)FRET cas s et t es . I n br i ef ,3μL of a 1/20 di l ut i on of t he CYP2A6 ‑s peci f i c PCR pr oduct s cont ai ni ng t he r egi on of T‑48G or t he no t ar get cont r ol( NTC:10 ng/μL t RNA)was added t o t he appr opr i at e wel l s ,f ol l owed by addi t i on of 1. 2 μL of s i gnal pr obes /I nvader Ol i go Mi x,1. 4μL of FRET Mi x and 0. 4μL of Cl eavas e/MgCls ol ut i on f or ampl i f i ed DNA ( Thi r d Wave Technol ogi es )and an over l ay of 6μL of mol ecul ar bi ol ogy‑gr ade mi ner al oi l( Si gma‑Al dr i ch,St .Loui s ,MO,USA) . The pl at es wer e t hen s pun f or 10 s econds at 1, 000 r pm ( 120 g) ,

i ncubat ed at 63° C f or 10 mi nut es i n a t her mal cycl er ( PTC‑100,MJ Res ear ch,Wal t ham,MA,USA) ,and t hen di r ect l y r ead wi t h a f l uor es cence pl at e r eader ( Cyt oFl our 4000,Appl i ed Bi os ys t ems )wi t h exci t at i on at 485 nm/20 nm ( wavel engt h/bandwi dt h)and emi s -

s i on at 530 nm/25 nm f or FAM ;exci t at i on at 560 nm/

20 nm and emi s s i on at 620 nm/40 nm f or RED.

CYP2A6 4 (deletion) Invader copy number assay :We us ed t he quant i t at i ve capabi l i t y of t he I nvader s ys t em t o det er mi ne gene copy number by compar i ng t he t ar get gene s i gnal( CYP2A6 )wi t h t hat of a r ef er ence gene s uch asα‑ actin. The CYP2A6 pr obe s et was des i gned t o r eact s peci f i cal l y wi t h onl y t he CYP2A6 s equence( Tabl e 1) . The r el at i ve r at i os

Ni cotine Kinetics after Cigarette Smoking

Table 1. PCR and Invader Signal Probes and Invader Oligonucleotide Sequences Oligonucleotide

Locus Reaction

type Oligonucleotide

type Sequence

9/s1 PCR PCR sense CAGGATTCATGGTGGGGCATGT ex1R PCR PCR antisens e CTTCATGAGGGAGTTGTACATC CYP2A6 9(T‑48G) Invader invader ol igo TGACGGCTGGGGTGGTTTGCCTTTA

Signal probe 1 cgcgccgaggATACTGCCTGAAAAAGAGG Signal probe 2 acggacgcggagCTACTGCCTGAAAAAGAGG

CYP2A64 Invader 2A6 Invader oligo GTATCTAGGGGTCTCAGAGCAGGAAATGATAGTCCGAATAG copy number assay α‑actin Invader oligo AAGAGTAGCCACGCTCGGTGAGGATCTTCATT

2A6 signal probe acggacgcggagGCAAAATGGGGTGG α‑actin signal probe cgcgccgaggCAGGTAGTCGGTGAGATC

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of t he CYP2A6 and r ef er ence gene s i gnal s f r om each as s ay al l owed us t o i dent i f y and quant i f y t he del et i on al l el es of CYP2A6.

Genomi c DNA was ext r act ed us i ng t he met hod des cr i bed above. I nvader r eact i ons wer e per f or med us i ng 384‑wel l pl at es wi t h r eagent s cont ai ni ng Cl eavas e XI enzyme f or genomi c DNA,and bot h FAM and RED FRET cas s et t es . I n br i ef ,3μL of pr edenat ur ed DNA s ampl es( 20 ng/μL)or NTC ( 10 ng/μL t RNA)was added t o t he appr opr i at e wel l s ,

f ol l owed by t he addi t i on of 1. 2μL of s i gnal pr obes / I nvader ol i go Mi x,1. 4μL of FRET Mi x and 0. 4μL of Cl eavas e/MgCl s ol ut i on f or Genomi c DNA and an over l ay of 6μL of mol ecul ar bi ol ogy‑gr ade mi ner al oi l . Fol l owi ng r eagent di s pens i ng,t he pl at es wer e s pun f or 10 s econds at 1, 000 r pm ( 120 g) ,i ncubat ed at 63° C f or 3 hour s i n a PTC‑100 t her mal cycl er ,and t hen di r ect l y r ead wi t h a Cyt oFl our 4000 f l uor es cence pl at e r eader us i ng t he s et t i ngs des cr i bed above . For t he genot ype det er mi nat i on,Fol d‑over ‑zer o( FOZ)val ues wer e us ed t o conf i r m t he val i di t y of each as s ay of t he s ampl es . The val ues wer e cal cul at ed wi t h a pr o- gr am pr ovi ded by Thi r d Wave Technol ogi es . For t he copy number as s ay,t he NET FAM and RED FOZ val ues( f or CYP2A6 andα‑act i n)wer e cal cul at ed,and t he r at i o of t he CYP2A6 t oα‑act i n NET FOZ was cal cul at ed t o i dent i f y CYP2A6 copy number .

Analysis of nicotine, cotinine, and their metabolites in biological fluids

Level s of ni cot i ne,cot i ni ne,and t hei r met abol i t es i n s er um,s al i va,and ur i ne wer e det er mi ned wi t h hi gh‑

per f or mance l i qui d chr omat ogr aphy ( HPLC) . The as s ay pr ocedur es wer e s l i ght l y modi f i ed f r om t he met hods r epor t ed by Zuccar o et al . and by Oddoze et al .. For t he ext r act i on pr ocedur e,a 200‑μL al i quot of ur i ne,600μL of s odi um hydr ochl or i de,and 200μL of 10 mg/mL 2‑phenyl i mi dazol e met hanol s ol ut i on ( i nt er nal s t andar d) ,f or a t ot al vol ume of 1 mL,wer e t r ans f er r ed t o Ext r el ut ‑1 gl as s col umns( Mer ck Shar p

& Dohme,Whi t ehous e St at i on,NJ,USA)t hat wer e pr epacked and f i l l ed wi t h 700 mg of di at omaceous ear t h and pr econdi t i oned wi t h 6 mL of di chl or omet h-

ane 1 day bef or e as s ay. Af t er 10 mi nut es ,t he compo- nent s wer e el ut ed under gr avi t y wi t h 5 mL of di chl or - omet hane‑i s opr opyl al cohol( 9:1,v/v) . The ext r act - ed f l ui d was mi xed wi t h 100μL of met hanol HCl( 25 mM)and t hen evapor at ed t o dr ynes s under ni t r ogen and r edi s s ol ved i n 200μL of wat er . For gl ucur oni de‑

conj ugat ed compound,250μL ur i ne was mi xed wi t h 20μL of 400 U β‑gl ucur oni das e. Af t er over ni ght r eact i on,200μL of t he r eact i on mi xt ur e was mea-

s ur ed i n a manner s i mi l ar t o t hat us ed f or t he con- j ugat ed compound. For HPLC as s ay( L‑6000,Hi t a- chi Medi cal Cor por at i on,Tokyo) ,an I ner t s i l C8 col - umn( 5‑μm par t i cl e s i ze s pher i cal s i l i ca,25 cm × 4. 6 mm I . D. ,GL Sci ences ,Tokyo)was us ed wi t h a mobi l e

Table 2. Subject Characteristics of Smoking Status and Putative Genetic Polymorphism of CYP2A6

Subject Age

(years) Weight

(kg) Smoking rate

(cigarettes/day) Total

(μg/24 hr) Copy number Effect

(T‑48G)Putative genotype

A 46 80 2 295.7 1 G 4/9

B 35 75 10 1,563. 2 1 G 4/9

C 37 71 30 3,504. 3 1 G 4/9

D 39 79 35 7,595.7 1 T 1/4

E 37 82 4 979. 5 2 T/G 1/9

F 46 82 15 1,932. 9 2 T/G 1/9

G 48 63 15 2,495. 2 2 T 1/1

H 43 63 20 3,359. 7 2 T 1/1

I 47 73 25 3,567. 9 2 T 1/1

J 27 52 15 2,981. 8 2 T 1/1

a)Total:Total of 24‑hour urinary excretion(μg/24 hours)of nicotine,cotinine,and 3 hydroxycotinine,measured as nicotine bioavailability:b)Data derived from Invader copy number assay;

c)The genotype in the present study was putative,derived from 4(copy number)and 9(T‑48G point mutation);therefore,other mutations cannot be ruled out

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phas e of wat er ‑acet oni t r i l e( 80:9,v/v)cont ai ni ng 5 mL of t r i et hyl ami ne,670 mg/L s odi um hept anes ul -

f onat e,and 0. 034 M each of K HPO and ci t r i c aci d ( pH＝4. 4) ,at a f l ow‑r at e of 1. 6 mL/mi n.

The s al i va s ampl e was meas ur ed i n a s i mi l ar manner as f or ur i ne. For ext r act i on of t he s er um s ampl es ,1. 5 mL of s er um s ampl e,1. 4 mL of s odi um hydr ochl or i de( 0. 5 M)and 100μL of i nt er nal s t andar d s ol ut i on,f or a t ot al of vol ume of 3 mL,wer e t r ans f er - r ed t o a Ext r el ut ‑1 gl as s col umn pr ewas hed wi t h 12 mL of di chl or omet hane. Af t er 15 mi nut es ,10 mL of t he ext r act ed f l ui d was added t o 300μL of met hanol HCl( 25 mM) ,evapor at ed t o dr ynes s under ni t r ogen, and r edi s s ol ved i n 100μL of wat er f or i nj ect i on i nt o t he HPLC s ys t em. The abs ol ut e r ecover y r at es of t he ext r act i on r anged f r om 85% t o 95% f or ni cot i ne, 95% t o 110% f or cot i ni ne,and 80% t o 90% f or t r ans ‑3′ ‑hydr oxycot i ni ne. The wi t hi n‑day coef f i - ci ent of var i at i on r anged bet ween 1. 9% and 4. 4% f or t he l ower l evel s and bet ween 0. 6% and 1. 6% f or t he hi gher l evel s .

Phar macokinetic and statistical analyses

Ni cot i ne and cot i ni ne concent r at i ons i n s er um and s al i va wer e f i t t ed t o a phar macoki net i c model by t he non‑l i near l eas t s quar es met hods us i ng t he mi cr o- comput er pr ogr am Wi ndow‑Nonl i ne( Ver s i on 4. 0,SCI Sof t war e,Phar s i ght Cor p. ,Lexi ngt on,KY,USA).

The hal f ‑l i f e was det er mi ned wi t h 0. 693/Kel . The peak pl as ma concent r at i on( Cmax)and t he t i me t o r each Cmax ( Tmax)wer e obt ai ned f r om r eal dat a poi nt s . The ar ea under t he concent r at i on t i me cur ve ( AUC )af t er ci gar et t e s moki ng was cal cul at ed wi t h l og‑l i near t r apezoi dal appr oxi mat i on f r om t i me 0 t o t he t i me of t he l as t obs er ved meas ur ement( Cpl as t ) and ext r apol at ed t o i nf i ni t y by addi ng Cpl as t /Kel t o obt ai n AUC . As a mar ker f or in vivo CYP2A6 act i vi t y, 24‑hour ur i nar y excr et i on of ni cot i ne met abol i t es( cot i ni ne＋tr ans ‑3′ ‑hydr oxycot i ni ne,f r ee and conj ugat ed)as a per cent age of t he t ot al ur i nar y excr et i on of ni cot i ne and met abol i t es( es t i mat ed ni co-

t i ne i nt ake)excr et ed was cal cul at ed. Fur t her mor e, as anot her mar ker f or in vivo CYP2A6 act i vi t y, met abol i c cl ear ance was cal cul at ed f r om t he 24‑hour ur i nar y excr et i on of cot i ni ne( f r ee and conj ugat ed)

and tr ans‑3′ ‑hydr oxycot i ni ne ( f r ee and conj ugat ed) di vi ded by s er um ni cot i ne AUC . We al s o cal cul at ed t he met abol i c i ndex as a s ur r ogat e f or met abol i c cl ear ance us i ng t he s al i var y ni cot i ne AUC i ns t ead of s er um ni cot i ne AUC. We at t empt ed t o us e s al i var y ni cot i ne AUC i ns t ead of t he s er um ni cot i ne AUC becaus e s er um ni cot i ne l evel s wer e ext r emel y l ow,

near t he det ect i on l i mi t ,and mi ght not be accur at e.

Mor eover ,t he s er um s ampl es f r om 1 s ubj ect( G)coul d not be meas ur ed becaus e of opaque s er um,pr obabl y caus ed by i ncr eas ed t r i gl ycer i des .

Dat a ar e s hown as means ±s t andar d er r or s of t he mean( S. E. M. )i f not ot her wi s e i ndi cat ed. The Mann‑

Whi t ney U t es t was per f or med f or t he s t at i s t i cal compar i s on bet ween t he I M and EM gr oups . A p val ue of＜ 0. 05 was cons i der ed t o i ndi cat e s t at i s t i cal s i gni f i cance.

R

ESULTS

Nicotine disposition in CYP2A6 genotype

Ten heal t hy mal e s ubj ect s wer e enr ol l ed i n pr es - ent s t udy;al l wer e s moker s ,but t hei r s moki ng habi t r anged f r om 2 t o 35 ci gar et t es per day ( Tabl e 2) . Af t er t he s moki ng of 3 ci gar et t es ,t ot al ni cot i ne bi oavai l abi l i t y es t i mat ed f r om 24‑hour ur i nar y excr e- t i on of ni cot i ne and i t s met abol i t es( nor ni cot i ne and nor cot i ni ne l evel s wer e not det ect ed i n any ur i ne s ampl e)was al s o var i abl e and r anged f r om 295. 7 t o 3, 567. 9μg/day. The genet i c anal ys i s of CYP2A6 r eveal ed CYP2A64/9( I M)i n 3 s ubj ect s , 1/4 i n 1 s ubj ect( s uppos ed EM) , 1/9 i n 2 s ubj ect s( s uppos ed EM) ,and 1/1 i n 4 s ubj ect s( s uppos ed EM)( Tabl e 3) .

Tabl e 3 s hows ur i nar y excr et i ons of ni cot i ne,cot i ni ne, and tr ans ‑3′ ‑hydr oxycot i ni ne( f r ee＋conj ugat ed)dur - i ng t he 24 hour s af t er s moki ng. Of t he t ot al ni cot i ne and met abol i t es excr et ed i n ur i ne over 24 hour s , ni cot i ne s howed s i gni f i cant l y hi gher per cent ages i n I Ms( 37. 0±6. 9)t han i n EMs ( 16. 3±2. 4%,p ＜0. 05) .

Conver s el y,t he per cent age of cot i ni ne＋ 3‑hydr ox- ycot i ni ne was l ower i n I Ms( 63. 0±6. 9%)t han i n EMs ( 83. 8±2. 4%, p ＝0. 30) . Bot h t he mean met abol i c cl ear ance ( 239. 8±98. 4 vs .959. 9±246. 4 mL/mi n, p＜

0. 05)and met abol i c i ndex ( 10. 4±21. 3 vs .39. 3±11. 7 mL/mi n, p ＜0. 05)wer e s i gni f i cant l y l ower i n I Ms t han

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i n EMs .

Nicotine and cotinine levels in ser um and saliva following smoking

Ni cot i ne and cot i ni ne l evel s i n s al i va wer e much hi gher t han t hos e i n s er um ( ni cot i ne:21 t o 37 t i mes hi gher dur i ng t he f i r s t 3 hour s;cot i ni ne:1. 9 t o 3. 4 t i mes hi gher up t o 24 hour s;Fi gur e 1) . The AUC

f or ni cot i ne was appr oxi mat el y 19 t i mes gr eat er i n s al i va t han i n s er um,wher eas t he AUC f or cot i ni ne was appr oxi mat el y 2. 7 t i mes gr eat er i n s al i va t han i n s er um. The Cmax f or ni cot i ne was 30 t i mes hi gher , and t he Cmax f or cot i ni ne was appr oxi mat el y 3 t i mes hi gher i n s al i va t han i n s er um ( Tabl e 4) . The mean hal f ‑l i f e f or ni cot i ne was 1. 20±0. 23 hour s i n s er um and 2. 02±0. 80 hour s i n s al i va,and t he hal f ‑l i f e f or

Table 3. Urinary excretion data of nicotine and nicotine metabolites,and metabolic clearance of nicotine

Group Subject Nicotine

(%) Cotinine(%)3‑Hydroxycotinine (%)

% of cotinine＋

% 3‑hydroxycotinine (%)

Metclearabol anceic

mL/min Metabolic index (mL/min) A 25.5 31.7 42.8 74.5 95.6 5.9 IM B 49.5 32.2 18. 3 50.5 196.0 13.3 C 36.1 53.6 10.2 63.9 427.9 11.9 Mean±S.E.M. 37.0±6.9 39.2±7.2 23.8±9.8 63.0±6.9 239.8±98.4 10.4±2.3

D 9.6 37.3 53.1 90.4 2,145.7 103.4 E 9.4 31.6 59.0 90.6 720.0 14.7 F 18.8 27.9 53.4 81.2 571.0 35.9

EM G 17.8 31.3 50. 9 82.2 − 25.0

H 11.8 35.5 52.7 88.2 851.5 17.6 I 26.3 38.7 35.0 73.7 951.4 26.9 J 20.0 56.2 23.9 80.0 519.5 51.9 Mean±S.E.M. 16.2±2.4 36.9±3.5 46.9±4.7 83.8±2.4 959.9±246.4 39.3±11.7

3‑hydroxycotinine:trans‑3′‑hydroxycotinine. a)The percentage data was calculated on the molecular basis of nicotine;b)metabolic clearance was obtained from urinary excretion of cotinine＋ 3 hydroxycotinine divided by the serum nicotine AUC on a molecular basis. c)The metabolic index was calculated in the same manner as metabolic clearance, but the salivary nicotine AUC was used instead of the serum AUC. p＜0.05 compared with the EM group(n＝7); :p＝ 0.020 compared with the EM group(n＝6)

Fig.1. Serum (●)and saliva(○)nicotine(left)and cotinine(right)concentration‑time curves following cigarette smoking in 10 healthy Japanese men. Data are means ±S.E.M.

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cot i ni ne was 20. 75±2. 11 hour s i n s er um and 23. 11±

2. 09 hour s i n s al i va( Tabl e 4) .

Ur inar y excr etions of nicotine and its metabolites : time r elationship

Fi gur e 2 s hows t he ur i nar y excr et i on of ni cot i ne, cot i ni ne,and tr ans‑3′ ‑hydr oxycot i ni ne over t i me f ol - l owi ng ci gar et t e s moki ng i n 10 heal t hy Japanes e men.

The ur i nar y excr et i on of tr ans ‑3′ ‑hydr oxycot i ni ne i ncr eas ed wi t h t i me t o 40. 4% of t ot al excr et i on,

wher eas ni cot i ne ur i nar y excr et i on decr eas ed gr adu- al l y f r om 37. 5% t o 20. 3% of t ot al excr et i on.

D

ISCUSSION

Of t he 10 Japanes e mal e s moker s enr ol l ed i n t he s t udy,3 had t he CYP2A64/9 genot ype. Accor di ng t o a r ecent s t udy by Yos hi da et al .of 92 Japanes e

s ubj ect s ,t he in vivo CYP2A6 enzyme act i vi t y ex- pr es s ed by t he bl ood cot i ni ne/ni cot i ne r at i o 2 hour s af t er chewi ng 2 mg of ni cot i ne gum i s l ower i n s ub- j ect s wi t h t hi s genot ype t han i n s ubj ect s wi t h ot her genot ypes( 1. 9±0. 9 f or 4/9 vs .4. 9±4. 4 f or 1/1;

3. 3±1. 7 f or 1/4;3. 1±0. 8 f or 1/9;3. 1±0. 8 f or 9/9) . The f r equency of t hi s genot ype i s 8. 7% i n t he Japanes e popul at i on. Fur t her mor e,Yos hi da et al . have al s o r epor t ed t hat t he expr es s i on l evel s of CYP2A6 mRNA and coumar i n 7‑hydr oxyl as e act i vi t y i n l i ver s peci ment s f r om s ubj ect s wi t h t hi s genot ype decr eas ed t o appr oxi mat el y 37% and 20%,r es pect i ve-

l y,of t hos e i n s peci mens f r om s ubj ect s wi t h t he wi l d

‑t ype genot ype(1/1) . I ndeed,no act i vi t y i s det ect - ed i n t he compl et e del et i on genot ype(4/4) . Ther e- f or e, whi l e s ubj ect s wi t h t he compl et e del et i on genot ype 4/4 ar e cl as s i f i ed as poor met abol i zer s , s ubj ect s wi t h t he 4/9 genot ype appear s t o be

Table 4. Pharmacokinetic Variables of Nicotine and Cotinine in Blood and Saliva

Variable Unit Blood Saliva

Nicotine Cotinine Nicotine Cotinine

AUC (μg・hr/mL) 0.08±0.01 1.59±0.06 1.49±0.28 4.28±0.36 Cmax (μg/mL) 0.02±0.01 0. 11±0.02 0.60±0.17 0.28±0.03 Tmax (hour) 1.16±0.16 2. 67±0.17 1.00±0.00 5.89±2.42 half‑life (hour) 1.20±0.23 20. 75±2.11 2.02±0.80 23.11±2.09 mean resident time (hour) 1.96±0. 31 30.67±3.02 3.31±1.01 33.85±3.44

Data are expressed as means±S.E.M. The above variables were obtained from observing the actual data or from Wi n‑Nonlin pharmacokinetic analysis or both.

Fig.2. Urinary excretion of nicotine(dark box),cotinine(gray box),and trans‑3′‑hydroxycotinine(white box)with the time following cigarette smoking in 10 healthy Japanes e men. Values in the boxes indicate the percentage of total excretion in each time interval cal culated on a molecular basis.

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I ms . On t he bas i s of t hi s cl as s i f i cat i on,t he 10 s ubj ect s of t he pr es ent s t udy wer e di vi ded i nt o 3 I Ms and 7 EMs ,and none had t he del et i on genot ype of poor met abol i zer s .

We as s es s ed t he in vivo CYP2A6 enzyme act i vi t y wi t h t he per cent age ur i nar y excr et i on and met abol i c cl ear ance or i ndex i n t he s ubj ect s af t er s moki ng.

The ur i nar y excr et i on of t he unchanged f or m of ni cot i ne i n I M s ubj ect s was 2 t i mes gr eat er t han t hat i n EM s ubj ect s . Conver s el y,I M s ubj ect s excr et ed l ower l evel s of ni cot i ne met abol i t es , whi ch ar e cat abol i zed by CYP2A6,i n ur i ne t han di d EMs . I n a r ecent r epor t us i ng met hods s i mi l ar t o our s ,t he met abol i c cl ear ance of ni cot i ne t ended t o be l ower i n I Ms t han i n EMs ,and t he met abol i c i ndex ( us i ng s al i var y dat a)was s i gni f i cant l y l ower t han t hat i n EMs . The r es ul t s of met abol i c cl ear ance or i ndex wer e al s o i n agr eement wi t h ur i nar y excr et i on dat a i n t he t wo gr oups . The s er um dat a f r om 1 EM s ubj ect wer e not avai l abl e f or t he cal cul at i on of met abol i c cl ear ance becaus e of s ampl e t ur bi di t y,pr obabl y due t o t r i gl ycer i des . Ther ef or e,we us ed t he s al i var y dat a t o obt ai n t he met abol i c i ndex. Addi t i onal l y,when s al i va col l ect i on i s an advant age becaus e bl ood col l ec- t i on i s i mpr act i cal or becaus e bl ood l evel s cannot be meas ur ed owi ng t o l ower s ens i t i vi t y,t he met abol i c i ndex us i ng s al i var y dat a may be an ef f ect i ve t ool t o i ndi cat e i ndi vi dual in vivo CYP2A6 act i vi t y. Many i nves t i gat or s have demons t r at ed t hat s er um ni cot i ne‑

t i me decay cur ves af t er admi ni s t r at i on of ni cot i ne t hr ough s ki n pat ch or gum di f f er among s ever al genot ypes . However ,i n a pr act i cal s et t i ng f or ci gar et t e s moki ng,t he bi oavai l abi l i t y of ni cot i ne var i es gr eat l y wi t h t he dept h of i nhal at i on,t he vol ume of each puf f ,and t he f or ce of dr awi ng,al l of whi ch depend of t he degr ee of ni cot i ne dependence or t he smoki ng behavi or. Therefore, the total bi oavai l abi l i t y of ni cot i ne i n our s ubj ect s var i ed wi de-

l y f r om 295. 7 t o 3, 567. 9μg of ni cot i ne ( Tabl e 3) . However ,t he pr es ent s t udy us ed t he per cent ur i nar y excr et i on dat a,and t he met abol i c cl ear ance or i ndex i ndi cat es t he i ndi vi dual met abol i c capabi l i t y vi a CYP2A6.

Accor di ng t o many pr evi ous s t udi es ,ni cot i ne and cot i ni ne l evel s i n s al i va ar e appr oxi mat el y 25

t i mes and 2. 3 t i mes hi gher ,r es pect i vel y t han t hos e i n s er um dur i ng t he f i r s t 12 hour s af t er s moki ng. The var i at i on i n t he s al i va/s er um concent r at i on r at i o i n di f f er ent s t udi es may be due t o di f f er ences i n s al i va f l ow r at es r es ul t i ng f r om di f f er ent col l ect i on met hods . A pr evi ous s t udy has s hown t hat t he cot i ni ne concent r at i on i n s al i va depends on t he s al i va f l ow r at e and i s l ower i n s t i mul at ed s al i va t han i n nons t i mul at ed s al i va . I n t he pr es ent s t udy,we col -

l ect ed s al i va s ampl es wi t hout s t i mul at i on. The pr es - ent phar macoki net i c di s pos i t i on dat a s howed l onger a hal f ‑l i f e f or cot i ni ne t han i n pr evi ous s t udi es . The r eas on f or t hi s di f f er ence i s uncl ear;however ,3 days ʼabs t ent i on f r om s moki ng may not be l ong enough t o det er mi ne t he hal f ‑l i f e of cot i ni ne pr eci s el y.

A l ong hal f ‑l i f e pr evi ous l y r epor t ed s ugges t s t hat t he pr es ent s ampl e col l ect i on s chedul e of up t o 24 hour s al s o may not be s uf f i ci ent t o det er mi ne t he cot i ni ne hal f ‑l i f e pr eci s el y. Becaus e a r ecent r epor t has s ugges t ed t hat cot i ni ne i s el i mi nat ed f r om mor e s l owl y f r om t he s al i va t han f r om t he bl ood or ur i ne , our dat a concer ni ng s al i va hal f ‑l i f e may be i n bet t er agr eement wi t h t hos e of pr evi ous s t udi es . Never t he- l es s ,t he above i s s ues s houl d be eval uat ed i n f ut ur e i nves t i gat i ons .

I n concl us i on,t he pr es ent r es ul t s s ugges t t hat ni cot i ne met abol i s m vi a CYP2A6 i s moder at el y s up- pr es s ed i n Japanes e s ubj ect s wi t h t he genot ype of CYP2A64/9 compar ed wi t h t hat i n EMs . Ni cot i ne and cot i ni ne appear t o be s ecr et ed i n l ar ge quant i t i es i n s al i va,and t he s al i var y l evel may be an excel l ent s ubs t i t ut e f or t he s er um l evel .

Acknowledgement :The aut hor s t hanks Dr s .Kazumi Kenmot s u and Tos hi kazu Yamaguchi( Di vi s i on of Devel opment of Cl i ni cal Genomi cs ,BML Res ear ch I ns t i t ut e,Kawagoe,Japan)f or t hei r s ci ent i f i c as s i s - t ance and genomi c anal ys i s ,and Dr s .Yumi e Har ada and Mi ki o Hos okawa( Ot s uka As s ay I ns t i t ut e,Toku- s hi ma,Japan)f or anal yt i cal as s i s t ance. The aut hor s t hank Dr .Gr ay D.Byr d( Tar gacept ,I nc. ,Wi ns t on‑

Sal em,NC,USA)f or t he ki nd gi f t of t r ans ‑3′ ‑hydr ox- ycot i ni ne.

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