Biomarkers of Tobacco Smoke Exposure Measured in

UK Smokers and Never-smokers

CORESTA Congress, Edinburgh

12-16th September 2010

Giles, L., Hosono, K., Sherwood, N.

Background

Clinical study started with Covance Laboratories in 2006

Clinical phase completed in 2007

2008 study independently audited by FGK/medicomp (Munich, Germany)

• ICH and GCP compliance

2008 analytical evaluation completed

• ABF GmBH, Munich, Germany (Prof. Gerhard Scherer)

Content

Objectives

Study population

Population characteristics

Study design

Ethics

Biomarker selection and analysis

Statistical analysis

Results

Conclusions

Objectives

Primary objective

• To determine the levels of various biomarkers in blood and urine samples obtained from healthy subjects smoking four commercially available brands of cigarettes, and never-smokers.

Secondary objective

• To perform a gender comparison of the levels of various biomarkers in blood and urine samples of smokers and never-smokers.

Study Population

Population = Aim 100 healthy subjects

GROUP CIGARETTE TYPE

A 10mg ISO tar Virginia style *premium brand

B 10mg ISO tar Virginia style *mid price brand

C 5mg ISO tar Virginia style

D 1mg ISO tar Virginia style

E Never-Smokers

Male to female ratio = 1:1 (no min. number from any gender)

Group E (never-smokers) closely matched to groups (A-D) in relation to age, gender, body mass index (BMI).

Population Characteristics

Recruited = 96 subjects

Analysis = 95 subjects (1 exclusion Group D)

Mean Age = 38 years

Mean BMI stable across groups (25 kg/m2)

Race = Caucasian (89%), Asian (7%), Other (4%)

Group Male Female Total

A 16 4 20

B 15 5 20

C 9 11 20

D 7 8 15

E 10 10 20

Study Design : Screening (non-residential)

One week prior to Day 1

Signing of consent form

Medical history

Smoking history

Demographic data: gender, age

Salivary cotinine test

Blood carboxyhaemoglobin

Spirometry

Subjects also obtained cigarettes and given butt collection tin & smoking diaries

Study Design : Residential Visit (Days 1 & 2)

24 hours in research unit

Saliva cotinine test on arrival

24 hour urine collection

Blood sample

Butt collection

Smoking topography measurements

Diaries of smoking behaviour

Questionnaires on nicotine dependence (or smoke exposure (ETS) for never-smokers)

Ethics

Approval

• Pre-study approval obtained from Local Research Ethics Committee (LREC), UK

Conduct

• Study conducted in accordance with the relevant articles of the “Declaration of Helsinki” and ICH GCP consolidated guidelines

Biomarker Selection

Biomarker Sample Constituent Nicotine Urine

Cotinine Saliva & Urine

Trans-3’-hydroxycotinine Urine Nicotine

Nicotine-N-glucuronide Urine

Cotinine-N-glucuronide Urine

Trans-3’-hydroxycotinine-Ο-glucuronide Urine

NNAL Urine NNK

NNN Urine NNN TSNA

NAB Urine NAB

NAT Urine NAT

MHBMA Urine 1,3-Butadiene

HPMA Urine Acrolein

SPMA Urine Benzene

Hydroxypyrene (1-OH-Pyrene) Urine Pyrene (PAH)

Creatinine Urine -

Carboxyhaemoglobin Blood Carbon monoxide

NIC

OTIN

E E

QU

IV

ALEN

TS

T

SN

AS

Biomarker Analysis

Biomarker analysis

• ABF GmbH, Munich, Germany (Prof. Gerhard Scherer)

Statistical Analysis

The formal statistical analyses on each of the biomarkers of interest consisted of two comparisons.

• The first comparison compared biomarker concentrations of smokers and never-smokers using the Student's t test.

• The second comparison was based on a standard two-way analysis of variance (ANOVA)

• Within this second comparison, the contrasts of interest between smoking groups (C versus A and B, D versus A and B, C versus D) were tested using the Bonferroni-Holm procedure for multiple comparisons.

Results: Carboxyhaemoglobin (CO)

At Screening: %COHb significantly higher in smokers as compared to never-smokers %COHb significantly higher for 10mg smokers as compared to 5mg smokers No significant gender differences observed for COHb concentrations

E

C,D,E

B,E

B,E

A,B,C,D

02

46

810

CO

Hb [%

]

M A

F M B

F M C

F M D

F M E

F

aa

a

a

b,c,d

02

46

810

CO

Hb [%

]

M a:non-smoker

F M b: 1 mg

F M c: 5 mg

F M d: 10 mg

F

Results: Nicotine Equivalents*Nicotine + 5 metabolites

• Nicotine uptake was significantly higher in smokers as compared to never- smokers (p<0.0001)

• Nicotine equivalents concentrations were significantly higher for 10mg smokers as compared to 5mg or 1mg smokers

• Nicotine equivalents concentrations marginally but consistently higher in females as compared to males

C,D,E

C,D,E

A,B,E

A,B,E

A,B,C,D

010

2030

Nic

otin

e e

quiv

ale

nts

[µg/m

g c

reatin

ine]

M A

F M B

F M C

F M D

F M E

F

b,c,d

a,d

a,d

a,b,c

010

2030

Nic

otin

e e

quiv

ale

nts

[µg/m

g c

reatin

ine]

M a:non-smoker

F M b: 1 mg

F M c: 5 mg

F M d: 10 mg

F

Results: NNAL (TSNA - NNK)

• NNAL uptake was significantly higher in smokers as compared to never-smokers (p<0.0001)

• NNAL concentrations were significantly higher for 10mg smokers as compared to 5mg or 1mg smokers • No significant gender differences observed for NNAL concentrations

b,c,d

a,d

a,d

a,b,c

0

200

400

600

800

NN

AL [p

g/m

g c

reatin

ine]

M a:non-smoker

F M b: 1 mg

F M c: 5 mg

F M d: 10 mg

F

C,E

C,D,E

A,B,E

B,E

A,B,C,D

0

200

400

600

800

NN

AL [p

g/m

g c

reatin

ine]

M A

F M B

F M C

F M D

F M E

F

Results: NNN (TSNA)

• NNN uptake was significantly higher in smokers as compared to never-smokers (p<0.0001) • NNN concentrations were significantly higher for 10mg smokers as compared to 5mg and 1mg smokers • No significant gender differences observed for NNN concentrations

E

C,D,E

B,E

B,E

A,B,C,D

010

2030

NN

N [p

g/m

g c

reatin

ine]

M A

F M B

F M C

F M D

F M E

F

b,c,d

a,d

a,d

a,b,c

010

2030

NN

N [p

g/m

g c

reatin

ine]

M a:non-smoker

F M b: 1 mg

F M c: 5 mg

F M d: 10 mg

F

Results: NAB (TSNA)

NAB uptake was significantly higher in smokers as compared to never-smokers (p<0.0001) NAB concentrations were significantly higher for 10mg smokers as compared to 5mg or 1mg smokers No significant gender differences observed for NAB concentrations

C,E

C,D,E

A,B,EB,E

A,B,C,D

050

100

150

200

NA

B [p

g/m

g c

reatin

ine]

M A

F M B

F M C

F M D

F M E

F

b,c,d

a,da,d

a,b,c

050

100

150

200

NA

B [p

g/m

g c

reatin

ine]

M a:non-smoker

F M b: 1 mg

F M c: 5 mg

F M d: 10 mg

F

Results: NAT (TSNA)

NAT uptake was significantly higher in smokers as compared to never-smokers (p<0.0001) NAT concentrations were significantly higher for 10mg smokers as compared to 5mg or 1mg smokers No significant gender differences observed for NAT concentrations

C,D,E

C,D,E

A,B,E

A,B,E

A,B,C,D

0

200

400

600

800

NA

T [p

g/m

g c

reatin

ine]

M A

F M B

F M C

F M D

F M E

F

b,c,d

a,d

a,d

a,b,c

0

200

400

600

800

NA

T [p

g/m

g c

reatin

ine]

M a:non-smoker

F M b: 1 mg

F M c: 5 mg

F M d: 10 mg

F

Results: MHBMA (1,3-Butadiene)

MHBMA uptake was significantly higher in smokers as compared to never-smokers (p<0.0001) MHBMA concentrations did not differ significantly with product tar level No significant gender differences observed for MHBMA concentrations

E

E

E

E

A,B,C,D

05

1015

MH

BM

A [n

g/m

g c

reatin

ine]

M A

F M B

F M C

F M D

F M E

F

b,c,d

a

a

a

05

1015

MH

BM

A [n

g/m

g c

reatin

ine]

M a:non-smoker

F M b: 1 mg

F M c: 5 mg

F M d: 10 mg

F

Results: HPMA (Acrolein)

HPMA uptake was significantly higher in smokers as compared to never-smokers (p<0.0001) HPMA concentrations did not differ significantly with product tar level HPMA concentrations were significantly higher in males as compared to females (p=0.002) even on correction for cigarette number

E

E

E

E

A,B,C,D

0

1000

2000

3000

4000

HP

MA

[ng/m

g c

reatin

ine]

M A

F M B

F M C

F M D

F M E

F

b,c,d

a

a

a

0

1000

2000

3000

4000

HP

MA

[ng/m

g c

reatin

ine]

M a:non-smoker

F M b: 1 mg

F M c: 5 mg

F M d: 10 mg

F

Results: SPMA (Benzene)

SPMA uptake was significantly higher in smokers as compared to never-smokers (p<0.0001) SPMA concentrations did not differ significantly with product tar level No significant gender differences observed for SPMA concentrations

E

E

E

E

A,B,C,D

05

1015

2025

SP

MA

[ng/m

g c

reatin

ine]

M A

F M B

F M C

F M D

F M E

F

b,c,d

a

a

a

05

1015

20

SP

MA

[ng/m

g c

reatin

ine]

M a:non-smoker

F M b: 1 mg

F M c: 5 mg

F M d: 10 mg

F

Results: OH-Pyrene (Pyrene – PAHs)

OH-pyrene uptake was significantly higher in smokers as compared to never-smokers (p<0.0001) OH-pyrene concentrations did not differ significantly with product tar level OH-pyrene concentrations were significantly higher in males as compared to females (p=0.04) even on correction for cigarette number

E

E

E

E

A,B,C,D

0.5

11.5

OH

-Pyr

ene [n

g/m

g c

reatin

ine]

M A

F M B

F M C

F M D

F M E

F

b,c,d

a

a

a

0.5

11.5

OH

-Pyr

ene [n

g/m

g c

reatin

ine]

M a:non-smoker

F M b: 1 mg

F M c: 5 mg

F M d: 10 mg

F

Conclusions

All biomarkers measured were significantly higher in smokers as compared to never-smokers (p<0.0001)

General association between increasing nicotine equivalents concentrations and increasing tar levels

Nicotine equivalents marginally but consistently higher in females as compared to males

In general, TSNA concentrations were significantly higher in 10mg smokers as compared to 5mg or 1mg smokers

Conclusions

MHBMA and SPMA concentrations did not differ significantly with product tar level or gender

HPMA and OH-pyrene concentrations did not differ significantly with product tar level

HPMA and OH-pyrene concentrations were found to be significantly higher in males as compared to females

Overall, smokers of lower ISO yield cigarette products (1-6mg) have reduced exposure to certain tobacco smoke constituents as compared to smokers of high ISO yield (9-10mg) cigarette products.

Acknowledgements

Clinical team & Co-authors

Covance

Ökolab

ABF